Features of structure lipoteichoic acids of Bifidobacteria according to TLC IR-FT
Abstract
Bifidobacteria are representatives of human normoflora, they control the qualitative and quantitative composition of the community, prevent the excessive growth of opportunistic bacteria. A detailed study requires molecular mechanisms for the formation of biofilms on the mucosa, as well as, in fact, the initial stages of interaction of microflora with the intestinal wall. The features of the structure of LTA, which are a component of the cell wall of representatives of the genus Bifidobacterium, have not been studied in full. The aim of the study was the analysis of LTA of different types of bifidobacteria in the TLC regime.
The subject of the study were cultures of Bifidobacterium bifidum 379 M, and Bifidobacterium longum - isolated from probiotic preparations and a strain of Bifidobacterium breve obtained from a person with immunodeficiency. Isolation and accumulation of microbial mass of bifidobacteria was carried out by a routine bacteriological method. For this, bifidobacteria were grown 24 hours on liquid Bifidum-environment. Anaerobic conditions and gas-generating packets were used to create anaerobic conditions. Identification of bacteria was carried out on the basis of morphological, tinctorial, cultural and biochemical properties. The latter were studied using the commercial test systems ANAERO-TEST 23 (Lachema).
Extraction of LTK was carried out as follows. The bacterial mass was washed three times with an apyrogenic, sterile sodium chloride solution - resuspended for 2 minutes at Vortex and centrifugation at 5000g 10min. Washed bacterial cells weighing 3.0 g (exact sample) were treated twice with 3 ml of chloroform: n-hexane (1:1), followed by the combination of the organic phase. The solvents were distilled off in a stream of nitrogen.
Chromatography was performed on Sorbfil plates. The extract in a volume of 50 μl was applied to the start line. Mobile phase: СHCI3: MeOH: H2O (60: 30: 5) and using as a developer 5% phosphoric-molybdenum acid solution in EtOH. The chromatographic zones were extracted with 3 ml of chloroform. The solvent was distilled off under a stream of nitrogen, in fractional application to a KBr disc. Fourier transform IR spectroscopy was performed on the FSM-1202 instrument. Chloroform extraction was fractionally applied to the KBr surface of the disk, (Fluka), the solvent was blown off with a nitrogen stream. The conditions for recording the spectra are as follows: the transmission mode, in the range 4000-400 cm-1 with a resolution of 4 cm-1 and the number of scans. 25. Air was used as a reference sample, a reference sample was recorded just before each sample was analyzed. Instrument control and processing of spectral data was carried out using the software Fspec (4.0.0.2) and Aspec (1.1).
For the samples studied, LTA is determined on the IR-spectra of all the chromatographic zones of the samples under investigation, in the interval 1755-1690 cm-1, the bands due to ν (C = O) appear. Attention is drawn to the fact that LTA B. bifidum 379 M in the chromatographic zones are most variable in this indicator. Thus, values from 1742 cm-1 (Rf 0.4) to 1712 cm-1 are traced. In turn, in all chromatographic zones of the B. breve sample, the absorption bands of 1744 and 1714 cm-1 are determined. The zone with Rf 0.95 is characterized by a band of 1744 cm-1. This variability of values is explained by the inductive effect of unsaturated fragments. The presence of a band of 1275 cm-1, which is present only in the sample of B. longum and corresponding phosphoric acid residues with three electronegative substituents, was observed. In the chromatographic zones corresponding to this sample, this band is traced at Rf values of 0.93 and 0.05. It should be noted that the band 760 cm-1 corresponding to the valence vibrations of C-O-P is found only in B. breve samples, both in native LTA and after TLC. This band is detected at Rf values of 0.25; 0.34; 0.97. Interestingly, for the LTA B. bifidum, three zones characteristic of phosphorus-bound bonds were found, with Rf values of 0.41; 0.63; 0.92. For these chromatographic zones, bands with a value of 800 and 772 cm-1 are characteristic. This bias in the B.bifidum sample is due to the presence of spatially close functional groups with which P = O can form a hydrogen bond. The resulted values of Rf and the affiliation of these bands to phosphorus-containing structures are consistent with the values of Rf obtained after TLC treatment with phosphomolybdic acid. It should be noted that the spectral characteristics of chromatographic zones are variable. This property is apparently due to differences in structural fragments of LTA within one sample, which leads to a difference in the chromatographic mobility of varieties of LTA. In turn, it should be noted that the spectral parameters of LTA of the B. breve sample isolated from the chromatographic zones of TLC are relatively low. Thus, phosphorus-containing structures can be traced only in zones with Rf values of 0.8-0.9. For this strain, For this strain, the lack of absorption bands in the range 1590-1530 cm-1 was rather specific. For a B. longum LTA sample in a range of Rf values of 0.6-0.9, a band of 1585 cm-1 was detected. For LTA B. bifidum, similar structures are found in the range Rf 0.3-0.95 with a maximum variation over the band 1587-1547 cm-1. It is known that strong absorption in the range 1650-1540 cm-1 can indicate an ionized carbonyl group, a planar deformation vibration of NH in amines, or stretching vibrations of C = O in amines. In turn, LTA probiotic strains contain one broad band with a maximum of 1635 cm-1, which is specific for polyene structures. Such strains of bifidobacteria can stimulate the immune system, which is the basis of the mechanisms of action of probiotic drugs.
Thus, a comparative analysis of LTA of representatives of the genus Bifidobacterium using the TLC method followed by analysis of IR-FT allowed to establish the distinctive chromatographic and spectral features of these structures. The distinctive features of the structure of LTA in a strain isolated from an individual with immunodeficiency were found. The application of the TLC version and the analysis of chromatographic zones in the IR spectroscopy mode can be used to study the component composition of samples obtained from plant objects and microorganisms, as well as to standardize probiotic preparations
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References
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