BG62168B1 - Strain aspergillus terreus, producent of hypocholesterolimic product - Google Patents

Abstract

The strain is used in the pharmaceutical industry for thepreparation of hypocholesterolimic product of the group of Alactones used in medicine as an agent for the adjustment of thecholesterin content in blood. Strain Aspergillus terreus isproduced by mutagenous treatment of the initial Aspergillusterreus, NBIMCC No. 3319, by treatment with UV light, selection ofspecific variant of the strain after resowing, natural selectionand subsequent treatment withN-methyl-N'-nitro-N-nitrosoguanadine. Mutant having considerablyincreased productive potentials of the desiredhypocholesterollimic product is produced.

Description

The invention relates to a strain of Aspergillus terreus, a producer of a hypocholesterolemic product, which is used in the pharmaceutical industry.

BACKGROUND OF THE INVENTION

Hypocholesterolemic products from group A Lactones are known to be used in human medicine as a means of regulating blood cholesterol. Their effect is achieved by competitive inhibition of 3-hydroxy3-methylglutaryl coenzyme A-reductase, a key enzyme in cholesterol biosynthesis. Such are the products lovastatin, dihydrolovastatin and the like.

Strains producing hypocholesterolemic products of the genus Aspergillus terreus, Penicillium citrinum, Monascus ruber / Proc are known. Natl. Acad. Sci USA, 1980, (77) 7, 39573961; US 4 231 938; US 4 376 863; J. Am. Chem. Soc., 1985 (107), 12, 3694-3701 /.

A known producer of Aspergillus terreus NBPMKK N3319 is known to produce the hypocholesterolemic products lovastatin and dihydrolovastatin in deep aerobic culture (US 4 376 863).

A disadvantage of the known strains is that, upon cultivation, the desired hypocholesterolemic product lovastatin is obtained as a minor component in the fermentation medium, rendering them ineffective for industrial use.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an Aspergillus terreus strain with increased production of the hypocholesterolemic product lovastatin in the culture fluid.

The task is solved by creating a strain of Aspergillus terreus, producing a hypocholesterolemic product registered in NBPMCK N3318, with selection of a source strain Aspergillus terreus NBPMKK N3319 by mutagenic treatment with ultraviolet light at 60 nm length-4 nm nm-80 certain strain variant after natural sieving and subsequent treatment with N-methyl-N'-nitro-N-nitrosoguanidine from 250 to 500 | Ag / l for 45 to 60 min.

The following nutrient media were used to characterize the parent parent strain Aspergillus terreus NBPMC N3319 and the Aspergillus terreus mutant NBPMCC N3318 derived from it:

1. Malt agar, composition by weight: Malt extract 3.0, soy peptone 0.3, agar 1.5 at pH 5.6 ± 0.1.

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

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

4. Combined agar with a composition by weight of: 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 at pH 4.8 ± 0.2.

5. Malt-glucose-peptone agar (MGP) with a composition by weight: malt extract 2.0, glucose 2.0, peptone 0.1, agar 1.5 at pH 5.6 ± 0.1.

The morphological and cultural characteristics of the two strains made on day 14 of their development are shown in Table 1:

Table 1

Comparative morphological characteristics of Aspergillus terreus strains

NBPCC No. N 3319 and 3318

Nutrient medium Parent strain A. terreus 3319, NBPCC Mutant strain A. terreus 3318, NBPCC Malt agar colonies 5 to 30 mm in size, fluffy aerial mycelium, not swollen with abundant sporulation; color of the aerial mycelium yellow sulfur in3 colonies 5 to 20 mm in size, fluffy aerial mycelium, not swollen with abundant sporulation; color of the aerial mycelium yellow k1 to pale sand k3 Malt-corn agar colonies over 40 mm in size, uncluttered, loose airy mycelium and abundant sporulation; color of aerial mycelium light shamoua d5 colonies 20-40 mm in size, uncluttered, fluffy air mycelium, normal sporulation; color of aerial micelles sand l7 Saburo-maltose extract colonies 30-40 mm in size, unobstructed, abundant sporulation; color of aerial mycelium pale sandy k3 to reddish brown h5 colonies 20-25 mm in size, uncluttered, fluffy air mycelium, abundant sporulation; aerial color of mycelium dark sandy blob to dark smoker m1 homogeneous population Combined agar colonies 10-40 mm in size, radially unclothed with fluffy aerial mycelium and abundant sporulation; color of aerial mycelium paleochra 65 + dZ colonies 7-30 mm in size, radially furrowed with fluffy mycelium and normal sporulation; color of air? micelles sulfur yellow i1 to amber yellow ~ k1 Malt-glucose peptone agar colonies 25-35 mm in size, radially ridged, abundant sporulation; color of air mycelium 3O ocher to sand l7 colonies 18-20 mm in size, flat uncut, abundant color air color sporulation? micelles dark sandy to garnet k5 high homogeneity

The color of aerial mycelium is determined by the color scale of Bondartsev / Bondartsev, AS, Color scale, Ed. at the Academy of Sciences of the USSR, 1954 /.

Data on absorption of different carbohydrates by the parent strain Asp. terreus NBPMCK N3319 and the mutant strain Asp. terreus NBPCC N3318 are listed in Table 2:

Table 2

Comparative characteristics of Asp strains. terreus NBPCC No. N 3319 and 3318

Substrate Parent strain A. terreus N 3319 Mutant strain A. terreus N 3318 1 2 3 Glucose +++ -H- + Fructose -n- + Maltose +++ +++ Sucrose ++ ++ Galactose ++ + Glycerol + ++ Starch ++ ++ Dextrin ++ + Xylose +++ +++ Manit ++ + Control + +

The markings in the table: "+" - positive growth; "±" - likely growth

The sugars used for comparative characterization are prepared as 10% solutions and fed sterile at 60 ° C to a control containing salts and a single carbon source at a concentration of 1%, with the following composition in g / Ι: ammonium sulphate 2.64, monocotassium phosphate anhydrous 2.38, dicalium phosphate trihydrate 5.65, magnesium sulphate 1, agar 15, brine 1 ml / l at pH 6.8 - 7.0. The saline solution used to prepare the control contained in g: CuSO ₄ 0.63, FeSO ₄ 0.11. MnCl ₂ 0.79, ZnSO ₄ 0.15 and up to 100 ml distilled water.

Stabilization of the resulting mutant strain and retention of its beneficial properties is achieved by sequential sieving of a natural selection to homogenize the population. After the fifth inoculation on malt-glucose-peptone agar, high homogeneity of the colonies was achieved, both in terms of physico-morphological characteristics and productivity.

The strain was stored on slanted agar at a temperature of + 4 ° C for no more than 6 months 45 or by lyophilization on skim milk or other protective medium.

The fermentation is carried out in two stages using the following nutrient media: inoculation medium with a composition of 50% by weight: corn extract 2, corn flour 0.1, glucose 0.1 and trace element solution 10 ml, pH 5.6; fermentation medium with the composition by weight: glucose 2.5, lactose 2.5, corn extract 2.5, soybean flour 1.5, mono-potassium phosphate 0.1, ammonium sulfate 0.1, magnesium sulfate 0.1, calcium carbonate 0.6 with pH 7.0. Cultivation was performed at 26-28 ° C under aerobic conditions for 140 to 168 hours.

The trace element solution in the composition of the inoculation medium contains in mg: FeSO ₄ .7H ₂ O 1000, MnSO ₄ .4H ₂ O 1000, CuCl ₂ .2H ₂ O 25, CaCl ₂ 100, H ₃ BO ₃ 56, (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O 19, ZnSO ₄ .7H ₂ O 200 and distilled water to 1 l.

Description of the attached figure

The production of hypocholesterolemic product in the culture fluid of the parent and mutant strain is presented relatively graphically in FIG. 1, where curve 1 depicts the differential rate of accumulation of hypocholesterolemic product in the culture fluid in the process of parental fermentation, and curve 2 of the resulting strain - mutant.

The advantages of the strain according to the invention are reflected in its greater productive capabilities of the hypocholesterolemic product lovastatin.

The invention is illustrated by the following exemplary embodiments:

Example 1. A strain of Aspergillus terreus NBPMCK N3319 was developed on a test tube with a MGP agar medium containing in g: difko malt malt 20.0, glucose 20.0, peptone 1.0, agar 20.0 and distilled water to 1 1, pH 5.6 ± 0.1, for 10-14 days. The spores are flushed with 10 ml of saline and 9.5 ml of the suspension is irradiated with ultraviolet light at a wavelength of 254 nm for 60-180 s with continuous stirring. Control seedlings were prepared with 0.5 ml of the stock suspension. Serial dilutions are made of the irradiated suspension, from which the plates are sown with MGP agar. Cultivation was performed at 26-28 ° C for 10-14 days. The surviving colonies were isolated on MGP agar tubes, and controls and productivity were checked by deep culture in two-step fermentation. At the end of the fermentation process, the accumulation of hypocholesterolemic product in the culture fluid is determined by HPLC method.

A natural selection is sown and a specific strain is isolated. The culture thus determined was washed with a test tube agar with 10 ml of distilled water. 0.5 ml of the suspension are inoculated with several 300 ml Erlenmeyer flasks containing 50 ml of MY medium with the following composition in%: malt extract 2, yeast extract 0,4, agar 0,1, pH adjusted with NaOH to 5 , 5 ± 0.1. The flasks were cultured for 24-36 h at 26-28 ° C on a circular shaker of 200-250 rpm. and 5 cm deviation, after which 1 ml of the most homogeneously developed flask was transferred to several 300 ml Erlenmiller flasks with the same nutrient medium and re-cultured to a mean growth phase.

The contents of one flask were sterile centrifuged, washed with saline twice, then resuspended in 50 ml MY medium and cultured for 4 h at 32 ° C on a 220-250 rpm circular shaker. and 5 cm deviation. The pH was then adjusted with NaOH to 8.5 and 250 to 500 mg / l of N-Memn-N'-HHTpo-N-HHTposoguanidine added. After 45-60 min, 1 ml was taken to count the colony forming units. The remainder was centrifuged and washed with MY medium. It was suspended in 50 ml of MY medium and cultured overnight at 26-28 ° C on a shaker. Serial dilutions were prepared from this culture, which were scattered on petri dishes with MY medium containing 2% agar.

The germinated colonies of the control and treated populations were isolated on the lean MGP agar, cultured and checked enzymatically to form a hypocholesterolemic product.

Example 2. For the fermentation check of colonies isolated from a naturally aspirated sample and a 500 ml Erlenmeyer flask containing 100 ml of the medium with a composition by weight: malt extract 0.2, yeast extract 0.4, arap “Difko 0.05, pH 5.6 sowed a rhetoric ear spores from a well sporulated culture of Aspergillus terreus strain. Cultivation was performed at 26-28 ° C for 24-26 h on a 200-250 rpm circular shaker. min. 510% of the well-developed seed is inoculated with a one-liter Erlenmeyer flask containing 200 ml of the same culture medium, which is cultivated under the same conditions. Well-developed seeds must have a pH in the range of 5.5 - 5.7, biomass 20-25%, age 24-36 h, microscopic hyphae with good basophilia and be sterile.

Transfer 5-10 vol% of the seed so prepared into a 500 ml Erlenmeyer flask onto a 100 ml fermentation medium containing the following ingredients by weight: glucose 4.0, maltose 2.0, monocotassium phosphate 0.2, dicalium phosphate 0.7, sodium citrate 0.05, ammonium sulfate 0.1, magnesium sulfate 0.1, casein peptone 1.0 with pH 5.5 ± 0.1. The fermentation process was carried out at a temperature of 26-28 ° C for 140 to 168 hours on a circular shaker of 200-250 rpm. and 5 cm deviation.

At the end of the fermentation process, the accumulation of hypocholesterolemic product in the culture fluid in acid form was determined by HPLC method of ethyl acetate extracts from the cells. The analysis was performed on a standard RP 18 chromatographic column, equilibrated with methanol and 0.1% phosphoric acid solution at a ratio of 70:30, flow rate 1.5 ml / min, wavelength 238 nm, against the USP standard also in acid form. The retention time of the acid form of the hypocholesterolemic product lovastatin is about 20 minutes.

As a result of the enzymatic verification and analysis of the colony's individual productivity, highly productive options are selected for the next stages of selection or for working under production conditions.

Example 3. A well sporulated culture of Aspergillus terreus strain NBPMCK N 3318 on a sown arap MGP was sown in a 500 ml Erlenmeyer flask, per 100 ml of culture medium with composition by weight%: corn extract 2, glucose 1.5, corn flour 1 and mineral salts solution 10 ml, pH 6.8, sterilized for 30 min at 120 ° C. 5-10% of the well-developed seed is inoculated into a one-liter Erlenmeyer flask containing 200 ml of the same culture medium cultivated under the same conditions. 200-400 ml of the seed so prepared was inoculated into a 50 l fermenter containing 35 l of the culture medium by weight. %: glucose 2.5, lactose 2.5, corn extract 2.5, soybean flour 1.5, monocalcium phosphate 0.1, ammonium sulfate 0.1, magnesium sulfate 0.1, and calcium carbonate 0.6 with pH 7 , 0. The cultivation is carried out at a temperature of 26-28 ° C, continuous stirring with a stirrer at 200 rpm, aeration with air in a ratio of fermentation medium 0.3: 1 to 24 h, and until the end of the fermentation process 0.6: 1 and a pressure of 0.5 atm for 140 to 160 h. Culture fluid activity 1300 MME * (1 MME - 1 g / ml) by HPLC method.

Claims (1)

Claims 1. A strain of Aspergillus terreus, registered in NBPMCC under No. 3318, producing a hypocholesterolemic product.