Cbm 20a

Biogenic amines, ammonia, and trimethylamine were analyzed with Özogul (2004) (link) method and measured as mg compound per liter broth. The rapid HPLC technique with a reversed phase column and a gradient elution program were used.
The HPLC instrument (Shimadzu, Kyoto, Japan) is equipped with a column oven (CTO-20AC), auto sampler (SIL 20AC), two binary gradient pumps (Shimadzu LC-10AT), SPD-M20A diode array detector, and a communication bus module (CBM-20A) with a valve unit FCV-11AL. A reverse-phase column, Spherisorb 5 Si C18 pH-St, 250 mm × 4.6 mm (Phenomenex, Macclesfield, Cheshire, United Kingdom) was used. Ammonia and TMA are measured using the same method with the other BA at the same injection.
BAs analysis were done using continuous gradient elution with acetonitrile (eluent A) and HPLC grade water (eluent B). The total separation time was 20 min and the injection volume was 10 μL. Detection wavelength was at 254 nm. A standard curve for ammonia and all amines in the range of 0–50 mg/mL was prepared. Correlation coefficient of peak area against amine standard concentrations for each compound was calculated after injecting five replicates of each standard solution of amine. The correlation coefficient (r2) in the curve was >0.99 for each benzoylated amine and ammonia.

High-performance liquid chromatography (HPLC) fingerprinting of ME and EA was conducted to detect the presence of patuletin. The purity of patuletin was also determined as described earlier (Li et al., 2010). Briefly, both samples and standards including patuletin, patulitrin, and methyl protocatechuate were prepared in the mobile phase. An HPLC-Prominence System Controller CBM-20A (Shimadzu) equipped with a degasser (DGU-20A5), quaternary pump (LC-20AT), autosampler (SIL-20A), and diode array detector (SPD-M20A) and software LC Solution were used for analysis.

The qualitative and quantitative analyses of chlorogenic acid (CGA) and rutin in DMLEs were conducted using an LC-MS 8050 chromatography system (Shimadzu, Kyoto, Japan) composed of a binary solvent delivery system (LC-30 AD), a controller (CBM 20A), an autosampler (SIL-30A), and a column thermostat (CTO-20AC). Tandem mass spectrometry (MS/MS) analysis was performed positively and negatively on a triple quadrupole equipped with a positive electrospray ionization (+ESI) source. The optimal parameters of ESI-MS were as follows: interface temperature of 340°C, DL temperature of 200°C, nebulizing gas flow at 2.8 L/min, heating gas flow at 8 L/min, and temperature of drying gas at 400°C. The active biological compounds were monitored using a scheduled multiple reaction monitoring mode and separated using a Kinetex C18 column (150 × 2.1 mm, 2.6 µm; Phenomenex, Torrance, CA, USA) at a flow rate of 0.3 mL/min, injection volume of 2 µL, and separation temperature of 40°C. The mobile phase consisted of water containing 5 mM ammonium acetate and 0.1% formic acid (A) and methanol containing 2.5 mM ammonium acetate (B) (gradient 0–18 min; 5%–100% B).

Shimadzu Prominence series UFLC system (Lyon, France), equipped with a CBM-20A controller bus module, a LC-20 AD liquid chromatograph, a CTO-20A column oven and an SPD-20A UV-visible detector was used for Gradient HPLC analysis. Wavelength was fixed at 295 nm for UV-VIS detection. The separation was performed using a C4 reverse-phase column (Jupiter^®, 5 µm, 300 A, 150 × 4.6 mm) at a flow rate of 1 mL min⁻¹. The gradient initial solution is 95% solvent A − 5% solvent B (A = H₂O/ACN/TFA: 98.9 v%/1 v%/0.1 v%, B = ACN/H₂O/TFA: 89.9 v%/10 v%/0.1 v%) over 5 min. In a second step, the samples were eluted by a gradient developed from 5 to 90% of solvent B in solvent A over 15 min. The concentration of solvent B was maintained over 5 min. Then, the concentration of solvent B was decreased to 5% over a period of 5 min to re-equilibrate the system, followed by an additional 5 min at this final concentration.

¹H- and ¹³C-NMR, COSY, HSQC, HMBC, and NOESY data were obtained using a superconducting FT-NMR 400 or 500 MHz spectrometer (Agilent Technologies, Santa Clara, CA, USA). HR-ESI mass spectra were recorded on an Agilent Technologies, 6530 Accurate-Mass Q-TOF LC/MS. The HPLC system (Shimadzu, Tokyo, Japan) consisted of a UV/VIS detector (model SPD-20A), two pumps (model LC-20AT), a system controller (model CBM-20A) and a workstation (model HW-2000 solution). Column chromatography was performed using Sephadex LH-20 gel (25–100 μM mesh, Pharmacia, Stockholm, Sweden) and silica gel (230–400 mesh, Merck, Darmstadt, Germany).

Preparative HPLC was performed using a Shimadzu LabSolutions system (Shimadzu, Kyoto, Japan), an SPD-M20A photodiode array detector, a CTO-20A column oven, a CBM-20A communications bus module, and an LC-20AD binary pump. HPLC separation was performed with a gradient system using 0.1% (v/v) trifluoroacetic acid in water as mobile phase A and 0.1% (v/v), trifluoroacetic acid in methanol as mobile phase B, a Tsk-gel ODS-80Ts (20 mm × 250 mm, 5 μm; #0018409; Tosoh Co., Ltd., Tokyo, Japan), and a flow rate of 8.0 ml/min. The injection volume was 500 μL and the temperature of the column oven was maintained at 40°C. C3G was separated using a linear gradient, commencing with 33% B over 0–22.5 min; then followed by 90% B over 22.5–35 min, and 33% B over 35–50 min, with elution between 16 and 22 min. The eluate was collected and evaporated to obtain purified-C3G. The gradient from 35 min onwards was used to re-equilibrate the system between samples. The absorbance of C3G was monitored at 280 nm and 513 nm using a UV detector. The HPLC chromatogram of purified-C3G was described in Supplementary Figure S7.