JPS63245666A - Novel bacterial strain capable of producing purple-blue pigment - Google Patents

Abstract

PURPOSE:To provide a novel bacterial strain, Janthinobacterium libidum KK-217 belonging to Janthinobacterium genus and capable of producing large amount of purple-blue pigment having excellent heat-resistance, light-resistance, color tone, etc. CONSTITUTION:The objective novel bacterial strain is Janthinobacterium libidum KK-217 (FERM P-9303) belonging to Janthinobacterium genus and capable of producing purple-blue pigment. The bacteria can be separated from silk stained in purple-blue color using a medium such as mannit yeast extract. The strain can produce a large amount of natural purple-blue pigment, which can be easily extracted from the cultured product. The pigment produced by this process has excellent heat-resistance, light-resistance, color tone, etc., compared with conventional natural purple-blue pigment and is suitable for the coloring of cosmetic and food or as a dye for textile, etc.

Description

Detailed Description of the Invention (Industrial Utilization) The present invention is a novel microorganism belonging to Yanthinobacterium.
Regarding. This bacterium can be used to produce a purple-blue natural pigment.

(Prior Art) Conventionally, purple-blue natural pigments have been obtained from gardenia and spirulina, but they lack #i heat resistance and light resistance.

There have been reports of pigments produced by microorganisms, but they have not yet been put to practical use.

(Purpose of the Invention) Therefore, the present inventors conducted intensive studies to obtain a purple-blue natural pigment with good heat resistance and light resistance, and found that the bacteria of the present invention efficiently produced a purple-blue pigment. The present invention was completed based on the discovery that the heat resistance and light resistance are excellent.

(Structure of the Invention) The present invention relates to Jantinobacterium lividum (Jantinobacterium lividum), which belongs to the genus Jantinobacterium and produces a purple-blue pigment.
hinobacterfum livfdum)
It is 217.

In Japan, it was isolated from purple-blue contaminated raw silk using a medium such as mannitol yeast extract, and as described below, it is a new bacterium.
) KK-217 (hereinafter abbreviated as KK-217)
Name it.

The national characteristics of Nini-217 are shown below.

A. Morphological properties (1) Cell shape and size: Short bacilli, (0.2-1.
2) X (2,5-6.0) μm (2) Cell pleomorphism: None (3) Motility: Motile, extremely hairy (4) @ No child growth: None 5) Durham Stainability: Negative (6) Acid-fast: Negative B, Growth status in each medium (1) When in contact with 1% peptone water or PI broth, a purple layered pattern is observed on the liquid surface. .

(2) Mannite yeast extract agar medium (25°C,
(Cultured for 5 days): Good growth, circular shape, 1 protruding surface, 1 smooth surface, 1'a maroon color.

(3) Agar medium (25°C, cultured for 5 days, good growth, circular shape, dark red color) (4) Heart Infusion medium, good growth, circular shape, color tone Dark blue.

C8 Physiological Properties (1) Reduction of nitrate: Positive (2) Utilization of citric acid Bi-positive (3) Formation of indole Eight phagocytic properties (4) Lysine decarboxylation: Negative (5) Arginine dihydrase: Negative (6)
Aesculin liquefaction: negative (7) Gelatin liquefaction: negative (8) Urease = negative (9) Catalase: positive CO oxidase: positive αυ OF test: positive 02 Oxygen requirement: aerobic C1 growth temperature: 4-36℃, Optimal growth temperature 20-26℃
.

α→ Growth pH: pI (5-10% pH 6-8° αQ Assimilation of carbon sources: nigerose, arabinose: +, mannose +, mannit +, fructose +.

1, II Pigment production: Yes (chord blue, mannitol yeast extract agar medium), not water soluble.

Based on the 8th edition (1974) of Bersey's Manual of Determinative Bacteriology, we searched for the properties of
Exercise with extreme hair.

It was confirmed to be Yanthinobacterium lividum because it is aerobic and has the ability to decompose sugars such as arabinose, mannose, and mannitol.

Next, known species of Yanthinobacterium lividum species, such as Yanthinobacterium lividum N0TO9796
, Doujin (8neath) * Same MB (Snea
th) *N0TO8661, HA (Sneath
) * Same PI, same Mu Too 14487゜ Same Ru (8
new) *Same NO'l'0 7150. Same HD (
When compared with A'l'OO6915 and the like, there is no one that produces a strong amber pigment very quickly like the one produced by Motomishi. That is, some other Yanthinobacterium lividum also produce violet pigment, but the color tone is not as strong. In addition, other strains have been observed to produce a pale yellow fluorescent pigment that is diffusible in the medium.

Furthermore, due to the difference in the mole content of guanine cytosine and other factors, the ministry recognized it as a new bacterium, Yanthinobacterium lividum.

The present inventors have deposited this bacterium with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Fiber Engineering Commission No. q3o3.

(Culture) Next, a method for culturing the microorganism of the present invention and a method for obtaining the produced pigment will be described.

As a medium for culturing the microorganism of the present invention, any ordinary bacterial culture medium can be used, but among them, a mannitol yeast extract agar medium is preferable.

As a carbon source, mannitol, glucose, arabinose, mannose, fructose, etc. can be used. Caciton powder, yeast extract, peptone, etc. can be used as natural nitrogen sources, and ammonium nitrate, potassium nitrate, ammonium sulfate, etc. can also be used in combination as inorganic nitrogen sources.

Inorganic components other than nitrogen include, in addition to sodium chloride,
Potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate,
Ferrous sulfate, manganese sulfate, calcium chloride, etc. are used.

No particular vitamins are required, but biotin, thiamin,
Yeast extract, cornstarch liquor, etc. may be added.

Culture is usually carried out under aerobic conditions, and liquid culture methods such as shaking culture method or aerated agitation culture method can also be used, but agar culture method is preferred, and it is best to carry out the culture at a culture temperature of 20 to 25°C.

The culture pH may be 6.0 to 8.0, and the culture time may be 24 hours or more, preferably 48 to 96 hours.

The dark blue pigment produced by the culture was produced using n-propyl alcohol, ethyl alcohol, ethylene glycol, 50%
%n-propyl alcohol, 50% ethyl alcohol,
It can be easily extracted with solvents such as dioxane and chloroform. Among them, 50% n-furobil alcohol%
Preferred are n-propyl alcohol, 50% ethyl alcohol, ethyl alcohol, and the like.

(Effects of the Invention) The microorganism of the present invention produces a larger amount of purple-blue pigment than conventionally known microorganisms, and furthermore, this can be easily extracted and collected. The obtained pigment has superior heat resistance and light resistance compared to conventional purple-blue natural pigments, and has a good color tone, and is useful as a textile dye or paint for cosmetics and food containers.

(Example) Examples will be described below.

Example 1 (Mannitol yeast extract medium) Mannnitol yeast extract liquid medium (composition excluding only agar from mannite yeast *1 mother extract agar medium) 5 m
7? The stock strain 1 Platinum Nu was inoculated into the cells and cultured at 25°C for 8 days with reciprocal shaking (100 times/min).

The bacteria grown in the bamboo cultivation folds were added to 15 bottles of 10 m of sterilized water at 0.0 ml.
It was diluted in 5 ml increments based on the dilution method.

0.05 ml of each dilution water was taken, dropped onto a yeast mannite agar plate prepared in advance, and spread evenly with a conlage rod. the plate at 26°
Cultured at C.

From the 2nd day, purple-colored bacterial cells began to proliferate, the raw stomach was in very good condition, and the colony was circular, smooth, and flat. After the 8th month, the purple-blue color became deeper, the colony surface rose parallel to it, and the colony itself became hard.

*1 Mannite yeast extract agar medium composition peptone 2.5
g, Na0Ji! 2.5 g, yeast extract 2.5
g, 5 g of mannitol, and 15 g of agar were dissolved in 11 g of tap water, and the pH was adjusted to 7.0 with NaOH.

Reference example! (Extraction method of pigment) Approximately 800 ml of ethanol was added to an agar petri dish (diameter f30 cm) covered with colored colonies on the 4th day after culturing, and extraction was performed by shaking reciprocally in a shaker for approximately 1 to 2 hours. After sterilizing this extract using a 0.45 μm membrane filter, the ethanol solvent was removed using a rotary evaporator to obtain about aomg of a purple-blue pigment.

The absorption spectrum of the dye purified by recrystallization using ethanol is shown in FIG. Furthermore, the following tests were conducted on the obtained dye.

(1) #Ifjl chromatographic analysis Analysis was conducted under the following conditions.

Plate used: Silica gel 70 FM (manufactured by Wako)
? X: n-propyl alcohol: water ±9 = 1 of purple-blue dye after development for 145 minutes (f value is 0.88, 1 spot).

Similarly, Avicel SF cellulose (manufactured by Funakoshi) was used, and the developing solvent was 0.2% ammonia:phenol = 1:4.
In the case of , one purple-blue spot was also observed.

(2) Light test The light resistance of this pigment was tested under the following conditions using the natural pigment Spirulina Blue (manufactured by Lina Bloom Dainippon Ink & Chemicals Co., Ltd.) as a comparison control. The dye solution was allowed to stand still, and its change in temperature was observed over time.

Spirulina blue is Mclluvaine Buffer
(Mix 0.1M citric acid and 0.2M disodium phosphate, dilute 10 times before use.)
．． The dye was prepared at a concentration of 2%, and the dye was mixed with ethanol and Mcllu.
Using a mixture of vaine Buffer and
．． It was prepared at 2% 1%. Evaluation was performed on a scale of 8 based on visual judgment.

The results are shown in Table-1.

Table-1 Light resistance test results O: No discoloration Δ: Slight discoloration ×: Complete discoloration (3) Heat resistance test Each dye solution was heated at 60°C, 160°C and 70°C, and the degree of discoloration was measured over time. observed. Spirulina blue is 0.2
% aqueous solution, this dye is a 0.2% water/ethanol mixture (1:
1). Evaluation was performed in four stages by visual judgment. The results are shown in Table-2.

←No change, +slightly changed (t, -changed, -completely changed) Example 2 (Normal agar medium) Preculture and main culture were carried out in the same manner as in Example 1 except that the following medium * was used.

*Normal agar medium (made in Japan) Meat extract 5g, peptone 10g, Mail 5g
.

15 g of agar was added to 1 g of water, and the pH was adjusted to 7.0 with NaOH.

White colonies appeared from the 2nd day, and from the 8th day onwards, the colonies arrived from the center of the colony toward the outside.

Example 8 (Heart Infusion Medium) The following medium*
Preculture and main culture were carried out in the same manner as in Example 2 except for using.

*Heart Infusion Medium (manufactured by Eiken) bovine heart infusion 500g% peptone 10g.

Add 5 g of Na0A' and 15 g of agar to water 11 and adjust the pH.
7.4 was prepared with NaOH.

The culture results were the same as in Example 2.

[Brief explanation of drawings]

Figure 1 shows the absorption spectrum of the dye extracted with ethanol. 400 soo
Soo l? o. -〉3Fll, Cotton No. 10

Claims (1)

[Claims] Janthinobacterium lividum belongs to the genus Janthinobacterium and produces a purple-blue pigment.
bacterium lividum) KK-217.