Way of the contact drying of microorganisms

 

(57) Abstract:

Usage: Microbiology, in particular the technology of production of dry biologics. The inventive method of drying of microorganisms involves mixing suspensions of microorganisms with sorbent moisture, and before mixing the microorganisms with sorbent last and/or suspension of microorganisms is mixed with a hydrophobic liquid containing or not containing a suspension stabilizer. 1 C. p. F.-ly.

The invention relates to the production technology of drugs, namely the production of dry microbial drugs.

Drying is the most popular method of preservation of microorganisms in the preparation of products with wide applications intended for storage in an ordinary household refrigerator (0. . . 4aboutC) or at room temperature. Occasionally in the practice of Museum storage of cultures of microorganisms used also the method of conservation under a layer of oil, which provides protection from exposure to oxygen. However, the preparations obtained in this way are stored worse dry and less convenient to use.

A number of known methods of preparation of dry preparations includes a step of disperser the efficiency of the heat sink, and in the case of freeze drying - speed freezing when the dispersion in cold liquid. Thermal drying methods, including using microwave heating, are not suitable for obtaining microbial biologics, because they lead to a strong inactivation of microorganisms. The main disadvantage of freeze-drying is the high complexity of hardware design; in addition, the product usually has a very high hygroscopicity. Finally, all the methods in this group are associated with a specific loss of drying substances and hydrophobic liquid during drying, as well as significant energy costs.

In the production of dry microbial drugs widely used method of contact drying, the essence of which consists in mixing a drying object (for example, suspensions of microorganisms) with sorbent moisture.

In the food industry for making contact-dried preparations as sorbents applied, dry starch, flour. In practice, the Museum store strains of microorganisms most popular sorbent is silica gel. The mentioned preparations are characterized by a low level of biological concentration. In the first drying of the suspension, and the product obtained is highly diluted. In the case of drying on silica gel substantial contribution to the decline in the concentration contributes to the inactivation of microorganisms during drying.

To obtain a highly concentrated biological products used vysokouglevodnye the adsorbents (ion exchange resin, zeolite, aluminum oxide), ensuring optimum storage level of residual humidity of the bio-component with a minimum content of sorbent in a biological product. However, the use of such adsorbents for drying of microorganisms usually leads to a significant inactivation of the latter due to rapid dehydration, heat the mixture during drying.

There is a method of preservation of microorganisms by mixing with dry silica gel. Tubes (h mm) half-filled with silica gel, cotton plug tubes, subjected to dry sterilization at 180aboutC for 1.5 h and placed in containers to cool. 0.5 ml of the suspension of microorganisms contribute to each tube, evenly distributing the volume. After standing for one week at room temperature away a small amount of the contents of each test tube for testing cotton tube replace their in vitro of living organisms. Their survival during drying is not defined.

This method has the following disadvantages:

survival of microorganisms during drying is very low, so this method cannot be widely used for the preparation of biological products;

microorganisms adhere to the surface of the particles obtained in this way form a biological product unsuitable for use in most practical cases.

The purpose of the invention is the improved survival of microorganisms during contact drying.

This is achieved by pre-mixing the suspension of microorganisms and/or sorbent moisture with a hydrophobic liquid containing or not containing a stabilizer suspension (emulsion). This hydrophobic liquid provides effective heat dissipation from the contact area of a drying object with desiccant, facilitates mixing, slows down the moisture transfer and prevents the suspension of microorganisms from exposure to oxygen and other damaging factors. In General this leads to improved survival of microorganisms during contact drying in comparison with the known method. The resulting preparation is a liquid or pasty appearance, easy to use and well kept. When the inside animals or people.

To increase the biological concentration of the drug after drying the hydrophobic liquid may be completely or partially removed.

P R I m e R 1. As sorbent moisture dried to a residual moisture content of less than 0.1% powdered alumina as a hydrophobic liquid corn oil. Drying is subjected to suspension of microorganisms Bifidobacterium bifidum pieces 1, containing 10% lactose obtained by submerged cultivation of microorganisms, centrifugation of the culture liquid and the subsequent suspension of the precipitate in 20% solution of lactose in a ratio of 1 ml per 1 g of sediment.

100 ml of corn oil was placed in a container homogenizer with a steel rotating knife. Under stirring introduced 30 g of aluminum oxide. After reaching its uniform distribution in the oil while continuing stirring injected 2 ml of the suspension of microorganisms. Continue stirring for 10 min, then the sample is transferred into a flask and placed in a refrigerator at a temperature of 4aboutC. in a day test, 0.5 ml of the mixture was placed in 50 ml of saline (0.15 M NaCl) containing 1% surfactant tween-20. Witkin agar (environment Blaurock with 1.5% additive agar). After incubation count the number of colonies.

Biological concentration was 1,30,3 108living cells per 1 g of the mixture, the survival rate of 61% . In a control sample obtained by mixing suspensions of microorganisms with alumina without the use of oil, the survival rate was 8% . Thus, in this case through the application of the proposed method for increasing the survival rate of 7.6 times. After storage for one month at a temperature of 20aboutWith the viability of the retained 85% of microorganisms, which confirms the efficiency of drying. In addition, to confirm the fact that microorganism really are in the dried state, the method of direct determination of the pressure of water vapor was measured activity in a mixture of water, amounting to 0.15, which corresponds to the dry state.

P R I m m e R 2. As sorbent moisture dried to a residual moisture content of less than 0.1% of granulated aluminum oxide, grain size of 0.7. . . 1.5 mm, as a hydrophobic liquid corn oil. Drying is subjected to suspension of microorganisms Salmonella enteritidis units 2 Isachenko, containing 10% lactose, prepared similarly as it opisanego. The oil is added 5 g of the emulsion stabilizer is a surfactant span-20 (lauryl-sorbitan). When mixing the injected 10 ml of the suspension of microorganisms. After obtaining a homogeneous suspension (2-3 min), the latter is poured into a beaker containing 150 g of aluminum oxide, cooled to 4aboutC. the Contents of the glass thoroughly stirred with a glass rod and placed 23 h in a refrigerator at 4aboutWith; then make a test. The drug is suspended according to the scheme outlined in example 1 and plated on solid nutrient medium, poured into Petri dishes. After incubation count the number of colonies.

Biological concentration was 6,70,4 108living cells per 1 g of the mixture, the survival rate of 79% . In a control sample obtained by mixing suspensions of microorganisms with alumina under the same conditions, but without the use of oil, the survival rate was 24% . Thus, the application of the proposed method gives an increase in the survival rate of 3.3 times. After storage for one month at 20aboutWith the viability of the retained 87% of microorganisms.

This drug can be used successfully to combat rodents.

P R I m e R 3. As adrianou liquid - polyethylsiloxane. Drying is subjected to suspension of microorganisms Escherichia coli K-12 containing 10% sucrose (30% sucrose solution was added at a ratio of 1: 2 to the liquid culture of microorganisms).

100 ml of polyethylsiloxane placed in the tank homogenizer with a steel rotating knife. In the liquid, add 5 g of surfactant span-80. When mixing the injected 10 ml of the suspension of microorganisms. After obtaining a homogeneous suspension add 30 g pre-cooled to -10aboutWith zeolite. Stirring is continued for 15 min, the sample was then placed in a refrigerator at 4aboutWith 20 h, after which testing is performed according to the method described in example 2.

Biological concentration was 5,40,5 x 108living cells per 1 g of the mixture, the survival rate of 35% . In the control sample (without the use of a hydrophobic liquid) survival rate was 2.5% . Thus, there is an increase in the survival rate 14 times.

P R I m e R 4. As sorbent moisture dried to a residual moisture content of less than 1% of the ion-exchange resin KB-P, as a hydrophobic liquid - perpendicular, as a stabilizer emulsion - Aerosil AM-1-300. Wyszukiwanie Kalina, pre-cooled in a water bath to a temperature of 3aboutWith, add 2 mg of Aerosil. Produce stirring using a mechanical stirrer. With stirring, add 4 ml of the suspension of microorganisms; forms a uniform emulsion. To the emulsion is added 20 g of ion-exchange resin KB-P, previously dried in a drying Cabinet at a temperature 1053aboutC to a residual moisture content of less than 1% and cooled to 0aboutC. Is the mixing of low intensity within 10 minutes the sample was Then kept in a refrigerator at a temperature of 4aboutC for 20 h, and is checked.

The bottom fraction of the mixture representing exfoliating perpendicular remove. The remaining part is thoroughly mixed, take 0.5 ml and diluted in 9.5 ml) cooled to 4aboutWith cultivation media, carefully re-mixed and produce seed on solid nutrient medium, poured into Petri dishes. After incubation count the number of colonies.

Biological concentration was 3,30,3x108living cells in 1 g of the sample (with the direct mixing of the ion exchange resin is first presented, and then with a suspension of microorganisms in the same quinno.

Thus, compared with the control sample there is a significant increase in survival.

P R I m e R 5. As sorbent moisture dried to a residual moisture content of less than 1% of the ion-exchange resin KB-P, as a hydrophobic liquid - perference, as a stabilizer suspension - Aerosil AM-1-300. Drying is subjected to suspension of the microorganism E. coli K-12 containing 10% sucrose.

In a beaker with 50 ml of perftorgeksan, cooled in a water bath to a temperature of 3aboutWith, paid 3 mg of Aerosil; is stirring using a mechanical stirrer. With stirring, is added 4 ml of a suspension of microorganisms, there is formed a uniform emulsion. To the emulsion with continued stirring, 20 g of dry resin KB-P. After 10 min after the injection of the resin mixing is stopped and the sample is placed in a refrigerator (4aboutC) for 21 hours After keeping in the fridge, the sample is transferred into an open Petri dish and give a hydrophobic liquid to evaporate at room temperature, after which produce a test sample according to the method described in example 4.

Biological concentration was 1.2 0,1x109living cells per 1 g, and survived

Thus, compared with the control sample there is a significant increase in survival.

As follows from the above examples, the proposed method can significantly improve the survival of microorganisms during contact drying and receive drugs that can be used in veterinary medicine and medicine, in the practice of Museum storage of strains for the control of rodents and other areas of the economy. (56) 1. Becker M. E. Dehydration microbial biomass, Riga, zinatne, 1967, 361 S.

2. Kalucki L. C. , Sidyakin T. M. the viability of microorganisms in nature and the main approaches to conservation collection cultures. Abstracts of the 2nd all-Union conference on animation, Riga, October 16-18, 1984 , C. 19-20.

3. Grivell A. R. Jackon J. F. Environ culture preservation with silica gell // J. Gen. Environ. , 1969, v. 58, No. 3, p. 423-425.

4. Perkins J. Preservation of Nenrospora stock cultures with anhydrous silica gel. //Can. J. Environ, 1962, v. 8, R. 591-594.

1. WAY of the CONTACT DRYING of MICROORGANISMS, involving the mixing of a suspension of microorganisms with sorbent moisture, characterized in that before mixing the microorganisms with sorbent last and/or suspension is mixed with a hydrophobic liquid.


 

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