KR820000373B1 - Process for producing steroidal alcohols - Google Patents

Abstract

Steroidal alcs. were prepd. by Mycobacterium species capable of producing I or II when provided with a steroidal substrate. Thus, a substrate culture was prepd. by inoculating a nutrient medium of glucose 10, meat ext. 1.0, and peptone 1.0% with M. parafortuitum and incubating at 30≰C, 72hr. The seed culture was then inoculated into a prodn. medium contg. soybean meal 4, K2HPO4 0.2, MgSO4.7H2O 0.1, fish oil residue 2, NaNO3 0.2, and cholesterol which was incubated at 30≰C, 160 hr. Extn. of the resulting fermn. broth with EtOAc followed by filteration of the ext. gave a filtrate having the steroid compn. cholesterol 0.05, I 2.65, and II 0.22g.

Description

Preparation of Steroid Alcohol

The present invention relates to a method for producing steroidal alcohol, and more particularly, to an improvement in a medium for producing a method for producing steroidal alcohol by a microorganism.

Microorganisms belonging to the genus Mycobacterium (hereinafter abbreviated as “M”) are 20α-hydroxymethylpregner-1,4-diene-3-one (= 22-hydroxy-22,23-bisnol-cora-). 1,4-diene-3-one (hereinafter abbreviated as "HPD") and 30α-hydroxymethylpregine-4-ene-3-one (= 22-hydroxy-22,23-bisnol core-4) The production of -en-3on (hereinafter abbreviated as "4HP") itself is, for example, A) Applied Microbiology, Vol. 23, No. 23, pp. 72-77, 1972, USA, B) US Patent No. 3,684,657 C, US Pat. No. 3,759,791, which is known.

However, the amount of HPD and 4HP (hereinafter collectively referred to as HP) described in the three documents A to C described above is trace and has little industrial significance. That is, about 20 µg / ml in the document A and 40 µg / ml in the document C was not more than 20 µg / ml in the amount of HPD, and in the case of 4HP, only one of the examples of the document C was found. It is only 40 µg / ml as much as possible.

However, HP is a very useful intermediate that can be easily converted into a steroid having an activity of corticosteroids (corticoids), progesterone, and anabolic hormones by conventional organic synthetic chemical reactions. Development of a manufacturing method is desired.

As a result of intensive studies in view of such circumstances, the present inventors have found that the use of any new medium can achieve the object, thereby reaching the present invention.

That is, the gist of the present invention uses a microorganism belonging to the genus M. and the production method of steroid alcohol, characterized in that using a medium containing at least 0.1% by weight of glycerides in the production of HPD or 4HP using the sterols as a substrate and In the preparation of HPD or 4HP using sterools as a substrate using microorganisms belonging to the genus M. genus, a medium containing at least 0.1% by weight of glycerides and vegetable fats and oils is used for producing a steroid alcohol.

Hereinafter, the present invention will be described in detail.

The microorganism used in the method of the present invention belongs to the genus M. and is a microorganism capable of producing HPD or 4HP.

Of course, it goes without saying that such microorganisms may produce HPD, 4HP, or HPD and 4HP simultaneously.

Such microorganisms include most species of M. genus that exhibit rapid development. For example M. phlei, M. rhodesiae, M. fortuitum (alternatives: lanae, M. giae, M. minetti), M. portoitum subspecise, M. peregrinum (nickname: M. anababanti), M. chelonei (nickname: M. borsi) Borstelense], M. chelonei Subsp. Abscessus [Tinys: M. Absessus, M. runyonii], M. Schneiss Megmatis (alternative name: M. lacticola), M. butyricum, M. specialty (sp) ＃ 607), M. flavescens (alternative name: M. lacticola) Acapulsencens], M. cubuense (nickname: M. Tokaiense), M. gilvum, M. themoresestibile, M. kita chitae, M. vaccae, M. duvalii, M. agri, M. lactae, M. obuense, M. ah. Aichiense, M. parafortuitum complex [M. Aurum, M. neoaurum, M. paraportuitum, M. dietenhoferi, Kanazawa-strains, and the like.

These products can be divided into mutant strains that do not require the presence of inhibitors such as chelating agents in the production of HP, and wild strains that require inhibitors such as chelating agents.

As a mutant strain which does not require the addition of an inhibitor such as a chelating agent, for example, Paraportitum Cupplex MCI-0617 bacteria (Japan Microbiological Technology Research Institute Accession No. FERM-P 4258), M. spaceids (sp.) NRRL B-3805 bacterium and M. speculum (sp.) NRRL B-383 bacterium.

Next, as a bacterium producing a small amount of HP under the addition of a chelating agent or an inhibitor such as Ni or Co, for example, M. bacca, ATCC 25954, M. paraportumum ATCC 25807, and M. duvari NCTC 8645 Can be mentioned.

It can be used in the present invention. The microorganism is not limited to the above, and any M. spp. Bacterium that exhibits rapid development by generating HP from sterols regardless of addition or no inhibitor is irrelevant. It is also possible to mix two or more microorganisms.

As a specific example, the above-mentioned M. paraportuitum complex MCI-0617 bacterium will be described in detail.

The bacterium was found to be Paraportuitum Complex ATCC 25790 [= M. NeoAutum ATCC 25790] is a mutant strain obtained by ultraviolet light irradiation from the bacterium.

M. Specialized NRRL B-3683 bacteria, B-3805 bacteria and allosterols 1,4-diene-3, 17-dione (hereinafter abbreviated as "ADD") or Androsto from sterols Methyl-0617 bacteria have the ability to convert and accumulate more than half of arsenic sterols into HP, while 4--4-en-3 and 17-dione (abbreviated as "4AD") are produced as the main product and HP is a by-product. Have

Regarding the parent strains of MCI-0617 bacteria, the parent strain ATCC 25790 was identified as a conclusion of a joint study by the International Working Group on Mycobacterial Taxonomy of 13 experts on M. It is said to belong. (See Saito Hajime Tabamura Michio, International Journal of Cycological Bacteriology, Vol. 27, pp. 75 to 85 (1977, USA)).

The strain ATCC 25790 was originally included in the new M. aureum Tabamura prepared as Tabamura # 309 and prepared as a reference strain of Tabamura # 358 (ATCC 23366). Michio, Medicine and Biology, Vol. 72, pp. 270 to 273, 1966]. Subsequently, Mr. Tabamura established M. neo-arum Tabamura as a different strain of M. aurum, based on Tabamura # 3503 (ATCC 25795), and ATCC 25790 was a genus of M. aurum. It was said. [Tabamura Michio, Medicine and Biology, Vol. 85, pp. 229 to 233, 1972].

However, according to the recent literature of Saito, Tabamura et al. (Transfer), the ATCC 25790 bacterium is not M. aurum but is a member of M. neo aurum. [See also Saito, Tabamura et al., Page 77, FIG. 1 of the transfer document] Also, in the same literature, M. parapruitum Tabamura, M. arum Lumba, M. neoarum, M. Diernhoferi Bonicke & Juhasz and "Kanazawa Strains" are extremely close to each other and are indistinguishable at this stage. It is concluded that these five species are to be treated collectively as M. paraportuitum complex without being dispersed.

Tabamura et al. Have reached almost the same conclusions in other literature that M. aurum and M. bioaurum are treated as members of the M. paraportuitum complex or subspecies of M. paraportuitum. (See Journal of General Microbiology, Vol. 98, pp. 511 517, (1977, United Kingdom)).

For the comparison of the general properties of the M. paraportumtum complex and their relatives, Saito Hajime (TB, 50, 402, Japan, 1975), Saito et al. (75, 80, 81, 1977, Japan) and See Taramura et al. (Ex. 515, Japan, 1977).

As described above, the taxonomic position of the parent strain ATCC 25790 (Tsukamura # 309) is appropriate to belong to the M. paraportuitum complex according to the conclusion of the chief authority in this field.

이며 신속 발육성, 암발색성 항산균(rapidly growing scotochromogenic mycobacteria) 혹은 Runyon의 IV군 항산균에 속한다.M. paraportuitum complex is a Gram-positive, acid-free, non-motor, nonspore-forming bacterium

It belongs to the rapidly growing, rapidly growing scotochromogenic mycobacteria or Runyon group IV.

However, compared to the parent strain, the mutant strain MCI-0917, which was dispersed in the parent strain ATCC 25790, had no difference in germs, except that the former formed a rough colony on the agar medium. As noted by the strain, it belongs to the M. paraportuitum complex, and therefore, the mutant strain MCI-0617 belongs to the M. paraportuitum complex. Thus, although not surprisingly, these mutant strains were introduced as U.S. Pat.Nos. 3,759,791 and 3,684,657 as 4AD and ADD producing mutants, M. sp. NRRL B-383 and NRRL B-3805. Bacillus [This strain is a new bacterium other than M. baccae (identified as vaccae) in Japanese Patent Publications Nos. 52-105 and 289. For a comparison of these taxa, the Bergi Handbook 8th Edition [ "Bergey's Manual of Determinative Bacteriology ed. 8" pages 695-696, William & Wilkins Co., 1974, USA], Saito Hajime (Ex., Japan, p. 402, 1975), Saito et al. See, e.g., pp. 80-81, Japan in 1977) and Tabamura et al. (Ex., P. 515, 1977).

Also, in addition to the taxonomic properties of MCI-0617 bacteria and NRRL B-3805 bacteria and B-3683 bacteria, the degradability of ADD, 4AD, 92-OH-4AD differs, and the former grows slightly as a substrate, but the latter does not grow. There is a difference.

The sterols used as the substrate in the present invention are collectively referred to as sterols by combining various sterols, their C-3 esters, ether derivatives or their oxidized intermediates. The various sterols are those having a hydroxyl group at C-3 of the perhydroxy clopentanophenanthrene nucleus, usually having a double bond at C-5, and a chain side chain having 8 to 10 carbon atoms at C-17. Therefore, you may have a double bond in C-7, C-8, C-9 (11), etc.

Examples of such various sterols include cholesterol, stiggrasterol, canfestosterol, cytosterol, ergosterol, brassica sterol, fucosterol, lanosterol, agosterol, dihydrolanosterol, and dihydroagnosterol. Preferred sterols are cholesterol, canfestosterol and cytosterol.

Moreover, it is used as a raw material of the method of this invention as C-3 ester derivative of 3 beta hydroxyl group of various sterols, and organic acids, such as inorganic acids, such as a sulfuric acid, or organic acids.

Examples of such c-3 ester derivatives include cholesteryl oleate, cholesteryl palmitate, and cholesteryl sulfate. Moreover, it is used as a raw material of the method of this invention as c-3 ether derivative obtained, for example by the method of adding alkylene oxide to the 3 (beta) hydroxyl group of various sterols.

Examples of such c-3 ether derivatives include polyoxyethylene cholesteryl ether and the like.

C-3 ether derivative obtained by reacting ethylene oxide with depressive alcohol and depressive alcohol containing wool obtained from the above-described c-3 ester derivatives of various sterols, lanolin and cholesterol obtained from hydrolysis of lanolin Of course, polyoxyethylene lanolin alcohol ether is also used as a raw material of the method of the present invention.

Alkali-washing oils from fish oil or squid oil, as well as natural products and processed products containing sterols such as deodorized scum, deodorized sludge and earth oil of vegetable oil are also used as raw materials of the method of the present invention.

In addition, oxidation intermediates of various sterols or c-3 esters or ester derivatives thereof are also used as the substrate of the method of the present invention. Examples of such oxidizing intermediates include various sterols, their c-3 esters, and 4-ene-3 silver or 1,4-diene-3-one derivatives of c-3 ether derivatives. For example, it is Cholesteron 4-en-3on, Cholester-1,4-diene-3-on, Cholester-4.22-diene-3-on, etc.

Glycerides added in the medium in the present invention include mono, di and triglycerides. It is also possible to add a single glyceride having the same fatty acid group to which it is bound and a mixed glyceride containing two or three fatty acid groups. Fatty acid groups include unsaturated fatty acid groups and saturated fatty acid groups. It is preferable that carbon number of the fatty acid group of glyceride is 26 or less normally from a hydrophilic viewpoint.

As such a glyceride, Monoglycerides, such as (alpha)-monoacetin, (beta) -monoacerine, (alpha)-monopalmitin, (alpha)-monostearine, (beta) -monostearin, (alpha)-monoolein, (beta)-monoolein; α, α'-diacetin, α, β-diacetin, α, α'-thipalmitin, α, β-dipalmitin, α, α-distearin, α, β-distearin, α, α'-diol And triglycerides such as diglyceride triacetin, trilaurin, trimyristin, tripalmitin, tristearin, and triolein such as lanes and α, β-diolein.

Said mono, di and triglycerides are all single glycerides, and other 1-acet-2,3-dipalmitin, 1-palmito-2,3-dicaprine, 1-lauro-2-myristo-3-palmitin And mixed glycerides such as 2-oleo-1,3-palmitine and 2-stearo-1,3-diolein can be used in the same manner.

In the method of the present invention, a fat or oil containing glycerides may be used instead of the glycerides themselves. Oils and fats include vegetable and animal fats and oils. Examples of plant fats and oils include linseed oil, eno oil, kerosene oil, sesame oil, corn oil, rapeseed oil, cottonseed oil, saplawa oil, soybean oil, soybean lecithin, camellia oil, rice bran oil, olive oil, castor oil, peanut oil, palm oil, green onion Rock oil, rock-nuclear oil, etc. are mentioned. Examples of animal fats and oils include fish oil, diesel oil, tallow, lard, parchment, parsley oil, kanji, and taro oil.

The natural fats and oils mentioned above do not necessarily have to be sufficiently purified, but it is preferable to remove substances which adversely affect the microbial oxidation of steroids in advance.

Preferred plant fats and oils include edible oils such as olive oil, soybean oil, soybean lecithin, cottonseed oil, corn oil, sesame oil, rapeseed oil, peanut oil, camellia oil, rock oil and palm oil. Particularly preferred are soybean oil, rapeseed oil, rock oil and the like whose feed is stable. Preferred animal fats and oils include lard, taro oil, and the like.

The glyceride-containing material may be used alone, or two or more homogeneous or heterogeneous mixtures may be used.

If the amount of glyceride-containing materials is low, the yield of HP is minimal. On the other hand, excessively increasing the amount of the glyceride-containing substance is inhibited, and the glyceride-containing substance becomes bulky, which is not preferable. Thus, the glyceride containing material is added to the medium such that the concentration of glycerides in the medium is usually about 0.1 to 10% by weight, preferably inverse 0.2 to 7% by weight, more preferably inverse 0.3 to 4% by weight relative to the medium weight.

Glyceride may be a seed oil or fruit oil so that its glyceride concentration may be contained within the above range.

Examples of such oilseed fruit are flax seed, rapeseed soybean, rapeseed, cottonseed, sesame, peanut, cherry, corn, rice bran oil and the like.

In the method of the present invention, the addition of vegetable oil to the glyceride-containing medium further increases the yield of HP.

Such vegetable oil and fat can use the dregs which extracted the fats and oils from various oilseed seeds or fruit trees. Specifically, linseed gourd, skim soybean meal, eno oil gourd, colza oil gourd, cottonseed oil gourd, sesame oil gourd, peanut, saflower oil gourd, copper gourd, skim corn moisture, camellia gourd, skim bran, olive oil gourd, palm oil gourd, Pale palm oil etc. can be mentioned.

In addition, the vegetable fat and oil foil is preferably pulverized sufficiently so as to be susceptible to magnetization by microorganisms.

Plant fats and oils may be used alone, or may be used by mixing two or more kinds.

And when there is little use of vegetable fats and oils, yield increase is small. On the other hand, when the usage-amount of vegetable fats and oils increases, the viscosity of a fermentation broth rises and it becomes difficult to stir.

Thus, the vegetable fats and oils are added to the medium so that the concentration is usually inverse 0.5 to 20% by weight, preferably about 1 to 15% by weight, more preferably about 1.5 to 10% by weight, based on the weight of the medium.

In the present invention, microorganisms require the addition of inhibitors to the production of HP.

As an inhibitor, a chelating agent; Salts of nickel, cadmium or cobalt; Or sodium selenium, methylene blue, naphthalin, α-naphthol, β-naphthol, phenantholin, anthracene and the like.

Representative examples of chelates include 1,10-phenantororine, 2,2-bipyridinyl, 8-hydroxyquinoline, juperon, isonicotinic acid, hydrazide, orofenylenediamine, N, N-diethyldithio Sodium cartbate.

In the medium used in the method of the present invention, a carbon source, a nitrogen source, and an inorganic substance are appropriately added in addition to the glycerides, fruit trees, seed seeds, and vegetable fats and oils.

Such carbon sources include, for example, hydrocarbons; Alcohols such as methanol and ethanol; Organic acids such as succinic acid and acetic acid and salts thereof; Sucrose such as starch, maltose, sucrose, glucose, and ramnose can be mentioned. Natural nutrients, including carbon sources, nitrogen sources and other nutritional substances, include molasses, including high test molasses, refined molasses and xylose molasses; Bacchus, Coon Cove, arp Alpa, Coons Photoquare, distiarasorble, liquid, fishmeal, bran, meat extract, yeast, yeast extract, potato extract, malt extract, Gurten, popton, glutamine, Asparaglycine, casein, caseinate, skim milk, etc. are mentioned.

Examples of the inorganic substance include nitrogen sources such as yuan and hydrochloric acid, potassium such as dipotassium hydrogen phosphate and phosphorus; Salts such as iron, copper, magnesium, manganese, cobalt, zinc and calcium; Natural products such as molasses may be mentioned. Vitamins can also be added as needed.

The composition of the medium is selected according to the type of fungal species used, and carbon source, nitrogen source, potassium, phosphorus and magnesium are indispensable as the medium component.

What is necessary is just to add a well-known antifoamer. Specifically, polyoxyalkyrenglycol and the like can be mentioned, but it is not necessary to add an antifoaming agent.

Surfactant is effective as an emulsifier of sterols, and it is preferable to add in surfactant. As surfactant, a nonionic and anionic thing is preferable. Specifically, polyoxyethylene sorbitan monostearate, sorbitan monopalmitate, polyethylene glycol monostearate, etc. are mentioned, for example.

The incubation temperature is usually 20 to 40 ° C., but the optimum is near 30 to 35 ° C.

The pH of the medium is usually adjusted to 5 to 10, with 6 to 9 being preferred. Since the microorganisms used in the present invention belong to M. genus, it can withstand a pH of around 10 as is well known.

Sterols which are raw materials of the method of the present invention are generally sterilized together with the medium, but may be added to the medium after the start of the culture. In addition, divided addition is also possible. In this case, it is added as it is after sterilization or wet heat sterilization, or dissolved in dimethylformamide or the like as a solution, or finely dispersed and suspended by sonication to be added as a suspension. The addition of a surfactant at the same time is preferable because emulsification of substrate sterols and the like is promoted.

The glyceride-containing material added according to the method of the present invention is usually added at the start of culturing since it is effective at all stages of the mythology of sterols and also promotes the growth of bacteria. However, the glyceride-containing substance is considered to be effective for the oxidation of the side chain at the 17 position of sterols, and therefore, the amount of HP production is greatly increased even when the raw material sterols are added or participated during the culture.

In addition, since glycerides and the like also act as antifoaming agents as described above, it is also preferable to add glycerides in the above-described concentration range by dividing during the culture.

The incubation time is not particularly limited. Usually, the amount of HP production increases from the third day after addition of the raw material sterols, but culturing for 20 days or more is rarely of industrial significance.

After the end of the culture, HP accumulated in the culture solution can be collected and separated and purified by a known method. For example, crude HP is obtained by extracting HP from the culture solution with an organic solvent which is difficult to be mixed with water such as several times the amount of ethyl acetate and distilling the solvent from the extract solution. If it is necessary to separate HPD, 4HP, substrate sterols or by-products, porous resin, silica gel, alumina, etc. are used as adsorbent and petroleum ether, benzene chloroform, ether, acetone, methanol, ethyl acetate, etc. are used as eluent. The product can be separated by column chromatography.

According to the method of the present invention, the amount of HP produced can be improved by 3 to 10 times as compared with the case of using a glyceride-free medium, and industrial production of HP can be advantageously performed.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples unless they exceed the gist of the present invention.

In the following examples, the qualitative and quantitative determination of sterols and derivatives thereof, PH, remaining sterols and by-product steroids was carried out by gas chromatography.

In the following examples,% is based on weight.

Example 1

100 ml of medium (pH7.2) consisting of 1.0% glucose, 1.0% hex extract, 1.0% peptone, and water were placed in a 500 ml shoulder flask and steam sterilized at 120 ° C. for 15 minutes, followed by M. paraportitum complex MCI. 0617 bacteria are inoculated with platinum and incubated at 30 ° C. for 120 hours at 120 round trips / minute and a round trip shaking condition of 7 cm in amplitude. 2 ml of this culture broth is 50% of skimmed soy flour, 1.0% of cottonseed oil, 0.2% of K ₂ HPO _4, 0.2% of NaOH, 0.2% of MgSO ₄ · 7H ₂ O, 0.8% of cholesterol, and 50 ml of this culture medium (pH 7.0, 10 ml 500 ml shoulder flasks were inoculated.

The cultivation was carried out at reciprocating shaking conditions of 120 reciprocation / minute and amplitude of 7 cm at 30 ° C., the culture was stopped 150 hours after the start of the culture, the cultures were combined, and extracted twice with 2 L of ethyl acetate. The extracts were combined and insoluble materials such as fungicides were filtered, and then the amount of HP was analyzed by gas chromatography.

The results are shown in Table 1.

Further, the extract was purified by silica gel chromatography, and eluted with 20% ethyl-n-hexane as a solvent. The HPD and 4HP were separated from each other and separated from the substrate cholesterol and by-products. HPD, 4HP was purified by recrystallization from 10% ethazole heptane of each fraction of eluted HPD and 4HP.

Instead of cholesterol, the above-mentioned incubation was carried out using a 2: 1 mixture of β-sitosterol and canfesterol, cholester-4-en-3one, cholester-1, 4-diene-3-one, or cholesterolate. It was repeated. The results are shown in Table 1.

In addition, the results without adding cottonseed oil for comparison are also presented.

Cottonseed oil used here is a mixture of almost 100% glycerides. The same holds true for the fats and oils used in the examples below.

TABLE 1

Example 2

In Example 1, Example 1 was repeated except that various glycerides and fats and oils were added to the culture medium instead of cottonseed oil. The results are shown in Table 2.

TABLE 2

Example 3

In Example 1, the stop of Example 1 was repeated using tristealine of various concentration instead of 1% cottonseed oil. The results are shown in Table 3.

TABLE 3

Example 4

In the same manner as in Example 1, the seed culture of Example 1 was repeated using M. sp. NRRL B-3693 and NRRL B-3805 for microorganisms. 2 ml of the cultivation contains 500 ml of this culture medium (pH7.0, 120 ° C., sterilized for 20 minutes) consisting of 8.0% skim cotton wool, 0.15% K ₂ HPO, 0.10% lean, 0.8% sardine oil, 1.0% cholesterol and water. Inoculate into a 5 L shoulder flask.

This culture was carried out at 30 ° C. under a reciprocating shaking condition of 100 round trips / minute and an amplitude of 7 cm. The culture was stopped 200 hours after the start of the culture, and the culture solution was extracted twice with 2 L of ethyl acetate. The combined extracts were filtered through insoluble materials such as cells, and the amount of double Hp was analyzed by gas chromatography. The results are shown in Table 4.

In addition, the results in the absence of sardine oil are also included.

TABLE 4

Example 5

The strains were cultured in the same manner as in Example 1 using M. duvalley NCTC 8645 bacteria and M. baccarat ATCC 25954 bacteria. 2 ml of this culture medium was prepared by using 6.0% degreasing soybean meal, 1.0% of yeast extract Difco, 0.15% K ₂ HPO₄, 1.0 cholesterol, 0.7% soybean oil, and water (pH 7.0, 120 ° C, 20%). Sterilize for 10 minutes) inoculate 10 500 ml shoulder colbene containing 50 ml. This is incubated at reciprocating shaking conditions of 120 reciprocation / min, 7 cm amplitude. 48 hours after the start of the culture, 3 mg of the chelating agent 2.2'-dipyridyl is aseptically added to each colben. After 140 hours of addition of the chelating agent, the incubation was stopped and all the cultures were combined for each strain and extracted with 1 L of ethyl acetate. Table 5 shows the results of analyzing the amount of HP in the extract by gas chromatography.

In addition, the results in the medium containing no soybean oil are shown in Table 5.

TABLE 5

Example 6

In Example 1, as a culture medium, 2% glycose, (NH ₄ ) SO 0.2%, K ₂ HPO ₄ 0.15%, MgSO ₂ 7H ₂ O 0.1%, rock oil 1.0%, yeast 1.0%, various Example 1 was repeated using a medium consisting of 4% plant fat and water. The results are shown in Table 6.

TABLE 6

Example 7

As a culture medium in Example 1, 2.0% of fish paste, 0.2% of K ₂ HPO, 0.2% of NaNO ₃ , 0.1% of MgSO ₄ · 7H ₂ O, 0.8% of cholesterol, circumferential soybean grits (glyceride content of about 17%) 60 Example 1 is repeated except using the medium consisting of% and water.

In addition, the same as using crushed ground grinding (glyceride content of about 35%) 3.0% or flat ground (glyceride content of about 40%) 2.5% or homa grinding (glyceride content of about 40%) in place of the soybean grinding The results of the cultures are also shown in Table 7.

TABLE 7

Claims (1)

Microorganisms belonging to Mycobacterium were cultured in a medium based on sterols, and 20-hydroxymethylpregner-1,4-diene-3-one or 20-hydroxydimeprene-4-ene-3 A method for preparing steroid alcohols, characterized in that in the preparation of ions, the medium contains at least 0.1% by weight of glycerides.