Thermo xcalibur software

The secondary metabolites of WT and ΔAfLaeA strains were analyzed by an LC-MS assay in this study. WT and ΔAfLaeA strains were cultured using TYGA liquid medium for 7 days, respectively. The fermentation broths were collected by filtration using a vacuum filter pump. Then, the fermentation broths were extracted three times by mixing them with the same volume of ethyl acetate. The extracts were evaporated under vacuum and dissolved in chromatography-grade methanol (SK, Korea). Finally, the samples were filtered through a 0.22-μm filter and subjected to LC-MS (Thermo Scientific Ultimate 3000; Thermo Fisher Scientific, USA). The metabolic profiles of the WT and ΔAfLaeA strains were compared using Thermo Xcalibur software (Thermo Fisher Scientific). Untargeted metabolomics was performed using Compounds Discoverer 3.0 software (Thermo Fisher Scientific).

Three hyphal mass discs (7 mm) of each strain were incubated in PD broth for 7 days at 28 °C and 180 rpm, followed by mass collection through vacuum filtration [44 (link)]. The biomass was dried, weighed (WT, 3.14 g; ΔAosec22, 2.89 g), and used to prepare extracts of the same concentration. Ethyl acetate (250 mL) was added to extract the fermentation broth and ultrasonic extraction was performed for 40 min. The fermentation broth was allowed to stand for 12 h and the crude extract was concentrated in ethyl acetate under vacuum [44 (link)]. The crude extract was dissolved in 500 µL of analytic-grade methanol and the solution was filtered through a 0.22 µm membrane filter for liquid chromatography–mass spectrometry (LC–MS) analysis. The metabolic profiles of the WT and mutant strains were compared using the Thermo Xcalibur software (Thermo Fisher Scientific). Untargeted metabolomics was performed using Compounds Discoverer 3.0 software (Thermo Fisher Scientific) [49 (link)].

Semi-untargeted qualitative and quantitative analyses were performed by an HPLC system (Dionex ultimate 3000 HPLC, ThermoFisher Scientific, Waltham, MA, USA) coupled via atmospheric-pressure chemical ionization (APCI) to a high-resolution tandem mass spectrometry instrument (HRMS²; LTQ Orbitrap, ThermoFisher Scientific, Waltham, MA, USA). Separation was carried out with Luna C18(2) 150 × 2 mm, 100 Å, 3 µm (Phenomenex, Torrance, CA, USA) using 0.1% (v/v) formic acid (solvent A) and 0.1% (v/v) acetonitrile (solvent B) as mobile phases. Chromatographic separation consisted of a solvent ramp from 5% to 100% solvent B for 30 min, followed by column reconditioning of 15 min. The flow rate was set to 0.2 mL min⁻¹ and the injection volume to 10 µL. Detection parameters were the following: negative ionization mode; capillary temperature: 250 °C; APCI vaporizer temperature: 450 °C; sheath gas: 35 Arb; auxiliary gas: 15 Arb; discharge needle: 5 kV; the acquisition was carried out in Dependent Scan mode with a mass range from 220 to 1000 m/z, normalized CE: 35V. Raw data obtained were analyzed with Thermo Xcalibur software (ThermoFisher Scientific, Waltham, MA, USA); molecules were identified using the MetFrag online tool [90 (link)]. Quantification of identified acidic triterpenes was performed with a calibration curve of moronic acid (TCI-Europe, Bruxelles, Belgium, EU).

Liver lipid profiling was performed on a LTQ XL mass spectrometer (Thermo Fischer Scientific, West Palm Beach, FL) equipped with an automated nanospray source (TriVersa Nanomate, Advion Biosciences, Ithaca, NY) using nanoelectrospray chips with 5.5-µm diameter spraying nozzles. The ion source was controlled using the Chipsoft 8.3.1 software (Advion Biosciences). Ionization voltage was −1.4 kV in negative mode and backpressure was set at 0.4 psi. Ion transfer capillary temperature and tube voltage were 200°C and 100 V, respectively. For the lipid analysis, five microliters of each sample were loaded into a 96-well plate (Eppendorf, Hamburg, Germany), and placed on the Nanomate cooling plate, which was set to 5°C to prevent solvent evaporation. Full scan spectra were collected at the m/z 400–1,000 in positive ion mode. The mass spectra of each sample were acquired in profile mode over 2 min. A collision-induced dissociation (CID) was performed with over an isolated width of 3 m/z units, with 35% collision energy. The tandem mass spectrometry (MS/MS) triggering threshold was set to 1,000, with a default charge state of 1. All spectra were recorded with the Thermo Xcalibur software (version 2.1., Thermo Fisher Scientific). MS/MS spectra were analyzed for the identification of lipid species using LipidBlast [16] (link) and in-house library.

After the GC-TOF-MS and UHPLC-LTQ-ESI-IT-MS/MS analyses, raw data files were converted into a computable document format (*.cdf) using LECO Chroma TOF software (Version 4.4, LECO Corp.) and using the thermo file converter program in Thermo Xcalibur software (version 2.1, Thermo Fisher Scientific). The acquired NetCDF format (*.cdf) files were processed to determine retention time, baseline correction, peak detection, and alignment using the MetAlign software package [35 ]. After alignment, the resulting data file (*.csv) including the corrected peak retention times, peak areas, and corresponding mass (m/z) data was exported to Microsoft Excel (Microsoft, Redmond, WA, USA) for further analysis. Multivariate statistical analysis using SIMCA-P+ 12.0 software (version 12.0, Umerics, Umea, Sweden) was performed to compare metabolite differences between commercial gochujang samples by unsupervised principal component analysis (PCA), supervised partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA (OPLS-DA). Significant differences (p-value < 0.05) between selected metabolites, their relative contents, and the values from the physicochemical characteristics assays were evaluated by one-way analysis of variance using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). The differences between metabolites were visualized by heat map analysis using MEV software [36 ].