Tryptophan Quantification in Prebiotic-Supplemented LAB

The tryptophan production by LAB was monitored as previously described (Vaitekūnas et al., 2020 (link)). In the prebiotic-supplemented LAB CFS samples, concentrations of tryptophan were determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS). First, the samples were mixed with an equal volume of acetonitrile and centrifuged for 10 min at 10,000 rpm. The samples were analyzed using the Shimadzu Prominence HPLC system (Shimadzu, Kyoto, Japan) equipped with a photodiode array (PDA) detector (Shimadzu, Kyoto, Japan) and LCMS-2020 mass spectrometer (Shimadzu, Kyoto, Japan) with an electrospray ionization (ESI) source. The chromatographic separation was conducted using a YMC Pack Pro C18 column (3 × 150 mm; YMC, Kyoto, Japan) at 40°C and a mobile phase that consisted of 0.1% formic acid water solution (solvent A) and acetonitrile (solvent B) delivered in the 5–95% gradient elution mode. Mass scans were measured from m/z 50 up to m/z 2,000 at a 350°C interface temperature, 250°C desolvation line (DL) temperature, ±4,500 V interface voltage, and neutral DL/Qarray, using N₂ as nebulizing and drying gas. Mass spectrometry data were acquired in both positive and negative ionization modes. The data were analyzed using LabSolutions software (Shimadzu, Kyoto, Japan).