JPWO2007013487A1 - Method for producing colistin powder - Google Patents

Abstract

Production of colistin powder without decolorization process, which solves the problems of conventional production methods such as increase in production time due to decolorization process, cost increase due to decrease in yield of colistin main component, and environmental impact due to generation of carbon waste Provide law. A method for producing Bacillus polymixer bar colistinius, high whiteness colistin powder, and high whiteness colistin powder capable of producing high whiteness colistin powder.

Description

  The present invention is characterized by obtaining a novel strain having a significantly reduced color of the culture solution compared to the production strain used for conventional colistin fermentation production, and using this as a colistin-producing strain, The present invention relates to a production method for obtaining a bulky colistin powder without performing a decolorization step.

  Colistin is a peptidic antibiotic that acts broadly on gram-negative bacteria. Its mechanism of action is by causing phospholipid degradation by activating phospholipases in the bacterial cytoplasmic membrane and causing lysis. Colistin has a low selective toxicity and is not absorbed from the intestinal tract, so it is widely used as a feed additive.

As a production method of colistin by fermentation production of microorganisms, the natural raw materials containing a large amount of such starchy available at low cost was the main raw material, Bacillus (Bacillus) bacteria belonging to the genus, especially the method of fermentation production by Bacillus polymyxa (Bacilluspolymyxa) Known (see Patent Documents 1 and 2).

  When fermentation of colistin is carried out using Bacillus bacteria, especially Bacillus polymixer, which is made from inexpensive natural raw materials, the color of the culture becomes darker as fermentation progresses, and the production of colistin, the target product, is the highest. The coloration increases until the end of the fermentation when the value is reached. Therefore, the purification process is carried out for the purpose of removing the nutrient components of the culture medium and metabolites other than the fermented colistin from the culture solution containing colistin to increase the purity of colistin. However, the coloring substances in the culture medium increased in the fermentation process cannot be completely removed in the purification process and remain in the product colistin bulk powder, resulting in a darkly colored colistin bulk powder. In particular, in the European Pharmacopoeia 5th edition (issued on June 15, 2004 and enforced on January 1, 2005), which is the quality standard used for pharmaceuticals in Europe (including veterinary drugs), the color standard for colistin powder is It is defined as “white or almost white”. In order to produce a colistin bulk powder that passes this standard, a conventional method for producing a colistin powder requires a decolorization process such as using carbon in the purification process. The conventional method for producing colistin powder, which performs a decoloring step with carbon, has problems such as an increase in the time required for production and a decrease in the recovery rate of colistin powder, which is a target substance.

JP 58-47493 A JP 58-129933 A

  An object of the present invention is to provide a method for producing a colistin bulk powder having a high degree of whiteness without performing a decolorization step required in the conventional method for producing a colistin bulk powder.

  In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, the conventional production strain is subjected to a mutation treatment to obtain a strain having a reduced degree of coloring, and by using this strain, a decolorization step is performed. The present inventors have found that a colistin powder having a high whiteness can be obtained without completing the present invention.

That is, the present invention is as follows.
(1) Bacillus polymixer bar colistinius, which can produce colistin powder having an absorbance of a 5% concentration (W / V) aqueous solution at 400 nm of 0.15 or less,
(2) The Bacillus polymixer bar colistinius M50 strain deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the deposit number FERM AP-20553,
(3) The Bacillus polymixer bar colitisius according to (1) or (2) above is cultured, the culture is adsorbed on an ion exchange resin, eluted, concentrated, and dried. A method for producing a colistin powder, wherein the absorbance of a 5% strength (W / V) aqueous solution at 400 nm is 0.15 or less,
(4) A colistin bulk powder obtained by the production method described in (3) above, wherein the absorbance of a 5% strength (W / V) aqueous solution at 400 nm is 0.15 or less.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel production strain useful for a method for producing colistin bulk powder can be provided. Furthermore, by using the strain of the present invention as a production strain, it is possible to provide a method for producing a colistin bulk powder having a high whiteness without performing a decolorization step that is essential for a conventional method for producing a colistin bulk powder.

The absorbance at 300 to 600 nm of the culture filtrate derived from 202-71 strain and the culture filtrate derived from M50 strain obtained in Example 1 is shown. The chart which analyzed the liquid which melted colistin bulk powder derived from M50 stock obtained in Example 3 in deionized water by liquid chromatography (HPLC) is shown.

Deposit of microorganisms Bacillus polymyxa var. Colistinus M50 strain is an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan No. 6) was deposited on May 31, 2005 under the deposit number FERM AP-20553.

Hereinafter, the present invention will be described in detail.
In the present invention, the production strain used for conventional colistin fermentation production was subjected to artificial mutation treatment to obtain the desired production strain.
The artificial mutation treatment method is not particularly limited as long as it is an artificial mutation treatment. For example, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate, etc. Chemical methods; physical methods such as ultraviolet irradiation and X-ray irradiation; mutation treatment methods such as biological methods such as genetic recombination and transposon can be used.
The bacterial group obtained by performing the mutation treatment was subjected to liquid culture using a flask and screened using the degree of coloration of the culture and the ability to produce colistin as indicators. Here, the degree of coloring is based on the value obtained by measuring the absorbance at 400 nm of the filter paper filtrate of the culture solution. The colistin production ability is based on a value obtained by analyzing the filter paper filtrate of the culture solution by HPLC.
In this way, the degree of coloring is greatly reduced compared to conventional production strain, and colistin producing ability is obtained equal or higher production strain Bacillus polymyxa bar Korisuchiniusu (Bacilluspolymyxa var. Colistinus).

  In the present invention, a strain having a clearly high whiteness in the colistin bulk obtained by producing colistin from Bacillus polymixer bar colistinius can be used. Specifically, a colistin bulk powder having an absorbance of a 5% concentration (W / V) aqueous solution at 400 nm of 0.15 or less, preferably 0.087 or less, more preferably 0.070 or more and 0.087 or less is obtained. Any Bacillus polymixer bar colistinius can be used in the present invention. Furthermore, examples of production strains that can be used in the present invention include subcultured strains, mutant strains, and genetically modified strains of these strains.

  The color of colistin powder produced without performing a decolorization step using carbon or the like from a culture solution produced by fermentation using the M50 strain, which is an example of the production strain of the present invention, is a decolorization step from a culture solution derived from a conventional production strain. Compared with the color tone of the colistin powder produced by the above, the whiteness is clearly higher. That is, by using the M50 strain obtained in the present invention as a production strain, it is possible to obtain a colistin bulk powder having a high whiteness without performing a decolorization step that was essential in the conventional method for producing a colistin bulk powder. It becomes.

  In the present invention, Bacillus polymixer bar colistinius is cultured, the culture is adsorbed on an ion exchange resin, eluted, concentrated, and dried to achieve 5% at 400 nm without performing a decolorization step. A colistin bulk powder having a concentration (W / V) aqueous solution with an absorbance of 0.15 or less can be produced. This makes it possible to produce a colistin powder that passes the color standard “substantially white” of the European Pharmacopoeia, which is a quality standard for colistin powder used in medicines (including veterinary drugs) in Europe. In addition, a decolorization step such as adding carbon such as activated carbon and stirring and filtering was usually required. It can be shortened by about half a day. Furthermore, loss of colistin powder due to carbon used for decolorization can be reduced, and disposal of carbon or the like used in the decoloring step of the conventional method for producing colistin powder can be avoided.

  In addition, the colistin powder in the present invention contains colistin generally known as a polymyxin peptide antibiotic. As for colistin, colistin A having the following structure and colistin B having the same amino acid structure are known.

  The colistin bulk powder of the present invention is a crude extract containing colistin to the extent that it can be used as a pharmaceutical product, and is obtained without going through a decolorization step as described above. The method of using the colistin bulk powder is not particularly limited, and can be used as it is, or further processed and used as a colistin preparation.

  EXAMPLES Hereinafter, although an Example and a test example demonstrate this invention further in detail, this invention is not limited to these Examples and a test example. Moreover, the measurement of the light absorbency in each Example and test example was performed using JASCO Corporation spectrophotometer V-560. Further, unless otherwise defined,% represents a weight to volume percentage (W / V).

<Example 1>
With respect to the Bacillus polymixer bar colistinius 202-71 strain conventionally used for the production of colistin bulk, it is allowed to stand at a temperature of 25 to 30 ° C. for 1 minute to 24 hours at a concentration of 10 to 1000 μg / mg. Alternatively, the mutation treatment was performed by shaking, and colonies were grown on an agar plate comprising 2% tryptone, 0.2% yeast extract, and 1% glucose. Each colony (strain) is inoculated into a liquid seed medium consisting of 2% normal broth, 0.2% yeast extract and 0.3% sodium chloride, and cultured at 28 ° C. for about 24 hours. Flour 4.5%, Cornmeal 1.5%, Wheat germ 0.2%, Non-fat soybean 0.2%, Ammonium sulfate 1%, Monopotassium phosphate 0.05%, Ferrous sulfate 0.005%, Carbonic acid Inoculated into a 250 mL Erlenmeyer flask containing 30 mL of liquid medium consisting of 0.05% calcium and 1.5% elutriate. After shaking culture at 28 ° C. for about 120 hours, the culture solution was filtered using filter paper, and the color of the obtained filtrate and colistin productivity were compared. From these fungal groups, the M50 strain was selected as a strain that is equivalent to or better than the colistin producing ability of the 202-71 strain and the color of the filtrate is clearly lighter than that of the 202-71 strain. About the culture filtrate of 202-71 stock | strain and M50 stock | strain, the light absorbency of 300-600 nm was measured with the spectrophotometer, and the result was shown in FIG. The absorbance at 400 nm was 3.61 in the culture filtrate derived from the 202-71 strain, and 1.42 in the culture filtrate derived from the M50 strain, and a clear difference was observed (Table 1).

<Example 2>
The M50 strain with a light color of the culture broth obtained in Example 1 and the 202-71 strain with a deep color of the culture broth conventionally used for colistin fermentation production were obtained by adding 2% normal bouillon, 0.2% yeast extract and After inoculating into a liquid seed medium consisting of 0.3% sodium chloride and culturing at 28 ° C. for about 24 hours, about 15 mL of each is corn flour 4.5%, corn meal 1.5%, wheat germ 0.2% 5L in 4L liquid medium consisting of 0.2% defatted soybean, 1% ammonium sulfate, 0.05% monopotassium phosphate, 0.005% ferrous sulfate, 0.05% calcium carbonate and 1.5% chicken pox Inoculated the jar fermenter. Each was cultured at 30 ° C. for about 120 to 144 hours at pH 5.0 to 7.0 with aeration and stirring. The obtained culture solution was filtered using a filter paper and the absorbance at 400 nm was measured. As a result, the culture filtrate from 202-71 strain was 4.50, and the culture filtrate from M50 strain was 1.39. A clear difference was observed similar to the test results in the flask of Example 1 (Table 2).

<Example 3>
The 5 L jar fermenter culture solution of M50 strain and 202-71 strain obtained in Example 2 was filtered with Nutsche covered with filter aid (body mix), and then ion exchange resin (Amberlite) packed in the column IRC-50 (Na ⁺ type) manufactured by Rohm and Haas, 300 mL). After elution of colistin from the ion exchange resin with about 0.4 mol / L sulfuric acid, it was washed with 600 mL of deionized water, and the resulting eluent and washing liquid mixture was concentrated to about 5% concentration (W / V). Thus, a colistin-rich solution was obtained. The absorbance at 400 nm of the colistin-rich solution is 0.359 for the colistin-rich solution derived from the 202-71 strain, and 0.07 for the colistin-rich solution derived from the M50 strain. The coloring of the high content liquid was light (Table 3).

  Furthermore, each colistin high content liquid was spray-dried and the target colistin powder was obtained. The colistin powder derived from 202-71 strain had a brownish color. In contrast, the colistin bulk powder derived from the M50 strain is slightly yellowish white, and the coloring of the colistin bulk powder derived from the M50 strain is obviously lighter and whiter than the colistin bulk powder derived from the 202-71 strain. Met. Each colistin powder was dissolved in deionized water to a concentration of 5% (W / V) to obtain a 5% concentration (W / V) aqueous solution. When the absorbance of a 5% concentration (W / V) aqueous solution at 400 nm was measured, it was 0.479 for the colistin powder derived from 202-71 strain and 0.087 for the colistin powder derived from M50 strain. A significant difference was observed (Table 4).

<Test Example 1>
The solution obtained by dissolving the M50 strain-derived colistin powder obtained in Example 3 in deionized water was analyzed by liquid chromatography (HPLC), and the results are shown in FIG. HPLC measurement conditions are as follows.
Measurement conditions Detector: Absorbance detector (measurement wavelength: 215 nm)
Column: YMC-Pack ODS-AM (AM-312) (manufactured by YMC)
150mm × Φ4.6mm
Column temperature: 30 ° C
Mobile phase: Dissolve 4.46 g of anhydrous sodium sulfate in 900 ml of pure water, add 2.5 ml of phosphoric acid, and make up to 100 ml (pH 2.3 to 2.5). This solution 78 is mixed with acetonitrile at a ratio of 22.
Flow rate: 1.0ml / min

<Test Example 2>
The colistin-rich solution derived from 202-71 strain obtained in Example 3 was tested for the amount of decolorized carbon necessary to make the color tone of the colistin-rich solution derived from M50 strain obtained in Example 3. Carbon equivalent to 2%, 5%, and 10% (each W / V) was added to the equivalent amount of colistin-containing liquid derived from 202-71 strain (refined birch, Takeda Pharmaceutical Co., Ltd.) After stirring for 30 minutes, it filtered with the Kiriyama funnel, and obtained filtrate was spray-dried. Each spray-dried powder was dissolved in deionized water to obtain a 5% strength (W / V) aqueous solution. As a result of measuring the absorbance of a 5% concentration (W / V) aqueous solution at 400 nm, the absorbance was 0.135 even in a 10% (W / V) carbon-added aqueous solution having the largest amount of decolorizing carbon, and was obtained in Example 3. The absorbance of the colistin-rich solution derived from the M50 strain not subjected to decolorization carbon treatment did not reach 0.087 (Table 5).

  In addition, the loss of colistin yield by the decolorization process in the addition of 10% (W / V) carbon is about 27% (Table 5), and the yield of colistin in the process for producing colistin including the decolorization process is clearly reduced. It was. In order to obtain colistin bulk powder with high whiteness in a conventional production strain, the ion exchange resin is washed with a large amount of deionized water, or a decoloration process is performed in which the eluent from the ion exchange resin is treated with a large amount of carbon. However, the method for producing colistin powder using the M50 strain, which does not require a decolorizing step with carbon because the culture solution is colored, is very advantageous.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel production strain useful for a method for producing colistin bulk powder can be provided. Furthermore, by using the strain of the present invention, an efficient method for producing a colistin bulk can be provided without going through the decolorization step that was essential in the conventional method for producing a colistin bulk.

Claims (4)

  Bacillus polymixer bar colistinius which can produce colistin powder having an absorbance of 0.15 or less in a 5% concentration (W / V) aqueous solution at 400 nm.   Bacillus polymixer bar colistinius M50 strain deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the deposit number FERM AP-20553.   5% concentration at 400 nm characterized by culturing Bacillus polymixer bar colistinius according to claim 1 or 2, adsorbing the culture on an ion exchange resin, elution, concentrating and drying (W / V) A method for producing a colistin bulk powder in which the aqueous solution has an absorbance of 0.15 or less.   A colistin bulk powder obtained by the production method according to claim 3, wherein the absorbance of a 5% concentration (W / V) aqueous solution at 400 nm is 0.15 or less.

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