FI82263C - PRODUKTER SOM AER ANVAENDBAR I KOSMETISKA SAMMANSAETTNINGAR OCH FRAMSTAELLNINGEN AV PRODUKTEN. - Google Patents

Description

1 82263

Product used in cosmetic compositions and its manufacture

Separated from Patent Application No. 802690 5

The invention relates to a product for use in cosmetic compositions and to a process for its preparation.

The product according to the invention is characterized in that its sole or main ingredient is a new strain of 10-strain, i.e. Trichosporo kashiwayama [deposited with the Fermentation Research Institute (FRI), 1-3, Higashi 1-chome, Yatabe-machi, Tsukuba-gun, Ibaraki-ken 305, Japan, under registration number FERM 4821, which has been converted into a deposit under the Budapest Treaty FERM BP-333], which is identifiable as

Geotricum or Endomycopsis, a culture product or culture medium, a sterile liquid of said culture product or culture medium, a sterile filtrate or a sterile supernatant from which the cells have been removed, or a concentrate or dry product thereof.

The novel product of the invention is used as an active ingredient in cosmetic compositions, whereby it is mixed with a carrier such as a conventional skin cream or ointment base.

The microorganism used in the present invention, or Trichosporo kashiwayama, is considered to belong to yeasts, but this strain is sometimes classified as fungi imperfect. The view that the strain is identifiable as belonging to the genus Geotricum or Endomycopsis is based on the scientific opinion of experts in the field.

The strain used in the invention is characterized in that the strain fermentation product, i. as such, the stock culture supernatant has an excellent effect on human skin. The active ingredient is not yet known. However, when a sterile supernatant or concentrate of a stock culture fluid or a dry product is applied to the skin as such or mixed with a suitable carrier in the form of a preparation suitable for human skin, such as tea, e.g. the following effects are achievable.

2 82263 1. The fermentation broth is slightly acidic (pH 5 4.5-5.0) and non-toxic to the skin and promotes the skin-protective effect of the fatty film.

2. Excessive peeling of keratin from the stratum corneum is prevented and at the same time the water content of the stratum corneum remains normal. Thus, the inherent properties of the skin, 10 such as moisture and elasticity, remain good.

As can be seen from the accompanying drawings, the strain used in the present invention is known to have pearlescent arthrospores and pseudomy gels.

The accompanying drawings show the growth of a Trichosporo kashiwayama strain used in the present invention with potato extract agar medium when cultured at an oblique surface for three days at 25 ° C. Figure 1 is a photomicrograph (magnification 75x) showing the adhesion of arthros-20 pores to hyphal cells under aerobic conditions. It can be seen from Figure 1 that the arthrospores do not adhere to the conidiophores but propagate laterally as chains from the septal sites of the hyphal cells. Figure 2 is an enlarged micrograph (magnification 300x) showing art-25 rospores. Figure 3 is a photomicrograph (magnification 300x) showing the cells and their contraction under anaerobic conditions.

The following describes the mycological properties of the Trichosporo kashiwayama strain (tal-30 flown to FRI under accession number 4821) used in the present invention.

(1) Morphological characteristics 1) Shape and size (malt extract-MY-liquid medium): 35 After culturing for three days at 25 ° C, cells of rectangular or oval shape and size (3-4 pm) x (3-20 pm). Continuing the culture for five days or more, the shape of the cells became 3 82263 irregular and finally the cells became mycelial.

2) Formation of pseudomycelles (oblique culture on potato extract agar medium):

The cells are pseudohyphic or pseudomycelic and have characteristic tortuous arthrospores but no conidia.

3) Ascospores: There are no ascospores in the cells.

4) Ballistic spores: There are no ballistic spores in the cells.

(2) Characteristic appearance of the media 10 1) Colony on an agar surface application medium (malt extract-MY agar medium):

Matt, white, eyelashed, rough surface, very soft, not elevated, but flat, periphery of kilos.

2) Membrane on liquid medium (malt extract-MY-nes-15 te medium):

A white, eyelashed, flocculating and tough film is formed.

(3) Physiological properties 1) Saccharide fermentation: 20 The strain does not ferment glucose, galactose, precipitate rose, lactose, trehalose, raffinose or inulin.

2) Use of saccharides (coupling) D-glucose +, D-galactose +, maltose, raffinose, sucrose, lactose, sodium lactate +, D-xylose-25 si +, erythritol, D-mannitol +, L-sorbose +, inositol -, trehalose, D-arabinose, L-arabinose, sodium succinate +, sodium sulphate +, sodium acetate +, inulin, soluble starch, ethanol, glycerol, cellobiose.

30 Note. n- - used, - * not used.

3) Use of nitrates: Not used.

4) Use of nitrites: Not used.

5) Use of ethylamine hydrochloride: Not used.

6) Glycoside (arbutin) degradation: Decomposes.

35 7) Mud formation of starch analogues: Not formed.

8) Pigment formation: Does not form.

4 82263 9) Formation of esters: Formed.

10) Litmus-milk reaction: Does not coagulate.

11) Vitamin need: No need.

12) Salt resistance: Critical concentration 6-8 5% w / v.

13) Optimum temperature: 25 ° C.

14) Optimal pH: 5.0.

15) Urea decomposition: Does not decompose.

16) Growth at 50% w / w glucose agar or 10 60% w / w yeast extract agar medium: No growth.

17) Growth at 37 ° C: No growth.

18) Acid formation from glucose: Not formed.

19) Durability of cyclohexamide: Durable.

15 20) decomposition of oil and fat: Decompose.

(4) Other mycological properties 1) Odor formation: The present strain is known to form an odor component. It was found by gas chromatography that the fragrance component consisted mainly of ethanol, ethyl acetate, isopropyl alcohol, isopropyl acetate, isobutyl alcohol, isobutyl acetate, isoamyl alcohol and isoamyl acetate. The scent is the so-called. apple aroma. Compared to known yeasts, the present strain is characterized in that not only the highest alcohols but also their esters are high in the formation of the odor.

The optimum conditions for the formation of these neutral fragrance ingredients are as follows: a) Initially, the pH is 4.0 to 5.0 and the low pH is maintained during fermentation.

b) The total nitrogen content of the medium is 400-500 mg / l.

Preferably, 35 ammonium sulfate, ammonium nitrate and ammonium chloride are used as the source of inorganic nitrogen.

II

5 82263 and preferably yeast extract, casamino acid and peptone are used as the source of organic nitrogen. In addition, good results are obtained by adding amino acids such as leucine, isoleucine, valine and phenylalanine.

5 c) Selection of carbon sources:

Suitable are glucose, galactose, ethanol and glycerol. The recommended saccharide concentration is 5%.

d) The number of cells inoculated is 1-2%.

e) The culture temperature is 25-30 ° C.

10 Stationary culture is recommended.

2) Formation of organic acids:

Glucose is formed from formic acid, propionic acid, n-butyric acid, succinic acid and citric acid.

Conventional yeast media can be used to cultivate the Trichosporon kashiwayma strain of the invention 15 used in the manufacture of the skin preparation. According to a preferred embodiment, the shaking or stationary culture is carried out at 20-30 ° C for about 24 hours in a solid or liquid medium containing 0.3% w / v glucose, 0.5% w / v skimmed milk and 0.05% w / v yeast extract and having a pH of 4.0 to 6.0. The same medium is inoculated with the formed culture product and stationary culture or shake culture is performed for 3-7 days at 20-30 ° C.

Using conventional methods, the culture medium is filtered and the cells are removed, e.g., by membrane filtration, and the cell-free supernatant is concentrated, e.g., in vacuo, with a concentration factor of preferably 4-25. The resulting concentrate is sterilized by conventional methods and stored at room temperature or in a refrigerator.

A sample of the cell-free supernatant prepared according to the present invention had the properties shown in Table 1: 6 82263

Table 1 The cell-free, purified filtrate of the supernatant of the culture product of the present strain contains small amounts of amino acids, saccharides and lipids. According to the quantitative determination of the filtrate, the total nitrogen content is 0.005 to 0.015% and the total phosphorus content is 0.004 to 0.014%.

1. Appearance and presence:

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

10 2. Detection tests: 1) Evaporate 20 ml of the filtrate in a water bath to a volume of about 5 ml. 1 ml of ninhydrin reagent solution is then added to the concentrate. When the mixture is heated for 30 minutes, the liquid turns purple.

2) To the concentrate obtained by evaporation in the same manner as in 1) above, 5 ml of nitric acid are added and the mixture is boiled for 20 minutes. After cooling, the mixture is neutralized with 10% sodium hydroxide solution. 2 ml of ammonium molybdate-20 reagent solution are then added to the mixture. When the mixture is heated, the liquid turns yellowish. Upon addition of 10% sodium hydroxide solution to the liquid, it becomes colorless.

3) Add 10 mg of indole and 2 ml of hydrochloric acid to the filtrate and shake sufficiently. After heating the mixture for 10 minutes, the liquid turns red.

3. Physical properties: 1) Density d ^: 1,000 - 1.015 2) Refractive index nj ^: 1.330 - 1.340 3) pH at 25 ° C: 4.5 - 5.5 30 4. Evaporation residue: 0.5 - 2.0%.

The analytical values of a sample of the cell-free supernatant concentrate (twice the concentration) of the present invention are shown in Table 2.

7 82263

Table 2

Water content (at normal pressure after drying): 98.7% Protein content (factor 6.25): traces Lipid content (after Soxhlet extraction): 0.2% 5 Fiber content: 0%

Ash content: 0.1%

Sugar content 1.0%

Total solids content *: 1.3%

Amino nitrogen content: 10 mg-% 10 Titrated acidity **: 0.6

Volatile base nitrogen content: 1 mg-% Arsenic content (as As20): not detected (detection limit 0.1 ppm) Heavy metal content (as Pb): not detected (detection limit 1 ppm) 15 Total mercury content: not detected (detection limit 0.01 ppm) Cadmium content: not detected (detection limit 0.01 ppm) Calcium content: 14.7 mg-%

Phosphorus content: 9.8 mg-%

Formic acid content: not detected (detection limit 0,1%) 20 Acetic acid content: not detected (detection limit 0,01%) Butyric acid content: not detected (detection limit 0,01%): Lactic acid content: 0,06%

Citric acid content: 0.02%

Number of viable cells: less than 30 cells / ml 25 E.coli: (-)

Staphylococcus aureus: (-)

Number of fungi: (-) / ml

Yeast count: (-) / ml

Propionic acid content: not determined (detection limit 0.02 g / kg) 30 Sorbic acid content: not determined (detection limit 0.005 g / kg) Benzoic acid content: 0.42 g / kg p-hydroxybenzoic acid ester content: not detected (detection limit 0.005 g / kg)

Vitamin Bj content: not detected (limit of detection 0,01 mg-%) 35 Vitamin B2 content: 0,02 mg-% β 82263 Total vitamin C content: not detected (limit of detection 2 mg-%)

Vitamin Bg content: not detected (detection limit 5 μg-%) 2-witarnin content · not detected (detection limit and 0.05 μg-%) 5 Pantothenic acid content: 0.09 mg-%

Choline content: not detected (detection limit 0.03%) Folic acid content: not detected (detection limit 1 ^, ug-%) Niacin content: not detected (detection limit 0.03 mg-%) Total carotene content: not detected (detection limit 0.02 mg-%) PH value: 4.9

*** The amino acid composition:

Arginine: less than 0.01 Lysine: less than 0.01 Histidine: less than 0.01 15 Phenylalanine: less than 0.01 Tyrosine: less than 0.01 Leucine: less than 0.01 Isoleucine: less than 0.01 Methionine: less than 0.01 20 Interlin: less than 0.01 Alanine: less than 0.01 Glycine: less than 0.01 Proline: less than 0.01 Glutamic acid: 0.01 25 Serine: less than 0.01 Threonine: less than 0.01 Aspartic acid: less than 0.01 Tryptophan: less than 0.01 Cystine: less than 0.01 30 Note.

* = percentage calculated on water ** = ml of 1-alkali solution neutralized by titration of 100 g of sample *** = g of amino acid / 100 g of sample (cystine: perch-35 acid oxidation method, tryptophan: microorganism assay) 9 82263

The cell-free, sterile supernatant can, of course, be used, even without concentration, as a cosmetic product, such as a skin preparation. But for storage, etc., it is recommended to concentrate the cell-free, sterile supernatant. For Li-5, a highly concentrated (more than 25-fold concentration) concentrate or dry product can be used. Such a concentrate, a highly concentrated concentrate or a dry product can be used as such as skin preparations. In addition, the skin preparation of the present invention can be prepared by mixing a concentrate, a highly concentrated concentrate or a dry product, and a cream, ointment base or other carrier.

When analyzing the concentrate obtained by concentrating four times the cell-free, sterile supernatant described above, it can be found that it contains various amino acids and vitamins. But since the analysis is general in nature, it is easy to understand that by mixing the components on the basis of the analytical values obtained, the product of the present invention 20 cannot be prepared. In other words, it is believed that the potency of the product of the present invention is due to unknown components that are excluded from the analysis.

The results of a toxicity study in a fetal chick show that the above-mentioned cell-free sterile 25 μl concentrate (four times the concentration) and non-toxic. In addition, the results of the human body patch test and the human skin continuous irritation test show that the concentrate is not toxic to human skin in the slightest degree. In addition, the results of the rabbit eye irritation test show that the 30 concentrate does not irritate in the slightest degree. And finally, the results of the antibiotic test show that the concentrate does not contain an antibiotic substance.

Example 1 A 1000 liter aerated and stirred fermentin-35 tank was charged with 800 L of medium having the composition described above, and after heat sterilization and self-cooling, this medium was inoculated with a culture previously obtained by culturing at 28 ° C for about 30 hours. In 10 liters of the same medium in a small aerated and 5 mixed fermentation tanks. At the end of the fermentation, the culture medium was drained through a filter machine model MP 293-16, manufactured by Naigai Shokuhin Kogyo Kabushiki Kaisha (filter paper GL-90, 0.5 pm, manufactured by Toyo Roshi) and a filter machine model MP 293-4, manufactured by the same company (mem-10 membrane filter TM -2, 0.45 μm, manufactured by Toyo Roshi) to remove solid particles and cells. The obtained filtrate was concentrated according to the reverse osmosis membrane method using a reverse osmosis device model RO-T10, manufactured by Bioengineering Co., whereby the volume was reduced to 200 liters.

The obtained concentrate was sterilized in a plate heat exchanger (manufactured by Iwai Kikai Kogyo Kabushiki Kaisha), sterile packed in glass containers and stored in a refrigerator (5 ° C).

Example 2

The sterilized concentrate obtained in Example 1 was freeze-dried and the resulting powder was mixed with a sterile ointment base to obtain a skin cream.

Esimerkki_3

A cosmetic product was prepared by mixing 1 g of the sterilized concentrate obtained in Example 1 with a cosmetic ointment base containing 1 g of stearic acid, 7 g of beeswax, 4 g of self-emulsifying glycerol monostearate, 30 g of liquid paraffin, 0.1 g of aqueous g. water and whisking the mixture by conventional methods. The composition was poured and packaged in ampoules and the packaged ampoules were marketed.

Example 4 :: A skin care preparation was prepared by mixing 0.001 to 1 g of pyridoxine hydrochloride, 0.001 to 1 g of γ-oryzanol, 0.001 to 1 g of ailantoin, 0.01 to 1 g of D1-OC tocopherol (E-vi-y '35 amine) and 0.01 to 1 g of mink oil per 100 g of the composition prepared in Example: 3.

Claims (5)

1. Appearance and condition: The filtrate is a colorless or slightly yellowish, translucent liquid and has a faint odor; 1. Product useful in cosmetic compositions, characterized in that it contains as a single component or as the main component a culture product or culture fluid of a new strain, ie. The trichosporon kashiwayama strain (deposited with FRI with deposit number FERM 4821), which can be identified as a strain belonging to the genus Geotricum or Endomycoplus, or a sterile liquid, sterile filtrate or sterile supernatant from which the cells were removed, of said culture product said culture fluid, or a concentrate or dry product thereof. 2. Identification Tests: (1) 20 ml of the filtrate was evaporated on a water bath until the volume was reduced to about 5 ml. Then, 1 ml of a ninhydride reagent solution was added to the concentrate. When the mixture was heated for 30 minutes, the liquid changed color to violet; (2) 5 ml of nitric acid were added to the concentrate formed by stripping off in the same manner as described in (1) above and boiling for 20 minutes 8 minutes. After the mixture was cooled, it was neutralized with 10% solution of sodium hydroxide. Then 2 ml of ammonium-molybdate reagent solution was added to the mixture. If the mixture is heated, the liquid turns yellowish. If a 10% solution of sodium hydroxide is added to the liquid, it becomes colorless; (3) 10 mg of indole and 2 ml of hydrochloric acid are added to the filtrate and the mixture is sufficiently shaken. After heating the mixture for 10 minutes, the liquid 10 turns red; 2. Product according to claim 1, characterized in that it contains as a single constituent or as the main constituent a sterile supernatant of the Trichosporon kashiwayama strain FERM 4821, which has the following characteristics: A cell-free purified filtrate of the supernatant of the culture product from the present strain comprises small amounts of amino acids, saccharides and lipids; When the filtrate is determined quantitatively, it should be noted that the total nitrogen content is 0.005 - 0.015% and the total phosphorus content is 0.004 - 0.014%; 3. Product according to claim 1, characterized in that it contains as a single constituent or as the main constituent a concentrate of the Tricho-sporon kashiwayama strain FERM 4821, which has the following analytical values, the concentrate being formed by double concentration: Water content (at normal pressure after drying): 98.7% Sample content (coefficient 6.25): barrier Lipid content (after Soxhlet extraction): 0.2% Fiber content: 0% Ash content: 0.1% Sugar content: 1.0% Total solids content *: 1.3% Nitrogen content, amino form: 10 mg% Titration acidity **: 0.6 Nitrogen content, volatile base form: 1 mg% Arsenic content (such as As203): not detected (limit of detection: 0.1 ppm) Heavy metal content (such as Pb): undetected (detection limit: 1 ppm) ice 82263 Total mercury content: undetected (detection limit: 0.01 ppm) Cadmium content: undetected (detection limit: 0.01 ppm) Calcium content: 14.7 mg % Phosphorus content: 9.8 mg% Acetic acid content: not detected (detection limit: 0.1%) Acetic acid content: not detected (detection limit: 0.01%) Butyric acid content: not detected (detection limit: 0.01%) Lactic acid content: 0.06% Citric acid content: 0.02% Number of living cells: less than 30 cells / ml E. coli: (-) Staphylococcus aureus: (-) Number of fungi: (-) / ml Number of yeast fungi: (-) / ml Propionic acid content: not detected (detection limit: 0.02 g / kg) Sorbic acid content: not detected (detection limit: 0.005 g / kg) Benzoic acid content: 0.42 g / kg p-hydroxybenzoic acid residue content: not detected (limit of detection: 0.005 g / kg) Vitamin B1 content: not detected (limit of detection: 0.01 mg%) Vitamin B2 content: 0.02 mg% Total Vitamin C content: not detected (detection limit: 2 mg%) Vitamin B6 content: not detected (detection limit: 5 pg%) Vitamin B12 content: not detected (detection limit: 0.05 30 pg%) Pantothenic acid content: 0.09 mg% Choline content: not detected (detection limit: 0.03%) Folic acid content: undetected (detection limit: 1 pg%) Niacin content: not detected (detection limit: 0.03 mg%) Total carotene content: not detected (detection limit: 0.02 mg%) pH 9.9 4.9 Amino acid composition ***: Arginine: less than 0.01 Lysine: less than 0.01 Histidine: less than 0 , 01 Phenylalanine: less than 0.01 Tyrosine: less than 0.01 Leuoin: less than 0.01 Isoleucine: less than 0.01 Methionine: less than 0.01 Valine: less than 0.01 Alanine: less than 0 , 01 Glycine: less than 0.01 Proline: less than 0.01 Glutamic acid: 0.01 Serine: less than 0.01 Treonine: less than 0.01 Aspartic acid: less than 0.01 Tryptophan: less than 0.01 Cystine: less than 0.01, the notes having the following meanings: *:% water-based **: ml of 1-n alkali solution titrated to neutralize 100 g of sample 25 ***: cystine: formic acid oxidation method, tryptophan: microorganism determination). 3. Physical Properties: (1) Density d 2: 1,000 - 1,015 (2) Refractive Index n ″: 1.330 - 1.340 (3) Process for the preparation of a product according to claim 1, characterized in that its only constituent or major constituent is obtained by culturing the Trichosporon kashiwayama strain FERM 4821 on or in a culture medium at a pH of 4.0 -6.0 and a temperature of 20-30 ° C to obtain a culture product or culture fluid, optionally the cells are removed from said culture fluid to obtain a cell-free, sterile fluid, sterile filtrate or sterile supernatant and optionally the sterile liquid, sterile filtrate or sterile supernatant from which the cells are removed is concentrated or dried. 4. Depreciation residuals: 0.5 - 2.0%. 5. A process according to claim 4, characterized in that the culture medium comprises glucose, skimmed milk and yeast extracts and that the strain is grown under aerobic conditions.