FI92938C - Process for forming crystals of amphotericin B in the fermentation medium - Google Patents

Description

92938

Method for forming amphotericin B crystals in fermentation broth

The present invention relates to a method for forming amphotericin-5 B crystals directly in a fermentation medium by culturing a Streptomyces nodosus strain in a fermentation medium to which no amphotericin B solvent is added in the isolation step until strong antifungal activity has developed in the medium. The process is variously adapted for use in high yield amphotericin B fermentation.

Hitherto, amphotericin B crystals have been prepared from fermentation broth by carefully performed recovery methods involving expensive filtration, solution extraction and crystallization steps.

According to the present invention, crystallization of amphotericin B is induced directly in a fermentation broth containing a water-soluble or water-insoluble fermentation medium, as described in U.S. Patent No. 2,908,611, which fermentation medium contains a nitrogen source, a carbon / energy source, and alternatively one or to control the process of several inorganic salts.

Also disclosed is a method of forming amphotericin B crystals directly in a fermentation broth, comprising providing a fermentation medium as described above, growing a Streptomyces nodosus strain in such a fermentation medium, and inducing amphotericin B crystallization and microbial cell autolysis. The crystallization and autolysis can be performed by heating the broth at a sufficient temperature, 70 ° C to 130 ° C, for a sufficient period of time, from 1 minute to 10 hours, and preferably about 10 to 45 minutes, to induce the formation of amphotericin B crystals.

Crystallization can also be initiated by seeding amphotericin B crystals in the fermentation broth during fermentation.

2,92938, preferably about 10 to about 40 hours after the start of fermentation.

Autolysis can also be performed using enzymes, such as lysozyme or another comparable enzyme, that has a lytic effect on the cell wall.

When using enzyme or seeding, less heat treatment may be necessary.

The fermentation medium used in the process contains 10 nitrogen sources in an amount ranging from about 0.1 to 20% and preferably from about 0.5 to 5% by weight of the medium. Examples of suitable nitrogen sources include casein hydrolyzate, cottonseed or derivatives thereof, corn steep liquor, soybean meal, or any comparable organic or inorganic N-sources, or soluble derivatives thereof.

The carbohydrate source is present in the fermentation medium in an amount ranging from about 0.5% to about 20%, and preferably from about 0.5% to about 10% by weight. Examples of suitable carbohydrate sources include starch, dextrin, sugars such as maltose, lactose and glucose, glycerol and the like.

The fermentation broth used in the process of the invention may alternatively contain other conventional fermentation broth components, such as one or more inorganic salts, which help to control the process. Examples of such inorganic salts include, but are not limited to, CaCO 3, KH 2 PO 4, (NH 4) 2 SO 4, MgSO 4, MnSO 4, or Na 2 HPO 4. The fermentation medium may also contain one or more antifoams, such as silicone antifoams.

The preferred fermentation medium composition contains from about 1% to about 6% by weight of an organic nitrogen source, preferably soybean meal, from about 2% to 10% by weight of glucose-35 as a carbohydrate source, alternatively from about 0.01% to 0.2% by weight of one or more inorganic

II

3,92938 salts, preferably calcium carbonate and potassium dihydrogen phosphate, and alternatively about 0.05% to 2% by weight of a silicone antifoam.

In a preferred embodiment of the method of the invention for forming separable crystals of a fermentation product (amphotericin B) in a fermentation broth, the fermentation medium may be completely water-soluble. The fermentation medium is used to ferment amphotericin using the method described in U.S. Patent No. 2,908,611.

The broth to be collected is then heated to at least 70 ° C for a time sufficient, for example 10-45 minutes, to cause microbial cell autolysis leading to the formation of amphotericin B crystals. In another embodiment, a lysozyme or other proteolytic ent-15 enzyme (such as proteases, glucanase, papain, trypsin, or pepsin) may be added to the fermentation broth, during collection, at a pH in the range of 5 to 7.5, and at a temperature of about 20 to about 80 ° C. minutes to about 5 hours. The temperature is then raised to 90 ° C or higher for 30 minutes to complete the formation of crystals.

In another preferred method of crystallization, about 0.01 to about 0.1% w / v sterile amphotericin 25 B crystals (based on broth volume) may be added to the fermenter during the fermentation 10 to 40 hours after the start of the fermentation, after which the fermentation is continued as has been described above with heating (as described in the autolysis step) or without heating. At the end of the fermentation, amphotericin B microcrystals are. 30 present in the fermentation broth, which crystals can be separated.

The use of heat is advantageous in addition to the sowing method.

Amphotericin crystals can be easily separated from the broth by any solid / liquid separation method, including the flotation technique.

4,92938

The following examples represent preferred embodiments of the present invention.

Example 1

Amphotericin B crystals were made as described below.

A 20 liter batch culture of Streptomyces nodosus strain ATCC 14899 was fermented for amphotericin B production using the inoculum preparation and fermentation conditions listed below.

10 Inoculation A. First stage

Inoculum source: Frozen tube of Streptomyces nodosus ATCC 14899 culture.

Substrate: 15 Glucose 1% N-Z Amine B 1.5%

Yeast extract 1%

NaCl 0.5%

CaCO 3 0.1% pH adjusted 6.8 to 7.0

Volume: 100 ml in a 500 ml bottle Sterilization: 30 minutes at 121 ° C Temperature: 25 ° C

Incubation: 48 hours in a reciprocating ra-25 shaker at 220 rpm.

B. The second stage

Inoculum source: 10% of the first stage Medium: Same as in the first stage Sterilization: 30 minutes at 121 ° C 30 Volume: 100 ml in a 500 ml bottle

Temperature: 25 ° C

Incubation: 48 hours back and forth on a shaker at 220 rpm.

Il 5 92938

germination:

Chassis:

Nutrisoy powder 5.0%

Corn steep liquor 1.6% 5 NaCl 0.5%

Silicone foam inhibitor 0.1%

Sterilization: 40 minutes at 121 ° C Temperature: 28 ° C

Shaking: 0.7 HP / 100 US gallons (= 380 1)

10 Aeration: 1 WM

Germination cycle: 28 hours

Inoculum size: 100 ml from the second stage bottle. Fermentation:

Substrate: 15 Glucose 7.0%

Soybean oil flour 2.5%

CaCO3 0.9% KH2PO4 0.01%

Silicone defoamer 0.02% Sterilization: 45 minutes at 121 ° C

Temperature: 25 ° C

Shaking: 3.5 HP / 100 US gallons (= 380 1)

Aeration: 1 WM Fermentation cycle: 136 hours * 25 Inoculum size: 14% of the germination stage

The resulting broth contained 2050 pg / ml amphotericin B and 390 pg / ml amphotericin A.

Microscopic examination of the broth to be collected did not show any crystals. The broth was heated at 60 ° C to 15: ·. minutes. By microscopic examination, the broth was found to be rich in amphotericin B crystals, which could be easily separated from the collected broth by flotation or other inexpensive separation techniques.

92938 6

Example 2

Recovery of amphotericin powder from fermentation broth

The fermentation side of the example was similar to that of Example 1, except that the fermentation medium was filtered to remove unused soybean residues using a Sparkler filter lined with cotton cloth. The resulting filtrate was then re-sterilized and used as a fermentation medium. The resulting collectible broth contained 960 pg / ml amphotericin B after 112 hours of fermentation. The fermentation broth was then treated for several minutes at 120 ° C to induce the formation of amphotericin crystals. After cooling to room temperature, the amphotericin crystals are then separated by centrifugation at 15 ° C and dried at 15 mmHg (= 2 kPa) at 25 ° C for 3 hours. Such recovered crystals were found to contain 70 pg amphotericin B / mg crystals in a total yield of 95%.

Example 3 20 Enzymatic treatment and heating

After the method of Example 2, after treating the fermentation broth for several minutes at 120 ° C to induce crystallization of amphotericin B, the broth was cooled and maintained at 35 ° C and a pH of about 7.0; then lysozyme was added to the broth 25 and allowed to react for 2 hours to partially lyse the mycelial cells. Lysozyme was added at a concentration of 4000 * units / ml of broth. The amphotericin B crystals were then separated from the broth by centrifugation and acetone dried. Such a powder was observed. 30 containing an even higher amphotericin B content than the product of Example 2.

* One unit is defined as the enzyme activity that produces 0.001 A450 per minute at pH 6.24 at 26 ° C.

n 7 92938

Example 4

seeding

The procedure of Example 2 was followed, except that 0.1 g of amphotericin B crystals / 1 was added 40 hours after the start of fermentation.

At the end of the heating step (after fermentation), amphotericin B crystals were recovered.

Example 5

Formation of Amphotericin B Crystals in a Water-Soluble Fermentation Medium The fermentation side of this example was similar to Example 1, except that 2.5% soybean oil in the fermentation medium was replaced with 1.25% spray-dried corn steep liquor in water-soluble Form 15 (supplied by Roquette Corporation ).

The broth was heated to about 75 ° C for 15 minutes. After heat treatment, the broth contained amphotericin B crystals and partially degraded mycelium. The amphotericin B crystals were then separated from the liquid by centrifugation and dried at 15 mmHg (= 2 kPa) at 25 ° C for 3 hours. The overall yield of amphotericin was 95%.

Claims (3)

92938 A method for forming amphotericin B crystals directly in a fermentation medium by culturing a Streptomyces nodosus strain in a fermentation medium until strong antifungal activity has developed in the medium, characterized in that no solvent for amphotericin B is added during the isolation step and that the medium is heated after growth. ° C to 130 ° C for 1 minute to 10 hours to form amphotericin B crystals and amphotericin B crystals are recovered in large amounts from the medium. A method according to claim 1, characterized in that the medium containing amphotericin B is heated for about 10 to 45 minutes to cause the formation of amphotericin B crystals. Process according to claim 2, characterized in that the fermentation broth containing amphotericin B is heated to about 121 ° C for about 20 to 20 minutes for the formation of amphotericin B crystals. 92938