Sttudy On Extraction And Purification Condutions Of Lipid Phosphorus Wall Acid From Enterococcus Faecium EF1

The aim of this study was to determine the optimum conditions for the extraction and purification of lipoteichoic acid from the cell wall of Enterococcus faecium EF1,and to provide a feasible method for obtaining lipoteichoic acid with high purity and stable quality.The growth rule of Enterococcus faecium EF1 was studied by counting viable bacteria.The bacterial biomass was studied by centrifugation.Lipoteichoic acid was extracted by water-saturated n-butanol,and the effects of cell wall breaking time and n-butanol extraction time on the extraction yield were studied.The purification effect of lipoteichoic acid by hydrophobic interaction chromatography and ion-exchange chromatography was studied,It lays a foundation for the study of the mechanism of obtaining high purity and stable fatty phosphorus wall acid in the future.The main results are as follows:(1)Enterococcus faecium EF1 reached the end of logarithmic growth in MRS medium at 37 ? for 24 hours,and then entered a stable phase after 36 hours.The biomass at the end of logarithmic growth was the highest(3.22g/L),which was 22.43%higher than that at the stable stage(P<0.01).The biomass obtained by centrifugation for 15 minutes was 12.20%higher than that obtained by 12 minutes(P>0.05).(2)The highest yield of lipoteichoic acid was obtained in Enterococcus faecium EF1 after 70 minutes of cell wall breaking and 60 minutes of n-butanol extraction(2.27%(P<0.01).(3)When the crude lipoteichoic acid from Enterococcus faecium EF1 was purified by octyl sepharose chromatography,the elution effect of 35%sodium n-propanol acetate buffer was better than that of 15-60%linear gradient buffer.DEAE ion exchange chromatography has no obvious effect on further purification of lipoteichoic acid.CONCLUSION:Enterococcus faecium EF1 should be harvested at the end of logarithmic growth and collected by centrifugation for 15 minutes.Cell wall breaking time and n-butanol extraction time had significant interaction effect on the yield of lipoteichoic acid.When using the octyl sepharose chromatography to purify the crude lipoteichoic acid,it is desirable to use isocratic elution.Needless to do DEAE ion exchange chromatography later.