Preliminary Study On Separation And Analysis Of Animal Intestinal Flora By The High Performance Ion-exchange Chromatograph

High performance ion exchange chromatography has been demonstrated to be capable of efficient separation of bacteria of different species with high resolution on the base of the differences in both electrostatic charge property and structure of the cell. Its application in the analysis of a complex microbial ecosystem such as animal intestinal microflora may effectively overcome the problems of low efficiency and fidelity of with the currently available approaches, and push the study of the influences of the microflora on the health of the host to a new height. The present work is the first effort to establish a methodology to separate and analyze the animal intestinal microflora by high performance ion exchange chromatography, and to apply the method to study the effect of probiotics on the animal intestinal microflora. Kunming white mouse was used as the first test animal in this study. Different methods of isolating bacteria from the animal feces were investigated. Based on the effect of the removal of the contaminants and the yield, filtration method was confirmed to be the appropriate method for bacteria isolation. TSKgel SuperQ-TOYOPEARL 650C resin was employed in ion exchange chromatography using an HPLC system connected to a laser light scattering monitor as well as a UV monitor. Each run of chromatography was carried out with a bacteria sample extracted from 0.1g of the feces. The absorbed bacteria cells were eluted by a linear gradient of 0-0.5M NaCl in 60 minutes followed by 0.5-.75M in 20 minutes and 1M NaCl for 15 minute 0.02M in Piperazine hydrochloride (pH8.0) buffer sequentially at a flow rate of 1ml/min. 20 fractions were obtained as was indicated by the Light scattering chromatograms. Judging by the comparison with UV chromatogram and further confirmation with microscopic observation of each fraction, 17 fractions were identified to be consisted of bacteria cells. The morphoric differences revealed by the microscopic studies of among bacteria cell fractions advocates the feasibility and validity of the chromatographic separation of the intestinal microflora basing the different properties of the bacteria cell surface. The conventional bacteria number count was carried out on each of the bacteria fractions, and a satisfactory linear correlation between the bacteria number count and its fraction's peak area was established, which indicates that light scattering chromatograms can be useful in quantitative analysis of microflora rendering an unprecedentedly efficient approach to the composition study. On the basis of the successful chromatographic separation of the mouse intestinal microflora, the method was applied to the study of the effect of the oral administration of probiotics on the intestinal microflora of a mouse. 300 μl of living bifidobacterium (Bifidobiogen-Livzon, 16.7mg/ml) were administered twice in the morning and evening respectively to the test mouse orally. Its feces was collected the next morning. The chromatographic data of the feces bacteria cells before and after the administration demonstrates a remarkable increase in all the bacteria fractions except fractions 15 and 16 with a logical increase of the fraction containing bifidobacterium, which is the first direct evidence of the effect of the bifidobacterium intake on the intestinal microflora composition, and is in good accord with the currently available assumption. The method was employed to analyze the intestinal microflora of a rabbit, obtaining a completely different chromatogram from that of a mouse, revealing the remarkable difference in microflora composition among different animals. In conclusion, the method of separating and analyzing animal microflora chromatographically established in this study has been proved to be feasible and effective. It can be used in the quantitative analysis and separation of the intestinal microflora, and in the study of the microflora of an individual animal under different conditions, as well as the comparative study of the microflora of different individuals, rendering a powerful tool for the elucidation of the relationship between microflora and its host's health and nutrition conditions.