Considering all criteria above, it was found that the most advanced method of the
obtainment of the extract of Сandidaalbicans fungus is the physical and chemical
method of combination of the extraction with 5.0% solution of sodium hydroxide with
the ultrasound disintegration on the equipment UZUU-21.
The determination of the concentration of proteins and polysaccharides for
the diagnostic operation of the allergen.
Grounding of the technology of purification of C. albicans fungus extract.
After the extract obtainment it was purified from the incidental low-molecular
substances, undesirable in the allergenic extracts (possible non-specific skin reactions
to the deferred formation of specific response).
For the purification of C.albicans fungus extract we decided to use the widely
known biochemical method of size exclusion column chromatography with Sephadex
G-100, according to State Pharmacopoeia of Ukraine, which ensures the deep
purification of the obtained allergen, through its subdivision into separate fractions
according to the size of molecules [7, 9].
The extract sonicated during 15 min. at the ratio of the C. albicans fungus biomass
(1g) and extracting agent with 5.0 % of the solution of sodium hydroxide (10 ml) -
1:10, was treated with 5.0% solution of hydrochloride acid, gradually bringing the
environment to the pH value 7.2 ± 0.2. The prior filtration on the membrane filters was
conducted with the pores diameter of 0.8 µm as well as the sterilization filtration on
the membrane filters with the pores diameters 0.22 µm. The obtained extract had the
protein concentration of 1.9 mg/ml and polysaccharides 6.05 mg/ml (i.e. The extract
contained the protein 38 mg and polysaccharides 121 mg).
The sterile extract of C. albicans fungus was introduced with the dropper along
the column wall with the gel Sephadex G-100 and gelled with the phosphate buffered
solution рН 7.2 ± 0.2. To prevent the contamination, all manipulations were conducted
in the laminar box under the fume hood with the maintenance of aseptic conditions.
The gel-fractionation curves of the extract on Sephadex G-100 demonstrated a
good reproducibility of subdivision into two fractions: I highly-molecular and II low-
molecular.
The determination of content of the obtained fractions of the extract of C. albicans
fungus was aimed at providing the biochemical characteristics and comparing the
biochemical composition of fractions. In I fraction, the volume of which amounted to
30 ml, the protein concentration amounted to 0.31 ± 0.02 mg/ml, polysaccharides – 3.
23 ± 0.21 mg/ml (i.e. Contained the protein 9.3 mg and polysaccharides 96.9 mg), in
II fraction, the volume of which amounted to 50 ml, the protein concentration
amounted to 0.57 ± 0.03 mg/ml, polysaccharides – 0.51 ± 0.03 mg/ml (i.e. contained
protein 28.5 mg and polysaccharides 25.5 mg).
When conducting the paper chromatography of acid hydrolyzates I and II
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