Method for production of powdered ice-forming biological reagent

FIELD: food industry.

SUBSTANCE: invention relates to food industry. According to the proposed method production of a powdered ice-forming biological reagent is performed by way of moisture evaporation (under vacuum) from liquid microbial or protein suspension with initial temperature equal to 26°C in flat-bottomed pans with walls 30-50 mm high containing porcelain balls with a 3 -7 mm diameter in a layer up to 50% of the pans walls height. The suspension poured into the pans in a layer height whereof is twice as little as that of the balls layer is divided by the balls into a multitude of small volumes with the pans subsequently covered with unwoven textile. Then follows the drying process during 9.0 - 12.5 hours with continual adjustable vacuumisation, controllable in terms of residual pressure in the chamber at four temporal stages of drying.

EFFECT: method allows to produce the end product in powdered form convenient for application and ensures high stability of the reagent during storage.

5 tbl, 2 ex

 

The invention relates to biotechnology, in particular to methods for biological powder loopazoid reagents, and can be used in applied Microbiology in the food industry, in the means of environmental monitoring.

The known method [Pushkar NS, Belous A.M., Flowers I.D. Theory and practice and cryogenic freeze preservation. Kyiv. Naukova Dumka, 1984; Mellor J.D. Fundamentals of freeze - driyng. Acad. Press., N.J. 1978], consists in obtaining a powder of heat-sensitive biological products by freeze drying used in applied Microbiology, biotechnology and food industries, providing for the freezing of microbial culture (biopharmaceutical, food and so on) followed by dehydration of biomass by removing moisture under vacuum and grinding the dried product.

The disadvantages of this method include damage to the structure of cells and biopolymers by freezing of biomaterials and grinding dehydrated products, leading to destruction of part of the microbial population and / or inactivation of biologically active molecules [Calcott R.N., Lee S.K., Macliod R.A. The effect of cooling and normino rotes on the survival of a variety of bacteria. - Can. J. Environ., 1976. V.22. No. 1., P.106-109; Belous A.M., Bondarenko V.A. Structural changes of biological membranes during cooling. Kyiv. Naukova Dumka, 1982, S-212].

Veshestvom, undergoes significant damage during freeze - thawing, and during grinding, biological loopazoid reagents representing biological products containing the bacteria Pseudomonas syringae, Pseudomonas fluorescens or Erwinia herbicola or isolated from them specific protein complexes that form heterogeneous nucleation centers (TSN) and has the ability to crystallize supercooled water.

Also the known method [RF Patent №2000111731. The method of dehydration of cultures of microorganisms in the flow of coolant], consisting in convective dehydration of microbial cultures using pryamostruynaya nozzle diameter of 0.5 mm, operating at a pressure of 0.12 PA and drip the filing of the original product to spray with a flow rate of approximately 120 drops/min

The disadvantages of this method include the death of a significant part of the microbial population, especially bacteria vegetative nature, due to thermoinactivation dehydrating coolant temperature above 50°C.

The techniques used to improve the survival of microbial cultures in the convective dehydration and consisting in the use of the concentrated source product, dispersing devices high pressure (speed), reduce the temperature and humidity of gas-Talanov the El, the use of two-stage drying, lead to a significant reduction in process performance, increase its duration and energonapryazhennosti, and, consequently, to the increase in the cost of drying equipment and produce our product [Karpov A.M., Alumiun A.A. Drying products of microbiological synthesis. M., 1982. - P.56-57].

Also the known method [RF Patent №2187053. Method for continuous freeze drying of thermolabile materials], consisting in the formation and freezing of granules capillary porous material in a vacuum, drying it in the field of infrared radiation prior to the formation of a dry crust. The drying of the material is carried out field infrared microwave in the upper part of the rolling layer during the drying process.

The disadvantages of this method include the inactivation of temperature-sensitive biomaterial during freezing, infrared irradiation and grinding granules (granules size up to 0.5 cm).

The closest in technical essence is [RF Patent №2108382. The method of drying biological product in capsules]composed:

- to use as a trap moisture condenser-memoratives with an operating temperature of minus 35°C;

in placing the ampoules in the cavity of the chamber;

in the gradual lowering of the pressure in the chamber: in the first stage for 30 minutes at 5-10 mbar, at the second stage within 60 minutes - 0.1 mbar;

- filling the chamber with gaseous nitrogen;

- in the secondary pressure in the chamber at 0.01 mbar for two hours.

The famous and the claimed invention are: dehydration of liquid by evaporation of moisture under vacuum; a phased reduction of the pressure in the chamber; the use for dehydration by sublimation chamber, condenser-memoratives and booster vacuum; filling the chamber with gaseous nitrogen.

The disadvantages of this method include:

- inability to obtain the final product in powder form and the necessity of using complicated and time-consuming operations for grinding;

- increased inactivation of microbial populations in a changing salt concentration and pH, resulting from interruption of the drying process to reduce foaming in ampoules [Konev S.V., Volotovski I.D. Structural adjustment of biological membranes. - Biomembranes. Riga. Zinatne, 1977. - P.42-46];

- working temperature of the condenser-memoratives minus 35°C does not capture a large amount of water at once.

The task of the invention to provide a method of manufacturing a biological powder Loubressac reagent cell-based Pseudomonas syringae, Pseudomonas fluorescens, Erwinia herbicola or isolated from proteins, initiating eh is obrazovanie (BEAT), by evaporation under vacuum.

The technical result is achieved due to the fact that in the method of drying biological product use:

for the placement of a drying material - flat-bottomed pan with a wall height of 30-50 mm, containing porcelain balls with a diameter of from 3 to 7 mm layer of up to 50% of the height of the wall ditch;

- layer suspension in misurova space ditch height, half-height of the layer of balls - this liquid is broken into many small volumes, which allows after drying to obtain a powder product;

- to cover the ditch - non-woven material having a surface density of 50-60 g/m2and breathability 300-320 l/DM2·min;

- dehydration - suspension with an initial temperature of 26-28°C and condenser-vigorazhival with the cooling temperatures of minus 52°C;

- the four-step lowering of the pressure in the chamber, which is controlled at the end of each of the four stages of drying: at the first stage after 0.5 hours to a residual pressure 260-220 PA, after a further 1.5 to 3.0 hours of drying (second stage) to a residual pressure 100-80 PA, at the end of the subsequent 4,0-6,0 hours of drying (the third stage) to a residual pressure 22-10 PA, in the fourth stage (final drying), lasting 3 hours, the residual pressure in the chamber 22-10 PA, the value of which is maintained until the end of the su is Ki;

continuous reduction of the pressure in sublimator and maintain it at an optimal level during drying, which ensures the continuity of the process;

laboratory sieve with mesh size 400 µm for screening the dried product from porcelain balls;

- gaseous nitrogen or argon to fill the chamber at the end of the drying process.

The essence of the invention is as follows.

Sublimation installation preparing to process by setting the capacitor-wykorzystane working temperature of minus 52°C, feeding shelves in the drying chamber the coolant with a temperature of 26°C and including a vacuum pump.

Flat-bottomed ditches filled with porcelain balls with a diameter of from 3 to 7 mm layer height of up to 50% of the height of the walls of the ditch.

Prepared microbial suspension, or suspension BEAT, pour into the prepared pan. The layer of suspension in a cell should not exceed half the height of the layer of porcelain balls. When this whole liquid volume is divided into many small volumes, which prevents foaming of the liquid when removing the air out of it when creating a vacuum in addition, the contact of the liquid with porcelain balls provides additional flow to heat it when creating vacuum and prevents its rapid and excessive cooling. The cell cover is non-woven filter material and establish on the shelves of the camera.

The chamber is pressurized and for the achievement of the suspension temperature 26°C (if necessary produce heating of the suspension due to the coolant supply to the shelf camera) generate a vacuum. With the creation of a vacuum in sublimator changes the partial pressure above the surface of the drying fluid and the volume of the chamber, resulting in evaporation of the liquid. The reduction of pressure in the chamber is adjustable and is constantly going down. The process of reducing the pressure in the chamber is divided into four stages.

Time 0.5 hours and the residual pressure 260-220 PA of the first stage due to the need to prevent foaming dryable liquid. Time of 1.5-3.0 hours and the residual pressure 100-80 PA of the second stage of drying is determined by the minimum temperature of the product that indicates the completion of the evaporation of free moisture. The end of the third (4,0-6,0) hours of stage of drying due to the minimum residual pressure (22-10 PA). The fourth stage - the final drying of the product to the desired residual moisture at a residual pressure of 22-10 PA, which duration is 3.0 hours.

The causal connection between the set of existing features of the proposed facility and achievable technical result is shown in table 1.

The invention allows for the placement of a drying material in michalopolous ditch their subsequent cover non-woven material, and due to the constant downward pressure in the chamber to conduct the dehydration process continuously and to obtain a dry powder of biological loopazoid reagent, containing in its composition polydisperse particles with a maximum diameter of not more than 400 μm, containing functionally active state from 78±2 to (79±3)% HIT from 77±1 to (91±1)% of bacteria at a residual moisture content of from 3.0 to 5.5 and from 3.2 to 5.5%, respectively.

Enablement of the claimed invention is shown in the following examples.

For the manufacture of biological powder Loubressac of reagent is used, the suspension of the bacteria Pseudomonas syringae, Pseudomonas fluorescens, Erwinia herbicola or a selection of them BEAT.

Example 1. Dehydration of the material culture of bacteria and isolated from them BEAT) was performed by evaporation under vacuum in cells with high wall 30 mm, containing porcelain balls with a diameter of from 3 to 7 mm layer height 15 mm, the Material was poured in the cell, filling megarave space layer height 7,5 mm Cuvette was covered with a non-woven material, placed on the shelves of sublimator and have sealed chamber. In shelf camera forcibly summed up the coolant and the heated material to a temperature of 26°C. Condenser-vigorazhival cooled to a temperature of minus 52°C, which was maintained during the whole process. Then create the Wali vacuum in the chamber, which is continuously decreased. Control over the drying was carried out at a residual pressure in the chamber created by the end of each of the four stages of drying, and the temperature change in the material. The parameters of the dehydration process are shown in table 2.

As can be seen from table 2, during the first stage of drying the residual pressure in the chamber was 220 PA, and for the next 1.5 hours drying (second phase) residual pressure reached 80 PA. At the same time for 2 hours drying temperature in the material dropped to minus 5°C. With the increase of vacuum in the chamber the temperature of the material is increased, and during the subsequent third stage of drying, its value amounted to 16°C at a residual pressure of 10 PA, which was maintained during the fourth stage of drying. The temperature of the material increased from 16 to 24°C. the Total drying time was 9 hours.

After drying the cavity of the camera fill technical argon or nitrogen and from the cell extract of the cell. The contents of the cuvettes poured into a sieve with a mesh size of 400 μm and spend sifting of dry product from porcelain balls. Through the mesh sifted all the dry product, which indicates the absence of the composition of particles of more than 400 μm. The resulting biological powdered reagent Packed in tight sealing.

After drying and UTS the Islands of the dry product was conducted qualitative analysis reagents obtained.

Stability BEAT when drying is calculated by the formula:

where WB is the stability of the BEAT when drying, percent;

LOALR, LOAm- Loubressac activity BEAT in the reagent (before drying) and the dry product, respectively, CN/cm3;

ρ is the density of the suspension, g/cm3;

OVER - dry the rest of the suspension, Rel. units.

Loopazoid activity, characterized by a content of nucleation centers (CN), active at the chosen temperature, estimated by the method based on the method G.Vali [Vali G. Quantative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids // J. Atmos. Sci. - 1971. - Vol.28. - P.402-409].

Residual humidity is determined by the number of lost moisture while drying samples Loubressac reagent to constant weight in a drying Cabinet at a temperature of from 102 to 105°C [kouzov P.A., Scriabin L.Y. Methods for determination of physico-chemical properties of industrial dusts. L. Chemistry. 1983. - C-84].

The results are presented in table 3.

Example 2. The results of example 1 were confirmed in experiments in the dehydration of the same material, but placed at a depth of 12.5 mm in misurova space a ditch with a wall height of 50 mm, containing porcelain balls with a diameter of from 3 to 7 mm thick 25 mm (50% of the height of the walls of the ditch), covered by non-woven material. Preparing for the dehydration of the material the wire is Dili, as described in example 1. The parameters of the dehydration process is shown in table 4.

As can be seen from table 4, the increase in the dried layer from 7.5 to 12.5 mm required an increase of 2 times the drying time on the second stage and 1.5 times in the third stage. However, the slower the creation of vacuum in the chamber resulted in lowering the temperature of the material only up to 3°C. the Process of the dried material at a temperature of 24°C was continued for 3 hours. The residual pressure in the chamber at the end of the third stage of drying was 22 PA, the total drying time is 12.5 hours. Received, screened by the balls, dry products represented powder loopazoid reagents with features whose values are given in table 5.

From the obtained data (table 5), it follows that the parameters of the dehydration process (table 4) allow to obtain a powdery loopazoid reagents with saving from 77 to 88% BEAT in the active state when a residual moisture content of from 4.5 to 5.3%.

Presented in table 3 and 5 one can see that the exhaust mode dehydration allows to obtain biological loopazoid reagents in convenient for operation (packing, storage, application) powder form, conditioned for humidity for storage, and ensures high stability of the reagent on the biological characteristic (LOA). When this is the procedure used conventional sublimation installation. Drying the liquid is placed in cuvettes in misurova space.

Table 1
A causal relationship between the set of essential features of the claimed object and achievable technical result
The technical result and their dimensionThe actual or estimatedAn explanation of what (hallmark and/or their combination) have made possible the improvement of the proposed facility compared to the prototype
prototypedeclare object
Getting Loubressac reagent in powder form without grinding stage, Yes/noNoYesDrying is carried out in flat-bottomed cells with a wall height of 30-50 mm, containing porcelain balls with a diameter of from 3 to 7 mm, the layer of balls is up to 50% of the height of the walls of the ditch, megarave space fill suspension layer half the height of the layer of balls.
For the Department of porcelain the balls from biological Loubressac reagent product drying proceviat through a sieve with cell 400 μm of the proposed facility compared to the prototype
The persistence of a biological product in a functionally active state after dehydrationLowAt least 70%The dehydration is carried out at the initial temperature of the suspension 26°C and minus 52°C in the condenser-wykorzystane; in the drying process continuously reduce the vacuum in the chamber, which is controlled by the residual pressure at the end of each of the four stages of drying: at the first stage after 0.5 hours to a residual pressure 260-220 PA, after a further 1.5 to 3.0 hours of drying (second stage) to a residual pressure 100-80 PA, at the end of the subsequent 4,0-6,0 hours of drying (the third stage) to a residual pressure 22-10 PA, which is supported at the fourth stage (final drying), 3 hours
Foaming dryable liquid biomaterial, Yes/noYesNoDrying half pans eliminates foaming liquid by reducing removal rate of the vapor mixture slurry, which is ensured by crushing it in small amounts when placed in misurova space, shelter, non-woven material having a surface density of 50-60 g/m2and breathability 300-320 l/DM2·min and slow the th creation of a vacuum in the first stage of drying (for 0.5 hours, the residual pressure in the chamber is only 260-220 PA)

Table 2
The parameters of the process of dehydration of the material contained in misurova space with a layer of 7.5 mm
stage № Duration, hIs ... at the end of phase
residual pressure in the chamber, PAthe temperature of the suspension, °C
10,522012
21,580Minus 5
34,01016
43,01024

Table 4
The parameters of the process of dehydration of the material contained in misurova space layer 12.5 mm
stage № Duration, hIs ... at the end of phase
residual pressure in the chamber, PAthe temperature of the suspension, °C
10,526015,0
23,01003,0
36,02219,0
43,02224,0

Table 5
Description powder reagent of the material contained in misurova space ditch layer 12.5 mm
Type of material, bacteria reagentResidual moisture, %Stability LOA during drying, %The maximum particle diameter, microns
The suspension of bacteria Pseudomonas syringae5,2±0,279±3400
Pseudomonas fluarenscens4,9±0,477±1The same
Erwinia herbicola5,3±0,284±4-//--//-
Suspension BILL4,5±0,578±2-//--//-

A method of manufacturing a biological powder Loubressac reagent from a liquid microbial or protein suspensions involving dehydration by evaporation under vacuum in the freeze plant, consisting of a camera, condenser-memoratives and the vacuum pump, when the gradual lowering of the pressure in the chamber, characterized in that the drying is carried out in flat-bottomed cells with a wall height of 30-50 mm, containing porcelain balls with a diameter of from 3 to 7 mm, the layer of balls is up to 50% of the height of the walls of the ditch, megarave space fill suspension layer half the height of the layer of balls; the cell is covered with a non-woven material having a surface density of 50-60 g/m2and breathability 300-320 l/DM2·min, the cell is placed the shelves and sublimation chamber; the dehydration is carried out at the initial temperature of the suspension 26°C and condenser-wykorzystane at minus 52°C; drying process continuously reduce the vacuum in the chamber, which is controlled by the residual pressure at the end of each of the four stages of drying: at the first stage after 0.5 h to a residual pressure 260-220 PA, after subsequent 1,5-3,0 h of drying, the second step to a residual pressure 100-80 PA, at the end of the next 4-6 hours drying, the third stage, to a residual pressure 22-10 PA, which is supported during the fourth phase is dried, 3 h; disqualified after drying biological powder loopazoid reagent with polydispersed composition of the particles, the maximum size of not more than 400 μm, containing functionally active state from 78±2 79±3% proteins, initiating icing and 77±1 to 91±1% of bacteria at a residual moisture content of from 3.6 to 5.5 and from 3.2 to 5.3%, respectively.



 

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