JP2837624B2 - Method for producing natural purple pigment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a natural violet pigment which becomes a blue dye. More specifically, the present invention relates to a method for producing a natural purple pigment comprising bioracein by culturing a microorganism having bioracein-producing ability belonging to the genus Chromobacterium (hereinafter referred to as bioracein-producing bacterium).

[0002]

2. Description of the Related Art Biolacein is contained in cells of microorganisms belonging to the genus Chromobacterium or the genus Janthinobacterium . This dye has a maximum absorption wavelength of 579 nm in an ethanol solution, and has a feature of having an absorption maximum at a wavelength of 700 nm in a sulfuric acid / ethanol mixed solution in which the mixing ratio of sulfuric acid to ethanol is 10% by volume. As a method for producing this dye, there is known a method in which tryptophan or peptone in which tryptophan is not decomposed is added to a culture medium for culturing bioracein-producing bacteria, followed by culturing. However, in this method, an expensive reagent such as tryptophan or peptone is used for the culture medium, so that the cost of producing the dye is naturally high, and even if these media are used, the extraction of the dye is not easy, and the production amount is also low. Is also low. For this reason, for industrial use, a more inexpensive and more efficient method has been desired.

[0003] In order to solve this problem, polyurethane foam or cellulose is added as an adsorption carrier to a medium for culturing bioracein-producing bacteria belonging to the genus Yantinobacterium, and the produced dye is adsorbed on the carrier. A method for producing this type of dye has been disclosed (JP-A-2-39892). Also, one of the present inventors has invented a production method for producing bioracein at low cost using a medium using animal hair degradation products instead of peptone, and has filed a patent application (Japanese Patent Application No. 5-46363).

[0004]

SUMMARY OF THE INVENTION However, Japanese Patent Application Laid-Open No. 2-3
The production method disclosed in Japanese Patent No. 9892 uses a relatively large amount of an expensive culture medium such as peptone, and the amount of the pigment produced is not industrially satisfactory. In addition, the bioracein-producing bacteria belonging to the genus Yanthinobacterium used in this production method have a markedly reduced pigment-producing ability at a normal culturing temperature of about 30 ° C., and the medium must be cooled in order to produce bioracein. is necessary. Therefore, this method has a problem that the production cost is high due to the increase in the cost of obtaining the culture medium and the cost of cooling.
Although the production method according to Japanese Patent Application No. 5-46363 has the advantage of greatly reducing the production cost of bioracein because a cheaply available animal hair degradation product is used for the culture medium, it is necessary to increase the production amount of bioracein. Animal hair degradation products must be added to the medium in large amounts. In this case, since foaming due to aeration, stirring and the like during the cultivation of the bioracein-producing bacterium becomes intense, an infinitely large amount of animal hair degradation products cannot be added, and there is a limit on the amount of bioracein produced.

On the other hand, it is known that tryptophan exists as a metabolic intermediate in the biosynthetic pathway of bioracein. In fact, it is known that when tryptophan is added to a culture solution, the production of bioracein is increased. ing. However, tryptophan is a very expensive reagent, and its use as a precursor to bioracein greatly increases production costs. The present inventors have focused on the fact that anthranilic acid contained in the intermediate of this tryptophan synthesis pathway can be obtained relatively inexpensively, and by adding anthranilic acid to the culture medium of conventional bioracein-producing bacteria, the yield of bioracein is reduced. The present inventors have found that they can be greatly improved, and have reached the present invention.

An object of the present invention is to add inexpensive anthranilic acid to the culture medium of bioracein-producing bacteria,
It is an object of the present invention to provide a method capable of significantly increasing the yield of bioracene and industrially inexpensively and stably producing a natural purple pigment composed of bioracein.

[0007]

According to the method for producing a natural purple pigment of the present invention, a bioracein-producing bacterium belonging to the genus Chromobacterium is inoculated in a liquid medium containing a tryptophan source, and the bioracein-producing bacterium is cultured in the liquid medium. This is a method in which a culture solution in which the bioracein-producing bacteria have grown is separated into cells and a liquid medium, and a natural purple pigment composed of bioracein is extracted from either or both the cells and the liquid medium. The characteristic configuration is that anthranilic acid or a salt thereof is sequentially or continuously added at a predetermined concentration to the liquid medium before or during the culture.

Hereinafter, the present invention will be described in detail. The microorganism used in the present invention may be any strain belonging to the genus Chromobacterium and producing bioracein, that is, a bioracein-producing bacterium, and is not particularly limited. As a bioracein-producing bacterium, there is a bacterium belonging to the genus Yantinobacterium, but a bacterium belonging to the genus Chromobacterium having a wide range of cultivation temperatures is selected. As a representative example of the bioracein-producing bacteria belonging to the genus Chromobacterium, Chromobacterium violaceum (Col-B) isolated from factory wastewater in Aichi Prefecture by the present inventors is described.
Strains. Next, the bacteriological properties of this strain will be described.

A. Morphological properties (a) Cell shape and size: 0.8 × 1.5 μm
(b) Motility: Yes (c) Presence or absence of spores: No (d) Epiphytic state of flagella: Periflagellate (e) Gram stain:-B. Physiological properties (a) Oxidase: + (b) Catalase: + (c) Lipase:-(d) Urease:-(e) Degradation of casein: + (f) Degradation of esculin:-(g) Degradation of starch:-(h) Formation of indole:-(i) Liquefaction of gelatin: + (j) arginine dihydrolase: + (k) assimilation of carbon source D-glucose: + starch: + D-sorbitol:-inositol:-D-xylose:-galactose:-D-arabinose: -Sucrose:-D-fructose:-Sodium acetate:-Lactose:-From the above bacteriological properties, "Virgie's Manual of Systematic Bacterio Gee "(BERGEY'S M
ANUAL of Systematic Bacteriology Volume 1)
Although the assimilation properties of fructose did not match, this strain was considered to belong to Chromobacterium bioraceum because of the other bacteriological properties. Therefore, this strain was named Chromobacterium violaceum Col-B strain and deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. The deposit number is FERM P-14136.

[0010] The medium for producing bioracein of the present invention is a liquid medium containing a tryptophan source. This liquid medium is prepared by adding a tryptophan source such as peptone or animal hair degradation product to an inorganic salt liquid medium. The inorganic salt medium is composed of an inorganic salt used for culturing a normal microorganism. Examples of the inorganic salt include a phosphate, a magnesium salt, a potassium salt, and a calcium salt, and the inorganic salt medium is used in combination. As the peptone to be added to the inorganic salt medium, a peptone in which tryptophan is not decomposed is used. In the present invention, when this peptone is used as a tryptophan source, a larger amount of bioracein can be produced by simply adding the peptone to the inorganic salt medium than in the past. Animal hair degradation products can also be used as a tryptophan source. As the animal hair source of this animal hair degradation product, wool is easily available and suitable, but is not particularly limited as long as it can extract keratin proteins such as angora, cashmere, mohair, and bird feathers. In order to obtain this animal hair degradation product, a commercially available hydrolyzed keratin solution can be purchased or a conventional method (P. Alexander, et al., Wool,
p.356, Reinhold, New York, 1954). When the animal hair is prepared according to a conventional method, the animal hair hydrolyzed with an oxidizing agent such as formic acid, peracetic acid or hydrogen peroxide is dissolved in 0.3N aqueous ammonia, and the solution is adjusted to pH 4
After the following, the insoluble protein in this solution is separated by filtration or centrifuged and dried.

[0011] The pH of the liquid medium for producing bioracein is determined by the growth rate of the microbial bioracein-producing bacteria.
Since H is in the range of 5 to 10, it is adjusted to a range from weakly acidic to weakly alkaline, and preferably around neutral. Specifically, considering the growth of bioracein-producing bacteria, the pH is 5
To 9 are preferable, and 6.5 to 8 are more preferable. The culture temperature is preferably from 20 to 35 ° C, more preferably from 25 to 30 ° C. Culture must be performed aerobically.

The anthranilic acid used in the present invention is added in advance to the liquid medium before culturing, or is added in a predetermined amount during culturing. The addition amount is preferably in a concentration range in which growth inhibition by anthranilic acid does not occur. For example Col-B
In the case of a bacterial strain, 0.0025 to 0.0150% is preferable, and particularly 0.0075 to 0.0100% is more preferable. If it is less than 0.0025%, the amount of increase in production of bioracein is small, and if it exceeds 0.0150%, the growth of bioracein-producing bacteria is inhibited, and the production of bioracein is delayed.
Since anthranilic acid is consumed with the production of bioracene, the anthranilic acid is added either sequentially or continuously at predetermined time intervals. The same effect can be obtained by adding anthranilic acid in the form of an anthranilic acid salt.

[0013] Bioracein is obtained by centrifuging a culture solution in which the bioracein-producing bacteria have accumulated and accumulated to separate the cells into a liquid medium, which is a supernatant, and is extracted from either or both of the cells and the liquid medium. You. Bioracein accumulated in the cells is easily extracted with ethanol. That is, after drying the cells, the cells were immersed in ethanol to extract crude bioracein in ethanol, the ethanol was removed from this extract by a rotary evaporator, the retained matter was dried to dryness, and extracted again with ethanol. Is filtered, concentrated and dried. As a result, purified bioracene, which is a black-purple fine powder pigment, is obtained. Bioracein in the supernatant liquid medium is extracted with ethyl acetate. That is, ethyl acetate is added at a ratio of 1 to 3 parts by volume to 1 part by volume of the medium to extract bioracein, and the extracted bioracein is concentrated by a rotary evaporator and purified by repeatedly performing silica gel column chromatography. . This purified bioracein is a black-purple fine powder pigment similar to that extracted from the cells. The maximum absorption wavelength of the purified bioracene in ethyl acetate solution is 550 nm
And its molar extinction coefficient is 926 l / mol ·
cm.

[0014]

[Effect] When anthranilic acid is added to a liquid medium containing a tryptophan source, and a bioracein-producing bacterium is inoculated and cultured therein, anthranilic acid is changed into tryptophan in the culture solution, and the bioracene-producing bacterium is converted into the original tryptophan source Bioracein is produced using the added tryptophan source as a metabolic intermediate. In the culture solution in which the bioracein-producing bacteria have proliferated, bioracein is excreted outside the cells. The mechanism of excretion of bioracein from the cells has not been completely elucidated, but is presumed as follows.
That is, the tryptophan source is used not only as a carbon source and a nitrogen source for the bioracein-producing bacteria, but is further decomposed into a water-soluble peptide by an enzyme of the bioracein-producing bacteria during the use process. Since bioracein is easily adsorbed to this water-soluble peptide, bioracein is easily transferred from the cells in the culture to the water-soluble peptide and accumulated in the medium. The extracellular medium, i.e., bioracein discharged into the culture solution, was extracted with a liquid medium by adding an extraction solvent immiscible with the medium to extract the solvent, and then the extract was dehydrated and concentrated, and further dried. By doing so, it is obtained in the form of a fine powder. On the other hand, the bioracein retained in the cells can be obtained in the form of a fine powder by drying the cells, immersing the dried product in alcohol to extract bioracein, and drying the extract to dryness.

[0015]

Next, embodiments of the present invention will be described in detail, but the embodiments described here are merely examples, and the present invention is not limited thereto.

<Example 1> First, 1 liter of tap water
Ri, bactotripton: 10 g, yeast extract: 5 g
, NaCl: 5 g, glucose: 1 g, 1N N
pH 7.2 prepared by uniformly mixing aOH: 4 ml.
50 ml of LB medium in a 300 ml Erlenmeyer flask
The solution was dispensed and sterilized at 120 ° C. for 15 minutes. This sterilization process
Chromobacte, a bioracein-producing bacterium,
Lium Bioraceum (Chromobacterium violaceu
m) Inoculation of Col-B strain, shaking at 25 ° C for 17 hours, and preculture
did.

In addition, K ₁ HP per liter of tap water
O ₄ : 2.0 g, MgSO ₄ .7H ₂ O: 0.5 g,
Glycerin: 10.0 g, wool protein: 30.0 g,
CaCl ₂ .2H ₂ O: 0.5 g, ZnSO ₄ .7H
50 ml of an inorganic salt liquid medium of pH 6.5, prepared by uniformly mixing 0.1 g of ₂ O and 1.0 g of KCl, was added to 30
Dispense into a 0 ml Erlenmeyer flask. After adding anthranilic acid to the liquid medium to the concentration shown in Table 1, 0.5 ml of the preculture suspension was inoculated into the liquid medium, and the medium was shaken at 25 ° C. for 72 hours. The production bacteria were cultured. The amount of bioracein produced was quantified by spectrophotometry. Table 1 shows the results.

Comparative Example 1 The amount of bioracein produced in the same manner as in Example 1 except that anthranilic acid was not added was quantified by a spectrophotometric method. Table 1 shows the results.

<Comparative Example 2> Instead of the inorganic salt liquid medium of Example 1, Bacto beef extract per liter of tap water:
50 ml of a commercially available broth medium containing 3 g and bactopeptone: 5 g was dispensed into a 300 ml Erlenmeyer flask. To this liquid medium, 0.5 ml of the same precultured bacterial suspension as in Example 1 was inoculated without adding anthranilic acid, and the medium was shaken at 25 ° C. for 72 hours to culture the bioracein-producing bacteria. The amount of bioracein produced was quantified by spectrophotometry. Table 1 shows the results.

[0020]

[Table 1]

As is apparent from Table 1, 0.0075%
The amount of bioracein produced by the method of Example 1 in which a concentration of anthranilic acid was added was about 1.8 times that of the method of Comparative Example 1 in which only anthranilic acid was not added, and the amount of bioracein produced without adding anthranilic acid was increased. The ratio was about 34 times that of the method of Comparative Example 2 using a commercially available broth medium containing a relatively large amount of peptone. Thus, the method of Example 1 was more effective than the method of Comparative Example 2 using an expensive medium using peptone as a tryptophan source because anthranilic acid was added in spite of using inexpensive wool protein as a tryptophan source. It was found that a large amount of bioracein could be produced.

[0022]

As described above, according to the method of the present invention, the production ability of the bioracein-producing bacterium can be remarkably increased only by adding a relatively small amount of inexpensive anthranilic acid to an inorganic salt liquid medium containing a tryptophan source. Can be improved. This makes it possible to produce bioracein in high yield without using a large amount of expensive culture medium such as peptone or animal hair degradation product as a tryptophan source. In particular, when using animal hair degradation products as a tryptophan source, while reducing the amount of animal hair degradation products added by adding anthranilic acid to the inorganic salt liquid medium, while limiting the amount of culture medium that tends to foam during culture, A large amount of bioracein can be produced in a relatively short time at low cost.

Claims (3)

(57) [Claims] 1. A bioracein-producing bacterium belonging to the genus Chromobacterium is inoculated into a liquid medium containing a tryptophan source, and the bioracein-producing bacterium is cultured and grown in the liquid medium. A method for producing a natural purple pigment, comprising separating cells and a liquid medium, and extracting a natural purple pigment comprising bioracein from either or both of the cells and the liquid medium, wherein the anthranilic acid or a salt thereof is cultured before or after culturing. A method for producing a natural purple pigment, wherein the natural purple pigment is sequentially or continuously added to the liquid medium at a concentration that does not inhibit the growth of baioylasein-producing bacteria. 2. The method according to claim 2, wherein the bioracein-producing bacterium is Chromobacterium violaceum C.
The method for producing a natural purple pigment according to claim 1, which is an ol-B strain. 3. The tryptophan source is peptone or animal hair degradation product in which tryptophan has not been degraded.
A method for producing the natural purple pigment according to the above.