JPS5846319B2 - Method for producing 5'-inosinic acid by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 5'-inosinic acid by fermentation.

More specifically, the present invention relates to a method for industrially and inexpensively producing 5'-inosinic acid, which is useful as a seasoning, by fermentation.

The inventors have continued to research a fermentation method for producing 5'-inosinic acid, which is useful as a seasoning, using mutant strains belonging to the genus Brevibacterium, and have established an industrial production method (APP 1 iedMicrobiology
, 16.981-987 (1968).

Applied Microbiology 18.
977-984 (1969)).

However, during the cultivation of 5'-inosinic acid in the production of 5'-inosinic acid by fermentation, the product 5'-inosinic acid is mixed with various enzymes (5'-inosinic acid).
It has been observed that inosine is easily degraded and converted into inosine and even hypoxatine by enzymes such as nucleotidase, alkaline phosphoatase, acid phosphorylase, nucleosidase, and nucleoside phosphorylase, and microorganisms possessing each of the above enzymes are widely distributed in nature. Therefore, if bacteria having such enzyme activity contaminate the 5'-inosinic acid fermentation, there have been cases where the accumulated 5'-inosinic acid is immediately decomposed.

As one of the measures to prevent a decrease in the production of 5'-inosinic acid due to contamination with bacteria, it became necessary to strengthen the sterilization conditions of the fermentation medium used for culture and exterminate the bacteria remaining in the fermenter. .

However, the sterilization conditions are, for example, 120℃, 30~6
When intensified to 0 minutes, the 5'-inosinic acid-producing bacteria belonging to the genus Brevibacterium that have been used so far have a 5'-
It became clear that the amount of accumulated inosinic acid was significantly reduced.

As a result of intensive study by the inventors to improve this point,
The present invention has been completed by successfully inducing and isolating a mutant strain in which the amount of 5'-inosinic acid accumulated does not substantially decrease even when the sterilization conditions of the medium are strengthened.

This invention has succeeded in industrially improving the production method of 5'-inosinic acid by fermentation.

The microorganism used in the present invention belongs to the genus Brevibacterium and is a mutant strain that requires at least adenine for growth and has the ability to accumulate 5' inosinic acid, and
Fermentation medium sterilized by pressure steaming for 0 minutes [Glucose 130♂
/l, KH2PO410'if/, e, 2HPO41
0? /l! , Mg504-7H2010P/l, j −
7 Sue f--7"! J Car 20 ?/1!, CaCl
2 ・2H200,1? /L FeSO4・7H201
oyno/, e, ZnSO4・7H2027n? /l,
MnCl2・4H202■/l, biotin 3oμfb'
l, vitamin B, 5■/L calcium pantothenate 10
■/L Nicotinic acid 5■/l, adenine 100■/l, guanine 100■/l, urea 4.0? /, e (separately 12
The production of 5'-inosinic acid in the above fermentation medium sterilized by pressure steaming at 120°C for 10 minutes is the same as that of the fermentation medium sterilized by pressure steaming at 120°C for 10 minutes. It is a mutant strain that has properties that do not substantially decrease compared to the amount.

Such a mutant strain belonging to the genus Brevibacterium requires at least adenine for growth and can be isolated from a mutant strain that accumulates 5'-inosinic acid by natural mutation or artificial mutation by mutation treatment using a mutagenic agent. I can do it.

A suitable mutant strain is Brevibacterium ammoniakenes strain KY13196, which was induced and isolated from Brevibacterium ammoniagenes strain KY13184, which requires adenine and guanine and accumulates 5'-inosinic acid, after mutation treatment with nitrosoguanidine. I can do it.

The results of the influence of the pressure steaming sterilization conditions of the fermentation medium on the accumulation of 5'-inosinic acid shown in Table 1 specifically and clearly demonstrate the characteristics of the microorganism used in the present invention.

That is, 25 of the above two strains, Brevibacterium ammoniagenes KY13184 strain and KY13196 strain, were
In a 0rrLl Erlenmeyer flask, glucose 2♂/dl,
Peptone 1g/dl, meat extract 1? /dl, yeast ff
4-su0.5? /di, salt 0.3? /di, (p
A loopful of the seed culture medium 201'Ll consisting of H7, 2) was inoculated and cultured at 30°C for 24 hours. The seed culture was then sterilized by changing the pressure steaming conditions of the fermentation medium with the above composition at 120°C for 10 to 60 minutes. With baffle plate 2 including culture medium pot 20rd
Inoculate a 50ml Erlenmeyer flask at a rate of 10% volume,
Culture was carried out with stirring (220 rpm) at 30° C. for 4 days.

0.29/d of separately sterilized urea at 48 and 72 hours.
It was fed at a rate of 1.

Table 1 shows the amount of 5'-inosinic acid accumulated as a result.

As is clear from this, the parent strain Brevibacterium ammoniagenes strain KY131.84 is
KY13196 when sterilized by pressure steaming for 10 minutes at 20℃
Although it shows the same accumulation of 5'-inosinic acid as the strain, the amount of accumulation decreases significantly when the steaming time is increased from 30 to 60 minutes.

In the KY13196 strain, the amount of 5'-inosinic acid accumulated did not substantially change even when the pressure-cooking time of the fermentation medium was increased.

This shows that, as mentioned above, it is very useful to use microorganisms having characteristics like the KY13196 strain in actual industrial production from the viewpoint of preventing bacterial contamination.

The above example shows the same sterilization effect as the steaming temperature and time of 120℃ and 30 to 60 minutes.
Table 2 shows the amount of 57-inosinic acid accumulated at 115° C. for 1 hour and 30 minutes.

The microorganism used in the present invention belongs to the genus Brevibacterium, and is a mutant strain that requires at least adenine for growth and accumulates 5'-inosinic acid. Although this is a mutant strain that has characteristics that are not affected by pressure steaming conditions, it is naturally possible to use mutant strains that have additional nutritional requirements, etc., as long as they have these characteristics.

In fact, the aforementioned Brevibacterium ammoniagenes K.
In addition to adenine auxotrophy, strain Y13196 also has guanine auxotrophy.

In this case, of course, substances required for growth must be added to the fermentation medium as appropriate.

Furthermore, the medium composition described in the claims is merely a medium composition used to clarify the characteristics of the microorganism used in the present invention, and is different from the fermentation medium composition used for the accumulation of 5'-inosinic acid. Of course, that is also possible.

That is, the medium composition used in the present invention includes a carbon source,
Either a synthetic medium or a natural medium can be used as long as the medium contains appropriate amounts of organic and inorganic nitrogen compounds, inorganic salts, and other nutrient sources.

As the carbon source and nitrogen source to be used in the culture medium, any type can be used as long as it is available to the strain used.

In other words, carbon sources include glucose, fructose,
Carbohydrates such as sucrose, glycerol, starch, starch hydrolyzate, molasses, molasses hydrolyzate, gluconic acid,
Various organic acids such as pyruvic acid, lactic acid and acetic acid, and amino acids such as glycine, glutamic acid, alanine and aspartic acid can also be suitably used.

Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium acetate, urea, peptone, NZ-amine,
Various substances such as meat extract, yeast extract, corn steep liquor, casein hydrolyzate, nitrogenous organic substances such as fish meal or its digested product, glycine, and glutamic acid are suitable.

Furthermore, preferred inorganic substances include phosphates, magnesium salts, calcium salts, iron salts, zinc salts, manganese salts, and the like.

The addition of external vitamins (vitamin B1, calcium pantothenate or β-alanine, nicotinic acid, biotin) is necessary depending on the microorganism used.

Also, as adenine sources, adenine, atesosine, 5'-
The addition of adenine-containing substances such as adenylic acid and ribonucleic acid or their hydrolysates is necessary.

When using microorganisms that exhibit auxotrophy, it is necessary to add to the medium a substance that satisfies their growth requirements.

Fermentation is carried out under aerobic conditions such as shaking culture or submerged culture with aeration and stirring.

The culture temperature is preferably 20 to 40°C.

The culture period is usually 2 to 7 days, and the pH of the culture medium is preferably kept near neutral using aqueous ammonia, urea solution, hydroxide solution, etc.

Thus, a significant amount of 57-inosinic acid accumulates in the culture solution and in the bacterial cells.

After the cultivation is completed, 5'-inosinic acid is recovered from the culture solution using an ion exchange resin as shown in Example 1.

Other known ion exchange resin treatment methods, adsorption methods, precipitation methods,
5'-inosinic acid can also be recovered by an extraction method.

Next, examples will be shown.

Example 1 Brevibacterium ammoniagenes KY as inoculum
13196 strain (Feikoken Bacterial Serial No. 3791) was used, and this bacterium was treated with glucose 2y/dl, peptone 1f/dl, and meat extract 1? /dl, yeast extract 0.5g/dl, salt 0
．． 3? /dl, biotin 20μf/L, adenine 10
Medium 3 having a composition of 0 ■/L and guanine 100 ■/L
Cultured in 21 Erlenmeyer flasks containing 00TLl at 30°C for 24 hours was inoculated into 51 jar fermenters containing 31 fermentation media.

The fermentation medium used had the following composition and was heated at 45°C at 120°C.
The material was sterilized by steaming under pressure for a minute.

Fermentation is carried out at 30°C with stirring at 600 revolutions per minute.
Culture was performed with aeration of 1.

The pH during fermentation was adjusted to around 6.8 with aqueous ammonia.

Glucose 15g/dL KH2PO41,Of/dl
, K2HPO41, Of /dl, MgSO4・7H
201°0? /d, l, CaC12・2 H200
-01? /lil, ZnSO4・7H205■/L,
FeSO4・7H20207V/L, MnCl2
・4 H205"9/L, biotin 30μP/L, calcium pantothenate 10■/L, vitamin B15■/L
, nicotinic acid 5 ■/L, adenine 100 ■/L, guanine 100 ■/L, corn stew liqueur - 20? /di,
Urea 0 or 2? /dl (separate sterilization at 120℃ for 5 minutes) pH
7.6° 28.4 m9/rul of 5'-inosinic acid was accumulated in the fermentation liquid cultured in this manner for 96 hours.

In addition, KY13184 strain, which is the parent strain of KY13196 strain (
5) was cultured under the same conditions.
The amount of accumulated '-inosinic acid was 16.4 μ/ml.

In addition, the fermentation medium was sterilized by pressure steaming at 120°C for 10 minutes, and cultured under the same conditions.
28.9 rrbg/ml and KY13184 for 96 strains
In the strain, 5'-inosinic acid accumulation of 29.1 cm/share was observed.

The clear liquid 11 obtained by removing bacterial cells from the culture solution of KY13196 strain was adjusted to pH 1.4 with hydrochloric acid, and then treated with Diaion SK#.
1 (H type) resin tower.

Immediately thereafter, distilled water is passed through the column, and the fraction containing 5'-inosinic acid of the aqueous solution at the initial stage of washing, which is passed following the P solution, is combined and the pH is adjusted to 7.2 with sodium hydroxide.

By concentrating this under reduced pressure, crude crystals of 5'-inosinate sodium salt 22.4S' were obtained.

Example 2 The bacterium used was Brevibacterium used in Example 1.
Ammoniagenes strain KY13196 was used, and the fermentation medium had the following composition, and sterilized by pressure steaming at 120° C. for 60 minutes.

Other conditions were the same as in Example 1.

Molasses hydrolyzate (glucose equivalent 17'?/d0, Na
H2PO40.5? /dl, K2 HPO41,5
? /dl, MgSO4・7H201,5g/dl, vitamin B15mp/L, β-alanine 10mg/l, nicotinic acid 5μ/L1 biotin 50μf/L, adenosine zoomq/j, guanosine 200m9/l, urea 0.
2 g/dl (separate sterilization at 120°C for 5 minutes), MnCl 2
・nH205rn9/L.

57-inosinic acid was 26.8 my/m after 120 hours of culture.
l accumulated.

When the KY13184 strain, which is the parent strain of the KY13196 strain, was cultured under the same conditions, the amount of 5'-inosinic acid accumulated was 14.2 μ/d.

In addition, when the fermentation medium was sterilized by pressure steaming at 120°C for 10 minutes, and other conditions were the same, KY1
27, 1 m9/ml and KY131 for 3196 strains
27, 31nI for 84 strains? /vLl accumulation of 5'-inosinic acid was observed.

Example 3 The bacteria used was Brevibacterium used in Example 1.
Ammoniagenes strain KY13196 was used, and the fermentation medium had the following composition, and sterilized by pressure steaming at 120° C. for 60 minutes.

Other conditions were the same as in Example 1.

Molasses hydrolyzate (glucose equivalent 17'/dl), N
aH2PO,o, 5? /dl, K2HPO41
,5? /d, l, MgSO4・7H201,5? /dl
, (NH4)2S041.8ft/dl, vitamin B,
5■/l, β-alanine 10■/l, nicotinic acid 5■/l
l, biotin 50 μ? /11 atenoshi720 omq
/l! , Liy/Schiff 2001ng/l, MnCL, -
5'-inosinic acid was accumulated at 26.0 μ/rrLl by culturing at 5 mg of nH2O/10120 hours.

When the KY13184 strain, which is the parent strain of the KY13196 strain, was cultured under the same conditions, the amount of 5'-inosinic acid accumulated was 14.0 μ/roll.

In addition, the fermentation medium was sterilized by pressure steaming at 120°C for 10 minutes, and the other conditions were the same.
26.9 m/? with 13196 plants? /ml and KY13
In strain 184, 57-inosinic acid accumulation of 27.1 yny/rnl was observed.

Claims (1)

[Scope of Claims] 1 A 5'-inosinic acid-producing strain belonging to the genus Brevibacterium and requiring at least adenine for growth is cultured in a nutrient medium, and 5'-inosinic acid is produced and accumulated in the culture. , 5'-
In the method for producing inosinic acid, a fermentation medium [Glucose 130? /
l, KH2PO410? /L K2HPO410te/
l, MgSO4・7H2010g/l, Corn Steep Liquor-20? /l. CaCl2・2H200,1g/11FeSO4・7H
2010? /L ZnSO4・7 H2O2m9/l,
MnMnCl2-4H202/l:, biotin 30d'
/J vitamin B, 5■/l, calcium pantothenate 1 omy/l, nicotinic acid 5m9/l, adenine 1
001ru? /l, guanine 1o omq/l:, urea 4.0? /l (separately sterilized at 120°C for 5 minutes; (1) H7, 6)). A mutant strain that does not substantially reduce the production amount of '-inosinic acid, and
It is characterized by using a nutrient medium that is sterilized by pressure steaming for 0 to 60 minutes, or sterilized by pressure steaming under conditions of temperature and time that can achieve the same sterilization effect, and that uses carbohydrates as the main carbon source. A method for producing 5'-inosinic acid.