高效阴离子交换色谱检测双歧杆菌胞外多糖的单糖组成

Determination of monosaccharide composition of exopolysaccharides from Bifidobacteria by high performance anion exchange chromatography with pulsed amperometric detectionby monascus

利用离子交换色谱柱(DEAE-Sepharose Fast Flow,2.6×20cm)和凝胶过滤色谱柱(SepharoseCL-6B,1.6×80cm)对双歧杆菌RH所产EPS进行分离纯化,得到EPSa和EPSb两个组分。然后,采用高效阴离子交换-脉冲安培检测色谱法(high-performance anion-exchange chromatography withpulsed amperometric detection,HPAEC-PAD)分别对2个组分的单糖组成进行了定性和定量测定。结果表明,用20%H2SO4于100℃水解2.5h,可将多糖完全水解。HPAEC-PAD检测结果表明EPSa和EPSb均为杂多糖,EPSa含有Rha(1.00%),Ara(3.54%),Gal(18.13%),Glc(42.84%),Man(33.47%)和Fru(1.03%),而EPSb含有Rha(9.41%),Ara(7.88%),Gal(34.82%),Glc(17.82%),Man(27.83%)和Fru(2.24%)。

Two fractions（EPSa and EPSb） were isolated and purified by ion-exchange chromatography on DEAE-Sepharose Fast Flow column and gel-filtration chromatography on Sepharose CL-6B column.And then their sugar components were quantified by high-performance anion-exchange chromatography with pulsed amperometric detection（HPAEC-PAD）.The results showed EPS sample could be hydrolyzed fully by adding 200 μL H2SO4 and 800 μL distilled water to hydrolyze for 2.5 h at 100 ℃.The monosacchrides analysis showed that EPSa was composed of Rha（1.00%）,Ara（3.54%）,Gal（18.13%）,Glc（42.84%）,Man（33.47%） and Fru（1.03%）;then EPSb Rha（9.41%）,Ara（7.88%）,Gal（34.82%）,Glc（17.82%）,Man（27.83%） and Fru（2.24%）.