Pack nh2 column

The supernatant from late exponential phase cultures of B. longum 105-A wild-type or its ∆cpsD mutant was filtrated using a 0.45-µm filter. The polysaccharide of culture supernatant was precipitated using 2 volume of 99.5% ethanol for an overnight at 4 °C. The ethanol-suspended polysaccharides were centrifuged at 7000 rpm for 30 min. The pellet was dried out at 55 °C for 1 h and re-suspended in water. The obtained polysaccharides were then acid hydrolyzed with 4 M trifluoroacetic acid at 100 °C for 3 h. The chromatography of monosaccharide analysis was conducted with an Asahipak GS-220 HQ column (300 × 7.5 mm, Showa Denko, Tokyo, Japan). The EPS was eluted in water at a flow rate of 0.4 ml/min at 30 °C, followed by post-column reaction with arginine/boric acid, and monitored by a fluorescent detector (Excitation: 331 nm, Emission: 383 nm). The chromatography of monosaccharide analysis to separate the glucose and galactose was performed with a YMC-Pack NH2 column (250 × 4.6 mm, YMC Co., LTD., Tokyo, Japan). The EPS was eluted with 25% water and 75% CH₃CN, at a flow rate of 0.5 ml/min at 50 °C with a fluorescent detection as above. For molecular weight analysis, the SUGAR KS-804 column (300 × 8.0 mm, Showa Denko, Tokyo, Japan) was used with a flow rate of 1.0 ml/min using a refractive index detector at 50 °C. The experiments were performed in duplicate.