JPS63112997A - Production of riboflavin - Google Patents

Abstract

PURPOSE:To produce the titled substance in high yield, by cultivating yeast belonging to the genus Saccharomyces by continuously adding acetic acid and a nitrogen source to a medium in a culture bath and maintaining concentration of calcium, etc., at <=a fixed value in the production of the titled substance using the yeast. CONSTITUTION:Yeast such as Saccharomyces cerevisiae, etc., belonging to the genus Saccharomyces, capable of producing riboflavin from acetic acid, is inoculated into a medium containing acetic acid, a nitrogen source such as urea, peptone, ammonia water, etc., a phosphoric acid source, etc. Culture is carried out under an aerobic condition by continuously or intermittently supplying the consumed acetic acid and nitrogen source. In the culture, calcium concentration in the medium is controlled to <=0.1.% and ammonium ion concentration to <=1,500ppm. Riboflavin is collected from the prepared culture solution by a well-known method.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing goflavin from acetic acid using yeast of the genus Satucharomyces.

(Prior Art) As for a method for producing riboflavin by a fermentation method, a method is known in which riboflavin is produced and accumulated in a liquid of Eremothecium (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.
l, Chem, ) vol, 28*p, 559.56
6+ 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 Laid-Open No. 60-241895), 3-amino-1,2t4-) belonging to the genus Satucharomyces Method using a mutant strain resistant to lyazole (Japanese Patent Application Laid-Open No. 60-241
No. 896), a method using a purine auxotrophic revertant strain belonging to the genus Satucharomyces (Special Patent No. 120119, 1986), and a method using a mutant strain resistant to ammonium ions belonging to the genus Satucharomyces (Special Patent No. 1, No. 120119, No. 1988). 234
No. 86) is known.

In addition, although Candida robusta (Candida robusta) is used as the name of the microorganism in the above-mentioned document, since spores were subsequently found in the standard method (tights strain) for Candida robusta, In the 1970 edition of ``De East,'' Candida robusta was classified as Saccharomyces cerevisiae.
siae).

In general culture methods, microorganisms are often inoculated into a medium with an appropriate composition, and culture is continued in a batch manner. In contrast, in recent years, a fed-batch culture method has been developed in which a carbon source such as methanol, which inhibits the growth of microorganisms if added in large amounts from the beginning, is added little by little as the culture progresses. When producing baker's yeast, a fed-batch culture method is used in which molasses is added little by little to suppress alcohol by-products.

(Problems to be Solved by the Invention) A method for producing boflavin by cultivating yeast of the genus Satucharomyces in a medium and collecting lysoflavin has already been proposed, such as a method using a mutant strain as described above by the present inventors. However, when producing riboflavin by such a fermentation method in a batch process, a major challenge for industrialization is to increase the concentration of riboflavin accumulated during culture as much as possible. However, when carrying out the method of the present inventors using a mutant strain in a batch method, poflavin production using acetic acid as a carbon source is often good. The ammonium ion concentration in the culture solution was 200.
When the concentration exceeds 0 ppm, yeast growth and riboflavin production tend to be extremely reduced. Furthermore, when acetic acid is used as a carbon source, an acetate such as calcium acetate must be initially added as a carbon source to keep the medium near neutrality, but the calcium concentration in the medium is 0.5% or more. In some cases, a large amount of calcium carbonate accumulates during culture, making it difficult to separate and purify riboflavin after culture.

(An unsuccessful means to solve the problem) In order to improve the method for producing riboflavin from acetic acid by fermentation, the present inventors conducted intensive studies on the above problems using a fed-batch culture method, and found that the In a method for producing lysoflavin from acetic acid using yeast,
By culturing by continuously or intermittently adding acetic acid and a nitrogen source to the medium in the culture tank, the calcium concentration in the culture solution can be reduced to 0.1% or less, and the ammonium ion concentration to 1500ppm or less. The present inventors recognized that lysoflavin can be accumulated in high concentration by maintaining the lysoflavin, and have completed the present invention.

That is, the present invention relates to a method for producing IJ, 35 flavin from acetic acid using yeast of the genus Satucharomyces, in which acetic acid and a nitrogen source are continuously or intermittently added to a culture medium in a culture tank, and culture is carried out. This is a method for producing riboflavin, which is characterized by maintaining the calcium concentration at 0.1% or less and the ammonium ion concentration at 1500 ppm or less.

(Microorganism used) The microorganism used in the present invention is a yeast of the genus Satucharomyces that produces IJ g flavin from acetic acid, and for example, wild strains and mutant strains described in the above-mentioned patents can be used. Specifically, Satsukaromyces cerevische AHU
3402. AHU 3405. P-154 (FE
RM BP-566), TPIOIO (FERM BP
-565), TW-573 (FERM BP-567)
, TR-29 (FERM BP-782), NH-26
8 (FERM BP-965) etc.

(Culture method) In carrying out the present invention, in order to accumulate IJ gflavin in the culture, the riboflavin-producing ability belonging to the genus Satucharomyces is injected into a medium containing acetic acid, a nitrogen source, a phosphoric acid source, and other necessary nutrients. Inoculate yeast and culture. As the acetic acid, any substance that produces acetate ions in an aqueous solution can be used, such as free acetic acid, calcium acetate, sodium acetate, potassium acetate, and ammonium acetate.

Various forms of nitrogen compounds can be used as the nitrogen source, such as ammonium sulfate, ammonium chloride, ammonium carbonate, ammonium nitrate, aqueous ammonia, urea, amino acids, peptone, corn styrene liquor, and the like.

Further, in addition to acetic acid and a nitrogen source, it is preferable to use inorganic salts such as potassium phosphate and magnesium sulfate. In addition, vitamins such as biotin, amino acids,
Should I add micronutrients such as nucleobases? It often increases the amount of furapin accumulated.

It is also possible to apply an improved manufacturing method (Japanese Patent Application Laid-Open No. 60-58088) which was previously invented by the present inventor and which improves the productivity of frapin by adding zinc ions to prevent inhibition by iron ions.

Aerobic conditions are preferred for culturing. - of medium is 2 to 10
However, adjusting it to 6 to 9 will give the most favorable results. A temperature suitable for the growth of the strain used and IJ g flavin productivity can be used within the range of 20°C to 37°C.

In the present invention, it is important to continuously or intermittently replenish and add the acetic acid and nitrogen sources consumed during the culture. This can significantly increase the amount of silipoflavin accumulated. As the nitrogen source used for addition, various ammonium salts such as ammonium sulfate, ammonium chloride, ammonium carbonate, aqueous ammonia, ammonium nitrate, ammonium acetate, and urea are used. In this case, it is preferable to control the calcium concentration in the medium to 0.1% or less and the ammonium ion concentration to 1500 ppm or less. The acetic acid and nitrogen source added during these steps may be added in powder form or in the form of an aqueous solution. When the calcium concentration in the medium is 0.1 h or more,
It becomes difficult to collect poflavin from the culture medium. Furthermore, if the ammonium ion concentration is 1500 ppm or more, yeast growth and lysoflavin production will be reduced, which is not preferable.

Known techniques can be applied to collect riboflavin from the culture fluid thus obtained. That is, the culture solution is heated to 60℃~
After heating to 120° C., the riboflavin is dissolved, centrifuged to separate the yeast cells and the filtrate, the filtrate is concentrated if necessary, and then reduced with hydrosulfide or titanium trichloride to precipitate the riboflavin. After oxidizing the IJ gflavin thus obtained in air,
It is possible to purify it by recrystallizing it using a solvent such as water or aqueous acetic acid solution.

Furthermore, the method for obtaining riboflavin using the present invention (Unexamined Japanese Patent Publication No. 6
1-21096) to make high-purity glue? It is possible to collect frapine crystals.

(Example) Next, examples will be shown.

Example 1 Satsuri Romyces Serepische NH-268 (F'ERM
BP-965) with 2% glucose and 0.0% polypeptone.
5 ml, inoculated into 100 rnl of a liquid medium containing 0.3% yeast extract and 0.3% malt extract, and cultured with shaking at 30°C for 43 hours. This pre-culture solution was mixed with ammonium acetate 0.58%
, caflucium acetate 0.13%, )G(2PO40,
1%, MgSO4・7H200,05%, ZnSO4・
15 to 3000 ml of fermentation medium containing 2011 ppm of 7H
%, and using a 7L melting jar fermentor, the aeration amount was Q, 5 v, v, m, and the rotation speed of the stirring blade was 600.
Culture was started at 30°C. Immediately after the start of culture, -1 was set to be maintained at 7 ± 0.5 - A mixture of 50% acetic acid and ammonium 1-thiacetate was added to the controller to keep the calcium concentration in the medium below 0.1%, and ammonium Culture was continued while keeping the ion concentration below 1500 ppm.

The total amounts of acetic acid and ammonium ions added during the entire period were 913.89.3.1.9, respectively. After 284 hours of culture, the amount of riboflavin accumulated was 5.8 fi/l.

Comparative Example 1 When the ammonium ion concentration in the medium was maintained at about 2000 to 1500 ppm in the same culture as in Example 1, the amount of IJ and pflavin accumulated was 2.911 after 237 hours of culture.
It became /l.

(Effects of the Invention) By continuously or intermittently adding acetic acid and a nitrogen source during the culture, the amount of riboflavin accumulated could be significantly increased. Lysoflavin is a very useful substance as a medicine, feed additive, food coloring agent, etc., and the present invention has made it possible to efficiently produce IJ and H flavins on an industrial scale.

Claims (2)

[Claims] (1) A method for producing riboflavin from acetic acid using yeast of the genus Saccharomyces, which comprises continuously or intermittently adding acetic acid and a nitrogen source to a medium in a culture tank. (2) The method according to claim 1, characterized in that the calcium concentration in the culture solution is maintained at 0.1% or less, and the ammonium ion concentration is maintained at 1500 ppm or less.