RU2220716C1 - Method for preparing microcapsule containing live microorganisms - Google Patents

Abstract

FIELD: medicine, microbiology. SUBSTANCE: lyophilized lactobacilli culture is covered by envelope that comprises polyvinylpyrrolidone tanned with tannic acid. As result of technological process forming aggregates of film-forming agent macromolecules occurs. Method provides the enhanced viability of microorganisms in the course of technological process due to using components that are safety for the host and microorganisms. Micro- capsulated lactobacilli can be placed then to different medicinal forms and used in complex with other preparations. EFFECT: improved preparing method. 1 tbl

Description

 The invention relates to medicine, namely to the technology of dosage forms with bacterial preparations.

 For the correction of dysbiotic conditions in medical practice, various drugs are widely used, based on live strains of microorganisms that make up the normal flora.

 Recently, various researchers have paid great attention to the use of microencapsulated forms of bacterial preparations. Substances used as an encapsulating coating for microorganisms should ensure their stability during long-term storage and uniform release when used directly in humid environments of the body.

 Of particular interest are microencapsulated forms of lactobacilli derived from polyvinylpyrrolidone. This polymer is non-toxic, not harmful to humans and microorganisms, approved for medical use. It has good solubility in inorganic solvents, including water, and its solubility does not depend on the pH of the medium, which allows it to be used to obtain microencapsulated forms of lactobacilli, intended for dissolution in various environments, including for the production of microcapsules for external use (in the form of powders).

A known method for producing microencapsulated forms of lactobacilli using aqueous solutions of crosslinked or linear polyvinylpyrrolidone (US patent 5733568, NPK 424-422, 1998). Its essence is as follows: a lyophilized culture of lactobacilli is dispersed at a temperature of about 0 ^o C in a solution of crosslinked or linear polyvinylpyrrolidone with constant stirring, and then the polymer is crosslinked with divinylbenzene. In this case, the bacteria are taken so that in the finished microcapsules there are at least 10 ³ bacteria in one microcapsule.

When reproducing this method in a laboratory, it was found that the number of viable lactobacilli preserved during microencapsulation and determined after destruction of the membrane by phosphate buffer is less than 10 ⁴ in 1.0 gram of microcapsules.

The disadvantages of this method include the high rate of loss of microorganisms during the process, which reaches 10 ⁴ cells or more, and the use of divinylbenzene, which is a toxic organic reagent, as a crosslinking agent. The use of this substance leads, on the one hand, to significant losses of microorganisms during the microencapsulation process, on the other hand, it is dangerous for humans, because is a moderately toxic substance.

 The objective of the invention was to reduce the loss of living microorganisms and increase the safety of the process by eliminating toxic reagents.

 The problem was solved when using a natural substance such as tannin obtained from plant materials (sumac, scumpium, etc.) and representing the sum of polysaccharides saturated with polyphenolic groups as a curing agent.

For the manufacture of microcapsules, a lyophilized culture of lactobacilli containing 10 ⁸ cells in 1.0 gram was taken.

 The essence of the invention lies in the fact that a lyophilized culture of microorganisms (in pure form or on a protective carrier of a polysaccharide structure) is dispersed upon cooling in a 30-50% aqueous solution of polyvinylpyrrolidone with a molecular weight of 15,000 (see table) in a ratio of from 1:10 to 1: 15 by weight with stirring to form a suspension containing individual particles of the desired size. A solution of a crosslinking agent (tanning agent) is added to the resulting suspension - a 10% -30% aqueous solution of tannin (GFX, Art. 658). The mixture is stirred for 30-180 minutes, after which the cured microcapsules are collected by filtration, washed and dried. The resulting microcapsules are particles of gray-yellow color ranging in size from 10 to 200 microns, depending on the rheological parameters of the process mixture.

 The invention is illustrated by specific examples, which are set forth below.

 EXAMPLE 1

For the manufacture of microcapsules, a culture of lyophilized lactobacilli of the strain L.fermentum 90TS4 containing 10 ⁸ living cells in 1.0 gram was taken.

Prepare 100.0 g of a 50% aqueous solution of polyvinylpyrrolidone, the process is carried out with constant stirring. Then, 10.0 g of lyophilized lactobacilli are dispersed in the indicated solution for 30 minutes until a uniform, stable suspension is formed (process temperature 4 ^{° C.} ). In parallel, when heated (water bath about 80 ^o C) prepare a 30% aqueous solution of tannin in an amount of 140 ml to obtain a clear solution of red-brown color. The solution is cooled to 4 ^{° C.} and fed through a separatory funnel to the working suspension at a rate of 1.8 ml / min. The system is left under constant stirring for 30 minutes, and the cured microcapsules are then collected by filtration, washed and dried. The obtained microcapsules are particles of gray-yellow color with a size of 50-150 microns. The yield of finished microcapsules was 70.5%. The number of viable lactobacilli was determined after the destruction of the polymer shell of the microcapsules in phosphate buffer at pH 7.6-7.8, followed by titration of the culture and plating of lactobacilli on agarized medium MPC. As a control, a standard culture in similar dilutions was used. The number of viable lactobacilli in this example was 1.5 • 10 ⁴ cells in 1.0 gram of microcapsules.

EXAMPLE 2
For the manufacture of microcapsules, a culture of lyophilized lactobacilli of the strain L.fermentum 90TS4 containing 10 ⁸ living cells in 1.0 gram was taken.

Prepare 120.0 g of a 40% aqueous solution of polyvinylpyrrolidone, cool to 4 ^{° C.} , then add 10.0 g of lyophilized lactobacilli to this solution and mix until a uniform, stable suspension is formed. After 30 minutes, 50 ml of a 10% aqueous solution of tannin cooled to 4 ^° C were fed into this system with a fine stream ^. The system was left under continuous stirring for 180 minutes. At the end of mixing, the cured microcapsules are precipitated, they are collected by filtration, washed and dried. The obtained microcapsules are rounded particles of gray-yellow color with a size of 50-120 microns. The output of microcapsules was 67.8%. The number of viable lactobacilli was determined according to the method described above. The number of viable lactobacilli in this case was 4.5 • 10 ⁴ cells in 1.0 gram of microcapsules.

EXAMPLE 3
For the manufacture of microcapsules, a culture of lyophilized lactobacilli of the strain L.fermentum 90TS4 containing 10 living cells in 1.0 gram was taken.

Prepare 100.0 g of a 50% aqueous solution of polyvinylpyrrolidone and cool it to 4 ^° C. 5.0 g of lyophilized lactobacilli are mixed with 5.0 g of an inert protective carrier of a polysaccharide structure and then dispersed in a solution of PVP for 30 minutes until a uniform stable suspension. Then, 50 ml of a 10% aqueous solution of tannin cooled to 4 ^° C are fed into this system with a fine stream ^. The system is left with constant stirring for 60 minutes. Cured microcapsules are collected by filtration, washed and dried. The resulting microcapsules are spherical particles of gray-yellow color with a size of 10-150 microns. The yield was 75.9%. The number of viable lactobacilli was determined according to the method described above. The number of viable lactobacilli was 5.3 • 10 ⁴ cells in 1.0 gram of microcapsules.

EXAMPLE 4
For the manufacture of microcapsules, a culture of lyophilized lactobacilli of the strain L.fermentum 90TS4 containing 10 ⁸ living cells in 1.0 gram was taken.

Prepare 75.0 g of a 30% aqueous solution of polyvinylpyrrolidone, cool it at 4 ^° C. In the resulting solution, 5.0 g of lyophilized lactobacilli are dispersed for 30 minutes until a uniform, stable suspension is formed. Then, 70 ml of a 30% aqueous solution of tannin cooled to 4 ^{° C.} at a rate of 1.8 ml / min are fed into this system through a separatory funnel. The system is left under constant stirring for 180 minutes, after which the cured microcapsules are collected by filtration, washed and dried. The obtained microcapsules are rounded particles of gray-yellow color with a size of 80-200 microns. The yield of finished microcapsules was 64.7%. The number of viable lactobacilli was determined according to the method described above. The number of viable lactobacilli was 3.7 • 10 ⁴ cells in 1.0 gram of microcapsules.

Claims (1)

A method of producing microcapsules containing living microorganisms based on polyvinylpyrrolidone, characterized in that the lyophilized culture of microorganisms is dispersed in a 30-50% aqueous solution of polyvinylpyrrolidone in a ratio of 1:10 - 1:15 by weight with stirring until a stable suspension is added, 10-30-30 is added % aqueous solution of tannin, the mixture is stirred until the formation of microcapsules.

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