NO159451B - PROCEDURE FOR THE PREPARATION OF A CULTIVATION PRODUCT OR A CULTIVATION LIQUID OF A TRICHOSPORON KASHIWAYAMA STOCK. - Google Patents

Description

The present invention relates to a method for producing a skin medication consisting exclusively of a culture product or a culture liquid of a Trichosporon Kashiwayama strain (deposited at FRI under deposit number 4821), which can be identified as a strain belonging to the genus Geotricum or Endomycopsis, a cell-free sterile liquid, sterile filtrate or sterile supernatant of specified culture product or liquid, or a concentrate or dry product thereof.

The method according to the invention is characterized by

that the strain is grown on or in a culture medium comprising glucose, skimmed milk and yeast extract, under aerobic conditions at a pH value of 4.0 - 6.0 and a temperature of 20 - 30°C, forming a culture product or a culture liquid, that possibly cells are removed from specified culture liquid to form a cell-free sterile liquid, sterile filtrate or sterile supernatant, and optionally that specified cell-free sterile liquid, sterile filtrate or sterile supernatant is concentrated or dried.

The microorganism used according to the present invention, i.e. Trichosporon Kashiwayama strain, is considered to belong to the yeast species, but the strain is sometimes classified as a fungi imperfecti. Accordingly, the indication that the strain can be identified as a strain belonging to the genus Geotricum or Endomycopsis is based on the scientific opinion held by the experts in the field.

As can be seen from the accompanying drawings (photographs), the strain used according to the invention is characterized in that it has ball-like arthrospores and pseudo-mycelium. Furthermore, the strain is characterized in that a fermentation product of the strain, i.e. a supernatant of a culture liquid of the strain, has in itself a conspicuous medicinal effect on human skin.

The effective substance that exhibits a medicinal effect is still unknown. If, however, a sterile supernatant of a culture fluid of the strain or a concentrate or dried product is applied to the skin as such or after it has been mixed with a suitable carrier and formed into a preparation suitable for application to the human skin, for example an ointment, the following medicinal effects are achieved. (1) The fermentation liquid is slightly acidic (has a pH value of 4.5 - 5.0) and is non-toxic to the skin, and it activates the skin-protective effect of the fat membrane, especially the bacteria-regulating effect inherent in the fat membrane, (2) Increasing desquamation of keratin pieces from the stratum corneum is prevented, and at the same time a suitable water content is maintained in the stratum corneum, and therefore the skin's characteristics such as moisture and elasticity can be maintained in good condition. (3) Lipid metabolism in the epidermal cells is regulated to activate appropriate formation of keratin and smooth proliferation and differentiation of the epidermal cells. (4) Especially for oily skin, increasing secretion of sebum will be regulated, and an increase in the thickness of the fat membrane will be prevented. Consequently, the diffusion of sebum in the skin fold surface from the hair follicle is activated, and a protective effect against fatty dermatitis such as acne is obtained. (5) As collagen fibers in the net layer are reinforced or swollen to maintain an appropriate water content in the upper layers, good and normal hardness and elasticity of the skin is maintained. (6) As the circulation of blood improves, the metabolism of the skin is activated, and the transport of skin waste to the vein and excretion thereof is activated. At the same time, excretion of melanin color to the outside of the body is accelerated. (7) For skin diseases caused by oxidation under actinic rays, the fermentation liquid or a sterile supernatant thereof acts as an antioxidant to the fat membrane and activates the decolorization of abnormally deposited melanin color in skin spots or the like. When the fermentation liquid or a sterile supernatant thereof is administered in connection with infrared irradiation treatment or oral administration of vitamin E, a preventive or curative synergistic effect can be achieved against contact dermatitis, skin spots and abnormal discoloration due to sun sensitivity.

The microbiological properties of the Trichosporon Kashiwayama strain (deposited at FRI with deposit number 4&21) which is used according to the invention, will be described in the following.

(1) Morphological characteristics:

(i) Shape and size (malt extract-MY liquid medium): After 3 days of cultivation at 25°C, cells with a rectangular or oval shape and a size of (3 to 4 µm) x (3 to 20 µm) were observed., and when the culture was continued for 5 days or more, the shape of the cells became irregular and finally became

the cells mycelioid.

2) Formation of pseudomycelia (slanted culture on potato extract-agar culture medium): The cells are pseudohyphal or pseudomycelial, and they have characteristic zigzag - arthrospores but

no conidia.

3) Ascospores:

The cells have no ascospores.

4) Ballisto spores:

The cells have no ballistospores.

(2) Characteristic properties of culture media:

1) Colony on agar streak culture medium (malt extract-MY agar culture medium): Dull, white, hairy and rough surface, very soft, not

raised but flat, periphery with f glue hairs...

2) Film on liquid culture medium (malt extract-MY liquid culture medium):

White, hairy, flocculent and rough film formed.

(3) Physiological properties:

1) Saccharide fermentation:

Non-fermentative towards glucose, galactose, sucrose, lactose, treharose, raffinose and inulin.

2) Saccharide utilization (assimilation):

D-glucose +, D-galactose + , maltose -, raffinose -, sucrose -, lactose -, sodium lactate +, D-xylose +, erythritol -, D-mannitol +, L-sorbose +, inositol -, treharose -, D -arabinose -, L-arabinose -, sodium succinate +, sodium citrate +, sodium acetate +,

inulin -, soluble stearin -, ethanol +, glycerol +, cellobiose -,

Footnotes

+: utilized

-: not utilized

3) Utilization of nitrates:

not utilized

4) Utilization of nitrites:

not utilized

5) Utilization of ethylamine hydrochloride: not utilized 6) Cleavage of glucoside (Arbutin): cleaved 7) Formation of starch analogues: not formed.

8) Formation of pigment:

not formed.

9) Formation of esters:

formed.

10) Litmus milk reaction:

not coagulated.

11) Vitamin needs:

not required.

12) Salt resistance:

critical concentration of 6 to 8 W/v %

13) Optimum temperature:

25° C.

14) Optimal pH value:

5.0.

15) Cleavage of urea:

not split

16) Growth on 50% (w/w) glucose agar or 60% (w/w) yeast extract agar medium:

no growth

17) Growth at 37° C:

no growth

18) Formation of acid from glucose: not formed 19) Resistance to cycloheximide: resistant 20) Cleavage of oil and fat:

the split

(4) Other mycological characteristics:

1) Production of fragrance:

The present strain is characterized by the production of a fragrance component. From the results of gas chromatographic analysis, it was found that the fragrance component is composed mainly of ethanol, ethyl acetate, isopropyl alcohol, isopropyl acetate, isobutyl alcohol, isobutyl acetate, isoamyl alcohol and isoamyl acetate. The scent is a so-called apple scent. The present strain is characterized in relation to known yeast varieties with fragrance-producing properties in that the formation conditions are high not only for higher alcohols, but also their esters.

Optimal conditions for the production of these neutral fragrance components are as follows. a) Initial pH value of 4.0 to 5.0 and maintenance of a low pH value during the fermentation. b) Total nitrogen content of 400 to 500 mg/litre of culture medium.

Ammonium sulfate, ammonium nitrate and ammonium chloride are preferably used as inorganic nitrogen sources, and yeast extract, casamino acid and peptone are preferably used as organic nitrogen sources. Furthermore, good results are obtained when amino acids such as leucine, isoleucine, valine and phenylalanine are added.

c) Selection of carbon credit sources.

Glucose, galactose, ethanol and glycerol are suitable.

A preferred concentration is 5%.

d) Cell inoculation ratio of 1 to 2%.

4) Cultivation temperature of 25 to 30° C.

Stationary cultivation is preferred.

2) Formation of organic acids:

Formic acid, propionic acid, n-butyric acid, succinic acid and citric acid are formed from glucose.

The accompanying drawings (photographs) illustrate growth of the Trichosporon Kashiwayama strain used according to the invention, on a potato extract agar culture medium when slide culture was carried out at 25° C. for 3 days. Fig. 1 is a microscope photograph (75 times magnification) showing the state of adhesion of arthrospores from hyphal cells under aerobic conditions. From fig. 1 it can be seen that the arthrospores do not adhere to conidiophores but extend laterally in chains from the dividing wall parts of hyphal cells.

Fig. 2 is an enlarged microscope photograph (300 times magnification) showing the arthrospores. Fig. 3 is a micro-scop autograph in (300x magnification) showing the cells and the state of occulation under aerobic conditions.

Ordinary yeast culture medium can be used for the cultivation of the Trichosporon Kashiwayama strain according to the invention. According to a preferred embodiment, stationary cultivation or shake cultivation is carried out at 20 - 30° C for approx. 24 hours in a solid or liquid medium comprising 0.3% (w/v) glucose, 0.5% (w/v) skimmed milk and 0.05% (w/v) yeast extract, and with a pH value of 4.0 - 6.0, and the same culture medium as stated above is inoculated with the resulting culture product as germ material, and stationary culture or shake culture is carried out at 20 - 30° C for 3 - 7 days.

The culture liquid is subjected to filtration and removal of the cells according to known procedures, for example using a membrane filter, and the cell-free supernatant is concentrated, for example under reduced pressure, preferably at a concentration rate of 4-25. The resulting concentrate is sterilized according to well-known procedures and stored at room temperature or in a freezer.

The cell-free supernatant is produced according to the invention and has the properties shown in table 1.

Table 1

A cell-free purified filtrate of the supernatant of the culture product of the present strain comprises small amounts of amino acids, saccharides and lipids. When the filtrate is determined quantitatively, it is seen that the total nitrogen content is 0.005 to 0.015%, and the total phosphorus content is 0.004 to 0.014%.

1. Type and condition:

The filtrate is a colorless to slightly yellowish translucent liquid and has a faint odor.

2. Confirmatory tests:

(1) 20 ml of the filtrate was evaporated on a water bath until the volume was reduced to approx. 50 ml. Then 1 ml of ninhydrin test solution was added to the concentrate. When the mixture was heated for 30 minutes, the liquid acquired a violet color. (2) 5 ml of nitric acid was added to the concentrate formed by evaporation in the same manner as described in point (1) above, and the mixture was boiled for 20 minutes. After the mixture had cooled, it was neutralized with a 10% solution of sodium hydroxide. Then 2 ml of an ammonium molybdate test solution was added to the mixture. If the mixture was heated, the liquid turned yellow. If a 10% solution of sodium hydroxide was added to the liquid, it became colorless. (3) 10 mg of indole and 2 ml of hydrochloric acid were added to 5 ml of the filtrate, and the mixture was sufficiently shaken. When the mixture was then heated for 10 minutes, the liquid turned red.

3. Physical properties:

(1) Specific gravity (d^ ): 1000 to 1015

(2) Refractive index (n^<5>): 1330 to 1340

(3) pH (25°C): 4.5 to 5.5

4. Evaporation residue:

0.5 to 2.0%

The analytical value of an example of the concentrate (concentration rate of 2) of the cell-free supernatant produced according to the invention is shown in table 2.

E. coli: ( - )

Staphylococcus aureus; ( - ) Number of fungi : ( - )/ml Number of yeast : ( - )/ml

Footnotes

x : % based on water

xx : number of ml IN alkali titrated for neutralization

of 10 0 g sample

xxx: g amino acid in 100 g sample (cystine: permauric acid oxidation method, tryptophan: microorganism testing).

Of course, the cell-free sterile supernatant can be used as a skin medication without concentration. From a storage point of view, however, it is preferred that the cell-free, sterile supernatant is concentrated. Furthermore, a hay concentrate with a degree of concentration of more than 25 or a dry product can be used. Such concentrates, high concentrate or dry product, can be used as skin medication as such. Furthermore, a skin medication can be formed

by mixing such a concentrate, highly concentrated or dry product with a cream, an ointment base or other carrier.

When a concentrate formed by concentrating the cell-free sterile supernatant to a concentration degree of 4 is analyzed, it is seen that various amino acids and vitamins are contained therein. However, since the analysis is done for common designations, it will be easily understood that even if the respective components are mixed according to the analytical values found, a skin medication according to the invention cannot be produced. In other words, it is assumed that the activities of the skin medication produced according to the invention are due to unknown components outside the analysis results.

Results of the in-use toxicity test

of chick embryos shows that the above-mentioned cell-free, sterile concentrate (concentration level of 4) has no toxicity. Also has results from patch test on

the human body and continuous irritation test on human skin showed that the concentrate is not toxic to human skin. Furthermore, results from eye irritation tests on rabbits have shown that the concentrate is not irritating. Results of the antibiotic test have shown that the concentrate does not contain any antibiotic substance.

It has been established that the skin medication produced according to the invention has a healing effect against skin spots, cracked skin, contact dermatitis and burnt skin. These playful effects will now be described with reference to the following experiments.

Patch test on the human body

50 women (suffering from some skin diseases or other and who went to dermatological hospitals), who were 23 to 44 years old, were selected for the test. Adhesive patches. with a size of 4 x 4 cm and containing approx. 0.2 g of concentrate (concentration level of 2) produced according to the invention was applied to the inside of the left upper arm and the area near the spine,

and after 48 hours the patches were removed and the applied areas were examined. In all patients, the inside of the left upper arm or the area near the spine was not adversely affected.

Continuous irritation test on human skin

Sterilized gas pieces with an area of 10 cm 2 were impregnated with approx. 5 ml of the concentrate (concentration level of 2) prepared according to the invention, and they were applied to the right cheek area of 45 women who were from 23 to 45 years old (35 women suffered from some skin disease or other and received medical treatment at a dermatological hospital, and the remaining 10 women were employed at this hospital) for 15 minutes. The treatment was carried out continuously for 40 days. No reaction was observed in any of these women, and problems such as erythema were not induced.

Eye irritation test on rabbits

3 white rabbits were used as test animals. At first glance, it was determined that the eyes had no abnormal signs. On each rabbit, 0.1 ml of the concentrate (concentration level of 2) prepared according to the invention was instilled in the right eye, and 0.1 ml of a blanking liquid was instilled in the left eye. Immediately after instillation, and 3, 6, 24, 48 and 72 hours

after instillation, both eyes were clouded using a slit lamp. Abnormal signs such as congestion of the cornea, iris or conjunctiva were not observed.

Skin patch test

621 women who had skin spots on their face, excepted

patients with liver diseases or gynecological disorders and pregnant women who received treatment against skin spots were selected. After washing the face, the skin pores were opened with ozone and steam, and gas impregnated with the concentrate (concentration level 2) of the cell-free supernatant produced according to the invention was applied to the area with spots. The applied gas was covered with a tape of "Saran-Wrap" and infrared irradiation was performed for 15 to 20 minutes to cause the concentrate to penetrate the skin. This treatment was carried out once a week and continued for 3 months. It was found that the condition was greatly improved in 363 women (58.4%), the condition was relatively improved in 122 women (19.8%), no improvement was observed in 133 women (21.4%), the condition was worsened in one case (0.01%) and contact dermatitis was observed in the remaining 2 women (0.03%)

(it was established that contact dermatitis had been induced for reasons not due to this treatment). The rate of patients in whom the condition significantly or relatively improved was thus as high as 80%. In 60% of 3 65 women in whom the condition was greatly improved, the skin spots also disappeared completely.

Effect on cracked skin and fine wrinkles

Generally, women of more than 25 to 30 years old are nervous about cracked skin and fine wrinkles, and especially in women of 35 to 40 years old, this tendency is conspicuous, and even women of more than 40 years old are nervous about cracked skin and fine wrinkles. In this test, 962 women who underwent dermatological treatment were selected (150 women aged 25 to 30, 218 women aged 30 to 35, 248 women aged 35 to 40, 172 women aged 40 to 50 and 174 women who were more than 45 years old). After washing the face, the skin pores were opened with ozone and steam, and gauze impregnated with the concentrate (concentration degree 2) of the cell-free supernatant prepared according to the invention was applied tightly to the skin and the gauze was covered with a tape of "Saran-Wrap". Infrared irradiation was performed for 15 to 20 minutes to cause the concentrate to penetrate the skin. It was found that after one treatment the cracked skin was significantly improved in 698 women (73%),

the cracked skin was relatively well improved in 156 women

(16%), the condition had not changed in 72 women (7%)

and the condition had not worsened in any of the tested women, the remaining 36 women (4%). In light of the fact that chapped skin leads to fine wrinkles, it is believed that the concentrate produced according to the invention, which is effective for the treatment of chapped skin, has a preventive effect with regard to the formation of fine wrinkles.

A test of the healing effect for contact dermatitis

A 21-year-old woman who had developed a skin rash and itching on both eyelids overnight, after switching from one eye shadow from Company A

to another eyeshadow from company B, and which was diagnosed as contact dermatitis, was chosen for the test. Gas-

pads impregnated with the concentrate (concentration level 2) of the cell-free supernatant produced according to the invention were applied to the affected areas by a dermatologist. Some steroid medication,

an anti-inflammatory agent or an antihistamine,

was not applied. About. 20 ml of the concentrate was given to the patient, and the concentrate was coated or applied for 30 minutes twice per day. day, i.e. morning and evening. 48 hours after the start of treatment the scabies had cleared, and after 96 hours the skin rashes had mostly disappeared. After 1 week, no skin rash was observed.

Activity against burns and scalds

A 47-year-old woman who had been scalded by hot water on the outer part of her left forearm (burn degree II) with a rash and swelling the size of a plum and with small blisters, came to the dermatologist approx. 1 hour after she was scalded. The affected area was immediately cooled with running water, and gas impregnated with the concentrate (concentration grade 2) of the cell-free, sterile supernatant prepared according to the invention was applied to the affected area and oil paper was coated thereon. Then bandage the affected area. After 24 hours, small blisters were found to have completely disappeared although the rash and swelling were still present, and the patient no longer complained of pain.

A 30-year-old man who had been scalded by hot water on the back of his right foot (burn degree II)

with a rash and swelling the size of a chicken egg, was treated with the concentrate (concentration level 2) of the cell-free, sterile supernatant produced according to the invention in the same way as described above. After 24 hours, the swelling had disappeared, although there was still a rash, and the patient no longer complained of any pain.

Thus, it was confirmed that the concentrate produced according to the invention has a healing effect against burns and scalds.

The skin medication and the cosmetic preparation produced according to the invention will be further described in the following examples.

Example 1

A fermentation tank with a stirrer and aeration and a capacity of 1 Kl was filled with 800 liters of a culture medium with the same composition as previously described, and after heat sterilization and natural cooling, this culture medium was inoculated with inoculum formed by carrying out a preliminary culture at 28° C in about. 30 hours in 10 liters of the same culture medium in a small fermentation tank with stirring and aeration. The cultivation was then carried out at 28° C for approx. 70 hours under stirring and aeration. After fermentation, the culture liquid was passed through a filter device model MP 293-16 (filter paper Gl-90; 0.5 pm) and through a filter device model MP 293-4 (membrane filter TM-2; 0.45 pm) to remove solid particles and cells. The filtrate obtained was concentrated according to the reverse osmosis membrane method using a reverse osmosis apparatus Model RO-T10 so that the volume was reduced to 200' litres. The resulting concentrate was sterilized with a plate-type heat exchanger, packed sterilely in shards of glass and stored in a refrigerator (5° C).

Example 2

The sterilized concentrate obtained in example 1 was freeze-dried, and the powder obtained was mixed with a sterile ointment base to form a skin ointment.

Example 3

A cosmetic preparation was prepared by incorporating 1 g of the sterilized concentrate obtained in Example 1 into a cosmetic base comprising 1 g of stearic acid, 7 g of beeswax, 4 g of self-emulsifiable glycerol monostearate, 30 g of liquid paraffin, 0.1 g of aqueous lanolin and 40 g of distilled water, after which the mixture was kneaded according to usual procedures. The material was poured into and packed into ampoules, and the packed ampoules were marketed.

Example 4

A drug for treating the skin was prepared by incorporating suitable amounts of pyridoxine hydrochloride, Y-oryzanol, allantoin, DL-a-tocophenol (vitamin E) and mink oil into the material prepared in example 3.

The skin medication produced according to the invention, which is derived from a naturally occurring substance, and which has been shown to be harmless, can be used for the treatment of skin diseases, for the treatment of diseases of the mucous membranes and for the protection of the epithelium against environmental pollution.

Claims (1)

Method for producing a culture product or a culture liquid from. a Thricosporon Kashiwayama strain (deposited at FRI under deposit number 4821), identifiable as a strain belonging to the genus Geotricum or Endomycopsis, a cell-free sterile liquid, sterile filtrate or sterile supernatant of specified culture product or liquid, or a concentrate or dry product thereof, characterized in that the strain is grown on or in a culture medium comprising glucose, skimmed milk and yeast extract, under aerobic conditions at a pH value of 4.0-6.0 and a temperature of 20 - 30°C, forming a culture product or a culture liquid, that possibly cells are removed from specified culture liquid to form a cell-free sterile liquid, sterile filtrate or sterile supernatant, and optionally that specified cell-free sterile liquid, sterile filtrate or sterile supernatant is concentrated or dried.