JPS60160888A - Preparation of ikarugamycin - Google Patents

Abstract

PURPOSE:To prepare ikarugamycin in high fermentation ratio, by cultivating a bacterium capable of producing ikarugamycin under specific conditions. CONSTITUTION:A bacterium such as Streptomyces Sp.S8201(FERM P-6856), etc. capable of producing ikarugamicyn is cultivated in a medium containing glycide or an organic acid as a carbon source, having a feed concentration of glycide in a starting medium of <=4wt%, glycide or an organic acid is intermittently or successively to the culture solution after logarithmic growth phase, and the culture solution is kept at 7-9pH, to carry out the cultivation.

Description

[Detailed description of the invention] The present invention relates to a method for producing icargamycin.

Icargamycin is also known as an antibiotic that destroys the anti-trichosonus effect and exhibits antibacterial activity against Durham-positive bacteria. In addition, in recent years, it has been discovered that icargamycin has an extremely excellent anti-ulcer effect (Japanese Patent Application Laid-open No. 189114/1982).
Since it is an important substance as a medicine, it is of great industrial significance to produce it cheaply and in large quantities. The conventional method for producing icargamycin is the fermentation method using microorganisms (Japanese Patent Publication No. 46-28833, The jo
Urnal of Antibloties Mol 2
5 (5)% 271.1972) is known, but
It has the disadvantage that the fermentation yield is extremely low, and is not necessarily satisfactory from an industrial standpoint.

The present inventors conducted various studies in order to establish a method for producing Ikarugamycin with a high fermentation yield in the fermentation production of Ikarugamycin using microorganisms. , the method of adjusting the medium pH during fermentation with acid or alkali is to add a part of the carbon source to the initial medium, and to add the rest in small amounts during the culture, intermittently or continuously, taking into account changes in the culture solution PU. The present invention was completed based on the new discovery that the production yield of icargamycin can be dramatically increased if the method is changed to a method in which icargamycin is added directly.

Therefore, the present invention provides a method for culturing a microorganism capable of producing icargamycin in a medium containing carbohydrates or organic acids as a carbon source, producing and accumulating icargamycin in the culture, and collecting icargamycin from the culture. The pi of the culture solution which has passed the growth phase is maintained in the range of 7 to 9 by adding carbohydrates or organic acids intermittently or continuously to the medium. A method for producing mycin is provided.

In the method of the present invention, carbohydrates among the carbon sources added to the medium include sugars [e.g., glucose, xylose,
Fructose, mannitol, arabinose, molasses, starch, starch hydrolysates (e.g. corn starch saccharification liquid)
etc.], sugar alcohols (e.g. glycerin, sorbitol)-A
-), and organic acids include halacetic acid, pyruvic acid, formic acid, citric acid, succinic acid, malic acid, and the like.

In the method of the present invention, carbohydrates or organic acids are added intermittently or continuously to a culture solution that has passed the logarithmic growth phase, and the pH of the culture solution is maintained at 7 to 9 during cultivation.

The concentration of carbohydrates as a carbon source in the initial culture medium may be about 1% or less or more than 1%, but the concentration of carbohydrates as a carbon source in the initial medium is 4% or less. The case is preferred.

In the method of the present invention, the p
H is controlled to a predetermined pH selected from a range of 7 to 9, but the pH in the culture solution may be controlled during the entire period from the start to the end of the culture, or when used. The period may start from the time when the microorganism to be cultured has passed the logarithmic growth phase and the pH reaches a predetermined value, until the end of the culture. Also, p in the culture solution
In order to control H, the pH in the culture solution can be determined using a pH sensor, and by linking these measuring devices with a carbohydrate or organic acid feed device, the pH in the culture solution can be adjusted to a predetermined value. It is possible to control automatically, which is convenient.

The microorganism used in the present invention may be any microorganism as long as it has the ability to produce icargamycin. Specific examples of the microorganism include Str as described in Japanese Patent Application No. 58-24489.
@ptomyc@m sp38201 (FERMP-
6856), which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

In the method of the present invention, all media that can be used for the production of icargamycin can be used.

These media usually contain nitrogen sources, etc., and specific examples include soybean flour, pezoton, cottonseed meal, fish meal,
In addition to organic substances such as corn staple liquor and yeast extract, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium chloride, and ammonium phosphate, organic compounds such as urea and amino acids can be mentioned. In addition, the culture medium contains #Roman crab, which is a carbon and nitrogen source.
Various metals, vitamins, and inorganic salts necessary for the growth of the microorganisms used and the accumulation of icargamycin are added as appropriate.

Cultivation in the method of the present invention is carried out by a conventional method including pH adjustment. That is, the culture is carried out under aerobic conditions such as shaking or submerged culture with aeration and stirring, and the culture temperature is usually 25 to 32°C, but in most cases it is preferable to culture at around 30°C. Also, silicone oil or Adekanol (
A general antifoaming agent such as (trade name) may be added as appropriate. When cultured under such conditions for usually 4 to 10 days, a significant amount of icargamycin is produced in the medium. The icargamycin thus accumulated in the culture solution can be easily collected by conventional RA methods such as precipitation, solvent extraction, ion exchange resin method, gel filtration method, adsorption or distribution column chromatography, etc. can.

There have been several reports on the fermentation production of icargamycin using microorganisms, but in those reports, carbohydrates, which are the main carbon source, are added to the initial medium at the beginning of culture.

The method of adding it to (Th・journa
l of Antlblotles Vol 2 5 (
5)% 271 pages, 1972, Special Publication No. 46-2883
No. 3).

Furthermore, in the fermentation production of some physiologically active substances, attempts have already been made to increase the amount of carbohydrates used by additionally adding carbohydrates during the culture. (for example,
Kotani et al., Agrle, Biol, Ch@m, vol. 42,
399, 1973), but even in such cases, this is to avoid situations where the sugar concentration in the culture solution deviates from the normal common sense concentration range and the growth of microorganisms is significantly inhibited. However, it is not intended to control the pH of the culture solution to a predetermined value.

In contrast, the method of the present invention involves culturing microorganisms capable of producing icargamycin while controlling the pH in the culture solution to a predetermined value, and is a novel method that is clearly different from the above methods. However, the present invention is an industrially advantageous method that can significantly improve the production amount of icargamycin.

The present invention will be explained in more detail with reference to Examples below.

Example 1 Strenotomyces sp. 88201 (FEBMP-6856) grown on yeast/malt agar medium was treated with 2.0% soluble starch, 2.0% cotton seed mill, 1.0% corn nuty IJ car, and 0 calcium carbonate. ．．
A sterilized seed medium consisting of 3296 (pI17.O) was inoculated and cultured with shaking at 30°C for 48 hours. 2,6
Add a liquid medium consisting of the above medium composition to the Zoya fermentor.1. OL was placed and 50ff1g of the above seed culture was transplanted. Culture was started at a temperature of 30°C, a rotation speed of 50°r, and a p0ml aeration rate of 1.017 mm. Addition of a glucose solution or an organic acid solution as a carbon source was carried out by the method shown below.

Method: Culture was continued for 190 hours without adding carbohydrates or organic acids from the start to the end of culture.

Method B: 36 hours after the start of culture, when the pH of the culture solution reached 7.5 after the logarithmic growth phase, a 25% glucose solution was added intermittently while adjusting the pH to 7.5 using a PI controller. Culture was continued for 190 hours.

Method C: After the logarithmic growth phase has passed after the start of culture, the pH of the culture solution is 8.
．． 45 hours after the pH reached 0, a 925% glucose solution was added intermittently while adjusting the pH to 8.0, and the culture was continued for 168 hours.

Method D: After the start of culture, after the logarithmic growth phase, the pH of the culture solution is
At the 50th hour when the pH value reached 8.5, the pH was adjusted to 8.5 using a 925% glucose solution, and the addition was continued intermittently, and the culture was continued for 144 hours.

Method E: After the start of culture, after the logarithmic growth phase has passed, the pH of the culture solution is 9.
．． After 60 hours when the pH reached 0, a 925% glucose solution was added intermittently while adjusting the pH to 9.0, and the culture was continued for 190 hours.

Method F: The culture was continued for 190 hours according to Method B except that the 25% glucose solution was replaced with a 6N acetic acid solution.

Method G: Cultivation was continued for 190 hours according to Method C except that the 25% glucose solution was replaced with a 6N acetic acid solution.

Method H: Culture was continued for 168 hours according to the method except that the 25% glucose solution was replaced with a 6N acetic acid solution.

Method X: Cultivation was continued for 190 hours according to Method E except that the 25% glucose solution was replaced with a 6N acetic acid solution.

Method 12 Culture was continued for 168 hours according to the method except that the 25% glucose solution was replaced with a 3N sulfuric acid solution.

The amount of icargamycin in each culture cultured by each of the above methods was measured using high performance liquid chromatography, and the production amount was investigated. The results are shown in Table 1.

Below margin

Claims (2)

[Claims] (1) A method in which a microorganism capable of producing icargamycin is cultivated in a medium containing carbohydrates or organic acids as a carbon source, 1 cargamycin is produced and accumulated in the culture, and icargamycin is collected from the culture. Icargamycin, which is characterized in that the pH of an aged culture solution is maintained within the range of 7 to 9 by adding carbohydrates or organic acids intermittently or continuously to the medium. manufacturing method. (2) The concentration of carbohydrates as a carbon source in the medium is approximately 4%
1. A method for producing icargamycin according to claim 1 below.