Thermo ics5000 ion chromatography system

RS, ERS, and SPI-WPI-ERS (10 mg) were dissolved in 5 mL of distilled water in a boiling water bath (HH-6, Shanghai Lichen Bangxi Instrument Technology Co., Ltd., Shanghai, China) for 60 min to prepare gelatinized samples. After cooling to room temperature, NaN₃ (10 μL 2% w/v), acetate buffer (50 μL, 0.6 mol/L, pH 4.4), 0.5% (w/v) sodium borohydride solution, and isoamylase (10 μL, 1400 U) were added to 2.5 mL gelatinized samples, and placed at 37°C for 24 h. The mixture (600 μL) was put into a centrifuge tube and dried under nitrogen at room temperature. Then, it was dissolved in NaOH (30 μL, 1 mol/L) for 60 min, and the solution was diluted with 570 μL of distilled water, followed by centrifugation at 12,000 r/min for 5 min. Eventually, the supernatant was collected. The chain length distributions of RS, ERS, and SPI-WPI-ERS were determined by a Thermo ICS5000 ion chromatography system (ICS5000+, Thermo Fisher Scientific, Waltham, MA, USA).

The molecular weight and purity of HPW were assayed with high-performance gel permeation chromatography (HPGPC) (Shimadzu, Japan) using dextrans (MW: 5 kDa, 12 kDa, 25 kDa, 50 kDa, 80 kDa, 150 kDa, 270 kDa, Sigma–Aldrich, USA) as standard samples in a TSK-gel GMPWXL (7.8 mm × 300 mm, 5 μm) [16 (link)]. The data showed that the average molecular weight of HPW was approximately 616 kDa, and the purity was 87% (Fig. 1C).
High-performance anion-exchange chromatography (HPAEC) was used to identify the monosaccharide composition of HPW. The chromatographic system used a Thermo ICS5000 ion chromatography system (Thermo Fisher Scientific, USA), and an electrochemical detector was used to analyse the monosaccharide components with the following parameters: flow rate, 0.5 mL/min; injection volume, 5 μL; solvent system, 0.1 M NaOH: (0.1 M NaOH, 0.2 M NaAc); gradient program, 95:5 V/V at 0 min, 80:20 V/V at 30 min, 60:40 V/V at 30.1 min, 60:40 V/V at 45 min, 95:5 V/V at 45.1 min, 95:5 V/V at 60 min.
The data showed that HPW was mainly composed of galactose (21.55%), glucose (20.77%), rhamnose (7.05%), mannose (7%), arabinose (5.02%) and xylose (3.21%). An ion chromatogram of the samples is shown in Fig. 1B, and the monosaccharide composition is detailed in Fig. 1C.

Carbohydrates were detected by Thermo ICS 5000+ Ion chromatography System (Thermo Fisher Scientific, Inc., Waltham. MA) equipped with Dionex CarboPac PA10 (4 × 250 mm) column and a pulsed amperometric detector. Samples were processed and detected according to the methods in previous studies (20 (link), 54 (link)).
Seventeen free amino acids were determined. Samples (1 g) were homogenized with 5% trichloroacetic acid and diluted to 25 mL and then ultrasonically treated for 30 min and maintained for 2 h. The suspension was filtered by double-layer filter paper and centrifuged at 15,000 × g for 30 min, and then the supernatant was filtered using a 0.22-μm membrane filter and applied to an automatic amino acid analyzer (Agilent 1100 Series; Palo Alto, CA) equipped with Agilent Hypersil ODS column (5 μm, 4.0 mm × 250 mm), according to the method by a previous study (55 (link)). Each amino acid was identified and quantified by the retention time and peak area from the instrument software in comparison to FAA standards (Sigma Chemical Co., St. Louis, MO).

The monosaccharide composition of lily polysaccharides was determined by high performance anion exchange chromatography (HPAEC) (24 (link)). Five milligrams crude LP samples were hydrolyzed in sealed ampoule with trifluoroacetic acid (2 M) at 121°C for 2 h. Then the samples were dried by nitrogen and washed by methanol, and the procedure was repeated three times. The residue was re-dissolved in deionized water and filtered by 0.22 μm microporous membrane for measurement. The chromatographic system was a Thermo ICS5000 ion chromatography system (ICS5000, Thermo Fisher Scientific, MA, USA). Chromatographic conditions included a CarboPac PA-20 Ionic Exchange column (3 × 150 mm) and a pulsed Amperometric detector (PAD; Dionex ICS 5000 system; Shanghai, China). The loading volume was 5 μl, and the flow rate was 0.5 ml/min. The solvent system included solvent system A: (ddH₂O), solvent system B: (0.1 M NaOH), solvent system C: (0.1 M NaOH, 0.2 M NaAc); The gradient program was shown in Table 1. In addition, the 11 monosaccharide standards needed in this experiment were purchased from Sigma Company (St. Louis, MO, USA), and the information of standard products is shown in Table 2.

The starch granules were digested with isoamylase (Sigma-Aldrich, Shanghai, China), and the supernatant of the sample was taken and used for the analysis of chain length distribution of amylopectin [42 (link)]. Thermo ICS5000 ion chromatography system (ICS500+, Thermo Fisher Scientific, Waltham, MA, USA) and Dionex^TM CarboPac^TM PA200 (250 × 4.0 mm, 10 μM) chromatographic column were used. Mobile phase A: 0.2 M NaOH, mobile phase B: 0.2 M NaOH/0.2 M NaAC, the flow rate was 0.4 mL/min, column temperature was 30 °C. The composition of enzymatic hydrolysate was analyzed by electrochemical detector.