Method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol. Microbiological oxidative elimination of side chain at atom C17 with formation of 9α-hydroxyandrost-4-en-3,17-dione is performed. Biomass is separated. 9α-hydroxyandrost-4-en-3,17-dione is extracted from clarified cultural liquid with aprotic organic solvent, selected from aromatic hydrocarbons or organochlorine hydrocarbons. After that, reaction of 9α-hydroxygroup of 9α- hydroxyandrost-4-en-3,17-dione dehydration is carried out in obtained extract. As dehydration agent applied is mineral acid, which contains water and is selected from group, which includes orthophosphoric, pyrophosphoric and chloric acids. Mineral acid is applied in quantity from 1 to 10 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. In the process of dehydration reaction removal of excessive water is carried out either in presence of effective quantity of pyrophosphoric acid or by azeotropic distillation.

EFFECT: invention makes it possible to intensify dehydration process with application of smaller quantity of mineral acid and exclude side product formation.

11 cl, 1 tbl, 1 ex

 

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to the field of biotechnology, specifically for the production of steroidal compounds androsta-4,9(11)-diene-3,17-dione from a phytosterol and can be used in the biotechnology and pharmaceutical industries for the production of steroid drugs.

The LEVEL of TECHNOLOGY

Androsta-4,9(11)-diene-3,17-dione (Δ9(11)HELL, 9(11)-dihydroanthracene, CAS 1035-69-4) is a key compound in the synthesis of corticosteroid drugs from sterols, such as hydrocortisone, prednisolone, and their 9-halogen-derivatives (for example, Cortine, 9-torpedinidae and the like), and compounds having also alternates With16in particular, alkyl groups and hydroxyl groups (e.g., triamcinolone, dexamethasone, betamethasone, mometazon etc.) [V. vanRheenen, K.P. Shepard,J. Org. Chem., 1979, 44(9), 1582-1584]. In addition, Δ9(11)-AD can be used as a starting substrate in the synthesis of androgenic drug peroxidation (11β-hydroxy-9α-fluoro-17α-methyltestosterone CAS 76-43-7) [CN 102040639, 2011]used to treat, for example, seborrhea [MXNL05000077, 2007], breast cancer [WO 0040230, 2000].

Methods of obtaining Δ9(11)HELL known.

Thus, the described method total synthesis of Δ9(11)HELL, on the basis of (1S, 7aS)-1-t-butoxy-7a-methyl-5,6,7,7a-tet is ahydrogen-5-she [U. Eder, G. Sauer, G. Haffer, J. Ruppert, R.Wiechert, A. Furst, and W. Meier.Helvetica Chimica Acta, 1976, 59(4), 999-1005]. The disadvantage of this method is a multi-stage, low product yield (27,8%), the use of chromatographic purification of intermediates and products, the use of expensive reagents, and reagents for use in industrial production is a serious threat.

In addition, known methods of obtaining Δ9(11)HELL dehydration of steroid alcohols, specifically derivatives of androst-4-ene-3,17-dione (AD), containing a hydroxyl group at the atom With9or11(11α - or 11β-configuration). It should be noted that in multistaging the synthesis of steroid drugs a modification of the 17-ketogroup hydroxyl-containing derivatives HELL previously conducted dehydration of the hydroxyl group with the formation of Δ9(11)HELL, who 9,11-double bond is more stable in terms of subsequent reactions than the hydroxyl group at C11. For example, in the synthesis of peroxidation at the first stage receives Δ9(11)HELL dehydration 11α-hydroxyandrost-4-ene-3,17-dione [CN 102040639, 2011].

Known methods for producing Δ9(11)HELL dehydration 11β-hydroxyandrost-4-ene-3,17-dione: in terms of in [Q. Zhao, Zh. Li.Synthetic Communications, 1993, 23(10), 1473-1478], N,N'-conidiomata in tetr is hydrofuran [S. Solyom, K. Szilágyi, L. Toldy.Journal für Praktische Chemie, 1988, 330 (2), 309 to 312] or SO2in pyridine [GB1081307, 1967, example 2]. However, the output Δ9(11)HELL does not exceed 45-50% in the first two and 77.3% in the last version.

In the synthesis of corticosteroid circuit based on the 17-ketogroup 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-BP) dehydration of 9α-hydroxy-group is usually carried out also in the first stage [J.G. Reid, T. Debiak-Krook,Tetrahedron Lett., 1990, 31(26), 3669-3672], as generated Δ9(11)HELL is more stable in the further synthesis. This avoids the following stages of the synthesis of possible rearrangements involving the tertiary hydroxyl group at C9especially rearrangements in the ring And [L.Chinn, R. Dodson,J. Org. Chem., 1959, 24(6), 879].

9-HE-HELL is an intermediate compound in the synthesis of Δ9(11)-AD from natural sterols (cholesterol or phytosterol). The word "phytosterol" means the sterols of plant origin (β-sitosterol, campesterol, stigmasterol and others), or a mixture thereof.

9-HE-HELL phytosterol derived from microbial transformation methods and cultures of the microorganisms described in the literature.

9-HE-AD may be obtained from the two-stage biotechnology process involving transformation of sterols in HELL and the stage of 9α-hydroxylation allocated to HELL with the application of known methods ([C. Perez, A. Falero, Duc, L.H., et al., . Ind. Environ. Biotechnol.,2006, 33(8),719-723; NV homeland, M.A. Molchanov,

N.E., Voishvillo, VA Andryushina, I.E. Stetsenko, Go active.biochem.microbiol.,http://elibrary.ru/issues.asp?id=7955&jyear=2008&selid=439695,http://elibrary.ru/issues.asp?id=7955&volume=44&selid=439695(1), 56-62] and [GB862701, 1961; US3065146, 1962; US4397947, 1983; B. Angelova, P. Fernandes, A. Cruz, H.M. Pinheiro, S. Mutafova and J.M.S. Cabra,Enzym. Microb. Tech.,2005, 37(7), 718-722; NV birthplace, VA Andryushina, I.E. Stetsenko, ETC. Turova, W. of Butlerov, A.N. Panteleyev, N.E., Voishvillo, Go active.biochem.microbiol.,http://elibrary.ru/issues.asp?id=7955&jyear=2008&selid=439695,http://elibrary.ru/issues.asp?id=7955&volume=44&selid=439695(4), 439-445; RU2351645, 2009], respectively).

where R=C2H5- β-sitosterol; R=CH3- campestrin; R=C2H5and ∆22(23)- stigmasterol)

Also known single-stage two-stage microbiological process consisting in the sequential use of sternthrusters strain and 9α-hydroxyperoxy culture without isolating the intermediate formed product of the degradation of the side chain - Androstenedione (AD) - from the culture fluid [VA Andryushina, NV homeland, I.E. Stetsenko, Lew Duk Hee,

ALEXANDER Druzhinin, V.V. Aderet, N.E., Voishvillo,Go active.biochem.microbiol.,2011, 47(3), 297-301].

Known one-step methods of microbiological transformation of phytosterol 9-HE-HELL [M .G. F. Wovcha J. Antosz, J. C. Knight, L. A. Kominek, and

T. R. Pyke,Biochim. Biophs. Acta, 1978, 539, 308; US4035236, 1977; US4175006, 1979; US4397946, 1983;DD298278, 1992; DD298279, 1992; RU2077590, 1997; M.V. Donova, S.A. Gulevskaya, D.V., Dovbnya, I.F. Puntus,Appl. Environ. Biotechnol., 2005, 67(5), 671-678; M.V. Donova, D.V., Dovbnya, G.V. Sukhodolskaya, S.M. Khomutov V.M. Nikolayeva, I. Kwon, K. Han,J. Chem. Technol. Biotechnol.,2005, 80, 55-60 and others].

It is known that the efficiency of the microbiological method of obtaining 9-HE-HELL, the transformation of sterols also depends on the efficiency of the extraction method 9-HE-HELL from the culture fluid. Typically, the extraction rate of product transformation is 70 to 90%.

For example, the known method [US 4035236, 1977], according to whichthe transformation of phytosterol 9-HE-AD spend cells bacteriaMycobacterium fortuitumNRRL B-8119 load source substrate 10 g/l of Cell culture after the transformation is separated, extracted with solvent (miscible or immiscible with water). The culture fluid is free from cells, is extracted with an immiscible with water, an organic solvent, for example methylene chloride (example 2). The extract is filtered through diatomaceous earth and evaporated in vacuum to dryness. The residue is dissolved in a mixture of methanol and chloroform, concentrated in a stream of nitrogen on a steam bath until the onset of crystallization. After cooling the residue to room temperature the precipitate nontransgenic Sterol is filtered (for example, sethost is Rina, example 2). From the supernatant after evaporation of the solvent receive crud 9-HE-HELL, in which there are traces of impurities 17-hydroxy-derivative. Additional amount of 9-Oh-AD may be obtained from the mother liquor further processing.

Also known is a method of obtaining 9-HE-AD of phytosterol [GB 2197869, 1988] with the load of the substrate 20 g/l, whereby cells of bacteria after fermentation is separated, the product is washed from the cells with methanol. Additional amount of 9-HE-HELL extracted from the supernatant liquid extraction after separation of the products of incomplete oxidation of the side chain of sterols. Clean 9-HE-AD spend chromatographytandem on a column of silica gel. Of 15.1 g crudités 9-HE-HELL get just 5.7 g of the pure product.

A known method of selective separation, purification and separation monohydroxylated 3,17-dietotherapy compounds from a solution obtained by microbiological method, containing a mixture of steroid compounds by extraction, purification, and selective crystallization [WO 2008032131, 2008]. Example 4 describes the selection 9-HE-HELL, obtained by biotransformation of β-sitosterol cultureMycobacterium fortuitumNRRL B-8119 with the load of the primary substrate 30 g/l in terms of the patent [US 40635236, 1977].According to HPLC analysis, the content of 9-HE-HELL in the culture liquid after transformation with the hat 12 g/l: 1.5 g/l of this amount is in the liquid phase, and the rest - on the surface of the filtered biomass. After separate product recovery from biomass and extraction of the supernatant, followed by purification and crystallization, 9-HE-HELL received with the content of the basic substance is 95.6% and a yield of 92%, considering the content in the culture fluid (the degree of extraction, thus, 88%).

A method of obtaining and allocating 9-HE-HELL [DD 298279, 1992], which consists in conducting microbial transformation of sterols cultureMycobacterium vaccaeZIMET 11053 with a load of 10 g/l in the presence of adsorption of the polymer (copolymer of divinylbenzene and ethylstyrene).

In addition described a similar allocation method 9-OH-AD from the culture fluid obtained by transformation sitosterol cellsMycobacteriumsp. 207 when the load of the substrate 5 g/l in the presence of synthetic resin, which is a modified copolymer of ethylstyrene and divinylbenzene (Porolas)

[E.A. Borman, Y.A Rediculas, K.A., Kasienka, A.M., Turuta, A.V., Komarnicki,Go active. Biochem. Microbiol.,1992, 28 (4), 551-56]. The method involves the separation of the resin with adsorbed product, washing, elution 9-HE-HELL with ethyl acetate, evaporation and crystallization. Exit at the stage of allocation of approximately 70%.

As fermentation proceeds in the presence of the adsorbent, the main drawback of these methods to obtain 9-HE-AD of the erased is new is the need to use special equipment to prevent the destruction of the resin with stirring and effective mass transfer between the polymer resin and steroid product. This limits the scalability and practical use of such processes.

From the foregoing it is obvious that the methods of extraction 9-HE-HELL of a fermentation medium a complex, multistage, and not without substantial loss of the basic substance.

Methods of obtaining Δ9(11)HELL dehydration 9-HE-HELL known. Feature dehydration 9-HE-AD is the formation of undesired isomer androsta-4,8(9)-diene-3,17-dione (Δ8(9)-AD).

This isomeric ∆9(11)and ∆8(9)-olefins have the same value of Rfon TLC and may not be separated by crystallization. Determination of impurities ∆8(9)isomer can be carried out, for example, by a spectroscope (1H NMR,13With NMR) [EP 0253415, 1988; EP 0294911, 1988] or chromatographic (HPLC [EP 0253415, 1988; EP 0294911, 1988] , GC [US 4102907, 1978]).

As the dehydrating agent used mineral and organic acids, anhydrides and acid chlorides of organic and mineral acids and other reagents.

The dehydration reaction of 9-HE-HELL with the formation ∆9(11)HELL can be conducted or in an environment dehydrating agent without solvent, or in the environment of an organic solvent.

The combination of essential features of the claimed invention is to conduct the reaction Digi is ratatsii 9-HE-HELL with the formation ∆ 9(11)-HELL in inert aprotic organic solvent, specifically, in the environment of aromatic hydrocarbon (e.g. benzene, toluene) or chlorinated hydrocarbons (e.g. dichloromethane, dichloroethane, chloroform), using as a dehydrating agent mineral acids, including those containing water, specifically, phosphoric or perchloric acid.

Known methods of carrying out dehydration 9-HE-HELL with the formation ∆9(11)-HELL in a medium of an organic solvent. It is known that as the environment can be used chlorinated hydrocarbons, aromatic hydrocarbons, cyclic ethers, organic bases.

There is a method of carrying out dehydration 9-HE-HELL in the environment of a cyclic ether, in particular tetrahydrofuran (THF).

So, it is known conducting dehydration 9-HE-HELL action N,N1-tional-diimidazole formedfrom thionyl chloride and imidazole, in an environment THF [S. Solyom, K. Szilágyi, L. Toldy,Journal für Praktische Chemie, 1988, 330(2), 309 to 312]. The product is obtained with a yield of 93%. However, the reaction takes place selectivity: formed a mixture of isomers Δ9(11)-HELL and Δ8(9)-HELL in a ratio of 69:31. And their ratio, as noted by the authors, is not affected by the change neither the temperature, nor the nature of the solvent.

Known methods of carrying out dehydration 9-HE-AD with images is of ∆ 9(11)-HELL in the environment of organic bases.

In the paper [C.G. Bergstrom, R.T. Nicholson, R. Dodson,J. Org. Chem., 1963, 28(10), 2633-2640] describes a method for ∆9(11)-AD from 9-HE-HELL action of the reagent formed from HF and pyridine, containing 30% pyridine. However, under these conditions, the formation of an equilibrium mixture containing 9α-fluoro-Androstenedione (9α-fluoro-AD).

Another well-known way to obtain ∆9(11)AD-processing 9-hydroxysteroids, including 9-HE-HELL, equimolar amount of thionyl chloride in the environment, organic bases (e.g. pyridine and triethylamine) [US 3065146, 1962]. However, stated only the possibility of obtaining ∆9(11)-HELL in General (example 7) without specifying output and product quality. Using these conditions, as noted in [V. VanRheenen, K. P. Shephard,J. Org. Chem., 1979, 44 (9), 1582-1584], does not allow the removal of a hydroxyl group at the 9-HE-HELL regioselective: formed a mixture of isomeric products ∆9(11)-HELL and ∆8(9)-HELL. Thus, in the patent [US 4102907,1978] describes how to obtain ∆9(11)-AD from 9-HE-HELL action of thionyl chloride in the environment pyridine (0-5°C, 2 min). It is shown that these conditions result in the formation of a mixture ∆9(11)-ADand ∆8(9)-The HELL with the content of 43.4-55,5% and 43.7-56%, respectively, according to GC analysis [Preparation 1 - 3].Unsatisfactory results are obtained by use of the Finance for the dehydration of a mixture of bromine and SO 2in the environment of pyridine [US 4102907,1978. Preparation of 4 - 5]. The product is a mixture of ∆9(11)-ADand ∆8(9)AD content 50-64,4% and 43.7-50% according to GC analysis.

A method of obtaining ∆9(11)-HELL [US 4102907,1978, A.25 formulas, example 1] a two-step synthesis, including 1) sulfinpyrasone 9α-hydroxy - Androstenedione action phenylsulfonylacetate with the formation of 9-phenolsulfonate 9α-hydroxyandrostenedione and 2) desulfonylation 9-phenolsulfonate 9α-hydroxyandrostenedione by heating to 40°C in the presence of silica gel and n-methyl-phenolsulfonic acid (p-toluenesulfonic acid, p-TAC).

Getting sulfinate ester 9-HE-AD spend in the environment pyridine (10-20ºC), the subsequent desulfonation - in the environment of chloroform at boiling point in the presence of silica gel and n-TJC. The maximum achievable output ∆9(11)HELL with a melting point of 191 to 201°C does not exceed 91,78%, and the content of side ∆8(9)HELL is not specified.

A method of obtaining ∆9(11)-HELL [US 3005834, 1961, p.6 formula], which includes the contacts 9-HE-HELL, 11α-hydroxy-HELL and 11β-hydroxy-HELL in anhydrous conditions in the presence of pyridine with 1) N-bromoacetamide and 2) anhydrous sulfur dioxide, and sulfur dioxide are added to complete the oxidation of the hydroxyl group with education is the W ∆ 9(11)-HELL. However, despite the mention of 9-HE-HELL as the starting compound in the reaction of dehydration with the formation ∆9(11)HELL, an example of its use is missing. It is therefore impossible to assess the effectiveness of this method as applied to 9-HE-HELL.

Known methods of carrying out dehydration 9-HE-HELL in the environment of aromatic hydrocarbons.

Thus, the described method of using p-toluenesulfonic acid in a medium dry benzene for dehydration 9-HE-HELL [C.G. Bergstrom and R.M.Dodson,Chem.and Ind., 1961, 1530]. However, instead of the expected ∆9(11)HELL by the authors was obtained a mixture containing 9,10-scoundrel-4-ene-3,9,17-Trion (a) and 9α-hydroxy-4-methylestr-4-ene-1,17-dione (B).

A method of obtaining 9(11)-dihydroergosterol dehydration of the corresponding 9α-hydroxyandrost, characterized in that the dehydration is carried out in the presence of aromatic sulfonic acids and silica gel [EP 0253415, 1988]. The reaction is performed in the environment of aromatic hydrocarbon with a boiling point of a solution of the steroid, the aromatic sulfonic acids can be used p-TJC or naphthalenesulfonic acid. The method is characterized by the fact that pre-receive adsorbate sulfonic acids (for example, the adsorbate monohydrate p-TJC) on silica gel chromatographic quality. Significant disadvantages of this way is as is the use of a substantial amount of silica gel (more than 10 times the weight of the original substrate) and a low yield of product: maximum achievable output ∆ 9(11)HELL does not exceed 65,8%.

The way to obtain 9(11)-dihydroceramides series of androstane and pregnane dehydration of the corresponding 9α-hydroxysteroid described in the patent [EP0294911, 1988]. The method is characterized by the fact that the dehydration is carried out in an inert organic solvent in the presence of a Lewis acid selected from the group consisting of ferric chloride, boron TRIFLUORIDEor their complexes, pentachloride antimony, titanium tetrachloride and mixtures of these compounds with silica gel. So, example 1 describes obtaining ∆9(11)-AD from 9-HE-HELL in a specified way in the medium of anhydrous benzene at the boiling point by the action of the reagent formed from ferric chloride and silica gel. The product does not contain impurities ∆8(9)-HELL. The disadvantages of this method are the use of chromatographic purification method and the low yield obtained ∆9(11)-HELL (from 0.4 g of 9-HE-HELL, the authors obtained 0.16 g ∆9(11)-HELL, which corresponds to a molar yield of 42.6%). Example 3 describes carrying out the dehydration reaction of 9-HE-HELL in the environment of methylene chloride at room temperature by the action of pentachloride antimony. The main disadvantages of this option are the use of toxic reagent, extremely low output ∆9(11)-HELL (604 mg 9-HE-HELL received 139 mg ∆9(11)-HELL, which corresponds to a molar yield of 24.5%). The best result obtained PR is the use of complexes of boron TRIFLUORIDE in an environment of benzene (epirate boron TRIFLUORIDE in example 4 at boiling point, complex of boron TRIFLUORIDE with methanol at room temperature in example 5, and a complex of boron TRIFLUORIDE with acetic acid at the boiling temperature of example 6). From 3,02 g 9-HE-HELL (example 4) was obtained 2.3 g ∆9(11)-HELL, which corresponds to a molar yield of 81%. Thus the melting point of the obtained ∆9(11)-HELL is 204-205,5°C, which confirms the purity of the product. Output ∆9(11)-HELL in example 5 was 92.4 per cent, but the content of the basic substance is just to 89.5%. Output ∆9(11)-HELL in example 6 was 97,5%. The authors noted that the presence of ∆8(9)-isomer was not detected (according to HPLC). A common shortcoming of the options described in examples 4-6, is the use of hazardous reagent boron TRIFLUORIDE having extremely high corrosion activity and toxicity.

A method of obtaining ∆9(11)HELL dehydration 9-HE-HELL chlorosulfonic acid in the environment chlorinated hydrocarbon, particularly methylene chloride [US 4127596,1978, examples 1-8]. The disadvantage of this method is that chlorosulfonic acid, which must dispose of the water generated by the dehydration of 9-hydroxy-group, thereby catalyzing the process, reacts with water which may be present in an organic solvent. In addition, to prevent the destruction of 9-HE-HELL or ∆9(11 HELL the reaction is carried out at low temperatures (specifically at a temperature of from minus 20 to 5°C). Output ∆9(11)-HELL is 85% (example 1) and ~ 95% (examples 2 to 7). When carrying out the reaction at a temperature of 20-27°C output ∆9(11)HELL falls to 84.4% (example 8).

One of the essential features of the present invention is used as a dehydrating agent mineral acids, including those containing water, more specifically, phosphoric or perchloric acid.

A method of obtaining ∆9(11)-steroids of some of the androstane, including ∆9(11)HELL, the reaction 9α-hydroxysteroid (in particular, 9-OH-BP) with nah kislorodsodyerzhascimi acid with pKa≤1,0 [US4127596,1978]. When this acid is selected from the group of chlorosulfonic, sulfuric, phosphoric, methansulfonate, Perlina and triperoxonane. The reaction is performed in the environment of oxygen-containing acids. So, for example, when using aqueous sulfuric acid (mixture of 3 parts of water and 7 parts of 70% sulfuric acid, which corresponds to ~55%) get ∆9(11)-The HELL with the yield of 97.4%. However, the duration of reaction is not specified, this product contains up to 1% of side ∆8(9)-HELL. Examples 12-15 describe the use methanesulfonic acid (MSC). It is shown that the highest yield ∆9(11)HELL get when carrying out the reaction without the addition of water (primer, the output is 95.6%, duration of response 80 min at 25°C). Conducting the reaction in a mixture of MSC and water with increasing water content increases the duration of the process and decreases the output ∆9(11)-HELL, and there is a need of carrying out the reaction at a higher temperature. So, at a volume ratio of MSC and water 4:1 the duration of the process when 31-39°C is 5 h, the output ∆9(11)AD-87,2%, TPL 198,5-201°C. (example 12); at a volume ratio of MSC and water 1:1 the duration of the process at a temperature of from 100 to 60°C is 2 h, the output crudités ∆9(11)-The HELL with TPL 170-175°C is 75,9% (example 15), and at a volume ratio of MSC and water 2:3 duration of the process at 68°C is 6 h, and the output ∆9(11)-The HELL with TPL 186-195°C is only 55,7% (example 14). Thus, the lack of application of the MSC is the long duration of the process and a significant reduction in yield and quality ∆9(11)-HELL in the presence of water and by increasing the temperature of the process above room temperature.

Also known is a method of obtaining ∆9(11)-AD from 9-HE-HELL dehydration with polyphosphoric acid [HUT36138, 1985]. The reaction is performed in the environment of polyphosphoric acid for 2 h 20 min at 40°C, and uses ~3-fold volumetric quantity of acid to 1 weight part 9-HE-AD. Upon completion of the reaction the reaction mass R is izbavlyayut water, the precipitate is filtered off. The product obtained with the yield 98,46% and TPL 199-201º.

The common disadvantage of the methods of dehydration in the environment dehydrating agent, especially in the presence of water, is the use of a large excess of dehydrating agent, the long duration of reaction, and in most cases, the necessity of using column chromatography for purification of product, and as a consequence or low yield or poor quality.

The closest analogue (prototype) one of the essential features of the claimed invention, which is nature used dehydrating agent, namely the use of mineral acids, including those containing water, more specifically the use of phosphoric acid or perchloric acid, is a method for ∆9(11)-steroids of some of the androstane, including ∆9(11)HELL, the reaction 9α-hydroxysteroid (in particular, 9-OH-BP) with non-aromatic oxygen-containing acid with pKa≤1,0 [US 4127596,1978, examples 10-11 and example 16, respectively (reprinted as USRE 33364, 1990].

Example 11 prototype [US 4127596,1978] describes how to obtain ∆9(11)HELL by the reaction of 9-HE-HELL with 85% phosphoric acid, and phosphoric acid also serves as dehydrating agent, and environment for the reaction. 1 mol 9-HE-AD used 12.2 m is La 85% phosphoric acid. The reaction mass is stirred at a temperature of 45-55°C for 24 h Then diluted with water three times, the precipitate is filtered off. Get ∆9(11)-HELL of a molar output 95,1%. Contents ∆9(11)-HELL in the sample is not specified. However, the melting point of the resulting product is just 193-195°C, which is an indicator of the low content of the basic substance and, probably due to prolonged heating of the reaction mass. For professionals working in the field of synthesis of steroid compounds, it is evident that the melting temperature is an important characteristic of the purity of the steroid compounds. It is known that the melting temperature ∆9(11)HELL, containing no impurities is in the range from 203 to 206°C. (for example, 204-205,5°C [EP0294911, 1988]). In addition, another significant drawback of this method is the long duration of the process. Significant dilution of the reaction mass phosphoric acid (up to 73-fold to the original steroid) can reduce the duration of reaction from 24 h to 7.5 h at a temperature of 35-48°C and obtain ∆9(11)-The HELL with the release of 96.2% [US 4127596,1978, example 10]. Although the melting point of the product rises to 197-201°C., GC analysis shows the presence of 1% of side ∆8(9)-HELL. The disadvantage of this alternative method is the use of a large number dehydrating the agent and the long duration of the process.

Example 16 prototype [US 4127596,1978] describes how to obtain ∆9(11)HELL by the reaction of 9-HE-HELL using dilute chloric acid, and perchloric acid also serves as dehydrating agent, and environment for the reaction. The reaction is carried out in the environment ~28% aqueous perchloric acid medium is a mixture of 5 ml water and 2 ml of 70% perchloric acid), and 1 mol 9-HE-HELL use 23,2 mole of perchloric acid. The method is extremely inefficient, because in addition to the long duration of the process (66 h) at a temperature of 54°C molar yield ∆9(11)HELL is only 69.6 per cent.

General shortcomings of the methods of obtaining ∆9(11)-The HELL with the use of mineral acids, including water used as a solvent medium, in particular, 85% phosphoric acid and 28% perchloric acid are:

• Large amounts of mineral acids (up to 73 times the weight of the original steroid);

• The long duration of the reaction (from 7.5 h to 24 h to 86% phosphoric acid and 66 h for 28% perchloric acid);

• Low product yield (69,6% when using perchloric acid).

Thus, the analysis methods of dehydration 9-HE-HELL, known from the available literature shows that when carrying out the reaction in the environment of an organic solvent, in particular in the environment of aromatic uglev Dorada or chlorinated hydrocarbon, the use of mineral acids, including water-containing, unknown. Describes how to carry out reaction with organic acids or their derivatives, anhydrides or acid chlorides of mineral acids, anhydrous environment is a condition for the reaction, providing a positive result. As follows from the prior art, the use of mineral acids, including those containing water, to obtain ∆9(11)HELL it is possible and known in the processes of dehydration 9-HE-HELL without the use of an organic solvent, however, these methods have significant shortcomings and the results obtained cannot be considered satisfactory.

In the available literature there is no information about the reaction of dehydration 9-HE-HELL with the formation ∆9(11)HELL, using as a dehydrating agent mineral acids, including water-containing environment aprotic organic solvent.

In addition, in the available literature there is no information about the intensification of the process of dehydration is the removal of excess water during the dehydration reaction, for example, by the method of azeotropic distillation.

In addition, in the available literature there is no information about the possibility of obtaining ∆9(11)-HELL of phytosterol through education 9-ABOUT THE-HELL without releasing the latter. Known methods, for example, described above [US 4035236, 1977; GB 2197869, 1988], provide for transformation of phytosterol emitting crystalline 9-HE-AD. Other known methods of obtaining ∆9(11)-HELL involve the introduction into the reaction dehydration of crystalline 9-HE-HELL.

In addition, in the available literature there is no information about the reaction of dehydration 9-HE-HELL with the formation ∆9(11)-HELL in the extract of the culture fluid containing 9-HE-AD and the resulting microbial transformation of sterols.

DISCLOSURE of INVENTIONS

The aim of the present invention is to simplify and reduce the cost of technology for ∆9(11)-HELL of phytosterol.

The essence of the invention is the combination of two stages of synthesis ∆9(11)-HELL of phytosterol - stage biotech, namely microbiological transformation of phytosterol with the formation of 9-HE-HELL, and chemical stages of dehydration 9-HE-HELL - in one manufacturing process to be carried out without the isolation of intermediate 9-HE-AD of the extract clarified culture fluid.

Such a technical solution is not known from the prior art. Sold by us for the first time.

The technical result of the present invention is provided by a set of essential features which which are:

• Conducting dehydration 9-HE-HELL, obtained from phytosterol microbiological transformation, in the extract of the culture fluid, from which previously removed bacterial cells of strain-transformant;

• Use for extraction of ∆9(11)-HELL of organic solvents, namely, aromatic hydrocarbons or chlorinated hydrocarbons;

• Use as a dehydrating agent mineral acids, including those containing water, more specifically, phosphoric acid or perchloric acid;

• Removing water from the reaction mass in the reaction, for example, azeotropic distillation.

The proposed solution is a new (not known from the prior art), is not obvious from the prior art, is industrially applicable.

The technical result of the proposed technical solutions are:

• Optimization and simplification of the method of obtaining ∆9(11)-HELL of phytosterol by combining two steps of the synthesis of ∆9(11)-HELL of phytosterol - stage biotech, namely microbiological transformation of phytosterol with the formation of 9-HE-HELL, and chemical stages of dehydration 9-HE-HELL - in one manufacturing process to be carried out without the isolation of intermediate 9-HE-AD of the extract clarified culture fluid;/p>

• Reducing the number of technological operations at the stage of biotechnological process of obtaining 9-HE-AD of phytosterol, namely the exclusion from the process the following operations: evaporation of the extract, the crystallization of 9-HE-HELL, filtering sediment technical 9-HE-HELL, drying technical 9-HE-HELL, cleaning technical 9-HE-HELL (includes crystallization/recrystallization, filtration, drying);

• Complete elimination of losses 9-HE-HELL on the operations of its allocation described above (from 10 to 30% of the basic substance), including mechanical losses and the loss of the basic substance in the mother solution;

• Conducting a dehydration reaction in the extract without loss of efficiency compared to the dehydration of crystalline 9-HE-HELL in a medium of an organic solvent;

• Reduction in the duration of the dehydration reaction of 9-HE-HELL, reducing the amount of dehydrating agent and thereby increase the yield and quality ∆9(11)HELL,

• Reducing the length of the production process and thus energy costs;

• Reduction of material costs.

The technical problem on which the invention is directed, is to simplify and improve the efficiency of the process of obtaining ∆9(11)-HELL of phytosterol through the formation of intermediate 9-HE-HELL due to lead the exception of losses 9-HE-HELL, increasing the yield of the target product ∆9(11)-HELL of phytosterol by the almost complete exclusion of the formation of by-product ∆8(9)-HELL on stage dehydration 9-HE-AD and obtain ∆9(11)-HELL of phytosterol way, devoid of the above disadvantages.

The implementation of the two complex processes in one technological stage determine the prospective use of the method according to the invention, to obtain ∆9(11)-HELL of phytosterol.

The technical problem of obtaining androsta-4,9(11)-diene-3,17-dione from phytosterol using as an intermediate compound 9α-hydroxyandrost-4-ene-3,17-dione, obtained by microbiological transformation is solved by a method of producing androsta-4,9(11)-diene-3,17-dione of the formula (I)

from phytosterol General formula (II)

where R=C2H5- β-sitosterol; R=CH3- campestrin; R=C2H5and ∆22(23)- stigmasterol, R=CH3and ∆22(23)- brassicasterol,

including microbiological elimination of the side chain with the formation of 9α-hydroxyandrost-4-ene-3,17-dione of formula (III),

branch biomass extraction 9α-hydroxyandrost-4-ene-3,17-dione (III) of the clarified culture fluid and subsequent dehydrate the s 9α-hydroxy group dehydrating agent, characterized in that the dehydration of 9α-hydroxy-group is carried out in an extract of the culture fluid, and as the dehydrating agent used mineral acids, including those containing water, and the removal of excess water may be carried out in the reaction of dehydration. Carrying out the dehydration reaction of 9-HE-HELL, obtained by microbiological transformation of phytosterol, without separating it from the extract of the culture fluid previously in the literature are not described.

Thus, the essence of the claimed invention, which includes receiving 9-HE-AD of phytosterol and then ∆9(11)-The HELL with it, is that phytosterol first subjected to microbiological transformation of the culture of the genusMycobacteriumwith the formation of 9-HE-HELL, then 9-HE-HELL is extracted from the pre-clarified culture fluid aprotic organic solvent followed by reaction of dehydration in the obtained extract, extract mineral acid is added, including those containing water, and excess water can be removed during the reaction, for example, azeotropic distillation.

While microbiological transformation of phytosterol order to obtain 9-HE IS HELL carried out using cells bacteriaMycobacteriumsp. ACM A-D.

In addition, as the aprotic organic solvent using an aromatic hydrocarbon (e.g. benzene or toluene) or chlorinated hydrocarbons (e.g. dichloromethane, or chloroform, or dichloroethane).

In addition, the concentration of phytosterol when conducting microbiological transformation is from 4 to 20 g/L.

The advantages of the proposed method are as follows:

• No need to remove from the extract, crystallization and purification 9-HE-HELL; no loss of 9-HE-HELL, taking place on these operations;

• High regiospecificity of the reaction of dehydration. The degree of transformation 9-HE-HELL in ∆9(11)HELL 98-100% (according to HPLC analysis); the degree of extraction of ∆9(11)-AD 96-98%.

• Total output ∆9(11)-HELL of phytosterol reaches 71%.

The method according to the present invention allows the process of dehydration 9-HE-AD:

• with a minimum amount of dehydrating agent, namely from 1.6 to 9 mol per 1 mol of 9-HE-HELL, in the extract of the culture fluid;

• without the use of dangerous and expensive dehydrating agents, such as anhydrides and acid chlorides of mineral and organic acids, complexes of boron TRIFLUORIDE;

• without the use of expensive anhydrous organic solvents;

• The method can be implemented on standard technology is Eskom equipment.

The IMPLEMENTATION of the INVENTION

Getting androsta-4,9(11)-diene-3,17-dione of the formula (I) of phytosterol formula (II) is carried out according to the scheme depicted in figure 1.

Figure 1 - Synthesis of Δ9(11)AD (I) of phytosterol (II) through 9-HE-AD (III) of the inventive method, where R=C2H5- β-sitosterol; R=CH3- campestrin; R=C2H5and ∆22(23)- stigmasterol, R=CH3and ∆22(23)- brassicasterol

Below cited as an example of a detailed description of the invention.

Phytosterol General formula (II)

where R=C2H5- β-sitosterol; R=CH3- campestrin; R=C2H5and ∆22(23)- stigmasterol, R=CH3and ∆22(23)- brassicasterol,

subjected to microbiological elimination of the side chain atom With17with the simultaneous formation of Δ4-3-closetime and 9α-hydroxylation culture of bacterial cells of the genusMycobacterium(preferably cells bacteriaMycobacteriumsp. VKM Ac-D), and the load of the source substrate is from 4 to 20 g/l, and the length of fermentation - from 72 to 144 hours; the process is carried out in aqueous medium selectivity of transformation in 9α-hydroxyandrost-4-ene-3,17-dione (III) to 72%, after which the cells of the strain-transformant separated. 9α-Hydroxyandrost-4-ene-,17-dione (III) is extracted from the received clarified culture fluid, practically does not contain phytosterol, an organic solvent (aromatic hydrocarbon or chlorinated hydrocarbon) and without separation from the extract is subjected to interaction with a dehydrating agent to form androsta-4,9(11)-diene-3,17-dione (I), which received the extract mineral acid is added.

Analysis of the products of microbial transformation carried out by thin layer chromatography (TLC) and HPLC. Determination of the contents of the products by HPLC performed on the chromatograph Agilent 1200 (USA) precolonial Symmetry C18, 5 μm, 3,9x20 mm and column Symmetry C18, 5 μm, 4,6x250 mm (Waters, USA) in the system with mobile phase (%): acetonitrile - 52, acetic acid 0.01 and water -47,99; at a flow rate of 1 ml/min, 50°C, with detection of the peaks in the optical absorption at 240 nm; with the calculation of concentrations according to the method of external standard. Typical retention times: 9-HE-HELL 4,27 min and for Δ9(11)-HELL of 8.25 minutes

Selection 9-HE-AD (III) of the clarified culture fluid can be carried out by extraction of water-immiscible solvent after the preliminary separation of the biomass from the water phase. To extract steroid can also be applied, for example, sorption method of extraction, as well as any other effective method. The selectivity of the formation of 9α-hydroxyandrost-4-ene-3,17-dione (III) at the stage of ICRI is the biological oxidation of phytosterol (II) is 68-72%. The selectivity of the formation of Δ9(11)AD (I) at the stage of dehydration is 98-100%. The degree of extraction of the crystalline Δ9(11)-HELL of crudités is 96-98%.

Total yield Δ9(11)AD (I) of phytosterol reaches 71%.

The claimed invention is illustrated by the following examples without limiting it.

For transformation can be used soy phytosterol content transformable sterols from 90 to 100%. When carrying out the invention is a method of producing androsta-4,9(11)-diene-3,17-dione in the following example used the phytosterol content transformable sterols 95,47%, including (%): β-sitosterol - 42,39; campestrin - 23,48; stigmasteryl - 26,08; brassicasterol - 3,52.

Microbiological oxidation of phytosterol in the implementation of the present invention, the method of obtaining androsta-4,9(11)-diene-3,17-dione can be carried out by the cells of bacteria of the genusMycobacteriumspecificallyMycobacteriumsp. VKM Ac-D.

As the organic solvent in carrying out the invention is a method of producing androsta-4,9(11)-diene-3,17-dione for the operation of extraction 9-HE-AD and subsequent dehydration can be used an aromatic hydrocarbon (e.g. benzene or toluene) or chlorinated hydrocarbons (e.g. dichloromethane, or chloroform, or dichloroethane).

As the dehydrating agent in the operation Degi is ratatsii 9-HE-AD can be used mineral acids, including those containing water, for example, perchloric acid with a water content of up to 43%, or phosphoric acid with a water content of up to 15%, or phosphoric acid with a content of up to 50% pyrophosphoric acid.

The intensification of the process of dehydration during implementation of the invention is a method of producing androsta-4,9(11)-diene-3,17-dione by the removal of excess water during the dehydration reaction can be carried out, for example, by the method of azeotropic distillation or by conducting the reaction in the presence of an effective amount of pyrophosphoric acid, which reacts with water, turns into the reaction conditions in the orthophosphoric acid.

Table 1 presents the results of dehydration 9α-hydroxyandrost-4-ene-3,17-dione with the formation ∆9(11)-HELL in the extract clarified culture fluid in the conditions of the present method and the methods described in the literature.

Examples

Example 1 Obtaining androsta-4,9(11)-diene-3,17-dione (I)

A) Growing the inoculum

BacteriaMycobacteriumsp. VKM Ac-D cultivated in a shaker-thermostat for 25 h at 30°C and a frequency of shaking at 220 rpm in katalozhnyh flasks with a capacity of 750 ml, containing 100 ml of liquid nutrient medium of the following composition (g/l): yeast extract dry - 10; (NH3)2HPO4- 1,5; KH2PO4-0,5; K2HPO4x 3H2O - 0,5; glycerol - 10; polyethylene glycol sorbitol monooleate - 3,0; MgSO4x 7H2O - 0,2; FeSO4x 7H2O - 0,005; ZnSO4x 7H2O - 0,002; distilled water to 1 l, pH of 6.8 to 7.2.

B) transformation

Received the seed material is introduced into the environment for transformation in the amount of 10% (by volume). Microbiological transformation of phytosterol is carried out in a shaker-thermostat for 72 hours at 30°C and a frequency of shaking at 220 rpm in katalozhnyh flasks with a capacity of 750 ml, containing 100 ml of medium for the transformation of the following composition (g/l): KH2PO4to 0.8; K2HPO4x 3H2O - 4,2; (NH3)2SO4- 3,0; glycerol - 5,0; urea - 0,13; MgSO4x 7H2O - 0,2; FeSO4x 7H2O - 0,01; ZnSO4x 7H2O - 0,002; polyethylene glycol sorbitol monooleate - 1.0; phytosterol (calculated on the transformed sterols) - 4,0; distilled water to 1 l, pH of 6.8 to 7.2. After sterilization, the medium phytosterol homogenized by ultrasound (42 kHz, 100 W) for 2 minutes.

C) Obtaining an extract of

After the transformation of the culture fluid centrifuged at

10 000 g for 40 min at 20-25°C. Receive the clarified culture fluid (supernatant)containing 9-HE-HELL (selectivity transformation 71,48%).

9-HE-HELL extrage the comfort of the supernatant liquid organic solvent. The extract containing 9-HE-HELL (the extract).

G) the reaction of dehydration

Option 1.The extract is placed in a flask equipped with a reflux condenser. To extract mineral acid is added. The reaction mass is heated to boiling and maintained under stirring until the end of the reaction. The mixture is then cooled to room temperature and add 5% aqueous NaCl solution. The organic layer is separated, the aqueous layer was extracted with the same solvent. The combined organic phases are washed with 5% NaCl solution until neutral and water, then dried with Na2SO4and evaporated in vacuum to dryness. Get crud containing ∆9(11)-HELL and not containing 9-HE-HELL according to HPLC analysis.

Option 2 (with azeotropic distillation of the water). The extract is placed in a flask equipped with nozzle Dean-stark and reflux condenser. Then to extract mineral acid is added. The reaction mass is heated to boiling and maintained under stirring until the end of the reaction, collecting the released water into the nozzle. The mixture is then cooled to room temperature. The treatment of the reaction mixture were carried out as described in the previous version.

The selectivity of the reaction of dehydration is assessed according to the HPLC analysis of the original extract (determination of the concentration of 9-OH-BP) and crudités (contents ∆9(11)-AD).

P the following selection of crudités get ∆ 9(11)HELL with a melting point from 203,5 to 206,5ºC and the degree of extraction of 96-98%.

An NMR spectrum1H (δ, ppm; CDCl3): 5,75 (l,J= 1.8 Hz, 1H, 4-CH); to 5.55 (m, 1H, 11-CH); to 1.35 (s, 3H, 19-CH3); to 0.87 (s, 3H, 18-CH3).

An NMR spectrum13With (δ, ppm; CDCl3): 221,0 (17-C(O)); 199,0 (3-C(O)); 168,9 (5-C); RUB 145.1 (9-C); to 124.2 (4-CH); 118,1 (11-CH); 48,0; 45,8; 41,1 36,8; 36,2; 34,2; 33,8; 33,4; 32,6; 31,1; 26,2; 22,6; 13,9 (19-FromN3).

Table 1

td align="center"> Toluene
MethodDissolve-telDehydrate-regulating agent (mol/mol 9-HE-AD)Temperature, duration of reactionThe selectivity of the formation ∆9(11)HELL, % (HPLC)Exit
9(11)HELL, %
(recovery,%)
Note
According to the method of US 4127596-85% H3PO4
12,2
45-55°C,
24 hours
-95,1Crystalline 9-HE-AD
According to the method of US 4127596-H3PO485%
73v/1w
35-48°C,
7,5 h
-96,2Crystalline 9-HE-AD
The inventive methodChloroform
85% H3PO4
1,6
Boiling,
1 h 25 min
~100-Extract
The inventive methodBenzene
85% H3PO4
1,6
Boiling,
1 h 10 min
~100(of 97.8) Extract
The inventive methodToluene
85% H3PO4
1,6
Boiling,
30 min
98,4
-Extract
The inventive methodDichloroethane85% H3PO4
8,9
Boiling
(78 °C), 30 min
~100(96,86)Extract
The inventive method85% H3PO4
1,6
Boiling,
5 min
~100-The extract. Carrying out the reaction with azeotropic distillation of the water
The inventive methodCH2Cl285% H3PO4
1,6
Boiling for 2 hours99(96,1)
The extract. Carrying out the reaction with azeotropic distillation of the water
The inventive methodToluene46.5% of H4P2O7/H3PO4Boiling,
40 min
98,52-Extract
The inventive methodChloroform
46.5% of H4P2O7/H3PO4
of 5.4
Boiling,
1 h 40 min

99,48
-Extract
By way
HUT36138
- n+2.(P)n.(O)3n+1
3v/1w
40°C,
2 h 20 min
-98,46Crystalline 9-HE-AD
The inventive methodBenzene
57% HClO4
1,83
Boiling,
10 min
~100(96,0)The extract. Carrying out the reaction with azeotropic distillation of the water
According to the method of US 4127596-28% HClO4
23,2
54 °C
66 h
-69,6Crystalline 9-HE-AD

1. The method of producing androsta-4,9(11)-diene-3,17-dione of the formula (I)

from phytosterol General formula (II),

where R=C2H5- β-sitosterol; R=CH3- campestrin; R=C2H5and ∆22(23)- stigmasterol; R=CH3and ∆22(23)- brassicasterol,
including microbiological elimination of the side chain atom With17with the formation of 9α-hydroxyandrost-4-ene-3,17-dione of formula (III),

the Department is giving biomass, extraction of 9α-hydroxyandrost-4-ene-3,17-dione (III) of the clarified culture fluid and subsequent dehydration of the 9α-hydroxy group dehydrating agent, characterized in that the compound (III) is extracted from the clarified culture fluid aprotic organic solvent selected from aromatic hydrocarbons or chlorinated hydrocarbons, followed by the reaction of dehydration of 9α-hydroxy-group of the compound (III) obtained in the extract, as the dehydrating agent used mineral acid, including water and selected from the group comprising phosphoric, pyrophosphoryl and perchloric acid, and the removal of excess water is carried out in the reaction of dehydration.

2. The method according to claim 1, characterized in that for transformation using soy phytosterol content transformable sterols from 90 to 100%.

3. The method according to claim 1, characterized in that the microbiological transformation of phytosterol spend cells bacteriaMycobacteriumsp. VKM Ac-D.

4. The method according to claim 1, characterized in that the aromatic hydrocarbon use benzene or toluene.

5. The method according to claim 1, characterized in that as a chlorinated hydrocarbon used dichloromethane, or chloroform, or dichloroethane.

6. The method according to claim 1, characterized in that the UD is excess of water can be carried out in the reaction of dehydration by azeotropic distillation.

7. The method according to claim 1, characterized in that the removal of excess water may be carried out in the reaction of dehydration in the presence of an effective amount of pyrophosphoric acid.

8. The method according to claim 6, characterized in that the use of mineral acid in an amount of from 1 to 10 mol per 1 mol 9α-hydroxyandrost-4-ene-3,17-dione, preferably from 2 to 5 mol per 1 mol 9α-hydroxyandrost-4-ene-3,17-dione.

9. The method according to claim 6, characterized in that the mineral acid is used 85% orthophosphoric acid.

10. The method according to claim 6, characterized in that the mineral acid is used 57% perchloric acid.

11. The method according to claim 1, characterized in that as the dehydrating agent used is phosphoric acid containing up to 50% pyrophosphoric acid.



 

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EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of obtaining preparation based on vaccine strain of plague microbe. Claimed invention includes preparing inoculation native culture of plague microbe, concentration of microbe suspension, preparing vaccine suspension and obtaining dry form of preparation, with process of preparing inoculation culture including cultivation of microbes in liquid nutritional medium in flasks for 48 h at temperature 26…28°C and contibuous aeration with not less than 10 l min-1. with passaged stabilised starting culture, obtained as a result of three successive passages through organism of guinea pigs and mixed with glycerol-lactose-polyglucinum liquid in ratio 2:1; for preparation of vaccine suspension used is optimised in component composition protective drying medium, lyophilisation being carried out with observance of the specified regimen.

EFFECT: claimed solution makes it possible to obtain product with higher activity with reduced duration of process of its manufacturing.

3 dwg, 6 tbl

FIELD: biotechnologies.

SUBSTANCE: nutritive medium includes lactoserum, yeast autolysate, acetic acid 70%, agar and cabbage brew at the specified component ratio.

EFFECT: invention allows increasing selectivity of nutritive medium and simplifying its production.

8 ex

FIELD: biotechnologies.

SUBSTANCE: method for obtaining spore material of bacteria of Clostridium type provides for production of inoculum of bacteria in a full synthetic growth medium, seeding of inoculum and cultivation under the corresponding conditions in growth medium including potato, glucose, ammonium sulphate and chalk. During the main fermentation process for 18-28 parts of bacterial culture growth depending on time of occurrence in one field of view of at least 80-100 thickened forms of cells, n-butanol in the amount of 0.16-0.81 wt % is added to growth medium. Number of formed spores is 1.08-2.86·108 in one millilitre of the medium.

EFFECT: increased content of spores in spore material.

1 tbl, 15 ex

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