RU2487542C2 - Entomopathogenic biopreparation for protecting plants from pests and method for preparation thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: entomopathogenic biopreparation for protecting plants from pests contains a dry mixture of a concentrate of a culture fluid based on infection units of entomopathogenic fungi of the type Verticillium lecanii or Beauveria bassiana, or Metarhizium anisopliae, aerosil and an additive which protects the infection units from loss of viability and biological efficiency when drying, with the following ratio of components, wt %: culture fluid concentrate 69.0-77.5, additive - 10.8-13.8, aerosil - 11.6-17.2. The additive is a polyatomic alcohol, carbohydrate, vegetable oil, sodium gluconate and water, with the following ratio of components, wt %: polyatomic alcohol 6.2-13.4, carbohydrate 38.0-41.9, vegetable oil 38.7-41.0, sodium gluconate 0.4-0.5, water 9.3-10.5. The biopreparation is obtained by mixing the culture fluid concentrate with the additive in ratio of 5-7:1, and then adding aerosil in ratio of 4.5-7.6:1 and drying the mixture. Efficiency of the biopreparation on white flies is 95-100% in a working concentration of 1·10⁷ spores/ml; the titre of viable units is (0.35-5.5)·10¹⁰ CFU/g.

EFFECT: invention increases biological efficiency of the biopreparation, preserves and increases viability of infection units of fungi during drying and storage.

8 cl, 3 tbl, 15 ex

Description

The invention relates to agricultural microbiology and relates to the production of plant protection products against pests, in particular a method for producing fungal entomopathogenic preparations. Biological products based on entomopathogenic fungi for protecting plants from pests and methods for their preparation by surface and deep cultivation of the microscopic fungus Verticillium lecanii, Beauveria bassiana, Metarhizium anisopliae on nutrient media containing carbon, nitrogen and mineral salts, followed by concentration and drying of the concentrate. A two-stage deep-surface method is used to obtain Verticillin grain TU 10.01.28-89 [1], Verticillin-K TU 10.01.29-89 [2], Boverin-concentrate Zh TU 10.01.26-88 [3]. The disadvantages of these drugs are the short shelf life of these drugs in liquid form or the high cost of dry drugs. The disadvantages of the methods for their preparation are the need for long-term cultivation of mushrooms (at least 14 days), multi-stage, reduced viability of spores during drying and storage.

The closest technical solution to the invention, taken as a prototype (RF Patent 2035144. VP Tretyakov. A method for producing an entomopathogenic preparation from 01/22/1993 [4]) is a biological product and a method for producing an entomopathogenic biological product in dry form by deep cultivation of microscopic fungi (Verticillium lecanii , Beauveria bassiana or Metarhizium anisopliae) in a nutrient medium containing glucose, glycerin, protein-vitamin concentrate hydrolyzate, chalk and water, concentration of culture fluid, addition of a NaX type zeolite filler and Montmore illonite or palygorskite and drying. To improve the quality of bipreparations and non-caking, they are granulated after drying. The preparations obtained by the described method have biological efficacy on a greenhouse whitefly: based on Verticillium lecanii blastospores - 70%, viable blastospore titer 1.5 · 10 ¹⁰ spores / g, Beauveria bassiana based - 85%, viable blastospore titer 9 · 10 ⁹ spores / g, based on Metarhizium anisopliae - 80%, titer of viable blastospores 2 · 10 ⁹ spores / g.

Thus, the disadvantages of the biological products of the prototype and methods for their preparation are the relatively low biological effectiveness of biological products and a decrease in the viability of infectious units due to the influence of adverse factors during drying and storage.

The objective of the invention is to increase the biological effectiveness of the biological product, preservation and increase the viability of infectious units in the process of drying and storage.

The problem regarding the proposed biological product is solved by the content of the culture fluid in the concentrate on the basis of infectious units of entomopathogenic fungi of the species Verticillium lecanii, or Beauveria bassiana, or Metarhizium anisopliae additives, which protects the infectious units from loss of viability and biological effectiveness during drying and storage, in the following ratio of components, wt.%: the concentrate of the culture fluid is 69.0-77.5; additive - 10.8-13.8; Aerosil - 11.6-17.2. The composition of the additive includes polyhydric alcohol, carbohydrate, vegetable oil, sodium gluconate in the following ratio, wt.%:

polyhydric alcohol - 6.2-13.4 carbohydrate - 38.0-41.9 vegetable oil - 38.7-41.0 sodium gluconate - 0.4-0.5 water - 9.3-10.5

As a polyhydric alcohol, the additive contains one of the following components: glycerin, polyethylene glycol, dulcite, or mixtures thereof. As a carbohydrate, the additive contains one of the following components: sucrose, glucose, rhamnose, inositol, sorbitol, corn dextrin, or mixtures thereof. As a vegetable oil, the additive contains one of the types of oils: sunflower, corn, olive, canola, peanut, palm or mixtures thereof. As a filler, the biological product contains aerosil.

The problem regarding the proposed method is solved in the following way: prepare a liquid sterile nutrient medium, inoculate with a fungal inoculum Verticillium lecanii, or Beauveria bassiana, or Metarhizium anisopliae and cultivate deeply, the stirring speed of 200-220 rpm for 4-5 days at 27 ° C to maximum spore formation. The resulting culture fluid is concentrated by filtration to a residual moisture content of not more than 85% and an additive is added to protect the infectious units from loss of viability and biological effectiveness of the mixture at a ratio of 5-7: 1, then a filler is added to the resulting mixture at a ratio of 4.5-7.6: one. As an additive, polyhydric alcohol, carbohydrate, vegetable oil, sodium gluconate are used in the following ratio of components, wt.%:

polyhydric alcohol - 6.2-13.4 carbohydrate - 38.0-41.9 vegetable oil - 38.7-41.0 sodium gluconate - 0.4-0.5 water - 9.3-10.5

Regarding the biological product, the following result was achieved: the biological product was obtained in the form of a dry wettable powder, uniform, stable for at least 6 months. The biological effectiveness of the biological product exceeds the prototype by 10-28%, moreover, it has a higher initial titer of viable infectious units.

Regarding the method of obtaining a biological product: the preservation of the viability of the infectious units during drying and further storage is achieved. There is also a simplification of the process of obtaining a biological product due to the absence of a granulation step.

Example 1. A dry biological product is a wettable powder containing 60 g of culture fluid concentrate, 10 g of additive, 12 g of aerosil. A culture fluid concentrate with a residual moisture content of 73% was obtained as a result of cultivation of the fungus Verticillium lecanii strain P-81 (Catalog of state collections of beneficial and harmful organisms, M.-SPb., RAAS, 2001, p.14 [5]), or Beauveria bassiana strain Bb 2 (Mitina et al., Entomopathogenic fungi of the genus Lecanicillium: systematics, virulence factors, toxin formation. Biological plant protection products, technologies for their manufacture and use, St. Petersburg, 2005, p. 277 [6]), or Metarhizium anisopliae strain MAK- 1 (Kryukov V.Yu. et al. Comparative analysis of two strains of entomopathogenic g riba Metarhizium anisopliae with different life strategies.Mycology and Phytopathology, 2011, 45 (2), p.164 [7]) in a liquid nutrient medium containing, by weight: 20 g glucose, 10 g peptone, 2 g yeast extract, H ₂ O - up to 1 l, pH 6.5 (Lednev et al. “Pathogens of insect mycoses”, St. Petersburg, 2003, p. 71 [8]). As a nutrient medium, any medium used for the cultivation of these species of entomopathogenic fungi can be used. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: glycerin - 0.6 ml or 0.76 g; 80% sugar syrup - 5.2 ml (4.16 g sugar in 1.04 ml of hot water), sunflower oil - 4.2 ml or 3.9 g and sodium gluconate - 0.04 g. Polyhydric alcohols, carbohydrates and vegetable oils listed in Table 3 can be used as components of the additive.

After applying aerosil and drying on pallets at a temperature of 35-37 ° C with forced ventilation to a residual moisture content of 23%, the biological product is a loose, wettable white powder that is not traced during storage and is easily suspended in water when used. The titer of viable units of samples of biological products is 8.5 · 10 ⁹ -1.0 · ¹⁰ spores / ml (table 1), which is higher than the prototype (table 2). The biological effectiveness of the preparations on the larvae of the greenhouse whitefly (Trialeuroides vaporariorum) under standard conditions in the working concentration of a spore suspension of 1 · 10 ⁷ pores / ml is from 95-100%, which is higher than in the prototype (table 1).

Table 1 Characteristics of entomopathogenic preparations obtained Type of fungus, strain The titer of viable infectious units after drying, CFU / g The biological effectiveness of the drug on whitefly,% Verticillium lecanii strain P-81 8.510 ⁹ one hundred Beauveria bassiana strain Bb 2 1,0 · 10 ¹⁰ 95 Metarhizium anisopliae strain MAK-1 8.510 ⁹ 95

Example 2. A dry biological product is a wettable powder containing 77 g of culture fluid concentrate, obtained as in example 1; 11.32 g of the additive and 11.8 g of aerosil. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: glycerin - 0.74 g, 80% sugar syrup - 5.9 ml (4.7 g sugar in 1.2 ml hot water) , sunflower oil - 4.64 g and sodium gluconate - 0.04 g. This example shows that at minimum concentrations of the components of the additive and aerosil, the properties of the obtained biological product do not change compared to example 1, and the number of viable units per gram of the drug and biological effectiveness higher than in the prototype (table 1).

Example 3. A dry biological product is a wettable powder containing 100 g of culture fluid concentrate obtained as in Example 1, as well as 15.5 g of an additive and 20.5 g of aerosil. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: PEG - 2.3 g, 6.6 ml of syrup (6.5 g of ramnose in 1.1 g of hot water), olive oil - 6, 5 g and sodium gluconate - 0.1 g. This example shows the possibility of using various components of the additives shown in table 3.

Example 4. A dry biological product is a wettable powder containing 100 g of culture fluid concentrate obtained as in Example 1, as well as 17.2 g of an additive and 22 g of aerosil. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: dulcite - 2.45 g, 6.6 ml of syrup (6.96 g of inositol in 0.45 g of hot water), rapeseed oil - 7, 08 g and sodium gluconate - 0.07 g. This example shows the possibility of using various components of the additives shown in table 3.

Example 5. A dry biological product is a wettable powder containing 100 g of a culture fluid concentrate obtained as in Example 1, as well as 15.7 g of an additive and 21 g of aerosil. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: a mixture, namely glycerin - 1.17 g, PEG - 1.1 g, 6.81 ml of syrup (6.44 g of glucose in 0, 37 g of hot water), rapeseed oil - 3.56 g, palm oil - 3 g and sodium gluconate - 0.06 g). This example shows the possibility of using mixtures of polyhydric alcohols as components of the additives shown in table 3.

Example 6. A dry biological product is a wettable powder containing 100 g of culture fluid concentrate obtained as in Example 1, as well as 14.37 g of an additive and 17 g of Aerosil. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: PEG - 2.08 g, 6.6 ml of syrup (a mixture of carbohydrates, namely 2.9 g of corn dextrin and 3.02 g of sorbitol in 0 , 28 g of hot water), peanut butter - 6.03 g and sodium gluconate - 0.06 g. This example shows the possibility of using mixtures of carbohydrates as components of the additives shown in table 3.

Example 7. The same as in examples 1-2, but a dry biological product is obtained with the maximum allowable amounts of the components of the additive and filler: for 69 g of the concentrate of the culture fluid make 13.36 g of the additive, 17.34 g of aerosil. An additive that protects infectious units from loss of viability and biological effectiveness during drying and storage contains: glycerin - 1.8 g, 80% sugar syrup - 6.25 ml (5 g sugar in 1.25 ml hot water), sunflower oil - 5.2 g and sodium gluconate - 0.07 g. This example shows that at the maximum concentrations of the components of the additive and aerosil, the properties of the obtained biological product do not change compared to example 1, and the number of viable units per gram of the drug and biological efficiency is higher, than in the prototype (table 1).

Example 8. The same as in examples 1-3, but a dry biological product is obtained from 100 g of culture fluid concentrate and with exorbitant amounts of components of the additive and filler. In this case, the claimed properties of the biological product are not achieved (table 3).

Example 9. The same as in example 1, receive a preparation based on the culture fluid of the fungus V.lecanii with a different titer (table 2). The titer of the concentrate of the culture fluid is (1.3-8.5) · 10 ⁸ CFU / g When drying with a protective mixture, the drug titer reaches up to (0.35-5.5) · 10 ¹⁰ CFU / g due to further concentration without loss of viability of the spores. In the prototype, the titer of the drug practically does not change (1.5 · 10 ¹⁰ spores / g) compared with the original titer of the concentrate (1.3 · 10 ¹⁰ spores / g), which indicates the loss of viable spores during drying of the drug.

The biological effectiveness of the drug based on the culture fluid of the fungus V.lecanii on whitefly 100% (table 1), in the prototype lower by 28%.

Example 10. The same as in example 1-9, receive the drug. For B.bassiana species, strain Bb 2 or M. anisopliae strain MAK-1 also leads to an increase in drug titers due to concentration without loss of viability of spores when dried with a protective mixture. The biological effectiveness of preparations based on the culture fluid of the fungus B.bassiana and M.anisopliae on whitefly 95% (table 1), in the prototype below 10 and 15%, respectively.

table 2 Kind of mushroom The titer of the culture fluid, spores / ml The titer of the culture fluid concentrate, CFU / g The titer of the drug, CFU / g V.lecanii (1 experiment) 2.310 ⁸ 5.310 ⁸ 2.510 ¹⁰ V.lecanii (2 experience) 5.010 ⁸ 8.510 ⁸ 5.510 ¹⁰ V.lecanii (3 experience) 3,510 ⁷ 1.310 ⁸ 3,510 ⁹ prototype V.lecanii 1.3 · 10 ¹⁰ 3,510 ⁹ B.bassiana 3,510 ⁸ 6.710 ⁹ 1,0 · 10 ¹⁰ prototype B.bassiana - 9 · 10 ⁹ M.anisopleae 3,510 ⁷ 5.310 ⁹ 8.510 ⁹ prototype M.anisopleae - - 2 · 10 ⁹

Example 11. A method of obtaining a biological product is as follows: prepare a sterile liquid nutrient medium of the following composition, masses: 20 g glucose, 10 g peptone, 2 g yeast extract, H ₂ O - up to 1 l, pH 6.5 (Lednev et al. “Causative agents of insect mycoses”, St. Petersburg, 2003, p. 71 [8]), inoculate strain P-81 with the fungus Verticillium lecanii (Catalog of state collections of beneficial and harmful organisms, M. - St. Petersburg, Russian Academy of Agricultural Sciences, 2001, p.14 [ 5]), or Beanveria bassiana strain Bb 2 (Mitina et al., Entomopathogenic fungi of the genus Lecanicillium: systematics, virulence factors, toxin formation. Biological agents plant protection, technologies for their manufacture and use, St. Petersburg, 2005, p.277 [6]), or Metarhizium anisopliae strain MAK-1 (Kryukov V.Yu. et al. Comparative analysis of two strains of the entomopathogenic fungus Metarhizium anisopliae with different life strategies Mycology and phytopathology, 2011, 45 (2), p. 164 [7]), and cultivated in depth, the mixing speed of 200-220 rpm. As a nutrient medium, any medium used for the cultivation of these species of entomopathogenic fungi can be used.

Cultivation is carried out for 4-5 days at 27 ° C to the maximum spore formation. The resulting culture fluid was concentrated to a residual moisture content of 73% by filtration. To obtain a dry entomopathogenic preparation, 14.3 g (7: 1) of a protective additive are introduced into 100 g of a culture fluid concentrate with the following content of each component: glycerin - 1.9 g; sucrose - 5.4 g; corn oil 5.5; sodium gluconate 0.07; water - 0.2 and then 15 g of aerosil (7.6: 1) and dried.

As a result of the addition, the infectious units were protected from the negative effects of the drying process, their titer was 8.5 · 10 ⁹ -1.0 · 10 ¹⁰ spores / ml (table 1), which is higher than the prototype (table 2) . The biological effectiveness of the preparations was determined on the larvae of the greenhouse whitefly (Trialeuroides vaporariorum) under standard conditions, the working concentration of the spore suspension is 1-10 spores / ml. It amounted, depending on the type of fungus, 95-100%, which is higher than the prototype (table 1).

Achieved the preservation of the viability of infectious units during further storage. This method does not require granulation, which simplifies the process of obtaining a biological product.

Table 3 Examples of a biological product based on culture fluid Verticillium lecanii strain P-81, or Beauveria bassiana strain Bb 2, or Metarhizium anisopliae strain MAK-1 with paltic salting of components in a protective additive Additive The amount of component per 100 g of concentrate (g or wt.%) Viable Spore Title Characterization of the process, drug Aerosil > 26.7 - Mixes poorly, lumps of aerosil 15-26.7 2.510 ¹⁰ Homogeneous fine-grained powder, stable during storage <15 2.510 ¹⁰ The adhesion of the mycelium during the drying process Glycerol or PEG, or dulcite, or mixtures thereof > 2.7 1.5 · 10 ¹⁰ Extending Drying Time 1.9-2.7 2.510 ¹⁰ Uniform, stable during storage <1.9 Mixing with mushroom biomass is difficult Sucrose, or rhamnose, or glucose, or inositol, or sorbitol, or corn dextrin, or mixtures thereof > 7, b - Stratification of the mixture, difficulties in mixing 5.4-7.6 2.510 ¹⁰ Uniform, stable during storage <5,4 1,0 · 10 ⁹ Reduced vitality Water > 1.9 Increase sample moisture 0.2-1.9 Uniform, stable during storage 0.2 Sugar dissolution difficult Sunflower oil, or corn, or olive, or rapeseed, or peanut, or palm, or mixtures thereof > 7.7 Emulsion separation, extension of drying time 5.5-7.7 2.510 ¹⁰ Stable emulsion for incorporation into culture fluid concentrate <5.5 1,0 · 10 ⁹ Reduced vitality Sodium Gluconate > 0.1 - Potentially reduced viability 0.05-0.1 2.510 ¹⁰ Uniform, stable during storage <0.05 2.510 ⁹ Reduced vitality

Example 12. The same as in example 7, receive a preparation based on the culture fluid Verticillium lecanii strain P-81, or Beauveria bassiana strain Bb 2, or Metarhizium anisopliae strain MAK-1. But they use a concentrate of culture fluid with a humidity of 80% and other components of the protective additive (table 3). To obtain a dry entomopathogenic preparation, 17.2 g (5.8: 1) of a protective additive are introduced into 100 g of culture fluid concentrate with the following content of each component: dulcite - 2.45 g; inositol - 6.96 g; rapeseed oil - 7.08 g; sodium gluconate 0.07; water — 0.45 g and then 22 g aerosil (5.3: 1) and dried.

Example 13. The same as in example 7, receive a preparation based on the culture fluid Verticiuium lecanii strain P-81, or Beauveria bassiana strain Bb 2, or Metarhizium anisopliae strain MAK-1. But they use a concentrate of culture fluid with a moisture content of 78% and a mixture of components of a protective additive (Table 3). To obtain a dry entomopathogenic preparation, 15.7 g (6.3: 1) of a protective additive is introduced into 100 g of the culture fluid concentrate, which includes mixtures of polyhydric alcohols and vegetable oils, with the following content of each component: glycerol - 1.17 g; PEG - 1.1 g; glucose - 6.44 g; rapeseed oil - 3.56 g; palm - 3 g; sodium gluconate - 0.06 g; water 0.37 g and then 21 g aerosil (5.5: 1) and dried.

Example 14. The same as in example 7, receive a preparation based on culture fluid Verticillwn lecanii strain P-81, or Beauveria bassiana strain Bb 2, or Meiarhizmm anisopliae strain MAK-1. But they use a concentrate of culture fluid with a humidity of 85%. To obtain a dry entomopathogenic preparation, 20 g (5: 1) of a protective additive are added to 100 g of culture fluid concentrate with the following content of each component: glycerin - 2.7 g; sucrose - 7.6 g; corn oil 7.7; sodium gluconate 0.1; water - 1.9 and then 26.7 g of aerosil (4.5: 1) and dried.

This example shows that at the maximum concentrations of the additive component and aerosil, the properties of the obtained biological product do not change compared to example 1, and the number of viable units per gram of drug and biological efficiency is higher than in the prototype (tables 1, 3).

Example 15. The same as in examples 7-8, but a dry biological product is obtained from 100 g of culture fluid concentrate and with exorbitant values of the ratios of QOL to the protective additive and filler (aerosil). In this case, the claimed properties of the biological product are not achieved (table 3). The amounts of componentosis in the recommended range provide an increase in the biological effectiveness of the biological product, preservation and increase the viability of infectious units during drying and storage.

The proposed entomopathogenic biological product and a method for producing it in the form of a wettable powder by deep cultivation of mushrooms (Verticillivm lecanii or Beauveria bassiana, or Metarhizium anisopliae) with a protective additive consisting of glycerol alcohols, carbohydrates, water, vegetable oil and sodium gluconate, and filler Aerosil (in comparison with the introduction of only fillers of the zeolite type NaX and montmorrillonite or palygorskite according to the prototype) have the following advantages: higher biological effectiveness, biological product, higher initial titer of viable units during long-term storage. This method ensures the preservation of the viability of infectious units in the process of drying and further storage, as well as simplifying the process of obtaining a biological product, because does not require additional granulation to prevent caking, which is more economical. This method is universal and applicable for the industrial production of biological products and has shown efficiency on an industrial scale.

Information sources

1. Verticillin grain TU 10.01.28-89.

2. Verticillin-K TU 10.0L29-89.

3. Boverin-concentrate Zh TU 10.01.26-88.

4. RF patent 2035144. Tretyakov VP A method of obtaining an entomopathogenic preparation from 01/22/1993.

5. Catalog of state collections of beneficial and harmful organisms, Moscow - St. Petersburg, RAAS Department. plant protection. 2001.

6. Mitina G.V., Sokornova S.V., Pavlyushin V.A. Entomopathogenic fungi of the genus Lecanicillium: systematics, virulence factors, toxin formation. Biological plant protection products, technologies for their manufacture and use. St. Petersburg, 2005, p. 273-281.

7. Kryukov V.Yu., Dubovsky I.M., Yaroslavtseva O.N., Levchenko M.V., Slyamova N.D., Belgibaeva A.B., Khodyrev V.P., Lednev G.R., Glupov VV. Comparative analysis of two strains of the entomopathogenic fungus Metarhizium anisopliae with different life strategies. Mycology and Phytopathology, 2011, 45 (2), pp. 164-176.

8. Lednev G.R., Borisov B.A., Mitina G.V. Pathogens of mycosis of insects, St. Petersburg. 2003.

Claims (8)

1. Entomopathogenic biological product for protecting plants from pests, containing a dry mixture formed from a culture fluid concentrate based on infectious units of entomopathogenic fungi of the species Verticillium lecanii, or Beauveria bassiana, or Metarhizium anisopliae with the addition of a filler, and dried, characterized in that the mixture is as a filler contains aerosil and additionally an additive that protects infectious units from loss of viability and biological effectiveness during drying, in the following ratio of components, wt.%:
culture fluid concentrate 69.0-77.5 additive 10.8-13.8 filler 11.6-17.2
while the additive contains a polyhydric alcohol, carbohydrate, vegetable oil, sodium gluconate and water in the following ratio of components, wt.%:
polyhydric alcohol 6.2-13.4 carbohydrate 38.0-41.9 vegetable oil 38.7-41.0 sodium gluconate 0.4-0.5 water 9.3-10.5 2. The entomopathogenic biological product according to claim 1, characterized in that the alcohol of the polyatomic series contains one of the following components: glycerin, polyethylene glycol, dulcite or mixtures thereof. 3. The entomopathogenic biological product according to claim 1, characterized in that it contains one of the following components as carbohydrate: sucrose, glucose, rhamnose, inositol, sorbitol, corn dextrin, or mixtures thereof. 4. Entomopathogenic biological product according to claim 1, characterized in that as a vegetable oil it contains one of the types of oils:
sunflower, corn, olive, canola, peanut, palm or mixtures thereof. 5. A method of producing an entomopathogenic biological product for protecting plants from pests, which consists in introducing a filler into the culture fluid concentrate on the basis of infectious units of entomopathogenic fungi of the species Verticillium lecanii, or Beauveria bassiana, or Metarhizium anisophae, characterized in that as Aerosil is used in the filler at a ratio of 4.5-7.6: 1, and previously, before the introduction of Aerosil, into the culture fluid concentrate as an additive that protects infectious units from loss of viability and biology eskoy efficiency of the mixture at a ratio of 5-7: 1 is made of a polyhydric alcohol series, carbohydrate, vegetable oil, sodium gluconate and water in the following ratio, wt.%:
polyhydric alcohol 6.2-13.4 carbohydrate 38.0-41.9 vegetable oil 38.7-41.0 sodium gluconate 0.4-0.5 water 9.3-10.5 6. The method according to claim 5, characterized in that one of the following components is used as the alcohol of the polyatomic series: glycerin, polyethylene glycol, dulcite, or mixtures thereof. 7. The method according to claim 5, characterized in that sucrose, glucose, rhamnose, inositol, sorbitol, corn dextrin, or mixtures thereof are used as carbohydrate. 8. The method according to claim 5, characterized in that as a vegetable oil use one of the types of oils: sunflower, corn, olive, rapeseed, peanut, palm or mixtures thereof.