JP7042544B2 - Method for preparing composition for hair loss treatment - Google Patents

Description

The present invention relates to methods for preparing various compositions and to the field of everyday chemicals.

Due to accelerated lifestyles, increased work pressure, and changes in diet and lifestyle, the incidence of hair loss in young and middle-aged men in China has increased more than 10 times compared to 20 years ago. At the same time, the incidence of hair loss in female residents is increasing year by year. However, the recognition rate that hair loss is a disease that requires going to the hospital is still very low. The main reasons are genetic factors, physiological dysfunction, work and research pressure, psychological stress, drug reactions, radiation therapy, environmental pollution, malnutrition, etc.

However, recent studies have shown that hair loss is associated with an imbalance in the body's gut flora. Disorders of the gut flora lead to an increase in lactic acid bacteria (Lactobacillus rhamnosus), which in turn leads to the lack of certain substances or enzymes most likely due to the secretion of gastrointestinal microbes. The human gut flora is a complex community. The total amount of microorganisms in the intestinal tract of a healthy adult is about 1-1.5 kg. There are more than 1,000 species, and the number of gut microbiota is ten times the number of human cells. This is called the "invisible human body super organ". The gut microbiota plays an important role in digestion, metabolism and immune function. Gut microbiota imbalance may be one of the important causes of various metabolic disorders such as obesity, diabetes, tumors and hair loss. Therefore, this patent aims to complement the diversity of the intestinal flora and improve the intestinal flora by oral ingestion of the compositions of the present invention.

It is an object of the present invention to improve the intestinal bacterial flora, thereby solving the technical problems of human alopecia, and to provide a method for preparing a composition for treating alopecia.

The method for preparing the first composition for treating hair loss is performed according to the following steps.

1. 1. Membrane separation of wood ear polysaccharides Black ear (black fungus) is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of black fungus. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, then ultrasonic power is 500 W, ultrasonic waves are applied for 15 minutes, extraction is performed in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifuge for a minute to remove the residue, and prepare the extract into a feed solution with a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. In order to obtain an Auricularia polysaccharide holding solution having a molecular weight cutoff of 300 kDa, filtration is performed under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar.

2. 2. Select perfect particles and uniform size soybeans, weigh and wash, mix with water at a volume ratio of 1: 3, soak at 30 ° C for 3 hours, cook well, cook and cook at 30 ° C. Cool to ~ 40 ° C. Inoculate 300 kDa Kikurage polysaccharide retention solution and Aspergillus inoculum solution at a mass ratio of 1: 3, keep the room temperature at 28 ° C, and stir the koji for 20 to 24 hours for the first time and 28 hours for the second time. Was done in. Jiuqu is formed after 34 hours. When the jiuqu is made, wash the jiuqu, seal it for 12 hours, put it in a tank, put it in a constant temperature room at 28 ° C to ferment it for 30 days or more, take it out and dry it. obtain.

The method for preparing the second composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. The dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and then ultrasonic waves having an ultrasonic power of 500 W are applied for 15 minutes. Extraction is performed in a water bath for 3 hours, and the extract is centrifuged at 4500 r / min for 10 minutes to remove the residue, and the extract is prepared as a feed solution having a black syrup polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Add the mucor strain to sterile physiological saline, take the bacterial solution with a pipette, inoculate the bran medium, incubate at 20 ° C for 96 hours, and add 500 mL sterile physiological saline to the cultured seed koji culture flask. Shake to homogenize, filter with double gauze, add 500 mL of sterile physiological saline to the filter residue, wash once and filter, mix the two filtrates to make a spore suspension, scab. Place the spore suspension in a refrigerator at 4 ° C.

3. 3. Whole-grain, uniform-sized soybeans are weighed, washed, mixed with water at a volume ratio of 1: 3, drained, steamed at 121 ° C. for 30 minutes in a high temperature autoclave, and cooled.

4. Mixed inoculation: Inoculate a mucor polysaccharide holding solution with a molecular weight cutoff of 300 kDa and a mucor spore suspension at a mass ratio of 1: 3, inoculate at 25 ° C. for 48 hours, and then aged for 48 hours to treat the mucor. Type-Kikurage polysaccharide 300 kDa holding liquid Tempeh fermented product is obtained.

The method for preparing the third composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Whole grain and uniform size soybeans are selected, weighed and washed, mixed with water at a volume ratio of 1: 3, soaked in water for 10-18 hours and sterilized at 120 ° C. to obtain a fermentation substrate.

3. 3. The fermentation substrate is inoculated with the complex solution until the concentration of the complex solution in the fermentation substrate reaches 2 ml / L. The complex solution is mixed with a Bacillus subtilis 300 kDa retention solution and a Bacillus subtilis inoculation solution at a mass ratio of 1: 3, shaken well, and cultured at 4 to 8 ° C. for 72 to 120 hours to retain Bacillus subtilis type-Bacillus polysaccharide 300 kDa. Obtain a liquid tempeh fermented product.

Tempeh (fermented soybeans) is rich in nutrients, mainly including proteins, free amino acids, essential fatty acids, soluble sugars, inorganic salts, vitamins and the like. In addition, tempeh also contains soy isoflavones, soy saponins, brown pigments, tempeh fibrinolytic enzymes, β-glucosidases, antibiotics and other physiologically active substances. These active substances in Tempe are partly derived from raw soybeans and are partly complex biochemical reactions such as the decomposition and reorganization of raw soybean organics by various microorganisms and enzymes involved in the fermentation process. Derived from.

Polysaccharides contained in black sardines have functions such as anti-ulcer, delayed aging, lowering blood lipids, anti-hepatitis, anti-mutation, anti-tumor, promotion of protein and nucleic acid metabolism, and promotion of body immunity. .. The co-fermentation of fungal polysaccharides with tempeh is truncated and separated by membrane separation, with a clear hair-fixing effect.

1. 1. Traditional hair growth uses ginger and kurobe leaves for epidermal massage to promote microcirculation of the scalp. Oral administration of the fermented product of the present invention can repair the structure of the intestinal flora and restore the microecological balance. It is still a blank area to achieve the hair-fixing effect by regulating the intestinal flora by diet.

2. 2. In animal experiments, the combination of the present invention showed excellent hair fixing effect and found a new method to solve the problem of large-scale hair loss in the future.

3. 3. The composition for treating hair loss of the present invention has a significant promoting effect on hair follicle growth and a significant improving effect on seborrheic alopecia.

The technical solutions of the present invention are not limited to the particular embodiments listed below, but include any combination of the particular embodiments.

[Specific Example 1]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Select soybeans of uniform size in whole grains, weigh and wash, mix with water at a volume ratio of 1: 3, soak at 30 ° C. for 3 hours (moisture content 45% -55%), and heat sufficiently. Cook and cool the cooked to 30 ° C-40 ° C. The Kikurage polysaccharide holding solution with a molecular weight cutoff of 300 kDa and the Aspergillus inoculum solution were inoculated at a mass ratio of 1: 3, the room temperature was kept at 28 ° C, and the inoculum was stirred for 20 to 24 hours for the first time and 28 hours for the second time. .. Jiuqu is formed after 34 hours. When the jiuqu is made, wash the jiuqu, seal it for 12 hours, put it in a tank, put it in a constant temperature room at 28 ° C to ferment it for 30 days or more, take it out and dry it. obtain.

The temperature of the water bath extraction in step 1 of the present embodiment is 70 ° C to 85 ° C.

In step 1 of the present embodiment, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

The cooked product in step 2 of the present embodiment is cooked soybeans.

In step 2 of the present embodiment, cooked soybeans of every 200 g are inoculated with 3 ml of a Kikurage polysaccharide holding solution and an Aspergillus inoculum, and the concentration of Aspergillus spores is 1.0 × 10 ⁷ to 1.0 × 10 ⁸ spores / ml. Is.

In step 2 of the present embodiment, "sealing" refers to processing in a closed incubator having a temperature of 30 ± 2 ° C.

[Specific Example 2]
The difference between the present embodiment and the first embodiment is that the water content in the step 2 is 50%. Others are the same as those in the first embodiment.

[Specific Example 3]
This embodiment differs from the specific Example 1 or 2 in that the cooked product in step 2 is cooled to 35 ° C. Others are the same as those of the first or second embodiment.

[Specific Example 4]
This embodiment differs from the specific Examples 1 to 3 in that the first stirring of the Jiuqu in step 2 is performed 22 hours after the production of the Jiuqu. Others are the same as those of the first to third embodiments.

[Specific Example 5]
The method for preparing the composition for hair loss treatment according to this example is carried out according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Add the mucor strain to sterile saline, pipette the bacterial solution, inoculate the bran medium, and incubate at 20 ° C. for 96 hours (the bran in the Erlenmeyer flask is covered with hyphae and gray spores and will be used later. Taken out to do). Add 500 mL of sterile saline to the cultured seed koji culture flask, shake well, filter with double gauze, add 500 mL of sterile saline to the filter residue, wash once and filter, and filter the two filtrates. Mix to make a spore suspension. Place the mucor spore suspension in a refrigerator at 4 ° C.

3. 3. Whole-grain, uniform-sized soybeans are weighed, washed, mixed with water at a volume ratio of 1: 3, drained, steamed at 121 ° C. for 30 minutes in a high temperature autoclave, and cooled.

4. Mixed inoculation A molecular weight cutoff of 300 kDa Kikurage polysaccharide holding solution and mucor spore suspension are inoculated at a mass ratio of 1: 3, and after culturing at 25 ° C. for 48 hours, they are aged for 48 hours (muturing treatment) to form a mucor type. -Obtain a mucor polysaccharide 300 kDa holding liquid tempeh fermented product.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of the present embodiment, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

In step 2 described in step 2 of the present embodiment, a total of 2 ml of a wood ear polysaccharide holding solution and a mucor spore suspension are inoculated for every 200 g of cooked soybeans.

The concentration of spores in the mucor spore suspension of the present embodiment is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

[Specific Example 6]
The method for preparing the composition for hair loss treatment according to the present specific embodiment is carried out according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Whole grain and uniform size soybeans are selected, weighed and washed, mixed with water at a volume ratio of 1: 3, soaked in water for 10-18 hours and sterilized at 120 ° C. to obtain a fermentation substrate.

3. 3. The fermentation substrate is inoculated with the complex solution until the concentration of the complex solution in the fermentation substrate reaches 2 ml / L. The complex solution is mixed with a Bacillus subtilis 300 kDa retention solution and a Bacillus subtilis inoculation solution at a mass ratio of 1: 3, shaken well, and cultured at 4 to 8 ° C. for 72 to 120 hours to retain Bacillus subtilis type-Bacillus polysaccharide 300 kDa. Obtain a liquid tempeh fermented product.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of the present embodiment, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

The spore concentration of the Bacillus subtilis inoculum of the present embodiment is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

The following experiments are used to verify the effectiveness of the invention.

[Example 1]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Select soybeans of uniform size for all grains, weigh and wash, soak in water at 30 ° C for 3 hours at a volume ratio of soybean to water 1: 3, drain, and steam at 121 ° C for 30 minutes in a high temperature autoclave. ,Cooling. A Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa is added, and the mixture is cultured at 25 ° C. for 48 hours to obtain a tempeh pre-fermented product. A soybean-Kikurage polysaccharide 300 kDa cut-off fermented product is obtained by aging treatment for 48 hours.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

[Example 2]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. For ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar, a 300 kDa permeate of Kikurage polysaccharide is selected.

2. 2. Soybeans of uniform size are selected, weighed and washed, soaked in water at 30 ° C for 3 hours at a volume ratio of soybean and water 1: 3, drained, and steamed in a high temperature autoclave at 121 ° C for 30 minutes. Cooling. A 300 kDa permeate of Kikurage polysaccharide is added and cultured at 25 ° C. for 48 hours to obtain a tempeh pre-fermented product. After that, a aging treatment for 48 hours is performed. In this way, a fermented soybean-Kikurage polysaccharide 300 kDa permeate is obtained.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

[Example 3]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Add the mucor strain to sterile saline, pipette the bacterial solution, inoculate the bran medium, incubate at 20 ° C. for 96 hours until the Erlenmeyer flask bran is filled with hyphae and gray spores, and use later. Take out for. Add 500 mL of sterile saline to the cultured seed koji culture flask, shake well, filter with double gauze, add 500 mL of sterile saline to the filter residue, wash once, filter and mix the two filtrates. To make a spore suspension. Place the mucor spore suspension in a refrigerator at 4 ° C.

3. 3. Select whole soybeans of uniform size, weigh and wash, mix with water at a mass ratio of 1: 3, soak at 30 ° C for 3 hours, drain, steam at 121 ° C for 30 minutes in a high temperature autoclave. And cool.

4. Mixed inoculation A molecular weight cutoff of 300 kDa Kikurage polysaccharide holding solution and mucor spore suspension are inoculated at a mass ratio of 1: 3, and after culturing at 25 ° C. for 48 hours, they are aged for 48 hours (muturing treatment) to form a mucor type. -Kikurage polysaccharide 300 kDa holding liquid Tempeh fermented product is obtained.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

In step 2 of the present embodiment, a total of 2 ml of a wood ear polysaccharide holding solution and a mucor spore suspension are inoculated for every 200 g of cooked soybeans.

The concentration of spores in the mucor spore suspension according to this example is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

[Example 4]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. For ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar, a 300 kDa permeate of Kikurage polysaccharide is selected.

2. 2. Add the mucor strain to sterile saline, pipette the bacterial solution, inoculate the bran medium, incubate at 20 ° C. for 96 hours until the Erlenmeyer flask bran is filled with hyphae and gray spores, and use later. Take out for. Add 500 mL of sterile saline to the cultured seed koji culture flask, shake well, filter with double gauze, add 500 mL of sterile saline to the filter residue, wash once, filter and mix the two filtrates. To make a spore suspension. The mucor spore suspension is placed in a refrigerator at 4 ° C. and stored as a strain.

3. 3. Select soybeans of uniform size for all grains, weigh and wash, soak in water at 30 ° C for 3 hours at a volume ratio of soybean to water 1: 3, drain, and use a high-temperature autoclave at 121 ° C for 30 minutes. Steam and cool. A 300 kDa permeate of Kikurage polysaccharide and a mucor spore suspension are inoculated at a mass ratio of 1: 3, and after culturing at 25 ° C. for 48 hours, a tempeh pre-fermented product is obtained. It is aged for 48 hours to obtain a mucor type-Kikurage polysaccharide 300 kDa permeate tempeh fermented product.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

In step 2 of the present embodiment, a total of 2 ml of a wood ear polysaccharide holding solution and a mucor spore suspension are inoculated for every 200 g of cooked soybeans.

The concentration of spores in the mucor spore suspension according to this example is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

[Example 5]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Whole grain and uniform size soybeans are selected, weighed and washed and mixed with water in a 1: 3 volume ratio to a moisture content of 45% -55%. Cook well and cool the cooked to 30 ° C-40 ° C. The Kikurage polysaccharide holding solution with a molecular weight cutoff of 300 kDa and the Aspergillus inoculum solution were inoculated at a mass ratio of 1: 3, the room temperature was kept at 28 ° C, and the inoculum was stirred for 22 hours for the first time and 28 hours for the second time. .. Jiuqu is formed after 34 hours. When the jiuqu is made, wash the jiuqu, seal it for 12 hours, and put it in a tank. ..

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

In step 2 of the present embodiment, cooked soybeans of every 200 g are inoculated with 3 ml of a Kikurage polysaccharide holding solution and an Aspergillus inoculum, and the concentration of Aspergillus spores is 1.0 × 10 ⁷ to 1.0 × 10 ⁸ spores / ml. Is.

“Sealing” in step 2 of the present embodiment refers to processing in a closed incubator having a temperature of 30 ± 2 ° C.

[Example 6]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. The dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and then ultrasonic waves having an ultrasonic power of 500 W are extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifuge for a minute to remove the residue, and prepare the extract into a feed solution with a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. For ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar, a 300 kDa permeate of Kikurage polysaccharide is selected.

2. 2. Select soybeans of uniform size for all grains, weigh and wash, soak in water at 30 ° C for 3 hours with a volume ratio of soybean and water of 1: 3 until the water content reaches 50%, and cook thoroughly. And cool the cooked food to 35 ° C. Inject Kikurage polysaccharide 300 kDa permeate and Aspergillus inoculum at a mass ratio of 1: 3, mix well, keep the room temperature at 28 ° C, stir the inoculum 22 hours after making the inoculum, and 28 in the second. It starts at the hour. Jiuqu is formed after 34 hours. After the jiuqu is produced, the jiuqu is washed, sealed for 12 hours, placed in a tank, and fermented in a constant temperature room at 28 ° C. for 30 days or more. Jiuqu-type-Kikurage polysaccharide 300 kDa permeate Tempeh To obtain a fermented product, take it out and dry it.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

In step 2 of the present embodiment, a total of 3 ml of the Kikurage polysaccharide holding solution and Aspergillus inoculum are inoculated for every 200 g of cooked soybeans. The concentration of Aspergillus spores is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

“Sealing” in step 2 of the present embodiment refers to processing in a closed incubator having a temperature of 30 ± 2 ° C.

[Example 7]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. Ultrafiltration is performed at a temperature of 40 ° C. and a pressure of 0.17 bar in order to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa.

2. 2. Whole grain and uniform size soybeans are selected, weighed and washed, mixed with water at a mass ratio of 1: 3, soaked in water for 10-18 hours and sterilized at 120 ° C. to give the fermentation substrate.

3. 3. The fermentation substrate is inoculated with the complex solution until the concentration of the complex solution in the fermentation substrate reaches 2 ml / L. The complex solution is mixed with a Bacillus subtilis 300 kDa retention solution and a Bacillus subtilis inoculation solution at a mass ratio of 1: 3, shaken well, and cultured at 4 to 8 ° C. for 72 to 120 hours to retain Bacillus subtilis type-Bacillus polysaccharide 300 kDa. Obtain a liquid tempeh fermented product.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant to dilute the black fungus polysaccharide of the supernatant to 4 g / L.

The spore concentration of the Bacillus subtilis inoculum according to this example is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

[Example 8]
The method for preparing the composition for treating hair loss is performed according to the following steps.

1. 1. Membrane Separation of Kikurage Polysaccharide The black ear is washed, impurities are removed, crushed and passed through a 40 mesh sieve to produce a dry powder of the black ear. Dry powder of black kikurage is added to distilled water at a mass ratio of 140: 1, and an ultrasonic wave having an ultrasonic power of 500 W is extracted over 15 minutes in a water bath for 3 hours, and the extract is extracted at 4500 r / min for 10 minutes. Centrifugation is performed to remove the residue, and the extract is prepared as a feed solution having a black sardine polysaccharide concentration of 4 g / L and a pH value of 5.6. For ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar, a 300 kDa permeate of Kikurage polysaccharide is selected.

2. 2. Whole grain and uniform size soybeans are selected, weighed and washed, mixed with water at a mass ratio of 1: 3, soaked in water for 10-18 hours and sterilized at 120 ° C. to give the fermentation substrate.

3. 3. The fermentation substrate is inoculated with the complex solution until the concentration of the complex solution in the fermentation substrate reaches 2 ml / L. The complex solution is mixed with a Bacillus subtilis 300 kDa holding solution and a Bacillus subtilis inoculation solution at a mass ratio of 1: 3, shaken well, and cultured at 4 to 8 ° C. for 72 to 120 hours. Obtain a tempeh fermented product.

In the present embodiment, the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

In step 1 of this example, distilled water is added to the centrifuged supernatant, and the black fungus polysaccharide in the supernatant is diluted to 4 g / L.

The spore concentration of the Bacillus subtilis inoculum according to this example is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

[Experiment 1] Inhibition experiment of the oral composition of the present invention for hair loss in seborrheic mice 1. Experimental material Testosterone propionic acid injection 25 mg / 1 ml, purchased from Ningbo No. 2 Hormone Factory, production batch number: 11025.
Salad oil 90 ml / bottle, Arowana Co., Ltd. Adult male B6CBAF1 / J mouse, weight 18-22 g, clean grade, Beijing Weitong Lihua Laboratory Animal Technology CO. Ltd. [License number: SC] (K computer) 2017-0011], room temperature 23 ° C, humidity 50-60%, twice a day, 1 hour each time According to the natural life pattern of the mice, the B6CBAF1 / J mice can move freely and freely. You can drink and eat.

2. 2. Experimental method 2.1 Experimental grouping From 100 male mice, 10 B6CBAF1 / J mice were randomly selected as the experimental control group, and the remaining 90 B6CBAF1 / J mice were used as experimental mice. , Raised in separate cages. That is, the hair loss model group and the oral composition examples 1 to 8 groups.

2.2 Preparation and administration method of animal model The depilatory animal model was created by injection. The injection site is subcutaneously on the back of the mouse, parallel to the back of the spine. In the case of the experimental control group, saline was injected, and the other 9 groups were injected with testosterone propionate. The injection dose is 5 mg / kg · d. The injection time is 4 weeks.

Drug treatment was started 4 weeks after the B6CBAF1 / J mouse model of hair loss was established, and the composition of each embodiment was orally administered to the hair loss model group for 6 weeks except for the control group. Observe the hair growth state of the mouse at the hair loss site and calculate the weight of the mouse hair loss. In combination with pathological observations, the ratio of hair / vellus hair under a microscope is calculated as an average compared to the model group.

3. 3. Statistical analysis of data SPSS 20.0 software was used for data processing. Differences between groups were analyzed by one-way ANOVA and were considered statistically significant with p <0.05 as the significant difference.

4. Experimental results (1) Determination of hair loss weight From Table 1, it can be seen that the degree of hair loss differs between the other experimental mice as compared with the experimental control group. Except for the significant difference (p <0.05) in Examples 3, 5 and 7, extreme significant difference (p <0.01) hair loss is seen in the other groups. Compared with the model group, the hair loss phenomenon of the mice of Examples 3, 5, and 7 was improved (p <0.01). There was no significant difference in the other example groups, and no improvement tendency was seen in hair loss.

(2) Determination of mouse hair / vellus hair ratio From Table 2, it was found that the ratio of hair / vellus hair in the model group was significantly reduced compared to the experimental control group (p <0.01), and the model. Compared with the group, Examples 3, 5 and 7 can significantly improve the hair / vellus hair ratio (p <0.01). The ratio, the ratio of hair / vellus hair in other experimental examples, did not change.

(3) Examples 3, 5, and 7 of the oral composition of the present invention have a significant improving effect on seborrheic alopecia, and soybean exerts a clear hair fixing effect after fermentation by a strain. It shows that it can be done.

[Experiment 2] Effect of oral composition on the number of hair follicles in hair-growth mice 1. The experimental material rosin was purchased from Shenzhen Jitian Chemical Co., Ltd. Paraffin wax was purchased from Aowei Plastic Wax Products. Adult female C57BL / 6 mice, body weight 18-22 g, clean grade, skin color pink, hair is interruption mice. Purchased from Beijing Weitong Lihua Laboratory Co. Ltd. [License No .: SCXK (Kyo) 2017-0149], room temperature 23 ° C, humidity 50-60%, 2 days a day Ventilation for 1 hour each time According to the natural life pattern of mice, mice are free to move, drink and eat freely.

2. 2. Experimental method 2.1 Experimental grouping 90 female C57BL / 6 mice were randomly divided into 9 groups, an experimental control group, and an oral composition example 1-8 group, and kept in separate cages.

2.2 Preparation of animal model and administration method After mixing rosin and paraffin wax at a ratio of 1: 1, heat and melt them, and apply them evenly to the back of the mouse in an area of about 2 cm × 3 cm. After cooling and solidifying, remove the hair on the back (make sure the hair growth is in the interruption period). Except for the control group to which distilled water was given by gavage, the other groups were administered once daily for 20 consecutive days at a gavage dose of 2 ml / 100 g by gavage. On day 20, the co-located skin tissue blocks on the backs of each group of mice are removed, fixed with 10% formalin and the sections are embedded with paraffin wax. Section thickness is about 4-5 microns, HE stain, number of hair follicles in high-power field (x400), averaged and statistically processed.

3. 3. Statistical analysis of data SPSS20.0 software was used for data processing. Differences between groups were analyzed by one-way ANOVA and were considered statistically significant with p <0.05 as the significant difference.

4. Experimental results (1) Effect of hair follicle density Table 3 shows the number of hair follicles in each group on the 20th day of the experiment. Compared to the experimental control group, Examples 3, 5, and 7 were able to increase the number of hair follicles in the experimental control group mice (p <0.01), and the examples in the other groups were C57BL / 6 mice. Does not affect the number of hair follicles (p> 0.05). The results suggest that fermented soybeans enter the hair follicle of C57BL / 6 mice, prolong the growth period, delay the transition to the degeneration phase, and promote the growth of the hair follicle.

(Additional note)
(Appendix 1)
The black sardines are washed, decontaminated, crushed and passed through a 40 mesh sieve to produce a dry black sardine powder, the dry black sardines are added to distilled water at a mass ratio of 140: 1 and ultrasonic. An ultrasonic wave having a power of 500 W was extracted over 15 minutes in a water bath for 3 hours, and the extract was centrifuged at 4500 r / min for 10 minutes to remove the residue. , Prepared in a feed solution with a pH value of 5.6, and subjected to ultrafiltration at a temperature of 40 ° C. and a pressure of 0.17 bar to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa. Step 1 of membrane separation of Kikurage polysaccharide. When,
Select whole-grain, uniform-sized soybeans, weigh and wash, mix with water at a volume ratio of 1: 3, cook well, and cool the heated to 30 ° C-40 ° C. Inject the Kikurage polysaccharide holding solution with a molecular weight cutoff of 300 kDa and the Aspergillus inoculum at a mass ratio of 1: 3, keep the room temperature at 28 ° C, and stir the Jiuqu for the first time when the Jiuqu is made for 20 to 24 hours. , When the koji making is 28 hours, the second stirring of the koji is performed, and when the koji making is 34 hours, the koji can be produced. Put it in a constant temperature room at 28 ° C, ferment it for 30 days or more, take it out and dry it to obtain a Jiuqu-type-Kikurage polysaccharide 300 kDa holding liquid tempeh fermented product.
A method for preparing a composition for treating hair loss, which comprises.

(Appendix 2)
The method for preparing a composition for hair loss treatment according to Appendix 1, wherein in step 2, the composition is immersed at 30 ° C. for 3 hours and has a water content of 50%.

(Appendix 3)
The method for preparing a composition for hair loss treatment according to Appendix 1, wherein the cooked product is cooled to 35 ° C. in step 2.

(Appendix 4)
The method for preparing a composition for hair loss treatment according to Appendix 1, wherein in step 2, the first stirring of the jiuqu is performed 22 hours after the production of the jiuqu.

(Appendix 5)
3 ml of Kikurage polysaccharide holding solution and Aspergillus inoculum are inoculated into the cooked soybeans every 200 g in Step 2, and the concentration of Aspergillus spores is 1.0 × 10 ⁷ to 1.0 × 10 ⁸ spores / ml. The method for preparing the composition for hair loss treatment according to Appendix 1.

(Appendix 6)
The black sardines are washed, decontaminated, crushed and passed through a 40 mesh sieve to produce a dry black sardine powder, the dry black sardines are added to distilled water at a mass ratio of 140: 1 and ultrasonic. An ultrasonic wave having a power of 500 W was extracted over 15 minutes in a water bath for 3 hours, and the extract was centrifuged at 4500 r / min for 10 minutes to remove the residue. , Prepared in a feed solution with a pH value of 5.6 and subjected to ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa. When,
Add the Kekabi strain to sterile physiological saline, take the bacterial solution with a pipette, inoculate it into bran medium, incubate at 20 ° C for 96 hours, add 500 mL of sterile physiological saline to the cultured seed koji culture flask, shake well, and shake well. Filter with heavy gauze, add 500 mL of sterile physiological saline to the filter residue, wash once and filter, mix the two filtrates to make a kekabi spore suspension, and prepare the kekabi spore suspension 4 Step 2 of putting in a refrigerator at ℃ and
Step 3 of selecting soybeans of uniform size in whole grains, weighing, washing, mixing with water at a volume ratio of 1: 3, draining, steaming at 121 ° C. for 30 minutes in a high temperature autoclave, and cooling. When,
Inoculate the mucor polysaccharide holding solution with a molecular weight cutoff of 300 kDa and the mucor spore suspension at a mass ratio of 1: 3, inoculate at 25 ° C for 48 hours, and then aged for 48 hours to treat the mucor type-mucor polysaccharide 30000 Da tempeh fermented product. In the mixed inoculation step 4 and
A method for preparing a composition for treating hair loss, which is performed according to the above.

(Appendix 7)
The method for preparing a composition for hair loss treatment according to Appendix 6, wherein a total of 2 ml of a wood ear polysaccharide holding solution and a mucor spore suspension is inoculated for every 200 g of soybean cooked in step 2.

(Appendix 8)
The black sardines are washed, decontaminated, crushed and passed through a 40 mesh sieve to produce a dry black sardine powder, the dry black sardines are added to distilled water at a mass ratio of 140: 1 and ultrasonic. An ultrasonic wave having a power of 500 W was extracted over 15 minutes in a water bath for 3 hours, and the extract was centrifuged at 4500 r / min for 10 minutes to remove the residue. , Prepared in a feed solution with a pH value of 5.6, and obtained a Kikurage polysaccharide holding solution having a molecular weight cutoff of 30,000 Da by ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar. When,
A step of selecting soybeans of uniform size in whole grains, weighing and washing, mixing with water at a volume ratio of 1: 3, soaking in water for 10 to 18 hours, and sterilizing at 120 ° C. to obtain a fermentation substrate. 2 and
The fermentation substrate is inoculated with the complex solution until the concentration of the complex solution in the fermentation substrate reaches 2 ml / L, and the complex solution is mixed with the syrup polysaccharide 300 kDa holding solution and the bacillus inoculum solution at a mass ratio of 1: 3. 3. Shake well and incubate at 4-8 ° C. for 72-120 hours to obtain a fermented product of Bacillus subtilis-Kikurage polysaccharide 300 kDa holding solution Tempe.
A method for preparing a composition for treating hair loss, which is performed according to the above.

(Appendix 9)
The method for preparing a composition for treating hair loss according to Appendix 8, wherein the spore concentration of the Bacillus subtilis inoculum is 1.0 × ¹⁰⁷ to 1.0 × 10 ⁸ spores / ml.

(Appendix 10)
The method for preparing a composition for hair loss treatment according to Appendix 1, 6 or 8, wherein the temperature of the water bath extraction in step 1 is 70 ° C to 85 ° C.

Claims (7)

The black sardines are washed, decontaminated, crushed and passed through a 40 mesh sieve to produce a dry black sardine powder, the dry black sardines are added to distilled water at a mass ratio of 140: 1 and ultrasonic. An ultrasonic wave having a power of 500 W was extracted over 15 minutes in a water bath for 3 hours, and the extract was centrifuged at 4500 r / min for 10 minutes to remove the residue. , Prepared in a feed solution with a pH value of 5.6, and subjected to ultrafiltration at a temperature of 40 ° C. and a pressure of 0.17 bar to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa. Step 1 of membrane separation of Kikurage polysaccharide. When,
Select whole-grain, uniform-sized soybeans, weigh and wash, mix with water at a volume ratio of 1: 3, cook well, and cool the heated to 30 ° C-40 ° C. Inject the Kikurage polysaccharide holding solution with a molecular weight cutoff of 300 kDa and the Aspergillus inoculum at a mass ratio of 1: 3, keep the room temperature at 28 ° C, and stir the Jiuqu for the first time when the Jiuqu is made for 20 to 24 hours. , When the koji making is 28 hours, the second stirring of the koji is performed, and when the koji making is 34 hours, the koji can be produced. Put it in a constant temperature room at 28 ° C, ferment it for 30 days or more, take it out and dry it to obtain a Jiuqu-type-Kikurage polysaccharide 300 kDa holding liquid tempeh fermented product.
A method for preparing a composition for treating hair loss, which comprises. The method for preparing a composition for treating hair loss according to claim 1, wherein in step 2, the composition is immersed at 30 ° C. for 3 hours and has a water content of 50%. The method for preparing a composition for hair loss treatment according to claim 1, wherein in step 2, the cooked food is cooled to 35 ° C. The method for preparing a composition for hair loss treatment according to claim 1, wherein in step 2, the first stirring of the jiuqu is performed 22 hours after the production of the jiuqu. 3 ml of Kikurage polysaccharide holding solution and Aspergillus inoculum are inoculated into the cooked soybeans every 200 g in Step 2, and the concentration of Aspergillus spores is 1.0 × 10 ⁷ to 1.0 × 10 ⁸ spores / ml. The method for preparing a composition for treating hair loss according to claim 1. The black sardines are washed, decontaminated, crushed and passed through a 40 mesh sieve to produce a dry black sardine powder, the dry black sardines are added to distilled water at a mass ratio of 140: 1 and ultrasonic. An ultrasonic wave having a power of 500 W was extracted over 15 minutes in a water bath for 3 hours, and the extract was centrifuged at 4500 r / min for 10 minutes to remove the residue. , Prepared in a feed solution with a pH value of 5.6 and subjected to ultrafiltration under the conditions of a temperature of 40 ° C. and a pressure of 0.17 bar to obtain a Kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa. When,
Add the Kekabi strain to sterile physiological saline, take the bacterial solution with a pipette, inoculate it into bran medium, incubate at 20 ° C for 96 hours, add 500 mL of sterile physiological saline to the cultured seed koji culture flask, shake well, and shake well. Filter with heavy gauze, add 500 mL of sterile physiological saline to the filter residue, wash once and filter, mix the two filtrates to make a kekabi spore suspension, and prepare the kekabi spore suspension 4 Step 2 of putting in a refrigerator at ℃ and
Step 3 of selecting soybeans of uniform size in whole grains, weighing, washing, mixing with water at a volume ratio of 1: 3, draining, steaming at 121 ° C. for 30 minutes in a high temperature autoclave, and cooling. When,
Soybeans that have undergone the above step 3 are inoculated with a kikurage polysaccharide holding solution having a molecular weight cutoff of 300 kDa and a mucor spore suspension at a mass ratio of 1: 3, cultured at 25 ° C. for 48 hours, and then aged for 48 hours to produce mucor. Type - Kikurage polysaccharide 300k Da Tempeh fermented product, mixed inoculation step 4 and
A method for preparing a composition for treating hair loss, which is performed according to the above. The method for preparing a composition for hair loss treatment according to claim 6, wherein a total of 2 ml of a wood ear polysaccharide holding solution and a mucor spore suspension is inoculated for every 200 g of soybean cooked in step 2.