CN116327884A - Saccharomyces cerevisiae polypeptide and plant extract composition and alopecia preventing composition - Google Patents

Abstract

The invention relates to the technical field of hair loss prevention. The invention provides a method for preparing a Saccharomyces cerevisiae polypeptide and plant extract composition. The Saccharomyces cerevisiae polypeptide and plant extract composition comprises Saccharomyces cerevisiae polypeptide and plant extracts, and the plant extracts are extracts of various plant materials. The preparation method comprises the steps of preparing plant extract, preparing brewer's yeast fermentation medium, brewing yeast's fermentation culture, brewing yeast's enzymolysis, filtering, purifying, concentrating and the like. The present invention also provides the prepared Saccharomyces cerevisiae polypeptide and plant extract composition, its use in preparing the anti-hair loss composition, and the anti-hair loss composition. The Saccharomyces cerevisiae polypeptide and the plant extract composition of the present invention have the effects of preventing hair loss and hair growth, and the anti-hair loss composition of the present invention has better anti-hair loss effect.

Description

Saccharomyces cerevisiae polypeptide and plant extract composition and anti-hair loss composition

technical field

The invention relates to the technical field of anti-hair loss, in particular to a Saccharomyces cerevisiae polypeptide, a plant extract composition and an anti-hair loss composition with functions of preventing hair loss and hair growth.

Background technique

Hair loss is a worldwide skin problem, and about 50% of adult men suffer from hair loss. In developed countries such as Europe and the United States, due to excessive dietary structure and mental stress, on average, 9 out of 10 adult men have hair loss or baldness. At present, among the 1.4 billion population in China, 250 million people suffer from hair loss, 163 million men suffer from hair loss, and 88 million women suffer from hair loss. 15% speed increment. According to data, among the hair loss population, the trend of feminization and youthfulness is obvious. In the same year, the post-80s and post-90s became the main force of hair loss, and the proportion of post-90s rose beyond that of post-80s. The probability of developing hair loss is the highest around the age of 30, which is a full 20 years earlier than the age of hair loss of the previous generation.

The factors that cause hair loss in modern people include the adverse effects of external environmental factors (such as pollution, microbial infection) and internal environmental factors (such as hormones, stress, staying up late, anxiety), and some people also have their own genetic factors. Due to the large number and rapid growth of the hair loss population, the problem of hair loss is slowly changing from a medical problem to a social problem. People with hair loss had significantly worse sleep quality than those without hair loss. Hair loss has a great impact on the appearance and image of the patient, and it is easy to cause troubles and anxiety in life. However, the existing anti-hair loss methods or products in the market are expensive, poor in effect, short in time, and high in hair care cost.

The scalp is an extension of the facial skin, with a thickness of only 1.476mm, which is 1/6 of the face, 1/12 of the body, and 1/50 of the soles of the feet. The thinness of the scalp makes its ability to resist changes in the external environment very weak. The metabolism of the scalp lasts 14 to 21 days. The strong metabolism makes the scalp the skin with the fastest replacement of the cuticle. This rapid metabolism also provides the basis for the continuous growth of hair follicles. Only a healthy scalp will have healthy hair follicles, and only healthy hair will grow from healthy hair follicles. Each hair follicle is connected with 300 tiny blood vessels, which supply almost all the nutrients needed for hair follicle growth. The scalp is the soil that nourishes the hair. When the scalp is unhealthy, symptoms such as scalp itching, oily scalp, dandruff, and hair loss will appear.

A follicular unit may contain 1 to 4 hairs. It is generally believed that the total number of normal adult hairs is about 80,000 to 100,000, with an average of about 150 hairs per square centimeter. Under normal circumstances, the growth of normal hair has a growth period-shedding period-repetitive cycle process of resting period. After the hair follicle has experienced a growth cycle of 2 to 6 years, it will enter a shedding period of about 4 weeks. After the shedding period, there will be a resting period. After that, the hair follicle stem cells will activate and differentiate into a complete hair follicle organ, and the scalp will grow hair again. In medicine, the hair loss is graded as follows: <50 hair loss per day is normal, 50-100 hair loss is mild hair loss, 100-150 hair loss is moderate hair loss, and >150 hair loss is severe hair loss. When a large number of hair follicles start to shrink, due to genetic factors and environmental factors, the hair follicle stem cells in the resting period cannot be activated and cannot enter a new physiological cycle, resulting in a large area of exposed scalp. As a result, patients with severe hair loss can eventually develop baldness or baldness.

There are a variety of hair growth products on the market, but the ideal product should be based on the activation of in situ hair follicle stem cells in the telogen phase to fundamentally solve the problem of hair loss and baldness. Yeast polypeptide has attracted the attention of the beauty industry as a hair growth active ingredient. Yeast hydrolyzate is made by fermenting yeast to obtain yeast cells, and then removing yeast cell walls and macromolecular proteins through bioengineering methods, which are rich in yeast polypeptides. Yeast peptides mainly exist in the form of small molecule active peptides, which are conducive to the absorption of the skin such as the scalp. At present, yeast extract (yeast polypeptide) is mainly used as a food additive in food and health products such as baked food, dairy products, and sports nutrition food. According to reports, some yeast hydrolyzates have been applied in cosmetics, such as Saccharomyces cerevisiae, Pichia anomala and Torulaspora delbrueckii, etc.

The name of the French SILAB application is "HYDROLYSATE OF PICHIA MINUTA AND COSMETIC USETHEREOF FOR CONTROLLING HAIR LOSS AND STIMULATING REGROWTH" (Pichiaminuta hydrolyzate and its cosmetic use in controlling hair loss and stimulating regeneration), and the publication number is WO2018127604(A1) The PCT application disclosed that the polypeptide contained in the hydrolyzate of Pichia minuta isolated from rhododendron has the effect of controlling hair loss and promoting hair regeneration. Its mechanisms of action include: effects on mitochondrial dynamics necessary for hair follicle growth, effects on "signaling" molecules regulating androgenetic alopecia, effects on epigenetic modifications associated with androgenetic alopecia, androgen It has an effect on the dermal papillae activity of androgenetic alopecia, has an effect on the growth of hair follicles in androgenetic alopecia, and can promote the growth of capillaries.

The PCT application has confirmed that only the hydrolyzate of Pichia minuta has the effect of controlling hair loss and promoting hair regeneration with comparative tests, while Saccharomyces cerevisiae, Torulaspora delbrueckii and even other Pichia Species of the genus Rhodotorula, such as Pichia anomala and Pichia heedii, did not have the above effects. The results of this experiment are somewhat unexpected. It seems that there are significant differences in sequence between the yeast polypeptides in Pichiaminuta hydrolyzate and the yeast polypeptides in other yeast hydrolyzates, which can explain the difference in its role from other yeasts. However, this PCT application does not give the sequence information of the yeast polypeptide of Pichia minuta. In addition, Pichia minuta is isolated from Rhododendron, and it is not a yeast that is easy to obtain and has mature fermentation technology like Saccharomyces cerevisiae, which may limit its application in hair growth and hair growth products.

Contents of the invention

Based on the existing plant extract anti-hair loss products of our company, the inventor hopes to combine Saccharomyces cerevisiae polypeptide with plant extracts to develop a new anti-hair loss product with better anti-hair loss effect.

Therefore, in one aspect, the present invention provides a method for preparing a Saccharomyces cerevisiae polypeptide and a plant extract composition, the Saccharomyces cerevisiae polypeptide and a plant extract composition comprising a Saccharomyces cerevisiae polypeptide and a plant extract, and the plant extract is a plant material Ginger (Zingiber officinale), Spatholobus suberectus, Arborvitae (Biotaorientalis), Polygonum multiflorum (Polygonum multiflorum), Angelica Sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Black mulberry (Morus nigra) fruit, Panax quinquefolius, Tropaeolum majus, Sapindus mukorossi, Betula alba, Angelica keiskei and Plukenetia volubilis extracts, the preparation The method includes the following steps:

(1) using the plant raw material to prepare a plant extract;

(2) preparing Saccharomyces cerevisiae fermentation medium components and the plant extract;

(3) Inoculate Saccharomyces cerevisiae into the Saccharomyces cerevisiae fermentation medium, and carry out fermentation culture to obtain Saccharomyces cerevisiae fermentation liquid;

(4) carrying out Saccharomyces cerevisiae enzymolysis with protease to this Saccharomyces cerevisiae fermentation liquid, obtains Saccharomyces cerevisiae enzymolysis liquid;

(5) Filtrating, purifying, and concentrating the Saccharomyces cerevisiae hydrolyzate to obtain the Saccharomyces cerevisiae polypeptide and plant extract composition.

In step (1) of the preparation method of the present invention, the plant material is pulverized. Pulverization facilitates the extraction of active substances in plant raw materials.

In a preferred embodiment, the plant raw material is pulverized into plant powder with a mesh size of 150 or less. The smaller the particle size of the plant powder, the more favorable the extraction of the active substances in the plant raw materials.

In step (1) of the preparation method of the present invention, the plant material is boiled with water for 25-35 minutes to prepare the plant extract.

In a preferred embodiment, the plant material is boiled in water for 30 minutes to prepare the plant extract.

In the step (2) of the preparation method of the present invention, the yeast fermentation medium components include malt extract, peptone, yeast extract, dipotassium hydrogen phosphate, potassium chloride, magnesium sulfate and iron sulfate.

In a specific embodiment, according to the preparation of 1 kg of the Saccharomyces cerevisiae fermentation medium, the weight of the plant material is 30-40 g, and the weight of the components of the Saccharomyces cerevisiae fermentation medium is: 25-35 g of malt extract, 15 g of peptone -20g, yeast extract 10-15g, dipotassium hydrogen phosphate 1.4-1.5g, potassium chloride 0.4-0.5g, magnesium sulfate 0.4-0.5g, iron sulfate 0.015-0.02g, and the balance is water. Malt extract contains amylase, invert sugar enzyme, vitamin B, fat, phospholipid, dextrin, maltose, glucose and other ingredients, which can provide nutrients such as carbon source and vitamins for the growth of Saccharomyces cerevisiae.

In a preferred embodiment, according to the preparation of 1 kg of the Saccharomyces cerevisiae fermentation medium, the weight of the plant material is 35 g, and the weight of the Saccharomyces cerevisiae fermentation medium components is: malt extract 30 g, peptone 15 g, yeast extract 15g, 1.5g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.02g of iron sulfate, and the balance is water.

In another preferred embodiment, based on the preparation of 1 kg of the Saccharomyces cerevisiae fermentation medium, the weight of the plant material is 30 g, and the weight of the Saccharomyces cerevisiae fermentation medium components is: 25 g of malt extract, 20 g of peptone, yeast Paste 15g, dipotassium hydrogen phosphate 1.5g, potassium chloride 0.5g, magnesium sulfate 0.5g, iron sulfate 0.02g, and the balance is water.

In yet another preferred embodiment, according to the preparation of 1 kg of the Saccharomyces cerevisiae fermentation medium, the weight of the plant material is 40g, and the weight of the Saccharomyces cerevisiae fermentation medium components is 35g of malt extract, 15g of peptone, yeast extract 10g, 1.5g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.02g of iron sulfate, and the balance is water.

In step (3) of the preparation method of the present invention, the fermentation culture is carried out under the following fermentation conditions: the inoculum of Saccharomyces cerevisiae accounts for 3%-5% of the Saccharomyces cerevisiae fermentation medium by weight, and the stirring speed of the fermenter is 120-5%. 180rpm, the temperature is controlled at 37°C, the ventilation rate is controlled at 0.70-0.80vvm, and the fermentation culture is 18-24h.

In a preferred embodiment, the fermentation culture is carried out under the following fermentation conditions: the inoculation amount of Saccharomyces cerevisiae accounts for 4% of the Saccharomyces cerevisiae fermentation medium by weight, the stirring speed of the fermenter is 150rpm, the temperature is controlled at 37°C, and the ventilation rate Control at 0.75vvm, and ferment for 21 hours.

In a preferred embodiment, the fermentation culture is carried out under the following fermentation conditions: the inoculum of Saccharomyces cerevisiae accounts for 5% of the Saccharomyces cerevisiae fermentation medium by weight, the stirring speed of the fermenter is 180rpm, the temperature is controlled at 37°C, and the ventilation rate Controlled at 0.70vvm, fermented and cultivated for 18h.

In another preferred embodiment, the fermentation culture is carried out under the following fermentation conditions: the inoculation amount of Saccharomyces cerevisiae accounts for 3% of the Saccharomyces cerevisiae fermentation medium by weight, the stirring speed of the fermenter is 120rpm, the temperature is controlled at 37°C, and ventilation The amount was controlled at 0.80vvm, and the fermentation was carried out for 24 hours.

In step (4) of the preparation method of the present invention, the protease is a mixed enzyme of neutral protease and papain.

In a specific embodiment, the Saccharomyces cerevisiae enzymolysis is carried out as follows: neutral protease and papain are added to the fermentation broth in a weight ratio of 1:1, and the amount of enzyme added accounts for 8-12% of the Saccharomyces cerevisiae cell weight, and Adjust the pH value of the fermentation broth to 4.8-5.2; then, adjust the temperature of the fermenter to 50-60°C, and perform enzymolysis treatment on the Saccharomyces cerevisiae cells in the fermentation broth for 2-3 hours; after enzymolysis, adjust the temperature of the fermenter to Incubate at 85°C for 1 hour to inactivate neutral protease and papain.

In a preferred embodiment, carry out Saccharomyces cerevisiae enzymolysis as follows: neutral protease and papain are added in the fermented liquid by the weight ratio of 1:1, and the addition amount of enzyme accounts for 10% of Saccharomyces cerevisiae cell weight, and The pH value of the fermentation broth was adjusted to 5; then, the temperature of the fermenter was adjusted to 55°C, and the Saccharomyces cerevisiae cells in the fermentation broth were subjected to enzymatic hydrolysis for 2.5 hours; after enzymolysis, the temperature of the fermenter was adjusted to 85°C, and kept for 1 hour , to inactivate neutral protease and papain.

In another preferred embodiment, carry out Saccharomyces cerevisiae enzymolysis as follows: neutral protease and papain are added in the fermented liquid by the weight ratio of 1:1, and the addition of enzyme accounts for 8% of Saccharomyces cerevisiae cell weight, and Adjust the pH value of the fermentation broth to 4.8; then, adjust the temperature of the fermenter to 50°C, and perform enzymatic hydrolysis on the Saccharomyces cerevisiae cells in the fermentation broth for 3 hours; , to inactivate neutral protease and papain.

In yet another preferred embodiment, Saccharomyces cerevisiae enzymolysis is carried out as follows: neutral protease and papain are added in the fermented liquid in a weight ratio of 1:1, and the addition of enzyme accounts for 12% of Saccharomyces cerevisiae cell weight, and Adjust the pH value of the fermentation broth to 5.2; then, adjust the temperature of the fermenter to 60°C, and perform enzymolysis treatment on the Saccharomyces cerevisiae cells in the fermentation broth for 2 hours; , to inactivate neutral protease and papain.

In step (5) of the preparation method of the present invention, filter, purify, and concentrate as follows: filter the enzymolysis solution with a filter cloth to obtain the filtrate; centrifuge the filtrate in a centrifuge to collect the supernatant; The microfiltration tube filters the supernatant to collect the first filtrate; the first filtrate is filtered with a 3kDa ultrafiltration tube to collect the second filtrate, and the second filtrate is filtered with a 1kDa ultrafiltration tube to collect the third filtrate; finally , and the third filtrate was dried under reduced pressure at 70-90°C.

In a preferred embodiment, the third filtrate is dried at 80°C under reduced pressure.

In another aspect, the present invention provides Saccharomyces cerevisiae polypeptide and plant extract composition prepared by the preparation method of the first aspect of the present invention.

In yet another aspect, the present invention provides the use of the Saccharomyces cerevisiae polypeptide and plant extract composition prepared by the preparation method of the first aspect of the present invention in the preparation of an anti-hair loss composition.

In still another aspect, the present invention provides an anti-hair loss composition, which comprises the Saccharomyces cerevisiae polypeptide and plant extract composition prepared by the preparation method of the first aspect of the present invention and skin cosmetically acceptable auxiliary materials .

Skin cosmetically acceptable excipients such as diluents, surfactants, carrier materials, other active agents, etc. are well known to those skilled in the art.

The anti-hair loss composition can be prepared into various product forms, including essence, lotion, cream, gel, ointment and the like.

The present invention has following beneficial effect:

The method of the present invention uses Saccharomyces cerevisiae, which is safe, reliable, easy to obtain, and has mature fermentation technology, to carry out co-fermentation with the plant raw materials of our existing plant extract anti-hair loss products, and the enzymes produced by Saccharomyces cerevisiae can promote the active substances in the plant raw materials. The further release of phytoactive substances can also promote the growth of S. cerevisiae and the production of yeast polypeptides.

Both plant active substances and yeast polypeptides have beauty and health care effects on growth and hair growth. Therefore, the Saccharomyces cerevisiae polypeptide and plant extract composition prepared by the method of the present invention have better anti-hair loss effect, and it is expected to be combined with skin cosmetically acceptable auxiliary materials to prepare a new type of anti-hair loss product.

The method of the present invention directly adopts the co-fermentation of plant raw materials and Saccharomyces cerevisiae for preparation. Compared with our existing plant extract anti-hair loss products, it is prepared by using commercially available high-priced extracts of various plant raw materials, which can reduce production costs and product quality. price.

Detailed ways

Our company has developed anti-hair loss essence, which is composed of ginger (Zingiber officinale) extract, spatholobus suberectus extract, arborvitae leaf (Biota orientalis) extract, Polygonum multiflorum (Polygonum multiflorum) extract, angelica (Angelica Sinensis) Extract, Chuanqiong (Ligusticumchuanxiong) Extract, Paeonia lactiflora (Paeonia lactiflora) Extract, Black Mulberry (Morus nigra) Fruit Extract, American Ginseng (Panax quinquefolius) Extract, Nasturtium (Tropaeolum majus) Extract, Sapindus mukorossi (Sapindus mukorossi) extract, White birch (Betula alba) bark/leaf extract, Ashitaba (Angelica keiskei) extract and Sapindus berry (Plukenetia volubilis) extract are formulated.

The anti-hair loss essence is a cosmetic preparation for external use, with a soft texture, soft and non-sticky, and a delicate smell. It has the function of hair growth and hair loss, and has a good effect on all kinds of hair loss. At the same time, it can improve the dryness and roughness of the hair, making the hair light, supple and elegant. It is easy to use. After cleaning the scalp and/or hair, directly press about a small amount of anti-fall hair growth essence on the scalp or hair, and gently massage until absorbed.

Based on the existing plant extract anti-hair loss products of our company, the inventor hopes to combine Saccharomyces cerevisiae polypeptide with plant extracts to develop a new anti-hair loss product with better anti-hair loss effect. After repeated research, the inventors proposed a method for preparing Saccharomyces cerevisiae polypeptide and plant extract composition, as summarized in the "Summary of the Invention" section above.

In the method of the present invention, firstly, the plant extract is prepared from the plant raw material of the existing plant extract anti-hair loss product of the company. The botanical extract is not filtered and still contains botanical material. Then, Saccharomyces cerevisiae and the plant extract are co-fermented and cultured. Saccharomyces cerevisiae is a common yeast, safe and reliable, easy to obtain, and mature fermentation technology. Saccharomyces cerevisiae fermentation can produce biologically active yeast polypeptides. During the fermentation and cultivation process of the plant extract containing plant raw materials, the active ingredients in the plant raw materials are further released under the action of amylase, cellulase, protease and other enzymes produced by Saccharomyces cerevisiae. These active ingredients may benefit the cultivation of yeast or stimulate yeast to produce polypeptides. After the fermentation culture, the Saccharomyces cerevisiae in the fermentation broth is enzymolyzed with protease, which can promote the release of polypeptides in Saccharomyces cerevisiae cells and increase the yield of polypeptides. Finally, the enzymatic hydrolysis solution is filtered, purified and concentrated to obtain the Saccharomyces cerevisiae polypeptide and plant extract composition. The composition comprises saccharomyces cerevisiae polypeptide and plant extract, wherein the saccharomyces cerevisiae polypeptide has the effect of beauty and health care, and the plant extract is also rich in various bioactive substances with beauty care and health care. The Saccharomyces cerevisiae polypeptide and plant extract composition prepared by the method of the present invention has better anti-hair loss effect through tests, and it is expected to be able to prepare novel anti-hair loss products in combination with skin cosmetically acceptable auxiliary materials.

In addition, our existing plant extract anti-hair loss products are prepared from commercially available extracts of various plant materials. These extracts have high purity, high biological activity content, and have good growth and hair growth effects. However, the price of these commercially available extracts is relatively high, which increases the production cost and product price. However, the method of the present invention directly adopts the co-fermentation of plant raw materials and Saccharomyces cerevisiae for preparation, which can reduce production costs and product prices, and the prepared Saccharomyces cerevisiae polypeptide and plant extract composition also has better anti-hair loss effect through tests.

In order to more fully describe the technical solution of the present invention, the present invention will be further illustrated through non-limiting examples below, combined with comparative examples and test examples.

In the embodiment, the dried plant materials of various traditional Chinese medicine extracts of the anti-hair loss essence of our company are used: ginger (Zingiber officinale), Caulis Spatholobus (Spatholobus suberectus), arborvitae leaf (Biotaorientalis), Polygonum multiflorum ( Polygonum multiflorum), Angelica Sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Morus nigra fruit, Panax quinquefolius, Tropaeolum majus, Sapindus mukorossi ), birch (Betula alba), ashitaba (Angelica keiskei) and vine fruit (Plukenetia volubilis). These dried plant raw materials were purchased from the local Chinese herbal medicine market.

Saccharomyces cerevisiae fermentation medium components used in fermentation culture Malt extract, peptone, yeast extract, dipotassium hydrogen phosphate, potassium chloride, magnesium sulfate and iron sulfate and Saccharomyces cerevisiae, and neutral protease used in Saccharomyces cerevisiae enzymatic hydrolysis and papain were purchased from Biochemical Reagent Company.

Example 1: Preparation of Saccharomyces cerevisiae polypeptide and plant extract composition

(1) Preparation of plant extract:

Mix the purchased dry plant raw materials in an equal weight ratio, wash and remove dust with tap water, drain and dry. Then, the plant raw material is placed in a traditional Chinese medicine grinder (Guangzhou Xulang Machinery Equipment Co., Ltd.) and ground into plant powder below 150 mesh.

Based on the preparation of about 1 kg of Saccharomyces cerevisiae fermentation medium, weigh 35 g of the prepared vegetable powder, add about 900 g of purified water, and stir thoroughly to uniformly disperse the vegetable powder in the water to obtain a vegetable powder dispersion.

Transfer the vegetable powder dispersion to a cooking pot, boil and extract for 30 minutes. After the extraction, the liquid was naturally cooled to room temperature, and purified water was added appropriately, so that the weight of the obtained plant extract was 937.5 g.

(2) Preparation of Saccharomyces cerevisiae fermentation medium:

Based on the preparation of about 1 kg of Saccharomyces cerevisiae fermentation medium, weigh the following yeast fermentation culture components: 30 g of malt extract, 15 g of peptone, 15 g of yeast extract, 1.5 g of dipotassium hydrogen phosphate, 0.5 g of potassium chloride, and 0.5 g of magnesium sulfate g, iron sulfate 0.02g. These components are added into the plant powder extract, and fully stirred, so that each component is completely dissolved in the liquid to obtain a saccharomyces cerevisiae fermentation medium.

(3) Fermentation culture

The prepared Saccharomyces cerevisiae fermentation medium was transferred to a laboratory glass fermenter (Beijing Jiade Precision Technology Co., Ltd., model JD-GFM-XL) for sterilization.

Then, aseptically inoculate Saccharomyces cerevisiae in Saccharomyces cerevisiae fermentation medium to carry out fermentation culture. The fermentation culture conditions are as follows: the inoculum amount of Saccharomyces cerevisiae accounts for 4% of the Saccharomyces cerevisiae fermentation medium by weight, the stirring speed of the fermenter is 150rpm, the temperature is controlled at 37°C, the ventilation rate is controlled at 0.75vvm, and the fermentation is carried out for 21 hours.

(4) Enzymolysis of Saccharomyces cerevisiae

After the fermentation, there will be polypeptides secreted by the yeast in the fermentation medium. In addition, the cultured yeast biomass is subjected to enzymatic hydrolysis to release yeast intracellular polypeptides.

After the fermentation broth is cooled to room temperature, open the fermenter, take the fermentation broth, count the number of Saccharomyces cerevisiae cells in the fermentation broth with the hemocytometer method, and convert it into the weight or concentration of Saccharomyces cerevisiae cells in the fermentation broth.

Neutral protease and papain are added to the fermentation broth at a weight ratio of 1:1, the amount of enzyme added accounts for 10% of the weight of the cells of Saccharomyces cerevisiae, and the pH value of the fermentation broth is adjusted to 5.

Then, the temperature of the fermenter was adjusted to 55° C., and the Saccharomyces cerevisiae cells in the fermentation broth were subjected to enzymatic hydrolysis for 2.5 hours.

After enzymatic hydrolysis, the temperature of the fermenter was adjusted to 85° C. and kept for 1 hour to inactivate neutral protease and papain.

(5) Filtration, purification, concentration

The enzymatic hydrolysis solution contains Saccharomyces cerevisiae fragments and plant powder residues. Filter the enzymolysis solution with a filter cloth to obtain a filtrate. The filtrate was centrifuged at 2500 rpm for 20 min in a centrifuge, and the supernatant was collected.

Then, the supernatant was filtered through a 0.22 μm microfiltration tube (Millipore), and the first filtrate was collected. The first filtrate was filtered with a 3 kDa ultrafiltration tube (Millipore) to collect the second filtrate, and the second filtrate was filtered with a 1 kDa ultrafiltration tube (Millipore) to collect the third filtrate.

Finally, the third filtrate was dried under reduced pressure at 80°C to obtain thick Saccharomyces cerevisiae polypeptide and plant extract.

Example 2: Preparation of Saccharomyces cerevisiae polypeptide and plant extract composition

(1) Preparation of plant extract:

Vegetable powder was prepared in the same manner as in Example 1.

Then, according to the preparation of about 1 kg of Saccharomyces cerevisiae fermentation medium, weigh 30 g of the prepared vegetable powder, add about 900 g of purified water, and fully stir to make the vegetable powder evenly dispersed in the water to obtain a vegetable powder dispersion.

Transfer the vegetable powder dispersion to a cooking pot, boil and extract for 30 minutes. After the extraction, the liquid was naturally cooled to room temperature, and purified water was added appropriately, so that the weight of the obtained plant extract was 937.5 g.

(2) Preparation of Saccharomyces cerevisiae fermentation medium:

Based on the preparation of about 1 kg of Saccharomyces cerevisiae fermentation medium, weigh the following yeast fermentation culture components: 25 g of malt extract, 20 g of peptone, 15 g of yeast extract, 1.5 g of dipotassium hydrogen phosphate, 0.5 g of potassium chloride, and 0.5 g of magnesium sulfate g, iron sulfate 0.02g. These components are added into the plant powder extract, and fully stirred, so that each component is completely dissolved in the liquid to obtain a saccharomyces cerevisiae fermentation medium.

(3) Fermentation culture

The prepared Saccharomyces cerevisiae fermentation medium was transferred to a laboratory glass fermenter (Beijing Jiade Precision Technology Co., Ltd., model JD-GFM-XL) for sterilization.

Then, aseptically inoculate Saccharomyces cerevisiae in Saccharomyces cerevisiae fermentation medium to carry out fermentation culture. The fermentation conditions are as follows: the inoculum amount of Saccharomyces cerevisiae accounts for 5% of the Saccharomyces cerevisiae fermentation medium by weight, the stirring speed of the fermenter is 180rpm, the temperature is controlled at 37°C, the ventilation rate is controlled at 0.70vvm, and the fermentation is cultivated for 18h.

(4) Enzymolysis of Saccharomyces cerevisiae

Similarly, after the fermented liquid is cooled to room temperature, open the fermenter, take the fermented liquid, count the number of Saccharomyces cerevisiae cells in the fermented liquid with the hemocytometer method, and convert it into the weight or concentration of Saccharomyces cerevisiae cells in the fermented liquid.

Neutral protease and papain are added to the fermentation broth in a weight ratio of 1:1, the amount of enzyme added accounts for 8% of the cell weight of Saccharomyces cerevisiae, and the pH value of the fermentation broth is adjusted to 4.8.

Then, the temperature of the fermenter was adjusted to 50° C., and the Saccharomyces cerevisiae cells in the fermentation broth were subjected to enzymatic hydrolysis for 3 hours.

After enzymatic hydrolysis, the temperature of the fermenter was adjusted to 85° C. and kept for 1 hour to inactivate neutral protease and papain.

(5) Filtration, purification, concentration: same as Example 1.

Example 3: Preparation of Saccharomyces cerevisiae polypeptide and plant extract composition

(1) Preparation of plant extract:

Vegetable powder was prepared in the same manner as in Example 1.

Then, according to the preparation of about 1 kg of Saccharomyces cerevisiae fermentation medium, weigh 40 g of the prepared vegetable powder, add 890 g of purified water, and fully stir to make the vegetable powder evenly dispersed in the water to obtain a vegetable powder dispersion.

Transfer the vegetable powder dispersion to a cooking pot, boil and extract for 30 minutes. After the extraction, the liquid was naturally cooled to room temperature, and purified water was added appropriately, so that the weight of the obtained plant extract was 937.5 g.

(2) Preparation of Saccharomyces cerevisiae fermentation medium:

Based on the preparation of about 1 kg of Saccharomyces cerevisiae fermentation medium, weigh the following yeast fermentation culture components: 35 g of malt extract, 15 g of peptone, 10 g of yeast extract, 1.5 g of dipotassium hydrogen phosphate, 0.5 g of potassium chloride, and 0.5 g of magnesium sulfate g, iron sulfate 0.02g. These components are added into the plant powder extract, and fully stirred to completely dissolve each component in the liquid to obtain a brewer's yeast fermentation medium.

(3) Fermentation culture

The prepared Saccharomyces cerevisiae fermentation medium was transferred to a laboratory glass fermenter (Beijing Jiade Precision Technology Co., Ltd., model JD-GFM-XL) for sterilization.

Then, aseptically inoculate Saccharomyces cerevisiae in Saccharomyces cerevisiae fermentation medium to carry out fermentation culture. The fermentation conditions are as follows: the inoculum amount of Saccharomyces cerevisiae accounts for 3% of the Saccharomyces cerevisiae fermentation medium by weight, the stirring speed of the fermenter is 120rpm, the temperature is controlled at 37°C, the ventilation rate is controlled at 0.80vvm, and the fermentation is cultivated for 24 hours.

(4) Enzymolysis of Saccharomyces cerevisiae

Similarly, after the fermented liquid is cooled to room temperature, open the fermenter, take the fermented liquid, count the number of Saccharomyces cerevisiae cells in the fermented liquid with the hemocytometer method, and convert it into the weight or concentration of Saccharomyces cerevisiae cells in the fermented liquid.

Neutral protease and papain are added to the fermentation broth in a weight ratio of 1:1, the amount of enzyme added accounts for 12% of the cell weight of Saccharomyces cerevisiae, and the pH value of the fermentation broth is adjusted to 5.2.

Then, the temperature of the fermenter was adjusted to 60° C., and the Saccharomyces cerevisiae cells in the fermentation broth were subjected to enzymatic hydrolysis for 2 hours.

After enzymatic hydrolysis, the temperature of the fermenter was adjusted to 85° C. and kept for 1 hour to inactivate neutral protease and papain.

(5) Filtration, purification, concentration: same as Example 1.

Comparative Example 1: Preparation of Saccharomyces cerevisiae polypeptide

In this comparative example, no plant material is used, and only Saccharomyces cerevisiae is fermented.

The components and contents of the yeast fermentation medium were the same as those in Example 1. Because no plant material was used, the yeast fermentation culture components were dissolved in 937.5 g of purified water to prepare the fermentation medium.

Fermentation treatment, enzymatic hydrolysis of Saccharomyces cerevisiae, filtration, purification, and concentration were all the same as in Example 1, and a thick Saccharomyces cerevisiae polypeptide liquid was finally obtained.

Comparative Example 2: Preparation of Plant Extract

In this comparative example, no yeast fermentation was carried out, and the plants were further extracted only under the yeast fermentation conditions of Example 1.

Prepare plant extract liquid 937.5g according to embodiment 1. The plant extract was transferred to a laboratory glass fermenter (Beijing Jiade Precision Technology Co., Ltd., model JD-GFM-XL). Under simulated fermentation conditions, the stirring speed of the fermenter was 150rpm, the temperature was controlled at 37°C, and the ventilation rate was The plant extract was treated for 21 hours under the condition of 0.75vvm.

Then, simulating the enzymatic hydrolysis conditions, the temperature of the fermenter was adjusted to 55° C., and the plant extract was treated for 2.5 hours, and then the temperature of the fermenter was adjusted to 85° C. and kept for 1 hour.

Finally, according to Example 1, the plant extract was filtered, purified, and concentrated to finally obtain a thick plant extract.

Test example: animal hair growth test

This application example uses our anti-hair loss essence, the Saccharomyces cerevisiae polypeptide and plant extract composition prepared in Example 1, the Saccharomyces cerevisiae polypeptide prepared in Comparative Example 1, and the plant extract prepared in Comparative Example 2 as test materials. Animal hair growth experiments were performed on mice.

Select 40 healthy adult SD mice with normal hair growth as experimental animals, and anesthetize the mice. Heat and melt rosin and paraffin in a ratio of 1:1, and mix well. After the mixture of rosin and paraffin was slightly cooled, it was evenly applied to the back of the mouse, and the area of application was about 2cm×2cm. The rosin and paraffin mixture hardens and then peels off to pluck out all the hair in the coated area to create a smooth epilated area.

40 mice were randomly divided into 4 groups, 10 in each group. The first group used the anti-hair loss essence of our company to test, the second group used the Saccharomyces cerevisiae polypeptide and plant extract composition prepared in Example 1 to test, and the third group used the Saccharomyces cerevisiae polypeptide prepared in Comparative Example Test, the fourth group uses the plant extract prepared in Comparative Example 2 to test.

On the second day after depilation, the corresponding test materials were applied to the depilatory areas of mice in each group for 15 consecutive days. During the experimental period, the mice were fed normally every day. On the 3rd, 6th, 9th, 12th and 15th day of smearing the test material, observe and record the long hair situation in the depilatory area of the mice respectively, and the results are as follows:

Table 1: Animal hair growth test results

The first group uses our anti-hair loss essence as the test material, which can be used as the control group. The results in Table 1 show that the hair growth effect of the first group is remarkable, which is also the basis for our company's anti-hair loss essence to gain a foothold in the market.

The third group used the Saccharomyces cerevisiae polypeptide prepared in Comparative Example 1 as the test material, which seemed to have little effect on mouse hair growth. The long hair in the depilated area of mice in this group basically grew naturally over time. This result is similar to the effect of Saccharomyces cerevisiae alone in WO2018127604 (A1) of SILAB, France.

The fourth group used the plant extract prepared in Comparative Example 2 as the test material, and the type of plant material was the same as that of the first group. However, the first group is made of commercially available plant extracts with high purity and high content of active substances, while the fourth group is made of plant raw materials through a cooking process and a soaking and stirring process that simulates brewing and fermentation conditions. The content should be inferior to that of the first group, so the mouse hair growth effect is also slightly inferior to the first group.

It can be seen from Table 1 that the mouse hair growth effect of the second group using the Saccharomyces cerevisiae polypeptide and plant extract composition prepared in Example 1 of the present invention is better than that of the first group. The inventor speculates that this should be due to the synergistic effect produced by the method of the present invention to test the plant material and Saccharomyces cerevisiae for fermentation. On the one hand, the enzymes produced by Saccharomyces cerevisiae can promote the further release of active substances in the plant material. On the other hand, the plant raw materials adopted are various Chinese herbal medicines, the active substances of which can also promote the growth of Saccharomyces cerevisiae and produce yeast polypeptides. It is more likely that these active substances of Chinese herbal medicine can regulate the growth and metabolism of Saccharomyces cerevisiae, and the polypeptides produced by Saccharomyces cerevisiae are different in sequence from those produced by Saccharomyces cerevisiae when fermented alone, and have higher biological activity.

Saccharomyces cerevisiae is a commonly used yeast. It is safe, reliable, easy to obtain, and has mature fermentation technology. The Chinese herbal plant materials used in our anti-hair loss and hair growth essence have also been verified by the market. Therefore, the inventors are convinced that the Saccharomyces cerevisiae polypeptide and plant extract composition prepared by the method of the present invention and the anti-hair loss composition prepared by it are not only safe and reliable, but also have low production cost, and can be developed to have better growth and development. A new anti-hair loss product with effective hair loss.

The specific examples above are used to illustrate the present invention, which are only used to help understand the present invention, and are not intended to limit the present invention. For those skilled in the technical field to which the present invention belongs, some simple deduction, deformation or replacement can also be made according to the idea of the present invention.

Claims (10)

1. A method for preparing a saccharomyces cerevisiae polypeptide and plant extract composition comprising saccharomyces cerevisiae polypeptide and plant extract, wherein the plant extract is an extract of ginger (Zingiber officinale), spatholobus stem (Spatholobus suberectus), cacumen biotae (Biota orientalis), polygonum multiflorum (Polygonum multiflorum), angelica (Angelica Sinensis), ligusticum chuanxiong (Ligusticum chuanxiong), paeonia (Paeonia lactiflora), black mulberry (Morus nigra), american ginseng (Panax quinquefolius), saussurea medusa (Tropaeolum majus), soapberry (Sapindus mukorossi), betula alba (Betula alba), angelica keiskei (Angelica) and vine fruit (Plukenetia volubilis) as plant raw materials, the preparation method comprises the following steps:

(1) Preparing a plant extract from the plant material;

(2) Preparing a saccharomyces cerevisiae fermentation culture medium by using a saccharomyces cerevisiae fermentation culture medium component and the plant extract;

(3) Inoculating saccharomyces cerevisiae into the saccharomyces cerevisiae fermentation medium, and carrying out fermentation culture to obtain a saccharomyces cerevisiae fermentation liquid;

(4) Carrying out Saccharomyces cerevisiae enzymolysis on the Saccharomyces cerevisiae fermentation liquor by using protease to obtain Saccharomyces cerevisiae enzymolysis liquor;

(5) Filtering, purifying and concentrating the Saccharomyces cerevisiae enzymatic hydrolysate to obtain the Saccharomyces cerevisiae polypeptide and plant extract composition.

2. The preparation method according to claim 1, wherein in step (1), the plant extract is prepared as follows: pulverizing the plant materials, and boiling the plant materials with water for 25-35min.

3. The production method according to claim 2, wherein the plant material is pulverized into plant powder of 150 mesh or less.

4. The production method according to claim 1, wherein in the step (2), the plant material has a weight of 30 to 40g based on 1kg of the s.cerevisiae fermentation medium prepared, and the s.cerevisiae fermentation medium has a composition of: 25-35g of malt extract, 15-20g of peptone, 10-15g of yeast extract, 1.4-1.5g of dipotassium hydrogen phosphate, 0.4-0.5g of potassium chloride, 0.4-0.5g of magnesium sulfate, 0.015-0.02g of ferric sulfate and the balance of water;

preferably, the weight of the plant raw material is 35g, based on 1kg of the saccharomyces cerevisiae fermentation medium, and the weight of the components of the saccharomyces cerevisiae fermentation medium is as follows: 30g of malt extract, 15g of peptone, 15g of yeast extract, 1.5g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.02g of ferric sulfate and the balance of water;

it is further preferred that the plant material weight is 30g based on 1kg of the Saccharomyces cerevisiae fermentation medium prepared, and the Saccharomyces cerevisiae fermentation medium comprises the following components in weight: 25g of malt extract, 20g of peptone, 15g of yeast extract, 1.5g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.02g of ferric sulfate and the balance of water;

still further preferably, the weight of the plant material is 40g, and the weight of the components of the Saccharomyces cerevisiae fermentation medium is 35g of malt extract, 15g of peptone, 10g of yeast extract, 1.5g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.02g of ferric sulfate, and the balance of water, based on 1kg of the Saccharomyces cerevisiae fermentation medium.

5. The production method according to claim 1, wherein in step (3), the fermentation culture is performed under the following fermentation conditions: the inoculation amount of the saccharomyces cerevisiae accounts for 3-5% of the weight of the saccharomyces cerevisiae fermentation medium, the stirring speed of a fermentation tank is 120-180rpm, the temperature is controlled at 37 ℃, the ventilation is controlled at 0.70-0.80vvm, and the fermentation culture is carried out for 18-24 hours;

preferably, the fermentation culture is performed under the following fermentation conditions: the inoculation amount of the saccharomyces cerevisiae accounts for 4% of the weight of the saccharomyces cerevisiae fermentation medium, the stirring speed of a fermentation tank is 150rpm, the temperature is controlled at 37 ℃, the ventilation is controlled at 0.75vvm, and the fermentation culture is carried out for 21 hours;

further preferably, the fermentation culture is performed under the following fermentation conditions: the inoculation amount of the saccharomyces cerevisiae accounts for 5% of the weight of the saccharomyces cerevisiae fermentation medium, the stirring speed of a fermentation tank is 180rpm, the temperature is controlled at 37 ℃, the ventilation is controlled at 0.70vvm, and the fermentation culture is carried out for 18 hours;

still further preferably, the fermentation culture is performed under the following fermentation conditions: the inoculation amount of the saccharomyces cerevisiae accounts for 3% of the weight of the saccharomyces cerevisiae fermentation medium, the stirring speed of a fermentation tank is 120rpm, the temperature is controlled at 37 ℃, the aeration rate is controlled at 0.80vvm, and the fermentation culture is carried out for 24 hours.

6. The production method according to claim 1, wherein in the step (4), saccharomyces cerevisiae enzymolysis is performed as follows: adding neutral protease and papain into the fermentation broth according to the weight ratio of 1:1, wherein the addition amount of the enzyme accounts for 8-12% of the weight of the saccharomyces cerevisiae cells, and regulating the pH value of the fermentation broth to 4.8-5.2; then, regulating the temperature of the fermentation tank to 50-60 ℃, and carrying out enzymolysis treatment on saccharomyces cerevisiae cells in the fermentation liquid for 2-3 hours; after enzymolysis, the temperature of the fermentation tank is adjusted to 85 ℃, and the temperature is kept for 1h so as to inactivate neutral protease and papain;

preferably, saccharomyces cerevisiae enzymatic hydrolysis is performed as follows: adding neutral protease and papain into the fermentation broth according to the weight ratio of 1:1, wherein the addition amount of the enzyme accounts for 10% of the weight of the saccharomyces cerevisiae cells, and regulating the pH value of the fermentation broth to 5; then, regulating the temperature of the fermentation tank to 55 ℃, and carrying out enzymolysis treatment on the saccharomyces cerevisiae cells in the fermentation liquid for 2.5 hours; after enzymolysis, the temperature of the fermentation tank is adjusted to 85 ℃, and the temperature is kept for 1h so as to inactivate neutral protease and papain;

further preferably, saccharomyces cerevisiae enzymatic hydrolysis is performed as follows: adding neutral protease and papain into the fermentation broth according to the weight ratio of 1:1, wherein the addition amount of the enzyme accounts for 8% of the weight of the saccharomyces cerevisiae cells, and regulating the pH value of the fermentation broth to 4.8; then, regulating the temperature of the fermentation tank to 50 ℃, and carrying out enzymolysis treatment on the saccharomyces cerevisiae cells in the fermentation liquid for 3 hours; after enzymolysis, the temperature of the fermentation tank is adjusted to 85 ℃, and the temperature is kept for 1h so as to inactivate neutral protease and papain;

still further preferably, saccharomyces cerevisiae enzymatic hydrolysis is performed as follows: adding neutral protease and papain into the fermentation broth according to the weight ratio of 1:1, wherein the addition amount of the enzyme accounts for 12% of the weight of the saccharomyces cerevisiae cells, and regulating the pH value of the fermentation broth to 5.2; then, regulating the temperature of the fermentation tank to 60 ℃, and carrying out enzymolysis treatment on the saccharomyces cerevisiae cells in the fermentation liquid for 2 hours; after enzymolysis, the temperature of the fermentation tank is adjusted to 85 ℃, and the fermentation tank is kept for 1h, so that neutral protease and papain are inactivated.

7. The production method according to claim 1, wherein in the step (5), filtration, purification, concentration are performed as follows: filtering the enzymolysis liquid by using filter cloth to obtain filtrate; centrifuging the filtrate in a centrifuge, and collecting the supernatant; filtering the supernatant with a 0.22 μm micro-filter tube, and collecting the first filtrate; filtering the first filtrate by using an ultrafiltration tube with the aperture of 3kDa, collecting the second filtrate, filtering the second filtrate by using an ultrafiltration tube with the aperture of 1kDa, and collecting the third filtrate; finally, the third filtrate is dried under reduced pressure at 70-90 ℃; preferably, the third filtrate is dried under reduced pressure at 80 ℃.

8. A saccharomyces cerevisiae polypeptide and plant extract composition prepared by the method according to any one of claims 1-7.

9. Use of a saccharomyces cerevisiae polypeptide and plant extract composition prepared by the method according to any of claims 1-7 for the preparation of anti-hair loss compositions.

10. An anti-hair loss composition comprising a saccharomyces cerevisiae polypeptide and plant extract composition prepared by the method according to any of claims 1-7 and cosmetically acceptable adjuvants to skin.