Method for the production of sodium salt of deoxyribonucleic acid from animal products and installation for its implementation

 

(57) Abstract:

Usage: biotechnology and pharmaceutical industry. The essence of the invention: animal raw materials (ROE of sturgeon, spleen, erythrocytes of chicken, and so on ) are ground and homogenized in citrate-saline solution, treated homogenate detergent and concentrated solution of sodium chloride at an elevated temperature; then the reaction mixture after the preliminary formation of a layer of a certain thickness is treated within 10 to 80 min ultrasound with a frequency of 8 - 35 kHz and an intensity of 0.2 to 2.0 W/cm2"voiced" the mass is mixed with diatomaceous earth in the ratio of 5 : 1 to 25 : 1 (vol/wt), the liquid phase is separated by filtration and concentrate baravilala; Na-salt DNA precipitated from the concentrate of ethyl alcohol, and the residue is dried at 20 - 60C. The scaling process is carried out through the creation and use of new industrial plants. 1 Il.

The invention relates to the technology of biologically active substances and can be used in biotechnology and pharmaceutical industry, and the obtained product is in medicine, veterinary medicine, perfumery and research the animal of origin.

The main drawback of most of them is that these methods are intended for use in laboratory conditions and are not designed to obtain the desired product in an industrial scale.

Among the few methods of DNA extraction from animal products, with the possibility of scaling process, i.e., with such factors as the availability of raw materials, ease of operations, no need for unique equipment and reagents, and so on , the closest to the offer in terms of total tricks is a process comprising grinding and homogenization of raw materials (milk sturgeon) in citrate-saline solution, treating the reaction mixture with the detergent and sodium chloride at 60-70aboutWith cooling, mixed with diatomaceous earth, the elution of DNA citrate-saline solution, separation of the eluate by filtration and precipitation of the final product with ethyl alcohol. This method uses the available raw materials, the possibility of processing large quantities includes simple operations that do not require the use of scarce equipment and reagents, however, its industrial use is clearly impractical due to nedostatochnoi> In addition, get this and other known methods, high molecular weight DNA has limited scope and is used in genetic engineering and research in molecular biology and genetics.

The use of such DNA in clinical practice is possible only after additional processing of the finished producta order restricted (controlled) fragmentation of macromolecules, which has the consequence of reducing the viscosity of the DNA solution, and the elimination of the inherent high-molecular form properties, toxicity and Carcinogenicity.

In many cases there is a significant degradation of native double-DNA structures (ibid), and consequently, her loss of some important biological properties.

The implementation of an industrial process comprising the fragmentation of DNA in the final stage of the process (after receipt of sediment molecular forms) prakticheski impossible because Tehnologichesky difficulties in the separation, dissolution and further processing of high-viscosity precipitation.

The present invention is to develop a method of production of sodium salt of DNA, causing not only the sa, namely characterized by high performance and allows to obtain a high yield of DNA preparation with characteristics that are necessary to significantly expand the scope of its application (limited decrease in molecular weight while maintaining the native structure and high quality cleaning).

This object is achieved due to:

a) introduction before mixing the reaction mixture with diatomaceous earth stage ultrasonic treatment under certain strictly defined modes;

b) the implementation of the "sound" of the reaction mixture in a layer thickness which provides uniform handling of all material in the selected mode;

C) selection of the optimal ratio of diatomaceous earth / reactive mixture;

g) introducing the procedure, the concentration of the liquid phase baromembrane method;

d) drying the precipitate in a "soft" conditions (20-60aboutC).

Reception "and" allows us not only controlled slicing of high molecular weight DNA, but this change in the structure of the reaction mass, which facilitates subsequent cleaning and improves its quality.

Method "b" increases the efficiency and uniformity of ULTRASONIC processing, and provides increased bandwidth of specialnote, the yield and quality of cleaning.

The change in the ratio of diatomaceous earth / reactive mixture in the direction of increasing reduces the speed of the process; "delay" on the sorbent not only ballast proteins and DNA, which is a consequence of the need to introduce additional operations elution; the decrease in the ratio leads to the rapid formation of sorbent protein film, deteriorating the quality of the separation of impurity compounds.

Reception "g" contributes to a more complete precipitation of the DNA, and therefore has a positive effect on the yield of the target product. Concentration using barometry in working with a full-sized DNA from animal products impossible.

Receiving "d" (drying at 20-60aboutC) protects the product from possible adverse effects of freeze drying, which is commonly used in similar methods; it is also possible in this case by reducing the molecular weight of the drug and the high level of treatment.

P R I m e R 1. 1 kg of sturgeon milk crushed in a grinder, homogenized in 2 l of citrate-saline solution (0.15 M NaCl and 0.015 M Na citrate) and placed in a reactor with heated citrate-solely the societal reaction mixture at 60aboutC for 1.5 h with constant stirring. Then add an equal volume of 5 M NaCl solution and continue stirring at the same temperature for further 1.5 hours After the reaction mass is then cooled to room to.

The cooled mass is then poured into the tub with a layer of 15 mm and treated with ultrasound at a frequency of 8 kHz and an intensity of 2 W/cm2within 80 minutes "Announced" the mass is mixed with diatomaceous earth powder in the ratio of 5: 1 (vol: wt), and then separating the liquid phase into a suction filter.

After filtration the filtrate is concentrated (baromembrane method) on the membrane with a pore size of 0.45 ám.

Get 8 l of concentrate containing 7.5 wt. % DNA in the form of Na-salt. Target product produce by its resultant deposition rates ethyl alcohol. The precipitate was separated by centrifugation and dried at 20-40aboutWith in 48 hours

Get 60 g of sodium salt of DNA that has the following characteristics: mol. m 400 100 KD, the protein content of not more than 1% , the content of RNA is not more than 2% , the content of polysaccharides not more than 2% , humidity 17% , hyperchromic effect 44% . The final product is a white amorphous powder.

P R I m m e R 2. 1 kg of pig spleen crushed and homogeniz dcuo phase concentrated on the membrane with a pore size of 0.8 μm.

Get 0.5 l of concentrate containing 3.6 wt. % of sodium salt of DNA. The target product is precipitated with ethyl alcohol, taken in relation to the concentrate 1: 2; the precipitate was separated and dried at 50-60aboutC.

Get 1.8 g Na-salt DNA with the following characteristics: mol. m 455 KD, the protein content of not more than 1.5% , the DNA content of not more than 2% , the content of polysaccharides not more than 2% , moisture 15% , hyperchromic effect 42% .

P R I m e R 3. The process is carried out as in example 1, but the exposure time of the ULTRASONIC is 10 min, a frequency of 35 kHz, the layer thickness of 70 mm, the intensity of 2.0 W/cm2; the ratio of diatomaceous earth and the reaction mass 1: 25, todrying 30-40aboutC.

Get 60 grams of Na-DNA, with the following characteristics: mol. m 400 100 KD, the protein content of RNA is not more than 2.0 wt. % , the moisture content of 16.5% , hyperchromic effect 44% .

P R I m e R 4. To the stage ultrasonic treatment as in example 1. Then the reaction mass immediately mixed with diatomaceous earth (5: 1) and filtered on a suction filter. From the filtrate without prior concentration produce DNA double presidenial in ethyl alcohol. Receive 40 mg of the drug with the following characteristics: mol. m 15000 CD; the protein content of 1.5 wt. % ; the content of RNA 2.0 wt. % ; HiPER is similar raw materials.

Controlled the size of the fragments, the safety of the secondary structure of the macromolecule and a high degree of purification give the possibility to use the claimed process, the preparation of Na-DNA in research practice and work on genetic engineering.

A limited decrease in molecular weight provides the possibility of using the obtained Na-salt DNA in medicine, veterinary medicine and perfumery (after receipt of the relevant lekarstennyh forms).

Thus, the claimed method for the production of Na-salt of DNA from animal products causes not only the possibility but also the feasibility of scaling process. The translation method on an industrial basis is due to the application of the proposed facility.

Analogues installation, providing a full technological cycle of production of sodium salt of DNA from animal products (from raw material preparation to obtain a dry powder product) in the scientific and patent literature is not found.

The creation of this setup is aimed at the solution of the translation method of obtaining sodium salt of DNA with the declared characteristics (high degree of purification, a certain amount of the fragm is designed to fulfill three basic conditions:

a) increase the amount of raw material processed during one cycle;

b) the exact observance of the sequence of operations of the method and mode of their performance;

C) to prevent additional losses normally associated with the scaling process (i.e. maintaining a high yield of the target product, the source provided by the proposed method).

At the device level, this is achieved respectively by:

a) the presence in the installation of suitable structural elements;

b) positioning in a strict order (in accordance with the apparatus of the scheme);

C) constructive solutions to individual items of equipment and/or their quantity in the "narrow" places of the process.

Regarding the latter conditions, it is dictated by the fact that due to significant differences in the nature and speed of certain operations of the technological process in the transition from stage high (due to high speed or large) "bandwidth" to the stage with a low may be a delay in the processing of the reaction mass, with the consequence of loss of the target product. These "outages" and, accordingly, additional is that such bottlenecks way any presence on these sites the number of elements required equipment selected from known means. In this case, the main "narrow" place when scaling method is ultrasonic treatment, because the device is usually applied for "sound" of biological objects, designed for a small amount of processed material. When creating this installation, this difficulty was overcome by the fact that the area of ULTRASONIC processing includes (depending on the amount of raw material in each cycle) baths with built-in bottom magnetostriction. Previously these baths were not used. The nature of the processed material and, most importantly, the need for uniform "study" the whole mass at strogogo certain modes, identified the following design features of the device as the minimum clearance between the side walls of the bath and magnetostriction, as well as the presence of construction baths roaming cooling coil and drain holes.

The drawing shows a functional-technical scheme of the proposed installation.

Installation for the production of sodium salt of DNA from animal products includes cutting table 1; minced 2 with the actuator; microi processing; the mixer 7 for mixing the reaction mixture with a powder of diatomaceous earth; a device for separation of the liquid phase, for example, suction filters 8; device 9 (a, b) for microfiltration; baromembrane device 10 for concentrating the liquid phase; the reactor for deposition of Na-salt DNA, equipment for separating and drying the precipitate, for example, stakanchikov centrifuge 12, SCHOTT funnel 13 and, if necessary, drying Cabinet 14.

In addition, the apparatus comprises a measuring tank 15 (see option 12 pieces ) for measuring liquids; mixers 16 (5 PCs ) for the preparation of solutions and suspensions at room temperature; reactor 17 (4 pieces ) to obtain solutions and mixtures at temperatures different from room temperature, and the tank 18 (4 pieces ) for collection of intermediate products and production waste.

The mixers 16 represent capacity, equipped with a mixing device; a mixer 6 for diatomaceous earth has a high-speed stirrer (8000 rpm). Mixers and measuring capacity, designed to work with alcohol and alcohol solutions, additionally sealed.

The reactors 17 - sealed containers, which are equipped with stirrer, jacket for under the low shear mixing device, for example, the stirrer of the anchor type.

Measuring tanks, mixers, reactors and collections of interconnected pipelines.

The device 6 for ultrasonic processing consists of several baths, each of which is equipped with a built-in bottom of the magnetostrictive transducer connected to an ultrasonic generator, while the gap between magnetostriction and side walls of the bath does not exceed 2 see cooling System made in the form of a tubular heat exchanger, which is placed inside of the bath so that the distance between him and magnetostriction to ensure the absence of interaction between them. Each tub has an opening for release of the reaction mass.

The process of obtaining a Na-salt DNA with the use of this installation is as follows.

1. Preparation of homogenate and solutions.

1.1. Preparation of homogenate.

1.1.1. Frozen raw (ROE of sturgeon) arrives at the cutting table 1 and thawed, and then manually cleaned from fat and films.

1.1.2. Purified raw materials are crushed with the grinder 2 and homogenized with citrate-saline solution (SSC) in the CLASS="ptx2">

1.2. Preparation of citrate-saline (SSC).

1.2.1. The addition of sodium citrate and sodium chloride are poured into the reactor 17A, pour from the volumetric capacity 15A necessary amount of the distillated water.

1.2.2. Include a stirrer, stirred until the salt is completely dissolved, the resulting solution was "perelavlivaet" in a volumetric tank 15B.

1.2.3. From the volumetric capacity 15B solution by gravity into the reactor 4 cooking and at the site of preparation of homogenate.

1.3. The preparation of an aqueous-alcohol solution of sodium dodecyl sulfate.

1.3.1. Ethyl alcohol from the volumetric capacity 15V and distilled water from a measuring vessel 15g gravity comes into the mixer 16A running stirrer in proporciii required to obtain 45% alcohol solution.

1.3.2. 45% alcohol solution is pumped into the measuring tank d, after which it flows under gravity into the reactor 17B.

1.3.3. In the reactor 17B filled with a water-alcohol solution, poured out a portion of sodium dodecyl sulfate, include the mixer and continue mixing until dissolved.

1.3.4. The resulting solution is pumped into the measuring capacity of the 15th and next by gravity into the reactor boiling 4.

1.4.2. The resulting solution was "perelavlivaet" in measuring capacity s, whence it flows by gravity into the reactor rollers 4.

1.5. Preparation of 70% ethanol.

1.5.1. In the mixer 16B in the required proportions distilled water from a measuring vessel 15 and 96% ethanol from the volumetric capacity 15K.

1.5.2. The resulting solution is fed to the mixer 16B.

2. Boiling of the reaction mixture

2.1. In the reactor 4 cooking after loading the reaction mixture sequentially receives SSC, saline sodium dodecyl sulfate and 5 M NaCl; processing each of the solutions (with constant stirring) hold when to= 60-70aboutWith within a certain time; at the end of the cooking process, the reaction mixture is pumped into the reactor cooling 5 and cooled to room to.

3. Sonication.

3.1. The cooled mass is served in a bath of the ULTRASONIC device 6 portions and treated with ultrasound for a certain period of time when required modes.

3.2. After the procedure, "scoring" with the BR> 4.1. Simultaneously with the "voiced" by the mass in the mixer 7 is served estimated number of diatomaceous earth powder; the mixture is stirred specified time.

4.2. The resulting suspension is portion on the vacuum suction filters 8, where the separation of the solid phase.

5. The filtering.

5.1. The collected liquid phase is pumped to the filter 9a and next to install to concentrate 10.

5.2. The concentrate flows into the collector 18b, and the permeate - collection 18V and then goes on regeneration.

6. The precipitation and washing of DNA.

6.1. The concentrate from the collection 18b passes through the fine filter 9b and is pumped into the measuring capacity of 15L.

6.2. From the measuring device 15A concentrate by gravity into the reactor planting 11, where the volumetric capacity 15m arrives 96% ethanol, Ogadenia DNA is carried out with constant stirring.

6.3. At the end of the deposition process, the suspension is rationed by gravity in a centrifuge 12A.

6.4. Separated by centrifugation DNA load in the mixer 16B and mixed with 70% ethyl alcohol coming from the mixer 16B.

6.5. The slurry from the mixer 16B is fed to the centrifuge 12B, the second container 15 nm.

6.6. After washing the precipitate DNA with ethanol, the suspension is again fed to the centrifuge 12B; the precipitate separated by centrifugation in the form of paste is transferred onto the filter 13, where the separation under vacuum residual moisture.

6.7. The supernatant from the position 12A, 12B and 13 enters the collection 18g, whence comes on regeneration.

6.8. Dehydrated powder is spread in a thin layer on trays and placed in a drying chamber 14, where it is dried to a certain moisture content.

6.9. The dried powder is then packing and packaging.

The proposed method for the production of sodium salt of DNA using the setup allows one cycle to process up to 10 kg of animal raw materials. Na-salt DNA obtained in an industrial setting has the following characteristics: mol. m 300-500 KD, hyperchromic effect at least 37% . The protein content of not more than 1.5% . The DNA content of not less than 80% . (56) AutoRAE's certificate of the USSR N 487897, C 07 H 21/04, 1976.

U.S. patent N 3899481, C 07 H 21/04, 1975.

1. Method for the production of sodium salt of deoxyribonucleic acid (DNA) from animal products, including grinding and homogenization of raw materials in citrate-saline restitute, cooling the reaction mass, mixing it with diatomaceous earth, the separation of the liquid phase by filtration and precipitation of the product with ethyl alcohol, characterized in that before being mixed with diatomaceous earth, the reaction mixture for 10 to 80 min, treated with ultrasound at a frequency of 8 - 35 kHz and an intensity of 0.2 to 2.0 W/cm2the impact of ultrasound is carried out in the layer of the reaction mass thickness, providing a complete and uniform treatment of all of the material in selected modes and temperature, when mixed with diatomaceous earth use the ratio of the sorbent and the reaction mass 1 : 5 - 25 (wt/vol. ), separated by filtration, the liquid phase is concentrated by baravilala, and the precipitate of sodium salt of DNA is dried at 20 - 60oC.

2. The method according to p. 1, characterized in that when the concentration of the liquid phase using membranes with a pore diameter of 0.05 - 1 mm.

3. Installation for the production of sodium salt of DNA from animal products containing a cutting table, a meat grinder, electric, microspherical tissue digester cooking and reactor cooling the reaction mixture, each of which is equipped with a low shear mixing device such as a stirrer of the anchor type, the heat-exchange jacket, thermoregulatory, the site of treatment of the reaction mass by ultrasound, consisting of several ultrasonic generators and baths, each bath is equipped with a built-in bottom of the magnetostrictive transducer, the gap between which and the side walls of the bath does not exceed 2 cm hole for the release of the reaction mixture and cooling system, made in the form of a tubular heat exchanger placed inside the bath over magnetostriction mixer for processing the reaction mixture sorbent installed inside a high-speed mixer, a device for separation of the liquid phase, the device for microfiltration, baromembrane device for concentrating liquid phase, the reactor for the deposition of the final product alcohol and the equipment for separating and drying the precipitate of sodium salt of DNA.

 

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FIELD: bioengineering.

SUBSTANCE: proposed device has housing with cover; side walls of housing are provided with light-reflecting surfaces. Housing is provided with micro-algae cultivation reservoirs which are mounted on grate; housing is also provided with filament lamps which are equidistant relative to reservoirs. Device is also provided with heating unit located under grate and sensors showing suspension concentration, suspension temperature and illuminance. Device is equipped with pipe line for delivery of carbon dioxide and discharge of oxygen.

EFFECT: reduction of power requirements; monitoring of technological parameters of process; enhanced efficiency.

4 dwg

FIELD: production methods.

SUBSTANCE: method of growing of 3-D cells cultures is foreseen the directed moving of cells and cells complexes mostly in vertical direction in the bottle, which has the cylinder profile. The moving is provided by the rotation of vertical plane to 180° with the pause of rotation during the coincidence of bottle axis with vector of gravity with many times cycle repeating. The device contains chamber of cultivation one as minimum, partly felled of gas penetrable material with cylinder profile and the gearing for rotating around the horizontal axis. Additionally the device contains the controlling device, connected with the gearing and provides the rotation to 180°. The chamber of cultivation is made as vertical situated high cylinder.

EFFECT: realization of this method provides good conditions for the cells during the process of growth the 3-D cultures.

3 cl, 1 dwg, 1 ex

FIELD: medicine; biology.

SUBSTANCE: bioreactor includes a vessel consisting of cylindrical middle part with upper lid and bottom in the form of a lid, both connected to it hermetically and featuring at least one orifice for inlet or outlet of nutrient medium or oxygen. The lids have rings with thread on the inner surface, while the cylindrical middle part has outer thread to form impermeable connection to the lid rings.

EFFECT: convenience of bioreactor maintenance and possibility to elicit cultivated cells safely.

10 cl, 13 dwg

FIELD: technological processes; chemistry.

SUBSTANCE: cells to be cultivated are located in hollow-fiber membranes and are alternately introduced into liquid nutrient medium and gas phase located in it. Device is a bioreactor with exposure in liquid and gas phases, which contains supply space, in which hollow-fiber membranes are located with internal diameter of not more than 5 mm, internal volumes of which form chambers for cultivation. After cells are introduced into chambers for cultivation, approximately half of supply space is filled with nutrient medium, and the other half is filled with gas phase. After connection of medium and gas perfusion, cyclical exposure of hollow-fiber membranes and cells located in them in gas and liquid phase starts.

EFFECT: possibility of efficient continuous cultivation of cells with high density and preparation of products from these cells at their simultaneous retention.

18 cl, 3 dwg

FIELD: biology.

SUBSTANCE: present invention relates to devices for growing tissue cells and microorganisms in null-gravity conditions and can be used in space biotechnology. The device has a cylindrical container with a cover and a device for aerating and moving suspension of the said biological objects, comprising connecting pipes for inlet of aerating gas and outlet of gaseous medium respectively, fitted in the cover of the container, and made with possibility of creating on the surface of the suspension, rotational flow of aerating gas with velocity field of the potential vortex on the periphery of the container and an axial counter-flow in the paraxial area and a 10-2000 Pa pressure drop between the periphery and the centre of the vortex. The inner surface of the container and the bottom are made from material with limiting wetting angle θ=90°, or part of the inner surface on the side of the bottom and the bottom itself are made from material soaked with a liquid substrate with limiting wetting angle θ<90°. The other part of the inner surface of the container, on the side of its cover, is made from material not soaked with liquid substrate with limiting angle θ>90°, where in degrees are values of wetting angles of aqueous substrates on solid surfaces. The inner surface of the container which is not wet, on the side of the cover, can be made in form of a cylindrical insert with a holder, holding it in position, the outer diametre of which corresponds to the inner diametre of the container and is put into the container with possibility of moving along its axis. In an alternative of making the device, the inner surface of the container which is not wet, made in form of a cylindrical insert, is made from Teflon, and the rest of the soaked part of the surface of the container is made from stainless steel.

EFFECT: process of growing cells or microorganisms in microgravity conditions due to provision for stable conditions of the boundary surface between liquid and gaseous phases.

7 cl, 7 dwg, 3 tbl

FIELD: biology.

SUBSTANCE: present invention relates to devices for growing tissue cells and microorganisms in null-gravity conditions and can be used in space biotechnology. The device has a cylindrical container with a cover and a device for aerating and moving suspension of the said biological objects, comprising connecting pipes for inlet of aerating gas and outlet of gaseous medium respectively, fitted in the cover of the container, and made with possibility of creating on the surface of the suspension, rotational flow of aerating gas with velocity field of the potential vortex on the periphery of the container and an axial counter-flow in the paraxial area and a 10-2000 Pa pressure drop between the periphery and the centre of the vortex. The inner surface of the container and the bottom are made from material with limiting wetting angle θ=90°, or part of the inner surface on the side of the bottom and the bottom itself are made from material soaked with a liquid substrate with limiting wetting angle θ<90°. The other part of the inner surface of the container, on the side of its cover, is made from material not soaked with liquid substrate with limiting angle θ>90°, where in degrees are values of wetting angles of aqueous substrates on solid surfaces. The inner surface of the container which is not wet, on the side of the cover, can be made in form of a cylindrical insert with a holder, holding it in position, the outer diametre of which corresponds to the inner diametre of the container and is put into the container with possibility of moving along its axis. In an alternative of making the device, the inner surface of the container which is not wet, made in form of a cylindrical insert, is made from Teflon, and the rest of the soaked part of the surface of the container is made from stainless steel.

EFFECT: process of growing cells or microorganisms in microgravity conditions due to provision for stable conditions of the boundary surface between liquid and gaseous phases.

7 cl, 7 dwg, 3 tbl

FIELD: engines and pumps.

SUBSTANCE: invention relates to studying technological processes in heterogeneous media using microorganisms, particularly, in biohydrometallurgical production of noble metals. The proposed laboratory unit represents a bioreactor mounted between the top and bottom frames. The bioreactor cylindrical housing and cover are screwed together. The top frame accommodates a motor, belt transmission and bearing assembly, the bearing being spaced apart on the shaft. The bioreactor cover is rigidly attached to the top frame via the said bearing assembly. The cover houses a tubular aerator and pickups. The bioreactor housing accommodates a tubular heat exchanger, deflectors and mixer driven by the motor via the bearing assembly shaft.

EFFECT: expanded performances, higher reliability, lower costs and improved ergonomic properties.

1 dwg

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