Vwd detector

Sample preparation. A 1 g sample with 5 mL distilled water was vortexed for 1 min, followed by 15 min ultrasonic treatment at room temperature, then centrifuged at 10,000 rpm for 10 min. The supernatant, containing extracted organic acids, was filtered through a nylon filter (pore size 0.45 μm), and 20 μL was injected into the high-performance liquid chromatography (HPLC) system.
Chromatographic condition. The HPLC analysis was performed utilizing an HP-1200 liquid chromatograph equipped with a quaternary pump, manual injector and VWD detector (Agilent-Technologies, USA, Santa Clara, CA, USA). The column was an Acclaim OA (5 μm; 4 × 150 mm i.d.) from Thermo Fisher Scientific, Waltham, MA, USA. The mobile phase was NaH2PO4 50mM solution at pH = 2.8, elution was done for 10 min at room temperature with a flow rate of 0.5 mL/min. Chromatograms were recorded at wavelength λ = 210 nm, and data acquisition was made with the Agilent ChemStation software.
Quantitative determination. The organic acid content was determined using a five-point calibration curve for each organic acid. The chromatograms of standards, frozen AP, and lyophilized AP are presented in Supplementary Figure S1.

Equal volume of methanol and culturing broth was mixed and standing for 30 min. Through this process, both supernatant SMX and SMX contained within the bacteria were extracted from the mixture. Then mixture of supernatants was obtained by centrifugation at 15,000 × g for 5 min and filtered for high-performance liquid chromatography (HPLC) quantification. The concentration of SMX was analyzed by HPLC (Agilent 1260 II, Santa Clara, CA, USA) equipped with an EC-C18 column (4 µm, 4.6 × 150 mm²) at 35°C. SMX was measured under the detection of a VWD detector (Agilent, Santa Clara, CA, USA) at 265 nm. The mobile phase was a 1:3 ratio of acetonitrile and 0.2% acetic acid with a flow rate of 0.6 mL/min.

Equal volume of methanol and culturing broth was mixed and standing for 30 min. Through this process, both supernatant SMX and SMX contained within the bacteria were extracted from the mixture. Then mixture of supernatants was obtained by centrifugation at 15,000 × g for 5 min and filtered for high-performance liquid chromatography (HPLC) quantification. The concentration of SMX was analyzed by HPLC (Agilent 1260 II, Santa Clara, CA, USA) equipped with an EC-C18 column (4 µm, 4.6 × 150 mm²) at 35°C. SMX was measured under the detection of a VWD detector (Agilent, Santa Clara, CA, USA) at 265 nm. The mobile phase was a 1:3 ratio of acetonitrile and 0.2% acetic acid with a flow rate of 0.6 mL/min.

Amino acids composition was determined using the method reported by Hou et al. [23 (link)] with some modifications, using an Agilent 1100 high-performance liquid chromatography (HPLC) instrument (Wilmington, DE, USA) coupled with a VWD detector (Agilent Technologies, Inc., Wilmington, DE, USA) and a column of Agilent Zoubax Elicpse AAA (4.6 × 150 mm, 3.5 μm). The determination of 17 hydrolysis AAs of 100 mg SYC and SYCHs was performed with 6 M HCl for 22 h, while Trp analysis of 100 mg SYC was performed by alkaline hydrolysis using 5 M NaOH for 20 h. After passing through a 0.22 μm filter, 10 μL of the sample was loaded into the column and eluted at a flow rate of 1.0 mL/min. The temperature was 40 °C, ultraviolet, 338 nm (0–19 min), 266 nm (19.01–25 min); mobile phase A (40 mM sodium dihydrogen phosphate (pH 7.8)); mobile phase B (acetonitrile: methanol: water = 45:45:10). All of the AAs were detected at 338 nm, except Pro, which was detected at 266 nm. The AAs were identified and quantified by authentic AA standards comparing the retention time and peak.

Glucosinolates were extracted and analyzed as previously described (1 (link)). Freeze-dried samples (100 mg) were boiled in 5 mL water for 10 min. The supernatant was collected after centrifugation, and the residues were washed once with water, centrifuged, and then combined with the previous extract. The aqueous extract was applied to a DEAE-Sephadex A-25 column (Sigma Chemical Co., Saint Louis, USA). The glucosinolates were converted into their desulpho analogs by overnight treatment with 100 μL of 0.1% aryl sulphatase (Sigma Chemical Co., Saint Louis, USA), and the desulphoglucosinolates were eluted with 1 mL water. HPLC analysis of desulphoglucosinolates was carried out using an Agilent 1260 HPLC instrument equipped with a VWD detector (Agilent Technologies, Inc., Palo Alto, USA). Samples were separated at 30°C on a Waters Spherisorb C18 column (250 × 4.6 mm i.d.; 5 μm particle size) using acetonitrile and water at a flow rate of 1.0 mL min⁻¹. Absorbance was detected at 226 nm. Glucosinolates were quantified by using ortho-Nitrophenyl β-D-galactopyranoside (Sigma Chemical Co., Saint Louis, USA) as the internal standard and considering the response factor of each glucosinolate. Result of glucosinolate content was expressed as μmol g⁻¹ of dry weight.

Ascorbic acid content was determined according to the previous report (1 (link)). Fifty milligram of sample powder was extracted with 5 mL 1.0% oxalic acid, subsequently centrifuged 5 min at 4,000 g. Each sample was filtered through a 0.45 μm cellulose acetate filter. HPLC analysis of ascorbic acid was carried out using an Agilent 1260 instrument with a variable wavelength detector (VWD) detector (Agilent Technologies, Inc., Palo Alto, USA). Sample were separated on a Waters Spherisorb C18 column (150 × 4.6 mm i.d.; 5 μm particle size), using a solvent of 0.1% oxalic acid at a flow rate of 1.0 mL min⁻¹. The amount of ascorbic acid was calculated from absorbance values at 243 nm, using authentic ascorbic acid (Sangon Biotech Co., Ltd., shanghai, China) as a standard. Result of ascorbic acid content was expressed as mg g⁻¹ of dry weight.