Chromatographic analyses of membrane fatty acid Bifidobacterium with different hydrophobicity

Abstract

Bifidobacteria are representatives of normal microflora of man, they control the qualitative and
quantitative composition of the community, prevent the overgrowth of opportunistic bacteria. A detailed
study are required molecular mechanisms of biofilm formation on the mucous membrane, and, in fact, the
initial stages of interaction of the microflora with the intestinal wall. The features of the composition of fatty
acids that form the cell wall of Bifidobacterium bifidum, not studied in full. The aim of the research is to
study the qualitative and quantitative composition of fatty acids of the membranes of bifidobacteria with
different hydrophobicity to establishing mechanisms of the adhesive activity of microorganisms.
The study used culture Bifidobacterium bifidum, isolated from the gut of healthy children. The
hydrophobicity of bifidobacteria was studed by Rosenberg et all. with modifications L-Q Wang et all. The
lipid fraction extracted from the washed isotonic NaCI broth culture of bifidobacteria by extraction with a
mixture of chloroform : n-hexane (1:1). The resulting extract was subjected to methylation. For this purpose,
a sample volume of 1 ml was placed in a vial with a volume of 1.5 ml, the solvent was evaporated under a
stream of inert gas to dryness. To the dry residue was added 500 µl of a 3% solution of H2SO4 in MeOH. The
resulting solution was added internal standard (10 µg undecenoic acid (Aldrich). Then the sample was heated
at 900 ° C within one hour. Further extraction was carried out with 700 µl of hexane. The volume selected
hexane fraction was concentrated by Stripping of solvent to volume of 200 µl. The obtained sample
containing fatty acids as methyl esters were used for the analysis. Methylated samples were analyzed on gas
chromatography / mass spectrometer Agilent 7000B using the column: ZB-WAX, 30m*0.25 mm*0.25 µm.
The sample volume was 2 µl enter without division of the flow, the flow rate of 1ml/min. For statistical
analysis used the software package Statistica version 6.1.
When analyzing the amount of saturated fatty acids in strains with different hydrophobicity were
observed statistically significant differences in the concentrations of the isoforms, methylated and long-chain
fatty acids, i.e., those forms that define the liquid-crystalline state of the membrane (p=0.00). It is established
that isopentadecanoic acid (isoС15:0) was present only in high hydrophobic bifidobacteria. Also they have a
large number of registered 13-methyl-tetradecanoic acid (me-C14:0), its content was 1.3 µg. In cultures with
medium hydrophobicity such acid is not detected, low hydrophobic noted a small the content is 0.1 µg. With
the medium hydrophobic of bifidobacteria were also absent 12-methyl-tetradecanoate (Мe-C14:0) and
palmitic (C16:0) acids, whereas at high and discovering strains their content varied in the range of 0.3-0.4
and 11.4 µg -12,5 µg, respectively. It was found that bifidobacteria with low hydrophobic content of all
unsaturated fatty acids was reduced in comparison with low hydrophobic cultures. Data of GH-MS of the
analysis of fatty acids of a membrane of B. bifidum allow to predict ability of a strain to adhesion that it is
necessary to consider at correction of microecological violations.
Thus, the mechanisms of changes in hydrophobicity, which plays a major role in nonspecific
adhesion of bacteria, determined by the regulation of the content of unsaturated and branched fatty acids in
the composition of cell walls of microorganisms