Quantitative Measurement Reveals Dynamic Volatile Changes and Potential Biochemical Mechanisms during Green Tea Spreading Treatment.

Quantitative data provide clues for biochemical reactions or regulations. The absolute quantification of volatile compounds in tea is complicated by their low abundance, volatility, thermal liability, matrix complexity, and instrumental sensitivity. Here, by integrating solvent-assisted flavor evaporation extraction with a gas chromatography-triple quadrupole mass spectrometry platform, we successfully established a method based on multiple reaction monitoring (MRM). The method was validated by multiple parameters, including the linear range, limit of detection, limit of quantification, precision, repeatability, stability, and accuracy. This method was then applied to measure temporal changes of endogenous volatiles during green tea spreading treatment. In total, 38 endogenous volatiles were quantitatively measured, which are derived from the shikimic acid pathway, mevalonate pathway, 2-C-methylerythritol-4-phosphate pathway, and fatty acid derivative pathway. Hierarchical clustering and heat-map analysis demonstrated four different changing patterns during green tea spreading treatment. Pathway analysis was then conducted to explore the potential biochemistry underpinning these dynamic change patterns. Our data demonstrated that the established MRM method showed high selectivity and sensitivity for quantitative tea volatile measurement and offered novel insights about volatile formation during green tea spreading.

Macro-composition quantification combined with metabolomics analysis uncovered key dynamic chemical changes of aging white tea.

It is a common belief in China that aging could improve the quality of white tea. However, the stored-induced compositional changes remain elusive. In this study, ten subsets of white tea samples, which had been stored for 1-, 2-, 3-, 4-, 5-, 6-, 7-, 10-, 11- and 13- years, were selected. Macro-compositions were quantified firstly. As the results showed, it was interesting to find total flavonoids, thearubigins (TRs), and theabrownines (TBs) increasing, accompanied with a gradual decrease of total polyphenols, which suggest a conversion of phenolic component in the aging process. Then, nontargeted metabolomics was further conducted on selected subsets of samples, including 1-, 7- and 13- years stored to profile their conversion. As a result, most different metabolites were related to flavonol glycosides and flavone glycosides, suggesting dynamic phenolic component changes were vital in aging. The partial least-squares-discriminant analysis (PLS-DA) also identified them as markers in distinguishing.

Widely Targeted Metabolomics Analysis Reveals Great Changes in Nonvolatile Metabolites of Oolong Teas during Long-Term Storage.

As a semifermented tea, oolong is exceedingly popular worldwide for its elegant, flowery aroma and mellow, rich taste. However, recent marketing trends for old oolong teas and their chemical quality largely remain unexplored. In this study, we applied widely targeted metabolomics using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with multivariate analysis to investigate the chemical change of oolong teas in the aging process. With the increasing of store time, most nongalloylated catechins; tannins, including TFs and proanthocyanidins; flavonols and glycosylated flavonols; amino acids and their derivatives; nucleotides and their derivatives; and lots of alkaloids and phospholipids declined, while most fatty acids and organic acids increased, and galloylated catechins, GA, and caffeine were almost stable. The result also suggested that approximately seven years (but not an infinite extension) was a special period for oolong tea storage, which brings about excellent taste.

Identification and comparison of oligopeptides during withering process of White tea by ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry.

Peptides could have specific tastes or bioactivities depending on the length and sequence of amino acids. Till date it remains unknown what peptides are formed during the white tea manufacturing process and whether they contribute to the flavor or bio-activities of white tea. As a first step to address these questions, we applied ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry (UPLC-Quadrupole-Orbitrap-UHRMS) to monitor peptides dynamic changes during the withering process. A total of 196 abundant peptides were identified. Most of them were oligopeptides within a molecular weight of 1000 Da. Four of them were randomly selected, synthesized peptides were applied for further confirmation and quantification. Sequence analysis suggested that some of them were potential taste contributors. Proteinase cleave site analysis identified two separate periods of active proteins degradation at 0-12 h and 30-42 h of the withering processes. Further analysis of cleavage sites also suggested that protein degradation during withering steps were random rather than a stepwise reaction.

Non-targeted metabolomics reveals distinct chemical compositions among different grades of Bai Mudan white tea.

So far, the chemical quality of different grades of white tea has largely remained unexplored. The objective of this study was to establish a model for quality evaluation of different grades of Bai Mudan white tea. We applied non-targeted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry in combination with multivariate analysis and analyzed four different grades of Bai Mudan white tea. We found that the metabolite composition from the super-grade and the first-grade shared higher similarity compared to the second-grade or the third-grade white tea, and the Partial Least Square-Discriminant Analysis model showed high capability to explain the sample variation (R2Y = 0.998, Q2 = 0.95 in negative ionization modes). In total, 93 metabolites were structurally identified, wherein 21 low abundant metabolites showed distinct changes in abundance that were closely correlated with tea grade variation. These findings suggested their potential as markers to discriminate different grades of Bai Mudan white tea.

Efficacy of gemifloxacin for the treatment of experimental Staphylococcus aureus keratitis.

PURPOSE: The objective of the present study was to evaluate the effectiveness of topically applied gemifloxacin for the treatment of experimental Staphylococcus aureus keratitis in a rabbit model. METHODS: Rabbit corneas were intrastromally injected with ~100 colony-forming units (CFU) of S. aureus ATCC25923. Eight hours (early treatment) or 16 h (late treatment) after the injection, 1 topical drop of balanced salt solution (BSS), gemifloxacin ophthalmic solution (0.5%), levofloxacin ophthalmic solution (0.5%), or gatifloxacin eye gel (0.3%) was applied to each eye every 15 min for 5 doses and then, every 30 min for 14 doses. The eyes were examined both before and after treatment. The corneas were harvested from treated and untreated rabbits for the quantitation of bacteria and histological observation. RESULTS: In the early-treatment groups, all 3 fluoroquinolones significantly lowered the clinical severity of infection and the median erosion area of the cornea compared with the BSS control (P=0.000). In the late-treatment groups, gemifloxacin and levofloxacin did not cause a significant reduction in clinical scores compared with the BSS control (P=0.107 and 0.531, respectively), but the gatifloxacin caused a significant reduction in clinical scores compared with the BSS control (P=0.011). The median erosion area significantly decreased with treatment with gemifloxacin, gatifloxacin, and levofloxacin in both early- and late-treatment groups, when compared with the control group (P≤0.022). In the early-treatment groups, the gemifloxacin, gatifloxacin, and levofloxacin groups had significantly lower CFU recovered from the corneas compared with the control group (P<0.01), while in the late-treatment groups, levofloxacin failed to reduce the CFU recovered from the corneas compared with the control group (P=0.695). The minimal inhibitory concentrations for gemifloxacin, gatifloxacin, and levofloxacin against S. aureus ATCC25923 were 0.0625, 0.0625, and 0.125 mg/L, respectively. CONCLUSIONS: Gemifloxacin, similar to gatifloxacin and levofloxacin, can significantly lower the clinical severity and CFU per cornea observed in S. aureus keratitis when early treatment is implemented. Significantly, gemifloxacin showed a significant efficacy improvement in reducing the bacterial load recovered from the corneas in the late-treatment experiment.