Thermo u3000 hplc system

The contents of total polysaccharides, proteins, and uronic acids in LLP-D and LLP-W were analyzed by previously reported colorimetric methods [19 (link)]. The molecular weights and molecular weight distributions of LLPs were investigated by using high performance size exclusion chromatography equipped with the multi angle laser light scattering and the refractive index detector (Wyatt Technology Co., Santa Barbara, CA, USA) [20 (link)]. Besides, the apparent viscosities of LLPs were determined at the concentration of 10.0 mg/mL by using a Discovery Hybrid Rheometer-1 (TA instruments, New Castle, DE, USA) with a parallel steel plate (40.0 mm diameter and 1.0 mm gap) [20 (link)]. The constituent monosaccharides of LLPs were analyzed by using Thermo U3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Phenomenex gemini 5 μ C18 (150 mm × 4.6 mm) column followed by a formerly reported method [20 (link)]. Moreover, the FT-IR spectra of LLPs were recorded to analyze their chemical structures by using a Nicolet iS 10 FT-IR (ThermoFisher scientific, Waltham, MA, USA) [20 (link)]. Furthermore, the ¹H and ¹³C NMR spectra of LLPs were also recorded to further analyze their chemical structures by using a Bruker Ascend 600 MHz spectrometer with a z-gradient probe (Bruker, Rheinstetten, Germany) [20 (link)].

Culture supernatant was collected at each time point, clarified by centrifugation, and stored at −30 °C until use. For analysis with HPLC, the samples were thawed and mixed with isomaltoheptaose (internal standard). The sugars were fluorescence-labelled with 2-AA, and the reaction mixtures were desalted by solid-phase extraction as described previously [40 (link), 73 (link)]. HPLC was performed using a Thermo U3000 HPLC system (Thermo Fisher Scientific, Waltham, MA). This was equipped with a TSKgel Amide-80 HR column (4.6 × 250 mm, φ = 5 μm) (Tosoh, Tokyo, Japan) at 65 °C, which was equilibrated with 85% solvent A (acetonitrile)/15% solvent B (100 mM ammonium formate buffer, pH 4.3). The elution was performed using a linear increase of solvent B (from 15 to 85%) over 90 min at a flow rate of 1 mL/min. Using a Waters 2475 Fluorescence Detector (Waters Corp., Milford, MA), the labelled sugars were detected at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The concentrations of mono- and oligosaccharides remaining in the spent medium were calculated based on the standard curves generated using similarly labelled standard sugars, and the data were normalized using the internal standard. The concentration of Fuc was measured separately with a colorimetric assay using fucose dehydrogenase (FDH) as described previously [74 (link)].

The F2 fraction was resuspended in methanol (1 mg/mL) and analyzed through Liquid Chromatography–High-Resolution Tandem Mass Spectrometry (LC HRMS/MS). Experiments were performed using a Thermo LTQ Orbitrap XL high-resolution ESI mass spectrometer coupled to a Thermo U3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) and equipped with a 5-µm C18 column (50 × 2.10 mm, Kinetex) (Phenomenex, Torrance, CA, USA), as already described [33 (link)]. The column was eluted at 200 µL × min⁻¹ with a gradient of 0.1% HCOOH supplemented with H₂O (solvent A) and CH₃CN (solvent B). The gradient program was set as follows: 3 min 5% A, 30 min from 5 to 99% B, and 7 min 100% B. A full MS scan event was acquired in positive ion mode. MS parameters were set as follows: spray voltage at 4.8 kV, capillary temperature at 285 °C, sheath gas rate at 32 units of N₂ (ca. 150 mL × min⁻¹), and the auxiliary gas rate at 15 units of N₂ (ca. 50 mL/min). High-resolution tandem mass spectrometry (HRMS/MS) data were acquired in data-dependent acquisition mode. CID fragmentation was employed to obtain HRMS/MS scans, setting an isolation width of 2.0, normalized collision energy of 35, activation Q of 0.250, and an activation time of 30 ms.