JPH02145189A - Production of tyrosinase inhibitor 3422 - Google Patents

Abstract

PURPOSE:To obtain colorless and odorless inhibitor excellent in stability and beautifying and whitening effects without any side effects on human bodies by culturing a microorganism, belonging to the genus Trichoderma and capable of producing a tyrosinase inhibitor in a culture medium. CONSTITUTION:(B) A strain of Trichoderma.harzianum 3422 (FERM P-10345), capable of producing a highly active tyrosinase inhibitor from (A) a strain of Trichoderma.harzianum IFO-30717 and having mycological properties of sufficiently growing in a potato dextrose agar culture medium with light green spherical conidia of colonies having 4-5X4.5-5mum size, etc., and properties of growth pH2-9, 20-30 deg.C growth temperature, utilizing glucose without utilizing cellulose, etc., is obtained by separation and collection from farmland soil in TOJO Town, KATO District in HYOGO Prefecture on September 8, 1987. The resultant strain (B) is then aerobically cultured in a culture medium containing 2-10wt.% glucose, etc., 0.1-1wt.% polypeptone, etc., 0.01-0.05wt.% Mg source, etc., at 20-30 deg.C for 2-7 days. The obtained culture is subsequently adsorbed, extracted and purified to recover the objective tyrosinase inhibitor 3422.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] This question relates to a method for producing a tyrosinase inhibitor that is effective for whitening the skin or removing spots or freckles, and a new strain that produces the inhibitor.

[Conventional technology]

Melanin pigment, which causes skin coloration, is produced in melanin-producing granules (melanocytes) within melanocytes (melanocytes) located between the epidermis and dermis, and the produced melanin diffuses to adjacent cells by osmosis. The biochemical reactions within melanocytes are currently estimated to be as follows. The process by which tyrosine, an essential amino acid, becomes dopaquinone through the action of the enzyme tyrosinase, which changes to black melanin via enzymatic or non-enzymatic oxidation through red pigment and colorless pigment, is the process by which melanin pigment is produced.

Therefore, it is thought that melanin production can be suppressed by suppressing the action of tyrosinase, which is the first step of the reaction, or by reducing quinones, which are the intermediate step. As a means of suppressing this, substances that bind to copper, which is the active center of tyrosinase (e.g., thiourea, cysteine, kojic acid), and substances that can compete with tyrosine, the substrate of tyrosinase (e.g., N-acetyl tyrosine,
γ-pyrone, hinokitiol), substances that prolong the induction period of the enzymatic reaction between tyrosinase and substrate (e.g. Tween 2),
0), substances that selectively bind to O-dihydroxy groups such as dopa (e.g., molybdenum ions), substances that bind to O-quinones (e.g., aniline), reducing agents for O-quinones (e.g., ascorbic acid, hydroquinone, and derivatives thereof) have been proposed. However, none of them are satisfactory in terms of toxicity, stability, sensory effects, etc. on humans. For example, cysteine suppresses the production of dopa pigment, but its sulfur odor makes it problematic to use in cosmetics. Furthermore, ascorbic acid is easily oxidized and causes browning of the formulation, which also poses a practical problem.

[Problems to be Solved by the Invention] The present inventors have spent many years researching to find a skin whitening agent that does not have any unfavorable side effects on the human body and has an excellent whitening effect. It has been found that the fermentation liquid obtained by culturing the bacterial strain exhibits a remarkable tyrosinase inhibitory effect superior to that of ascorbic acid. Moreover, the crude product obtained from this culture solution has a pH of
The present inventors have discovered that it has very good stability against heat and the like, and is also colorless and odorless, so it can be used for many purposes, leading to the completion of the present invention.

[Means to solve the problem]

That is, the present invention relates to Trichoderma spp.
Tyrosinase inhibitor 3, characterized in that a tyrosinase inhibitor-producing bacterium belonging to P. derma is cultured, preferably aerobically, and a tyrosinase inhibitor is collected from the culture.
422 manufacturing method.

The present invention will be explained in detail below.

In the method of the present invention, any tyrosinase inhibitor-producing bacteria belonging to the genus Trichoderma can be used. Microorganisms that can be used in the method of the present invention include, for example, Trichoderma ha
rizianum) IFD-30717,) Trichoderma viride
) IFD-31137, and a new microorganism discovered by the present inventor. The new microorganism was isolated and collected from field soil in Tojo Town, Kato District, Hyogo Prefecture on September 8, 1987.

The mycological properties of this microorganism will be described later, but its properties and Rifai's A Revision of G+S! J COF'LUMA (A REVISI
口NOF THEGENIJS TRIClll0DE
RI, IA), 1969, etc., the microorganism of the present invention is Trichobul'? - Trichoderma harzian
um) or Trichoderma harzianum, so here it is referred to as Trichoderma harzianum (Tr+choderma harzianum).
It was named 3422. In addition, this microorganism has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken) (Feikoken Bacterial Submission No. 10345).

Next, Trichoderma harucianum 3 used in the present invention
The mycological properties of 422 (Feikoken Bibori No. 10345) are shown.

1. Morphological properties (1) Potato dextrose agar medium Growth is rapid on potato dextrose agar medium (Kyokuto Pharmaceutical Industries, Ltd.).

Colonies grow rapidly and initially appear white on the surface, forming sparse mats of hyphae. Woolly hyphae soon elongate, cover the colony surface, and rise as it approaches the center. Groups of conidia are formed from the center of the colony and are initially pale colored. It then becomes bright green and finally dark green. The conidiophore branches from the aerial hyphae and produces many side branches, giving it a tree-like appearance. Therefore, the colony appears to be a wheel-shaped, somewhat sparse, lawn-like colony. Conidia are spherical or spherical, pale green in color, and have a diameter of 4.0 to 5.5 mm. Op-
x 4.5~5.3 厁, and the conidial wall surface is smooth.

The underside of the colony is white and does not produce any pigment.

(2) Malt extract agar medium Growth on malt extract agar medium is rapid.

Aerial hyphae extend to a length of <8n+m, sparsely cover the front surface of the medium, and exhibit white fluffy colonies. Conidiophores arise from aerial hyphae, and the sporophores branch into wheel shapes. Short stalks are produced at the tips of the stalks, and conidia are produced in clusters from the tip of each stalk, giving a tree-like appearance. Conidia are spherical or spherical, green in color and 3 in diameter.
．． 5-4.51MX 4.5-5.5-, and the conidial wall surface is smooth.

Therefore, as the settlement of conidia progresses, a band of green conidia forms in a ring shape in the center of the colony.

The underside of the colony is white with no pigment production.

2. Growth conditions pH: Grows well in a wide range of pH 2 to pH 9.

It grows particularly well at pH 3 to pH 6 and prefers acidic conditions.

Temperature: Grows well at 20°C to 30°C. Growth at 15°C is somewhat good, and growth at 10°C is weak.

On the other hand, growth is somewhat good at 37°C, but no growth occurs at 45°C.

3. Utilization of carbon sources xylose Arabinose + Glucose ÷ Mannose ÷ Fructose ← Galactose Sucrose Azusa Cellobiose → Trehalose ← Maltose + Lactose → Raffinose ± Starch + Cellulose ± Orange: Very useful.

It can be used often.

+ Can be used.

± is rarely available.

4. Phenol oxidase reaction: positive In the method of the present invention, the above-mentioned microorganism is used to react with tyrosinase inhibitor 3.
422 will be produced. The medium composition of the microorganism used to produce this tyrosinase inhibitor 3422 is usually about 2 carbon sources such as xylose, arabinose, glucose, mannose, fructose, galactose, sucrose, cellobiose, trehalose, maltose, lactose, or starch. ~10% (wt%, same hereinafter), about 0.1-1% nitrogen source such as ammonium sulfate, polypeptone, sodium nitrate, baker's yeast extract, brewer's yeast extract or potato extract, magnesium source such as magnesium sulfate Approximately 0.01 to 0.05%, 0.01 to 0.1% of phosphorus and potassium sources such as potassium monohydrogen phosphate and potassium dihydrogen phosphate, and other sources such as ferric sulfate, ferric chloride, sodium chloride, etc. Inorganic salt of about 0.001~0
．． A medium containing 0.005% can be used.

In the present invention, it is preferable to inoculate a culture medium with the bacteria used and carry out the reaction under aerobic conditions in which an oxygen-containing gas such as air is introduced by means such as aeration. In addition, culture is carried out at 10°C to 4°C.
2 to 7 days at a temperature of 0°C, preferably 20°C to 35°C.
It is appropriate to do this for several days. After culturing, the bacterial cells were removed and the culture solution was filtered to obtain a fermentation solution. Even when the obtained fermentation liquid was diluted 100 times, it showed a strong tyrosinase inhibitory effect. After removing impurities such as proteins and pigments from this fermentation liquid using acidic alumina, the active substances are adsorbed onto activated carbon.

After washing this activated carbon with water, the active substance is extracted with aqueous methanol. By drying this extract, a crude product of tyrosinase inhibitor 3422 is obtained.

〔Example〕

Next, the present invention will be specifically explained with reference to Examples.

Tyrosinase activity inhibition test method Tyrosinase inhibitory activity was evaluated by the following method.

(1) Reaction system reagents The following reaction reagents were used.

0.05% aqueous solution of L-Dopa (Wako Pure Chemical Industries) 1/15M
Phosphate buffer (pH 6,8) Inhibitor (sample solution) Tyrosinase (pine mushroom tyrosinase manufactured by Sigma) 2000 units/mg, 0.7 mg/mn (
2) Tyrosinase activity measurement sample solution and measurement reagent should be kept chilled on ice until measurement.

Preparation of reaction solution When measuring tyrosinase activity, C1. Prepare three test tubes each of four reaction solutions, C2, TI, and T2, and leave them at room temperature for 10 minutes or more to return to room temperature.

Measurement C1, C2, T1. Transfer the reaction solution to be measured for T2 to a spectrophotometer cell (3 mj).

Add 0.05m of tyrosinase solution to the transferred solution! ! ,
was added, and 90 seconds after the addition, the absorbance was 475 nm (
optical density; below 0. D) is measured using a spectrophotometer.

(3) How to determine the tyrosinase activity inhibition effect Calculation of the tyrosinase activity inhibition rate Three pieces of data are obtained for C+, C2, T+, and T2, so calculate the average value for each, and calculate 2. The inhibition rate of tyrosinase activity is calculated according to the following formula.

The concentration of the sample solution in the measurement reaction solutions T1 and T2 is changed at three or more points to determine the inhibition rate of tyrosinase activity, and using a semi-log graph, the inhibition rate is plotted on the vertical axis and the sample concentration (logarithm) is plotted on the horizontal axis.
Create a graph, determine the sample concentration that shows 50% inhibition rate, and calculate the IDs. shall be.

The lower the concentration indicating IDso, the higher the tyrosinase activity inhibition effect.

Example 1 Medium: 3% sucrose, 0 potassium dihydrogen phosphate
．． 1%, magnesium sulfate 0.05%, calcium chloride 0505%, ferric sulfate 0.001% and yeast extract 0
．． After preparing an aqueous solution containing 5% and adjusting the pH to 7, it was filled into an Erlenmeyer flask to 115 volumes and sterilized under high pressure in an autoclave for 2D minutes. This medium was added to Trichoderma hartiatum strain 3422
No. 45) was inoculated and cultured with shaking at 25°C for 3 days. After culturing, the bacterial cells were removed and the culture solution was filtered to obtain a fermentation solution. A freeze-dried product was obtained by freeze-drying a portion of this fermentation liquid. 50 g of acidic alumina was added to the fermented liquid 1β, stirred for about 1 hour, and then filtered. Activated carbon 5m in the filtrate
g/m and stirring for about 0.5 h.
Tyrosinase activity inhibitors in the fermentation liquid were adsorbed onto activated carbon. After washing this activated carbon five times with distilled water, tyrosinase activity inhibitor 3422 was extracted from the activated carbon with 80% methanol. After removing methanol from this extract using an evaporator, it was freeze-dried to obtain 0.58 g/β of crude tyrosinase inhibitor 3422.

Next, the degree of 50% inhibition of tyrosinase activity of this crude product was measured together with vitamin C, a known tyrosinase inhibitor. The results are shown in Table 2.

From the above results, it is clear that the crudely purified tyrosinase inhibitor 3422 exhibits a strong tyrosinase inhibitory effect.

This crude product of tyrosinase inhibitor 3422 is a white, flocculent solid, and its aqueous solution is colorless, transparent, and odorless, so it is suitable as a tyrosinase inhibitor used in cosmetics.

Next, the crude 1! of this inhibitor 3422! ! We investigated the thermal stability of materials. Crude purified product of inhibitor 3422 was heated at 25℃50
℃, 60℃, 80℃, and 90℃, and the tyrosinase inhibitory effect was measured at regular intervals. The results are shown in FIG.

As shown in FIG. 1, the crude product of inhibitor 3422 was
Even when heated at ℃ for 90 minutes, the tyrosinase inhibitory activity hardly decreased. Furthermore, even after heating at 90°C for 90 minutes, approximately 60% of its tyrosinase inhibitory activity remains, indicating that it has extremely good thermostability.

Tricoble? Physiologically active substances produced by molds belonging to Trichoderma include trichoviridin and pyridine.
Viridin), Gliotoxi
n), Trichodermin,
Suzukashiri:/ (Suzukac i 11 in)
etc. have been reported. However, all of these are antibiotics and have antibacterial properties. In contrast, this substance (
When the antibacterial activity of tyrosinase inhibitor 3422 (crude purified product) was investigated using the paper disc method, it was found that even at a 2% concentration of this substance, Bacillus subtilis,
1is) I AM1213, Staphylococcus
Staphylococcus aureus
us) 209P, Pseudomonas aeruginosa (
Pseudomonas erginosa) ATC
C15442, Candidaa albicans (Cand
ida albicans) and Aspergillus niger. Therefore, this substance was found to be a novel tyrosinase inhibitor that is clearly different from the previously reported physiologically active substances.

Example 2 Medium: 3% sucrose, 0 potassium dihydrogen phosphate
．． 1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferric sulfate 0.001% and yeast extract 0
．． An aqueous solution containing 5% was prepared and adjusted to I]H7, then filled into an Erlenmeyer flask to a volume of 115, and sterilized under high pressure in an autoclave for 20 minutes. In this medium, Trichoderma harzianum, which was provided by the Institute for Fermentation Research (IFO), was added.
IFO-30717 was inoculated and cultured with shaking at 25°C for 3 days. After culturing, the bacterial cells were removed and the culture solution was filtered to obtain a fermentation solution. A freeze-dried product was obtained by freeze-drying this fermentation liquid.

Example 3 Medium: 3% sucrose, 0 potassium dihydrogen phosphate
．． 1%, magnesium sulfate 0.05%, calcium chloride 0.05%, ferric sulfate 0.001% and yeast extract 0
．． After preparing an aqueous solution containing 5% and adjusting the pH to 7, it was filled into an Erlenmeyer flask to a volume of 115 and sterilized under high pressure in an autoclave for 20 minutes. In this medium, Trichoderma viride (Trichoderma viride), which was provided by Fermentation Research Institute, was used.
derma viride) IFD-31137 was inoculated and cultured with shaking at 25°C for 3 days. After culturing, the bacterial cells were removed and the culture solution was filtered to obtain a fermentation solution. A freeze-dried product was obtained by freeze-drying this fermentation liquid.

Next, the degree of 50% inhibition of tyrosinase activity of the freeze-dried products obtained in Examples 1 to 3 was measured.

The results are shown in Table 3.

FIG. 1 is a graph showing the thermal stability of crudely purified tyrosinase inhibitor 3422.

hand Continued Supplementary Positive calligraphy (spontaneous) January 1999

Claims (1)

[Scope of Claims] 1. A method for producing tyrosinase inhibitor 3422, which comprises culturing a tyrosinase inhibitor-producing bacterium belonging to the genus Trichoderma and collecting the tyrosinase inhibitor from the culture. 2. A tyrosinase inhibitor-producing bacterium belonging to the genus Trichoderma is Trichoderma halcyanum.
aharzianum) 3422 (Microtechnical Laboratory No. 103
45), the manufacturing method according to claim 1. 3. Trichoderma halcyanum
Trichoderma harzianum (IFO-30717), which produces a more active tyrosinase inhibitor than Trichoderma harzianum (IFO-30717)
ianum) 3422 (Feikoken Bibori No. 10345).