JPS6225996A - Production of riboflavin - Google Patents

Abstract

PURPOSE:To obtain in high productivity riboflavin without adding an expensive demanding substance to a medium, by cultivating a purine demanding reverse variant belonging to the genus Saccharomyces, derived from a purine demanding mold capable of producing riboflavin. CONSTITUTION:A purine demanding reverse variant (variant having lost purine demanding properties) belonging to the genus Saccharomyces, derived from a purine demanding mold capable of producing riboflavin, is cultivated in a medium, riboflavin is formed, accumulated and collected. Saccharomyces cerevisiae TR-29 (FERM P-782) may be cited as the preferable strain. This strain is derived by a conventional proceduere by NTG treatment of purine demanding 3-amino-1,2-4-triazole resistant strain TP-1010 (FERM BP-565) as a parent strain.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing riboflavin by fermentation. More specifically, riboflavin is produced and accumulated by culturing in a medium a mutant strain that has lost purine auxotrophy (referred to as a purine auxotrophic revertant strain) derived from a riboflavin-producing bacterium that belongs to the genus Satucharomyces and has purine auxotrophy. The present invention relates to a method for producing riboflavin that involves the collection of riboflavin.

With this method, riboflavin can be efficiently produced by fermentation using acetic acid as a carbon source.

Riboflavin is a substance useful as a medicine, feed additive, food coloring agent, etc.

[Conventional technology]

As a method for producing riboflavin by fermentation, Eremothecium ashb
yii), Ashbya go
ssypii), Candida Fralerii (Can
dida flareri), or Clostridium
Clostridium acet
A method is known in which riboflavin is produced and accumulated in the culture solution by culturing A. obtylicum) in a carbohydrate medium (Progress Industrial Microbiology Vol. 1, p. 139, 1959).

Some of the present inventors have reported a method for producing riboflavin by a fermentation method using acetic acid as a carbon source [Agricultural
and Biological Chemistry (Agr, B
iol, Chem,) vol, 28+ p.

559, p, 566+ p, 765 (1964))
.

Further, as a method using a mutant strain, a method using a purine auxotrophic mutant strain belonging to the genus Satucharomyces by the present inventors (Japanese Patent Application No. 59-99096), 3-amino-1,2,4 -) Method using a mutant strain resistant to lyazole (Patent application 1990!199097)
No.) is known.

In addition, in the above literature, Candida robusta is used as the name of the microorganism, but since spores have since been found in the type strain of Candida robusta, In the 1970 edition of The East J, Candida robusta is referred to as Saccha-romyces cerevisiae.
siae).

However, since spore formation has not been observed in the strain used in the above literature, it is considered to be a non-spore type of Satucharomyces cerevisiae, and in this specification, it is referred to as Satucharomyces cerevisiae (Candida robusta). Describe it.

[Problem that the invention seeks to solve]

Although the above-mentioned bacteria are known, there are still many problems to be solved in order to industrially produce riboflavin by fermentation, and it is of course difficult to obtain bacteria that can accumulate and produce riboflavin at a high concentration. In particular, when conventionally known auxotrophic bacteria are used, it is necessary to add expensive required substances to the culture medium, and reducing or eliminating the amount of the required substances is of great industrial importance.

Based on this viewpoint, the present invention aims to obtain a bacterial strain with high riboflavin productivity and to provide a new method for producing riboflavin using this strain.

[Means for solving problems]

As a result of intensive studies aimed at improving the method for producing riboflavin by fermentation, the present inventors derived a purine-auxotrophic revertant strain of a riboflavin-producing bacterium belonging to the genus Satucharomyces and having purine-auxotrophic properties. The present invention was completed based on the recognition that riboflavin production ability was improved in addition to eliminating the need to add riboflavin to the culture medium.

That is, the present invention is characterized in that a purine auxotrophic revertant strain derived from a riboflavin-producing bacterium belonging to the genus Satucharomyces and having purine auxotrophy is cultured in a medium to produce and accumulate riboflavin, and then collected. This is a method for producing poflavin.

(Microorganism used) The microorganism used in the present invention belongs to the genus Satucharomyces, and any purine auxotrophic revertant strain derived from a riboflavin-producing bacterium having purine auxotrophy can be used.

A preferred specific example is Satucharomyces cerevisiae (Sa
ccharomyces cerevisiae) TR
-29 (Feikoken Jokyo No. 782). This mutant strain is purine auxotrophic, 3-amino-1,2゜4-triazole resistant strain TP-1010 (FERM BP-565
) is a 3-amino-1゜2.4-) lyazole-resistant mutant strain induced by NTG treatment in a conventional manner using the parent strain TP. -1010 can be clearly distinguished.

(Method for Obtaining a Bacterial Strain) The bacterial strain used in the present invention belongs to the genus Satucharomyces, and can be obtained relatively easily by using a riboflavin-producing bacterium with purine auxotrophy as a parent strain and applying a conventional mutation induction treatment method.

For example, using Satucharomyces cerevisiae TP-1010, a 3-amino-1,2,4-triazole-resistant, purine-requiring strain, as a parent strain, after treatment with ultraviolet irradiation or a drug such as NTG, the purines listed in Table 1 were produced. The colonies that grow are selected as purine auxotrophic, 3-amino-L2,4-) lyazole-resistant strains.

Table 1 Minimal medium composition In order to confirm the reversion of purine auxotrophy of the obtained mutant strain, a growth experiment was conducted as follows.

The parent strain Satucharomyces cerevisiae TP-1010 and the purine auxotrophic revertant strain Satucharomyces cerevisiae TR-29 derived from it were plated on the medium shown in Table 1 and the medium to which 0.1% adenine was added.
The cells were cultured at 0°C for one week, and growth was observed. The results are shown in Table 2.

Table 2+: Sufficient growth m: No growth As shown in Table 2, Satucharomyces cerevisiae TR-
29 has obviously lost his pudding-requiring nature (and has returned).
It was confirmed that there is.

(Culture method) A method for culturing the microorganism used in the present invention will be explained. Carbon sources include organic acids such as acetic acid and gluconic acid, carbohydrates such as glucose, sucrose, and xylose, ethanol,
Alcohols such as glycerin and others can be used.

Various forms of nitrogen compounds can be used as the nitrogen source, such as ammonium sulfate, ammonium chloride, ammonium carbonate, urea, amino acids, polypeptone, and the like.

In addition to the carbon source and nitrogen source, it is preferable to use inorganic salts such as potassium phosphate and magnesium sulfate. Furthermore, if necessary, the amount of accumulated riboflavin can be increased in many cases by adding micronutrients such as vitamins such as biotin, amino acids, and nucleobases. It is also possible to apply an improved manufacturing method (Japanese Patent Application No. 165245/1983) which was previously invented by the present inventor and which improves riboflavin productivity by adding zinc ions to prevent inhibition by iron ions.

Aerobic conditions are preferred for culturing. The pH of the medium is between 2 and 1.
The value is set to 0, but the most preferable results can be obtained by adjusting it to 6 to 9. A temperature suitable for the growth of the strain used and riboflavin productivity can be used within the range of 20°C to 37°C.

Known techniques can be applied to collect riboflavin from the culture fluid thus obtained.

That is, the culture solution is heated to 60°C to 120°C to dissolve riboflavin, and then centrifuged to separate yeast cells and filtrate. The filtrate is concentrated as necessary, and then reduced with hydrosulfide or titanium trichloride to dissolve riboflavin. Let it settle. After the riboflavin thus obtained is oxidized in the air, it can be purified by recrystallization using a solvent such as water or an acetic acid aqueous solution.

Furthermore, it is possible to collect highly pure riboflavin crystals using the method for obtaining riboflavin proposed by the present inventors (Japanese Patent Application No. 143953/1982).

〔Example〕

Example 1 Satucharomyces cerevisiae Tl? -29 and its parent strain Satucharomyces cerevisiae TP-1010 were mixed with 2% glucose, 0.5% polypeptone, and 0.0% yeast extract.
100 ml of preculture medium containing 3% malt extract and 0.3% malt extract.
and cultured with shaking at 30°C for 37 hours. This pre-culture solution was inoculated into the fermentation medium shown in Table 3 at a 10% inoculation amount, and 30
The cells were cultured with shaking at ℃ for 10 days.

The amount of riboflavin accumulated in the culture medium was 2.56 g/12 when using the parent strain Satucharomyces cerevisiae TP-1010, whereas when using Satucharomyces cerevisiae TR-29, the amount of riboflavin accumulated in the medium In addition to eliminating the need for purines, which are required substances in the composition, 2.
79g/j! improved.

Example 2 Using Satucharomyces cerevisiae TR-29, 132g of calcium acetate was added to the fermentation medium composition shown in Table 3.
/β, add 6 g/j of ammonium sulfate! Culture was carried out for 8 days in the same manner as in Example 1 using a medium having the same composition as shown in Table 3, except for the following.

As a result, 3.02 g/II of riboflavin was accumulated in the culture solution.

Table 3 Fermentation medium composition [Effects of the invention] According to the present invention, riboflavin could be produced at a high production rate and at a high concentration without adding expensive required substances.

Claims (1)

[Claims] A purine-auxotrophic revertant strain derived from a riboflavin-producing bacterium belonging to the genus Saccharomyces and having purine auxotrophy is cultured in a medium to produce and accumulate riboflavin, which is then collected. Production method.