RU2720099C1 - Method of producing a dialdehyde derivative of a gel film of bacterial cellulose - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method of producing a dialdehyde derivative of a gel film of bacterial cellulose. Method involves cultivation of a strain of bacteria Gluconacetobacter sucrofermentans VKPM B-11267 on a post-alcohol grain brew or on native milk whey during 3–5 days at 28±2 °C. Produced bacterial cellulose is dried at 80 °C and treated with 0.2–0.5 N sodium periodate solution for 6 hours.

EFFECT: method provides higher binding capacity of the dialdehyde derivative of the gel film of bacterial cellulose without disturbing the film structure and strength properties.

1 cl, 1 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of cosmetics and medicine, specifically to the preparation of reactive biodegradable and biocompatible cellulose matrices for grafting (immobilizing) enzymes and other biologically active compounds (BAS) for the manufacture of cosmetic masks, wound dressings and dressings, as well as for obtaining aerogels with high binding ability.

It is known that for the manufacture of medical dressings worldwide, materials from natural cellulose (C) are used. Products from C have excellent sanitary and hygienic properties without chemically interacting with biological fluids and tissues. Thus, medical dressings from natural TS passively participate in the wound cleansing process, sorbing tissue breakdown products, protect the wound from aerogenic contamination and maintain the thermal regime in the wound environment. Immobilization of ALS through the formation of new chemical bonds is currently the dominant way to obtain long-acting dressings. The most important condition for obtaining highly active preparations of immobilized ALS is the participation in the formation of covalent bonds with the carrier of those ALS functional groups that are not responsible for its biological properties (in the case of immobilized enzymes, catalytic properties), that is, are not part of the ALS active center and are not located in close proximity to it. The advantage of this method is that BAS, as a rule, does not pass into solution, even with very prolonged use. For covalent binding of ALS with carriers (matrices) such as C is required; so that the carrier is preactivated, so that it would be possible to chemically bind it with BAS molecules. For the chemical binding of proteins to carriers, a large arsenal of various methods is used. The choice of Ts as carriers is associated with widespread use in medicine, biocompatibility, and biodegradability. Cellulose fibers still dominate the textile market (52%), synthetic polyamide fibers (9%) also occupy a significant place. A known method for the production of dialdehyde cellulose (DAC) by the selective oxidation of periodate with gauze, tissue, and cotton wool ions The resulting DAC is an excellent carrier of ALS due to the formation of azomethine bonds between the aldehyde groups of DAC and ALS (enzymes) (RU 2323748, IPC A61L 15/16, A61L 15 / 32, A61L 15/38, published May 10, 2008; RU 2380117, IPC A61L 15/28, A61L 38/36, A61L 15/32, published January 27, 2010; RU 2426558, IPC A61L 15/18, A61L 15 / 38, A61L 15/28, publ. 08/20/2011; RU 2448738, IPC A61L 15/28, A61L 15/32, A61L 31/717, publ. 04/27/2012; RU 2203684, IPC A61L 15/48, A61L 15 / 20, published May 10, 2003).

A disadvantage of the known carriers is the high adhesion to the wound, the impossibility of visual observation of the healing process. Alternatively, a gel film of bacterial cellulose can be used, which is transparent and, moreover, adhesion to the wound is virtually eliminated.

The closest in technical essence is the method of producing bacterial cellulose, which involves the cultivation of the bacterial strain Gluconacetobacter sucrofermentans H-110 (VKPM B-11267) in the liquid phase of the post-alcohol grain stillage obtained after mechanical separation of the solid phase for 3-5 days at a temperature of 28 ± 2 ° C and pH 3.9-4.4, as well as separating the resulting bacterial cellulose from the culture medium and drying at a temperature of 80 ° C to constant weight (RU 2536973, IPC C12N 1/20, C12P 19/04, C12R 1 / 01, publ. 12/27/2014). A known method for producing bacterial cellulose, which provides for the cultivation of the bacterial strain Gluconacetobacter sucrofermentans H-110 on native milk serum for 3-5 days at a temperature of 28-30 ° C. The separation of the resulting bacterial cellulose from the culture medium and drying at a temperature of 80 ° C to constant weight (RU 2536257, IPC C12N 1/20, C12R 1/01, publ. 20.12.2014).

The known gel film can be used as a carrier for immobilized biologically active compounds, without prior modification, for immobilization by adsorption. Adsorption is a weak method of immobilization, which, as a rule, weakly affects the activity of immobilized ALS. The main disadvantage of this method is that the ALS may not bind to the carrier well enough. In addition to salt (ionic) bonds, other weak interactions, for example, hydrogen or Van der Waals bonds, can also participate in the interaction of BAS with a carrier. Even a slight change in experimental conditions: pH, ionic strength, temperature and nature of the solvent, as well as the substrate itself, can cause the desorption of UAS. As is known, the sorption abilities of bodies are determined not only, and in some cases not so much by chemical nature as by their physical structure.

These disadvantages can be eliminated if the gel film of bacterial cellulose is activated by the method of periodic oxidation. In this case, aldehyde groups are formed in the gel film, capable of forming strong azomethine bonds with the amino groups of many ALS, for example, with trypsin, pepsin, antibiotics, etc.

The technical result consists in increasing the binding ability of the dialdehyde derivative of the bacterial cellulose gel film without violating the film structure and maximally preserving the strength properties.

The essence of the invention lies in the fact that in the method for producing a dialdehyde derivative of a bacterial cellulose gel film, the bacterial strain Gluconacetobacter sucrofermentans is cultured in the liquid phase of the post-alcohol grain stillage or on native milk serum for 3-5 days at a temperature of 28 ± 2 ° C, separating the resulting bacterial cellulose from the culture medium and drying at a temperature of 80 ° C to constant weight. Obtained by static cultivation in an amount of 0.10-0.15 g, the gel film is treated with 20 ml of 0.2-0.5 N sodium periodate solution for 6 hours

In the table. 1 shows the physicomechanical characteristics of gel films; the figure shows the effect of the modifier on the structure (appearance) of the gel film: a - control without treatment; b is the exposure time of 6 hours at a modifier concentration of 0.2 N; c is the exposure time of 6 hours at a modifier concentration of 0.5 N; d is the exposure time of 24 hours at a concentration of modifier of 0.5 N.

Example. A gel film of bacterial cellulose obtained according to one of the known methods (RU 2536973, IPC C12N 1/20, C12P 19/04, C12R 1/01, publ. 12/27/2014; RU 2536257, IPC C12N 1/20, C12R 1 / 01, publ. 20.12.2014), in an amount of 0.1-0.15 g is treated with 20 ml of 0.2-0.5 N sodium periodate solution for 6 hours

The strain of the bacterium Gluconacetobacter sucrofermentans H-110 was deposited in the All-Russian Collection of Industrial Microorganisms under registration number VKPM: B-11267 (RU 2523606, IPC C12N 1/20, C12P 19/04, C12R 1/01, publ. 07.20.2014).

The presence of the formed aldehyde groups is determined by the appearance of the absorption band in the region of 870-900 cm ^-1 , the number of formed aldehyde groups is determined by the iodometric method. The strength and tensile strength of protein-polysaccharide biodegradable films in MPa (N / mm ² ) are determined in accordance with GOST 17035-86 and ASTM using a universal testing machine with electromechanical drive XLW (PC) Auto (GOST 7855-84; ASTM). The film thickness is determined on an automatic high-resolution thickness gauge CHY-C2. The film retains its structure and with minimal loss of strength with the content of aldehyde groups, 18-22%, degree of substitution, 120.8-185.4 rel. units). The tensile strength of gel films is 50–25 (5–6) MPa, and tensile strength is 8–6 (1.5–3.0)%. During a longer treatment with the modifier, the films did not retain their structure and a significant decrease in strength and elongation was observed (Table 1, Fig.).

Compared with the known solution, the claimed invention allows to increase the reactivity of the gel film due to the formed aldehyde groups, while maintaining the structure, and minimizing the decrease in strength and tension.

Claims (1)

A method of obtaining a dialdehyde-derivative gel film of bacterial cellulose, which consists in cultivating a strain of the bacterium Gluconacetobacter sucrofermentans VKPM B-11267 in the liquid phase of the post-alcohol grain stillage or on native milk serum for 3-5 days at a temperature of 28 ± 2 ° C, separating the resulting bacterial cellulose from culture medium and drying at a temperature of 80 ° C to constant weight, characterized in that the gel film obtained by static cultivation in an amount of 0.10-0.15 g is treated with 20 ml of 0.2-0.5 N periodate solution on tria for 6 hours

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