Hp1100

In order to identify and quantify the main components of the extracts, a series of HPLC-DAD analyses were performed. The HPLC system consisted of a quaternary pump (HP 1100 gradient pump), a degasser (HP 1100), an autosampler (Agilent Infinity 1260), and a Diode Array Detector (Hewlett Packard, Waldbronn, Germany). A ZORBAX Eclipse XDB-C18 column (5 μm, 250 × 4.6 mm, Agilent, Santa Clara, CA, USA) was used at room temperature, while the samples were injected after filtration (0.45 μm, PVDF syringe filters, Teknokroma, Barcelona, Spain). The gradient method, including three solvents (water, methanol, acetonitrile, acidified with TFA 0.2% v/v), has been extensively described in previous papers [81 (link),84 (link),85 (link)]. The detection was performed at 230, 280, and 360 nm, and the elaboration of chromatographic data was performed on a ChemStation for LC 3D software version B.04.06 (Agilent Technologies, Santa Clara, CA, USA). Hydroxytyrosol (Extrasynthese, France) and luteolin (Extrasynthese, France) standards were used for the development of the respective calibration curves for the quantification of the compounds in extracts.

Bacterial growth was monitored by measuring the cell density at 600 nm (TU-1901, Persee, Beijing, China). Samples from the flask cultures were centrifuged at 13,000 rpm for 10 min, and the supernatant was stored at -20°C for future analysis. The sugar and fermentation products were determined using a high-performance liquid chromatograph (HP1100, Agilent Technologies, Palo Alto, USA) equipped with an ion exclusion Aminex HPX 87-H column (Bio-Rad, Richmond, USA), and 5 mM H₂SO₄ (0.4 ml/min) at 65°C was used as the mobile phase. The forms of D-xylose, L-arabinose and D-glucose were detected using a refractometer (Agilent, HP1047A), and acetoin was detected using an UV absorbance detector (Agilent, G1315D).

Merck 60 F254 TLC plates were used in monitoring the reactions.
¹H NMR spectra were recorded with an INOVA 400 instrument with a 5 mm probe. All chemical shifts were quoted relative to deuterated solvent signals (δ in ppm and J in Hz).
HPLC-MS analysis was carried out with an Agilent Technologies HP1100 instrument, equipped with a ZOBRAX-Eclipse XDB-C8 Agilent Technologies column (flow: 0.4 mL/min; mobile phase: CH₃CN/H₂O gradient from 30 to 80% CH₃CN in 8 min and then 80% CH₃CN until 25 min), coupled with an Agilent Technologies MSD1100 single-quadrupole mass spectrometer (full-scan mode from m/z 50 to 2600; scan time of 0.1 s in positive ion mode, ESI spray voltage of 4500 V, nitrogen gas of 35 psi (1 psi = 6894.7 Pa), drying gas flow of 11.5 mL/min, fragmentor voltage of 20 V).

The compounds used for fluorescent labeling were resuspended in DMSO to a final concentration of 50 nM. Then, 4 µL (200 nmol) of these compounds were dissolved in 95.3 µL of DMSO + 0.7µL of DIPEA and labeled via the amino group to DY647-Peg4 (0.2 mg, 200 nmol) (cat. no 647P1-01, Dyomics, Germany) utilizing a reactive NHS ester group. The mixture was incubated in the dark at room temperature (RT) for 2 h. The conjugate was purified on a reversed-phase high-performance liquid chromatography (HP-1100, Agilent, USA) using an ACN/H₂O gradient from 20% to 80% ACN (BH296) or 40% to 60% ACN (BH267.meta), allowing separation of unlabeled and labeled compounds. Then, the labeled compound was lyophilized, dissolved in DMSO, and stored at −20 °C.

Semipreparative
HPLC was conducted on an HP 1100 liquid chromatograph (Agilent Technologies,
Waldbronn, Germany) fitted with a Zorbax Phenyl-Hexyl reverse-phase
(9.4 × 250 mm) C18 column (Agilent Technologies, Waldbronn, Germany).
A mobile phase (3 mL/min) consisting of 2% acetic acid in water (solvent
A) and acetonitrile (solvent B) was used. A solvent gradient was applied
over a total run time of 50 min: initially 100% A for 10 min, reducing
to 90% A over 1 min, then reducing to 80% A over 9 min, then reducing
to 60% A over 10 min, then reducing to 40% A over 10 min and then
finally to 0% A over 10 min.