The determination of BAs was conducted based on the work of Wang et al. [26 (link)]. H. alvei H4 strains (wild type, ΔluxI, ΔluxR and ΔluxIR) were cultivated in LB supplemented with 0.005% pyridoxal-5-phosphate and 0.1% precursor amino acid for 24 h. Then 1 mL of culture was mixed with 9 mL 10% trichloroacetic acid in a centrifuge tube. After standing for 2 h at 4 °C, the mixture was homogenized for 10 min (3000× g). A 200 μL volume of supernatant was derivatized using 80 μL 2 mol/L NaOH and 800 μL 10 mg/mL dansyl chloride dissolved in acetone. After water-bath heating at 45 °C for 40 min, 50 μL ammonium hydroxide and 550 μL acetonitrile were added into the dansyl derivatives, homogenized for 5 min (3000× g) and filtrated through a 0.22 μm filter. Finally, 10 μL aliquots were injected for HPLC analysis.
The concentrations of BAs were determined by an HPLC system (ZORBAX, Agilent, Tokyo, Japan). An SB-C18 reversed-phase column (5 μm, 4.6 mm × 125 mm; Agilent, Tokyo, Japan) was used for chromatographic separation. The gradient elution program was operated with acetonitrile/water as the mobile phase.
The concentrations of BAs were determined by an HPLC system (ZORBAX, Agilent, Tokyo, Japan). An SB-C18 reversed-phase column (5 μm, 4.6 mm × 125 mm; Agilent, Tokyo, Japan) was used for chromatographic separation. The gradient elution program was operated with acetonitrile/water as the mobile phase.
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