采用高效离子交换色谱分离分析动物肠道菌群方法初探

Separation and Analysis of Animal Intestinal Flora by High Performance Ion-exchange Chromatograph

报道了利用高效离子交换色谱直接分离动物粪便肠道菌群的有关研究结果.人与兔的粪便样品稀释后经离心分离岀细菌,通过DEAE弱阴离子交换色谱(30cm×2cmi.d)进一步除去杂物,将所得细菌的完整细胞样品直接上样高效液相阴离子交换色谱进行分离.色谱柱采用TSKgelSuperQ-TOYOPEARL650C强阴离子交换树脂,平衡缓冲液为0.02mol/L的哌嗪-HCl缓冲液(pH8.0),吸附于色谱柱上的细菌完整细胞用0～1mol/LNaCl线性梯度进行洗脱,洗岀液由260nm紫外吸收与光散射强度检测器检测.人与兔粪便细菌样品分别获得8个与5个组分,经镜检与培养实验确认为细菌,并且光散射仪洗脱峰峰面积同细菌计数结果具有线性相关性.实验表明,高效离子交换色谱能够应用于肠道菌群这样的复杂细菌体系的分离分析.

The animal intestinal microflora were separated and analyzed by high performance ion -exchange chromatography. The bacteria were separated from diluted human and rabbit feces by centrifugation, the contaminants were further removed by DEAE weak-anion exchange chromatography （30 cm × 2 cm i. d）. A strong-anion exchange column （TSKgel SuperQ - TOYOPEARL 650C） was employed for the chromatographic separation of the obtained bacterial samples with a 20 mmol/L piperazine -hydrochloric acid buffer （pH 8.0） as the equilibrium solution. The absorbed bacterial cells on the column were eluted with a linear gradient of 0 - 1 mol/L NaCl, and the eluate was detected by a UV detector at 260 nm and a laser light scattering monitor. The bacterial samples of human and rabbit feces were fractionated into 8 and 5 fractions respectively, and each fraction was confirmed to be the bacteria by means of microscope and plate culture. And in light scattering spectrum, the correlation between peak area and quantity of bacteria was linear. The results suggested that HPLC could be a useful tool to reveal the composition and status of intestinal flora with high fidelity. Fig 5, Tab 2, Ref 11