Co-fermentation product of aureobasidium pullulans and beauveria bassiana, skin care composition containing co-fermentation product and application of co-fermentation product

Abstract

The invention relates to a co-fermentation product of aureobasidium pullulans and beauveria bassiana, a skin care composition containing the co-fermentation product and application of the co-fermentation product. The co-fermentation product of aureobasidium pullulans and beauveria bassiana is prepared by a preparation method comprising the following steps: inoculating a beauveria bassiana liquid into a sterilization culture medium for fermentation to obtain a beauveria bassiana fermentation liquid; inoculating the aureobasidium pullulans liquid into the beauveria bassiana fermentation liquid for fermentation, and sterilizing to obtain mixed fermentation liquid; centrifuging the mixed fermentation liquor, collecting supernate, carrying out alcohol precipitation treatment on the supernate, and filtering to obtain filtrate; and concentrating and drying the filtrate to obtain the co-fermentation product of aureobasidium pullulans and beauveria bassiana. The aureobasidium pullulans can be inhibited from generating melanin by adopting a way of co-fermenting the beauveria bassiana and the aureobasidium pullulans, so that the quality and the stability of an aureobasidium pullulans fermentation product are improved. And the product has excellent effects in the aspects of whitening skin, preserving moisture, maintaining stability and slowing skin aging.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and relates to a co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, a skin care composition containing the co-fermentation product, and application of the co-fermentation product. Background Art

[0002] Aureobasidium is a yeast-like fungus that is widely distributed in nature. In recent years, Aureobasidium has been continuously isolated from high-salt environments, deep sea waters, and Antarctic and Arctic soils. Aureobasidium has a unique metabolic ability and can produce a variety of fermentation products that are beneficial to the skin, such as pullulan, glucan, β-polymalic acid, and L-sorbitol. Among them, pullulan has adhesive properties, the ability to form fibers, excellent formability, and strong oxygen barrier properties. It also has the functions of anti-oxidation, isolation of static electricity and dust, and provides comprehensive protection for the skin. Among them, glucan is an ingredient that can enhance the activity of skin cells and promote cell proliferation and metabolism. It can delay skin aging, efficiently repair the skin, and has a certain antibacterial effect. It can prevent bacterial growth and avoid skin infections and allergies. Among them, β-polymalic acid can promote the shedding of aged stratum corneum, accelerate the renewal rate of keratinocytes and a small number of upper epidermal cells, promote the proliferation of elastic fibers in the dermis, and can also reduce photoaging, increase the synthesis of mucopolysaccharides and collagen, reduce fine lines and wrinkles, and restore the youthful state of the skin. Among them, L-sorbitol can absorb and retain skin moisture, help improve skin dryness, peeling and other problems, and keep the skin soft and smooth. Therefore, the fermentation products of Aconitum have excellent skin care effects and broad application prospects.

[0003] However, during the fermentation process of A. pullulans, melanin or similar substances will inevitably be produced as byproducts. These substances will adhere to the fermentation products, affecting the quality and appearance of the A. pullulans fermentation products and giving the fermentation liquid a darker color. If A. pullulans is used in the industry to produce skin care products, it will lead to an additional decolorization process and increase costs, thus affecting its application in the field of skin care products. Currently, there are few types of skin care products containing A. pullulans fermentation products on the market. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, a skin care composition containing the same and applications thereof.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, wherein the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana is prepared by a preparation method comprising the following steps:

[0007] (1) inoculating a Beauveria bassiana bacterial liquid into a sterile culture medium for fermentation to obtain a Beauveria bassiana fermentation liquid;

[0008] (2) inoculating the budding Aureobasidium pullulans liquid into the Beauveria bassiana fermentation liquid for fermentation, sterilization, and obtaining a mixed fermentation liquid;

[0009] (3) centrifuging the mixed fermentation broth, collecting the supernatant, subjecting the supernatant to alcohol precipitation, and filtering to obtain a filtrate;

[0010] (4) The filtrate is concentrated and dried to obtain the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana.

[0011] The fermentation product of budding mildew has the effect of keeping skin moisture, delaying skin aging, and efficiently repairing skin, and it can form a thin film on the skin, effectively isolating gases such as oxygen and carbon dioxide, while maintaining the normal breathing of the skin, can lock the moisture and nutrients of the skin, and make the skin care ingredients better absorbed and utilized by the skin. But its fermentation product contains melanin, and the present invention creatively adopts the mode of co-fermentation of beauveria bassiana and budding mildew as mentioned above, can suppress the activity of budding mildew tyrosinase, and then suppress budding mildew to produce melanin, thereby improve the quality and stability of budding mildew enzyme fermentation product. In addition, beauveria bassiana fermentation product can also suppress the activity of human skin tyrosinase, slow down the generation of melanin, thus have the effect of whitening skin, removing spots, beauveria bassiana fermentation product can also eliminate free radicals, help to slow down the process of skin aging. And the fermentation product of budding mildew and beauveria bassiana fermentation product cooperate with each other, and have synergistic effect on the effect of whitening skin, moisturizing and maintaining stability, slowing skin aging.

[0012] Preferably, the number of viable bacteria in the Beauveria bassiana liquid is (1-5)×10 8 CFU / mL, for example 1×10 8 CFU / mL, 2×10 8 CFU / mL, 3×10 8 CFU / mL, 4×10 8 CFU / mL, 5×10 8 CFU / mL, etc.; the inoculum amount is 0.5-2%, for example 0.5%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, etc.

[0013] Preferably, the formula of the culture medium is: sucrose 100-150 g / L, magnesium sulfate 0.1-0.5 g / L, dipotassium hydrogen phosphate 1-10 g / L, ammonium sulfate 0.5-1 g / L, sodium chloride 0.5-1.5 g / L, and the balance is water.

[0014] The content of sucrose can be selected from 100g / L, 110g / L, 115g / L, 120g / L, 125g / L, 130g / L, 140g / L, 150g / L, etc.; the content of magnesium sulfate can be selected from 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, etc.; the content of dipotassium hydrogen phosphate can be selected from 1g / L, 2g / L, 3g / L, etc. g / L, 4g / L, 5g / L, 7g / L, 8g / L, 9g / L, 10g / L, etc.; the content of ammonium sulfate can be selected as 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, etc.; the content of sodium chloride can be selected as 0.5g / L, 0.7g / L, 0.8g / L, 1g / L, 1.2g / L, 1.5g / L, etc.

[0015] Preferably, the fermentation in step (1) is carried out at 24-28°C (eg, 24°C, 25°C, 26°C, 27°C, 28°C, etc.) for 1-4h (eg, 1h, 2h, 3h, 4h, etc.).

[0016] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0017] Preferably, the viable count of the budding short-stalked mold liquid is (3-10)×10 9 CFU / mL, for example 3×10 9 CFU / mL, 4×10 9 CFU / mL, 5×10 9 CFU / mL, 6×10 9 CFU / mL, 7×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL, 10×10 9 CFU / mL, etc.; the inoculum amount is 3-5%, for example 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0018] Preferably, the fermentation in step (2) is carried out at 25-30°C (e.g., 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc.) for 20-170h (e.g., 24h, 36h, 48h, 72h, 96h, 108h, 120h, 148h, 170h, etc.).

[0019] Preferably, the sterilization conditions in step (2) are 70-90°C (e.g., 70°C, 75°C, 80°C, 85°C, 90°C, etc.) for 10-20 min (e.g., 10 min, 12 min, 14 min, 15 min, 16 min, 17 min, 18 min, 20 min, etc.).

[0020] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0021] Preferably, the centrifugation in step (3) is performed at 6000-9000 rpm (e.g. 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, etc.) for 10-20 min (e.g. 10 min, 12 min, 14 min, 15 min, 16 min, 17 min, 18 min, 20 min, etc.).

[0022] Preferably, the alcohol precipitation treatment is to add 1-3 times (e.g., 1, 2 or 3 times) the volume of 75-100% (e.g., 75%, 80%, 85%, 90%, 95%, 100%, etc.) ethanol or ethanol aqueous solution, and let it stand at 2-10°C (e.g., 2°C, 4°C, 5°C, 6°C, 7°C, 8°C, 10°C, etc.) for 5-12h (e.g., 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, etc.).

[0023] Preferably, after the drying process in step (4), the following steps are repeated 1-3 times:

[0024] The product is subjected to alcohol precipitation treatment, filtered to obtain a filtrate, and the filtrate is concentrated and dried.

[0025] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0026] In a second aspect, the present invention provides a skin care composition, the components of which include: the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to the first aspect, the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen.

[0027] The jasmine fermentation product of Lactobacillus rhamnosus can increase the vitality of fibroblasts exposed to ultraviolet radiation and free radical stress, helping skin cells to resist the pressure of the external environment. At the same time, it reduces cytotoxicity, the production of intracellular reactive oxygen species, reduces skin damage, and can inhibit the degradation of collagen in human skin fibroblasts to maintain skin elasticity and health.

[0028] Hyaluronic acid has excellent moisturizing and lubricating effects. At the same time, it can also enhance the film-forming properties of the co-fermentation products of Aureobasidium pullulans and Beauveria bassiana, making the film more stable. Therefore, the molecular chains of hyaluronic acid can interact with the molecular chains of pullulan in the co-fermentation products, and enhance the connection between pullulan molecules through hydrogen bonds and other binding methods, thereby improving the stability of the pullulan film. The stability of the film can further promote the absorption and full play of various effective ingredients.

[0029] Collagen has the effect of tightening the skin and reducing wrinkles. At the same time, it can also improve the film-forming speed and stability of the co-fermentation product of Aureobasidium and Beauveria bassiana, because the aldehyde group of pullulan in the co-fermentation product can undergo a grafting reaction with the amino group of collagen to generate a new amide bond, which enhances the binding force between the two and makes the pullulan film more stable and firm. The molecular structure of collagen helps to increase the viscosity and elasticity of the pullulan film, making it easier to form a uniform and tight film on the skin, which can effectively lock the moisture and nutrients of the skin, prevent moisture loss and external environmental damage, strengthen the skin barrier, and further promote the absorption and full play of various functional ingredients.

[0030] In summary, the present invention creatively compounds the co-fermentation product of the aforementioned Aureobasidium pullulans and Beauveria bassiana with the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid and collagen to obtain a new skin care composition. The four components work synergistically with each other and have excellent comprehensive skin care effects in whitening the skin, moisturizing and stabilizing, and slowing down skin aging.

[0031] Preferably, the jasmine fermentation product of Lactobacillus rhamnosus is prepared by a preparation method comprising the following steps:

[0032] (1) mixing jasmine powder with water to obtain a jasmine powder aqueous solution;

[0033] (2) inoculating a Lactobacillus rhamnosus bacterial liquid into a jasmine pollen aqueous solution for fermentation and sterilization to obtain a fermentation liquid;

[0034] (3) crushing the fermentation liquid, and then mixing it with an ethanol aqueous solution to perform diafiltration extraction to obtain an extract;

[0035] (4) filtering the extract, concentrating and drying the filtrate to obtain the jasmine fermentation product of Lactobacillus rhamnosus.

[0036] The jasmine fermentation product of Lactobacillus rhamnosus produced under the above-mentioned specific preparation process conditions has a higher degree of compatibility with the other three components in the composition, making the skin care effect of the product more excellent.

[0037] Preferably, the mass ratio of the jasmine pollen to water is 1:(5-15), for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:13, 1:14, 1:15, etc.

[0038] Preferably, the viable count of the Lactobacillus rhamnosus bacterial solution is (5-8)×10 9 CFU / mL, for example 5×10 9 CFU / mL, 6×10 9 CFU / mL, 7×10 9CFU / mL, 8×10 9 CFU / mL, etc.; the inoculation amount is 1-3%, for example 1%, 1.5%, 2%, 2.5%, 3%, etc.

[0039] Preferably, the fermentation is carried out at 10-45°C (e.g., 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, etc.) for 20-90h (e.g., 24h, 36h, 48h, 72h, 84h, 90h, etc.).

[0040] Preferably, the sterilization conditions are 70-90°C (e.g., 70°C, 75°C, 80°C, 85°C, 90°C, etc.) for 10-20 min (e.g., 10 min, 12 min, 14 min, 15 min, 16 min, 17 min, 18 min, 20 min, etc.).

[0041] Preferably, the crushing treatment is high-pressure homogenization at 50-70 MPa (e.g., 50 MPa, 55 MPa, 60 MPa, 65 MPa, 70 MPa, etc.) for 5-15 min (e.g., 5 min, 7 min, 8 min, 10 min, 12 min, 14 min, 15 min, etc.).

[0042] Preferably, the volume fraction of the ethanol aqueous solution is 60-80%, such as 60%, 65%, 70%, 75%, 80%, etc.; the mass is 5-10 times that of the fermentation broth, such as 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0043] Preferably, the percolation extraction is carried out at 30-60°C (e.g., 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc.) for 3-8h (e.g., 3h, 4h, 5h, 6h, 7h, 8h, etc.).

[0044] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0045] Preferably, the components of the skin care composition include, by mass: 25-38 parts of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to the first aspect, 12-18 parts of jasmine fermentation product of Lactobacillus rhamnosus, 2-10 parts of hyaluronic acid, and 1-10 parts of collagen.

[0046] The mass proportions of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana can be selected to be 25 parts, 28 parts, 30 parts, 32 parts, 34 parts, 35 parts, 37 parts, 38 parts, etc.

[0047] The mass proportion of the jasmine fermentation product of Lactobacillus rhamnosus can be 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, etc.

[0048] The mass fraction of the hyaluronic acid can be selected from 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc.

[0049] The mass proportion of the collagen can be selected from 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc.

[0050] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0051] In a third aspect, the present invention provides a cosmetic composition having whitening, anti-aging and barrier repair effects, wherein the cosmetic composition comprises the skin care composition described in the second aspect, a skin conditioner, an emulsifier and water.

[0052] Preferably, the skin conditioning agent comprises lavender essential oil and / or bisabolol.

[0053] Lavender essential oil has antibacterial, anti-inflammatory and antioxidant effects, can improve skin conditions, can enter the skin follicles, remove deposits and excess oil in the follicles, regulate the water and oil balance of the skin, and enhance the skin barrier function to resist the invasion of the external environment. On the one hand, the film formed by the pullulan polysaccharide in the co-fermentation product can lock the active ingredients in the lavender essential oil, so that it stays on the skin surface for a longer time and continues to play a role. On the other hand, lavender essential oil can also further enhance the antioxidant and isolation effects of the pullulan polysaccharide film and improve its ability to protect the skin. Therefore, adding lavender essential oil can further improve the anti-aging, moisturizing, whitening and other effects of the product.

[0054] Preferably, the emulsifier comprises soy lecithin.

[0055] In a fourth aspect, the present invention provides a method for preparing the cosmetic composition according to the third aspect, the preparation method comprising the following steps:

[0056] The skin care composition is mixed with water to obtain a water phase; the skin conditioner is mixed with an emulsifier to obtain an oil phase; the water phase and the oil phase are mixed, emulsified and homogenized to obtain the cosmetic composition.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] On the one hand, the present invention creatively adopts the method of co-fermenting Beauveria bassiana with Aureobasidium pullulans, inhibiting the production of melanin by Aureobasidium pullulans, and improving the quality and stability of the Aureobasidium pullulans fermentation product. Moreover, the Aureobasidium pullulans fermentation product and the Beauveria bassiana fermentation product can also cooperate with each other, and have a synergistic effect in the effects of whitening the skin, moisturizing and stabilizing, and slowing down skin aging.

[0059] On the other hand, the present invention also creatively develops a skin care composition, which compounds the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana with the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen. The four components also have potential to promote each other, and have excellent comprehensive skin care effects in whitening the skin, moisturizing and stabilizing, and slowing down skin aging. DETAILED DESCRIPTION

[0060] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0061] The Beauveria bassiana involved in the following preparation examples is a product purchased from the Authentic Source Chinese Medicine Store in Bozhou City with the model number of Bombyx batryticatus; the budding Aureobasidium pullulans is a product purchased from Shanghai Keborui Biotechnology Co., Ltd. with the model number of Aureobasidium pullulans; and the Lactobacillus rhamnosus is a product purchased from Shanghai Keborui Biotechnology Co., Ltd. with the model number of Lactobacillus rhamnosus.

[0062] Preparation Example 1-1

[0063] This preparation example provides a co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, and the preparation method is as follows:

[0064] (1) Add Beauveria bassiana liquid (1×10 8 CFU / mL) was inoculated into a sterile culture medium at 1%, and fermented at 28°C for 2 hours. The formula of the culture medium was: sucrose 120 g / L, magnesium sulfate 0.3 g / L, potassium hydrogen phosphate 5 g / L, ammonium sulfate 0.7 g / L, sodium chloride 1.1 g / L, and the balance was water to obtain Beauveria bassiana fermentation liquid.

[0065] (2) Add the budding short-stalked mold liquid (5×10 9 CFU / mL) was inoculated into Beauveria bassiana fermentation broth at 3%, and the mixture was fermented at 28°C for 6 days and sterilized at 80°C for 15 min to obtain a mixed fermentation broth.

[0066] (3) The mixed fermentation broth was centrifuged at 8000 rpm for 15 min, the supernatant was collected, and the supernatant was subjected to alcohol precipitation treatment. Two volumes of anhydrous ethanol were added, and the mixture was allowed to stand at 4°C for 8 h and filtered to obtain a filtrate;

[0067] (4) The filtrate is concentrated and freeze-dried to obtain a crude fermentation product, which is then added with 2 times the volume of anhydrous ethanol, allowed to stand at 4° C. for 8 h, and filtered. The filtrate is concentrated and dried at 60° C. to obtain the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, which has a light yellow appearance.

[0068] Preparation Example 1-2

[0069] This preparation example provides a co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, and the preparation method is as follows:

[0070] (1) Add Beauveria bassiana liquid (5×10 8 CFU / mL) was inoculated into a sterile culture medium at 0.5%, and fermented at 26°C for 1 hour. The formula of the culture medium was: sucrose 110 g / L, magnesium sulfate 0.5 g / L, potassium hydrogen phosphate 3 g / L, ammonium sulfate 0.8 g / L, sodium chloride 1.0 g / L, and the balance was water to obtain Beauveria bassiana fermentation liquid.

[0071] (2) Add the budding short-stalked mold liquid (3×10 9 CFU / mL) was inoculated into Beauveria bassiana fermentation broth at 5%, and the mixture was fermented at 30°C for 5 days and sterilized at 90°C for 15 min to obtain a mixed fermentation broth.

[0072] (3) The mixed fermentation broth was centrifuged at 9000 rpm for 10 min, the supernatant was collected, and the supernatant was subjected to alcohol precipitation treatment. 3 volumes of 95% ethanol aqueous solution were added, and the mixture was allowed to stand at 4°C for 8 h and filtered to obtain a filtrate;

[0073] (4) The filtrate is concentrated and freeze-dried to obtain a crude fermentation product, which is then added with 3 times the volume of 95% ethanol aqueous solution, allowed to stand at 4° C. for 8 h, and filtered. The filtrate is concentrated and dried at 60° C. to obtain the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, which has a light yellow appearance.

[0074] Preparation Example 1-3

[0075] This preparation example provides a co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, and the preparation method is as follows:

[0076] (1) Add Beauveria bassiana liquid (1×10 8 CFU / mL) was inoculated into a sterile culture medium at 2%, and fermented at 28°C for 2 hours. The formula of the culture medium was: sucrose 130 g / L, magnesium sulfate 0.2 g / L, potassium hydrogen phosphate 7 g / L, ammonium sulfate 0.5 g / L, sodium chloride 1.3 g / L, and the balance was water to obtain Beauveria bassiana fermentation liquid.

[0077] (2) Add the budding short-stalked mold solution (10×10 9 CFU / mL) was inoculated into Beauveria bassiana fermentation broth at 3%, and the mixture was fermented at 28°C for 7 days and sterilized at 80°C for 15 min to obtain a mixed fermentation broth.

[0078] (3) The mixed fermentation broth was centrifuged at 6000 rpm for 20 min, the supernatant was collected, and the supernatant was subjected to alcohol precipitation treatment. Two volumes of anhydrous ethanol were added, and the mixture was allowed to stand at 4°C for 10 h and filtered to obtain a filtrate;

[0079] (4) The filtrate is concentrated and freeze-dried to obtain a crude fermentation product, which is then added with 2 times the volume of anhydrous ethanol, allowed to stand at 4° C. for 10 h, and filtered. The filtrate is concentrated and dried at 60° C. to obtain the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, which has a light yellow appearance.

[0080] Comparative Preparation Example 1-1

[0081] This preparation example provides a co-fermentation product of Aureobasidium and Beauveria bassiana. The preparation method is different from that of Preparation Example 1-1 only in that the Beauveria bassiana culture liquid and the Aureobasidium pullulans culture liquid are simultaneously introduced into the culture medium for fermentation, specifically:

[0082] (1) Add Beauveria bassiana liquid (1×10 8 CFU / mL) and 1% Aureobasidium pullulans solution (5×10 9 CFU / mL) was inoculated into a sterile culture medium at 3%, and fermented at 28°C for 6 days. The culture medium formula was: sucrose 120g / L, magnesium sulfate 0.3g / L, potassium dihydrogen phosphate 5g / L, ammonium sulfate 0.7g / L, sodium chloride 1.1g / L, and the balance was water. The mixture was sterilized at 80°C for 15min to obtain a mixed fermentation liquid.

[0083] (2) The mixed fermentation broth was centrifuged at 8000 rpm for 15 min, the supernatant was collected, and the supernatant was subjected to alcohol precipitation treatment. Two volumes of anhydrous ethanol were added, and the mixture was allowed to stand at 4°C for 8 h and filtered to obtain a filtrate.

[0084] (3) The filtrate is concentrated and freeze-dried to obtain a crude fermentation product, which is then added with 2 times the volume of anhydrous ethanol, allowed to stand at 4° C. for 8 h, and filtered. The filtrate is concentrated and dried at 60° C. to obtain the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, which has a light yellow appearance.

[0085] Comparative Preparation Example 1-2

[0086] This preparation example provides a fermentation product of Aureobasidium pullulans, and the preparation method is as follows:

[0087] (1) Add budding short-stalked mold liquid (5×10 9 CFU / mL) was inoculated into a sterile culture medium at 3%, fermented at 28°C for 6 days, and sterilized at 80°C for 15 minutes to obtain a fermentation broth; the formula of the culture medium was: sucrose 120 g / L, magnesium sulfate 0.3 g / L, potassium dihydrogen phosphate 5 g / L, ammonium sulfate 0.7 g / L, sodium chloride 1.1 g / L, and the balance was water.

[0088] (2) The fermentation broth was centrifuged at 8000 rpm for 15 min, the supernatant was collected, and the supernatant was subjected to alcohol precipitation treatment. Two volumes of anhydrous ethanol were added, and the mixture was allowed to stand at 4°C for 8 h and filtered to obtain a filtrate.

[0089] (3) The filtrate is concentrated and freeze-dried to obtain a crude extracted fermentation product, which is then added with 2 times the volume of anhydrous ethanol, allowed to stand at 4° C. for 8 h, and filtered. The filtrate is concentrated and dried at 60° C. to obtain the budding Aureobasidium pullulans fermentation product, which has a dark reddish-brown appearance.

[0090] Preparation Example 2-1

[0091] This preparation example provides a Lactobacillus rhamnosus fermentation product, and the preparation method thereof is as follows:

[0092] (1) mixing jasmine powder and water in a mass ratio of 1:10 to obtain a jasmine powder aqueous solution;

[0093] (2) Add Lactobacillus rhamnosus culture (5×10 9 CFU / mL) was inoculated into a jasmine pollen aqueous solution at 2%, fermented at 35°C for 72h, and sterilized at 80°C for 15min to obtain a fermentation liquid;

[0094] (3) The fermentation broth was homogenized under high pressure at 60 MPa for 10 min, and then mixed with 10 times the mass of 75% ethanol aqueous solution and subjected to diafiltration extraction at 50° C. for 5 h to obtain an extract;

[0095] (4) filtering the extract, concentrating and drying the filtrate to obtain the jasmine fermentation product of Lactobacillus rhamnosus.

[0096] Preparation Example 2-2

[0097] This preparation example provides a Lactobacillus rhamnosus fermentation product, and the preparation method thereof is as follows:

[0098] (1) mixing jasmine powder and water in a mass ratio of 1:8 to obtain a jasmine powder aqueous solution;

[0099] (2) Add Lactobacillus rhamnosus culture (8×10 9 CFU / mL) was inoculated into a jasmine pollen aqueous solution at 1%, fermented at 40°C for 68h, and sterilized at 90°C for 10min to obtain a fermentation liquid;

[0100] (3) The fermentation broth was homogenized under high pressure at 70 MPa for 10 min, and then mixed with 8 times the mass of 65% ethanol aqueous solution and subjected to diafiltration extraction at 60° C. for 8 h to obtain an extract;

[0101] (4) filtering the extract, concentrating and drying the filtrate to obtain the jasmine fermentation product of Lactobacillus rhamnosus.

[0102] Preparation Example 2-3

[0103] This preparation example provides a Lactobacillus rhamnosus fermentation product, and the preparation method thereof is as follows:

[0104] (1) mixing jasmine powder and water in a mass ratio of 1:12 to obtain a jasmine powder aqueous solution;

[0105] (2) Add Lactobacillus rhamnosus culture (5×10 9 CFU / mL) was inoculated into a jasmine pollen aqueous solution at 3%, and fermented at 30°C for 90h, and sterilized at 90°C for 20min to obtain a fermentation liquid;

[0106] (3) The fermentation broth was homogenized under high pressure at 50 MPa for 20 min, and then mixed with 10 times the mass of 80% ethanol aqueous solution and subjected to diafiltration extraction at 45° C. for 8 h to obtain an extract;

[0107] (4) filtering the extract, concentrating and drying the filtrate to obtain the jasmine fermentation product of Lactobacillus rhamnosus.

[0108] Comparative Preparation Example 2-1

[0109] This preparation example provides a Lactobacillus rhamnosus fermentation product, and the preparation method thereof is different from that of Preparation Example 2-1 only in that the fermentation liquid is not subjected to cell wall breaking treatment, as follows:

[0110] (1) mixing jasmine powder and water in a mass ratio of 1:10 to obtain a jasmine powder aqueous solution;

[0111] (2) Add Lactobacillus rhamnosus culture (5×10 9 CFU / mL) was inoculated into a jasmine pollen aqueous solution at 2%, fermented at 35°C for 72h, and sterilized at 80°C for 15min to obtain a fermentation liquid;

[0112] (3) mixing the fermentation broth with 10 times the mass of a 75% ethanol aqueous solution and performing percolation extraction at 50° C. for 5 h to obtain an extract;

[0113] (4) filtering the extract, concentrating and drying the filtrate to obtain the jasmine fermentation product of Lactobacillus rhamnosus.

[0114] The weight percentages of the hyaluronic acid, collagen, lavender essential oil and bisabolol mentioned below are all calculated based on the actual content of the effective ingredients contained in the commercially available raw materials. The commercially available raw materials may also selectively contain any one or a combination of at least two of solvents, fillers, diluents, stabilizers, pH adjusters, antibacterial agents, antioxidants or impurities within the permitted range.

[0115] The collagen involved in the following embodiments and application examples is a product purchased from Shanxi Ruisi Biotechnology Co., Ltd. with the model number of recombinant human collagen powder; the lavender essential oil is a product purchased from Lvyuan Natural Spice Oil Refinery in Qingyuan District, Ji'an City with the model number of lavender essential oil; and the soybean lecithin is a product purchased from Shaanxi Qionghua Biotechnology Co., Ltd. with the model number of soybean lecithin.

[0116] Example 1

[0117] This embodiment provides a skin care composition, which is composed of the following components:

[0118] 35 parts of the co-fermentation product prepared in Preparation Example 1-1, 15 parts of the Lactobacillus rhamnosus fermentation product prepared in Preparation Example 2-1, 5 parts of hyaluronic acid, and 1 part of collagen.

[0119] Example 2

[0120] This embodiment provides a skin care composition, which is composed of the following components:

[0121] 25 parts of the co-fermentation product prepared in Preparation Example 1-2, 18 parts of the Lactobacillus rhamnosus fermentation product prepared in Preparation Example 2-2, 10 parts of hyaluronic acid, and 5 parts of collagen.

[0122] Example 3

[0123] This embodiment provides a skin care composition, which is composed of the following components:

[0124] 38 parts of the co-fermentation product prepared in Preparation Example 1-3, 12 parts of the Lactobacillus rhamnosus fermentation product prepared in Preparation Example 2-3, 3 parts of hyaluronic acid, and 3 parts of collagen.

[0125] Comparative Example 1

[0126] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in that an equal amount of the co-fermentation product prepared in Preparation Example 1-1 is replaced by an equal amount of the co-fermentation product prepared in Comparative Preparation Example 1-1, and other components and contents remain unchanged.

[0127] Comparative Example 2

[0128] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in that an equal amount of the co-fermentation product prepared in Preparation Example 1-1 is replaced by an equal amount of the co-fermentation product prepared in Comparative Preparation Example 1-2, and other components and contents remain unchanged.

[0129] Comparative Example 3

[0130] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in that an equal amount of the Lactobacillus rhamnosus fermentation product prepared in Preparation Example 2-1 is replaced by an equal amount of the Lactobacillus rhamnosus fermentation product prepared in Comparative Preparation Example 2-1, and other components and contents remain unchanged.

[0131] Comparative Example 4

[0132] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in the lack of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, and the reduced mass thereof is proportionally distributed to the mass of the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen.

[0133] Comparative Example 5

[0134] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in the lack of the jasmine fermentation product of Lactobacillus rhamnosus, and the reduced mass is proportionally distributed to the mass of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, hyaluronic acid, and collagen.

[0135] Comparative Example 6

[0136] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in the lack of hyaluronic acid, and the reduced mass thereof is proportionally distributed to the mass of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, the jasmine fermentation product of Lactobacillus rhamnosus, and collagen.

[0137] Comparative Example 7

[0138] This comparative example provides a skin care composition, the components of which differ from those of Example 1 only in the lack of collagen, and the reduced mass thereof is proportionally distributed to the masses of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, the jasmine fermentation product of Lactobacillus rhamnosus, and hyaluronic acid.

[0139] Application Example 1

[0140] This application example provides a cosmetic composition, the components of which are composed of the following:

[0141] 45 parts of the skin care composition of Example 1, 5 parts of lavender essential oil, 3 parts of bisabolol, 3 parts of soybean lecithin, and 30 parts of water.

[0142] The preparation method comprises the following steps: mixing the skin care composition with water at 35°C for 15 minutes to obtain a water phase; mixing lavender essential oil, bisabolol and soybean lecithin at 45°C for 15 minutes to obtain an oil phase; mixing the water phase and the oil phase, and emulsifying and homogenizing them at 35°C for 30 minutes to obtain the cosmetic composition.

[0143] Application Example 2-3

[0144] This application example provides two cosmetic compositions, the components of which differ from those of application example 1 only in that the skin care composition of example 1 is replaced by the skin care composition of examples 2-3 in equal amounts, and the other components and contents remain unchanged. The preparation method is similar to that of application example 1.

[0145] Application Example 4

[0146] This application example provides a cosmetic composition, the components of which differ from those of application example 1 only in that lavender essential oil is missing, and the reduced mass is supplemented by the skin care composition of example 1, while other components and contents remain unchanged. The preparation method is similar to that of application example 1.

[0147] Comparative Application Examples 1-7

[0148] This comparative application example provides seven cosmetic compositions, the components of which differ from those of Application Example 1 only in that the skin care composition of Example 1 is replaced by the skin care compositions of Comparative Examples 1-7 in equal amounts, and the other components and contents remain unchanged. The preparation method is the same as that of Application Example 1.

[0149] Test Example 1

[0150] Skin safety test:

[0151] The cosmetic composition products prepared in Application Examples 1-4 and Comparative Application Examples 1-7 were placed in a patch tester, and the patch tester with the test substance added was attached to the flexion side of the forearm of the test persons (20 subjects), and the patch was lightly pressed with the palm to evenly apply it on the skin for 24 hours; 30 minutes, 24 hours, and 48 hours after the removal of the test substance patch tester, the skin irritation and sensitization were observed according to Table 1 (Skin Reaction Grading Standard for Skin Closure Patch Test), and the observation results were recorded.

[0152] Table 1

[0153]

[0154] The results show that no adverse skin reactions occurred in the cosmetic composition products prepared in Application Examples 1-4 and Comparative Application Examples 1-7, indicating that the eye repair composition and eye mask involved in the present invention are safe and non-irritating to the skin.

[0155] Test Example 2

[0156] Skin barrier function test:

[0157] Normal skin has a strong barrier function that can prevent harmful substances from entering the skin. When the barrier function is damaged, the connection between keratinocytes becomes loose, forming pathways, causing water to be lost from the pathways. Therefore, when the skin barrier function is weakened, the transepidermal water loss increases, and vice versa. The better the skin's protective layer, the higher the moisture content and the lower the value of skin water loss TEWL. This test uses transepidermal water loss (TEWL) to judge the strength of the skin's barrier function.

[0158] 110 female subjects with dry and dehydrated facial skin, aged 20-45 years old, were selected and randomly divided into 11 groups, 10 people in each group. Three days before the test, no cosmetics were used on the face. Each group of subjects took 0.2g of the test sample (50% dilution of application examples 1-4 and comparative application examples 1-7) and applied it on the facial skin. The samples were used once in the morning and evening every day for 28 consecutive days. The samples were stopped on the 29th and 30th days. The TEWL values ​​of the face were measured by an instrument on the first day and the 31st day after the samples were used. The results were repeated 5 times and the average value was taken. The results are shown in Table 2.

[0159] Table 2

[0160] Group TEWL value on day 1 TEWL value on day 31 Rate of change (%) Application Example 1 18.99 5.72 -69.88 Application Example 2 18.37 6.57 -64.24 Application Example 3 17.73 7.13 -59.79 Application Example 4 19.35 7.72 -60.10 Comparative application example 1 20.62 9.26 -55.09 Comparative Application Example 2 18.74 9.78 -47.81 Comparative Application Example 3 19.36 8.69 -55.11 Comparative Application Example 4 17.95 11.51 -35.88 Comparative Application Example 5 18.56 10.84 -41.59 Comparative Application Example 6 19.33 8.36 -56.75 Comparative Application Example 7 19.37 9.98 -48.48

[0161] It can be seen from the data results in Table 2 that the skin care composition involved in the present invention has an excellent effect of improving the skin barrier. Compared with the data of Application Example 1 and Comparative Application Examples 4-7, it can be seen that the four components of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen have a synergistic effect in the above-mentioned effects; Compared with the data of Application Example 1 and Comparative Application Examples 1-3, it can be seen that the preparation process of the co-fermentation product and the jasmine fermentation product of Lactobacillus rhamnosus affects the effect of the product on improving the skin barrier.

[0162] Test Example 3

[0163] Skin elasticity test:

[0164] The skin elasticity tester (MPA580, CK Electronic GmH, Germany) was used to test the elasticity of the cheek skin of each group of subjects after using each group of products in Test Example 2. The test site was the zygomatic region. After thoroughly cleaning the face with a uniform cleansing product, the face was gently wiped dry with a paper towel. The room temperature was 22℃±2℃ and the relative humidity was 40±2%. The measurement was performed after waiting for 15 minutes. During the measurement, the probe repeated the measurement 3 times in the same tested area and the average value was taken. The test parameter is R2 (the ratio of the rebound amount Ua of the skin without negative pressure to the maximum stretch amount Uf when there is negative pressure). The closer R2 is to 1, the better the elasticity of the skin. The results are shown in Table 3.

[0165] The facial skin of each group of subjects after using each group of products in Test Example 2 was tested for wrinkles using the VISIA facial image analysis system (CANFIELD Technology, USA). After thoroughly cleaning the face with a uniform facial cleanser, gently wipe it dry with a paper towel. In a room with a room temperature of 22°C ± 2°C and a relative humidity of 40 ± 2%, wait for 15 minutes before taking a facial photo of the test subject with the VISIA facial image analyzer. The analysis area should try to avoid interference from reflective areas and hair. The face of the subject (including the front, left side, and right side) was photographed, and the absolute scores obtained were statistically analyzed to record the detection data of the wrinkles on the face of the subject. The wrinkle reduction rate (%) was calculated, and the results are shown in Table 3.

[0166] Table 3

[0167]

[0168]

[0169] It can be seen from the data results in Table 3 that the skin care composition involved in the present invention has excellent anti-wrinkle and skin elasticity enhancing effects. Compared with the data of Application Example 1 and Comparative Application Examples 4-7, it can be seen that the four components of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen have a synergistic effect in the above-mentioned effects; Compared with the data of Application Example 1 and Comparative Application Examples 1-3, it can be seen that the preparation process of the co-fermentation product and the jasmine fermentation product of Lactobacillus rhamnosus affects the above-mentioned effects of the product.

[0170] Test Example 4

[0171] Whitening efficacy test:

[0172] Prepare 5mM L-tyrosine solution (L-tyrosine can be dissolved with a small amount of hydrochloric acid and then fixed to volume with PBS buffer) and tyrosinase solution (diluted to 100U / mL with deionized water). Use a micropipette to transfer 0.4mL sample, 1mL 0.05mol / L phosphate buffer (pH=6.8) to the cuvette, then add 0.8mL 0.5mmol / L tyrosine solution, and finally add 0.40mL tyrosinase solution, mix quickly, measure the growth curve of the optical density value with a wavelength of 475nm over time under a constant temperature of 30°C, and obtain the inhibition rate from the slope of the curve. Measure the inhibition rate of the test samples with different concentrations (products of application examples 1-4 and comparative application examples 1-7) respectively, draw a straight line with concentration as the horizontal coordinate and inhibition rate as the vertical coordinate, and after obtaining the linear equation, calculate the product concentration when the inhibition rate is 50%, which is IC50. The results are shown in Table 4.

[0173] Table 4

[0174] Group IC50(mg / mL) Application Example 1 1.16 Application Example 2 1.09 Application Example 3 1.21 Application Example 4 1.33 Comparative application example 1 1.52 Comparative Application Example 2 1.89 Comparative Application Example 3 1.68 Comparative Application Example 4 2.14 Comparative Application Example 5 2.05 Comparative Application Example 6 1.69 Comparative Application Example 7 1.72

[0175] It can be seen from the data results in Table 4 that the skin care composition involved in the present invention has an excellent tyrosinase inhibition effect, that is, a whitening effect. Comparing the data of Application Example 1 with those of Comparative Application Examples 4-7, it can be seen that the four components of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen have a synergistic effect in the above-mentioned efficacy; Comparing the data of Application Example 1 with those of Comparative Application Examples 1-3, it can be seen that the preparation process of the co-fermentation product and the jasmine fermentation product of Lactobacillus rhamnosus affects the above-mentioned effects of the product.

[0176] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0177] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0178] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A co-fermentation product of Aureobasidium pullulans and Beauveria bassiana, characterized in that: The co-fermentation product of Aureobasidium pullulans and Beauveria bassiana is prepared by a preparation method comprising the following steps: (1) inoculating a Beauveria bassiana bacterial liquid into a sterile culture medium for fermentation to obtain a Beauveria bassiana fermentation liquid; (2) inoculating the budding Aureobasidium pullulans liquid into the Beauveria bassiana fermentation liquid for fermentation, sterilization, and obtaining a mixed fermentation liquid; (3) centrifuging the mixed fermentation broth, collecting the supernatant, subjecting the supernatant to alcohol precipitation, and filtering to obtain a filtrate; (4) The filtrate is concentrated and dried to obtain the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana.

2. The co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to claim 1, characterized in that: The number of live bacteria in the Beauveria bassiana liquid is (1-5)×10 8 CFU / mL, inoculum size 0.5-2%; Preferably, the formula of the culture medium is: sucrose 100-150 g / L, magnesium sulfate 0.1-0.5 g / L, dipotassium hydrogen phosphate 1-10 g / L, ammonium sulfate 0.5-1 g / L, sodium chloride 0.5-1.5 g / L, and the balance is water; Preferably, the fermentation in step (1) is carried out at 24-28°C for 1-4 hours.

3. The co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to claim 1 or 2, characterized in that: The viable bacteria count of the budding short-stalked mold liquid is (3-10)×10 9 CFU / mL, inoculum size 3-5%; Preferably, the fermentation in step (2) is carried out at 25-30° C. for 20-170 h; Preferably, the sterilization conditions in step (2) are 70-90° C. for 10-20 min.

4. The co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to any one of claims 1 to 3, characterized in that: Step (3) centrifugation at 6000-9000 rpm for 10-20 min; Preferably, the alcohol precipitation treatment is to add 1-3 times the volume of 75-100% ethanol or ethanol aqueous solution and let it stand at 2-10°C for 5-12h; Preferably, after the drying process in step (4), the following steps are repeated 1-3 times: The product is subjected to alcohol precipitation treatment, filtered to obtain a filtrate, and the filtrate is concentrated and dried.

5. A skin care composition, characterized in that: The components of the skin care composition include: the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to any one of claims 1 to 4, the jasmine fermentation product of Lactobacillus rhamnosus, hyaluronic acid, and collagen.

6. The skin care composition according to claim 5, characterized in that The jasmine fermentation product of Lactobacillus rhamnosus is prepared by a preparation method comprising the following steps: (1) mixing jasmine powder with water to obtain a jasmine powder aqueous solution; (2) inoculating a Lactobacillus rhamnosus bacterial liquid into a jasmine pollen aqueous solution for fermentation and sterilization to obtain a fermentation liquid; (3) crushing the fermentation liquid, and then mixing it with an ethanol aqueous solution to perform diafiltration extraction to obtain an extract; (4) filtering the extract, concentrating and drying the filtrate to obtain the jasmine fermentation product of Lactobacillus rhamnosus.

7. The skin care composition according to claim 6, characterized in that The mass ratio of the jasmine pollen to water is 1:(5-15); Preferably, the viable count of the Lactobacillus rhamnosus bacterial solution is (5-8)×10 9 CFU / mL, inoculum size 1-3%; Preferably, the fermentation is carried out at 10-45°C for 20-90h; Preferably, the sterilization condition is 70-90°C for 10-20 min; Preferably, the crushing process is high pressure homogenization at 50-70 MPa for 5-15 min; Preferably, the volume fraction of the ethanol aqueous solution is 60-80%, and the mass is 5-10 times that of the fermentation broth; Preferably, the percolation extraction is carried out at 30-60°C for 3-8h.

8. The skin care composition according to claim 5 or 6, characterized in that The components of the skin care composition include, by mass: 25-38 parts of the co-fermentation product of Aureobasidium pullulans and Beauveria bassiana according to any one of claims 1 to 4, 12-18 parts of jasmine fermentation product of Lactobacillus rhamnosus, 2-10 parts of hyaluronic acid, and 1-10 parts of collagen.

9. A cosmetic composition having whitening, anti-aging and barrier repair effects, characterized in that: The cosmetic composition comprises the skin care composition according to any one of claims 5 to 8, a skin conditioner, an emulsifier and water; Preferably, the skin conditioning agent comprises lavender essential oil and / or bisabolol; Preferably, the emulsifier comprises soy lecithin.

10. The method for preparing the cosmetic composition according to claim 9, characterized in that: The preparation method comprises the following steps: The skin care composition is mixed with water to obtain a water phase; the skin conditioner is mixed with an emulsifier to obtain an oil phase; the water phase and the oil phase are mixed, emulsified and homogenized to obtain the cosmetic composition.

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