Thermophilic bacteria play an important role in aroma formation during pile-fermentation process of dark tea. With the aim to reveal the impact of thermophilic bacteria on volatile compounds in dark tea, Bacillus licheniformis (thermophilic bacteria) were inoculated into sun-dried green tea for spontaneous fermentation. In this study, headspace solid phase microextraction combined to gas chromatography mass spectrometry (HS-SPME-GC-MS), odor activity value (OAV), principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) were employed to investigated the characteristics of volatile compounds during thermophilic bacteria pile-fermentation. According to HS-SPME-GC-MS, a total of 64 volatile compounds were identified. PCA revealed that tea samples could be clearly discriminated from each other. Furthermore, sulcatone, hexanoic acid, linalool, 2-methyl-trans-decalin, myrtenal, and α-ionone were found to play an important role in discrimination of tea samples, based on OPLS-DA. In addition, the OAV could effectively characterize the aroma contribution of volatile compounds during thermophilic bacteria pile-fermentation, and (Z)-linalool oxide (furanoid), α-ionone, (E)-2-nonenal, and linalool were the critical volatile compounds of aroma quality in dark tea. This study provides insight into volatile compounds characteristics during thermophilic bacteria pile-fermentation in dark tea.
The gene-encoding carboxylesterase (TM1022) from the hyperthermophilic bacterium Thermotoga maritima (T. maritima) was cloned and expressed in Escherichia coli Top10 and BL21 (DE3). Recombinant TM1022 showed the best activity at pH 8.0 and 85 °C and retained 57% activity after 8 h cultivation at 90 °C. TM1022 exhibited good stability at pH 6.0–9.0, maintaining 53% activity after incubation at pH 10.0 and 37 °C for 6 h. The esterase TM1022 exhibited the optimum thermo-alkali stability and kcat/Km (598.57 ± 19.97 s–1mM–1) for pN–C4. TM1022 hydrolyzed poly(ethylene terephthalate) (PET) degradation intermediates, such as bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). The Km, kcat, and kcat/Km values for BHET were 0.82 ± 0.01 mM, 2.20 ± 0.02 s–1, and 2.67 ± 0.02 mM–1 s–1, respectively; those for MHET were 2.43 ± 0.07 mM, 0.04 ± 0.001 s–1, and 0.02 ± 0.001 mM–1 s–1, respectively. When purified TM1022 was added to the cutinase BhrPETase, hydrolysis of PET from drinking water bottle tops produced pure terephthalic acids (TPA) with 166% higher yield than those obtained after 72 h of incubation with BhrPETase alone as control. The above findings demonstrate that the esterase TM1022 from T. maritima has substantial potential for depolymerizing PET into monomers for reuse.
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