Sensory-directed isolation and identification of an intense salicin-like bitter compound in infected teas with bird's eye spot disease.

Teas infected with bird's eye spot disease generally exhibited a lingering and long-lasting, salicin-like bitter taste, which was unpalatable to consumers. Sensory-directed isolation processes have been performed in this study to investigate the salicin-like bitter compounds in infected teas. Results showed that infected teas were extracted using a 70% methanol aqueous solution to produce methanol extract, which was then further separated by sequential solvent extraction (SSE) to obtain dichloromethane extract, which contained the salicin-like bitter compounds. The dichloromethane extract was then isolated by flash chromatography to produce two salicin-like bitter fractions, eluted using 60% and 65% methanol aqueous solution. Finally, these two salicin-like bitter fractions were analyzed by RP-HPLC using 60-68% and 70-75% methanol aqueous solution, respectively, affording the location of the salicin-like bitter compounds in RP-HPLC chromatograms. Moreover, a new ursane-type triterpenoid, camellisin A methyl ester, was identified from infected teas. This study has provided preliminary isolation methods of salicin-like bitter compounds from the infected teas, which were essential to designing targeted debittering strategies for infected teas and improving the quality of the finished tea and the effective utilization of fresh tea leaves.

[Method for Japanese encephalitis virus NS3 protease activity analysis and high-throughput screening assay for inhibitors].

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.

Biodegradation of bensulphuron-methyl by a novel Penicillium pinophilum strain, BP-H-02.

A fungal strain able to rapidly degrade bensulphuron-methyl (BSM), called BP-H-02, was isolated for the first time from soil that had been contaminated with BSM for several years. BP-H-02 can use BSM as the sole carbon and energy source for growth in a mineral salt medium. Based on morphological and internal transcribed spacer (ITS) analysis, BP-H-02 was identified as Penicillium pinophilum. Under optimal conditions (pH 6.5, temperature 30 °C and 200 mg/L VSSinoculum), more than 87% of the initially added BSM (50mg/L) was degraded after 60 h. Metabolites were identified as 2-amino-4,6-dimethoxypyrimidine and 1-(4,6-dimethoxypyrimidin-2-yl) urea by liquid chromatography-mass spectrometry (LC-MS), and a possible degradation pathway was deduced. In a soil bioremediation experiment, inoculation of soil with BP-H-02 promoted the degradation of BSM more effectively than did the control. These results revealed that BP-H-02 can biodegrade bensulphuron-methyl efficiently and could potentially be used to bioremediate sulphonylurea herbicides contamination.