Preparation method and application of small molecule schizophyllan with soothing and repairing effects

Abstract

The present invention provides a preparation method and application of a small molecule schizophyllan with soothing and repairing effects. The preparation method of the schizophyllan of the present invention includes the steps of strain activation, cultivation, fermentation, extraction and post-treatment. By culturing and fermenting the schizophyllan, the fermentation environment, fermentation temperature and fermentation time during the fermentation process are strictly controlled to regulate the growth of microorganisms, the synthesis and secretion of polysaccharides, and the consumption and conversion of nutrient substrates; and by further extracting and post-treating the fermentation product, the yield of schizophyllan is greatly improved. The schizophyllan prepared by the present invention is applied to cosmetics, has a small molecular weight, is easily absorbed by the skin, is mild and non-irritating, has excellent moisturizing, soothing and repairing, and anti-inflammatory effects, and has good compatibility with raw materials, and is suitable for use as a raw material for various cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a preparation method and application of a small molecule schizophyllan with soothing and repairing effects. Background Art

[0002] As living standards improve, people pay more and more attention to facial skin care. According to survey results, the top three demands for skin care are moisturizing / hydrating, whitening and anti-aging.

[0003] Schizophyllan, also known as schizophyllan polysaccharide or schizophyllan polysaccharide, is produced by schizophyllan and is one of the main active substances of schizophyllan. It is composed of a (1-3)-linked β-D-pyranose glucose residue main chain and a (1-6)-linked β-D-pyranose glucose residue side chain. Its unique triple helix structure and appropriate branching degree (0.33) make it very effective in regulating skin immunity, promoting skin cell proliferation, clearing free radicals, and anti-inflammation. When applied to cosmetics, it has multiple skin care effects and physiological activities such as moisturizing and anti-oxidation.

[0004] In the prior art, there are two main methods for obtaining schizophyllan. One is to obtain fruiting bodies through the cultivation of schizophyllan, and then extract, separate and purify the fruiting bodies to obtain schizophyllan. The other is a fermentation method, which is to extract from the fermentation broth and mycelium. The fermentation method is a common method for preparing schizophyllan. A suitable strain is selected from schizophyllan for cultivation, and then the strain is prompted to produce schizophyllan by controlling the culture conditions such as temperature, pH, culture medium, dissolved oxygen value, etc., and then schizophyllan is obtained through extraction and purification steps.

[0005] The method of obtaining the fruiting body through cultivation and then extracting to obtain schizophyllan has the disadvantages of long cycle, large production space, low yield and difficult product post-processing, so the fermentation method is more favored by researchers. The fermentation method is divided into solid fermentation and liquid fermentation. Although the solid fermentation has simple equipment and low cost, its culture cycle is long. The advantage of liquid fermentation is that the cycle is short and the yield is large. However, the fermentation preparation of schizophyllan is to carry out the corresponding growth and metabolism process in its production environment. Under the influence of the fermentation environment and fermentation conditions provided by humans, the growth of its microorganisms, the synthesis and secretion of polysaccharides, the consumption and conversion rate of nutrient matrix, etc. will change accordingly. Therefore, it is also necessary to conduct in-depth research on the fermentation to obtain schizophyllan polysaccharides, in order to obtain a fermentation technology with short cycle, low cost, high yield and high product quality. Especially when applied to the field of cosmetics, how to improve the utilization rate of the product, improve the compatibility with other raw materials, improve the absorption effect of the skin and avoid irritation are still the current research focus. Summary of the invention

[0006] In view of the above problems, the present invention provides a preparation method and application of a small molecule schizophyllan with soothing and repairing effects. In the preparation method of the present invention, the schizophyllan is cultured and fermented, and the fermentation environment, fermentation temperature, fermentation time and other conditions in the fermentation process are strictly controlled to regulate the growth of microorganisms, the synthesis and secretion of polysaccharides, and the consumption and conversion of nutrient substrates; and the fermentation product is further extracted and post-processed to greatly improve the yield of schizophyllan. The schizophyllan prepared by the present invention is applied to cosmetics, has a small molecular weight, is easily absorbed by the skin, is mild and non-irritating, has excellent moisturizing, soothing and repairing, and anti-inflammatory effects, and has good compatibility with raw materials, and is suitable for use as a raw material for various cosmetics.

[0007] The first aspect of the present invention provides a method for preparing a small molecule schizophyllanthus having a soothing and repairing effect, and the specific steps of the preparation method are as follows:

[0008] Step S1: strain activation: inoculate the original strain into PDA medium and culture at 25°C for 5-10 days;

[0009] Step S2: strain culture: picking mycelium to a liquid culture medium, the liquid volume of the liquid culture medium is 100mL / 250mL; placing the liquid culture medium on a constant temperature shaker, culturing at 25-27°C for 4-6 days, to obtain a strain seed liquid;

[0010] Step S3: fermentation: inoculate the bacterial seed liquid into the fermentation medium, the volume of the fermentation medium is 100mL / 250mL, and culture in a shaking incubator at 28-29°C for 5-8 days to obtain a fermentation liquid;

[0011] Step S4: extraction: centrifuging the fermentation liquid at a rate of 8000-10000 r / min for 20-30 min to obtain a supernatant and mycelium; adding water at a solid-liquid ratio of 1:3-4 to the mycelium, treating at 70-90° C. for 6-8 h, centrifuging, and obtaining a supernatant; mixing the above supernatants to obtain a mixed solution;

[0012] Step S5: Post-treatment: add activated carbon to the mixed solution and treat it at 40-45°C for 40-45min, filter and remove the activated carbon, add 25-30% volume of chloroform and n-butanol mixed solution and treat it for 30-40min; centrifuge at 3000-4000r / min for 10-15min, and take the supernatant; add β-glucanase, react at 40-45°C for 4-6h, and then treat it in a boiling water bath for 15min, cool to room temperature, and concentrate under reduced pressure to 30-40%; add 2-3 times the volume of ethanol, precipitate at 4°C for 24-28h, and then take the precipitate, and vacuum freeze-dry to obtain the schizophyllanthus. Preferably, in step S1, the components of the PDA culture medium are: 200g potato, 20g glucose, 20g agar, and 1000mL water.

[0013] In order to make the cultured bacteria have higher activity, preferably, in step S2, the components of the liquid culture medium are: 180g potato, 10g glucose, 4g yeast extract powder, 10g peptone, 3g KH2PO4, 1g MgSO4·7H2O, 0.01g vitamin B1, and 1000mL water.

[0014] Preferably, in step S2, the rotation speed of the constant temperature shaker is 150-170 r / min.

[0015] Preferably, the inoculation amount of the bacterial seed liquid in step S3 is 8-12% (v / v).

[0016] In order to further improve the yield of schizophyllan and obtain schizophyllan with a smaller molecular weight, preferably, in step S3, the components of the fermentation medium are: 35.00 g / L glucose, 4.00 g / L yeast extract, 1.00 g / L KH2PO4, 0.50 g / L MgSO4·7H2O, 0.1 g / L oleic acid, 0.05 g / L citric acid, and 0.1 g / L hydroxymethyl cellulose.

[0017] During the fermentation process of Schizophyllum, sufficient carbon sources, nitrogen sources and inorganic salts are needed to provide the nutrients required for reproduction, but the initial concentration cannot be too high, otherwise it will inhibit the synthesis of metabolites. For example, excessive nitrogen sources will promote mycelial growth, but will be detrimental to the accumulation of polysaccharides and reduce the yield of schizophyllum.

[0018] The present invention further improves the yield of schizophyllan by using a specific fermentation medium, using glucose as a carbon source, yeast extract as a nitrogen source, and adding specific contents of inorganic salts, growth factors and other components at the same time. The inventors found that when the fermentation medium contains 35.00 g / L glucose, 4.00 g / L yeast extract, 1.00 KH2PO4, 0.50 MgSO4·7H2O, 0.1 g / L oleic acid, 0.05 g / L citric acid, and 0.1 g / L hydroxymethyl cellulose, the best fermentation effect is achieved. This may be because the fermentation medium provides sufficient initial growth nutrients for schizophyllan, and growth factors and the like can further promote the mycelial reproduction and the production of extracellular polysaccharides of schizophyllan. For example, a certain amount of citric acid can allow more carbon sources to enter the metabolic pathway and increase the polysaccharide production. However, if the citric acid content is too high, the metabolism will be too strong, and the bacteria will begin to reproduce rapidly, which will weaken the polysaccharide synthesis pathway and reduce the polysaccharide content. Ingredients such as hydroxymethylcellulose may also make the bacteria grow more uniformly and densely, and have higher metabolic activity. The inventors unexpectedly discovered that under this condition, the obtained schizophyllanthus also has a smaller molecular weight distribution.

[0019] Preferably, in step S3, the rotation speed of the shaking table is 160-180 r / min. Preferably, in step S3, 5.00 g / L glucose, 0.5 g / L yeast extract, and 1 g / L NH4Cl are added on the 5th to 6th day of fermentation.

[0020] The inventors found in the experiment that carbon sources and nitrogen sources such as glucose and yeast extract were consumed, the propagation of mycelium slowed down, and the sugar content was different, the bacteria would secrete extracellular enzymes to decompose extracellular polysaccharides for self-propagation, so glucose and other ingredients can be added in the late fermentation to achieve more sustained growth, and the inventors also found that when adding glucose 5.00g / L, yeast extract 0.5g / L, NH4Cl 1g / L, not only can the polysaccharide content be increased, but also the prepared schizophyllan has a smaller molecular weight distribution. The inventors analyzed that although the addition of glucose and yeast extract in the late fermentation can make the bacteria grow rapidly, there is a certain competitive relationship between the rapid growth of the bacteria and the production of extracellular enzymes, and a balance needs to be achieved between the growth of the bacteria and the accumulation of polysaccharides and the production of extracellular enzymes. If too much glucose and yeast extract are added at this time, it is not conducive to the accumulation of polysaccharides and the production of extracellular enzymes, thereby reducing the yield of schizophyllan and affecting its molecular weight.

[0021] In order to fully extract the schizophyllanthus obtained by fermentation, preferably, in step S4, the steps of adding water with a solid-liquid ratio of 1:3-4 to the mycelium, treating at 70-90° C. for 6-8 h, centrifuging, and taking the supernatant are repeated 2-3 times.

[0022] There are many gaps between the mycelium of schizophyllum. After the fermentation of schizophyllum, in addition to the schizophyllum in the fermentation liquid, some polysaccharides are also wrapped inside the thalli. In the prior art, the fermentation liquid is usually further treated to obtain schizophyllum after centrifugation, but this method cannot fully obtain the schizophyllum obtained by fermentation. The inventor found that the yield of schizophyllum can be greatly improved by further treating the mycelium. It may be because the centrifugation operation after fermentation cannot separate the extracellular polysaccharides and thalli wrapped inside the mycelium, resulting in partial polysaccharide loss. In the present invention, the extracellular polysaccharides inside the mycelial ball are released by further water treatment, thereby greatly improving the polysaccharide yield. Too few water treatments will lead to inadequate extraction, but too many times will lead to increased costs, prolonged cycles, and increased production costs.

[0023] Preferably, the mass of the activated carbon added in step S4 is 0.3-0.5% of the mass of the mixed liquid.

[0024] Preferably, the volume ratio of chloroform to n-butanol in step S4 is 1:3-5.

[0025] Preferably, the concentration of ethanol in step S4 is 95% by mass.

[0026] Preferably, in step S4, after adding ethanol, the concentration of ethanol needs to be maintained at > 70%.

[0027] Preferably, in step S4, the vacuum freeze-drying conditions are: vacuum degree <10 Pa, temperature is -50°C, and drying time is 12-24h.

[0028] Preferably, in step S5, the amount of β-glucanase added is 200-300 μL / mL.

[0029] The second aspect of the present invention provides the use of the above-mentioned small molecule schizophyllan in cosmetics. When the above-mentioned schizophyllan is used in cosmetics, it has excellent moisturizing, anti-oxidation and soothing and repairing effects.

[0030] The third aspect of the present invention provides a hydrodynamic repair spray. The raw materials of the hydrodynamic repair spray include, by weight percentage: 5-10% moisturizer, 0.3-0.7% hexylene glycol, 0.3-0.7% dimethyl isosorbide, 1-3% schizophyllan, 0.1-0.5% anti-allergic agent, 1-2% trehalose, 0.1-0.2% parahydroxyacetophenone, 0.1-0.3% fragrance, and water as the remainder; the schizophyllan is a small molecule schizophyllan with a soothing and repairing effect prepared according to the above-mentioned preparation method.

[0031] Preferably, the moisturizing agent is one or a mixture of methyl propanediol, sodium hyaluronate, glycerin, and polyether polyol.

[0032] Preferably, the molecular weight of the sodium hyaluronate is 1100-1500 KDa.

[0033] In order to further improve the moisturizing effect, feel and stability of the product, preferably, the moisturizing agent is methylpropanediol and sodium hyaluronate. Preferably, the mass ratio of methylpropanediol to sodium hyaluronate in the present invention is 8:0.005-0.015.

[0034] In the present invention, methylpropanediol and sodium hyaluronate in a mass ratio of 8:0.005-0.015 are used as moisturizers, and ingredients such as schizophyllan and trehalose are added to further improve the moisturizing, repairing, and anti-oxidation effects of the product. Sodium hyaluronate can form a moisturizing film on the skin surface to effectively replenish water and moisturize, and trehalose can also form a special protective film on the skin surface, which can not only play a good role in moisturizing and nourishing the skin, but also protect the skin; while ingredients such as methylpropanediol and hexylene glycol have moisturizing and penetration effects at the same time, and can promote the rapid absorption of active ingredients by the skin.

[0035] At the same time, the inventors found that under the raw material ingredients and content conditions of the present invention, not only can it have excellent moisturizing and repairing effects, but also the use feeling and stability of the product can be improved. The inventors analyzed that the repair spray of the present invention uses ingredients such as schizophyllan and trehalose at the same time. The polysaccharide component is very easy to form aggregates in aqueous solution, and it contains many active groups (such as hydroxyl, amino, etc.), which can form complexes with metal ions, thereby affecting the stability and service life of the product. In the present invention, it is used together with specific ingredients such as sodium hyaluronate. Due to its longer molecular chain, it can avoid the intermolecular aggregation of ingredients such as polysaccharides to a certain extent, and make the small molecule components stably dispersed, so that each component in the system can be stably present, and the stability of the system is enhanced. However, the content of sodium hyaluronate cannot be too much, and the molecular weight cannot be too large, otherwise it may form self-agglomeration, which will affect the stable storage of the product. In particular, when used as a spray product, its viscosity is relatively large, the use feeling is relatively sticky, and the fineness of the spray is affected.

[0036] Preferably, the flavor is one of grapefruit flavor, lemon flavor, jasmine flavor, rose flavor, cherry flavor, and pomegranate flavor. The present invention does not specifically limit the selection of the flavor, and those skilled in the art can select it according to actual experimental conditions.

[0037] A fourth aspect of the present invention provides a method for preparing a hydrodynamic repair spray, wherein the specific steps of the preparation method are as follows:

[0038] Step S1: add the moisturizer to water, homogenize for 1-2 minutes until transparent and granular, stir and heat to 65-70°C, add hexylene glycol and p-hydroxyacetophenone, stir until transparent and granular, then cool down;

[0039] Step S2: After cooling to 40-45° C., add isosorbide dimethyl ether, schizophyllan, antiallergic agent and trehalose, stir until transparent, add flavor, stir evenly to obtain a mixture, and filter through a 300-500 mesh filter cloth to obtain the hydrodynamic repair spray.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) In the preparation method of the present invention, the fermentation environment, fermentation temperature, fermentation time and other conditions are strictly controlled during the fermentation process to regulate the growth of microorganisms, the synthesis and secretion of polysaccharides, and the consumption and conversion of nutrient substrates; and the yield of schizophyllan is greatly improved by further extraction and post-treatment of the fermentation product. The prepared schizophyllan has a small molecular weight and is easily absorbed and utilized by the skin, and has excellent moisturizing, soothing, repairing and anti-inflammatory effects.

[0042] (2) The preparation method of the present invention is simple and has the advantages of short cycle, low cost and high output.

[0043] (3) The schizophyllan prepared by the present invention is applied to cosmetics, which is mild and non-irritating, has good compatibility with raw materials, and is suitable for use as a raw material for various cosmetics.

[0044] (4) The hydrodynamic repair spray prepared by the present invention is easily absorbed and utilized by the skin by controlling the ingredients and contents of various raw materials, especially using small molecular weight mycophenolate mofetil, and can reduce the sticky feeling. When used as a spray, the spray is fine and can form a film evenly and quickly on the skin surface. It not only has excellent moisturizing and repairing effects, but also has excellent product usage feeling and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a picture of the water-soluble product of schizophyllan prepared in Example 1 of the present invention;

[0046] Figure 2 This is a product picture of the hydrodynamic spray prepared in Example 2 of the present invention;

[0047] Figure 3 This is the GPC test chart of the mycophenolic acid prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. It should be noted that, in the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0049] Example

[0050] Example 1

[0051] This embodiment provides a method for preparing a small molecule mycoplasma with soothing and repairing effects. The specific steps of the preparation method are as follows:

[0052] Step S1: strain activation: the original strain was inoculated into PDA medium and cultured at 25°C for 8 days; the components of PDA medium were: 200 g potato, 20 g glucose, 20 g agar, and 1000 mL water.

[0053] Step S2: strain culture: picking mycelium to a liquid culture medium, the liquid volume of the liquid culture medium is 100mL / 250mL; placing the liquid culture medium on a constant temperature shaker (the speed of the constant temperature shaker is 160r / min), culturing at 26°C for 5d to obtain a strain seed liquid; the components of the liquid culture medium are: 180g potato, 10g glucose, 4g yeast extract powder, 10g peptone, 3g KH2PO4, 1g MgSO4·7H2O, 0.01g vitamin B1, and 1000mL water.

[0054] Step S3: fermentation: inoculate the bacterial seed liquid into the fermentation medium at an inoculum amount of 10% (v / v), the liquid volume of the fermentation medium is 100mL / 250mL, and culture on a shaking table at 28°C for 5d (the speed of the shaking table is 170r / min), add 5.00g / L of glucose, 0.5g / L of yeast extract, and 1g / L of NH4Cl; continue to culture until the 7th day to obtain a fermentation liquid; the components of the fermentation medium are: 35.00g of glucose, 4.00g of yeast extract, 1.00g of KH2PO4, 0.50g of MgSO4·7H2O, 0.1g of oleic acid, 0.05g of citric acid, 0.1g of hydroxymethyl cellulose, and 1000mL.

[0055] Step S4: extraction: centrifuge the fermentation liquid at a centrifugal speed of 10000r / min for 20min to obtain a supernatant and mycelium; add water at a solid-liquid ratio of 1:4 to the mycelium, treat at 80°C for 7h, centrifuge, take the supernatant, repeat the above steps of adding water, treating, centrifuging, and taking the supernatant twice; mix the above supernatants to obtain a mixed solution;

[0056] Step S5: post-treatment: add 0.4% activated carbon to the mixed solution and treat it at 45°C for 40 minutes. After filtering out the activated carbon, add 25% volume of a mixed solution of chloroform and n-butanol (the volume ratio of chloroform to n-butanol is 1:4) and treat it for 40 minutes; centrifuge at 4000r / min for 10 minutes, and take the supernatant; add 250μL / mL of β-glucanase, react at 45°C for 5 hours, and then treat it in a boiling water bath for 15 minutes. After cooling to room temperature, concentrate it under reduced pressure to 35%; add 3 times the volume of 95% ethanol, precipitate it at 4°C for 26 hours, take the precipitate, and vacuum freeze-dry it (vacuum freeze-drying conditions are: vacuum degree <10Pa, temperature is -50°C, and drying time is 20 hours) to obtain the said mycobacterium.

[0057] The original strain was Schizophyllum GIM 5.43, purchased from Guangdong Institute of Microbiology. β-Glucanase was purchased from Wuhan Xinhuayang Biological Co., Ltd.

[0058] Example 2

[0059] The present embodiment provides a hydrodynamic repair spray. The raw materials of the hydrodynamic repair spray include, by weight percentage: 9% moisturizer, 0.5% hexylene glycol, 0.5% dimethyl isosorbide, 2% schizophyllan, 0.3% anti-allergic agent, 1.5% trehalose, 0.2% p-hydroxyacetophenone, 0.17% grapefruit essence, and the balance water.

[0060] The moisturizer is methyl propanediol and sodium hyaluronate in a mass ratio of 8:0.01. The molecular weight of sodium hyaluronate is 1300 KDa, purchased from Chengdu Ounrui Chemical Reagent. The anti-allergic agent is JS-PRE anti-allergic agent, purchased from Shanghai Ruidian Biotechnology Co., Ltd.

[0061] This embodiment also provides a method for preparing the above-mentioned hydrodynamic repair spray, and the specific preparation steps are as follows:

[0062] Step S1: add the moisturizer to water, homogenize for 2 minutes until transparent and granular, stir and heat to 70°C, add hexylene glycol and p-hydroxyacetophenone, stir until transparent and granular, and then cool down;

[0063] Step S2: After cooling to 45° C., add isosorbide dimethyl ether, schizophyllan, antiallergic agent and trehalose, stir until transparent, add grapefruit essence, stir evenly to obtain a mixture, and filter through a 400-mesh filter cloth to obtain the hydrodynamic repair spray.

[0064] Example 3

[0065] The present embodiment provides a hydrodynamic repair spray. The raw materials of the hydrodynamic repair spray include, by weight percentage: 10% moisturizer, 0.7% hexylene glycol, 0.5% dimethyl isosorbide, 3% schizophyllan, 0.3% anti-allergic agent, 2% trehalose, 0.2% p-hydroxyacetophenone, 0.1% grapefruit essence, and the balance water.

[0066] The specific implementation of this embodiment is the same as that of Embodiment 2.

[0067] Comparative Example 1

[0068] This comparative example provides a method for preparing a small molecule schizophyllanthus with soothing and repairing effects, and the specific steps of the preparation method are as follows:

[0069] Step S1: strain activation: the original strain was inoculated into PDA medium and cultured at 25°C for 8 days; the components of PDA medium were: 200 g potato, 20 g glucose, 20 g agar, and 1000 mL water.

[0070] Step S2: strain culture: picking mycelium to a liquid culture medium, the liquid volume of the liquid culture medium is 100mL / 250mL; placing the liquid culture medium on a constant temperature shaker (the speed of the constant temperature shaker is 160r / min), culturing at 26°C for 5d to obtain a strain seed liquid; the components of the liquid culture medium are: 180g potato, 10g glucose, 4g yeast extract powder, 10g peptone, 3g KH2PO4, 1g MgSO4·7H2O, 0.01g vitamin B1, and 1000mL water.

[0071] Step S3: fermentation: inoculate the bacterial seed liquid into the fermentation medium at an inoculum amount of 10% (v / v), the liquid volume of the fermentation medium is 100mL / 250mL, and culture on a shaking table at 28°C for 5 days (the speed of the shaking table is 170r / min), add 5.00g / L of glucose, 0.5g / L of yeast extract, and 1g / L of NH4Cl; continue to culture until the 7th day to obtain a fermentation liquid; the components of the fermentation medium are: 35.00g of glucose, 4.00g of yeast extract, 1.00g of KH2PO4, 0.50g of MgSO4·7H2O, and 1000mL.

[0072] Step S4: extraction: centrifuge the fermentation liquid at a centrifugal speed of 10000r / min for 20min to obtain a supernatant and mycelium; add water at a solid-liquid ratio of 1:4 to the mycelium, treat at 80°C for 7h, centrifuge, take the supernatant, repeat the above steps of adding water, treating, centrifuging, and taking the supernatant twice; mix the above supernatants to obtain a mixed solution;

[0073] Step S5: post-treatment: add 0.4% activated carbon to the mixed solution and treat it at 45°C for 40 minutes. After filtering out the activated carbon, add 25% volume of a mixed solution of chloroform and n-butanol (the volume ratio of chloroform to n-butanol is 1:4) and treat it for 40 minutes; centrifuge at 4000r / min for 10 minutes, and take the supernatant; add 250μL / mL of β-glucanase, react at 45°C for 5 hours, and then treat it in a boiling water bath for 15 minutes. After cooling to room temperature, concentrate it under reduced pressure to 35%; add 3 times the volume of 95% ethanol, precipitate it at 4°C for 26 hours, take the precipitate, and vacuum freeze-dry it (vacuum freeze-drying conditions are: vacuum degree <10Pa, temperature is -50°C, and drying time is 20 hours) to obtain the said mycobacterium.

[0074] The original strain was Schizophyllum GIM 5.43, which was purchased from Guangdong Institute of Microbiology.

[0075] Comparative Example 2

[0076] This comparative example provides a method for preparing a small molecule mycobacterium schizophylline with soothing and repairing effects. The specific implementation steps of this comparative example are the same as those of Example 1. The difference from Example 1 is that step S3 is: fermentation: inoculate the bacterial seed liquid into the fermentation medium at a 10% inoculation amount (v / v), the liquid volume of the fermentation medium is 100mL / 250mL, and culture on a shaking table at 28°C for 7 days (the shaking table speed is 170r / min) to obtain a fermentation liquid.

[0077] Comparative Example 3

[0078] This comparative example provides a method for preparing a small molecule mycoplasma with soothing and repairing effects. The specific implementation steps of this comparative example are the same as those of Example 1. The difference from Example 1 is that step S4 is: extraction: the fermentation broth is centrifuged at a centrifugal rate of 10000r / min and a centrifugal time of 20min to obtain a supernatant and mycelium.

[0079] Comparative Example 4

[0080] This comparative example provides a hydrodynamic repair spray, and the specific implementation method is the same as that of Example 2. The difference from Example 2 is that the moisturizing agent is methyl propanediol.

[0081] Comparative Example 5

[0082] This comparative example provides a hydrodynamic repair spray, and the specific implementation method is the same as that of Example 2. The difference from Example 2 is that the moisturizing agent is methyl propanediol and sodium hyaluronate in a mass ratio of 8:0.03, and the molecular weight of sodium hyaluronate is 2500KDa.

[0083] Performance Testing

[0084] 1. Polysaccharide content

[0085] Preparation of standard curve: measure 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 ml of 1 mg / ml β-1,3-glucan standard solution into graduated test tubes respectively, add distilled water to 2 ml, add 1 ml of 20 g / L phenol solution and 10 ml of concentrated sulfuric acid, mix well, boil in boiling water bath for 2 min, quickly cool to room temperature, and measure the ultraviolet absorption value at a wavelength of 485 nm to obtain the standard curve.

[0086] Take 1 ml of the sample to be tested, add 1 ml of distilled water, 1 ml of 20 g / L phenol solution, and 10 ml of concentrated sulfuric acid, mix well, boil in a boiling water bath for 2 min, quickly cool to room temperature, measure the ultraviolet absorption value at a wavelength of 485 nm, and obtain the polysaccharide content in the sample from the standard curve.

[0087] 2. Molecular weight determination: The molecular weight was determined by high performance gel chromatography.

[0088] 3. Soothing effect test: Tested according to the laboratory method (GDYJJC-SOP-03-GX004) hyaluronic acid inhibition method.

[0089] The hyaluronic acid inhibition rate of the test sample group was better (higher) than that of the blank control group, and the statistical difference P value was <0.05 (significant difference), indicating that the test sample has a soothing effect.

[0090] The above polysaccharide content and molecular weight tests were performed on the schizophyllan of Example 1 and Comparative Examples 1-3; the above soothing effect test was performed on the schizophyllan of Example 1, the hydrodynamic spray of Examples 2-3 and Comparative Examples 4-5, and the hyaluronic acid inhibition rate of the blank group was measured to be 0.71%; the measured results are shown in Table 1 below.

[0091] Table 1

[0092]

[0093] 4. Soothing effect test: The test is conducted according to SN / T 2329-2009 “Chicken chorioallantoic membrane test for eye irritation / corrosion of cosmetics”.

[0094] The above soothing test was performed on the schizophyllan in Example 1, and the irritation score IS was measured to be 0, which is non-irritating.

[0095] 5. Repair effect test: The repair effect test is carried out based on the zebrafish neutrophil inhibition rate test.

[0096] Zebrafish embryos at the third day of development were selected as experimental organisms.

[0097] A blank control group, a model group (1 μM), a positive drug group (0.02 g / L) and a sample group (0.1%) were set up. Three replicates were set up for each experimental group. Ten zebrafish embryos were placed in a 24-well plate in each replicate. 2 mL of the working solution corresponding to each concentration group was added to each well. The 24-well plate to be tested was placed in a constant temperature incubator at 28.5±0.5°C and samples were taken after incubation for 4 hours.

[0098] After incubation, no less than 12 zebrafish embryos with normal phenotypes were randomly selected from each group and fixed with 4% paraformaldehyde overnight. The juveniles were washed with PBS 4 times, 10 min / time. Sudan black solution was added for staining, incubated in the dark for 40 min, washed with 70% ethanol in the dark until the body color was transparent, washed with PBS 3 times, and the fish were uniformly adjusted to the lateral position and photographed using a stereo microscope. The number of neutrophils on the skin surface of the yolk sac area of ​​each fish was counted.

[0099] The neutrophil aggregation inhibition rate was calculated using the following formula:

[0100] Inhibition rate (%) = (MT) / M × 100%------------(1)

[0101] (1) Where: T is the average number of neutrophils in the yolk sac region of the fish embryos in the test group; M is the average number of neutrophils in the yolk sac region of the fish embryos in the model group;

[0102] T test was performed on the neutrophils in the yolk sac region of the fish embryos in the test group and the neutrophils in the yolk sac region of the fish embryos in the model group, and p<0.05 was considered to be significantly different.

[0103] The above repair effect test was performed on the schizophyllan in Example 1. Compared with the model group, the sample had a neutrophil aggregation inhibition rate of 62.19% in the zebrafish yolk sac region at a concentration of 0.1%, which was significantly different (p<0.05).

[0104] 6. Moisture test

[0105] A 0.5% aqueous solution of schizophyllan and sodium hyaluronate was placed in a constant temperature and low humidity (dry silica gel) desiccator, and the water loss rate of each was measured after 75 hours.

[0106] The moisturizing performance of the schizophyllan in Example 1 was tested, and the results were shown in Table 2 below.

[0107] Table 2

[0108] Schizophyllin Sodium Hyaluronate Initial Value 30.05 30 After 75 hours 20.78 23.56 Water loss rate 30.1％ 21.47％

[0109] 7. Stability test

[0110] Test conditions: The samples were packaged in white polyethylene plastic bottles and placed in a 40°C constant temperature and humidity chamber, room temperature (25°C), and refrigerated (-8 to -4°C). The composition was observed in the first and second months to see if there was discoloration, stratification, solid precipitation, or other adverse phenomena. Normal samples were recorded as A, slight discoloration and stratification were recorded as B, and stratification or partial solid precipitation were recorded as C.

[0111] The hydrodynamic spray of Examples 2-3 and Comparative Examples 4-5 was subjected to stability test. The test results are shown in Table 3 below.

[0112] Table 3

[0113]

Claims

1. A method for preparing a small molecule schizophyllan with soothing and repairing effects, characterized in that: The specific steps of the preparation method are as follows: Step S1: strain activation: inoculate the original strain into PDA medium and culture at 25°C for 5-10 days; the original strain is Schizophyllum GIM 5.43; Step S2: strain culture: picking mycelium to a liquid culture medium, the liquid volume of the liquid culture medium is 100mL / 250mL; placing the liquid culture medium on a constant temperature shaker, culturing at 25-27°C for 4-6 days, to obtain a strain seed liquid; Step S3: fermentation: inoculate the bacterial seed liquid into the fermentation medium, the volume of the fermentation medium is 100mL / 250mL, and culture in a shaking incubator at 28-29°C for 5-8 days to obtain a fermentation liquid; Step S4: extraction: centrifuging the fermentation liquid at a rate of 8000-10000 r / min for 20-30 min to obtain a supernatant and mycelium; adding water at a solid-liquid ratio of 1:3-4 to the mycelium, treating at 70-90° C. for 6-8 h, centrifuging, and obtaining a supernatant; mixing the above supernatants to obtain a mixed solution; Step S5: post-treatment: adding activated carbon to the mixed solution and treating it at 40-45°C for 40-45 minutes, filtering to remove the activated carbon, adding 25-30% volume of chloroform and n-butanol mixed solution and treating it for 30-40 minutes; centrifuging at 3000-4000r / min for 10-15 minutes, and taking the supernatant; adding β-glucanase, reacting at 40-45°C for 4-6 hours, and then treating it in a boiling water bath for 15 minutes, cooling to room temperature, and concentrating under reduced pressure to 30-40%; adding 2-3 times the volume of ethanol, precipitating at 4°C for 24-28 hours, and then taking the precipitate, and vacuum freeze-drying to obtain the schizophyllanthus; In step S3, the components of the fermentation medium are: glucose 35.00 g / L, yeast extract 4.00 g / L, KH2PO4 1.00 g / L, MgSO4·7H2O 0.50 g / L, oleic acid 0.1 g / L, citric acid 0.05 g / L, hydroxymethyl cellulose 0.1 g / L; In step S3, 5.00 g / L of glucose, 0.5 g / L of yeast extract, and 1 g / L of NH4Cl are added on the 5th to 6th day of fermentation culture; In step S3, the rotation speed of the shaking table is 160-180 r / min; The inoculation amount of the bacterial seed liquid in step S3 is 8-12% (v / v); In step S4, repeat the steps of adding water with a solid-liquid ratio of 1:3-4 to the mycelium, treating at 70-90° C. for 6-8 hours, centrifuging, and taking the supernatant 2-3 times; In step S5, the amount of β-glucanase added is 200-300 μL / mL.

2. The method for preparing the small molecule schizophyllan with soothing and repairing effects according to claim 1, characterized in that: In step S2, the liquid culture medium comprises: 180 g potato, 10 g glucose, 4 g yeast extract powder, 10 g peptone, 3 g KH2PO4, 1 g MgSO4·7H2O, 0.01 g vitamin B1, and 1000 mL water.

3. The method for preparing the small molecule schizophyllan with soothing and repairing effects according to claim 1, characterized in that: The mass of the activated carbon added in step S5 is 0.3-0.5% of the mass of the mixed solution.

4. A hydrodynamic repair spray, characterized by: The raw materials of the power repair spray include: 5-10% moisturizer, 0.3-0.7% hexylene glycol, 0.3-0.7% isosorbide dimethyl ether, 1-3% schizophyllan, 0.1-0.5% anti-allergic agent, 1-2% trehalose, 0.1-0.2% p-hydroxyacetophenone, 0.1-0.3% flavor, and the balance of water; the schizophyllan is prepared according to the preparation method according to any one of claims 1 to 3; The moisturizing agent is methyl propanediol and sodium hyaluronate in a mass ratio of 8:0.005-0.

015.

5. The method for preparing a hydrodynamic repair spray according to claim 4, characterized in that: The specific steps of the preparation method are as follows: Step S1: add the moisturizer to water, homogenize for 1-2 minutes until transparent and granular, stir and heat to 65-70°C, add hexylene glycol and p-hydroxyacetophenone, stir until transparent and granular, then cool down; Step S2: After cooling to 40-45° C., add isosorbide dimethyl ether, schizophyllan, antiallergic agent and trehalose, stir until transparent, add flavor, stir evenly to obtain a mixture, and filter through a 300-500 mesh filter cloth to obtain the hydrodynamic repair spray.

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