RU2754927C1 - Method for immobilizing microorganisms on montmorillonite clays - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and is a method for immobilizing microorganisms on montmorillonite clays. The method includes mixing pre-enriched and activated 10% hydrochloric acid montmorillonite clay with biological material in the form of biomass of microorganisms Lysobacter sp. Wherein, after activation, montmorillonite clay is washed with purified distilled water to a neutral medium, dried at a temperature of no more than 105°C and ground in a ball mill to a particle size of no more than 10 mcm; and biological material in the form of biomass of microorganisms Lysobacter sp. pre-fill with 0.05 M tris-hydrochloride buffer solution with pH 7.5 and mix, wherein, with stirring, the mixture is gradually heated to 45°C for 5 minutes, then cooled to room temperature and centrifuged, after which the supernatant is decanted and to the remainder of the treated biomass in the form of L-form Lysobacter sp. with constant mechanical stirring, the crushed clay is gradually added in the ratio of the carrier : biomass equal to 1:(2-4) at a temperature of 25°C; the mixture is thoroughly mixed for at least 40 minutes and then freeze-dried at a temperature of minus 40-45°C for 24 hours to a level of 3-7% moisture content of the composition.EFFECT: invention allows extending the shelf life of bacterial cells of Lysobacter sp. on a carrier made of montmorillonite clay, with the possibility of ensuring the restoration of their activity when applied to the wound surface.2 cl, 1 tbl, 5 ex

Description

The invention relates to biotechnology and can be used in agriculture to obtain an agent for treating wounds in animals.

Numerous methods are now known for immobilizing microorganisms and cells. Various carriers are used for immobilization: gels, Ca-alginate gel, n-alkanes with C14-C16, natural sorbents. But all the described methods have one big drawback, they are mainly used for wastewater treatment or for cleaning the aquatic environment from pollution with oil products.

There is a known method of immobilizing cells by mixing cells with a gel, characterized in that, in order to lengthen the life of mammalian cells, a suspension of cells is mixed with an aqueous solution containing methylcellulose 0.3-1.5%, sodium alginate 0.2-1% and galactan sulfate 0.1 - 0.5%, in a ratio of 1: (1 - 4), then the resulting mixture is mixed with collagen, taken in an amount of 0.3 - 1.5% by weight of the formed helium. (patent SU No. 1 264 575 A1 dated 1984.10.23)

A known method for immobilizing cells of microorganisms with β-tyrosinase activity by including them in a gel, immobilization is carried out in the presence of a stabilizer - ammonium acetate or sodium pyruvate or tyrosine, while a polyacrylamide gel is used as a gel, and Citrobcioter reund No. 62 is used from microorganisms. Immobilization of cells is carried out in phosphate buffer pH 7-8 with the addition of ammonium acetate, or in acetate buffer at pH 8. Cell suspension, solutions of acrylamide and methylene bisacrylamide, tetramethylenediamine and ammonium persulfate are mixed in the buffer. The reaction vessel is immersed in an ice bath. Polymerization ends in a minute. The block of the obtained polyacrylamide gel with included cells is rubbed through a polyethylene sieve with a pore size of 1 * 1 mm and washed from free cells. 89% of cells are included in the gel (patent SU 922142 A1 from 1982.04.23).

A known method of obtaining a biological product for cleaning the aquatic environment from oil pollution. The method of obtaining a biological product consists in the fact that the substance of the carrier, the substance-growth factor of microorganisms and the biomass of microorganisms-oil destructors, immobilized by dispersing the biomass of microorganisms-oil destructors in the mass of the substance of the carrier is physically combined with the substance of the carrier. A composition of a Ca-alginate gel, n-alkanes with C14-C16 and a substance-growth factor for microorganisms was used as a carrier substance. The immobilization of oil-degrading microorganisms is carried out by dispersing the biomass of oil-degrading microorganisms in the mass of the carrier substance with their physical connection with the carrier substance, for which a 1% aqueous mixture with sodium alginate is prepared (for example, in an amount of 100 ml for the mentioned amount of biomass suspension), it is thoroughly mixed and incubated for 2-3 hours to complete the absorption of sodium alginate fragments. Growth factors, for example corn extract in an amount of 10 g per 100 ml of the mixture, are added to the mixture thus obtained with sodium alginate. The physical connection of microorganisms-oil destructors with the substance of the carrier is carried out through the formation of drops from a mixture of suspended biomass with the ingredients of the composition of the carrier substance and the polymerization of the drops with the formation of granules in an aqueous solution containing Ca ions. The granules are filtered, washed and placed in saline. (RF patent No. 2255052 dated 2003.01.17)

A known method for producing a biosorbent consists in immobilizing biomass strains of micromycete Fusarium lateritium NK-204 or Gliocladium deliquescens NK-205 or Gliocladium deliquescens NK-206 or a consortium of these strains into a hydrophobic oil sorbent, by means of overgrowing the mycelium of fungi placed on the sorbent. Next, the resulting preparation is dried. The sorbent is made of peat-based hydrophobic oil sorbent. The mycelium of mushrooms is 20-50% by dry weight. (patent RU (11) No. 2299181, No. 2318736, No. 2299181 dated 2005.08.03)

A known method of producing a biosorbent, including immobilization on an oil hydrophobic sorbent yeast fungi Candida lipolytica, Candida guilliermondii, Pichia guilliermondii and bacterial cultures Rhodococcus erythropolis, Arthrobacter sp. in an amount from 10 to 50% (by dry weight) by means of overgrowing with mycelium of fungi of the sorbent, followed by air drying. (patent RU No. 2318736 dated 2006.02.10)

There is a known method of immobilizing cells of microorganisms in a hydrophobic oleophilic sorbent, which consists in mixing two aerosols to a given degree of saturation of the sorbent with a bioagent. The first aerosol consists of a gaseous medium in which sorbent particles are suspended. The second aerosol consists of a gaseous medium in which a mixture of either oil or oil product and culture liquid with a bioagent is in suspension. The sorbent particles are 1 ÷ 3 mm in diameter. The droplet size of oil or oil aerosol is 15-25 microns. Drops of oil or oil aerosol fall on the surface and into the pores of the hydrophobic sorbent, providing quick contact and introduction of microbial cells into the branched structure of the hydrophobic sorbent. The process of immobilization of microbial cells contained in oil or oil is performed in a chamber where flows of sorbent and emulsion are supplied with a given intensity, temperature, and pressure. Immobilization is continued until the content of microbial cells in the sorbent is not less than 109 living cells per 1 g of sorbent. This value is determined by design and is provided in production through sampling and subsequent laboratory control. (RF patent No. 2420579 C2 dated 2009.08.11)

A known method of obtaining a composition containing dried bacteria, which involves the cultivation of one or more species of living bacteria; mixing the cultured bacteria with one or more carriers; treatment of bacteria with pulsed electromagnetic fields of 2 mV / cm at 50 Hz and 55 V; incubating the culture: vehicle mixture for at least about 6 hours; and drying the bacteria so as to reduce the moisture level to about 1 to about 6 wt%.

Preferred carriers according to the invention are one or more of the following: zeolite carrier, clay carrier, natural silicon compound with a natural water content similar to the final moisture content of the mixture culture: carrier after drying, generally in the range from 3 to 7.5% (w / w). The cultured bacterium and the carrier are mixed in such a way that the ratio of culture to carrier is from about 1: 3 to about 1: 5. Treatment with PEMF (pulsed electromagnetic fields) is carried out at one or more of the following stages: during the cultivation of the bacteria; while mixing the cultured bacteria with the carrier; after mixing the cultured bacteria with the carrier; during incubation of the mixture, culture: vehicle; during the drying of the mixture, culture: vehicle; at any time after applying the specified mixture to the seed or seed component; at any time after the mixture has dried, culture: vehicle; at any time after rehydration of the dried culture: vehicle mixture.

Preferably, the microorganism and / or the culture of microorganism: carrier mixture is dried with room air in trays or similar containers. Preferably, room air drying is carried out at a temperature of about 10-30 ° C, usually about 20-24 ° C, and at a relative humidity of less than 75%, preferably about 30-60%, more preferably about 32.5- 35%. When drying with room air, drying can take from 1 to 5 days, preferably from 1 to 4 days, suitably 3-4 days. ^{Accordingly, during drying with room air, the presence of Ca 2+} ions in the atmosphere may be beneficial for the survival of the microorganism. Alternatively, drying can be accomplished by placing the culture: carrier mixture in a container, such as a Milli-Wrap ^RTM container, where the container allows moisture to evaporate.

The moisture level is gradually reduced to about 1% to about 6% (w / w).

The drying step can be carried out under non-sterile conditions.

The dried product can be milled using an air mill to a final particle size of about 0.1 to about 150 microns.

Accordingly, in one embodiment, the culture: carrier mixture can be incubated at about 10-15 ° C and at a moisture content of about 18-33% (wet weight), followed by drying at 20 ° C over saturated calcium chloride for 3- 4 days at approximately 32.5% RH or rapid drying in less than 24 hours. The moisture content of the culture: carrier mixture can be reduced to about 4% to about 7%.

The resulting composition can be used for application to seeds or other reproductive material of a plant, for introduction into a medium for plant growth, for purification of waste water and / or purification of chemical / biological waste, for purification of contaminated soils, for introducing an acceptable microorganism into food products and / or animal feed to provide lactic acid bacteria for medical purposes. The combination of mixing the cultured bacterium with one or more carriers and treating the microorganisms in the composition significantly increases the initial survival and shelf life of the composition. (RF patent No. 2370525 C2 dated 2005.03.30)

The disadvantage of this method is the need for processing by pulsed electromagnetic fields. In addition, the resulting products are not intended for the treatment of wounds in animals.

For the prototype, a technical solution was chosen according to patent US5695541 (A) (published 1997-12-09), which discloses a method for immobilizing dried microorganisms, namely, species of microorganisms selected from B. japonicum, R. meliloti, R. leguminosarum biovar trifolii, Viceae and phaseoli, Bradyrhizobium species for peanuts and B. lupini, by mixing the microorganisms with a carrier, namely a mixture of kaolinite and montmorillonite, having a pH range of about 5 to 8. The weight ratio of culture to carrier in the mixing step ranges from 1: 2 to 1: 4. Then incubate the mixture of the culture-carrier for at least one day at a temperature in the range from 20 to 30 ° C and a humidity level in the range from about 25 to 33 wt. % by wet weight of the mixture so that the number of microorganisms in said mixture is increased; then dry the resulting mixture for at least about one day at a temperature in the range from 20 to 30 ° C to a moisture content of not less than about 15 wt. % on a wet basis. Additionally, the mixture is kept for about 3-14 days at a relative humidity in the range from 35 to 60%, so that the moisture level of the mixture gradually decreases to about 1-5 wt. % on a wet basis. It is crushed to form particles of the specified composition having a size in the range from about 0.1 to 150 microns. This invention relates to an improved slow drying process for bacterial agricultural inoculants and other compositions that enhance the viability of bacteria to control insects, fungi, and the like, or microorganisms that have growth promoting effects.

The disadvantage is that the sorption activity of the clay is not fully utilized, as well as the fact that the resulting product cannot be used to treat animal wounds.

The task of the group of inventions is to develop a method for the immobilization of microorganisms Lysobacter sp. on montmorillonite clays in order to obtain a remedy for the treatment of animal wounds.

The technical result of the invention is to extend the shelf life of bacterial cells of Lysobacter sp. on a carrier made of montmorillonite clay, with the possibility of ensuring the restoration of their activity when applied to the wound surface.

Bacterial cells of Lysobacter sp. selected because they are known in the art to produce antibacterial and antimicrobial agents (Bonner, D.P., J. O'Sullivan, S.K. Tanaka, J.M. Clark and R.R. Whitney. 1988. Lysobactin is a novel antibacterial agent produced by Lysobacter sp. II. Biological properties. Judo (Tokyo) 41: 1745-51) (Hashizume, H., S. Hirosawa, R. Sawa, Y. Muraoka, D. Ikeda, H. Naganawa and M. Igarashi 2004. Tripropeptins, novel antimicrobial agents, manufactured by Lysobacter sp J Antibiot (Tokyo) 57: 52-8.

The problem is solved by the method proposed below for the immobilization of microorganisms Lysobacter sp. for montmorillonite clays (hereinafter MSG), which may include illite, quartz, muscovite:

- in this case, MSG is pre-enriched and then activated with 10% hydrochloric acid, after which it is washed with purified distilled water to a neutral medium, dried at a temperature of no more than 105 ° C and ground in a ball mill to a particle size of no more than 10 microns;

- biological material in the form of biomass of microorganisms Lysobacter sp. pour 0.05 M tris - hydrochloride buffer solution with pH 7.5 and mix, while stirring, the mixture is gradually heated to 45 ° C for 5 minutes. Then it is cooled to room temperature, then centrifuged and the supernatant is discarded. Such treatment causes a stop of the cell cycle, disruption of intracellular transport processes, and most importantly contributes to the accumulation of trehalose, which contributes to the formation of the L-form of the microorganism, which has bacteriolytic activity against living opportunistic and pathogenic bacteria, including multiply resistant strains (Internet source http: //mbio.bas-net.by/wp-content/uploads/2015/09/07_Kudryakova_2015.pdf);

- then to the remainder of the treated biomass in the form of L-form Lysobacter sp. with constant mechanical stirring, the enriched and activated MSH is gradually added in a carrier: biomass ratio of 1: (2-4) at a temperature of 25 ° C;

- the mixture is thoroughly mixed for at least 40 minutes;

- then subjected to freeze drying at a temperature of minus 40-45 ° C for 24 hours to a level of 3-7% moisture content of the composition.

The invention can be illustrated by the following examples of its specific implementation.

Example 1.

Biological material in the form of biomass of microorganisms Lysobacter sp. 20 g is poured with 40 ml of 0.05 M tris - hydrochloride buffer solution pH 7.5, mixed, centrifuged and the supernatant is discarded. Then it is subjected to freeze-drying for 24 hours to a level of 3-7% moisture content of the composition.

Example 2.

Biological material in the form of biomass of microorganisms Lysobacter sp. 20 g is poured with 40 ml of 0.05 M tris - hydrochloride with a buffer solution of pH 7.5, stirred and gradually heated to 45 ° C for 5 minutes. Then it is cooled to room temperature, then centrifuged and the supernatant is discarded. Such treatment causes a stop of the cell cycle, disruption of intracellular transport processes, and most importantly contributes to the accumulation of trehalose, which contributes to the formation of the L-form of the microorganism. Then it is subjected to freeze-drying for 24 hours to a level of 3-7% moisture content of the composition.

Example 3.

Immobilization of microorganisms on montmorillonite clays in a carrier: biomass ratio of 1: 2.

Preparation of montmorillonite-containing clay is carried out, for which sedimentation enrichment of montmorillonite-containing clay in water is carried out by holding the suspension for 24 hours, stirring up for one minute and settling the suspension for 20 minutes, then a supernatant suspension with a clay particle size of less than 10 μm is taken from the upper 10 cm layer, defended for another 10 minutes and after sedimentation of the suspension, clarified water is decanted, then the precipitate of the enriched MSG is dried in an oven at 70-105 ° C. With the appropriate equipment, the drying process can be carried out at temperatures up to 900 ° C, which shortens the duration of the process, but increases its energy consumption. After the enriched MSG is crushed to a homogeneous consistency. Then MSG is activated with 10% hydrochloric acid, after which it is washed with purified distilled water to a neutral medium, dried at a temperature of no more than 105 ° C and ground in a ball mill to a particle size of no more than 10 microns.

Biological material in the form of biomass of microorganisms Lysobacter sp. in the amount of 20 g pour 40 ml of 0.05 M tris - hydrochloride buffer solution pH 7.5, mix and gradually heat up to 45 ° C for 5 minutes. Then it is cooled to room temperature, then centrifuged and the supernatant is discarded. Such treatment causes a stop of the cell cycle, disruption of intracellular transport processes, and most importantly contributes to the accumulation of trehalose, which contributes to the formation of the L-form of the microorganism. Then 10 g of MSH is gradually added to the remainder of the treated biomass at a temperature of 25 ° C with constant mechanical stirring. The mixture is thoroughly mixed for at least 40 minutes. Then it is subjected to freeze-drying for 24 hours to a level of 3% moisture content of the composition.

Example 4.

Immobilization of microorganisms on montmorillonite clays in a carrier: biomass ratio of 1: 3.

Montmorillonite clay is prepared as in example 3.

Biological material in the form of biomass of microorganisms Lysobacter sp. in an amount of 30 g, pour 60 ml of 0.05 M tris - hydrochloride with a buffer solution of pH 7.5, mix and gradually heat up to 45 ° C for 5 minutes. Then it is cooled to room temperature, then centrifuged and the supernatant is discarded. Such treatment causes a stop of the cell cycle, disruption of intracellular transport processes, and most importantly contributes to the accumulation of trehalose, which contributes to the formation of the L-form of the microorganism. Then 10 g is gradually added to the remainder of the treated biomass at a temperature of 25 ° C. MSG with constant mechanical stirring. The mixture is thoroughly mixed for at least 40 minutes. Then it is freeze-dried for 24 hours to a 5% moisture content of the composition.

Example 5.

Immobilization of microorganisms on montmorillonite clays in a carrier: biomass ratio of 1: 4.

Montmorillonite clay is prepared as in example 3.

Biological material in the form of biomass of microorganisms Lysobacter sp. in an amount of 40 g, 80 ml of 0.05 M tris - hydrochloride are poured with a buffer solution of pH 7.5, stirred and gradually heated to 45 ° C for 5 minutes. Then it is cooled to room temperature, then centrifuged and the supernatant is discarded. Such treatment causes a stop of the cell cycle, disruption of intracellular transport processes, and most importantly contributes to the accumulation of trehalose, which contributes to the formation of the L-form of the microorganism. Then 10 g of MSH is gradually added to the remainder of the treated biomass at a temperature of 25 ° C with constant mechanical stirring. The mixture is thoroughly mixed for at least 40 minutes. Then it is subjected to freeze-drying for 24 hours to a level of 7% moisture content of the composition.

Initial survival after immobilization of microorganisms Lysobacter sp. on the montmorillonite clays of Examples 1-5 was determined by the Pour Plate method, in which the sample is suspended in a Petri dish using molten agar cooled to about 40-45 ° C (just above the solidification point to minimize heat-induced cell death). After the nutrient agar has solidified, the plate is incubated.

The results (based on CFU colony forming units) are presented in the table below.

* p≤0.05 by Student's t test

It follows from the table that the temperature shock before the immobilization of the microorganism by gradually heating the biomass to 45 ° C followed by combination with the proposed carrier increases the initial survival over a long shelf life.

Thus, the proposed method for the immobilization of microorganisms Lysobacter sp. on enriched and activated montmorillonite clays leads to increased viability of the microorganism during a long shelf life. The limitation of the vital activity of microorganisms before application to montmorillonite clays allows maintaining the qualitative and quantitative composition of the original biomaterial and ensuring the restoration of their activity when applied to the wound surface.

Claims (2)

1. A method for immobilizing microorganisms on montmorillonite clays, including mixing montmorillonite clay, pre-enriched and activated with 10% hydrochloric acid, with biological material in the form of biomass of microorganisms Lysobacter sp. not more than 105 ° C and ground in a ball mill to a particle size of not more than 10 microns; and biological material in the form of biomass of microorganisms Lysobacter sp. pre-pour 0.05 M tris-hydrochloride with a buffer solution with pH 7.5 and stir, and with stirring, the mixture is gradually heated to 45 ° C for 5 minutes, then cooled to room temperature and centrifuged, after which the supernatant is poured and the treated residue is biomass in the form of L-form Lysobacter sp. with constant mechanical stirring, crushed clay is gradually added in a carrier: biomass ratio of 1: (2-4) at a temperature of 25 ° C; the mixture is thoroughly mixed for at least 40 minutes and then freeze-dried at a temperature of minus 40-45 ° C for 24 hours to a level of 3-7% moisture content of the composition. 2. The method according to claim 1, characterized in that the enriched montmorillonite-containing clay is obtained using the sedimentation method by keeping a suspension of montmorillonite clay in water for 24 hours, then the suspension is stirred up for one minute and settled for 20 minutes, then the supernatant suspension is taken from the size of clay particles less than 10 microns from the upper 10-cm layer, settle for another 10 minutes and after sedimentation of the suspension, clarified water is decanted, then the enriched clay sediment is dried and ground to a uniform consistency.