Method of determining antibacterial activity of chitosan

FIELD: chemistry.

SUBSTANCE: disclosed is a method of determining antibacterial properties of chitosan by estimating its minimum bacteriostatic and/or bactericidal concentration. Complex buffer solutions based on three organic acids MES, ACES and TES with different pH values are prepared. The ready buffer solutions are poured into a vessel. Double dilutions of chitosan are then prepared in vessels with the buffer solutions. Aliquots of a bacterial suspension in a fluid medium are added to the chitosan solutions in the buffer. The solutions are incubated for 24 hours at temperature which is optimum for bacterial growth. The minimum bacteriostatic and/or minimum bactericidal concentration of chitosan is then determined after incubation by determining growth of the culture or a drop in the number of living cells, respectively.

EFFECT: invention enables to determine antibacterial properties of chitosan in a wide pH range from 5,50 to 8,00 without the need to use buffers of different chemical composition.

5 dwg, 2 ex

 

The invention relates to Microbiology and can be used to determine the antibacterial properties of chitosan by estimating the minimum bacteriostatic and/or bactericidal concentrations in a wide range of levels of acidity in the environment.

Standard determination of antibacterial activity of substances is reduced to the evaluation of their minimum inhibitory and minimum bactericidal concentrations against bacteria, which is in preparation in containers of solutions of the investigated substances with different concentrations by two-fold dilutions in a pre-selected buffer solutions, then add in the capacity of a bacterial suspension in a liquid nutrient medium, incubation at the optimum for growth of bacterial culture temperature, the determination after incubation, the presence or absence of growth of the culture to evaluate the bacteriostatic effect of the substance and/or decrease the number of living cells for the evaluation of bactericidal effect of the substance. Thus for the minimum bacteriostatic concentration of the substance take the minimum concentration of a substance in which there is visual growth of the bacterial culture solution is not turbid, do not receive sediment from bacterial cells, etc. For the minimum bactericidal concentration of the substances take the minimum concentration of a substance when the number of live bacterial cells in comparison with their number in the initial inoculum is reduced to 1000 or more times (99.9% or more), or a different number of times depending on the methodological requirements. The number of live cells determined by sowing aliquot of the suspension on a solid nutrient medium with subsequent counting of grown colonies.

However, current methods of estimating the minimum bacteriostatic and/or minimum bactericidal concentration against bacteria are unsuitable for studies of antibacterial activity of chitosan - natural polycation, composed of residues of D-glucosamine and N-acetyl-D-glucosamine, United 1,4-β-glycosidic bonds. Because, firstly, chitosan is polyaminoacids, as a result of its antibacterial properties are strongly dependent on the acidity, which determines the positive charge of its free amino groups. Secondly, the chitosan molecules Dier in their degree of polymerization, which determines their dissociation constant pKa, therefore, under the same conditions of pH of the medium is a different degree of charge of molecules with different molecular weight. Thirdly, chitosan, a natural copolymer of acetylglucosamine and glucosamine, represents heteroge the ing group of substances differing in molecular weight (or degree of polymerization), the degree of acetylation, the location of acetylated units along the polymer chain, the viscosity [2, 3]. Thus, the antibacterial properties depend on combinations of a few basic physico-chemical characteristics of the chitosan and the acidity, but at the same time for different samples antibacterial activity may have a different character changes with the change of the pH. In addition, typically the antibacterial properties of substances in practice are investigated at pH values in the range from slightly acidic (pH of 5.50) to slightly alkaline (pH of 8.00), however, studies on antibacterial activity of chitosans are not used buffer systems, continuously overlapping this range. The most commonly used are sodium acetate or potassium acetate buffer systems, which have a buffer properties only up to a pH of 6.5 [4]. Sometimes use a buffer system based on sodium salt morpholinepropanesulfonic acid, which creates a buffer system in the pH range from 5.50 to 6.70 [5]. The use of sodium or potassium phosphate solutions, which have a good buffer properties in weakly acidic and weakly alkaline region, prevents the tendency of chitosan to precipitate when interacting with the phosphate is I. Thus, to date there is no unified approach to the evaluation of bacteriostatic and/or minimum bactericidal concentrations of chitosan, which would take into account the high degree of variation of its antibacterial properties depending on the pH of the medium.

Objective of the claimed invention is to develop a method allowing to determine the antibacterial properties of chitosan by estimating the minimum bacteriostatic and/or bactericidal concentrations in a wide range of levels of acidity (from 5.50 to 8.00).

This object is achieved by developing a method for determining the antibacterial properties of chitosan by estimating the minimum bacteriostatic and/or bactericidal concentration, which involves the preparation of a complex of the buffer solutions on the basis of the three organic acids - morpholinepropanesulfonic - MES (morpholineethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic-ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), 2-[Tris(hydroxymethyl)methylamino]-1-econsultancy - TES (2-[Tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid) in equimolar ratio (1 part MES, part 1 ACES, 1 part TES) with different levels of pH in the range from 5.50 to 8.00, which are created by adding water to the solution data of organic acids necessary amount of concentrated sodium hydroxide solution,add the prepared buffer solutions in the vessel (for example, wells), prepare two dilutions of chitosan in containers with a buffer solution, adding a solution of chitosan in the buffer aliquot of bacterial suspension in a liquid nutrient medium, incubation solutions for 24 h under optimal for bacterial growth temperature, the assessment after the incubation, the minimum inhibitory and minimum bactericidal concentrations of chitosan by determining the availability of crop growth or reduce the number of surviving cells, respectively.

Integrated buffer system based on MES, ACES and TES allows you to maintain a pH in the range of pH values from 5.50 to 8.00 (Fig.1)consisting of intervals of pH values, which consistently support these acids separately (Fig. 2-4). Thus, the use of integrated MES buffer solution-ACES-TES-Na allows to evaluate the antibacterial activity of chitosan in the range of pH of the medium, which is not available for the formation of other known buffer systems based on other components and suitable to create solutions of chitosan polymer without dropping it into the sediment.

The technical result of the claimed invention is to develop a method for determining the antibacterial properties of chitosan by estimating the minimum bacteriostatic and/or BA is aricidea concentration in a wide range of levels of acidity, with good reproducibility and without using a different chemical composition of buffers to create a different level of acidity of solutions.

Example 1. Determination of minimum inhibitory concentrations depending on the pH of the environment for chitosans with different degrees of polymerization against Staphylococcus epidermidis. In 96-well round-bottom tablet make 50 μl of 0.15 M MES-ACES-TES-Na buffer. In the vertical strip No. 1 make a buffer with a pH of 5.50; in the vertical strip No. 2 make a buffer with a pH of 5.75; in the vertical strip No. 3 make a buffer with a pH 6,00; in the vertical strip No. 4 make a buffer with a pH of 6.25; in the vertical strip No. 5 make a buffer with a pH of 6.50; in the vertical strip No. 6 make a buffer with a pH of 6.75; in the vertical strip No. 7 make a buffer with a pH of 7.00; in the vertical strip No. 8 make a buffer with a pH of 7.25; in the vertical strip No. 9 make a buffer with a pH 7,50; vertical strip No. 10 make a buffer with a pH of 7.75; in the vertical strip No. 11 make a buffer with a pH of 8.00. In the upper holes of each strip make 50 ál of an aqueous solution of chitosan with a degree of polymerization equal to 4 (final concentration of 1000 µg/ml). Samples of chitosans with a degree of polymerization 34 and 116 apply to other tablets, filled with buffer solutions according to the same scheme. Produced by serial twofold dilution of chitosan through lane the nose 50 ál of the mixture from the first hole to the second and so on. The bottom holes of each strip is not used in the breeding of chitosan and leave them as a negative control. Then added to all wells 100 ál mycopathologia broth containing a suspension of bacteria S. epidermidis (final concentration of 2x105CFU/ml). Incubate the plates for 24 h at 37°C. After incubation evaluate the minimum inhibitory concentration for each of the samples of chitosan by visual (or spectrophotometer) registration of the absence/presence of the growth of bacterial cultures in the wells with different concentrations of polymer. The results are shown in figure 5.

Example 2. Determination of minimum bactericidal concentrations depending on the pH of the environment for chitosans with different degrees of polymerization against Staphylococcus epidermidis carried out analogously to example 1, but after incubation estimate the number of live bacterial cells in the wells and sravnivaut with their number in the initial inoculum.

LITERATURE

1. Liu X.F., Y.L. Guan, D.Z. Yang, Z. Li, K.D. Yao Antibacterial action of chitosan and carboxymethylated chitosan. J. Appl. Polym. Sci. 2001. V.79. P.1324-1335.

2. Tharanathan R.N., Kittur F.S. Chitin - the undisputed biomolecule of great potential. Crit. Rev. Nutr. 2003. V.43. P.61-87.

3. Kulikov S., Tyurin Y.A., Albulov A.I., Lopatin S.A., Varlamov V.P. Antibacterial activity of chitosan: practice and theory. / Proceedings of the 9th the international conference "Modern perspectives in the study of chitin and chitosan", Stavropol, October 13-17, 2008, P.184-187.

4. Gerasimenko D.V., Avdienko I.D., G. Bannikov, Zueva O.Y, Varlamov V.P. Antibacterial activity of water-soluble low molecular weight chitosans against various microorganisms. Go active. biochem. microbiol. 2004. T. No. 3. S-306.

5. Raafat D., Bargen K.,Haas, A., Sahl H. Insight into the mode of action of chitosan as an antibacterial compound. Appl. Env. Environ. 2008. V:74. No. 12. P.3764-3773.

The way to determine the antibacterial properties of chitosan by estimating the minimum bacteriostatic and/or bactericidal concentration, characterized by the fact that the use of the integrated buffer solutions on the basis of the three organic acids MES, ACES and TES, which is added in the tank, then prepare two-fold dilution of chitosan in containers with a buffer solution, is added to the chitosan solutions in the buffer aliquots of bacterial suspension in a liquid nutrient medium, incubated solutions for 24 h under optimal for bacterial growth temperature, determined after incubation minimum bacteriostatic and/or minimum bactericidal concentration of chitosan by determining the presence of a growth culture or decrease the number of survivors cells, respectively.



 

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