WO2020057451A1 - 治疗脱发组合物的制备方法 - Google Patents

治疗脱发组合物的制备方法 Download PDF

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WO2020057451A1
WO2020057451A1 PCT/CN2019/105861 CN2019105861W WO2020057451A1 WO 2020057451 A1 WO2020057451 A1 WO 2020057451A1 CN 2019105861 W CN2019105861 W CN 2019105861W WO 2020057451 A1 WO2020057451 A1 WO 2020057451A1
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hair loss
polysaccharide
preparing
fungus
retentate
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PCT/CN2019/105861
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English (en)
French (fr)
Inventor
王伟明
霍金海
李梦雪
董文婷
刘华石
韩德强
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黑龙江省中医药科学院
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Priority to KR1020207030744A priority Critical patent/KR102549413B1/ko
Priority to JP2021500332A priority patent/JP7042544B2/ja
Publication of WO2020057451A1 publication Critical patent/WO2020057451A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/08—Antiseborrheics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/82—Preparation or application process involves sonication or ultrasonication
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/92—Oral administration

Definitions

  • the invention relates to a method for preparing various compositions, and relates to the field of daily chemicals.
  • the human intestinal flora is a complex community.
  • the total number of microorganisms in the intestine of healthy adults is about 1-1.5 kg, and there are more than 1,000 types.
  • the number of intestinal microorganisms is 10 times the number of human cells.
  • the intestinal flora plays an important role in digestion, metabolism and immune function.
  • Intestinal flora imbalance may be one of the important reasons for various metabolic abnormalities such as obesity, diabetes, tumors, and hair loss. Therefore, the patent intends to supplement the intestinal flora diversity and improve the intestinal flora through oral administration of the composition of the invention.
  • the purpose of the present invention is to improve the intestinal flora, thereby solving the technical problem of human alopecia, and provides a method for preparing a composition for treating hair loss.
  • the preparation method of the first hair loss treatment composition is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • the preparation method of the second hair loss treatment composition is performed according to the following steps:
  • Mucor species to sterile physiological saline, pipette the bacteria solution, inoculate the bran medium, incubate at 20 ° C for 96h, add 500mL of sterilized physiological saline to the cultured seed koji culture bottle, and shake thoroughly. Evenly, filter with double-layer gauze, wash the filter residue with 500mL of sterilized physiological saline and wash once. The two filtrates are mixed to make a Mucor spore suspension. This Mucor spore suspension is placed in a refrigerator at 4 ° C.
  • the third method for preparing a hair loss treatment composition is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • the fermentation substrate is inoculated with a complex solution to a concentration of 2 ml / L in the fermentation solution.
  • the complex solution is composed of a fungus polysaccharide 300 kDa retentate and a Bacillus subtilis inoculum according to a mass ratio of 1: 3. After incubation at -8 ° C for 72-120 hours, a Bacillus subtilis-Agaric polysaccharide 300 kDa retentate fermented fermented product was obtained.
  • Douchi (fermented soybeans) is rich in nutrients, including proteins, free amino acids, essential fatty acids, soluble sugars, inorganic salts, vitamins, and so on.
  • tempeh also contains physiologically active substances such as soybean isoflavones, soybean saponins, brown pigments, tempeh fibrinolytic enzymes, ⁇ -glucosidase, and antibiotics.
  • active substances in tempeh are partly derived from raw soybeans, and partly from complex biochemical reactions such as the decomposition and reorganization of organic matter in raw soybeans by various microorganisms and enzymes involved in the fermentation process.
  • the polysaccharides contained in Auricularia auricula have the functions of anti-ulcer, delay aging, reduce blood lipids, anti-hepatitis, anti-mutation, anti-tumor, enhance protein and nucleic acid metabolism, and promote immune system. , The effect of solid hair is obvious.
  • the combination of the present invention shows excellent hair-fixing effect in animal experiments, and finds a new way to solve the problem of hair loss on a large scale in the future.
  • composition for treating hair loss of the present invention has a significant promotion effect on the growth of hair follicles and a significant improvement effect on seborrheic hair loss.
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • the temperature of the water bath extraction in step 1 of this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • the clinker in step 2 of this embodiment is cooked soybeans.
  • step 2 of this embodiment every 200 g of cooked soybeans are inoculated with 3 ml of fungus polysaccharide retentate and Aspergillus inoculum, and the concentration of Aspergillus spores is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the "boring" in step 2 of this embodiment is to be processed in a closed incubator with a temperature of 30 ⁇ 2 ° C.
  • Embodiment 2 The difference between this embodiment and Embodiment 1 is that the water content in step 2 is 50%. Others are the same as the first embodiment.
  • Embodiment 5 The method for preparing the hair loss treatment composition according to this embodiment is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • Mucor mold to sterile physiological saline, pipette the bacterial solution, inoculate the bran culture medium, and incubate at 20 ° C for 96h (the bran is full of hyphae and gray spores in a triangle flask and removed for future use).
  • 500mL of sterilized physiological saline to the cultured seed koji culture flask, shake well, filter with double gauze, and wash the filter residue with 500mL of sterilized physiological saline once to filter.
  • the two filtrates are mixed to make a Mucor spore suspension. Mucor spore suspension is placed in a refrigerator at 4 ° C;
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • step two described in step two of this embodiment every 200 g of cooked soybeans is inoculated with agaric polysaccharide retentate and Mucor spore suspension for a total of 2 ml.
  • the spore concentration in the Mucor spore suspension of this embodiment is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the method for preparing the composition for treating hair loss according to the present embodiment is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • the fermentation substrate is inoculated with a composite solution to a concentration of 2ml / L in the fermentation solution.
  • the composite solution is composed of a fungus polysaccharide 300kDa retentate and Bacillus subtilis according to a mass ratio of 1: 3, and shaken. Culturing at 72 ° C for 120-120 hours, a 300-kDa retentate fermented tempeh fermented by Bacillus subtilis-Agaric polysaccharide was obtained.
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • the spore concentration of the B. subtilis inoculum is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • Example 1 A method for preparing a composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus polysaccharide retentate;
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • Example 2 The method for preparing a composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration at a temperature of 40 ° C and a pressure of 0.17 bar, and a 300 kDa permeate of agaric polysaccharide was selected;
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • Example 3 The method for preparing a composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • Mucor species to sterile physiological saline, pipette the bacterial solution, inoculate the bran culture medium, and incubate at 20 ° C for 96 hours. After the bran is full of hyphae and gray spores in the triangle flask, remove it for later use.
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • step two of this embodiment every 200 g of cooked soybeans is inoculated with agaric polysaccharide retentate and Mucor spore suspension for a total of 2 ml.
  • the spore concentration in the Mucor spore suspension is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the method for preparing the composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration at a temperature of 40 ° C and a pressure of 0.17 bar, and a 300 kDa permeate of agaric polysaccharide was selected;
  • Mucor species to sterile physiological saline, pipette the bacterial solution, inoculate the bran culture medium, and incubate at 20 ° C for 96 hours. After the bran is full of hyphae and gray spores in the triangle flask, remove it for later use.
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • step 2 of this embodiment every 200 g of cooked soybeans are inoculated with agar polysaccharide permeate and Mucor mold spore suspension for a total of 2 ml.
  • the spore concentration in the Mucor spore suspension is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the method for preparing the composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • step 2 of this embodiment every 200 g of cooked soybeans is inoculated with 3 ml of agaric polysaccharide retentate and Aspergillus inoculum, and the concentration of Aspergillus spores is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the "boring" is processed in a closed incubator with a temperature of 30 ⁇ 2 ° C.
  • the method for preparing the composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration at a temperature of 40 ° C and a pressure of 0.17 bar, and a 300 kDa permeate of agaric polysaccharide was selected;
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • step 2 of this embodiment every 200 g of cooked soybeans are inoculated with 3 ml of agaric acid polysaccharide permeate and Aspergillus inoculum, and the concentration of Aspergillus spores is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the "boring" is processed in a closed incubator with a temperature of 30 ⁇ 2 ° C.
  • the method for preparing the composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration under the conditions of a temperature of 40 ° C and a pressure of 0.17 bar to obtain a 300 kDa molecular fungus retentate polysaccharide retentate;
  • the fermentation substrate is inoculated with a complex solution to a concentration of 2 ml / L in the fermentation solution.
  • the complex solution is composed of a fungus polysaccharide 300 kDa retentate and a Bacillus subtilis inoculum according to a mass ratio of 1: 3. Incubate at -8 ° C for 72-120 hours to obtain a 300 kDa retentate fermented tempura of Bacillus subtilis-Agaric polysaccharide.
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this example distilled water was added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant was diluted to 4 g / L.
  • the spore concentration of the B. subtilis inoculum is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • the method for preparing the composition for treating hair loss is performed according to the following steps:
  • the black fungus was washed, decontaminated, and smashed through a 40-mesh sieve to make a black fungus dry powder.
  • the black fungus dry powder was added to the distilled water according to a mass ratio of dry black fungus powder to distilled water of 140: 1, and then the ultrasonic power was Ultrasonication at 500W for 15min, water bath extraction for 3h, then the extract was centrifuged at 4500r / min for 10min, the residue was removed, and then the extract was formulated into a feed solution with a polysaccharide concentration of 4g / L and pH 5.6, and then Ultrafiltration at a temperature of 40 ° C and a pressure of 0.17 bar, and a 300 kDa permeate of agaric polysaccharide was selected;
  • Fermentation substrate inoculation compound solution to a concentration of 2ml / L in the fermentation solution.
  • the compound solution is composed of a fungus polysaccharide 300kDa permeate solution and a Bacillus subtilis inoculation solution in a mass ratio of 1: 3.
  • the temperature of the water bath extraction in step 1 in this embodiment is 70 ° C to 85 ° C.
  • step 1 of this embodiment distilled water is added to the centrifugal supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.
  • the spore concentration of the B. subtilis inoculum is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 cells / ml.
  • Testosterone propionate injection 25mg / 1ml purchased from Ningbo Second Hormone Factory, production batch number: 11025
  • mice Among 100 male mice, 10 B6CBAF1 / J mice were randomly selected as the experimental blank control group, and the remaining 90 B6CBAF1 / J mice were used as experimental mice, and they were reared in cages. That is, the hair loss model group and the oral composition examples 1-8 group.
  • the animal model of hair loss was modeled by injection.
  • the injection site was the mouse's back parallel to the spinal region subcutaneously. Except for the blank control group, physiological saline was injected, and the remaining 9 groups were injected with testosterone propionate at an injection dose of 5 mg / kg ⁇ d.
  • the injection time is 4 weeks.
  • Drug treatment was started at the 4th week of B6CBAF1 / J mouse hair loss animal model successfully. Except for the blank group, the hair loss model group was orally administered with the composition of each example for 6 weeks. Observe the hair growth status of the mice at the hair removal site, and calculate the hair loss weight of the mice. Combined with the pathological observation, compared with the model group, the ratio of terminal hair to hair in the microscope was used to calculate the average.
  • SPSS 20.0 software was used for data processing. Differences between groups were analyzed by one-way analysis of variance. Significant differences were considered at p ⁇ 0.05, which was considered statistically significant.
  • Examples 3, 5, and 7 of the oral composition of the present invention have a significant effect on improving seborrheic alopecia, which indicates that after fermenting soybeans, they can exhibit a significant hair-fixing effect.
  • Rosin was purchased from (Shenzhen Jitian Chemical Co., Ltd.); paraffin was purchased from (Aowei Rubber Wax Products); adult female C57BL / 6 mice, weighing 18-22g, clean grade, choose pink skin color, hair belongs to rest Mice. Purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. [License No .: SCXK ( ⁇ ) 2017-0149], room temperature 23 °C, humidity 50-60%, ventilation 2 times a day, each time lasting 1 hour, according to mice Natural lifestyle, free movement, drinking and eating.
  • mice Ninety female C57BL / 6 mice were randomly divided into 9 groups, a blank control group, an oral composition group of Examples 1-8, and divided into cages.
  • the other groups were administered orally, with a regular administration once a day, with an intragastric volume of 2ml / 100g for 20 consecutive days.
  • the skin tissue pieces at the same position on the back of each group of mice were taken, fixed with 10% formalin, and the sections were embedded in paraffin. The sections were about 4-5 microns thick, HE stained, and the number of hair follicles at high magnification ( ⁇ 400). , Take the mean value and perform statistical processing.
  • SPSS 20.0 software was used for data processing. Differences between groups were analyzed by one-way analysis of variance. P ⁇ 0.05 was significant and statistically significant.
  • Examples 3, 5, and 7 can increase the number of hair follicles in blank mice (p ⁇ 0.01), and the other groups of examples have no effect on the number of hair follicles in C57BL / 6 mice (p> 0.05); It is suggested that soybean ferment can induce the growth of hair follicles in C57BL / 6 mice, prolong the growth period, and delay the entry into the degenerative phase, which can promote the growth of hair follicles.

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Abstract

治疗脱发组合物的制备方法，其涉及三种治疗脱发组合物，属于日用化学品领域。该组合物是为了解决人群中脱发现象日益突出，并且呈现年轻化趋势的技术问题。该组合物的制备方法如下：一、膜分离木耳多糖；二、制备发酵基质、三、在发酵基质接种曲霉、毛霉或枯草芽孢杆菌，得到治疗脱发组合物。该治疗脱发的组合物对毛囊生长有明显促进作用，对脂溢性脱发有明显改善作用。

Description

治疗脱发组合物的制备方法 技术领域
本发明涉及多种组合物制备方法，涉及日用化学品领域。
背景技术
随着生活节奏的加快，工作压力的增大，饮食及生活习惯的改变，我国中青年男性脱发的发病率较20年前增长了近10倍以上。同时女性居民的脱发发病率也在逐年上升。但对脱发是种病需要去医院就诊的认知率仍然很低。主要的原因有遗传因素、生理功能失调、工作和学习压力大、精神紧张、药物反应、放射化疗、环境污染、营养不良等。
但是最新的研究显示脱发与机体的肠道菌群不平衡有关，肠道菌群紊乱导致鼠乳杆菌增多，进而导致某些物质或酶的缺乏，而该物质又极有可能必须通过胃肠道微生物分泌。人类肠道菌群是一个复杂的群落，健康成人肠道内的微生物总量大约1-1.5kg，有1000多种，肠道微生物的数量是人类细胞数的10倍，被称为“人体隐形的超级器官”。肠道菌群在消化、代谢和免疫功能中起到重要的作用。肠道菌群失衡可能是造成肥胖、糖尿病、肿瘤、脱发等多种代谢异常的重要原因之一。所以本专利想通过口服该发明组合物能够起到补充肠道菌群多样性，改善肠道菌群的目的。
发明内容
本发明的目的是为了改善肠道菌群，从而解决人类脱发现象的技术问题，提供了一种治疗脱发组合物的制备方法。
第一种治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h，煮熟，将熟料冷却至30℃-40℃，然后将分子截留量300kDa的木耳多糖截留液和曲霉接种液按1:3的质量比接种，保持室温28℃，制曲至第20-24小时进行第一次翻曲，制曲28小时进行第二次翻曲，制曲34小时后出曲，出曲后洗曲，闷12小时入罐， 置于28℃恒温室中保温发酵30天以上，取出晾干，即得曲菌型-木耳多糖300kDa截留液豆豉发酵物。
第二种治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、将毛霉菌种加入无菌生理盐水，用移液器吸取菌液，接种于麸皮培养基，20℃下培养96h，在培养好的种曲培养瓶中加入500mL灭菌生理盐水充分摇匀，双层纱布过滤，滤渣再加500mL灭菌生理盐水洗涤一次过滤，两次滤液混合制成毛霉菌孢子悬液，将此毛霉菌孢子悬浮液置于4℃冰箱中；
三、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h，沥干，在高温高压灭菌锅下，于121℃蒸煮30min，冷却，
四、混合接种：将分子截留量300kDa的木耳多糖截留液和毛霉菌孢子悬液按1:3质量比接种，在25℃的温度下培养48h，然后经后熟处理48h，制得毛霉菌型-木耳多糖300kDa截留液豉发酵物。
第三种治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1：3的体积比混合后在水中浸泡10-18小时，于120℃灭菌，得到发酵基质；
三、发酵基质接种复合液至复合液在发酵基质中的浓度为2ml/L，所述复合液由木耳多糖300kDa截留液和枯草芽孢杆菌接种液按照1:3质量比混合，摇匀，于4-8℃培养72-120小时，得到枯草芽孢杆菌型-木耳多糖300kDa截留液豉发酵物。
豆豉(经发酵的黄豆)中含有丰富的营养物质，主要包括蛋白质、游离氨基酸、必需脂肪酸、可溶性糖、无机盐、维生素等。除此之外，豆豉中还含有大豆异黄酮、大豆皂甙、褐色色素、豆豉纤溶酶、β-葡萄糖苷酶、抗菌素等生理活性物质。豆豉中这些活性物质部分来源于原料大豆中，部分来源于参与发酵过程中的各种微生物及酶对原料大豆中有机物的分解、重组等复杂的生化反应。
黑木耳所含的多糖体具有抗溃疡、延缓衰老、降低血脂、抗肝炎、抗突变、抗肿瘤、增强蛋白质和核酸代谢及促进机体免疫等功能，采用膜分离法截断分离木耳多糖与豆豉联合发酵，固发效果明显。
本发明的特点：
1、传统的生发多以生姜、侧柏叶等用于表皮按摩，促进头皮微循坏为主，而口服本发明发酵物，能够修复肠道菌群结构，恢复微生态平衡。通过吃-肠道菌群调节达到固发作用的，还是空白领域。
2、本发明的组合在动物实验中，表现了极好的固发效果，为今后的大规模解决脱发问题，寻找了新的途径。
3、本发明的治疗脱发组合物对毛囊生长有明显促进作用，对脂溢性脱发有明显改善作用。
具体实施方式
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。
具体实施方式一：治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h(含水量45％-55％)，煮熟，将熟料冷却至30℃-40℃，然后将分子截留量300kDa的木耳多糖截留液和曲霉接种液按1:3的质量比接种，保持室温28℃，制曲至第20-24小时进行第一次翻曲，制曲28小时进行第二次翻曲，制曲34小时后出曲，出曲后洗曲,闷 12小时入罐，置于28℃恒温室中保温发酵30天以上，取出晾干，即得曲菌型-木耳多糖300kDa截留液豆豉发酵物。
本实施方式步骤一中所述水浴浸提的温度为70℃～85℃。
本实施方式步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施方式步骤二中所述熟料为煮熟的黄豆。
本实施方式步骤二中每200g煮熟的黄豆接种木耳多糖截留液和曲霉接种液共3ml，曲霉菌液孢子的浓度为1.0×10 7～1.0×10 8个/ml。
本实施方式步骤二中“闷”为置于温度30±2℃的封闭培养箱中处理。
具体实施方式二：本实施方式与具体实施方式一不同的是步骤二中至含水量50％。其它与具体实施方式一相同。
具体实施方式三：本实施方式与具体实施方式一或二不同的是步骤二中将熟料冷却至35℃。其它与具体实施方式一或二相同。
具体实施方式四：本具体实施方式与具体实施方式一至三之一不同的是步骤二中制曲至第22小时进行第一次翻曲。其它与具体实施方式一至三之一相同。
具体实施方式五：本具体实施方式治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、将毛霉菌种加入无菌生理盐水，用移液器吸取菌液，接种于麸皮培养基，20℃下培养96h(三角瓶内麸皮长满菌丝和灰色孢子后取出备用)，在培养好的种曲培养瓶中加入500mL灭菌生理盐水充分摇匀，双层纱布过滤，滤渣再加500mL灭菌生理盐水洗涤一次过滤，两次滤液混合制成毛霉菌孢子悬液，将此毛霉菌孢子悬浮液置于4℃冰箱中；
三、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h，沥干，在高温高压灭菌锅下，于121℃蒸煮30min，冷却；
四、混合接种：将分子截留量300kDa的木耳多糖截留液和毛霉菌孢子悬液按1:3质量比接种，在25℃的温度下培养48h，然后经后熟处理48h，制得毛霉菌型-木耳多糖300kDa 截留液豆豉发酵物。
本实施方式中步骤一所述水浴浸提的温度为70℃～85℃。
本实施方式中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施方式步骤二中所述的步骤二中每200g煮熟的黄豆接种木耳多糖截留液和毛霉菌孢子悬液共2ml。
本实施方式毛霉菌孢子悬液中孢子浓度为1.0×10 7～1.0×10 8个/ml。
具体实施方式六：本具体实施方式治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1：3的体积比混合后在水中浸泡10-18小时，于120℃灭菌，得到发酵基质；
三、发酵基质接种复合液至复合液在发酵基质中的浓度为2ml/L，所述复合液由木耳多糖300kDa截留液和枯草芽孢杆菌按照1:3质量比混合，摇匀，于4-8℃培养72-120小时，得到枯草芽孢杆菌型-木耳多糖300kDa截留液豆豉发酵物。
本实施方式中步骤一所述水浴浸提的温度为70℃～85℃。
本实施方式中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施方式中枯草芽孢杆菌接种液的孢子浓度为1.0×10 7～1.0×10 8个/ml。
采用下述实验验证本发明效果：
实施例1：治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar 的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比于30℃下浸泡3h，沥干，在高温高压灭菌锅下，121℃蒸煮30min，冷却，加入分子截留量300kDa的木耳多糖截留液，在25℃的温度下培养48h，即得豆豉前发酵产物；然后经后熟处理48h，即得黄豆-木耳多糖300kDa截流液发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
实施例2：治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，选取木耳多糖300kDa透过液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比于30℃下浸泡3h，沥干，在高温高压灭菌锅下，121℃蒸煮30min，冷却，加入木耳多糖300kDa透过液，在25℃的温度下培养，48h，即得豆豉前发酵产物；然后经后熟处理48h。即得黄豆-木耳多糖300kDa透过液发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
实施例3：治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、将毛霉菌种加入无菌生理盐水，用移液器吸取菌液，接种于麸皮培养基，20℃下 培养96h，至三角瓶内麸皮长满菌丝和灰色孢子后取出备用，在培养好的种曲培养瓶中加入500mL灭菌生理盐水充分摇匀，双层纱布过滤，滤渣再加500mL灭菌生理盐水洗涤一次过滤，两次滤液混合制成毛霉菌孢子悬液，将此毛霉菌孢子悬浮液置于4℃冰箱中；
三、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的质量比混合后于30℃下浸泡3h，沥干，在高温高压灭菌锅下，于121℃蒸煮30min，冷却，
四、混合接种：将分子截留量300kDa的木耳多糖截留液和毛霉菌孢子悬液按1:3质量比接种，在25℃的温度下培养48h，然后经后熟处理48h，制得毛霉菌型-木耳多糖300kDa截留液豆豉发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施例步骤二中每200g煮熟的黄豆接种木耳多糖截留液和毛霉菌孢子悬液共2ml。
本实施例毛霉菌孢子悬液中孢子浓度为1.0×10 7～1.0×10 8个/ml。
实施例4：
治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，选取木耳多糖300kDa透过液；
二、将毛霉菌种加入无菌生理盐水，用移液器吸取菌液，接种于麸皮培养基，20℃下培养96h，至三角瓶内麸皮长满菌丝和灰色孢子后取出备用，在培养好的种曲培养瓶中加入500mL灭菌生理盐水充分摇匀，双层纱布过滤，滤渣再加500mL灭菌生理盐水洗涤一次过滤，两次滤液混合制成毛霉菌孢子悬液，将此毛霉菌孢子悬浮液置于4℃冰箱中，作为菌种保存；
三、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比于30℃下浸泡3h，沥干，在高温高压灭菌锅下，121℃蒸煮30min，冷却，待用，将木耳多糖300kDa透过液和毛霉菌孢子悬按1:3的质量比接种，在25℃的温度下培养48h，即得豆豉前发酵产物；然后经后熟处理48h，制得毛霉菌型-木耳多糖300kDa透过液豆豉发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施例步骤二中每200g煮熟的黄豆接种木耳多糖透过液和毛霉菌孢子悬液共2ml。
本实施例毛霉菌孢子悬液中孢子浓度为1.0×10 7～1.0×10 8个/ml。
实施例5：
治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h，至含水量45％-55％，煮熟，将熟料冷却至30℃-40℃，然后将分子截留量300kDa的木耳多糖截留液和曲霉接种液按1:3的质量比接种，保持室温28℃，制曲至第22小时进行第一次翻曲，制曲28小时进行第二次翻曲，制曲34小时后出曲，出曲后洗曲,闷12小时入罐，置于28℃恒温室中保温发酵30天以上，取出晾干，即得曲菌型-木耳多糖300kDa截留液豆豉发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施例步骤二中每200g煮熟的黄豆接种木耳多糖截留液和曲霉接种液共3ml，曲霉菌液孢子的浓度为1.0×10 7～1.0×10 8个/ml。
本实施例步骤二中“闷”为置于温度30±2℃的封闭培养箱中处理。
实施例6：
治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件 下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，选取木耳多糖300kDa透过液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比于30℃下浸泡3h，至含水量50％，煮熟，将熟料冷却至35℃，将木耳多糖300kDa透过液和曲霉接种液按1:3的质量比接种，拌匀入室，保持室温28℃，制曲至第22小时进行第一次翻曲，制曲28小时进行第二次翻曲，制曲34小时后出曲，出曲后洗曲，闷12小时入罐，置于28℃恒温室中保温发酵30天以上。取出晾干即得曲菌型-木耳多糖300kDa透过液豆豉发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施例步骤二中每200g煮熟的黄豆接种木耳多糖透过液和曲霉接种液共3ml，曲霉菌液孢子的浓度为1.0×10 7～1.0×10 8个/ml。
本实施例步骤二中“闷”为置于温度30±2℃的封闭培养箱中处理。
实施例7：
治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1：3的质量比混合后在水中浸泡10-18小时，于120℃灭菌，得到发酵基质；
三、发酵基质接种复合液至复合液在发酵基质中的浓度为2ml/L，所述复合液由木耳多糖300kDa截留液和枯草芽孢杆菌接种液按照1:3质量比混合，摇匀，于4-8℃培养72-120小时，得到枯草芽孢杆菌型-木耳多糖300kDa截留液豆豉发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至 4g/L。
本实施例中枯草芽孢杆菌接种液的孢子浓度为1.0×10 7～1.0×10 8个/ml。
实施例8：
治疗脱发组合物的制备方法按照以下步骤进行：
一、膜分离木耳多糖:
将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，选取木耳多糖300kDa透过液；
二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3比例在水中浸泡10-18小时，于120℃灭菌，得到发酵基质；
三、发酵基质接种复合液至复合液在发酵基质中的浓度为2ml/L，所述复合液由木耳多糖300kDa透过液和枯草芽孢杆菌接种液按照1:3质量比混合，摇匀，于4-8℃培养72-120小时，得到枯草芽孢杆菌-木耳多糖300kDa透过液豆豉发酵物。
本实施例中步骤一所述水浴浸提的温度为70℃～85℃。
本实施例中步骤一中向离心上清液中加入蒸馏水，将上清液中的黑木耳多糖稀释至4g/L。
本实施例中枯草芽孢杆菌接种液的孢子浓度为1.0×10 7～1.0×10 8个/ml。
实验一：本发明口服组合物对脂溢性小鼠脱发的抑制实验
1、实验材料
丙酸睾酮注射液25mg/1ml，购自宁波第二激素厂，生产批号：11025
色拉油90ml/瓶，金龙鱼有限公司
成年雄性B6CBAF1/J小鼠，体重18-22g，清洁级，购自北京维通利华实验动物技术有限公司【许可证号：SCXK(京)2017-0011】，室温23℃，湿度50-60％，每天通风2次，每次持续1小时，按照小鼠的自然生活模式，使B6CBAF1/J小鼠自由进行自由活动、饮水和进食。
2、实验方法
2.1实验分组
100只雄性小鼠中随机选出10只B6CBAF1/J小鼠作为实验空白对照组，其余90 只B6CBAF1/J小鼠作为实验小鼠，分笼饲养。即脱发模型组、口服组合物实施例1-8组。
2.2动物模型制备和给药方法
脱发动物模型采用注射给药造模方式，注射部位为小鼠背部平行于脊柱区皮下，除空白对照组给予注射生理盐水外，其余9组给予注射丙酸睾酮，注射剂量为5mg/kg·d，注射时间为4周。
在B6CBAF1/J小鼠脱发动物模型成功造模第4周时，开始给予药物治疗，除空白组外，脱发模型组均灌胃给予各实施例组合物，给药治疗6周。观察小鼠脱毛部位小鼠的毛发生长状况，统计小鼠脱毛重量；并结合病理学观察，与模型组相比，显微镜下终毛/毳毛的比值，计算出平均数。
3.数据统计分析
采用SPSS 20.0软件进行数据处理，组间差异采用单因素方差分析，p＜0.05显著性差异，认为具有统计学意义。
4实验结果
(1)脱毛重量测定
由表1可知：与空白对照组相比，其他各实验小鼠均发生不同程度的脱毛现象，除实施例3、5、7存在显著性差异外(p<0.05)，其余各组均存在极显著性差异(p<0.01)；与模型组相比，实施例3、5、7组小鼠脱毛现象均有所改善(p<0.01)，其他各实施例组均没有显著差异，对脱发没有改善趋势。
(2)小鼠终毛/毫毛比例测定
由表2可知，与空白对照组相比，模型组的终毛/毫毛的比例显著下降(p<0.01)；与模型组相比，实施例3、5、7能明显提高终毛/毫毛的比例(p<0.01)，其他实验例终毛/毫毛比例没有改变。
(3)本发明口服组合物实施例3、5、7对脂溢性脱发有明显改善作用，表明大豆经菌种发酵后，可发挥明显的固发作用。
表1小鼠脱毛重量测定结果
Figure PCTCN2019105861-appb-000001
Figure PCTCN2019105861-appb-000002
**与空白对照组相比p<0.01； △△与模型组相比p<0.01。
表2小鼠终毛/毫毛比例测定结果
Figure PCTCN2019105861-appb-000003
**与空白对照组相比p<0.01； △△与模型组相比p<0.01。
实验二：口服组合物对毛发生长期小鼠毛囊数目的影响
1、实验材料
松香购自于(深圳市吉田化工有限公司)；石蜡购自于(奥伟胶业蜡制品)；成年雌性C57BL/6小鼠，体重18-22g，清洁级，选择皮色粉红，毛发属于休止期的小鼠。购自北京维通利华实验动物技术有限公司【许可证号：SCXK(京)2017-0149】，室温23℃，湿度50-60％，每天通风2次，每次持续1小时，按照小鼠的自然生活模式，进行自由活动、饮水和进食。
2、实验方法
2.1实验分组
90只雌性C57BL/6小鼠随机分为9组，空白对照组，口服组合物实施例1-8组，分笼饲养。
2.2动物模型制备和给药方法
将松香与石蜡1:1混合加热融化后，均匀涂于小鼠背部，涂抹面积约2cm×3cm，冷却凝固后揭去，以去除背毛(确认毛发生长期处于休止期)。脱毛次日，除空白组灌胃给予蒸馏水外，其他各组分别灌胃给药，每天定时给药1次，灌胃体积为2ml/100g，连续给药20天。第20天时，取各组小鼠背部相同位置皮肤组织块，用10％福尔马林固定，石蜡包埋切片，切片厚约4-5微米，HE染色，高倍视野(×400)的毛囊数，取其均值，并进行统计学处理。
3.数据统计分析
采用SPSS 20.0软件进行数据处理，组间差异采用单因素方差分析，p＜0.05显著性差异，具有统计学意义。
4实验结果
(1)毛囊密度的影响
实验第20天，各组小鼠毛囊数目见表3。与空白对照组相比，实施例3、5、7可增加空白小鼠的毛囊数目(p<0.01)，其他各组实施例对C57BL/6小鼠的毛囊数目没有影响(p>0.05)；提示大豆发酵物其可诱导C57BL/6小鼠毛囊进入生长期，延长生长期时间，并推迟进入退行期，具有促进毛囊生长的作用。
表3小鼠脱毛重量测定结果
Figure PCTCN2019105861-appb-000004
*与空白对照组相比p<0.05， **与空白对照组相比P<0.01。

Claims (10)

  1. 治疗脱发组合物的制备方法，其特征在于治疗脱发组合物的制备方法按照以下步骤进行：
    一、膜分离木耳多糖:
    将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
    二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h，煮熟，将熟料冷却至30℃-40℃，然后将分子截留量300kDa的木耳多糖截留液和曲霉接种液按1:3的质量比接种，保持室温28℃，制曲至20-24小时进行第一次翻曲，制曲至28小时进行第二次翻曲，制曲至34小时后出曲，出曲后洗曲，闷12小时入罐，置于28℃恒温室中保温发酵30天以上，取出晾干，即得曲菌型-木耳多糖300kDa截留液豆豉发酵物。
  2. 根据权利要求1所述治疗脱发组合物的制备方法，其特征在于步骤二中至含水量50％。
  3. 根据权利要求1所述治疗脱发组合物的制备方法，其特征在于步骤二中将熟料冷却至35℃。
  4. 根据权利要求1所述治疗脱发组合物的制备方法，其特征在于步骤二中制曲至第22小时进行第一次翻曲。
  5. 根据权利要求1所述治疗脱发组合物的制备方法，其特征在于步骤二中每200g煮熟的黄豆接种木耳多糖截留液和曲霉接种液共3ml，曲霉菌液孢子的浓度为1.0×10 7～1.0×10 8个/ml。
  6. 治疗脱发组合物的制备方法，其特征在于治疗脱发组合物的制备方法按照以下步骤进行：
    一、膜分离木耳多糖:
    将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的 条件下超滤，得到分子截留量300kDa的木耳多糖截留液；
    二、将毛霉菌种加入无菌生理盐水，用移液器吸取菌液，接种于麸皮培养基，20℃下培养96h，在培养好的种曲培养瓶中加入500mL灭菌生理盐水充分摇匀，双层纱布过滤，滤渣再加500mL灭菌生理盐水洗涤一次过滤，两次滤液混合制成毛霉菌孢子悬液，将此毛霉菌孢子悬浮液置于4℃冰箱中；
    三、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1:3的体积比混合后于30℃下浸泡3h，沥干，在高温高压灭菌锅下，于121℃蒸煮30min，冷却，
    四、混合接种：将分子截留量300kDa的木耳多糖截留液和毛霉菌孢子悬液按1:3质量比接种，在25℃的温度下培养48h，然后经后熟处理48h，制得毛霉菌型-木耳多糖30000Da豆豉发酵物。
  7. 根据权利要求6所述治疗脱发组合物的制备方法，其特征在于步骤二中所述的步骤二中每200g煮熟的黄豆接种木耳多糖截留液和毛霉菌孢子悬液共2ml。
  8. 治疗脱发组合物的制备方法，其特征在于治疗脱发组合物的制备方法按照以下步骤进行：
    一、膜分离木耳多糖:
    将黑木耳洗净、除杂、粉碎后过40目筛，制成黑木耳干粉，按照黑木耳干粉与蒸馏水质量比为140:1的比例将黑木耳干粉加入到蒸馏水中，然后在超声功率为500W的条件下超声15min，水浴浸提3h，之后将提取液以4500r/min离心10min，除渣，然后将提取液配制成黑木耳多糖浓度为4g/L、pH值5.6的料液，然后在温度为40℃、压力为0.17bar的条件下超滤，得到分子截留量30000Da的木耳多糖截留液；
    二、选取颗粒饱满，大小均匀的黄豆，称量洗净，与水以1：3的体积比混合后在水中浸泡10-18小时，于120℃灭菌，得到发酵基质；
    三、发酵基质接种复合液至复合液在发酵基质中的浓度为2ml/L，所述复合液由木耳多糖300kDa截留液和枯草芽孢杆菌接种液按照1:3质量比混合，摇匀，于4-8℃培养72-120小时，得到枯草芽孢杆菌-木耳多糖300kDa截留液豆豉发酵物。
  9. 根据权利要求8所述治疗脱发组合物的制备方法，其特征在于枯草芽孢杆菌接种液的孢子浓度为1.0×10 7～1.0×10 8个/ml。
  10. 根据权利要求1、6或8所述治疗脱发组合物的制备方法，其特征在于步骤一中所述水浴浸提的温度为70℃～85℃。
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