JP2675047B2 - Method for forming amphotericin B crystals in fermentation medium - Google Patents

Description

TECHNICAL FIELD The present invention includes a method for forming amphotericin B crystals in a fermentation medium, and more specifically, a direct microbial cell autolysis step for promoting crystal formation, which is costly. It relates to a fermentation method which allows crystals to be easily separated without the use of solvent extraction techniques and is particularly suitable for use in high yield amphotericin B fermentations.

Conventional technology and problems to be solved by the invention So far, crystals of amphotericin B have been
It was manufactured through costly filtration, elaborate recovery operations including solvent extraction and crystallization steps.

Structure and Effect of the Invention The present invention is a method for directly forming crystals of amphotericin B in a fermentation broth containing a water-soluble or water-insoluble fermentation medium described in US Pat. No. 2,908,611, the fermentation medium comprising: It contains at least one of a nitrogen source, a carbohydrate source (carbon / energy source), and optionally an inorganic salt for process control.

In addition, the present invention provides a method for directly producing a crystal of amphotericin B in a fermentation medium, which comprises preparing the above-mentioned fermentation medium and streptomyces nodosus in the medium. It consists of allowing growth and then inducing crystallization of amphotericin B and microbial cell autolysis. This crystallization and autolysis causes the fermentation medium to be at least 50 ° C, preferably about 70-130.
Amphotericin B is heated at a sufficient temperature of ℃ for a sufficient period of time, for example, about 1 minute to 10 hours, preferably about 10 to 45 minutes.
This can be done by inducing the formation of crystals.

Crystallization can also be initiated by adding a seed of amphotericin B crystals to the fermentation medium during fermentation, preferably about 10-40 hours after the start of fermentation.

Autolysis can also be performed by the use of lysozyme or other similar enzymes that have a lytic effect on the cell wall.

If the addition of enzyme or crystal seed is employed, heat treatment may be omitted.

The fermentation medium used in the production method of the present invention contains about 0.1 to 20% by weight, preferably about 0.5 to 5% by weight of a nitrogen source.
Specific examples of suitable nitrogen sources include casein hydrolysates,
Mention may be made of cottonseed or its derivatives, corn steep liquor, soybean meal or other similar organic or inorganic nitrogen sources or their soluble derivatives.

The fermentation medium contains about 0.5 to 20% by weight, preferably about
There is 0.5 to 10% by weight carbohydrate source. Specific examples of suitable carbohydrate sources include starch, sugars such as dextrin, maltose, lactose and glucose, glycerol and the like.

Fermentation medium, other normal fermentation medium components, if necessary,
For example, you may contain at least 1 sort (s) of the inorganic salt which assists process control. Specific examples of the inorganic salt include
CaCO ₃ , KH ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ , MnSO ₄ or Na ₂ H
Examples include, but are not limited to, PO ₄ . Further, the fermentation medium may contain at least one antifoaming agent such as a silicone antifoaming agent.

A preferable fermentation medium composition is about 1 to 6% by weight of an organic nitrogen source (preferably soybean meal), about 2 to 10% by weight of glucose as a carbohydrate source, and optionally about 0.01 to
0.2% by weight of at least one inorganic salt (preferably CaCO ₃
And KH ₂ PO ₄ ) and, if necessary, about 0.05-2% by weight
Of silicone antifoam.

In a preferred embodiment of the production method of the present invention for producing isolated crystals of a fermentation product (amphotericin B) in a fermentation medium,
The fermentation medium may be completely water soluble. In such an amphotericin fermentation, the fermentation medium is used according to the procedure described in US Pat. No. 2,908,611.

The resulting culture is then heated at a temperature of at least 50 ° C. for a sufficient period of time, for example 10 to 15 minutes, to allow autolysis of the microbial cells, which results in the formation of amphotericin B crystals. In another embodiment, the fermentation medium at harvest has pH
Lysozyme or other proteolytic enzyme (eg, protease, glucanase, papain, trypsin or pepsin) may be added over a period of about 30 minutes to 5 hours at 5 to 7.5 and a temperature of about 20 to 80 ° C. The temperature is then raised to 90 ° C or above for 30 minutes to complete the crystal formation.

In another preferred method of performing crystallization, during fermentation, 10
Approximately 0.01-0.1% W / V (relative to medium volume) of sterile amphotericin B crystals can be added to the fermentation vessel during ~ 40 hours fermentation and then heated (as in the autolysis process).
Continue the above fermentation under or without heating. After fermentation, amphotericin B is present in the fermentation medium in the form of microcrystals, which can be separated. Heating in addition to the addition of crystal seeds is advantageous.

Amphotericin B crystals can be easily separated from the medium, but this is carried out by a solid / liquid separation operation including a flotation method.

The following examples are preferred specific examples of the present invention.

Example 1 Amphotericin B crystals are produced by the following procedure.

20 batches of Strept for the production of amphotericin B
omyces nodosus (ATCC14899) was fermented, and the inoculum growth length and fermentation conditions in this case are listed below.

Fermentation A. First step Inoculum: Streptomyces nodosus (ATCC14899) frozen product Medium: Glucose 1% NZ amine B 1.5% Yeast extract 1.0% NaCl 0.5% CaCO ₃ 0.1% (adjusted to pH 6.8-7.0) Volume: 100 ml in a 500 ml flask Sterilization: 121 ° C for 30 minutes Temperature: 25 ° C Culture: 48 hours at 220 rpm on a reciprocal shaker B. Second stage Inoculum: 10% concentration obtained in the first stage Medium: First Same as steps Sterilization: 30 min at 121 ℃ Volume: 500 ml 100 ml in flask Temperature: 25 ℃ Cultivation: 48 hours at 220 rpm on a reciprocal shaker Growth: Medium: Nutrisoy Flour 5.0% Corn steep liquor 1.6% NaCl 0.5% Silicone antifoam 0.1% Sterilization: 121 ° C for 40 minutes Temperature: 28 ° C Stirring: 0.7HP / 100U.S. Gallon Aeration: 1VVM Development cycle: 28 hours Inoculum: 100ml from the second flask stage Fermentation conditions : Medium: Glucose 7.0% Soybean oil meal 2.5% CaCO ₃ 0.9% KH ₂ PO ₄ 0.01% Silicone defoamer 0 .02% Sterilization: 45 min at 121 ° C Temperature: 25 ° C Agitation: 3.5 HP / 100 U.S. gallons Aeration: 1 VVM Fermentation cycle: 136 hours Inoculum: 14% from development stage The resulting culture is 2050γ / ml amphotericin B and 390 γ / ml amphotericin A.

Microscopic examination of the culture showed no crystals in the culture. When the culture solution was heated at 60 ° C for 15 minutes and examined under a microscope, it was found that the culture solution contained a large amount of amphotericin B crystals, and these crystals were separated from the culture solution by the flotation method or another inexpensive method. It can be easily separated by various separation methods.

Example 2 Recovery of Amphotericin Powder from Fermentation Medium: -The fermentation mode of this example uses a Sparkler filter lined with cotton fabric, except that the fermentation medium is passed to remove undigested soybean residues. This is the same as in the first embodiment. The resulting liquid is sterilized again and used as a fermentation medium. The culture broth obtained after 112 hours of fermentation contains 960 γ / ml amphotericin B. The fermentation broth was then added to 120
Incubate for a few minutes at ° C to induce the formation of amphotericin crystals. After cooling to room temperature, the amphotericin crystals are separated by centrifugation and dried at 15 mmHg / 25 ° C for 3 hours. Such recovered crystals were 79γ / mg of amphotericin B (total recovery 9
5%) is included.

Example 3 Enzyme treatment and heating: -The procedure of Example 2 is followed. The fermentation broth was cured at 120 ° C for several minutes to induce the crystallization of amphotericin B, and then the broth was cooled to 35 ° C and maintained at the same temperature and pH of about 7.0, and then lysozyme was added to the broth. The reaction is carried out for 2 hours to partially dissolve the mycelial cells. Add lysozyme at a concentration of 4000 units ^* / ml of medium.

*) 1 unit means the enzyme activity to produce 0.001 / min ΔA ₄₅₀ at pH 6.24 and 26 ° C.

The amphotericin B crystals were then centrifuged from the culture,
Dry with acetone. It is noted that such powder has a higher amphotericin B content than the product of Example 2.

Example 4 Addition of crystal seeds: −0.1 g / amphotericin B crystals 40 from the start of fermentation
The procedure of Example 2 is carried out except that it is added after a period of time.

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

Example 5 Production of Amphotericin B Crystals in Soluble Fermentation Medium: -The fermentation aspect of this example is 1.25% soluble spray dried corn steep liquor (obtained from Roquette corp.) Instead of 2.5% soybean oil meal in the fermentation medium. The same as Example 1 except that is used.

The culture is heated at about 75 ° C for 15 minutes. The culture solution after the heat treatment contains amphotericin B crystals and partially dissolved mycelium. Then from that solution amphotericin B
The crystals are centrifuged and dried at 15 mmHg / 25 ° C for 3 hours. The total recovery of amphotericin is 95%.

Claims (13)

(57) [Claims] 1. A fermentation medium in which a solvent for amphotericin B is substantially absent, and Streptomyces nodosus (Stre) is used.
ptomyces nodosus) strain is cultivated until substantial antibacterial activity is imparted to the medium, the medium is heated to a temperature of 50 ° C or higher for at least 1 minute to form amphotericin B crystals, and then a substantial amount of amphotericin is cultured from the medium. A method for directly producing amphotericin B crystals in a fermentation medium, which comprises recovering B crystals. 2. The method further comprising the step of inducing crystal formation by adding crystals of amphotericin B to the fermentation medium.
The production method described in the section. 3. A medium containing amphotericin B in an amount of about 70-
The method according to claim 1, wherein the amphotericin B crystals are formed by heating at a temperature of 130 ° C. for about 1 minute to 10 hours. 4. A culture medium containing amphotericin B in an amount of about 121.
The method according to claim 3, wherein the crystals of amphotericin B are formed by heating at 0 ° C for about 20 minutes. 5. After the heating step, lysozyme, protease,
The method according to claim 1, further comprising the step of adding glucanase, papain, trypsin or pepsin to complete the crystal formation. 6. The method according to claim 5, wherein lysozyme is added. 7. The method according to claim 2, wherein crystals of amphotericin B are added to the fermentation medium about 10 to 40 hours after the start of the culture. 8. The method according to claim 7, wherein the amphotericin B crystals are added in an amount of about 0.01 to 0.1% W / V with respect to the volume of the fermentation medium. 9. A fermentation medium comprising about 0.1 to 20% by weight of a nitrogen source,
A process according to claim 1 which comprises 0.5 to 20% by weight of a carbohydrate source, optionally at least one inorganic salt, and optionally at least one antifoaming agent. 10. The method according to claim 9, wherein the nitrogen source is soybean oil meal or corn steep liquor, and the carbohydrate source is glucose. 11. The process according to claim 9, wherein the fermentation medium contains one or more inorganic salts to help control the process. 12. The method according to claim 10, wherein the inorganic salt is CaCO ₃ and KH ₂ PO ₄ . 13. The method according to claim 9, wherein the fermentation medium contains a silicone antifoaming agent.