BG108752A - Fermentation method for compactine production - Google Patents

Abstract

Compactine is an antihypercholesterol and antiatherosclerotic agent of the group of stearines and is used as an intermediate product for the preparation of pravastatin. A mutant strain is used, by means of which the product titre is increased in the cultural medium and relatively small byproducts are accumulated in the fermentation process. By the method for its preparation, the strain-producer Penicillium citrinum, registered in NBIMCC under No. 8359 is cultivated in depth in the nutritive medium containing, in wt.%: glucose from 3 to 15; peptone from 1 to 1.5; Soya bean oil cake flour from 3 to 5; sodium nitrate from 0.1 to 0.7; magnesium sulphate from 0.05 to 0.5; glycerol from 4 to 12 and Soya bean oil from 1 to 3. The fermentation process occurs in aerobic conditions at temperatures ranging from 22 to 28 degrees C, pressure from 0.4-0.6 bar and pH from 5.3 to 8.0.

Description

Fermentation Method for Preparation of Compact

Technique

The invention relates to a fermentation method for the preparation of compacts, which is used in the pharmaceutical industry.

BACKGROUND OF THE INVENTION

Compactin was isolated for the first time from a culture fluid of Penicillium brevicompactum and reported by Brown et al. in 1975 it was an anti-fungal metabolite (Brown A.G. et al., 1976, Cristal and molecular structure of Compactin, a new antifungal metabolite from P. brevicompactum, J. C. S. Perkin I, 1165-1170).

Later, Endo et al. find that compact is also produced by Penicillium citrinum. They have also identified its hypocholesterol activity, attributing it to the statin group - substances that inhibit 3-hydroxy-3-methylglutaryl coenzyme A-reductase - a key enzyme in cholesterol biosynthesis (Endo MK, Y.Tsjita, 1976, J. Antibiot., 29, 13461348; Endo MK, Y.Tsjita, 1976, FEBS Lett., 72, 323-326).

A fermentation method is known for the preparation of compacts by strain Penicillium citrinum. Hosobuchi et al. describe the conditions of cultivation of a strain on a fermentation medium containing a large amount of carbon source (glycerol), suitable nitrogen sources, inorganic salts, and glycerol feeding and maltose during fermentation. Fermentation takes place over 12 days under aerobic conditions and at + 24 ° C. (Hosobuchi M., et al., Biosci.Biotech.Biochem., 57 (9), 1414-1419, 1993)

There is a known fermentation method for the preparation of compacts by the producer strain Penicillium adametzioides, which is cultivated on a culture medium containing nitrogen and carbon sources and £ - · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ♦ ·? · · · · · · · · · · · · · · · · · · · · · · · Inorganic salts, at a temperature of 23 -: - 28 ⁰ C for 7-11 days with maximum yield 1 , 6 g / l. (US 5691173)

The disadvantage of this method is the low productivity of the strain used.

A fermentation method is known for the preparation of compacts from strain Gliocladium sp.YJ-9515, which is cultured on a culture medium containing carbon, nitrogen and inorganic salts sources. The fermentation process was carried out at a temperature of 10-36 ° C and a pH of 4.08.0 (US 6204032).

The disadvantage of this method is the low yield of the target product.

There is a known fermentation method for the preparation of compacts by the immobilization of spores from Penicillium cyclopium. The fermentation process is carried out in a bioreactor on a solid food matrix. (WO 0181611)

The disadvantage of this method is the need to create a suitable installation for its application.

A fermentation method for the preparation of compacts is known in which the strain Penicillium citrinum ATCC 38065 is sown on a culture medium containing 7% glucose, 3% glycerol, 1% pharmamedia, 1% corn extract and inorganic salts. The fermentation process is carried out at pH 4-9, temperature 20-35 ° C for 5 days. (1186323021)

The disadvantage of this method is the low yield of compact.

A fermentation process is known for the preparation of compacts at which a Penicillium citrinum SANK 18767 producer strain (NBPMKK 3426) is cultured under aerobic conditions on a culture medium containing malt extract, glucose, peptone and agar at pH 3-9 and temperature 20-30 ° C (US 3983140, US 4049495).

The disadvantage of this method is the low yield of the target product, which makes it ineffective for industrial applications.

SUMMARY OF THE INVENTION

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It is an object of the invention to create a strain micro-organism in which a high titer of compacts is accumulated on a suitably selected culture medium.

According to the method of the invention, for the preparation of the compact strain Penicillium citrinum registered in NBPMCC No. 8359, it is cultured in deep aerobic conditions on a culture medium containing absorbable sources of nitrogen, carbon and inorganic salts. The fermentation process was carried out at a temperature of 22-28 ° C, a pressure of 0.4-0.6 bar and a pH of 5.3 to 8.0 for 260-300 hours.

Penicillium citrinum strain NBPMCK 8359 was obtained from Penicillium citrinum strain NBPMKK 3426 when irradiated with UV light and treated with NTG (nitrosoguanidine). The resulting variant of the strain is stabilized by natural selection.

The following culture media were used to characterize the parent parent strain Penicillium citrinum NBPMCK 3426 and the improved strain obtained during selection:

.Saburo-dextrose agar, in percent by weight composition: glucose 4.0; peptone -1.0; agar-1.5; pH -5.6 ± 0.1.

2. Malt-corn agar, composition by weight: malt extract -2.0; corn extract -1.0; agar-1.5; pH -6.5 ± 0.2.

3. Saburo-maltose agar, composition by weight: meat peptone 0.5; casein-0.5; maltose- 2.0; agar-1.5; pH -5.6 ± 0.1.

4. Combined agar, composition by weight: malt extract 1.5; peptone -0.075; maltose-1.3; dextrin -0.3; glycerol -0.25; monocotassium phosphate -0.1; ammonium chloride -0.1; agar -1.5; pH -4.8 ± 0.2.

5. Malt agar, by weight composition: Malt extract 3.0; soy peptone-0.3; agar-1.5; pH -5.6 ± 0.1.

Morphology, cultural characteristics of the two strains were made at 14 ^'th day of development, and are presented in Table 1.

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Table 1. Comparative morphological characteristics of the strains

Penicillium citrinum NBPMCC 3426 and 8359

No Nutrient medium Parental strain R. citrinum NBPCC 3426 Improved strain of P. citrinum NBPCC 8359 2 3 4 1 Saburodextrose agar Large circular colonies 25-30 mm radially folded with apex in the middle; fluffy BMl4; CM-PB Round colonies 5-10 mm, strongly raised with wrinkled surface, reddish-brown pigment BM-14; CM- g1 2 Malt corn agar Large colonies 25-30 mm, without radial folds with apex in the middle; fluffy BMl4; CM-3. Round colonies 5-10 mm, raised slightly furrowed surface; reddish-brown pigment; VM-l7; CM -y1 3 Saburomaltose agar Large colonies 30-40 mm, without radial folds with apex in the middle; fluffy BM-a7; CM-d2. 5-10 mm circular colonies raised with a crater in the middle; light brown pigment; fluffy VM-o5; CM-MB. 4 Combined agar Large colonies 20-25 mm, radial creased without apex in the middle, fluffy VM and 5; CM- L. 3-5 mm round colonies, strongly raised with radial folds in the base; does not separate pigment in the middle; BM-r2; CM- k1. 5 Malt agar Large colonies 20-25 mm, radial curved with apex in the middle, fluffy BM 14; CM- LZ Flat flat colonies 3-8 mm with radial folds; pale yellow pigment; fluffy VM-i2; CM-MB.

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The color of the aerial mycelium was determined on the Bondartsev scale (Bondartsev AS, Scale of Flowers, USSR Published, 1954).

The absorption data for different carbohydrate sources from the two strains are presented in Table. 2. The assay was performed on a single carbohydrate source medium at a concentration of 1%, with a composition in percent by weight: ammonium sulfate - 0.026; mono-potassium phosphate - 0.024; dicalium phosphate - 0.56; magnesium sulfate - 0.1; saline solution * - 0.1; agar -1.5; pH 6.8-7.0.

Saline solution: copper sulphate - O.bZd; ferrous sulfate - 0.11g; manganese sulfate - 0.79g; zinc sulfate - 0.15g; distilled water to 100 ml. The sugars are added in the form of sterile solutions at about 60 ° C.

Table 2. Absorption of carbohydrate sources by the parent strain

Penicillium citrinum NBPMKK 3426 and Penicillium citrinum strain NBPMKK 8359

No Substrate Penicillium citrinum NBPCC 3426 Penicillium citrinum NBPCC 8359 1 Control + + 2 Glucose +++ ++ 3 Fructose +++ + 4 Sucrose ++ ++ 5 Maltose ++ ++ 6 Lactose ++ ++ 7 D-alactose ++ I ¹ 1 8 Raffinose ++ + 9 Xylose +++ ++

Markings in the table: "+" - positive growth; "++" - good growth; "+++" - very good growth.

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The strain is stored on a slanted agar at a temperature of up to + 4 ° C, while maintaining its properties for up to 2 months. For a longer period, the strain is stored as a lyophilized culture using skimmed milk.

Penicillium citrinum strain NBPMK 8359 was developed on a CSM agar slurry with a weight percentage composition: Difco 1.0 1.5 mg; soybean meal flour 1.0 - 1.5; sodium nitrate 0.2 to 0.4; magnesium sulfate heptahydrate 0.1-0.3; glucose, anhydrous 3 - 5; glycerol 5 - 7; agar 1.5 - 1.8. The pH was adjusted in the range 6.3-6.5 and the medium was sterilized at 121 ° C for 30 min. From the fresh culture, sterile about 2 cm ² of the tube slurry was transferred to 30 ml of the culture medium, with a weight percent composition: Difco baptopeptone. 1.0-1.5; soybean meal 2.0 - 2.5; sodium nitrate 0.2 to 0.4; magnesium sulfate heptahydrate 0.1 - 0.3; anhydrous glucose 5-10. The pH is adjusted to 6.2-6.4 and the medium is sterilized at 121 ° C for 30 minutes. The cultivation process is carried out at 24 ° C and stirring continuously for 48-72 hours.

From 7 to 12% by volume of the seed is added to the fermentation medium containing by weight: carbon sources 15-25%, organic nitrogen sources 3-5% and mineral salts 0.1-1%. The pH of the fermentation medium is maintained in the range 5.3-8.0. The vegetative phase can be two-stage. The fermentation process was carried out at a temperature of 22-28 ° C and a pressure of 0.4-0.6 bar for a period of 260-300 hours.

The following may be used as carbon sources in the fermentation medium: glucose, sucrose, glycerol and soybean oil. The following may be used as sources of organic nitrogen: peptone, proflo, soybean meal, dry yeast, pharmamedy Specific to the strain Penicillium citrinum NBPMCK 8359 is that glucose, peptone, soybean meal and soybean meal are better absorbed by food substrates. Sodium nitrate and magnesium sulfate are used as mineral salts in the composition of the culture medium.

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The advantage of the method is the high product tiger in the culture medium of the improved strain compared to the parent strain.

Explanation of the annexed figures

The compactin obtained by the method of the invention was quantified by HPLC method. The results of the analysis are shown as follows:

1. Figure 1 shows the results of HPLC analysis of compactin standard solution.

2. Figure 2 shows the results of an HPLC analysis of a culture fluid sample from the original strain.

3. Figure 3 shows the results of HPLC analysis of a compactin culture fluid sample prepared by the method described in Example 1.

Examples of carrying out the invention

The invention is illustrated by the following examples:

EXAMPLE 1

Culture of Penicillium citrinum strain NBPMCK 8359 was grown for 10-12 days at 24 ° C on a CSM agar tube with the following composition:

No Component The amount of g / l 1. Bactopeptone Difco 15 2. Soybean meal 15 3. Sodium nitrate 4 4. Magnesium sulfate, heptahydrate 3 5. Glucose, crystalline 45 6. Glycerol 65 7. Arab Difco 15 8. Distilled water K1 I

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Transfer from a well-developed tube about 2 cm ² of slant into a 300 ml Erlenmeyer flask containing 30 ml of seed medium of the following composition:

No Component Quantity, g / l 1. Bactopeptone Difco 15 2. Soybean meal 25 3. Sodium nitrate 2 4. Magnesium sulfate, heptahydrate 3 5. Glucose, crystalline 80 6. Distilled water K1 I

The pH of the seed medium was adjusted to 6.2 ± 0.2 and sterilized for 30 minutes at 121 ° C. The pH of the medium after sterilization is 6.0 ± 0.2. Glucose is sterilized separately and then added to each flask before sowing. It develops at 24 ° C in a circular cap for 48-72 hours.

up to 12% by volume of the seed is fed to a fermentation medium with the following composition:

No Component Quantity, g / l 1. Bactopeptone Difco 15 2. Soybean meal 35 3. Sodium nitrate 5 4. Magnesium sulfate heptahydrate 1 5. Glucose, crystalline 30 6. Sucrose, crystalline 100 7. Glycerol 100 8. Distilled water Up to 11

pH of the medium including Difco bactopeptone, soybean meal, sodium nitrate, magnesium sulfate heptahydrate, crystalline glucose,

Adjust to 6.4 ± 0.2, then pour 30 ml into 300 ml Erlenmeyer flasks, sterilize for 30 minutes at 121 ° C. The pH of the medium after sterilization is 6.2 ± 0.2. Sucrose and glycerol are sterilized separately and added to each flask before sowing. The fermentation was carried out at 24 ° C on a circular shaker with 250 rpm and 5 cm deviation for 260300 hours. After completion of the fermentation process, each flask is analyzed for qualitative and semi-quantitative content of compact by TLC method against the reference substance. The highest yield flasks were analyzed for quantitative content by compact HPLC method. Productivity - 5-7 d / 1.

EXAMPLE 2

A culture of Penicillium citrinum strain NBPMCK 8359 was grown for 10-12 days at 24 ° C on a slanted CSM agar tube with the composition indicated in Example 1.

Transfer from a well-developed tube about 2 cm ² of slurry into a 500 ml Erlenmeyer flask containing 100 ml of the seed medium with the composition specified in Example 1.

It develops at 24 ° C on a circular shaker for 48-72 hours.

ml of the developed crop was transferred to a 500 ml Erlenmeyer flask containing 100 ml of the same seed medium. Development continues under the same conditions for about 46-60 hours. The thus obtained seed (1D ^nd vegetative), and prepared by him under the same conditions NG ™ vegetative, their use for sowing 50 I sowing apparatus containing the following seed medium.

No Component Quantity, g / l 1. Bactopeptone 15 2. Soybean meal 25 3. Sodium nitrate 2 4. Magnesium sulphate, heptahydrate 3

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5. Glucose, crystalline 80 6 Silicone 2 7. Tap water Up to 11

Cultivation was carried out at 24 ° C, aeration 1: 1-1 / 1 / min, pressure 0.4-0.6 bar for 48-68 hours. The finished seed has a pH of at least 5.8 and a biomass of at least 18-20%.

up to 12 volume percent of the seed is fed to a 50 I working apparatus with the following fermentation medium:

N Component Quantity, g / l 1. Bactopeptone 15 2. Soybean meal 35 3. Sodium nitrate 5 4. Magnesium sulfate heptahydrate 1 5. Glucose, crystalline 110 6. Glycerol 120 7. Soybean oil 25 8. SAG silicone 1.8 9. Tap water Up to 11

The medium was sterilized under standard conditions, followed by the addition of the separately sterilized glucose solution; The pH after sterilization is 5.8 ± 0.2. Cultivation was carried out at 24 ° C, aeration 0.5: 1- l / l / min, pressure 0.4-0.6 bar for 260-300 hours. The following parameters are monitored during the fermentation process: pH, glucose level, foaming, dissolved oxygen and biomass accumulation. Compactin formation begins after 40 hours and is monitored by HPLC until the end of fermentation.

The product accumulated at the end of the process is in the amount of 12-14 g / l.

The quantitative content of the compact is determined by the following HPLC method:

To prepare a standard solution of octylamine salt of compact acid, 0.010 g of standard substance is dissolved in 25 ml of H2O. The results of the HPLC analysis of this solution are shown in Figure 1. A working standard of 73% activity was used.

The culture fluid resulting from the fermentation process was diluted 5 or 10 times (depending on the age of the culture) with phosphate buffer pH 12, stirred on a magnetic stirrer for 1 hour, then filtered through Millipor 0.45 μ. The resulting solution was chromatographed on a Lichrosorb (Merck) RP-8/5 µm / 125-4 column, mobile phase containing 235 ml of methyl cyanide, 365 ml of water, 0.45 ml of glacial acetic acid and 0.45 ml of triethylamide, flow rate 1.00 ml /. min, detection 238 nm and an injection volume of 20 μΙ. The results of the analysis of this sample by the HPLC method are shown in Figure 2 and Figure 3.

The product was proven qualitatively by the TLC method against the reference substance using Merck 5553 sorbent chromatography plates, mobile phase: dichloroethane: acetic acid = 7: 3, saturation of the chamber for about one hour, distance of travel 15 cm.

Application solutions:

A solution of 1: 5 ml of culture fluid was extracted with 25 ml of ethyl acetate for 30 minutes. Starting point "a" was applied from 15 to 20 μΙ depending on the expected activity.

Solution 2: dilute to μο / μΙ from a standardized aqueous compactinic acid solution. Starting point “b” is applied 20 μΙ of the standard solution.

Manifestation: after complete removal of solvents (2 min at 100 ° C). The plate was sprayed with a solution of bromophenol blue (dissolve 0.1 g of bromophenol blue with 20 ml of ethanol and bring to 100 ml with distilled water). Heat for 5 min at 100 ° C.

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Assessment: The observed base spot above the starting point "a" must be of Rf value, magnitude and intensity equal to that above the starting point "b". The Rf value is 0.66.

Claims (1)

Patent Claims A fermentation method for the preparation of a compact comprising culturing a Penicillium citrinum producer strain on a culture medium containing carbon, nitrogen and mineral salts, characterized in that Penicillium citrinum registered in the producer strain is used NBPCC under No. 8359 and cultivation is carried out on a culture medium containing by weight: glucose from 3 to 15; glycerol from 4 to 12; soybean oil from 1 to 3; peptone from 1 to 1.5; soybean meal from 3 to 5; sodium nitrate from 0.1 to 0.7; magnesium sulphate from 0.05 to 0.5, under aerobic conditions at a temperature of 22 © 28 ° C, a pressure of 0.4-0.6 bar and a pH of 5.3 to 8.0.