JPH02312595A - Production of 5'-inosinic acid by fermentation method - Google Patents

Abstract

PURPOSE:To obtain the subject compound, utilizable as a seasoning and useful in the field of food industry by culturing a specific microorganism, belonging to the genus Corynebacterium and having tolerance to osmotic pressure in a culture medium. CONSTITUTION:A microorganism [e.g. a variant strain obtained by subjecting Corynebacterium Ammoniagenes KY13184 (FERM P-3790) as a parent strain to varying treatment, such as by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet rays or X-rays], belonging to the genus Corynebacterium and having tolerance to >=2000mOSMOL/kg osmotic pressure is cultured in a culture medium to produce and accumulate 5'-inosinic acid in the resultant culture and collect the resultant 5'-inosinic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing 5'-inosinic acid by fermentation. 5'-inosinic acid is widely used as a seasoning and is a useful substance in the food industry.

BACKGROUND ART Conventionally, as a method for directly fermenting and producing 5'-inosinic acid from saccharides, a 5'-inosinic acid, which belongs to the genus Corynebacterium, has an adenine auxotrophy, and is resistant to decoinine or sulfa drugs,
Furthermore, a method for culturing microorganisms capable of producing 5'-inosinic acid and collecting 5'-inosinic acid accumulated in the medium (JP-A-55-150899), which belongs to the genus Corynebacterium and has an adenine auxotrophy, 5′-
Method for producing and accumulating inosinic acid (Japanese Patent Publication No. 58-463
19) etc. are known.

There is no known method for producing 5'-inosinic acid using a bacterial strain that is resistant to osmotic pressure.

In this specification, the strain conventionally named Brevibacterium ammoniagenes will be changed to Corynebacterium ammoniagenes. This bacterial species name change was published in the International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology).
Journal of SeitematicBa
ctoriology) 442-443, October (1987).

Problems to be Solved by the Invention The purpose of the present invention is to solve the problem by solving the following problems:
- An object of the present invention is to provide a method for industrially producing inosinic acid at low cost and efficiently by direct fermentation.

Means for Solving the Problems According to the present invention, 200 microorganisms belonging to the genus Corynebacterium
Resistant to osmotic pressure of 0mO3MOL/KG or more and 5
By culturing a microorganism capable of producing '-inosinic acid in a medium, 5'-inosinic acid can be produced and accumulated in the culture, and 5'-inosinic acid can be collected from the culture.

The present invention will be explained in detail below.

The microorganism to be used may be either a wild strain or a mutant strain as long as it belongs to the genus Corynebacterium and is resistant to an osmotic pressure of 2000 mO3MOL/KG or more and has the ability to produce 5'-inosinic acid. The mutant strain described in ``1'' confers osmotic pressure resistance of 2000 mO5MOL/KG or more to 5'-inosinic acid-producing bacteria belonging to the genus Corynebacterium, and
It can be obtained by imparting the ability to produce 5'-inosinic acid to a strain that is resistant to osmotic pressure of 00 mO3MOL/KG or higher.

For example, the mutant strain used in the examples of the present invention is Corynebacterium ammoniagenes (Corynebacterium ammoniagenes).
cterium Ammoniagenes) KY1
3184 (FERM P-3790) (hereinafter KY 13
There are 184 stocks. ) was used as the parent strain, and N-methyl-
It can be obtained by a conventional mutation treatment using N'-nitro N-nitrosoguanidine, ultraviolet rays, X-rays, etc.

To select mutants with high osmolarity, add NaCCKCj! to the selective medium. , salts such as ammonium sulfate and sodium succinate, and sugars such as sorbitol, glucose, and sinicrose.

The amount added is preferably 20 to 100 g/j'.

A specific method for obtaining the mutant strain used in the present invention is shown below.

KY13184 strain was used as the parent strain, and 100 μg of N-methyl=N'-nitro N-nitrosoguanidine was added to the strain.
/- for 30 minutes at 30°C, the parent strain was grown on a minimal medium agar plate containing NaCl at a concentration (70 g/j?) that inhibited growth (glucose 20 g/β, ammonium chloride 2 g).
/Il, KH2PO, 0.1 g/L K2HP[].

0.3g/R, MgCl2 ・2L00.3g/I, P
e5o<'7H2010mg/I! , MnSO4・4
~6LO1mg#, ZnSO4・lH2O1mg/j2
, CuSO4・5H200,2mg#! , Ca
CA 2・21120 10mg/L L-cystine 40mg#! , I, -asparagine 0.5g #, thiamine hydrochloride 10mg/C calcium pantothenate 20m
g/j! , biotin 60μg/β, nicotine rJ13g
/It, adenine 20mg/I! , guanine 20mg/
j, agar 20g/i', pf 17.2).

After culturing at 30°C for 7 to 10 days, the mutant strain that grows
Among them, a strain with particularly excellent ability to produce 5'-inosinic acid is selected. Corynebacterium ammoniagenes H-
7466 (hereinafter referred to as H-7466 strain) is the − strain.

In addition, a strain with excellent 5'-inosinic acid production ability can be obtained by carrying out the same method as above except for using K(l instead of NaCl2. Corynebacterium ammoniagenes H-7467 ( Below, H-7
There are 467 stocks. ) is - stock among them.

The H-7466 strain and the H-7467 strain were each submitted to the Institute of Microbiology, Agency of Industrial Science and Technology as of May 18, 1989.
7> and No. 2428 (F[!RM BP-2428
) has been deposited under the Budapest Treaty.

KY13184 strain, H-7466 strain and H-7467
Tables 1 and 2 show the growth rates of the strains when they were cultured on minimal medium agar plates containing NaCl2 and KCl at 30°C for 10 days. The osmotic pressure was measured using an osmometer (SMOMETBR) 0M-801 (manufactured by Wogel).

Table 1 Drugs Bacterial Strains To
2542-++ *: Incorrect measurement of 7i in the solution before adding agar.

Table 2 Drug strain 70
2108-++ When culturing the microorganisms used in the present invention, a medium generally used for the fermentation production of nucleic acids is used, and any medium containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc. that can be assimilated by the microorganisms may be used. For example, any medium such as a synthetic medium or a natural medium can be used.

As carbon sources, in addition to hydrolysates such as glucose, fructose, sucrose, molasses, and starch, various organic acids such as acetic acid, fumaric acid, and citric acid, and alcohols such as ethanol and glycerol can be used.

Nitrogen sources include various inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate, amines such as ethylamine, nitrogen-containing compounds such as urea, and peptone, meat extract,
Yeast extract, corn steep liquor, casein hydrolyzate, soybean meal or its hydrolyzate, various types of fermented microbial cells such as amino acid fermentation, nucleic acid fermentation, and their digested products are used.

Inorganic substances include primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, and chloride)
Jum, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used.

Vitamins such as biotin, thiamine, nicotinic acid, and β-alanine, and amino acids such as glutamic acid are also used.

Culture is carried out under aerobic conditions such as shaking and aeration at a temperature of 20°C.
The reaction is carried out at a temperature of -40°C, preferably 25-38°C, and a pH of 5-9, preferably around neutrality, and is usually completed in 2-7 days. The pH of the medium is adjusted using calcium carbonate, inorganic or organic acids, alkaline solutions, ammonia, pH buffers, and the like.

After culturing, 5'-inosinic acid can be collected from the culture solution by removing precipitates such as bacterial cells from the culture solution and using ion exchange treatment, adsorption, extraction, precipitation, etc. can.

Examples of the present invention are shown below.

Example I KY13184 strain, ) I-7466 strain and H-746
Each of the seven strains was inoculated into 300 ml of a seed medium having the following composition in a 21-volume Erlenmeyer flask, and cultured at 30°C for 24 hours.

Seed medium composition Nigelcose 20g/A, peptone 10g/
j! , meat extract 10g/β, yeast extract 5g/β, Na
Cl13g/C biotin 20ug/II, adenine 100mg/It, guanine 100mg/I
, pH 7.2, adjusted with H2SO1, 10 at 120°C.
Sterilized by pressure steaming for minutes.

The resulting seed culture solution was 300 mf! Production medium 3I consisting of the following composition in a 51-capacity culture tank! 96 inoculated at 30℃
Time culture (rotation speed 600 rpm, aeration rate 3β/m1n
) did. The pH during the culture was adjusted to around 6.8 with aqueous ammonia.

Production medium composition Nigelcose 150g #, K) I2P0
゜10g/ffl, 2HPO-10g/II 5
Mg5O<・7) 12010g/R, CaCj! 2
・2H200,1g/j2SZnSO-・78205
mg/j! , Pe504 ・71120 2 button g/Il, MnCA 2 ・4H4H2O
5/L biotin 30J1g/A, calcium pantothenate 10mg/β, vitamin B+ 5mg/β, nicotinic acid 5mg/A, adenine 100mg/R, guanine 100mg/II, corn steep liquor 20
g/β, urea 2g/β (separately sterilized by pressure steaming at 120℃,
5 minutes) Adjust the pH to 7.2 with NaOH or H2SO4, and sterilize by steaming under pressure at 120°C for 10 minutes.

As a result, KY13184 strain, H-7466 strain and H
The 5'-inosinic acid-producing teeth in the culture solution of strain -7467 were 26.5 g/C28, 9 g/β and 29.1, respectively.
g/β.

) The supernatant liquid IIl obtained by removing bacterial cells from the cultured liquid of strain 1-7466 was adjusted to pH 4 with hydrochloric acid, and then added to a resin tower of Diaion SK#1 (H type, manufactured by Mitsubishi Kasei Corporation). After passing through the column, distilled water was immediately passed through the column to obtain an eluate. Thereafter, the resin column was washed with water, and both parts of the aqueous solution containing 5'-inosinic acid at the initial stage of washing were combined with the eluate already obtained, and the pH was adjusted to 7.2 with NaOH. This was concentrated under reduced pressure using a rotary evaporator to obtain 20.3 g of crude crystals of 5'-inosinate sodium salt.

Effects of the Invention According to the present invention, 5'-inosinic acid can be produced industrially and efficiently at low cost.

Patent Applicant (102> Kyowa Hakko Kogyo Co., Ltd. Procedural Amendment (Voluntary) Relationship to the patent applicant's case Postal code 100 Address 6-1 Otemachi, Chiyoda-ku, Tokyo Name Name
(102) Contents of amendment in Column 5 of Detailed Description of the Invention in the specification of Kyowa Hakko Kogyo Co., Ltd. (1) “Seitem-at
Correct icB to r Systematic J.

Claims (1)

[Claims] Belongs to the genus Corynebacterium, 2000mOSMOL/
A microorganism that is resistant to osmotic pressure of KG or more and has the ability to produce 5'-inosinic acid is cultured in a medium, and 5'-inosinic acid is added to the culture medium.
A method for producing 5'-inosinic acid by a fermentation method, which comprises producing and accumulating inosinic acid and collecting 5'-inosinic acid from the culture.