Ics 5000 system

Weighing about 5 mg of CMPP was hydrolyzed with 1 mL 2.5 mol/L trifluoroacetic acid (TFA) at 121 °C for 2 h, and the TFA was removed three times with methanol and nitrogen gas. Then, the dissolved hydrolytic product was injected into high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The equipment consisted of the ICS-5000 system (Thermo Fisher Scientific, Waltham, MA, USA) with Dionex™ CarboPac™ PA20 (150 mm × 3 mm, 6.5 μm) and a pulsed amperometric detector. The work of quantification was operated by professional staff from San-Shu Biotech Co. (Shanghai, China).

Using the phenol-sulfuric acid method with glucose as the standard, the total saccharide content was calculated [26 (link)]. The protein concentration was measured by a BCA protein assay kit (P0012S, Beyotime Biotechnology Corp., Shanghai, China), using BSA as the standard [74 (link)]. The total polyphenol content was determined by the Folin–Ciocalteau method [75 (link)] with gallic acid as a standard.
The composition of the monosaccharides was assessed via the HPAEC-PAD method [76 (link)]. The samples (5 mg) were hydrolyzed for 2 h at 121 °C with 1 mL of 2 M trifluoroacetic acid (TFA) in a sealed tube. At 50 °C, the residual trifluoroacetic acid was removed by a rotary evaporator. The residue was redissolved in methanol (4 mL) and dried five times, after which it was dissolved in distilled water and injected into the HPAEC-PAD, which consisted of an ICS-5000 system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Dionex™ CarboPac™ PA20 (150 mm × 3 mm, 10 μm, Dionex Corp., Sunnyvale, CA, USA) and a pulsed amperometric detector.

LcITF (Frutafit TEX! Sensus, Roosendaal, the Netherlands) isolated from chicory roots contained oligomers and polymers with degree of polymerization from 10 to 60, linked by β(2–1) bonds. LcITF was characterized by high-performance anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PED), which was performed on an ICS5000 system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a Dionex CarboPac PA-1 column (2 × 250 mm) in combination with a Carbopac PA-1 guard column (2 × 50 mm) (Supplementary Fig. S7).
LaW37 (Winclove, Amsterdam, The Netherlands) was produced anaerobically at 37 °C in media adapted from Man Rogosa Sharpe broth.
STM strain DT12 (B; O1, 4, 5, 12) was isolated from a pig mesenteric lymph node^{75 (link)}. Inocula were prepared as previously described^{76 (link)} and were used to challenge the piglets. In short, bacteria were grown from glycerol stocks in Brain-Heart Infusion medium at 37 °C until stationary phase. Cell count was confirmed with plating on Columbia Blood Agar medium.
Salmoporc STM is an oral live attenuated porcine vaccine licensed in Europe (IDT Biologica, Dessau-Roßlau, Germany). The lot number used was 0161213, and vaccine suspension was prepared freshly, according to manufacturer’s instructions, prior to administration.

Alcoholysis with 1-hexanol was performed in the same way as described with methanol, except that 25% (v/v) 1-hexanol was used instead of methanol, and prior to the sampling of the reaction mixture, the tube was shaken to mix the two phases. Reactions with TrMan5A were also performed with varying enzyme concentrations (0.2, 2, and 4 μM) and M₄ concentrations (5, 25, and 50 mM). Duplicate incubations were analyzed with MALDI-TOF MS as described above, except that the samples were diluted 10-fold in Milli-Q water before being applied on the target plate due to the presence of hexanol. In addition, reactions with 5, 25, and 50 mM M₄ were analyzed with high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) in order to analyze the rate of M₄ degradation in these reactions, using an ICS-5000 system and a CarboPac PA200 column (Thermo Fisher Scientific, Waltham, MA, USA).

Both analytical procedures used an ICS-5000+ system (Thermofisher, Sunnyvale, CA, USA), consisting of a gradient pump, conductivity detector, eluent generator module equipped with KOH cartridge and AS autosampler. A Thermo Scientific Dionex IonPac AS11-4 µm (2 × 250 mm, 4 µm particle size) column with a guard column, Dionex IonPac AS1 µm (2 × 50 mm), served as an ion exchange resin, held at a temperature of 30 °C for HPIEC–HRMS method, while for the HPIEC–SCD the AS16-4 µm was chosen.