JP2558525B2 - Process for producing pyrimine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for efficiently producing pyrimine useful as an intermediate for ferropyrimine which is a natural pigment.

[Conventional technology]

In recent years, natural pigments have attracted attention from the viewpoint of safety with respect to synthetic pigments. Among them, ferropyrimine, which is a natural red pigment, cultivates a GH strain that belongs to the genus Pseudomonas in the past, produces pyrimine, and at the same time binds iron ions present in the culture to form ferropyrimine, It is carried out by the method of collecting ferropyrimine from the culture (Biochemistry Vol.4 NO.10, 1965).
Year pages 2233 to 2236). Pyrimine has the following structural formula: And has the structural formula described in the above biochemistry.

[Problems to be Solved by the Invention] It is an object of the present invention to provide a method capable of producing pyrimine efficiently.

[Means for solving the problem]

The present invention, by culturing a pyrimine-producing bacterium in a medium containing nutrients such as meat extract and a specific element, it is possible to efficiently produce pyrimine without mixing plants, and to achieve the above-mentioned object efficiently. It was made based on the knowledge that it was possible.

That is, the present invention provides a pyrimine-producing bacterium belonging to the genus Pseudomonas with a pH of 3.8 to 5 containing an organic nitrogen-containing compound, a sugar, an inorganic nitrogen-containing compound, a phosphorus compound and an iron compound, but no calcium ion and chlorine ion. Provided is a method for producing pyrimine, which comprises culturing in a medium and then recovering the produced pyrimine.

Examples of the pyrimine-producing bacterium used here include those having the following mycological properties.

Mycological properties 1. Morphological properties Morphology: Single or not forming bacillus spores that form chains Reactivity: Yes, with polar flagella Large: 0.6-0.8 μx 1.0-3.5 μ Gram stain: Negative 2 .Growth condition in each medium (1) Broth culture Growth: Medium, Film: Ring formation Sediment: Slightly cloudy: Yes (2) Slope culture of broth Shape: Thread, Surface: Smooth and glossy Edge: All Edge, Color: Milky white Transparency: Opaque (3) Broth agar plate culture Shape: Round, Peripheral: All edges, Surface ridge shape: Lenticular Surface: Smooth and glossy, Color: Milky white (4) Broth agar puncture culture Grows on the surface and upper puncture 3. Physiological properties (1) Attitude toward oxygen: aerobic (2) Catalase: + (3) Oxidase: + (4) Arginine dehydrolase: − (5) Lipase (hydrolysis of Tween 80 Solution): + (6) Lecithinase (yolk decomposition) :-( 7) Hydrolysis of gelatin :-( 8) Hydrolysis of starch :-( 9) Hydrolysis of extracellular poly-β-hydroxybutyric acid (PHB): - (10) utilize to independently nutritionally growing H _2: - (11) generated in the dye: King a, B fluorescence medium, glutamate medium, Pseudomonas F agar, Pseudomonas P agar, TSI agar, potato dextrose agar address Does not produce pigment.

(12) Reducibility of nitrate :-( 13) Denitrification reaction :-( 14) Degradation position of protocatechurate: Ortho (15) Intracellular accumulation of PHB: + (16) Levan formation from sucrose :-( 17) ) Growth temperature: 11-35 ℃ Optimum temperature 18-35 ℃ (18) Growth pH: 4.0-8.5 Optimum pH 4.5-8.0 (19) Carbon source assimilation: D-Glucose: + D-Fructose: + D -Arabinose: + Trehalose: ± Mannitol: + Inositol: + Glycerol: + L-Lysine: + L-Valine: ± β-Alanine: + Glycolate: + p-Hydroxybenzoate: + According to the above mycological properties, The categorical status of the strains of the present invention is described in "Bergey's Manual of Systematic Bacteriolog" by Vergie.
y) As a result of volume 1 (1984), this strain is a Gram-staining bacillus that exhibits motility due to polar hairs and is aerobic, catalase-positive, and oxidase-positive, so it is considered to belong to the genus Pseudomonas. It was

Of the genus Pseudomonas, poly-β-hydroxybutyric acid accumulates intracellularly as a carbon source storage substance, cannot grow at 40 ° C., and Pseudomonas solanaceram (as Pseudomonas solanaceram has the property of decomposing protocatechuic acid at the ortho position). Pseudomonas solanacearum) exists.

However, as shown below, D as a denitrification reaction and carbon source
-The property is different from this strain in that arabinose cannot be used.

Therefore, this strain was judged to be appropriate as a new strain belonging to the genus Pseudomonas, and was named Pseudomonas sp. K strain. This strain has been deposited as Microindustrial Research Institute No. 9628.

Also, it has the above-mentioned mycological properties similar to Pseudomonas sp. Pseudomonas sp. K-1 strain (Microtechnology Research Institute
FERM BP-2932) can also be used.

Furthermore, Pseudomonas sp. K strain means that pyrimine is produced at 400 µg / ml or more by culturing with a plant in L-lysine- and LS basic medium, and the period from the culture to the start of producing pyrimine is within 8 days. Pyrimine-producing bacteria of the genus Pseudomonas, for example, Pseudomonas sp. K-2 strain (FERM BP-2933, Institute for Microbiology Research) can be used.

The present invention is characterized by culturing the bacterium by culturing in a specific medium containing an organic nitrogen compound such as meat extract.

Specifically, the medium used in the present invention, organic nitrogen compounds, such as meat extract, proteose peptone, casamino acid, at least one selected from defatted soybean flour and corn stapler, sugar, inorganic nitrogen-containing compounds, Contains phosphorus compounds and iron compounds but no calcium ions. Here, the sugar acts as a carbon source, and examples thereof include sugars such as glucose and fructose, and sugar alcohols such as glycerols. Glucose and fructose are particularly preferable. Examples of the inorganic nitrogen-containing compound include ammonium salts and nitrates. The phosphorus compound is a compound that supplies phosphorus ions, and examples thereof include potassium phosphate and sodium phosphate. The iron compound is a compound that supplies iron ions, such as iron sulfate. If divalent iron ions are allowed to exist in the medium, ferropyrimine can be directly obtained from pyrimine.

Specific examples of the medium used in the present invention include sugar 20 to 80 g, preferably 25 to 75 g, inorganic nitrogen-containing compound 1 to 5 g, preferably 1.5 to 3.5 g, and phosphorus compound 0.2 to 1 g, preferably 0.25 per sugar. ~ 0.6 g and iron compounds 0.01 to 0.2 g, preferably 0.02 to 0.15 g, organic nitrogen-containing compounds 0.1 to 4 g, preferably
An example of the medium is 0.2 to 2 g and the rest is water. In addition, components other than calcium ion and chloride ion can be added to this medium. For example, magnesium compounds such as magnesium sulfate 0 to 0.45 g, EDTA alkali metal salt 0 to 0.2 g, and agar 0 to 20 g. When 1M potassium phosphate buffer (pH 5) is used as the phosphorus compound, the above amount is 100 to 200 g.

The medium for producing pyrimine may be solid or liquid, and can be cultured by various methods, but rotary shaking culture and aeration and stirring culture are preferable. Culture conditions are appropriately selected and adjusted so as to maximize the target amount of accumulated ferropyrimine, and the following conditions are preferable.

Culture medium pH 3.8 to 5.0, culture temperature 15 to 30 ° C, more preferably 20 to 25 ° C, culture time 8 to 20 days. Inoculate approximately 10 ⁵ to 10 ⁷ / ml of bacteria in the medium. Is good. In order to obtain the pyrimine produced in the medium by the above method, separation and purification means can be adopted by a conventional method.

In addition, according to the method of the present invention, the presence of a plant is not necessary for producing pyrimine, but a plant can be present. Plants used here include lilies such as green onions, crucifers such as lettuce, cabbage, wasabi, radish and watercress, Lamiaceae such as perilla, basil and mint, and Polygonaceae such as sorel. Plants, legumes such as pods, plants of the Solanaceae such as tomatoes, eggplants and capsicums, and plants of the Tobacco family such as Saintpaulia can be preferably used.

Here, as the plant, α-naphthyl acetic acid, the mesophyll of the plant on an LS agar medium containing hormones such as kinetin,
It is preferable to use those obtained by aseptically planting sections of stems, rhizomes, etc. and then inducing differentiation.

It is also possible to use the callus or plant cells (tobacco etc.) obtained by sterilizing the plant body and aseptically implanting the explant into the medium (callus induction). The amount of plant added is arbitrary, but 0.01 to 5 g, preferably 0.1 to 0.5 g, per 100 parts by weight of the medium.
It is good to do.

〔The invention's effect〕

According to the present invention, without the presence of plants during culture,
Provided is a novel method for producing pyrimine which is industrially obtained and has high production efficiency. Therefore, the red natural pigment ferropyrimine can be easily obtained only by adding an iron salt to the obtained pyrimine in an aqueous solution.

 Next, the present invention will be described with reference to examples.

Example 1 (a) Glucose 36000 mg dissolved in distilled water 300 ml, (b) NH ₄ NO ₃ 1980 mg, MgSO ₄ .7H ₂ O 222 mg, KH ₂ PO _{4
300mg, FeSO 4 · 7H 2 O} 16.68mg, Na 2 -EDTA 22.38mg
Was dissolved in 300 ml of distilled water and the pH was adjusted to 6.0 with 1N-KOH. (C) 1200 mg of meat extract (manufactured by Difco) was added to 120 ml of distilled water.
(D) 1M potassium phosphate buffer (pH50) 120m
l, after sterilizing (a) to (d) above at 121 ° C for 15 minutes, 2
A volume of jar fermenter (sterilized) was aseptically filled and mixed. The medium of the same composition prepared in advance in the same manner as the above was placed in a 300 ml Erlenmeyer flask, and 2
Inoculate 84 ml of the pre-cultured solution of Pseudomonas sp.
/ Min, 300-400 rpm).

After 1 day of culture, the culture solution was cultured with 1N-KOH and 1N phosphoric acid while maintaining the pH at 4.2 to 4.5.

After 13 days, the culture supernatant obtained by removing the bacterial cells by centrifugation was filtered with a 0.45 μm membrane filter. After the FeSO ₄ .7H ₂ O solution (278 mg /) was added to this solution, the absorbance at 558 nm was measured and the content of ferropyrimine in the culture solution was determined from the calibration curve to be 163 μg / ml.

Example 2 Pyrimine was prepared in the same manner as in Example 1 except that Pseudomonas sp. K-1 strain was used as a pyrimine-producing bacterium and the cells were removed by centrifugation after 8 days instead of after 13 days. Was produced, the content of ferropyrimine in the culture was 1950 μg / ml.

Example 3 Pyrimine was prepared in the same manner as in Example 1 except that Pseudomonas sp. K-2 strain was used as a pyrimine-producing bacterium and the cells were removed by centrifugation after 12 days instead of after 13 days. Was produced, the content of ferropyrimine in the culture was 1940 μg / ml.

Claims (1)

(57) [Claims] 1. A medium containing a pyrimine-producing bacterium belonging to the genus Pseudomonas, containing an organic nitrogen-containing compound, a sugar, an inorganic nitrogen-containing compound, a phosphorus compound and an iron compound, but containing no calcium ion and chloride ion and having a pH of 3.8 to 5. A method for producing pyrimine, which comprises culturing in, and then recovering the produced pyrimine.