JPH06113824A - New microorganism and production of red pigment thereby - Google Patents

Abstract

PURPOSE:To provide a new microorganism and a new red pigment produced by the microorganism. CONSTITUTION:There are provided a new microorganism belonging to Norcardioides and a method for producing a new red pigment by isolating and purifying a cultured material of the above microorganism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism and a novel method for producing a red pigment by the microorganism. More specifically, the present invention relates to Nocardioides (N
The present invention relates to a novel microorganism belonging to the genus ocardioides) and a method for producing a novel red pigment obtained by isolation and purification from a culture of the above microorganism.

[0002]

2. Description of the Prior Art L.C. Mark Ant in 1835
(LCMarquant) first purified and structurally determined anthocyanin, a pigment of cornflower, to the present,
Only a few flavonoid compounds of microbial origin have been discovered, the majority being quinonoids, carotenoids and chlorophyll.

In the systematic evolutionary process, the secondary metabolic pathways from phenylalanine and tyrosine to flavonoid compounds are sometimes considered to have been acquired after the evolution from fungi to higher plants, and anthocyanins are not only flavonoids. It is not equivalent, and a novel microorganism that can be produced using a conventional culture method and that can be easily isolated and purified, and a novel method for producing a red pigment obtained from a culture of the microorganism are known. Not not.

[0004]

Accordingly, it is an object of the present invention to provide new microorganisms that produce new red pigments.

Another object of the present invention is to provide a novel method for producing a red pigment produced by isolating and purifying from the culture of the above microorganism.

[0006]

The above objects can be achieved by a microorganism belonging to the genus Nocardioides and producing a purple or red pigment on and between basal hyphae.

In addition, the above-mentioned various purposes are the Nocardioides
It is also achieved by a method for producing a red pigment, which is purified from a culture of a microorganism belonging to the genus (Nocardioides) .

[0008]

The microorganisms of the present invention are obtained from the surface soil in Matsuyama City, Ehime Prefecture (Ehime University Faculty of Agriculture) by using the YEPG agar medium (1% yeast extract, 2% peptone, 2 glucose).
%, Agar 2%), and the obtained colonies were separated to obtain the colonies.

In addition, regarding the microorganism of the present invention, Bergey's Manual of Determinative Ba
When a mycological test was conducted based on the method described in Cteriology), the microorganism of the present invention showed that Nocardioides (Noc
ardioides) can see that belong to the genus, microorganisms Nocardioides sp · R-001 strain of the present invention (Nocard
ioides sp. R-001) (hereinafter, referred to as R-001 strain). The R-001 strain was deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology on February 20, 1992, and the deposit number is Micromachine Research Institute No. 12784 (FERM).
P-12784).

The bacteriological properties of the microorganism R-001 strain of the present invention are shown below.

(1) Morphology a) Branching method of spore-forming hyphae Simple branching (sporulation is only division of hyphae, according to the branching method of hyphae) b) Morphology of spore-forming hyphae Only sporulation due to hyphal breakage c) Number of spores 10 or more d) Surface structure of spores smooth e) Spore size 1.0-1.2 μm × 0.
8 to 1.0 μm f) Presence or absence of flagella spores g) Presence or absence of sporangia h) Location of spore stalk spores not present i) Division of mycelium 3 to 7 days after inoculation on agar medium, aerial hyphae And the basal hyphae are almost completely divided. J) No sclerotia formation (2) Growth state in medium (indicated by parenthesis) a) Poor growth of sucrose colonies (±) Nitrate Agar medium The growth of basal mycelium is also poor (±) The surface of the colony is white b) The growth of glucose colonies is poor (±) The growth of the asparagine agar medium basal mycelium is also poor (±) The surface of the colony is white C) Glycerin colonies that show good growth (+
＋) Asparagine agar basal hyphae are reddish with growth (++) White on the surface of the colony d) Starch agar broth very well (++) basal hyphae are reddish purple , Grows darker with growth (++
＋) The surface of the colony has a powdery gray color e) Tyrosine agar medium The colony grows very well (++) The basal hyphae show a reddish purple color and become darker with the growth (++
＋) The surface of the colony is powdery gray f) Nutrient agar medium The colonies grow well (＋)
＋) Basal hyphae appear white and become egg-colored with growth (＋
+) The surface of the colony is white g) Yeast colonies grow (+) Malt agar medium The basal hyphae are cream colored and become white with growth (+) The surface of the colony is white h ) Oatmeal agar medium Good colony growth (+
＋) Basal hyphae are pale red and become purple with growth (++) The surface of the colony is gray (3) Physiological properties a) Growth temperature range 10 to 35 ℃ Optimal growth temperature is 26 ℃ Does not grow above 4 ° C and above 37 ° C. It is difficult to form aerial hyphae above 30 ° C b) Liquefaction of gelatin Liquefaction of gelatin on glucose / peptone gelatin medium c) Hydrolysis of starch Starch on starch agar medium Hydrolyze d) Coagulation and peptone of defatted milk Coagulate defatted milk, then peptone e) Formation of melanin-like pigments No melanin-like pigment formation (4) Assimilation of carbon source on Pridham-Godlieve agar medium A) L-arabinose is not assimilated (-) b) D-xylose is assimilated (+) c) D-glucose is assimilated (+++) ) D) D-fructose assimilate (+) e) Sucrose not assimilate (-) f) Inositol somewhat assimilate (±) g) L-rhamnose somewhat assimilate (±) h) Raffinose not assimilate (-) i) D-mannite assimilation (++) The medium used for culturing the bacterium may be either solid or liquid, and needs to contain a carbon source that can be assimilated by the bacterium. Either a synthetic medium or a natural medium may be used as long as it is a medium containing a nitrogen source, an inorganic salt and the like.

The carbon source is not particularly limited as long as it can be assimilated by the bacterium, but D-xylose, D-glucose, D-fructose, inositol, L-rhamnose, D-mannitol, D-galacto-. It is possible to use sugar raw materials such as sugarcane, D-mannose, maltose, salicin, starch and glycerin, and natural products such as peptone and meat extract.

Further, the present bacterium produces a large amount of purple or red pigment on and between basal hyphae. This bacterium has a dark purple pigment on glucose / nitrate agar, Bennett's agar, and Pridham's / Godlieve agar, and red-purple on other media except yeast agar, iron agar, and peptone yeast / iron agar. Produces the pigment. However, the yeast agar medium, iron agar medium, and peptone-yeast-iron agar medium hardly produce pigments, and the basal hyphae show a white to ocher color. In yeast agar medium, a trace amount of salt solution (for example, prideham.
Addition of Godleybe's trace saline) or tap water produces a red to magenta dye. The aerial mycelium has a yellowish white to pale gray color,
Very good growth on any medium, but no soluble pigment is produced. However, when the agar medium becomes old, the red pigment diffuses into the agar medium. In addition, the colony before forming aerial hyphae has wrinkles on the surface and has little gloss.

The culturing is carried out under aerobic conditions, and the suitable temperature for culturing is 10 to 35 ° C., but it is desirable to cultivate at about 26 ° C. Also, the pH of the medium suitable for culturing
Is 3 to 9, but preferably 6 to 7.5.

The novel red dye produced by the microorganism of the present invention exhibits the following physicochemical properties.

(1) Elemental analysis (C ₆₈ H ₇₀ O ₂₉ N) _n (2) Integer multiple of molecular weight 1364 (3) Melting point 360 ° C. to 400 ° C. (4) Ultraviolet absorption spectrum (5) Infrared absorption Spectrum (6) Proton NMR (3) Solvent solubility Soluble in pyridine, dimethylsulfoxide, tetrahydrofuran, acetone, dioxane Insoluble in n-hexane, benzene, ether, methanol, acid Dilute aqueous alkaline solution , Slightly dissolved in chloroform (8) Color reaction Reddish purple due to aluminum ion (causing a bathochromic shift) (9) Color of substance Red crystalline substance Furthermore, extraction and purification of red pigment produced by the microorganism of the present invention The method is as follows. This strain R-001 strain is prepared by preparing YM medium containing 2% of glucose (0 to 2% yeast extract, 0.5 to 2% malt extract, 0 to 5% glucose using tap water, or a trace salt). (The solution is added) and the mixture is cultivated with shaking at 26 ° C. At this time, if necessary, pre-culture with the present strain R-001 strain is carried out in the same medium, and a part or all thereof is inoculated into the culture solution of the main culture. This is 96 ~
After culturing for 120 hours, 0.05 to 0.5% ascorbic acid was added to the whole culture solution, and concentrated hydrochloric acid was added to 1/15 to 1/1.
Add 0 volume. Next, 400 ml per liter of culture solution
Tetrahydrofuran (THF) containing 0.01-0.1% of t-butylquinone was added, and then saturated with sodium chloride to give T.
Separate the HF layer. The separated THF layer was centrifuged (1000
g, 5 minutes), collect the supernatant, and concentrate by distillation on a rotary evaporator. Furthermore, 2-10 in the concentrated liquid
Double volume of water is added to precipitate the crude dye. The precipitate is collected by centrifugation (1000 g, 10 minutes), washed with distilled water, and then dissolved in acetone. The dye is precipitated by adding 10 to 20 volumes of distilled water to this solution, and the precipitate is collected by centrifugation (1000 g, 10 minutes) and washed with water. Furthermore, this precipitate is suspended in n-hexane and then with ether, and washed by centrifugation (1000 g, 5 minutes). The precipitate washed in this way was dried under reduced pressure, dissolved in a small amount of acetone, and then n-hexane was added little by little to precipitate the dye.
-Wash well with hexane and dry under reduced pressure. In this way, a crude pigment is obtained from the culture solution.

Next, the crude dye was thoroughly dried under vacuum or in a phosphorus pentoxide desiccator, dissolved in a small amount of acetone, applied to a Wakogel C-200 column, and dioxane, water, hydrochloric acid (100: 5: 0. Develop with the aqueous solution of 7), collect the strongest red fraction, and distill to dryness. Further, the dried product is washed with n-hexane, then dissolved in acetone, and distilled water is added little by little to precipitate crystals. The precipitated crystals are further washed with water, collected by centrifugation (1000 g, 5 minutes) or filtration, dried under reduced pressure, and redissolved in acetone. In addition, n
-Hexane is added little by little to precipitate crystals. The crystals are collected, washed with n-hexane, and vacuum dried to a constant weight to obtain a purified dye preparation.

The yield of red pigment produced by this strain is calculated based on the following values by measuring the absorbance at 510 nm with a spectrophotometer. The purified dye preparation has an absorption of 10D _{510 in} 80 ug / ml dioxane solution and the crude preparation has an absorption of 10D ₅₁₀ per 150-200 ug / ml dioxane solution.

[0019]

The present invention will be described in more detail and specifically below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Nocardioides sp. R-001 strain ( Nocard
ioides sp. R-001) aerial hyphae 1-2 platinum loops were scraped from the slant and YM containing 10 ml of 2% glucose added.
Medium (0.5% yeast extract, 1% malt extract, 2%
Prepare glucose with tap water or
Godleybe's trace amount of salt solution) and incubate at 26 ° C for 4
Incubate with shaking for 8 hours. After culturing, add 0.5 ml of culture solution to 35
A shake flask containing 0 ml of the same YM medium as described above was inoculated and main-cultured. The amount of bacteria in this main culture is OD8
It was measured at 00 nm.

The growth curve at this time is shown in FIG.

As shown in FIG. 4, the R-001 strain was
Vigorous growth was observed between 6 and 48 hours, and thereafter became almost steady.

Example 2 In the same manner as in Example 1, culture solution 1 of Nocardioides sp. R-001 strain ( Nocardioides sp. R-001)
After culturing 96 liters for 96 to 120 hours, the entire culture solution was adjusted to 0.
1% ascorbic acid is added, and 0.08 volume of concentrated hydrochloric acid is further added. Next, 400 ml of THF containing 0.03% t-butylquinone is added to this culture solution, and then saturated with sodium chloride to separate the THF layer. Centrifuge the separated THF layer (1
000 g, 5 minutes), collect the supernatant, and concentrate by distillation on a rotary evaporator. In addition, add 1 to the concentrated liquid.
Add 0 volume of water to precipitate the crude dye. The precipitate is collected by centrifugation (1000 g, 10 minutes), washed with distilled water, and then dissolved in acetone. The dye is precipitated by adding 10 volumes of distilled water to this solution, and the precipitate is collected by centrifugation (1000 g, 10 minutes) and then washed with water. Furthermore, this precipitate is suspended in n-hexane and then with ether, and washed by centrifugation (1000 g, 5 minutes). Further, the washed precipitate is dried under reduced pressure, dissolved in a small amount of acetone, and then n-hexane is added little by little to precipitate the dye, and the precipitate is sufficiently washed with n-hexane, and then reduced in pressure. Dry under. By the above process,
0.2 to 0.3 g of crude pigment is obtained per liter of culture.

Next, the crude dye was thoroughly dried under vacuum or in a phosphorus pentoxide desiccator, dissolved in a small amount of acetone, applied to a Wakogel C-200 column, and dioxane, water, hydrochloric acid (100: 5: 1). The solution is developed with the above aqueous solution, and the strongest red fraction is collected and distilled to dryness. Further, the dried product is washed with n-hexane, then dissolved in acetone, and distilled water is added little by little to precipitate crystals. The precipitated crystals are further washed with water, collected by centrifugation or filtration, dried under reduced pressure, and then redissolved in acetone. Further, n-hexane is added little by little to precipitate crystals. After collecting these crystals and washing with n-hexane,
Vacuum-dry until a constant weight is obtained to obtain a purified dye preparation. The yield of purified dye at this time was 80 mg per liter of culture solution.
It is a degree.

The yield of the red pigment produced by this strain is measured by the same method as described above.

The production of the dye at this time is shown in FIGS. 5 and 6. 5 and 6, the horizontal axis represents the culture time and the vertical axis represents the pigment production amount. In FIG. 5, white and black triangles indicate the amount of pigment contained in the cells,
White and black circles indicate the amount of pigment contained in the medium supernatant. Further, FIG. 6 shows the total amount of pigment contained in the bacterial cells and medium supernatant shown in FIG.

As shown in FIGS. 5 and 6, the pigment production started after 24 hours both in the medium supernatant and in the bacterial cell fraction, and the amount of pigment in the medium supernatant was 72 to 168 hours. It becomes almost constant. Further, the amount of pigment in the cell fraction rapidly increased after 48 hours, reached the maximum at 72 to 96 hours, and hardly increased thereafter.

Also, the total amount of dye reaches its maximum in 96 to 120 hours.

[0029]

INDUSTRIAL APPLICABILITY As described above, the microorganism belonging to the genus Nocardioides according to the present invention is a novel microorganism, and a novel red color is obtained by isolating and purifying a culture of the microorganism according to the present invention. A dye can be obtained.

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a red dye produced by a microorganism according to the present invention.

FIG. 2 shows an infrared absorption spectrum of a red dye produced by a microorganism according to the present invention.

FIG. 3 shows proton NMR of red dye produced by a microorganism according to the present invention.

FIG. 4 is a graph showing a growth curve of a microorganism according to the present invention.

FIG. 5 is a graph showing the amount of red pigment produced by the microorganism of the present invention.

FIG. 6 is a graph showing the amount of red pigment produced by the microorganism of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 365) (C12P 1/06 C12R 1: 365)

Claims (2)

[Claims] 1. A microorganism which belongs to the genus Nocardioides and produces a purple or red pigment on and between basal hyphae. 2. A method for producing a red pigment, which is purified from a culture of a microorganism belonging to the genus Nocardioides .