Pack ods a column

Glucose, glutamic acid and lactic acid were analyzed using the SBA-40D biosensor (Biology Institute, Shandong Academy of Sciences, Jinan, Shandong, China). Acetic acid, xylose, furfural, and HMF were analyzed on HPLC (LC-20AD, refractive index detector RID-10A, Shimadzu, Kyoto, Japan) with a Bio-Rad Aminex HPX-87H column (Bio-rad, Hercules, CA, USA) at 65 °C and 5 mM H₂SO₄ solution as the mobile phase at the flow rate of 0.6 mL/min. Phenolic compounds were analyzed on HPLC (UV/Vis detector SPD-20A, at 270 nm, Shimadzu, Kyoto, Japan) with a YMC-Pack ODS-A column (YMC Co., Kyoto, Japan) at 35 °C as mentioned before [13 (link)].
Cell growth was indicated by optical density at 600 nm (OD₆₀₀) by the spectrophotometer Beckman Coulter DU800 (Beckman, Brea, CA, USA). The dry cell weight (DCW) was transformed 1 unit of OD₆₀₀ to approximately 0.4 mg/mL of the dry cell weight.

All the transformation samples were analyzed by HPLC-UV on a Shimadzu Analytical Instrument (Shimadzu, Japan). The separation was performed on a YMC-pack ODS-A column (4.6 × 250 mm; i.d., 5 μm; YMC Co., Ltd., Japan) using a mobile phase of water with 0.05% phosphoric acid (A) and acetonitrile (B) at a flow rate of 1 mL/min. The isocratic profile of gastrodin was as follows: 0–25 min, 3% B. The gradient profile of esculin was as follows: 0–20 min, 20% B; 20–25 min, 20–50% B. The gradient profile for the separation of daidzin and baicalin was as follows: 0–1 min, 5% B; 1–25 min, 5–70% B. The column temperature was set at 30°C. The detection wavelength of gastrodin, esculin, daidzin, and baicalin was set at 220, 348, 250, and 280 nm, respectively (Yan et al., 2016 (link); Chang et al., 2018 (link)).

IR spectra were recorded with a NICOLET iS50 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA). NMR data were collected on Bruker Avance-400FT and Avance-600 NMR spectrometers (Bruker Corporation, Billerica, MA, USA). GC–MS analyses were performed on an Agilent 7890A GC system connected to an Agilent 5975 mass spectrometer with a HP-5MS column (30 m × 250 µm × 0.25 µm, Agilent Technologies, Santa Clara, CA, USA). HREIMS spectrum was collected on a Hybrid Quadrupole-Orbitrap GC-MS/MS System (Thermo Scientific, Waltham, MA, USA) equipped with a TraceGOLD TG-5HT column (30 m × 250 µm × 0.1 µm, Thermo Scientific, Waltham, MA, USA). Size-exclusion chromatography was carried out with Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) columns. HPLC analysis was performed on an Agilent 1260 series (Agilent Technologies, Santa Clara, CA, USA) with a C18 RP-column (Extend-C18, 250 × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA). Semi-preparative HPLC was performed on an SSI 23201 system (Scientific Systems Inc., State College, PA, USA) with a YMC-Pack ODS-A column (250 × 10 mm, 5 µm, YMC CO., LTD. Shimogyo-ku, Kyoto, Japan). All fermentations were carried out in MQD-B1R shakers (Minquan Instrument Co., Ltd., Shanghai, China).

An HPLC system (Shimadzu Co., Kyoto, Japan) equipped with a multi-channel pump (LC-20AD) and a diode array detector (SPD-M20A) was used to analyze the extracts from P. cretica obtained by UAE under optimized conditions and HRE. A YMC-Pack ODS-A column (5 μm, 250 mm × 4.6 mm id; YMC Co., Tokyo, Japan) was employed for all separations at 25 °C. The mobile phase was composed of 0.5% TFA in ultrapure water (solvent A) and methanol (solvent B). The elution procedure was as follows: 0–25 min, 25–45% B; 25–40 min, 45–65% B; 40–60 min, 65–90% B; 60–65 min, 90–25% B, with a flow rate of 0.5 mL/min. Detection was carried out at the wavelength of 254 nm. The flavonoids in extracts from P. cretica were identified on the basis of comparison of the retention time and ultraviolet spectrum of standards, and the quantification of flavonoids was performed by the external standard method.

Seven phenolic acids, including p-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, and salicylic acid, were extracted from the soil after passing through a 0.3-mm mesh and assessed by high-performance liquid chromatography (HPLC, Waters, TM 2695 pump/2996 UV-DAD detector/7752 injector, Milford, MA), following the method of Zhang et al.⁴⁸ . These compounds were separated by a YMC-Pack ODS-A column (250 mm × 4.6 mm, 5 µm) and identified and quantified by comparison to their corresponding standards (obtained from Alfa Aesar Co., UK) (Table S7).

CD spectra were determined on a JASCO J-810 spectropolarimeter (JASCO Corporation), and UV data were recorded using a PERSEE UV-VIS spectrophotometer T9 (Beijing, China). NMR experiments were carried out on a Bruker AVANCE III 400 NMR spectrometer (Bruker, Germany), using tetramethylsilane (TMS) or solvent signals as an internal reference. HRESIMS data were collected on an Agilent 6250 TOF LC/MS, and ESIMS data were acquired on an Agilent 1200 series LC/MS system. Semipreparative HPLC was run on a Calmflow^plus system that was equipped with a YMC Pack ODS-A column (10 mm × 250 mm 5 μm, Japan) and a 50D UV-vis Detector (Lumiere Tech Ltd) and with a flow rate of 2.0 ml/min. Packing materials for column chromatography were silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China), ODS (YMC, Japan), and Sephadex LH-20 (GE Healthcare BioSciences AB, Sweden). All chemicals used in the study were of analytical grade.