WO2020177390A1 - Method for preparing l-ergothioneine-containing cosmetic stock solution by means of fermenting hericium erinaceus - Google Patents

Abstract

A method for preparing an L-ergothioneine-containing cosmetic stock solution by means of fermenting Hericium erinaceus, the method comprising the following steps: step 1: inoculating Hericium erinaceus hyphae into a liquid seed culture medium for culturing to obtain a seed solution; step 2: inoculating the seed solution into a fermentation culture medium for fermentation and adding an L-ergothioneine precursor substance, and fermenting to an end point to obtain a fermentation broth; step 3: processing the fermentation broth containing mycelium to obtain an L-ergothioneine-containing fermentation stock solution and using same as the L-ergothioneine-containing cosmetic stock solution. The described stock solution product contains higher than 300 mg/L of L-ergothioneine, and has strong antioxidant, anti-radiation and anti-inflammatory effects; moreover, the described stock solution can be added as a raw material into water, milk, creams, and so on, and is widely applied in the field of cosmetics.

Description

Method for preparing ergothioine-containing cosmetic stock solution by fermentation of Hericium erinaceus Technical field

The invention relates to the field of cosmetic raw materials, in particular to a method for preparing ergothioine cosmetic stock solution by Hericium erinaceus fermentation and a fermentation stock solution prepared therefrom.

Background technique

Hericium erinaceus belongs to the phylum Basidiomycota and Hericium erinaceus, and is a famous medicinal and edible fungus. Both the fruit body and the mycelium can be used as medicine, the nature is flat and the taste is sweet, it has the functions of helping digestion and benefiting the five internal organs, and is rich in nutrition. It is known as "mountain hericium, seafood bird's nest". The medicinal ingredients of Hericium erinaceus are mainly polysaccharides, oligosaccharides, sterols, fatty acids, hericin and hericin, etc., which can protect the liver and stomach, lower blood sugar, protect nerves, enhance human immunity, and fight cancer. Antioxidant and other functions. In addition, Hericium is also rich in sulfur, and sulfur is an important nutrient element that is indispensable to the human body. In Hericium, sulfur is mainly present in the form of thioneine Once the ergothioine in Hericium enters the human body, most of it will eventually become inorganic sulfate, sulfate and neutral sulfur into the blood circulation. One of the effects is to invigorate the spleen and stomach, increase the gastric mucosal barrier function, and affect various Chronic gastritis has a good therapeutic effect. In the field of skin care products, ergothioneine can also resist radiation, promote embryonic growth and development, resist fatigue, and improve the survival ability of the body. Ergothioneine (L-Ergothioneine, EGT), whose chemical name is 2-mercaptohistidine trimethyl inner salt, is the only natural 2-thioimidazole amino acid known so far. It has clear antioxidant and anti-oxidant properties. Inflammation, when applied to cosmetics, it has anti-inflammatory, anti-aging and radiation protection effects. Many foreign cosmetics have used ergothioine as the main functional ingredient in their formulations. As early as 2014, ergothioine was under the National Food and Drug Supervision The Administration is listed in the list of used cosmetic raw materials, and the EGT content in cosmetics reaches 0.01% to 2%, which can achieve better results. EGT is added to various whitening cosmetics, and the addition amount of EGT for creams, creams and lotions ranges from 0.01% to 1.0%, 0.01% to 2.0%, and 0.01% to 3.0%, respectively. American OXIS company has launched two cosmetics based on EGT: Reverge and Prograce. The former can be used as an emollient to improve aging skin, the latter can effectively remove wrinkles and promote the regeneration of new skin. Estee Lauder uses this ingredient as a treasure of the town, adding it to Clinique, the source of wood, and the mystery of the sea And other lines of products.

Although it has such excellent effects, the human body cannot synthesize the substance and can only ingest it from external food. In food, the content of edible fungi is the highest. It is generally recognized that edible fungi are mostly planted and cultivated. Although many mushroom products have reached the scale of commercial production, the cultivation of fruit bodies requires a lot of labor and space. Equipment, time, etc. In addition, the cultivation of some edible fungi can easily absorb heavy metal ions in the soil, so how to further accumulate mycelium is the key to efficient synthesis of thioneine. Using liquid fermentation technology for edible fungus fermentation culture can greatly reduce fermentation costs, shorten the fermentation cycle, and use fermentation metabolism to control the fermentation process with ergot sulfide as the target product, which can greatly increase the content of the active product.

Patent Document 1 discloses a "product containing ergothioneine and its preparation method and use of mushroom extracellular fermentation broth", which is characterized in that the mushroom is fermented and the mycelium in the fermentation broth is removed to obtain extracellular The fermentation broth or the concentrate of the extracellular fermentation broth is provided. The disadvantage is that the mycelium rich in high concentration of ergothione is removed during the process, resulting in the loss of active ingredients, and the mycelium is in addition to ergothione. It contains active substances such as polysaccharides and sterols, which can be used as important functional ingredients in cosmetics.

Patent Document 2 discloses a "method for preparing thioneine", which is characterized by optimizing the fermentation process of Pleurotus ostreatus by using a method of fermentation metabolism regulation, and then leaching the thioneine in the mycelia by hot water extraction When it comes to fermentation broth, the disadvantage is that hot water extraction is used in the purification process. As cosmetic raw materials, the appearance, color and smell of the product are very important. On the one hand, this method will make the fermentation broth darker and produce peculiar smells. On the other hand, other heat-sensitive active ingredients in the fermentation broth, such as fungal polysaccharides, may also be degraded and inactivated, especially ergothione may be degraded to a certain extent, which affects the efficacy of the product and is difficult to be used as a cosmetic raw material.

Patent Document 3 discloses "a method of preparing thioneine", which is characterized in that mushrooms are used as a raw material to obtain thioneine extract through ethanol extraction and resin separation and purification. The disadvantage is that it relies on a pure plant extraction process, which is low in efficiency. In addition, organic solvents are involved in the process, and there is a risk of residual organic solvents and may cause environmental pollution.

Patent Document 4 discloses "microbial thioneine biosynthesis", which is characterized in that genes related to synthetic thioneine are transcribed into E. coli by genetic means to be fermented to obtain thioneine. The disadvantage is that Escherichia coli is a pathogenic bacterium, which is difficult to apply in cosmetics.

Patent Document 5 discloses a "preparation method of a functional oral agent rich in thiothioine", which is characterized by mixing edible fungus mycelium rich in thiothioine with water, and stirring and leaching at 80°C to 100°C. Concentrated to obtain a concentrated liquid, the concentrated liquid is added with food-acceptable additives to make a liquid oral preparation. Its products are mainly used in the field of food and health products, and cannot be directly used as cosmetic raw materials.

In summary, the current development of cosmetic raw materials for edible fungus ergothioine mainly has the following shortcomings:

1. Hericium erinaceus is mostly used in the food field, and the active ingredients fermented or extracted from it have not been reported in the field of cosmetics.

2. The existing fermentation and extraction processes applied to the ergothioine fermentation broth are not sufficient to prepare products that meet the requirements of cosmetic specifications.

3. In the existing research, the content of thioneine obtained by fermentation of strains is generally not high.

Prior art literature

Patent literature

Patent Document 1: CN107708443A

Patent Document 2: CN 105296559

Patent Document 3: CN 106831596 A

Patent Document 4: CN 106661585 A

Patent Document 5: CN 103181933

Summary of the invention

In view of the problems in the prior art, the present invention aims to provide a method for preparing ergothione cosmetic stock solution by fermentation of Hericium erinaceus. The method is simple in process, good in physical condition, stable in product, high in active substance content, safe and healthy. Raw material requirements. The invention adopts Hericium erinaceus for the first time to ferment, with stable fermentation and high output, which can reach more than 300mg/L, which is a leading level at home and abroad.

In order to achieve the above objective, the present invention adopts the following technical solutions.

1. A method for preparing a fermentation stock solution containing ergothioneine by fermentation of Hericium erinaceus, characterized in that it comprises the following steps:

Step 1: Inoculate the hyphae of Hericium erinaceus into the liquid seed medium and cultivate to obtain the seed liquid;

Step 2: Inoculate the seed liquid into the fermentation medium for fermentation and add ergothione precursor materials, and ferment to the end to obtain the fermentation liquid;

Step 3: The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution.

2. The method according to item 1, characterized in that the preservation number of the Hericium erinaceus is CCTCC NO: M 2018567.

3. The method according to item 1, characterized in that the seed culture medium comprises the following components: 1-10% by mass of carbon source, 1-5% by mass of organic nitrogen source and 0.01-1% by mass of inorganic salt.

4. The method according to item 1, characterized in that in step 1, shaking culture is performed at 15-30°C, pH 4.0-7.0, 50-300 rpm, preferably 100 rpm-200 rpm for 5-10 days to obtain seed liquid.

5. The method according to item 1, wherein the fermentation medium comprises the following components: 1 to 5 mass% of carbon source, 1 to 10 mass% of organic nitrogen source, 0.01 to 1 mass% of inorganic Salt, 0.0001 to 0.001% by mass of trace elements and 0.0001 to 0.001% by mass of coenzyme.

6. The method according to item 1, characterized in that, in step 2, shaking fermentation is performed at 15-30°C, pH 4.0-7.0, 50-300rpm, preferably 100rpm-200rpm, for 7-15 days, and fermented to the end to obtain fermentation liquid.

7. The method according to item 1, wherein in step 2, the precursor substance is at least one of aspartic acid, glutamine and betaine, and the addition amount is 1-20 mM.

8. The method according to item 1, characterized in that, in step 2, the inoculation amount of the seed liquid into the fermentation liquid is 1-10% by volume.

9. The method according to item 1, characterized in that, in step 2, the fermentation end point is residual sugar in the fermentation broth≤0.5% by mass.

10. The method according to item 1, characterized in that, in step 3, the treatment includes at least one of homogenization, ultrasound, and activated carbon adsorption.

11. The method according to item 10, characterized in that the conditions of the homogenization are a spindle speed of 1000 r/min to 3000 r/min, and a homogenization time of 10 to 30 min.

12. The method according to item 10, characterized in that the ultrasound conditions are that the ultrasound power is 100w to 1000w, the ultrasound time is 5 to 60 min, and the ultrasound endpoint is that the content of ergothioneine no longer increases.

13. The method according to item 10, characterized in that the amount of activated carbon added in the activated carbon adsorption is 0.1 to 1% of the volume of the fermentation broth, and the adsorption time is 10 to 60 minutes.

14. The method according to item 1, characterized in that it further comprises step 4: removing impurities in the processed fermentation broth.

15. The method according to item 14, characterized in that, in step 4, the removal of impurities is performed by centrifuging the fermentation broth at 1000-5000 rpm for 10 to 30 minutes, and then using the resulting supernatant with a filter element with a pore size of 0.22 μm to 0.45 μm Filtered.

16. The method according to any one of items 1-15, wherein the content of ergothioine in the obtained ergothioine-containing cosmetic stock solution is 300 mg/L or more.

17. The method according to any one of items 1-15, characterized in that the obtained ergothioine-containing cosmetic stock solution contains 2.5 g/L or more of fungal polysaccharides and a total amount of 5.0 mg/100 mL or more of amino acids Preferably, the polysaccharide content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, preferably the total amino acid content is 5.5 mg/100 mL or more, and more preferably 5.7 mg/100 mL or more.

18. The method according to any one of items 1-15, characterized in that the 0.5v/v%-2v/v% dilution of the obtained ergothioine-containing cosmetic stock solution is 1,1-two The clearance rate of phenyl-2-trinitrophenylhydrazine DPPH is over 30%,

19. The method according to any one of items 1-15, characterized in that cells treated with a 0.5v/v%-2v/v% dilution of the obtained ergothioine-containing cosmetic stock solution are subjected to ultraviolet rays The relative proliferation rate after injury is more than 70%.

20. A fermentation stock solution containing ergothioneine, characterized in that it contains: 0.3g/L or more ergothioine, 2.5g/L or more fungal polysaccharides, and a total amount of 5.0mg/100mL or more of amino acids, preferably polysaccharides The content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, and the total amino acid content is preferably 5.5 mg/100 mL or more, and more preferably 5.7 mg/100 mL or more.

21. The ergothioine-containing fermentation stock solution according to item 20, characterized in that it is obtained by using the method according to any one of items 1-19.

22. The ergothioine-containing fermentation stock solution according to item 20 or 21, characterized in that the 0.5v/v%-2v/v% dilution of the fermentation stock solution is 1,1-diphenyl-2 -The clearance rate of trinitrophenylhydrazine DPPH is more than 30%,

23. The ergothioine-containing fermentation stock solution according to item 20 or 21, wherein the relative proliferation rate of cells treated with a dilution of 0.5v/v%-2v/v% of the fermentation stock solution after being damaged by ultraviolet rays It is more than 70%.

24. An application of the ergothioine-containing fermentation stock obtained by the method according to any one of items 1-19 or the ergothioine-containing fermentation stock solution according to any one of items 20-23 in cosmetics .

25. A fermentation medium composition for the production of ergothioine-containing cosmetic stock solution, characterized in that it comprises the following components: 1 to 5 mass% carbon source, 1 to 10 mass% organic nitrogen source, 0.01 to 1% by mass of inorganic salts, 0.0001 to 0.001% by mass of trace elements, and 0.0001 to 0.001% by mass of coenzyme.

26. A cosmetic stock solution composition containing ergothioneine, characterized in that it comprises ergothioine above 300mg/L, fungal polysaccharides above 2.5g/L and amino acids above 5.0mg/100mL, preferably the polysaccharide content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, preferably the total amino acid content is 5.5 mg/100 mL or more, more preferably 5.7 mg/100 mL or more.

The invention has the following advantages:

One aspect of the present invention adopts the Hericium erinaceus mycelium fermentation method to extend the application scope of the Hericium erinaceus fermentation active product to the field of cosmetics;

Another aspect of the present invention is to physically disrupt cells to maximize the retention and release of intracellular ergothioneine and other active substances of edible fungus mycelium to the outside of the cell without any damage to physical properties and biological functions.

In another aspect of the present invention, a mild decolorization process is adopted to better remove the odor and pigment that affect the physical characteristics of the product in cosmetics, and can better meet the requirements of different cosmetic formulations.

In addition to the higher content of thioneine after fermentation, the present invention also contains other active ingredients, such as fungal polysaccharides, small molecular amino acids and the like.

Description of the drawings

Figure 1 is the HPLC chart of the determination of thioneine content, in which a) is the HPLC chart of the standard product (the concentration of thioneine is 100mg/L), b) is the HPLC chart of the fermentation broth of Example 5, and c) is The HPLC profile of the fermentation broth of Example 6, e) is the HPLC profile of the fermentation broth of Example 7, and d) is the HPLC profile of the fermentation broth of Example 8;

Figure 2 is a graph showing the results of fermentation stock promoting cell repair, where a is the initial control treatment group, b is the control treatment group 24h, c is the S0 group initial, d is the S0 treatment 24h, e is the S1 treatment group initial, f is S1 Treatment group 24h.

Specific embodiments of the invention

The present invention will be described in detail below.

The method for preparing ergothioine-containing cosmetic stock solution by fermentation of Hericium erinaceus provided by the present invention includes the following steps:

Step 1: Inoculate the hyphae of Hericium erinaceus into the liquid seed medium and cultivate to obtain the seed liquid;

Step 2: Inoculate the seed liquid into the fermentation medium for fermentation and add ergothione precursor materials, and ferment to the end to obtain the fermentation liquid;

Step 3: The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution.

The invention adopts the Hericium erinaceus mycelium fermentation method to extend the application scope of the Hericium erinaceus fermentation active product to the field of cosmetics. The method has simple process, good physical condition, stable product, high active substance content, safety and health and meets the requirements of cosmetic raw materials.

step 1

Step 1: Inoculate the hyphae of Hericium erinaceus into a liquid seed culture medium to obtain a seed liquid.

Among them, Hericium erinaceus is a fungus, Basidiomycetes, Polyporaceae, Hericium erinaceus (Rull ex F.) Pers., which is a saprophytic fungus and a famous edible fungus. It looks like a hedgehog or hericium, so it is commonly called hericium or monkey mushroom. Among them, Hericium erinaceus is preferably Hericium erinaceus CCTCC NO: M 2018567, which has been deposited at the China Type Culture Collection (CCTCC) on August 23, 2018. This strain is a new strain discovered by the applicant. Hericium erinaceus species, and found that it can be used for the biosynthesis of thioneine.

A single wire mesh cell is called hyphae, including vegetative hyphae and aerial hyphae. The hyphae gather together to form a certain macroscopic structure called mycelium.

In a specific embodiment, the Hericium erinaceus mycelium is taken from a test tube with a size of about 5*5cm to inoculate the liquid seed culture medium, which can be inoculated at 15-30°C, pH 4.0-7.0, 50-300rpm, preferably 100rpm~ Shake culture on a 200 rpm shaker for 5-10 days to obtain seed liquid.

In a specific embodiment, the above-mentioned seed culture medium includes the following components: 1-10% by mass carbon source, 1-5% by mass organic nitrogen source, and 0.01-1% by mass inorganic salt.

The above-mentioned carbon sources are commonly used carbon sources for microorganisms, and are not particularly limited, and may include one or a combination of the following: glycerol, sucrose, fructose, glucose, maltose, etc., preferably sucrose,

The above-mentioned nitrogen source is a commonly used nitrogen source for microbial cultivation, and is not particularly limited, and may include one or more of the following combinations: beef extract, peptone, yeast powder, soybean meal powder, etc., preferably soybean meal powder,

The above-mentioned inorganic salts are commonly used inorganic salts for microbial cultivation, and are not particularly limited, and can include one or more of the following combinations: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, etc., preferably Sodium dihydrogen phosphate and sodium sulfate.

Common organic or inorganic acids, such as phosphoric acid, hydrochloric acid, acetic acid, and lactic acid, can be used to adjust the pH of the seed culture medium, preferably acetic acid. By culturing Hericium erinaceus under the above-mentioned conditions, it is more suitable for the growth of strains and can obtain strains with good activity.

Step 2

Step 2: Inoculate the seed liquid into the fermentation medium for fermentation and add the ergothione precursor material to ferment to the end to obtain the fermentation liquid.

Fermentation refers to the process in which people prepare microbial cells themselves, or direct or secondary metabolites through the life activities of microorganisms under aerobic or anaerobic conditions.

In a specific embodiment, shake fermentation at 15-30° C., pH 4.0-7.0, 50-300 rpm, preferably 100 rpm-200 rpm, for 7-15 days, and ferment to the end to obtain fermentation broth.

The pH of the fermentation medium can be adjusted with common organic or inorganic acids, such as phosphoric acid, hydrochloric acid, acetic acid, and lactic acid, preferably acetic acid.

In a specific embodiment, the fermentation medium includes the following components: 1-5% by mass carbon source, 1-10% by mass organic nitrogen source, 0.01-1% by mass inorganic salt, 0.0001-0.001% by mass trace Element and 0.0001 to 0.001 mass% of coenzyme.

The carbon source mentioned above is a carbon source commonly used by microorganisms, and is not particularly limited, and may include one or a combination of the following: glycerol, sucrose, fructose, glucose, maltose, etc., preferably glycerol.

The above-mentioned nitrogen source is a commonly used nitrogen source for microbial cultivation, and is not particularly limited, and may include one or more combinations of the following: beef extract, peptone, yeast powder, soybean cake powder, etc., preferably beef extract.

The above-mentioned inorganic salts are commonly used inorganic salts for microbial cultivation, and are not particularly limited, and can include one or more of the following combinations: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, etc., preferably Sodium dihydrogen phosphate and sodium sulfate.

The above-mentioned trace elements may include one or a combination of the following: magnesium sulfate, ferrous chloride, zinc chloride, etc., preferably zinc chloride.

The aforementioned coenzyme may include one or a combination of the following: biotin, niacin, pyridoxal phosphate, betaine, vitamin B12, riboflavin, etc., preferably pyridoxal phosphate.

The present invention optimizes the carbon source, nitrogen source, coenzyme and other factors in the fermentation medium to increase the yield of ergothioneine.

In a specific embodiment, the amount of seed liquid inoculated into the fermentation liquid is 1-10% by volume.

In a specific embodiment, the aforementioned precursor substance is at least one of aspartic acid, glutamine and betaine, and the addition amount is 1-20 mM. By adding precursor substances, the Hericium erinaceus can be provided with better nutrition, which is beneficial to its fermentation and production of thioneine.

In a specific embodiment, the fermentation end point is that the residual sugar in the fermentation broth is ≤0.5% by mass. Preferably, there is no glucose in the fermentation broth, and the above-mentioned glucose content can be determined by common chemical methods, such as DNS method, Fehling reagent method, indirect iodometry or optical rotation method.

Step 3

Step 3: The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution.

In a specific embodiment, the above-mentioned treatment includes at least one of physical crushing treatment and decolorization treatment. The physical crushing treatment can be homogenization and/or ultrasound, and the decolorization treatment can be activated carbon adsorption. The mycelium of Hericium erinaceus in the fermentation broth is fully broken by homogenizing and sonicating the fermentation broth. The fermentation broth is fully adsorbed and decolorized by adding activated carbon to the fermentation broth.

The above-mentioned homogenization conditions are the spindle speed of 1000r/min～3000r/min, and the homogenization time of 10～30min.

The above-mentioned ultrasonic conditions are that the ultrasonic power is 100w-1000w, the ultrasonic time is 5-60min, and the end point of the ultrasonic is that the content of thioneine in the fermentation broth no longer increases. Preferably, high-performance liquid phase is used to detect the content of thioneine in the fermentation broth.

The amount of activated carbon added in the above activated carbon adsorption is 0.1 to 1% of the volume of the fermentation broth, and the adsorption time is 10 to 60 minutes.

By adopting the method of physically disrupting cells, the present invention retains and releases edible fungus mycelium intracellular ergothioneine and other active substances to the outside of the cell to the greatest extent without any damage to physical properties and biological effects. By adopting a mild decolorization process, the present invention can better remove the odor and pigment that affect the physical characteristics of the product in cosmetics, and can better meet the requirements of different cosmetic formulations.

Step 4

According to needs, the method of the present invention may further include step 4: removing impurities in the fermented broth after treatment.

In a specific embodiment, the removal of impurities is performed by centrifuging the fermentation broth at 1000-5000 rpm for 10-30 minutes, and then filtering the resulting supernatant with a filter element with a pore size of 0.22 μm to 0.45 μm. The centrifugation can remove bacterial cell debris and impurities in the culture medium, and the filtration can remove microorganisms. By using centrifugation and filtration, it is possible to obtain ergothioine-containing cosmetic stock solutions with better performance and meeting the needs of different cosmetic formulations.

The present invention also provides a fermentation stock solution (cosmetic stock solution) containing ergothioneine. The ergothioneine fermentation stock solution (cosmetic stock solution) preferably contains more than 300 mg/L of ergothioneine, and the fermentation stock solution has antioxidant properties in cosmetics. , Cell repair effect.

In a preferred embodiment, the ergothioine-containing fermentation stock solution contains: ergothioine above 300 mg/L, fungal polysaccharides above 2.5 g/L, and amino acids with a total amount of 5.0 mg/100 mL or above.

In a specific embodiment, the ergothioine-containing fermentation stock solution of the present invention is obtained by the method for preparing the ergothioine-containing cosmetic stock solution by fermentation of the Hericium erinaceus of the present invention. The content of ergothioine in the ergothioine-containing cosmetic stock obtained by the method for preparing the ergothioine-containing cosmetic stock solution by fermentation of Hericium erinaceus of the present invention is 300 mg/L or more. Moreover, the obtained ergothioine-containing cosmetic stock solution contains not only a higher content of ergothioine, but also other active ingredients, such as fungal polysaccharides, small molecular amino acids, and the like. For example, it contains more than 2.5g/L of fungal polysaccharide and a total amount of more than 5.0mg/100mL of amino acids. Among them, amino acids include aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine Acid, phenylalanine, lysine, histidine, arginine, etc. It is further preferred that the polysaccharide content is 2.6g/L or more, and the polysaccharide content is more preferably 2.7g/L or more, and the total amino acid content is preferably 5.5mg/100mL or more, and even more preferably 5.7mg/100mL or more

The obtained ergothioneine-containing cosmetic stock solution also has the effects of anti-oxidation and radiation protection. The 1,1-diphenyl-2-trinitrophenylhydrazine DPPH clearance rate of the 0.5v/v%-2v/v% dilution of the obtained ergothioine-containing cosmetic stock solution is more than 30%,

The relative proliferation rate of the cells treated with the 0.5v/v%-2v/v% dilution of the obtained ergothioneine-containing cosmetic stock solution after being damaged by ultraviolet rays is 70% or more.

The cosmetic stock solution of the present invention has extremely high oxidation resistance, has a good protective effect on UV damage, and has a good effect of repairing damaged cells.

The present invention will be further described below in conjunction with the embodiments and drawings, but the present invention is not limited by the following embodiments.

Example

The experimental methods used in the following examples are conventional methods unless there are special requirements.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

(1) Test tube preservation of bacterial strains, inoculate the bacterial clumps into 100ml seed culture medium, shaker at 150rpm, culture at 25℃ for 5 days;

The composition of the seed medium is as follows:

Sucrose 20g/L, soybean meal powder 15g/L, sodium sulfate 0.5g/L, disodium hydrogen phosphate 0.5g/L, acetic acid to adjust pH 5.0.

(2) Seed culture medium was inoculated into 1L fermentation culture medium, 200rpm shaker, 25℃ fermentation culture for 10 days until no residual sugar;

The fermentation medium components are as follows:

Glycerin 25g/L, beef extract 20g/L, sodium sulfate 1g/L, disodium hydrogen phosphate 1g/L, zinc chloride 0.005g/L, pyridoxal phosphate 0.001g/L, acetic acid to adjust pH 5.0, aspartame Acid 10mM, Glutamine 5mM, Betaine 10Mm;

(3) The fermentation broth is homogenized for 30 minutes, the homogenization speed is 3000 rpm/min, and the ultrasonic power is 500w for 15 minutes, which can release ergothioneine and other active ingredients into the fermentation broth to obtain a preliminary fermentation broth.

Example 2

(1) Test-tube preservation of bacterial strains to inoculate bacterial clumps into 200ml seed culture medium, 200rpm shaker, 25℃ for 7 days;

The composition of the seed medium is as follows:

Glucose 25g/L, soybean meal 15g/L, sodium sulfate 0.5g/L, disodium hydrogen phosphate 0.5g/L, acetic acid to adjust pH 5.5

(2) Seed culture medium was inoculated into 2L fermentation culture medium, 200rpm shaker, 25℃ fermentation culture for 9 days until no residual sugar;

The fermentation medium components are as follows:

Glycerin 25g/L, beef extract 20g/L, sodium sulfate 0.75g/L, disodium hydrogen phosphate 1g/L, zinc chloride 0.005g/L, pyridoxal phosphate 0.001g/L, acetic acid to adjust pH 5.5, Asparagus Acid 8mM, glutamine 5mM, betaine 15Mm;

(3) The fermentation broth is homogenized for 40 minutes, the homogenization speed is 3000 rpm/min, and then ultrasonic for 30 minutes, the ultrasonic power is 800w, which can release ergothioneine and other active ingredients into the fermentation broth to obtain a preliminary fermentation broth.

Example 3:

(1) Test tube preservation of bacterial strains, inoculate the bacterial clumps into 500ml seed culture medium, shake at 150rpm, culture at 30℃ for 8 days;

The composition of the seed medium is as follows:

Maltose 50g/L, beef extract 35g/L, potassium chloride 0.75g/L, disodium hydrogen phosphate 0.5g/L, pH 4.5 adjusted by lactic acid.

(2) Seed culture medium was inoculated into 5L fermentation culture medium, 100 rpm shaker, 30 ℃ fermentation culture for 15 days to no residual sugar;

The fermentation medium components are as follows:

Glycerin 50g/L, peptone 30g/L, sodium dihydrogen phosphate 1g/L, disodium hydrogen phosphate 1g/L, ferrous chloride 0.0075g/L, vitamin B12 0.002g/L, acetic acid to adjust pH 4.5, aspartame Acid 10mM, glutamine 5mM, betaine 15Mm;

(3) The fermentation broth is homogenized for 30 minutes, the homogenizing speed is 3000 rpm/min, and then ultrasonicated for 45 minutes with an ultrasonic power of 500w, which can release ergothioneine and other active ingredients into the fermentation broth to obtain a preliminary fermentation broth.

Example 4:

(1) Test tube preservation of bacterial strains to inoculate the bacterial clumps into 300ml seed culture medium, 200rpm shaker, 20℃ for 9 days;

The composition of the seed medium is as follows:

Glucose 35g/L, yeast powder 15g/L, potassium chloride 0.75g/L, disodium hydrogen phosphate 0.5g/L, pH 4.2 adjusted by hydrochloric acid.

(2) Seed culture medium was inoculated into 2L fermentation culture medium, 150rpm shaker, 20℃ fermentation culture for 15 days until no residual sugar;

The fermentation medium components are as follows:

Glucose 40g/L, peptone 25g/L, sodium dihydrogen phosphate 1g/L, disodium hydrogen phosphate 1g/L, zinc chloride 0.0075g/L, vitamin B12 0.002g/L, biotin 0.001g/L, adjusted by acetic acid pH 4.3, aspartic acid 15mM, glutamine 10mM, betaine 5Mm;

(3) The fermentation broth is homogenized for 20 minutes at a homogenizing speed of 3000 rpm/min, and then ultrasonicated for 60 minutes with an ultrasonic power of 1000w, which can release ergothione into the fermentation broth to obtain a preliminary fermentation broth.

Example 5

(1) Add 5 g of activated carbon to the 1L fermentation broth prepared in Example 1, and adsorb and decolorize for 30 minutes.

(2) The fermentation broth is centrifuged at 3000 rpm for 30 minutes, the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 μm pore size filter element to filter and sterilize the final product.

(3) Add 9 ml of phenoxyethanol, 1 ml of ethylhexyl glycerol, and 50 ml of 1,3-butanediol as preservatives to the 1L final product of fermentation broth obtained in step (2). Liquid phase detection of thioneine content is 319.8mg/L

Example 6

(1) Add 20 g of activated carbon to the 2L fermentation broth prepared in Example 2, and adsorb and decolorize for 45 minutes.

(2) The fermentation broth is centrifuged at 3000 rpm for 30 min, and the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 μm pore size filter element to obtain the final product.

(3) Add 18 ml of phenoxyethanol, 2 ml of ethylhexyl glycerol, and 100 ml of 1,3-butanediol as preservatives to the final product of 2L fermentation broth obtained in step (2), and mix them aseptically. The content of ergothioneine detected by liquid phase was 308.5mg/L.

Example 7

(1) Add 4 g of activated carbon to the 500 mL fermentation broth prepared in Example 3, and adsorb and decolorize for 30 min.

(2) The fermentation broth is centrifuged at 4000 rpm for 30 min, and the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 μm pore size filter element to obtain the final product.

(3) Add 5 ml of phenoxyethanol, 0.5 ml of ethylhexyl glycerol, and 25 ml of 1,3-butanediol as preservatives to the 500 ml final product of fermentation broth obtained in step (2), and then mix them aseptically. The content of ergothioneine detected by liquid phase was 331.1mg/L.

Example 8

(1) Add 1 g of activated carbon to the 300 mL fermentation broth prepared in Example 4, and adsorb and decolorize for 15 minutes.

(2) The fermentation broth is centrifuged at 5000 rpm for 30 min, and the precipitated activated carbon and bacterial fragments are taken out, and then filtered through a 0.22 μm pore size filter element to filter and sterilize the final product.

(3) Add 2 ml of phenoxyethanol, 0.3 ml of ethylhexyl glycerol, and 15 ml of 1,3-butanediol as preservatives to the 300 mL final product of fermentation broth obtained in step (2), and mix them aseptically. The content of ergothioneine detected by liquid phase was 300.5mg/L.

Example 9 Physical and chemical properties detection

1. Ergothioneine content

The samples in Examples 5 to 8 were named S1, S2, S3, and S4, respectively, and were detected by the external standard method. HPLC conditions: Hypersil ODS C18 column (250mm×4.6mm, particle size 5μm); mobile phase: acetonitrile-water (3:97); flow rate: 1.0mL/min; column temperature: 30°C; detection wavelength: 254nm; injection Quantity: 20μL. The chromatogram is shown in Figure 1, where a) is a standard product (the concentration of ergothioneine is 100mg/L), b) is S1, c) is S2, d) S3, and e) S4 results are shown in Table 1. .

Table 1 The content of thioneine in the products of Examples 5-8

To S1 S2 S3 S4 Content (mg/L) 319.8 308.5 331.1 300.5

2. Fungal polysaccharides

The present invention adopts phenol sulfuric acid method to detect polysaccharide content.

(1) Instruments: Visible-UV spectrophotometer, analytical balance (precision 0.0001g), vortex mixer (2) Reagents:

2.1 Standard solution: accurately weigh 100mg of dry and constant-weight glucose (analytical purity) into a volumetric flask, add water to make the volume up to 250mL, shake well, draw up 10mL of this solution, and add water to make the volume up to 100mL.

2.2 Preparation of 80% phenol solution: accurately pipet 80 mL of re-distilled phenol, add distilled water to make the volume to 100 mL, and then obtain 80% phenol solution. Store it in a brown bottle away from light.

2.3 Preparation of 6% phenol solution: Dilute 80% phenol solution with water to 6%, and prepare it for immediate use.

2.4 Concentrated sulfuric acid (superior grade pure)

(3) Detection:

3.1 Make a standard curve: draw 0.0, 0.4, 0.8, 1.2, 1.6, 2.0 mL of glucose standard solution into a test tube with a stopper, and add water to each to make up to 2.0 mL. Add 1 mL of 6% phenol solution, mix well and quickly add 5.0 mL of concentrated sulfuric acid (when concentrated sulfuric acid is added in the air, it cannot stick to the wall), shake it immediately, react at room temperature for 20 minutes, and measure the absorbance at 490nm. Use 0 tube as a blank control. The coordinate is the concentration of polysaccharide, and the abscissa is the absorbance to obtain a standard curve.

3.2 Sample preparation: Weigh 0.2ml each of the samples S1-S4 in Examples 5-8 and place them in a 50mL volumetric flask, add an appropriate amount of water, and after completely dissolving, add water to the mark as a stock solution and shake well. Measure 5mL of the stock solution before use, put it in a 50mL volumetric flask, add water to the mark, and then dilute it 10 times in the same way. Take 2 mL into a test tube with a stopper, follow the same method as the above "add 1.0 mL of 6% redistilled phenol", obtain the polysaccharide concentration of the sample to be tested from the standard curve, and calculate the polysaccharide content according to the dilution factor.

Table 2. Polysaccharide content of Examples 5-8:

Sample serial number Polysaccharide content (g/L) Example 5 (S1) 2.92 Example 6 (S2) 2.74 Example 7 (S3) 2.94 Example 8 (S4) 2.87

3. Amino acids

Using samples S1 to S4 in Examples 5 to 8 as experimental samples, the amino acid content was determined using an automatic amino acid analyzer. The results are shown in the following table.

Table 3. Amino acid content of Examples 5-8

In order to show that the fermented product has a unique high-activity effect, S0 was selected as a comparative example.

In order to further illustrate that the activity of the fermentation broth product prepared by this process is a combination of multiple active substances, 350 mg of pure ergothione (content greater than or equal to 98%) is taken, and 1L purified water is added to fully dissolve the thioneine solution S0. Comparative example.

4. Antioxidant activity

Precisely measure 5.0 mL of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution and 5.0 mL of S0-S4 sample solution of different concentrations in a test tube with a stopper, and mix. Take an equal volume of water and 95% ethanol mixed solution as a blank control. Leave it at room temperature for 30 minutes, and measure the absorbance of the solution at 523nm. Another set was set up to accurately measure 5.0 mL of DPPH solution and 5.0 mL of purified water and mix them with the same operation as above. The calculation method is as follows:

Select S1 as the test sample, and the results are shown in Table 3. From the data in the table, it can be seen that under the concentration gradient of 0.5%-2%, the sample S1 has a higher free radical scavenging ability, up to 70% at a concentration of 2%, and has extremely high oxidation resistance.

Table 4 Scavenging DPPH free radical activity of samples S0～S4

Concentration of stock solution (%) 0.5 1 2 S1 clearance rate (%) 31% 54% 70% S2 clearance rate (%) 28% 46% 61% S3 clearance rate (%) 39% 57% 76% S4 clearance rate (%) 27% 44% 60% S0 clearance rate (%) 17% 35% 51%

5. Protection against UVA and UVB

5.1 Preparation of sample solution: Dissolve the samples S1-S4 and sample S0 obtained in Examples 5-8 in serum-free medium, prepare a solution with a final concentration of 2.0%, filter and sterilize with a 0.22μm filter membrane, and use serum-free before use The medium is diluted to the specified concentration.

5.2 Plating: Take HaCaT cells (human immortalized epidermal cells) in the logarithmic growth phase, after trypsinization, adjust the cell density to 1×10 ⁵ /mL, inoculate the flat bottom 96-well cell culture plate, 100μL cell suspension per well , Place a carbon dioxide incubator at 37°C and 5% CO _{2 for} regular culture overnight.

5.3 Dosing: Discard the culture solution, and the test groups are as follows. Each well of the test group is added with 100 μL of sample solution with a concentration of 0.5%, 1.0%, and 2.0%. The normal group and the model group are added with the same amount of serum-free medium and placed in the culture Continue to incubate for 24h in the box.

Table 5 The protective effects of samples S1-S4 and S0 on UVA and UVB

5.4 of irradiation: using 7.2J / cm ² UVA plus 126mJ / cm ² UVB irradiated HaCaT cells.

After irradiation, discard the old culture medium, add serum-free culture medium, and continue culturing for 24 hours, add 10 μL of WST-1 to each well, and place it in the cell culture incubator to continue incubating for 4 hours. Measure the light absorption value with a microplate reader at 450nm wavelength.

It can be seen from Table 6 that after combined irradiation of UVA and UVB, compared with the normal group (100%), the proliferation rate of HaCaT cells in the model group decreased by 45.58%, indicating that the model was successful. Before UV irradiation, the test group was pretreated with different concentrations of HaCaT cells. Compared with the untreated model group, the cell proliferation rate was significantly increased, indicating that the fermentation stock solution has a better protective effect against UV damage, and the protective effect As the concentration of the fermentation broth increased, the UV protection effect of the fermentation broth group S1 was significantly better than that of the S0 group containing only ergothioneine solution.

Table 6 The relative proliferation rate (%) of HaCaT cells pretreated with different concentrations of samples after UV damage

6. Promote cell repair effect

Cell culture medium: DMEM culture medium containing 10% FBS. Sample solution: S1 and S0 are prepared into 4% mother liquor with cell culture solution.

HaCaT cells were seeded in a 24-well plate at a density of 5×10 ⁴ cells/mL, and cultured at 37°C and 5% CO ₂ for 24 hours. On the monolayer of cells close to confluence, use a 200 μL pipette tip to scribble vertically in each well of the 24-well plate. Discard the liquid in the well, add the sample solution (final serum content 2.5%), continue to incubate for 24 hours and take pictures for observation.

The results are shown in Figure 2, where a is the initial control treatment group, b is the control treatment group 24h, c is the S0 group initial, d is the S0 treatment 24h, e is the S1 group initial, and f is the S1 treatment group 24h. At a concentration of 2.0%, the S1 group and the S0 group can better promote cell migration and self-repair after contacting damaged cells for 24 hours, and the effect of the S1 group is better than that of the S0 group containing only ergothioine solution. .

As mentioned above, the cosmetic stock solution obtained by the method of the present invention contains high content of ergothioneine, fungal polysaccharides and amino acids at the same time, so it has good antioxidant activity, better protection against UV damage, and has a Good effect of repairing damaged cells.

The above embodiments of the present invention are only used to illustrate specific implementation manners for implementing the present invention, and these implementation manners should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention are regarded as equivalent replacement methods and fall within the protection scope of the present invention.

Claims (26)

A method for preparing a cosmetic stock solution containing ergothioine by fermentation of Hericium erinaceus is characterized in that it comprises the following steps: Step 1: Inoculate the hyphae of Hericium erinaceus into the liquid seed medium and cultivate to obtain the seed liquid; Step 2: Inoculate the seed liquid into the fermentation medium for fermentation and add ergothione precursor materials, and ferment to the end to obtain the fermentation liquid; Step 3: The fermentation broth containing mycelium is processed to obtain the thiothioine-containing fermentation stock solution as the thiothioine-containing cosmetic stock solution. The method according to claim 1, wherein the preservation number of the Hericium erinaceus is CCTCC NO: M 2018567. The method according to claim 1, wherein the seed culture medium comprises the following components: 1-10% by mass of carbon source, 1-5% by mass of organic nitrogen source, and 0.01-1% by mass of inorganic salt . The method according to claim 1, wherein in step 1, the seed solution is obtained by shaking culture at 15-30°C, pH 4.0-7.0, and 50-300 rpm for 5-10 days. The method according to claim 1, wherein the fermentation medium comprises the following components: 1 to 5 mass% of carbon source, 1 to 10 mass% of organic nitrogen source, 0.01 to 1 mass% of inorganic salt , 0.0001 to 0.001 mass% of trace elements and 0.0001 to 0.001 mass% of coenzyme. The method according to claim 1, characterized in that in step 2, shaking fermentation is carried out at 15-30°C, pH 4.0-7.0, 50-300 rpm for 7-15 days, and fermented to the end to obtain fermentation broth. The method according to claim 1, wherein in step 2, the precursor substance is at least one of aspartic acid, glutamine and betaine, and the addition amount is 1-20 mM. The method according to claim 1, wherein in step 2, the inoculation amount of the seed liquid inoculated into the fermentation liquid is 1-10% by volume. The method according to claim 1, wherein in step 2, the fermentation end point is that the residual sugar in the fermentation broth is ≤0.5% by mass. The method according to claim 1, wherein in step 3, the treatment includes at least one of homogenization, ultrasound, and activated carbon adsorption. The method according to claim 10, characterized in that the conditions of the homogenization are the spindle speed of 1000 r/min to 3000 r/min, and the homogenization time of 10 to 30 min. The method according to claim 10, characterized in that the conditions of the ultrasound are that the ultrasound power is 100w to 1000w, the ultrasound time is 5 to 60 minutes, and the ultrasound endpoint is that the content of ergothioneine no longer increases. The method according to claim 10, wherein the amount of activated carbon added in the activated carbon adsorption is 0.1 to 1% of the volume of the fermentation broth, and the adsorption time is 10 to 60 minutes. The method according to claim 1, further comprising step 4: removing impurities in the fermented broth after the treatment. The method according to claim 14, characterized in that, in step 4, the removal of impurities is performed by centrifuging the fermentation broth at 1000-5000 rpm for 10 to 30 minutes, and then filtering the resulting supernatant with a filter element with a pore size of 0.22 μm to 0.45 μm And proceeded. The method according to any one of claims 1-15, wherein the content of ergothioine in the obtained ergothioine-containing cosmetic stock solution is more than 300 mg/L. The method according to any one of claims 1-15, wherein the obtained ergothioine-containing cosmetic stock solution contains more than 2.5 g/L of fungal polysaccharide and a total amount of more than 5.0 mg/100 mL of amino acids, Preferably, the polysaccharide content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, and the total amino acid content is preferably 5.5 mg/100 mL or more, and more preferably 5.7 mg/100 mL or more. The method according to any one of claims 1-15, characterized in that the 0.5v/v%-2v/v% dilution of the obtained ergothioine-containing cosmetic stock solution is 1,1-diphenyl The clearance rate of 2-trinitrophenylhydrazine DPPH is over 30%,

The method according to any one of claims 1-15, characterized in that the cells treated with the 0.5v/v%-2v/v% dilution of the obtained ergothioine-containing cosmetic stock solution are damaged by ultraviolet rays The relative proliferation rate afterwards is more than 70%. A fermentation stock solution containing ergothioneine, characterized in that it contains: ergothioine above 300mg/L, fungal polysaccharides above 2.5g/L, and amino acids with a total amount of 5.0mg/100mL or more, preferably the polysaccharide content is 2.6 g/L or more, more preferably the polysaccharide content is 2.7 g/L or more, preferably the total amino acid content is 5.5 mg/100mL or more, more preferably 5.7 mg/100mL or more. The ergothioine-containing fermentation stock solution according to claim 20, characterized in that it is obtained by using the method according to any one of claims 1-19. The fermentation stock solution containing ergothioine according to claim 20 or 21, wherein the 0.5v/v%-2v/v% dilution of the fermentation stock solution is 1,1-diphenyl-2- The clearance rate of trinitrophenylhydrazine DPPH is over 30%,

The fermentation stock solution containing ergothioneine according to claim 20 or 21, wherein the relative proliferation rate of cells treated with a dilution of 0.5v/v%-2v/v% of the fermentation stock solution after UV damage is 70% or more. A kind of ergothioine-containing fermentation stock obtained by the method of any one of claims 1-19 or the use of the ergothioine-containing fermentation stock of any one of claims 20-23 in cosmetics . A fermentation medium composition for producing ergothioine-containing cosmetic stock solution, which is characterized by comprising the following components: 1 to 5 mass% of carbon source, 1 to 10 mass% of organic nitrogen source, 0.01 to 1 Mass% of inorganic salt, 0.0001 to 0.001% by mass of trace elements, and 0.0001 to 0.001% by mass of coenzyme. A cosmetic stock solution composition containing ergothioneine, which is characterized in that it comprises ergothioine above 300mg/L, fungal polysaccharides above 2.5g/L and amino acids above 5.0mg/100mL, preferably the polysaccharide content is 2.6g/L L or more, more preferably the polysaccharide content is 2.7 g/L or more, preferably the total amino acid content is 5.5 mg/100 mL or more, more preferably 5.7 mg/100 mL or more.

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