JPH0566115B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) This invention relates to a method for producing riboflavin by a fermentation method. More specifically, the present invention relates to a production method in which a riboflavin-producing bacterium belonging to the genus Satucharomyces and having resistance to 3-amino-1,2,4-triazole is cultured in a medium, and the produced and accumulated riboflavin is collected. 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. (Prior art) As a method for producing riboflavin by a fermentation method, Eremothecium acyubii, Acyuvia gossytpii, Candida fullerii, Clostridium acetobutylicum, etc. are cultured in a carbohydrate medium to produce and accumulate riboflavin in the culture solution. method is known. (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.
(Agr.Biol.Chem.vol.28, p.559, p.56, p.765
(1964)) 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, the Author The East 1970
In the 2015 edition, Quyandida robusta was replaced by Saccharomyces cerevisiae.
cerevisice). However, regarding the bacterial strains used in the above literature,
Since no sporulation has been observed, it is considered to be a non-spore type of Satucharomyces cerevisiae, and is referred to herein as Satucharomyces cerevisiae (Candeida robusta). (Objective of the Invention) Although the above-mentioned bacteria are known, there are still many problems to be solved in order to industrially produce riboflavin by the fermentation method. It is important to obtain Based on this viewpoint, an object of the present invention is to obtain a bacterial strain with high riboflavin productivity and to provide a new method for producing riboflavin using this strain. (Structure of the Invention) As a result of intensive research into the improvement of the above-mentioned Satucharomyces cerevisiae (Candeida robusta), the present inventor found that a mutant strain endowed with 3-amino-1,2,4-triazole resistance produced riboflavin. The present invention was completed based on the discovery that a bacterial strain with high virulence can be obtained. That is, the present invention belongs to the genus Satucharomyces 3
This is a method for producing riboflavin, which comprises culturing riboflavin-producing bacteria having -amino-1,2,4-triazole resistance in a medium, producing and accumulating riboflavin, and collecting the same. (Microorganisms used) The microorganisms used in the present invention can be any riboflavin-producing bacteria that belong to the genus Satucharomyces and have resistance to 3-amino-1,2,4-triazole. , 4-triazole resistance, which distinguishes it from the bacteria used in the prior art. A specific example of a suitable strain is 3-amino-1, derived from Satucharomyces cerevisiae (Candeida robusta AHU3405).
A 2,4-triazole resistant mutant, Satucharomyces cerevisiae TW573
), Satucharomyces cerevisiae P-154 (Feikoken Bibori No. 7562), and Satucharomyces cerevisiae TP-1010 (Feikoku Kenbibi No. 7564). Satsukaromyces cerevisiae TP-1010 is
This is a mutant strain derived from Satucharomyces cerevisiae P-154 that has purine auxotrophy. (Method for Obtaining a Bacterial Strain) The strain used in the present invention can be obtained relatively easily by using a riboflavin-producing bacterium belonging to the genus Satucharomyces as a parent strain and applying a conventional mutation induction treatment method. For example, using Saccharomyces cerevisiae (Candeida robusta AHU3405) (this bacterium is a preserved bacterium listed by the Hokkaido University Faculty of Agriculture) as a parent strain, UV irradiation or N-methyl-
After treatment with a drug such as N'-nitro-N-nitrosoguanidine, an agar medium containing the composition shown in Table 1 plus an amount of 3-amino-1,2,4-triazole that makes it impossible for the parent strain to grow. The colonies that grow are selected as 3-amino 1,2,4-triazole resistant mutants. When the parent strain used exhibits auxotrophy, 3-amino 1,2,4-triazole resistant mutants can be selected in the same manner by adding auxotrophic components to the medium composition shown in Table 1.

[Table] In order to confirm the 3-amino-1,2,4-triazole resistance of the obtained mutant strain, 3-amino-
A growth experiment on 1,2,4-triazole was conducted as follows. That is, the parent strain and the 3-amino-1,2,4-triazole-resistant strain derived from it were washed with physiological saline, and the suspension was mixed with 3-amino-1,2,4-triazole in the amount shown in Table 2. - They were each inoculated into 5 ml of the medium listed in Table 1 supplemented with triazole, cultured at 30°C for 2 days, and the degree of growth at this point was measured by absorbance at 610 nm.
Table 2 shows the relative growth rate when the growth rate without the addition of 3-amino-1,2,4-triazole is set as 100. Regarding Satucharomyces cerevisiae P-154 and Satucharomyces cerevisiae TP-1010 derived from it, please refer to Part 1.
A medium with the composition shown in the table to which 0.005% of Aden was added was used. From Table 2, Satscharomyces cerevisiae
It is confirmed that TW573, S. cerevisiae P-154, and S. cerevisiae TP-1010 are clearly endowed with 3-amino-1,2,4-triazole resistance.

[Table] (Culture method) The method for culturing the microorganisms used in the present invention will be explained. As carbon sources, acetic acid, gluconic acid, etc., organic acids, carbohydrates such as glucose, sucrose, xylose, etc., alcohols such as ethanol, glycerin, etc. 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 carbon sources and nitrogen sources, inorganic salts such as potassium phosphate and magnesium sulfate are used. Also 3
It is of course necessary to add an auxotrophic substance to mutant strains that have properties other than -amino-1,2,4-triazole resistance, such as auxotrophy. 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 has been found that zinc addition has no particular effect on the production of riboflavin when yeast stored on agar media are inoculated directly into the riboflavin production medium. However, when producing riboflavin after pre-cultivating yeast on agar medium in a liquid medium, unlike the case of direct inoculation, the productivity of riboflavin is significantly improved by adding a small amount of zinc. The inhibitory effect of ions can be prevented. This improved manufacturing method (Japanese Unexamined Patent Publication No. 58-
165245) can also be applied. The amount of the preculture solution inoculated into the riboflavin production medium is preferably 3 to 25%. The amount of zinc ions added is between 0.1 and 100 mg, but the optimal concentration varies depending on the iron ion concentration in the medium. For example, if iron ions are less than 0.1 mg, zinc ions of about 0.5 mg/are sufficient; When ions are present at 5 mg, it is necessary to add about 10 to 30 mg/. Aerobic conditions are preferred for culturing. The pH of the medium is 2
The range is set between 6 and 10, but the most favorable results are obtained by adjusting the range between 6 and 9. Temperature is 20℃ to 37℃
A temperature suitable for the growth of the strain used and the productivity of riboflavin can be used within the range of °C. Known techniques can be applied to collect riboflavin from the culture fluid thus obtained. That is, after heating the culture solution to 60°C to 120°C to dissolve riboflavin, it is separated into yeast cells and filtrate by centrifugation, the filtrate is concentrated as necessary, and then reduced with hydrosulfite or titanium trichloride. and precipitate riboflavin. After the riboflavin thus obtained is oxidized in air, it can be purified by recrystallization using a solvent such as water or an acetic acid aqueous solution. Unlike the case of using molasses as a substrate, the present invention does not contain impurities with a complex composition in the culture solution.
It is also possible to collect riboflavin crystals of good purity by the following simple process.
That is, water is added to the culture solution or a mixture of bacterial cells and riboflavin crystals obtained by cooling and centrifuging the culture solution, heated to 60°C to 120°C to dissolve the riboflavin, and then the bacterial cells are removed by hot filtration. and filtrate. Crystalline riboflavin separated from bacterial cells can be obtained by concentrating the filtrate as necessary and then cooling it again. High purity riboflavin crystals can be obtained by recrystallizing these crystals using a solvent such as water, aqueous acetic acid, or aqueous hydrochloric acid. This will be explained below using examples. Example 1 Satucharomyces cerevisiae TW-573 was mixed with 2% glucose, 0.5% polypeptone, and yeast extract.
100 ml preculture medium containing 0.3% malt extract and 0.3% malt extract
and cultured with shaking at 30°C for 30 hours. This preculture solution was inoculated into the following fermentation medium at an inoculation amount of 12.6%, and cultured with shaking at 30°C for 6 days. The amount of riboflavin accumulated in the culture solution was 1.47g/. For comparison, Satsukaromyces cerevisiae TW-
573, its parent strain Satsucharomyces
Cerevisiae (Quillandida Robusta)
AHU3405), the amount of riboflavin accumulated in the culture solution was 0.85 g/. Fermentation medium composition Calcium acetate 103g/(NH ₄ ) ₂ SO ₄ 3g/ KH ₂ PO ₄ 2g/ MgSO ₄・7H ₂ O 1g/ ZnSO ₄・7H ₂ O 2.2mg/ PH 7.0 Culture solution thus obtained Cooling 1.2,
By centrifugation, a mixture of yeast cells and riboflavin crystals was obtained as a precipitate. Water 2 was added to this precipitate, extracted at 80℃ for 1.5 hours, the hot water extract was concentrated to 1, and then cooled to obtain a purity of 97.3%.
895 mg of riboflavin crystals were obtained. Example 2 Satucharomyces cerevisiae TW-573, which had been liquid-precultured in the same manner as in Example 1, was cultured with shaking at 30°C for 9 days to produce riboflavin. The composition of the fermentation medium was the same as in Example 1 except that ammonium sulfate was 3.8 g/min and the zinc concentration was changed. The result is the third
It was as shown in the table.

[Table] Example 3 Satucharomyces cerevisiae TP-1010 was cultured in the same manner as in Example 1. The fermentation medium had the composition of Example 1 plus 0.1% adene. As a result, TP-1010 accumulated 2.5 g/riboflavin in the culture medium. Example 4 Satucharomyces cerevis pre-cultured in the same manner as in Example 1 was cultured on S. TP-1010 at 30° C. for 10 days to produce riboflavin. The composition of the fermentation medium was the same as in Example 3 except that ammonium sulfate was 3.8 g/min and the zinc concentration was changed. The results were as shown in Table 4.

[Table] Example 5 Satucharomyces cerevisiae P-154 was mixed with 2% glucose, 0.5% polypeptone, and 0.3 yeast extract.
%, and 100 ml of preculture medium containing 0.3% malt extract, and cultured with shaking at 30°C for 30 hours. This pre-culture solution was inoculated into the following fermentation medium at an inoculation amount of 12.6%, and 30
The cells were cultured with shaking at ℃ for 6 days. The amount of riboflavin accumulated in the culture solution was 1.55g/. Fermentation culture composition Calcium acetate 103g / (NH ₄ ) ₂ SO ₄ 3g / KH ₂ PO ₄ 2g / MgSO ₄・7H ₂ O 1g / ZnSO ₄・7H ₂ O 2.2mg / Adenine 1g / PH 7.0

Claims (1)

[Claims] 1. A mutant strain belonging to the genus Saccharomyces that is resistant to 3-amino-1,2,4-triazole and capable of producing riboflavin is cultivated to produce and accumulate riboflavin. ,
A method for producing riboflavin, which comprises collecting riboflavin from a medium. 2 The mutant strain is Satucharomyces cerevisiae
The method for producing riboflavin according to claim 1, which is TW573, Satucharomyces cerevisiae P-154, or Satucharomyces cerevisiae TP-1010.