JPH07227290A - Production of natural purple pigment - Google Patents

Abstract

PURPOSE:To extremely improve a yield of violacein and to produce a natural purple pigment industrially, inexpensively and stably by adding inexpensive anthranilic acid to a medium of a bacterium capable of producing violacein. CONSTITUTION:Before or while a bacterium capable of producing violacein is cultured in a liquid medium containing a tryptophan source, anthranilic acid or its salt in a given concentration is successively or continuously added to the medium to provide a method for producing a natural purple coloring matter. A bacterium which belongs to the genus Chromobacterium, capable of producing violacein is inoculated into the liquid medium and multiplied. The culture solution having multiplied bacterium capable of producing violacein is separated into the cell and the liquid medium and a natural purple pigment comprising violacein is extracted from the cell or the liquid medium or both.

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 purple pigment which becomes a blue dye. More specifically, it relates to a method for producing a natural purple pigment composed of biolacein by culturing a microorganism belonging to the genus Chromobacterium having biolacein-producing ability (hereinafter referred to as bioracein-producing bacterium).

[0002]

2. Description of the Related Art Violacein is retained in the cells of microorganisms belonging to the genus Chromobacterium or the genus Janthinobacterium . This dye has a feature that it has a maximum absorption wavelength of 579 nm in an ethanol solution and has 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 pigment, a method is known in which tryptophan or peptone in which tryptophan is not decomposed is added to a medium for culturing a biolacein-producing bacterium and culturing. However, in this method, since expensive reagents such as tryptophan or peptone are used for the culture medium, the production cost of 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 high. Is also low. Therefore, for industrial use, a cheaper and more efficient method has been desired.

To solve this problem, polyurethane foam or cellulose is added as an adsorption carrier to a medium for culturing biolacein-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). Further, one of the inventors of the present invention invented a manufacturing method for inexpensively producing biolacein in a medium using a decomposed product of animal hair in place of peptone, and filed a patent application (Japanese Patent Application No. 5-46363).

[0004]

However, JP-A-2-3
The manufacturing method disclosed in Japanese Patent No. 9892 uses a relatively large amount of an expensive culture medium such as peptone, and the amount of pigment produced is not industrially satisfactory. In addition, the biolacein-producing bacterium belonging to the genus Yantinobacterium used in this production method has a markedly reduced pigment-producing ability at a normal culture temperature of about 30 ° C., and the medium must be cooled in order to produce biolacein. is necessary. Therefore, this method has a problem that the manufacturing cost is high due to the increase of the cost for obtaining the culture medium and the cost for cooling.
The production method according to Japanese Patent Application No. 5-46363 has the advantage of greatly reducing the production cost of biolacein because it uses a cheaply available animal hair degradation product as the culture medium, but in order to increase the production amount of biolacein A large amount of animal hair degradation products must be added to the medium. In this case, since foaming due to aeration, stirring, etc. during the culture of the biolacein-producing bacterium becomes vigorous, it is impossible to add a large amount of animal hair decomposed product indefinitely, and there is a limit in the production amount of biolacein.

On the other hand, it has been clarified that tryptophan exists as a metabolic intermediate in the biosynthesis pathway of biolacein, and it is known that the production of biolacein is increased when tryptophan is added to the culture medium. ing. However, tryptophan is a very expensive reagent, and when it is used as a precursor of biolacein, the production cost is significantly increased. The present inventors focused on that the anthranilic acid contained in the intermediate of the synthetic pathway of this tryptophan is relatively inexpensively available, and by adding anthranilic acid to the medium of the conventional biolacein-producing bacterium, the yield of biolacein was increased. They have found that they can be greatly improved, and have reached the present invention.

The object of the present invention is to add inexpensive anthranilic acid to the medium of biolacein-producing bacteria,
It is an object of the present invention to provide a method capable of greatly improving the yield of bioracein and industrially inexpensively and stably producing a natural purple pigment composed of bioracein.

[0007]

The method for producing a natural purple pigment of the present invention comprises inoculating a biolacein-producing bacterium belonging to the genus Chromobacterium into a liquid medium containing a tryptophan source, and culturing the biolacein-producing bacterium in the liquid medium. In this method, the culture solution in which the biolacein-producing bacterium has grown is separated into cells and a liquid medium, and a natural purple pigment composed of biolacein is extracted from either or both of the cells and the liquid medium. Its characteristic constitution is that anthranilic acid or a salt thereof is added to the above liquid medium before or during the culture at a predetermined concentration successively or continuously.

The present invention will be described in detail below. The microorganism used in the present invention is not particularly limited as long as it is a strain belonging to the genus Chromobacterium and producing biolacein, that is, a biolacein-producing bacterium. As a biolacein-producing bacterium, there is a bacterium belonging to the genus Yantinobacterium, but a bacterium belonging to the genus Chromobacterium having a wide range of application of the culture temperature is selected. As a representative example of this biolacein-producing bacterium belonging to the genus Chromobacterium, the present inventors separated Chromobacterium violaceum ( Chromobacterium violaceum ) Col-B isolated from industrial wastewater in Aichi prefecture.
Examples include strains. Next, the mycological properties of this strain will be described.

A. Morphological properties (a) Cell shape and size: 0.8 × 1.5 μ
bacilli of m (b) Motility: Yes (c) Presence or absence of spores: No (d) Epiphytic state of flagella: Periflagellates (e) Gram stain: -B. Physiological properties (a) Oxidase: + (b) Catalase: + (c) Lipase:-(d) Urease:-(e) Casein decomposition: + (f) Esculin decomposition:-(g) Starch decomposition:-(h) Indole formation:-(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-mentioned mycological properties, "Bergie's Manual of Systematic Bacterio Gee "(BERGEY'S M
From the ANUAL of Systematic Bacteriology Volume 1),
This strain is considered to belong to Chromobacterium violaceum due to the fact that the assimilation of fructose did not match, but other mycological properties matched. Therefore, this strain was designated as Chromobacterium violaceum Col-B strain and deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology. Its deposit number is FERM P-14136.

The medium for producing biolacein of the present invention is a liquid medium containing a tryptophan source. This liquid medium is made 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 inorganic salts used for culturing ordinary microorganisms. Examples of the inorganic salt include phosphate, magnesium salt, potassium salt, calcium salt and the like, and the inorganic salt medium is used in combination. The peptone added to the inorganic salt medium is one in which tryptophan is not decomposed. In the present invention, when this peptone is used as the tryptophan source, more biolacein can be produced by adding a small amount of the medium to the inorganic salt medium as compared with the conventional method. Animal hair degradation products can also be used as a source of tryptophan. As the animal hair source of this animal hair decomposed product, wool is easily available and suitable, but is not particularly limited as long as it can extract keratin protein such as angora, cashmere, mohair or bird feather. To obtain this animal hair hydrolyzate, a commercially available hydrolyzed keratin solution is purchased, or a conventional method (P. Alexander, et al., Wool,
p.356, Reinhold, New York, 1954). When 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 ammonia water, and the solution is adjusted to pH 4
After the following, the insoluble protein in this solution is separated by filtration or centrifuged to dry.

[0011] The pH of the liquid medium for producing biolacein depends on the growth application of microbial biolacein-producing bacteria.
Since H is in the range of 5 to 10, it is adjusted to a weakly acidic to weakly alkaline range, preferably near neutral. Specifically, considering the growth of biolacein-producing bacteria, the pH should be 5
-9 are preferable, and 6.5-8 are more preferable. The culture temperature is preferably 20 to 35 ° C, more preferably 25 to 30 ° C. The culture must be aerobic.

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

Biolacein is obtained by centrifuging a culture solution in which biolacein-producing bacteria have grown and accumulated to separate cells and a liquid medium that is a supernatant, and extracted from either or both of the cells and the liquid medium. It Biolacein accumulated in the cells is easily extracted with ethanol. That is, after immersing the cells in ethanol after drying, to extract crude biolacein in ethanol, remove ethanol by rotary evaporator from this extract to dry the retained material, extract again with ethanol, extract liquid Is filtered, concentrated and dried. As a result, a purified biolacein of a black-purple fine powder pigment is obtained. Further, biolacein in the liquid medium as the supernatant is extracted with ethyl acetate. That is, 1 to 3 parts by volume of ethyl acetate is added to 1 part by volume of the medium to extract biolacein, and then the extracted biolacein is concentrated by a rotary evaporator and purified by repeating silica gel column chromatography. . This purified biolacein is a black-purple fine powder pigment like that extracted from bacterial cells. The maximum absorption wavelength of the purified biolacein solution in ethyl acetate is 550 nm.
And its molar extinction coefficient is 926 liter / mol.
cm.

[0014]

[Function] When anthranilic acid is added to a liquid medium containing a tryptophan source, and the biolacein-producing bacteria are inoculated and cultured in the liquid medium, the anthranilic acid is changed to tryptophan in the culture solution, and the biolacein-producing bacteria are used as the original tryptophan source and this. Biolacein is produced using the added tryptophan source as a metabolic intermediate. Biolacein is excreted outside the cells in the culture solution in which the biolacein-producing bacterium grows. The mechanism by which this biolacein is excreted out of the cells has not been completely clarified, but it is presumed as follows.
That is, the tryptophan source is not only used as a carbon source and a nitrogen source of the biolacein-producing bacterium, but is also decomposed into a water-soluble peptide by the enzyme possessed by the biolacein-producing bacterium during the utilization process. Since biolacein is easily adsorbed to this water-soluble peptide, biolacein is easily transferred from the bacterial cells in culture to the water-soluble peptide and accumulated in the medium. Extracellular medium, that is, biolacein discharged into the culture medium, is extracted by adding an extraction solvent immiscible with the medium to the liquid medium to extract biolacein by solvent, and then the extract is dehydrated and concentrated, and further dried to dryness. By doing so, it can be obtained in the form of fine powder. On the other hand, the biolacein retained in the microbial cells is also obtained in the form of fine powder by drying the microbial cells, immersing the dried product in alcohol to extract biolacein, and drying the extract.

[0015]

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

<Example 1> First, bactotryptone: 10 g and yeast extract: 5 g per liter of tap water.
, NaCl: 5 g, glucose: 1 g, 1N N
pH 7.2 made by uniformly mixing aOH: 4 ml
50 ml of the LB medium of 3 was dispensed into a 300 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes. In this sterilized medium, Chromobacterium violaceu which is a biolacein-producing bacterium is added.
m ) Col-B strain was inoculated and shaken at 25 ° C. for 17 hours for preculture.

Also, K ₂ HP per liter of tap water
O _4: 2.0 g and, MgSO ₄ · 7H ₂ O: and 0.5g,
Glycerin: 10.0 g, wool protein: 30.0 g,
CaCl ₂ · 2H ₂ O: 0.5 g and ZnSO ₄ · 7H
50 ml of an inorganic salt liquid medium having a pH of 6.5 prepared by uniformly mixing ₂ O: 0.1 g and KCl: 1.0 g with 30 ml
Dispense into an Erlenmeyer flask with a volume of 0 ml. After adding anthranilic acid to this liquid medium to a concentration shown in Table 1, 0.5 ml of the above preculture suspension was inoculated into this liquid medium, and this medium was shaken at 25 ° C. for 72 hours to prepare biolacein. The producing strain was cultured. The amount of bioracein produced was quantified spectrophotometrically. The results are shown in Table 1.

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. The results are shown in Table 1.

Comparative Example 2 Instead of the inorganic salt liquid medium of Example 1, per 1 liter of tap water, Bacto beef extract:
50 ml of a commercially available broth medium containing 3 g and 5 g of bactopeptone: 5 g was dispensed into an Erlenmeyer flask having a volume of 300 ml. This liquid medium was inoculated with 0.5 ml of the same preculture suspension as in Example 1 without adding anthranilic acid, and this medium was shaken at 25 ° C. for 72 hours to culture biolacein-producing bacteria. The amount of bioracein produced was quantified spectrophotometrically. The results are shown in Table 1.

[0020]

[Table 1]

As is clear from Table 1, 0.0075%
The amount of biolacein 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 anthranilic acid was not added, and the anthranilic acid was not added. The amount 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 superior to the method of Comparative Example 2 in which an expensive medium containing peptone as a tryptophan source was used by adding anthranilic acid in spite of using an inexpensive wool protein as a tryptophan source. It was found that a large amount of biolacein can be produced.

[0022]

As described above, according to the method of the present invention, it is possible to remarkably improve the production ability of biolacein-producing bacteria 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 biolacein in high yield without using a large amount of expensive culture medium such as peptone or animal hair decomposed product as a tryptophan source. In particular, when using animal hair hydrolyzate as a tryptophan source, while reducing the amount of animal hair hydrolyzate added by adding anthranilic acid to the inorganic salt liquid medium to limit the amount of the culture medium that easily foams during the culture, A large amount of bioracein can be produced inexpensively in a relatively short time.

Claims (3)

[Claims] 1. A culture medium in which a biolacein-producing bacterium belonging to the genus Chromobacterium is inoculated into a liquid medium containing a tryptophan source, and the biolacein-producing bacterium is grown in the liquid medium to grow the culture medium. A method for producing a natural purple pigment that separates cells and a liquid medium, and extracts a natural purple pigment composed of biolacein from either or both of the cells and the liquid medium, before or after culturing anthranilic acid or a salt thereof. A method for producing a natural purple pigment, which comprises sequentially or continuously adding to the liquid medium therein at a concentration that does not inhibit the growth of baoilasein-producing bacteria. 2. The biolacein-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 is not decomposed.
A method for producing the described natural purple pigment.