JPS635080B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing pyruvic acid by fermentation, and more particularly to a method for efficiently producing pyruvic acid in large quantities by using a specific yeast.

[Prior art and problems to be solved by the invention]

Fermentation methods for producing pyruvic acid include methods using bacteria and filamentous fungi, as well as methods using Saccharomyces cerevisiae.
Methods using some yeasts, such as, are also known.

However, conventional methods have been problematic in industrially mass-producing pyruvic acid.

[Means for solving problems]

Therefore, as a result of repeated research to solve the above problems, the present inventors discovered that Torulopsis
The present inventors have discovered that yeast belonging to the genus produce pyruvate, and in particular, that when the concentration of thiamine in the medium is restricted, they produce a significant amount of pyruvate, and based on this knowledge, they have completed the present invention.

That is, the present invention is a method for producing pyruvic acid, which is characterized by culturing salt yeast that belongs to the genus Torulopsis and has the ability to produce pyruvic acid, and collecting pyruvic acid from the culture.

The microorganism used in the present invention is Torulopsis.
T. etchellsii S-9 strain, T. etchellsii F
-8 strains can be mentioned. These microorganisms were identified by Seiichi Imai et al. (Japan Brewing Association Magazine, Vol. 70, No. 6,
413-415, 1975), and the mycological properties of these strains are as follows.

Growth in glucose-yeast extract-peptone water: After culturing at 25°C for 3 days, it becomes spherical or oval, (2-
4)×(2.5-5) μ, forming a ring on the surface Growth on glucose-yeast extract-peptone agar medium: After culturing at 25°C for 1 month, grayish cream-colored colonies are formed. Pseudohyphae are not formed. figure.

Fermentable glucose + lactose - galactose - melibiose - seuucrose - ruffinose - maltose + meletitose - cellobiose - inulin - trehalose - assimilated carbon compound glucose + D-xylose - galactose - L-arabinose - L-sorbose + D-arabinose - Seuucrose - D-ribose - Maltose + L-rhamnose - Cellobiose - Glycerol + Trehalose - Erythritol - Lactose - Ribitol - Merebiose - Galactitol - Roughinose - Citric acid - Meletitose - Inositol - Inulin - Soluble starch - Assimilation of potassium nitrate: Growth in vitamin-deficient medium: Poor salt tolerance: Growth up to 21% (w/v) Maximum growth temperature: 35°C The mycological properties of the S-9 strain and F-8 strain described above were When compared with the description in "The Yeasts", 2nd edition, it was found that these strains corresponded to Torulopsis ethylci, and they were named Torulopsis ethylci S-9 and Torulopsis F-8, respectively. These strains have been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and their accession numbers are FERM P-8304 for the former and FERM for the latter.
It is P-8331.

Since Torulopsis et al. is halophilic, similar results were predicted for Torulopsis versatilis (T.versatilis), which is also known as a salt-tolerant yeast belonging to the genus Torulopsis, and an experiment was conducted to determine its ability to produce pyruvate. It has become clear that there is a In particular, the D-5 strain and T-38 strain (Seiichi Imai et al., Journal of the Japan Brewing Association, Vol. 70, No. 6, No.
413-415, 1975) had excellent pyruvate production ability.

Growth on glucose-yeast extract-peptone: spherical or short oval shape after 3 days of culture at 25°C;
(2.5-3.5) x (3-4.5) μ, forming a ring on the surface Growth on glucose-yeast extract-peptone agar medium: Colonies produced by slant culture at 25°C for 1 month are slightly brownish milk. Color: Pseudohyphae are not formed.

Fermentability Glucose + melibiose - Galactose + Roughinose - Maltose + Meletitose - Trehalose + Inulin- Lactose+ Assimilation of carbon compounds glucose + L-rhamnose- Galactose + ethanol- L-sorbose- glycerol+ Maltose + Erythritol - Trehalose + Ribitol- Lactose + Galactitol - Inulin-D-glucitol- Soluble starch - citric acid - D-xylose-inositol- D-arabinose- Assimilation of potassium nitrate: Assimilated.

Growth on vitamin-deficient media: poor Growth is improved in the presence of thiamine.

Salt tolerance: Growth at 13% (w/v) Maximum growth temperature: 35℃ The mycological properties of the D-5 strain and T-38 strain described above are described in "The Yeasts", 2nd edition. When compared with Torulopsis, these strains
It was found that they correspond to Torulopsis bellsateilis, and Torulopsis bellsateilis strain D-5 and Torulopsis T-, respectively.
It was named 38 strains. These strains have been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and their accession numbers are FERM P-8329 for the former and FERM P- for the latter.
It is 8330.

Halophile (or salt tolerance) of the above Torulopsis species
The medium used for culturing yeast may be any medium that allows these microorganisms to grow and produce a sufficient amount of pyruvic acid; the carbon source is preferably glucose, maltose, etc., and the nitrogen source is meat extract,
Organic nitrogen compounds such as polypeptone, casamino acids, and amino acids, and inorganic nitrogen compounds such as ammonium sulfate and potassium nitrate are used. Other inorganic substances such as phosphates, magnesium salts, calcium salts, barium salts, etc. are added as appropriate. Furthermore, it is preferable to add a small amount of NaCl as a medium component. In addition,
By controlling the amount of thiamine in the medium, the amount of pyruvate accumulated can be increased. In the present invention, the amount of thiamin in the medium is 0 to 0.
Production of pyruvic acid was observed at 400μg/
In general, when static culture is used, thiamine may not be present or may be contained in a small amount, but when culture is performed with aeration and agitation, a certain amount of thiamine must be present. Normally, it is desirable that the amount of thiamine be 0 to 40 μg/approximately.

Cultivation can be carried out until the amount of pyruvate accumulated is at its maximum, usually at 20-35°C, preferably at 25-35°C.
C. for 2 to 8 days, preferably 3 to 6 days under aerobic conditions.

Confirmation and recovery of the generated pyruvate can be carried out using conventional methods, such as an enzymatic method, i.e., quantification based on the change in absorbance at 340 nm due to the reaction between reduced nicotinamide adenine dinucleotide (NADH) and pyruvate using lactate dehydrogenase. can do.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

Example 1 Glucose 30g, ammonium chloride 2.7g, potassium nitrate 4.0%, thiamine hydrochloride 400μg, potassium phosphate 0.85g, dipotassium phosphate 0.15g,
Magnesium sulfate 0.5g, sodium chloride 80.0g,
Add 0.1 g of calcium chloride and distilled water to make 1 and put 8 ml of culture medium into a test tube with a diameter of 16 mm and a depth of 160 mm, sterilize it at 121°C for 15 minutes, cool it, and prepare Torulopsis et al. S-9 (FERM P- 8304),
Torulopsis F-8 (FERM P-8331), Torulopsis versatheilis D-5 (FERM P-8329) and Torulopsis T-38 (FERM P-8330) were each inoculated with one platinum loop and left at 30°C for 3 days. Seed cultured. Put 100ml of a medium with the same composition as that used for seed culture into a 200ml Erlenmeyer flask, sterilize it under the same conditions, cool it, and then
0.3 ml of the seed culture solution was inoculated and statically cultured at 30°C for 14 days. The amount of pyruvate accumulated is 1.08g/strain S-9.
, F-8 strain 0.88g/, D-5 strain 0.75g/
, T-38 strain was 0.92 g/.

Example 2 Torulopsis ETLC S-9 (FERM P-8304) was seed cultured under the same conditions as Example 1, and 500 ml of a medium with the same composition as that used for seed culture was placed in a 1-volume glass jar fermenter. After sterilizing and cooling under the same conditions, 0.75 ml of the seed culture solution was inoculated and cultured at 30°C for 88 hours with an aeration volume of 0.5 VVM and a stirrer rotation speed of 700 rpm. The amount of pyruvate accumulated is
It was 1.62g/.

Example 3 Seed culture of Torulopsis ETCH S-9 (FERM P-8304) was carried out under the same conditions as in Example 1, and the thiamin content in the medium of the main culture used in Example 1 was changed to 40 μg instead of 400 μg. 500 ml of the culture medium changed to / was placed in a 1-volume glass jar fermentor and sterilized at 121°C for 15 minutes.The cooled medium was inoculated and cultured at 30°C with an aeration rate of 1.0 VVM and 500 rpm.
Accumulated amount of pyruvate when cultured for 146 hours: 5.1g/
It was hot.

〔Effect of the invention〕

According to the present invention, pyruvic acid can be produced in high yield using halophilic yeast belonging to the genus Torulopsis. The obtained pyruvic acid is useful as a flavor component of foods, and is expected to be used in the food manufacturing field.

Claims (1)

[Scope of Claims] 1. A method for producing pyruvic acid, which comprises culturing halophilic yeast belonging to the genus Torulopsis and having the ability to produce pyruvic acid, and collecting pyruvic acid from the culture. 2. The halophilic yeast is Torulopsis ethylci S-9 (FERM P-8304) or Torulopsis ethylci F-8 (FERM P-8331)
The method according to claim 1.