Asparagus cochinchinensis fermented extract, preparation method and application thereof, soothing and anti-aging cosmetic composition and cosmetics

Abstract

The application belongs to the technical field of cosmetic preparation, and particularly relates to a kind of asparagus fermentation extract and its preparation method and application, soothing anti-aging cosmetic composition and cosmetics. The preparation method comprises the following steps: the yeast liquid of Saccharomyces cerevisiae with preservation number CCTCC KY 2008613 is fermented with asparagus extract as raw material, and the asparagus fermentation extract is obtained. The asparagus fermentation extract provided by the application can not only maximize the natural characteristics of asparagus, but also further enhance the efficacy of asparagus in cosmetics through the metabolic reaction of Saccharomyces cerevisiae, so that the cosmetics have significant antioxidant, anti-aging and soothing skin ability. The preparation method of the asparagus fermentation extract is simple, green and environmentally friendly. The whole fermentation and enzymolysis process is carried out at low temperature, which helps to protect the stability of active ingredients, saves energy and reduces production cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetic preparation, and in particular relates to an asparagus cochinchinensis fermentation extract, a preparation method and application thereof, a soothing and anti-aging cosmetic composition, and cosmetics. Background Art

[0002] Asparagus cochinchinensis is a plant rich in active ingredients, including saponins, polysaccharides, amino acids, and various antioxidants. Due to its significant moisturizing, antioxidant, and anti-inflammatory properties, it has broad potential for application in cosmetics. Asparagus extract can help improve skin hydration, reduce free radical damage, and delay skin aging. With increasing consumer demand for natural and safe cosmetics, cosmetic manufacturers are increasingly demanding ingredients derived from natural plants. Asparagus, a traditional Chinese herbal medicine, has attracted attention from the cosmetics industry for its mild and natural properties. However, the direct use of asparagus extract in cosmetics may limit the absorption and bioavailability of active ingredients.

[0003] Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) is widely used in the food industry due to its strong fermentation capacity, short growth cycle, potential low cost, and ease of large-scale cultivation. Saccharomyces cerevisiae's metabolites have a strong affinity for the skin, not only enhancing skin cell vitality and metabolism but also significantly repairing, moisturizing, whitening, and delaying skin aging. Existing research shows that extracts prepared from plants fermented with Saccharomyces cerevisiae can effectively enhance the activity of plant components, enhancing their antioxidant, moisturizing, and anti-inflammatory properties.

[0004] For example, Chinese invention patent publication number CN110772460A discloses a preparation method and application of a Rhodiola rosea fermentation extract, which comprises the following steps: taking Rhodiola rosea roots, drying, crushing, and sieving to obtain Rhodiola rosea root powder; adding water to the Rhodiola rosea root powder and mixing evenly, then adding enzyme for enzymolysis, enzymolysis to the end point, and then heating to inactivate the enzyme to obtain Rhodiola rosea root powder enzymolysis solution; inoculating yeast seed liquid into a fermentation culture medium containing the Rhodiola rosea root powder enzymolysis solution and fermenting to obtain a fermentation liquid; removing impurities and sterilizing the fermentation liquid to obtain the Rhodiola rosea fermentation extract. The Rhodiola rosea fermentation extract prepared by the method provided by the invention has good whitening and spot-removing, anti-oxidation and anti-inflammatory effects.

[0005] However, scientific research on the potential efficacy of products obtained by fermentation with Saccharomyces cerevisiae in a culture medium enriched with Asparagus extract in the cosmetic field is still relatively limited.

[0006] Therefore, there is an urgent need in the art to provide a natural, low-irritating cosmetic raw material with multiple functions such as moisturizing, anti-aging and soothing, and to provide a new raw material preparation method and application scheme for the cosmetic industry. SUMMARY

[0007] The present application provides a kind of asparagus fermentation extract and its preparation method and application, soothing anti-aging cosmetic composition and cosmetics to solve the problems of the prior art, which can improve the stability of effective components in conventional asparagus extract, and the problem of severe loss of effective components, thereby improving the comprehensive performance of asparagus cosmetic raw materials.

[0008] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0009] A preparation method of asparagus fermentation extract, comprising the following steps: fermenting the bacterial liquid of Saccharomyces cerevisiae with preservation number CCTCC KY2008613 Saccharomyces cerevisiae ) with asparagus extract as raw material, and obtaining it directly.

[0010] Preferably, the preparation process of the bacterial liquid comprises inoculating Saccharomyces cerevisiae into YPD liquid medium and activating it at 25-35℃ for 10-14 h, and obtaining it directly.

[0011] Preferably, the preparation process of the asparagus extract comprises: first placing asparagus in water, adding pectinase and cellulase for enzymolysis, then centrifuging and filtering, adding anhydrous glucose to the filtrate, sterilizing, and obtaining it directly.

[0012] Preferably, the solid-liquid ratio of asparagus to water is 1 g:8-12 mL.

[0013] More preferably, the solid-liquid ratio of asparagus to water is 1 g:9-10 mL.

[0014] Further preferably, the solid-liquid ratio of asparagus to water is 1 g:10 mL.

[0015] Preferably, the total amount of pectinase and cellulase added is 1-2% of the mass of asparagus.

[0016] Preferably, the amount of cellulase added is 0.5-1.5% of the mass of asparagus.

[0017] More preferably, the amount of cellulase added is 0.8-1.2% of the mass of asparagus.

[0018] Further preferably, the amount of cellulase added is 1% of the mass of asparagus.

[0019] Preferably, the enzymolysis temperature is 40-60℃, the enzymolysis time is 1.5-3 h, and the enzymolysis needs to be stirred at 800-1200 rpm.

[0020] More preferably, the enzymolysis temperature is 50℃, the enzymolysis time is 2 h, and the enzymolysis needs to be stirred at 1000 rpm.

[0021] Preferably, the centrifugation speed is 1800-2200 rpm, and the centrifugation time is 12-18 min.

[0022] More preferably, the centrifugation speed is 2000 rpm, and the centrifugation time is 15 min.

[0023] Preferably, the mass concentration of anhydrous glucose in the filtrate is 0.8-1.2%.

[0024] More preferably, the mass concentration of anhydrous glucose in the filtrate is 1%.

[0025] Further preferably, the mass concentration of anhydrous glucose in the filtrate is 1.0%.

[0026] Preferably, the volume concentration of the Saccharomyces cerevisiae bacterial solution in the asparagus extract is 0.5-2%.

[0027] More preferably, the volume concentration of the Saccharomyces cerevisiae bacterial solution in the asparagus extract is 1.5%.

[0028] Preferably, the fermentation process comprises inoculating the Saccharomyces cerevisiae bacterial solution into the asparagus extract, adjusting the initial pH to 6-7, and then fermenting at a speed of 120-180 rpm and a temperature of 25-35℃ for 36-48 h.

[0029] More preferably, the fermentation process comprises inoculating the Saccharomyces cerevisiae bacterial solution into the asparagus extract, adjusting the initial pH to 7, and then fermenting at a speed of 150 rpm and a temperature of 30℃ for 40 h.

[0030] Preferably, the treatment comprises sterilizing at 120-122℃ for 28-32 min, centrifuging at 1500-2500 rpm for 10-20 min, and then filtering.

[0031] More preferably, the treatment comprises sterilizing at 121℃ for 30 min, centrifuging at 2000 rpm for 15 min, and then filtering.

[0032] The application also provides the asparagus fermentation extract prepared by the above preparation method.

[0033] The application also provides a soothing anti-aging cosmetic composition, comprising magnesium ascorbyl phosphate and the above-mentioned asparagus ferment extract.

[0034] Preferably, the mass percentage of the magnesium ascorbyl phosphate in the cosmetic composition is 0.2-1.5%.

[0035] More preferably, the mass percentage of the magnesium ascorbyl phosphate in the cosmetic composition is 0.2%, 0.3%, 0.4%, 0.5% or 1.5%.

[0036] Further preferably, the mass percentage of the magnesium ascorbyl phosphate in the cosmetic composition is 1.5%.

[0037] Preferably, the mass percentage of the asparagus ferment extract in the cosmetic composition is 10-30%.

[0038] More preferably, the mass percentage of the asparagus ferment extract in the cosmetic composition is 10%, 15%, 20%, 25% or 30%.

[0039] Further preferably, the mass percentage of the asparagus ferment extract in the cosmetic composition is 30%.

[0040] The application also provides a soothing anti-aging cosmetic, comprising the above-mentioned cosmetic composition.

[0041] Preferably, the mass percentage of the cosmetic composition in the cosmetic is 10-50%.

[0042] More preferably, the mass percentage of the cosmetic composition in the cosmetic is 10.2%, 15.3%, 20.4%, 25.5% or 31.5%.

[0043] Further preferably, the mass percentage of the cosmetic composition in the cosmetic is 31.5%.

[0044] Preferably, the cosmetic comprises any one of toner, serum, eye cream, emulsion, cream, mask, scalp serum, shampoo, body milk, sunscreen and shower gel.

[0045] The application also provides the use of the asparagus ferment extract prepared by the above-mentioned preparation method in the preparation of a cosmetic with the effects of anti-oxidation, anti-inflammation, soothing and anti-aging.

[0046] The present application utilizes Saccharomyces cerevisiae to ferment asparagus, combines the metabolic advantages of the yeast, and further decomposes and transforms the active ingredients in asparagus to obtain a novel asparagus fermented cosmetic raw material. Yeast fermentation can release a variety of metabolites, including amino acids, polysaccharides, and polyphenols, etc. These components, in combination with the unique plant active ingredients of asparagus, make the prepared cosmetic raw material have remarkable effects in terms of antioxidant, anti-aging, anti-inflammatory, and skin soothing. The small molecule substances generated during fermentation can be more easily absorbed by the skin, improving the bioavailability of asparagus active ingredients and making the product more effective.

[0047] Using asparagus fermented extract as a cosmetic raw material in cosmetics can not only exert the natural activity of asparagus, but also take advantage of the additional effects produced by yeast fermentation, allowing the components in skin care products to interact and achieve a 1+1>2 effect. After asparagus fermented extract is applied as a raw material in skin care products, not only does it make the skin moist and smooth, but it also effectively resists the invasion of external pollution on the skin, delays skin aging, provides soothing and anti-inflammatory effects, and has the effect of improving skin quality and improving skin health.

[0048] Compared with the prior art, the present application has the following beneficial effects:

[0049] (1) The asparagus fermented extract provided by the present application not only maximizes the retention of the natural properties of asparagus, but also further enhances the efficacy of asparagus in cosmetics through the metabolic reaction of Saccharomyces cerevisiae, making the cosmetic have remarkable antioxidant, anti-aging, and skin soothing ability.

[0050] (2) The preparation method of the asparagus fermented extract provided by the present application is simple, green and environmentally friendly, avoiding the addition of exogenous chemicals, and simultaneously using natural enzymes in the basic medium for biological transformation. The entire fermentation and enzymolysis process is carried out at low temperature (below 40℃), which helps to protect the stability of active ingredients, saves energy and reduces production costs. In addition, the asparagus fermented extract is light yellow-green in color, meeting the natural and pure requirements of cosmetic raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The asparagus fermented extract prepared in Example 1 and Comparative Example 1 was tested for its toxicity to RAW264.7 cells at different dilution ratios.

[0052] Figure 2 The asparagus fermented extract prepared in Example 1 and Comparative Example 1 was tested for its effect on the expression of inflammatory factors. IL-6

[0053] Figure 3 The asparagus fermented extract prepared in Example 1 and Comparative Example 1 was tested for its antioxidant capacity.​

[0054] Figure 4 Toxic effects of cosmetic compositions numbered 1-15 on RAW264.7 cells.

[0055] Figure 5 Effects of cosmetic compositions numbered 1-15 on expression of inflammatory factors IL-6 .

[0056] Figure 6 Effects of cosmetics prepared for Examples 17-21 and Comparative Examples 6-10 on zebrafish neutrophil aggregation.

[0057] Figure 7 Effects of cosmetic compositions numbered 1-15 on expression of human fibroblast type I collagen genes COL1A1 and COL1A2 and elastin genes ELNA (where A is the effect on expression of the gene COL1A1 , B is the effect on expression of the gene COL1A2 , and C is the effect on expression of the gene ELNA ).

[0058] Figure 8 Effects of cosmetics prepared for Examples 17-21 and Comparative Examples 6-10 on average lifespan of Caenorhabditis elegans.

[0059] Figure 9 Effects of cosmetics prepared for Examples 17-21 and Comparative Examples 6-10 on maximum lifespan of Caenorhabditis elegans.

[0060] Significance analysis:

[0061] Figure 1 , Figure 4 and Figure 7 , ns means P > 0.05 compared to the control group.

[0062] Figure 2 , Figure 5 and Figure 7 , ns means P > 0.05 compared to the model group, * means P <0.05, ** means P <0.01, *** means P <0.001; and ### means P <0.001 compared to the control group.

[0063] Figure 6 , Figure 8 and Figure 9 , a means Example 17 compared to Comparative Example 6,P <0.001; b represents Example 18 compared with Comparative Example 7, P <0.001; c represents Example 19 compared with Comparative Example 8, P <0.001; d represents that Example 20 is compared with Comparative Example 9, P <0.001; e represents that Example 21 is compared with Comparative Example 10, P <0.001. DETAILED DESCRIPTION

[0064] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which asparagus cochinchinensis is produced in Neijiang City, Sichuan Province; Saccharomyces cerevisiae (deposit number CCTCC KY 2008613) and Pichia pastoris (deposit number CCTCC KY 2008612) are both purchased from China Center for Type Culture Collection; YPD liquid culture medium is purchased from Beijing Biolab Technology Co., Ltd.; DMEM culture medium and serum-free low-glucose DMEM culture medium are both purchased from Thermo Fisher Scientific; pectinase (CAS No. 9032-75-1) and cellulase (CAS No. 9001-73-4) are both purchased from Jiangsu Jiujia Biotechnology Co., Ltd.; avocado extract is purchased from Fujian Shengshi Jiatai Biotechnology Co., Ltd.; Centella asiatica extract is purchased from Shaanxi Xinyanghe Biotechnology Co., Ltd.; green tea extract is purchased from Xi'an Tianbao Biotechnology Co., Ltd.; dioscin, content 9 8%; ethanol, content ≥99.5%; concentrated sulfuric acid, content 99.99%; mouse monocyte / macrophage cells (RAW264.7 cells) were purchased from Shanghai Chuanqiu Biotechnology Co., Ltd.; SOD activity detection kit was purchased from Beijing Solebeau Technology Co., Ltd.; human dermal fibroblast primary cells (generation P2) were purchased from Guangdong Boxi Biotechnology Co., Ltd.; wild-type AB strain zebrafish were purchased from China National Zebrafish Resource Center; N2 wild-type Caenorhabditis elegans were purchased from the Caenorhabditis elegans Genetics Center; nematode growth medium was purchased from Shandong Top Bioengineering Co., Ltd.; Escherichia coli OP50 was purchased from Hubei APT Bioengineering Co., Ltd.

[0065] Example 1

[0066] A method for preparing an asparagus fermentation extract comprises the following steps:

[0067] (1) First, the brewer's yeast with the deposit number of CCTCC KY 2008613 ( Saccharomyces cerevisiae ) were inoculated into YPD liquid culture medium and activated at 30℃ for 12 h to obtain the bacterial liquid of Saccharomyces cerevisiae.

[0068] (2) Then cut the asparagus off both ends to take the middle, evenly cut into small pieces, according to the ratio of 1:10, 50 g of asparagus is placed in 500 mL of deionized water, 0.5% of pectinase and 1% of cellulase of the mass of asparagus are added for enzymolysis, the enzymolysis is stirred at 1000 rpm, the enzymolysis temperature is 50°C, and the time is 2 h. After the enzymolysis is completed, filter with gauze, centrifuge at 2000 rpm for 15 min, take the supernatant and cool, filter, add 1% of anhydrous glucose of the mass of the solution to the filtrate, sterilize at high temperature, and obtain the asparagus extract.

[0069] (3) Finally, the yeast liquid is inoculated in the asparagus extract at a volume concentration of 1.5%, the initial pH is adjusted to 7, and is placed in a shaker at 150 rpm and 30°C for fermentation for 40 h. After the fermentation is completed, the fermentation product is sterilized at 121°C for 30 min, centrifuged at 2000 rpm for 15 min, and filtered, and the asparagus fermentation extract is obtained.

[0070] Example 2

[0071] A preparation method of an asparagus fermentation extract, compared with example 1, the only difference is that the addition amount of pectinase is 0.5% of the mass of asparagus, and the cellulase is 0.5% of the mass of asparagus.

[0072] Example 3

[0073] A preparation method of an asparagus fermentation extract, compared with example 1, the only difference is that the addition amount of pectinase is 0.5% of the mass of asparagus, and the cellulase is 1.5% of the mass of asparagus.

[0074] Example 4

[0075] A preparation method of an asparagus fermentation extract, compared with example 1, the only difference is that the enzymolysis temperature in step (2) is 40°C.

[0076] Example 5

[0077] A preparation method of an asparagus fermentation extract, compared with example 1, the only difference is that the enzymolysis temperature in step (2) is 60°C.

[0078] Example 6

[0079] A preparation method of an asparagus fermentation extract, compared with example 1, the only difference is that the enzymolysis time in step (2) is 1.5 h.

[0080] Example 7

[0081] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the enzymolysis time is 3 h in step (2).

[0082] Example 8

[0083] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the rotation speed is 800 rpm in step (2).

[0084] Example 9

[0085] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the rotation speed is 1200 rpm in step (2).

[0086] Example 10

[0087] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the volume concentration of saccharomyces cerevisiae bacteria solution in asparagus extract solution is 0.5% in step (3).

[0088] Example 11

[0089] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the volume concentration of saccharomyces cerevisiae bacteria solution in asparagus extract solution is 2% in step (3).

[0090] Example 12

[0091] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the initial pH of fermentation is 6 in step (3).

[0092] Example 13

[0093] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the rotation speed of the shaker is 120 rpm in step (3).

[0094] Example 14

[0095] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the rotation speed of the shaker is 180 rpm in step (3).

[0096] Example 15

[0097] A preparation method of asparagus fermentation extract solution, compared with example 1, the only difference is that the fermentation time is 36 h in step (3).

[0098] Example 16

[0099] A preparation method of asparagus ferment extract, the difference from example 1 is only that the fermentation time in step (3) is 48 h.

[0100] Example 17

[0101] A soothing anti-aging serum, the ingredient content is shown in Table 1 in mass percentage.

[0102] Table 1 A soothing anti-aging serum formula

[0103]

[0104] The preparation process of the serum is as follows:

[0105] First, mix glycerin, sodium hyaluronate, panthenol and purified water; then add the asparagus ferment extract prepared in example 1, niacinamide, magnesium ascorbyl phosphate and vitamin E in turn, and stir well to ensure uniform dispersion of the ingredients; then slowly add recombinant type III human collagen, stir until completely dissolved; finally add phenoxyethanol and mix well.

[0106] Example 18

[0107] A soothing anti-aging eye cream, the ingredient content is shown in Table 2 in mass percentage.

[0108] Table 2 A soothing anti-aging eye cream formula

[0109]

[0110] The preparation process of the eye cream is as follows:

[0111] First, heat jojoba oil and polysorbate-60 to complete melting in a 60℃ water bath to obtain an oil phase; then mix glycerin, sodium hyaluronate, caffeine and purified water, and stir evenly, and heat in a water bath to 60℃ to obtain an aqueous phase; gradually add the oil phase to the aqueous phase and continue to stir to form a uniform emulsion, cool to 40℃, then add the asparagus ferment extract prepared in example 1, magnesium ascorbyl phosphate, shea extract and ceramide, and stir evenly; finally add phenoxyethanol and mix well.

[0112] Example 19

[0113] A soothing anti-aging moisturizing emulsion, the ingredient content is shown in Table 3 in mass percentage.

[0114] Table 3 A soothing anti-aging moisturizing emulsion formula

[0115]

[0116] The preparation process of the moisturizing emulsion is as follows: first, heat the jojoba oil, squalane, ceramide, sorbitan stearate and cetylstearyl alcohol to 85℃, stir until completely dissolved to obtain the oil phase; then, stir the glycerol, carbomer, panthenol and purified water uniformly, heat to 85℃ in a water bath to obtain the water phase; gradually add the oil phase to the water phase and homogenously stir to form a uniform emulsion, cool to 40℃, then add the asparagus ferment extract prepared in Example 1 and magnesium ascorbyl phosphate, triethanolamine, stir uniformly; finally, add phenoxyethanol and ethylhexylglycerin, mix uniformly to obtain the product.

[0117] Example 20

[0118] A soothing anti-aging sunscreen, the ingredient content is shown in Table 4 in mass percentage.

[0119] Table 4 Formulation of a soothing anti-aging sunscreen

[0120]

[0121] The preparation process of the sunscreen is as follows:

[0122] First, heat the titanium dioxide, zinc oxide, jojoba oil, polysorbate-80 and cetylstearyl alcohol to 80℃ and grind uniformly to obtain the oil phase; then, stir the glycerol, nicotinamide, purified water and carbomer uniformly, heat to 85℃ in a water bath to obtain the water phase; gradually add the oil phase to the water phase and homogenously and continuously stir to form a uniform emulsion, cool to 40℃, then add the asparagus ferment extract prepared in Example 1 and magnesium ascorbyl phosphate, stir uniformly; finally, add phenoxyethanol, mix uniformly to obtain the product.

[0123] Example 21

[0124] A soothing anti-aging repair mask, the ingredient content is shown in Table 5 in mass percentage.

[0125] Table 5 Formulation of a soothing anti-aging repair mask

[0126]

[0127] The preparation process of the repair mask is as follows:

[0128] First, stir the glycerol, panthenol and purified water uniformly, heat to 60℃ in a water bath to obtain the water phase; add vitamin E to the oil phase, cool to 40℃, then add the asparagus ferment extract prepared in Example 1, magnesium ascorbyl phosphate, centella asiatica extract, green tea extract and dipotassium glycyrrhizinate in sequence, stir uniformly; finally, add the preservative phenoxyethanol, mix uniformly to obtain the product.

[0129] Comparative Example 1

[0130] The difference compared with Example 1 is that the fermentation medium does not contain the asparagus extract.

[0131] A preparation method of an asparagus extract, comprising the following steps:

[0132] The asparagus is cut into small pieces in the middle, 50 g of the asparagus is placed in 500 mL of deionized water according to a solid-liquid ratio of 1:10, 2% of pectinase and 1% of cellulase of the mass of the asparagus are added for enzymolysis, the enzymolysis is stirred at a speed of 1000 rpm, the enzymolysis temperature is 50°C, and the enzymolysis time is 2 h. After the enzymolysis, the asparagus is filtered with gauze, centrifuged at a speed of 2000 rpm for 15 min, the supernatant is taken out and cooled, and the filtrate is filtered under suction. 1% of anhydrous glucose of the mass of the solution is added to the filtrate, high-temperature sterilization is performed, and the asparagus extract is obtained.

[0133] Comparative Example 2

[0134] The difference compared with Example 1 is that the fermentation medium does not contain the asparagus extract.

[0135] A preparation method of a Saccharomyces cerevisiae bacterial solution, comprising the following steps:

[0136] The Saccharomyces cerevisiae bacteria with a preservation number of CCTCC KY 2008613 are inoculated in YPD liquid medium, activated at 30°C for 12 h, and the Saccharomyces cerevisiae bacterial solution is obtained. Saccharomyces cerevisiae

[0137] The Saccharomyces cerevisiae bacterial solution is inoculated in YPD liquid medium at a volume concentration of 1.5%, and placed in a shaker at 150 rpm and 30°C for fermentation for 40 h. After the fermentation, the fermentation product is sterilized at 121°C for 30 min, centrifuged at 2000 rpm for 15 min, and filtered under suction, and the Saccharomyces cerevisiae bacterial solution is obtained.

[0138] Comparative Example 3

[0139] The difference compared with Example 1 is that the Saccharomyces cerevisiae bacteria are replaced by Pichia pastoris bacteria.

[0140] A preparation method of an asparagus fermentation extract, comprising the following steps:

[0141] (1) The Pichia pastoris bacteria with a preservation number of CCTCC KY 2008612 are inoculated in YPD liquid medium, activated at 30°C for 12 h, and the Pichia pastoris bacterial solution is obtained. Pichia pastoris

[0142] ​​(2) Cut off both ends of the asparagus and take the middle, cut into small pieces evenly, and place 50 g of asparagus in 500 mL of deionized water at a material-liquid ratio of 1:10. Add 0.5% pectinase and 1% cellulase to the asparagus for enzymatic hydrolysis. Stir at 1000 rpm, keep the enzymatic hydrolysis temperature at 50°C, and perform the enzymatic hydrolysis for 2 h. After the enzymatic hydrolysis is completed, filter with gauze and centrifuge at 2000 rpm for 15 min. Remove the supernatant, cool it, and filter it. Add 1% anhydrous glucose to the filtrate and sterilize it at high temperature to obtain the asparagus extract.

[0143] (3) Finally, the Pichia pastoris culture was inoculated into the Asparagus cochinchinensis extract at a volume concentration of 1.5%, and the initial pH was adjusted to 7. The culture was then shaken at 150 rpm and 30°C for 40 h. After the fermentation, the fermentation product was sterilized at 121°C for 30 min, centrifuged at 2000 rpm for 15 min, and filtered.

[0144] Comparative Example 4

[0145] Compared with Example 1, the only difference is that the initial pH of the fermentation is 5.5.

[0146] A method for preparing an asparagus fermentation extract comprises the following steps:

[0147] (1) First, the brewer's yeast with the deposit number of CCTCC KY 2008613 ( Saccharomyces cerevisiae ) were inoculated into YPD liquid culture medium and activated at 30°C for 12 h to obtain the bacterial liquid of Saccharomyces cerevisiae.

[0148] (2) Cut off both ends of the asparagus and take the middle, cut into small pieces evenly, and place 50 g of asparagus in 500 mL of deionized water at a material-liquid ratio of 1:10. Add 0.5% pectinase and 1% cellulase to the asparagus for enzymatic hydrolysis. Stir at 1000 rpm, keep the enzymatic hydrolysis temperature at 50°C, and perform the enzymatic hydrolysis for 2 h. After the enzymatic hydrolysis is completed, filter with gauze and centrifuge at 2000 rpm for 15 min. Remove the supernatant, cool it, and filter it. Add 1% anhydrous glucose to the filtrate and sterilize it at high temperature to obtain the asparagus extract.

[0149] (3) Finally, the yeast culture of Saccharomyces cerevisiae was inoculated into the asparagus extract at a volume concentration of 1.5%, and the initial pH was adjusted to 5.5. The culture was then placed in a shaker at 150 rpm and 30°C for 40 h. After the fermentation, the fermentation product was sterilized at 121°C for 30 min, centrifuged at 2000 rpm for 15 min, and filtered.

[0150] Comparative Example 5

[0151] Comparative Example 5 is the same as Example 1 except that the enzymatic hydrolysis temperature is 30°C.

[0152] A preparation method of an asparagus fermentation extract, comprising the following steps:

[0153] (1) First, the Saccharomyces cerevisiae bacteria with the preservation number CCTCC KY 2008613 are inoculated in a YPD liquid culture medium and activated at 30°C for 12 h to obtain a Saccharomyces cerevisiae bacteria liquid. Saccharomyces cerevisiae (2) Then, the asparagus is cut into small pieces in the middle, 50 g of the asparagus is placed in 500 mL of deionized water at a solid-liquid ratio of 1:10, 0.5% of pectinase and 1% of cellulase are added to the asparagus for enzymatic hydrolysis, the enzymatic hydrolysis is stirred at a speed of 1000 rpm, the enzymatic hydrolysis temperature is 30°C, and the enzymatic hydrolysis time is 2 h. After the enzymatic hydrolysis is completed, the asparagus is filtered with gauze, centrifuged at a speed of 2000 rpm for 15 min, the supernatant is taken out and cooled, and filtered, 1% of anhydrous glucose is added to the filtrate, high-temperature sterilization is performed, and an asparagus extract is obtained.

[0154] (3) Finally, the Saccharomyces cerevisiae bacteria liquid is inoculated in the asparagus extract at a volume concentration of 1.5%, the initial pH is adjusted to 7, and the fermentation is performed in a shaker at 150 rpm and 30°C for 40 h. After the fermentation is completed, the fermentation product is sterilized at 121°C for 30 min, centrifuged at 2000 rpm for 15 min, and filtered, and the asparagus fermentation extract is obtained.

[0155] (3) Finally, the Saccharomyces cerevisiae bacteria liquid is inoculated in the asparagus extract at a volume concentration of 1.5%, the initial pH is adjusted to 7, and the fermentation is performed in a shaker at 150 rpm and 30°C for 40 h. After the fermentation is completed, the fermentation product is sterilized at 121°C for 30 min, centrifuged at 2000 rpm for 15 min, and filtered, and the asparagus fermentation extract is obtained.

[0156] Comparative Example 6

[0157] A soothing anti-aging serum, which is the same as Example 17 except that equal amounts of purified water are used instead of the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1.

[0158] Comparative Example 7

[0159] A soothing anti-aging eye cream, which is the same as Example 18 except that equal amounts of purified water are used instead of the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1.

[0160] Comparative Example 8

[0161] A soothing anti-aging moisturizing emulsion, which is the same as Example 19 except that equal amounts of purified water are used instead of the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1.

[0162] Comparative Example 9

[0163] A soothing anti-aging sunscreen formula, compared with Example 20, the only difference is that the asparagus fermented extract prepared in Example 1 and magnesium ascorbyl phosphate are replaced with an equal amount of purified water.

[0164] Comparative Example 10

[0165] A soothing anti-aging repair mask, compared with Example 21, the only difference is that the asparagus fermented extract prepared in Example 1 and magnesium ascorbyl phosphate are replaced with an equal amount of purified water.

[0166] Test Example 1 Evaluation of the soothing efficacy of asparagus fermented extract

[0167] 1. Effect on RAW264.7 cell activity

[0168] 1.1 Experimental method:

[0169] (1) First, mouse monocyte / macrophage cells (RAW264.7 cells) were inoculated in DMEM medium containing 10% fetal bovine serum at a cell density of 1 x 10 4 6 cells / well, and cultured in a 37°C incubator with 5% CO2 for 24 h.

[0170] (2) Set up experimental groups: Example 1 group, Comparative Example 1 group, and control group, wherein the Example 1 group was added with 200 μL of the asparagus fermented extract prepared in Example 1 diluted 1000-fold, 800-fold, 600-fold, 400-fold, 300-fold, 200-fold, 100-fold, 50-fold, and 25-fold with cell culture medium, respectively; the Comparative Example 1 group was added with 200 μL of the asparagus extract prepared in Comparative Example 1 diluted 1000-fold, 800-fold, 600-fold, 400-fold, 300-fold, 200-fold, 100-fold, 50-fold, and 25-fold with cell culture medium, respectively; the control group was added with 200 μL of cell culture medium.

[0171] (3) After treatment, each group was cultured at 37°C with 5% CO2 for 24 h, and the cell viability was determined by the MTT method.

[0172] 1.2 Experimental results: As Figure 1 shown, the cell viability of each group diluted from 1000-fold to 25-fold was maintained at more than 80%, and with the increase of the dilution fold, the cell activity of Example 1 showed a trend of increasing → decreasing → increasing → decreasing, while the cell activity of Comparative Example 1 showed a trend of increasing → decreasing → increasing. Among them, the cell viability of the 25-fold and 50-fold dilution groups of Example 1 and Comparative Example 1 was still more than 80%, indicating that the asparagus fermented extract and the asparagus extract had no obvious toxicity to cells and had good biological safety, and could be used as a cosmetic raw material to test its efficacy.

[0173] 2. Inflammatory cytokines in cells IL-6 Effects of expression

[0174] 2.1 Experimental method:

[0175] (1) RAW264.7 cells were first inoculated in DMEM medium containing 10% fetal bovine serum, and the cell density was controlled at 1×10 4 cells / well to ensure uniform distribution of cells. The inoculated cells were placed in a 37℃, 5% CO2 incubator for 24 h for adhesion growth.

[0176] (2) Set up experimental groups: sample group, model group and control group. The control group was used as the baseline, the model group was treated with pro-inflammatory agent LPS to induce inflammatory response, so that the expression level of IL-6 increased.

[0177] Among them, the sample group was 194 μL cell culture medium + 2 μL LPS (1 mg / mL) + 4 μL Asparagus racemosus fermentation extract prepared in Example 1 (diluted 50 times), 190 μL cell culture medium + 2 μL LPS (1 mg / mL) + 8 μL Asparagus racemosus fermentation extract prepared in Example 1 (diluted 25 times), 194 μL cell culture medium + 2 μL LPS (1 mg / mL) + 4 μL Asparagus racemosus fermentation extract prepared in Comparative Example 1 (diluted 50 times), and 190 μL cell culture medium + 2 μL LPS (1 mg / mL) + 8 μL Asparagus racemosus fermentation extract prepared in Comparative Example 1 (diluted 25 times). The model group was 180 μL cell culture medium + 2 μL LPS (1 mg / mL). The control group was 200 μL cell culture medium.

[0178] (3) After treatment, each group was further incubated at 37℃ with 5% CO2 for 48 h, with GAPDH as the internal reference, the expression of IL-6 in RAW264.7 cells was detected by qPCR experiment, and the primer sequence is shown in Table 6, in order to evaluate the inhibitory effect of different concentrations of extract on IL-6 .

[0179] Table 6 Primer sequence

[0180]

[0181] 2.2 Experimental results: As shown in Figure 2 , Comparative Example 1 at a dilution of 25 and 50 times, the expression of inflammatory cytokines IL-6 was similar to that of the model group, and the Asparagus racemosus extract under this concentration condition had no soothing effect. The expression of IL-6The level was similar to the model group, indicating that the inflammatory response was not effectively inhibited at this concentration; at a higher concentration, the 25-fold dilution group significantly reduced the expression level of IL-6 by 32.8%, indicating that the active ingredients in the asparagus ferment extract effectively inhibited IL-6 at this concentration, showing an anti-inflammatory soothing effect.

[0182] Test Example 2: Antioxidant capacity detection of asparagus ferment extract

[0183] 1. Superoxide dismutase (SOD) activity determination

[0184] 1.1 Experimental method: The supernatant of the asparagus ferment extract prepared in Example 1 and the asparagus extract prepared in Comparative Example 1 was diluted 25 times with distilled water as the sample to be tested. The SOD activity was determined using an SOD activity detection kit, and the operation was performed according to the kit instructions.

[0185] 1.2 Experimental results: As shown in Figure 3 , the SOD activity of the asparagus ferment extract of Example 1 was 70.9 U / mg, and the SOD activity of the asparagus extract of Comparative Example 1 was 55 U / mg. This indicates that the fermentation treatment significantly improves the SOD activity of the asparagus extract, and its ability to scavenge superoxide radicals is enhanced.

[0186] 2. ABTS + radical scavenging capacity determination

[0187] 2.1 Experimental method:

[0188] (1) The supernatant of the asparagus ferment extract prepared in Example 1 and the asparagus extract prepared in Comparative Example 1 was diluted 25 times with distilled water as the sample to be tested.

[0189] (2) Prepare 7 mM ABTS solution and 2.45 mM potassium persulfate solution, mix them, and place them in the dark for 12-16 h to generate ABTS radical cations. Before use, dilute the ABTS radical solution to an absorbance of 0.70±0.02 at 734 nm wavelength.

[0190] (3) Sample tube: 0.2 mL of sample to be tested was taken into 2.8 mL of ABTS radical solution, and placed in the dark at room temperature for 6 min. The absorbance was determined at 734 nm wavelength using a spectrophotometer. The blank tube used distilled water instead of the sample solution, and the control tube used distilled water instead of the ABTS + working solution, and were recorded as A 样品 , A 空白 , and A 对照 , respectively. The ABTS+ Free radical scavenging rate.

[0191] ABTS + Free radical scavenging rate (%) = [[A 空白 -(A 样品 -A 对照 )]÷A 空白 ]×100%

[0192] Where: A 对照 Represents the absorbance of ABTS free radical solution; A 样品 Indicates the absorbance after adding the sample to be tested.

[0193] 2.2 Experimental results: Figure 3 As shown, the ABTS of the Asparagus cochinchinensis fermentation extract of Example 1 + The free radical scavenging rate was 86%, and the ABTS of the asparagus extract of comparative example 1 was + The free radical scavenging rate was 61%. This indicates that fermentation treatment significantly improved the free radical scavenging ability of Asparagus cochinchinensis extract, especially in ABTS + In the free radical scavenging experiment, the effect was significantly improved after fermentation treatment.

[0194] 3. DPPH free radical scavenging ability determination

[0195] 3.1 Experimental methods:

[0196] (1) The supernatant of the fermented asparagus extract prepared in Example 1 and the asparagus extract prepared in Comparative Example 1 after centrifugation and filtration was diluted 25 times with distilled water as the test sample.

[0197] (2) Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 515 nm, and zero with anhydrous ethanol. Dilute a 1 mg / mL DPPH anhydrous ethanol solution with anhydrous ethanol to an absorbance of 0.8-1.0 at a wavelength of 515 nm.

[0198] (3) Vortex mix, and let stand at room temperature in the dark for 30 min. The absorbance at 515 nm is recorded as A 空白 、A 测定 、A 对照 Each test tube needs to have a control tube, and the blank tube only needs to be tested once. DPPH free radical scavenging rate calculation formula: DPPH free radical scavenging rate (%) = [[A 空白 -(A 测定 -A 对照 )]÷A 空白 ]×100%.

[0199] 3.2 Experimental results: Figure 3As shown, the DPPH free radical scavenging rate of the asparagus officinalis fermented extract of Example 1 was 83.4%, while that of Comparative Example 1 was 63.1%. This indicates that the asparagus officinalis extract after fermentation treatment has stronger free radical scavenging ability in the DPPH free radical scavenging experiment, and has higher antioxidant activity, which helps to resist skin aging.

[0200] In summary, fermentation treatment significantly improves the performance of asparagus officinalis extract in multiple antioxidant indicators, especially in superoxide dismutase activity, ABTS + and DPPH free radical scavenging ability. The fermentation process promotes the release and transformation of antioxidant components in asparagus officinalis extract, improves its antioxidant activity, and the fermented asparagus officinalis extract can more effectively scavenge free radicals, delay cell aging, reduce inflammation, and enhance its value as a cosmetic raw material in antioxidant and anti-inflammatory applications.

[0201] Test Example 3 Detection of active ingredients in asparagus officinalis fermented extract

[0202] 1. Determination of total polyphenol content

[0203] Polyphenols, as a key ingredient in cosmetics, have strong antioxidant and anti-inflammatory ability, can effectively scavenge free radicals, reduce inflammation, and delay skin aging.

[0204] 1.1 The total polyphenol content in the sample was determined by Folin-phenol reagent method, and the experimental method was as follows:

[0205] (1) The asparagus officinalis fermented extract prepared in Examples 1-16, the asparagus officinalis fermented extract prepared in Comparative Examples 3-5, the asparagus officinalis extract prepared in Comparative Example 1, and the Saccharomyces cerevisiae liquid prepared in Comparative Example 2 were diluted 25 times with distilled water as the sample to be tested.

[0206] (2) Prepare gallic acid standard solution with concentrations of 0, 20, 40, 60, 80 and 100 µg / mL, respectively. Add 2.5 mL of Folin-phenol reagent, dilute them by volume ratio 1:10, and after standing for 5 min, add 2 mL of 7.5% sodium carbonate solution. After reaction for 30 min at room temperature in the dark, measure the absorbance at 760 nm wavelength using a spectrophotometer, and draw a standard curve.

[0207] (3) Take 0.5 mL of the sample to be tested, respectively, and place it in a 25 mL volumetric flask. Dilute with 5 mL of distilled water, shake well, add 2.5 mL of Folin-phenol reagent, and after standing for 5 min, add 2 mL of 7.5% sodium carbonate solution. After reaction for 30 min in the dark, measure the sample absorbance at 760 nm wavelength. Calculate the total polyphenol content in the sample according to the following formula by the standard curve:

[0208] Total polyphenol content (mg / mL) = (C x V1) ÷ V2.

[0209] In the formula: C represents the concentration of gallic acid equivalent in the sample to be tested obtained according to the standard curve, mg / mL; V1 represents the total volume of asparagus extract, mL; V2 represents the total volume of asparagus fermentation extract, mL.

[0210] 1.2 Experimental results: As shown in Table 7, the polyphenol content in the asparagus fermentation extract was as high as 48.5 mg / mL, indicating that fermentation treatment increased the polyphenol content in the asparagus extract, and the Saccharomyces cerevisiae in the YPD liquid medium without asparagus extract (Comparative Example 2) did not produce polyphenols during fermentation. The metabolic action of Saccharomyces cerevisiae during fermentation in Example 1 promoted the release and activation of polyphenols in the asparagus fermentation extract, which had the potential to enhance the anti-inflammatory, antioxidant, and anti-aging functions of the asparagus extract in cosmetics. In addition, the fermentation process of the asparagus fermentation extract may also improve the bioavailability of polyphenols, making them more easily absorbed and utilized by the human body.

[0211] Table 7 Total polyphenol content

[0212]

[0213] Note: Compared with Example 1, *** indicates P <0.001.

[0214] 2. Saponin content determination

[0215] 2.1 The vanillin-sulfuric acid method was used to determine the saponin content in the sample, and the experimental method was as follows:

[0216] (1) The asparagus fermentation extract prepared in Examples 1-16, the asparagus fermentation extract prepared in Comparative Examples 3-5, the asparagus extract prepared in Comparative Example 1, and the Saccharomyces cerevisiae broth prepared in Comparative Example 2 were subjected to saponin extraction at a solid-liquid ratio of 1 g:10 mL using ethanol as the solvent, and extracted in a 50°C water bath for 2 h. After filtration, the ethanol was removed by rotary evaporation, and the concentrated supernatant was diluted 25 times with distilled water as the sample to be tested.

[0217] (2) Dioscin was prepared into saponin standard solutions with concentrations of 0, 10, 20, 40, 60, 80, and 100 µg / mL. 1 mL of the saponin standard solution was taken, 1 mL of a 1% vanillin solution (vanillin dissolved in glacial acetic acid) was added first, and then 5 mL of concentrated sulfuric acid was quickly added. After standing at room temperature for 15 min, the absorbance was measured at a wavelength of 560 nm using a spectrophotometer, and a standard curve was plotted.

[0218] (3) Take 1 mL of the sample to be tested, add 1 mL of 1% vanillin solution and 5 mL of concentrated sulfuric acid, mix well, and stand at room temperature for 15 min to generate a red compound. The absorbance is detected at a wavelength of 560 nm. The total saponin content in the sample is calculated according to the standard curve according to the following formula.

[0219] Total saponin content (mg / mL) = (C x V1) ÷ V2

[0220] In the formula, C represents the concentration of saponin in the sample to be tested obtained from the standard curve, mg / mL; V1 represents the total volume of asparagus extract, mL; V2 represents the total volume of asparagus fermented extract, mL.

[0221] 2.2 Experimental results: As shown in Table 8, the saponin content of the asparagus fermented extract was as high as 19.5 mg / mL, higher than 10.3 mg / mL of Comparative Example 1, indicating that fermentation treatment increased the saponin content in the asparagus extract, and the Saccharomyces cerevisiae in the YPD liquid medium without asparagus extract (Comparative Example 2) did not produce saponin during fermentation. This indicates that the metabolic action of Saccharomyces cerevisiae during fermentation promotes the release and activation of saponin in the asparagus fermented extract, making it have stronger potential anti-inflammatory and anti-aging effects in cosmetics.

[0222] Table 8 Total saponin content

[0223]

[0224] Note: Compared with Example 1, *** indicates P <0.001.

[0225] 3. Determination of polysaccharide content

[0226] 3.1 Experimental method:

[0227] (1) The asparagus fermented extract prepared in Examples 1-16, the asparagus fermented extract prepared in Comparative Examples 3-5, the asparagus extract prepared in Comparative Example 1, and the Saccharomyces cerevisiae liquid prepared in Comparative Example 2 were extracted with distilled water as the solvent at a solid-liquid ratio of 1 g:10 mL, and extracted in a water bath at 50°C for 2 h. After filtration and centrifugation at 4000 rpm for 10 min, the supernatant was concentrated and diluted 25 times with distilled water as the sample to be tested.

[0228] (2) Prepare glucose standard solutions with concentrations of 0, 10, 20, 40, 60, 80, and 100 μg / mL, take 1 mL of the glucose standard solution, first add 1 mL of a phenol solution with a volume concentration of 5%, then quickly add 5 mL of concentrated sulfuric acid, and let stand at room temperature for 15 min to generate a yellow to orange compound. Use a spectrophotometer to measure the absorbance at a wavelength of 490 nm and draw a standard curve.

[0229] (4) Take 1 mL of the sample to be tested, add 1 mL of a phenol solution with a volume concentration of 5% and 5 mL of concentrated sulfuric acid, mix well, and let stand at room temperature for 15-30 min to generate a yellow to orange compound. Detect the absorbance at a wavelength of 490 nm. Calculate the polysaccharide content in the sample according to the following formula based on the standard curve.

[0230] Polysaccharide content (mg / mL) = (C x V1) ÷ V2

[0231] In the formula, C represents the concentration of glucose in the sample to be tested obtained from the standard curve, mg / mL; V1 represents the total volume of the asparagus extract, mL; and V2 represents the total volume of the asparagus fermentation extract, mL.

[0232] 3.2 Experimental results: As shown in Table 9, the polysaccharide content of the asparagus fermentation extract was as high as 135.6 mg / mL, indicating that fermentation treatment significantly increased the polysaccharide content of the asparagus extract. The Saccharomyces cerevisiae in the YPD liquid medium without asparagus extract (Comparative Example 2) only metabolized a small amount of polysaccharide during fermentation. This indicates that the metabolic action of Saccharomyces cerevisiae during fermentation promotes the release and activation of polysaccharides in the asparagus fermentation extract, which has potential effects of moisturizing, antioxidant, immune regulation, and promoting collagen production.

[0233] Table 9 Polysaccharide content

[0234]

[0235] Note: Compared with Example 1, *** indicates P <0.001.

[0236] Test Example 4 Evaluation of soothing and anti-aging efficacy of the cosmetic composition

[0237] 1. Effect on RAW264.7 cell activity

[0238] 1.1 Experimental method:

[0239] (1) Seed RAW264.7 cells in DMEM medium containing 10% fetal bovine serum, with a cell density of 1 x 10 4Each well was seeded with 1 x 10

[0240] (2) Set up experimental groups: sample group and control group. The sample group was added with 200 μL of cell culture medium containing the cosmetic compositions numbered 1-15 in Table 10, and the control group was added with 200 μL of cell culture medium containing PBS. When the cell confluence reached 60%, the cells were treated according to the above grouping.

[0241] (3) After treatment, each group was cultured at 37°C with 5% CO2 for 24 h, and the cell viability was determined by MTT method.

[0242] Table 10 Composition of cosmetic compositions

[0243]

[0244] 1.2 Experimental results: As shown in Table 11, no changes in cell morphology and no obvious toxicity were observed in each sample group within the test range. Figure 4

[0245] 2. Effect on expression of cell inflammatory factors IL-6

[0246] 2.1 Experimental method:

[0247] (1) RAW264.7 cells were first inoculated in DMEM medium containing 10% fetal bovine serum, and the cell density was controlled at 1 x 10 4 cells / well to ensure uniform distribution of cells. The inoculated cells were placed in a 37°C, 5% CO2 incubator for 24 h to adapt to the environment and adhere to the wall.

[0248] (2) Set up experimental groups: sample group, model group and control group. The sample group was added with 200 μL of cell culture medium containing the cosmetic compositions numbered 1-15 in Table 10, and the control group was added with 200 μL of cell culture medium containing PBS. When the cell confluence reached 60%, the cells were treated according to the above grouping. The control group was used as the baseline, and the model group was treated with pro-inflammatory agent LPS to induce inflammatory response and increase the expression level. IL-6

[0249] (3) After treatment, each group was cultured at 37°C with 5% CO2 for 48 h, and the expression of GAPDH in RAW264.7 cells was detected by qPCR experiment with IL-6 as the internal reference. The primer sequences are shown in Table 6, and the inhibition rate of IL-6 expression was calculated according to the following formula.

[0250] ​​​Inhibition rate (%) = (1- IL-6 expression level ÷ IL-6 expression level in the model group) × 100%

[0251] 2.2 Experimental results: Inflammatory factors in each group IL-6 Relative expression levels Figure 5 As shown, compared with the control group, the inflammatory factors in the model group IL-6 The content of TNF-α was significantly increased, indicating that the RAW264.7 cell inflammation model has been successfully established.

[0252] Compared with the model group, the Asparagus cochinchinensis fermentation extract groups (No. 1-5) had a negative effect on inflammatory factors. IL-6 The inhibition rates were 60.6%, 53.6%, 46.0%, 38.0% and 33.8% respectively. The 10-30% concentration range of Asparagus cochinchinensis fermentation extract could significantly inhibit the inflammatory factors. IL-6 The expression ( P <0.001), and the inhibitory effect was dose-dependent with the extract of Asparagus cochinchinensis fermentation; the magnesium ascorbyl phosphate group (No. 6-10) had no significant effect on inflammatory factors. IL-6 The inhibition rates of ascorbyl phosphate were 18.9%, 13.8%, 11.2%, 10.5% and 10.1% respectively. Magnesium ascorbyl phosphate in the concentration range of 0.2-1.5% could inhibit the expression of inflammatory factors to a certain extent. The combination groups of asparagus fermentation extract and magnesium ascorbyl phosphate (No. 11-15) had no significant effect on the expression of inflammatory factors. IL-6 The inhibition rates were 71.6%, 68.6%, 56.5%, 42.7% and 38.5% respectively, and compared with the soothing effect of the asparagus fermentation extract group (No. 1-5), the compound group (No. 11-15) showed a significant improvement in the soothing effect.

[0253] 3. Anti-aging efficacy evaluation

[0254] Type I collagen has an antioxidant effect, which can prevent free radicals from damaging skin cells, thus helping to maintain the health and youthfulness of the skin. Type I collagen has a triple helical structure, consisting of two α1 chains and one α2 chain. These three polypeptide chains are tightly bound together by hydrogen bonds to form a stable triple helical structure. The human type I collagen gene mainly includes COL1A1 and COL1A2 . COL1A1 The gene encodes the α1 chain of type I collagen, which is an important component of the triple helical structure of type I collagen; COL1A2 Gene encoding the α2 chain of type I collagen COL1A1The alpha 1 chains encoded by genes are synthesized together in a ratio of 2:1 to form a triple helix structure of type I collagen. Type I collagen and elastin are distributed in the dermis, and after being affected by hydrogen peroxide and other factors, the content of collagen and elastin in the extracellular matrix decreases, showing that the skin appearance structure degenerates and the function declines. The anti-aging effect of the cosmetic compositions numbered 1-15 in Table 10 is verified by inducing human skin fibroblasts (HSF) to age by H2O2.

[0255] 3.1 Experimental method:

[0256] (1) After human dermal fibroblasts (P2 generation) were cultured to 2×10 5 cells / well, the cells were cultured for 24 h, and when the cell confluence was 60%, the experimental groups were set: sample group, model group and control group. Among them, the sample group was 200 μL of serum-free low-glucose DMEM medium containing the cosmetic compositions numbered 1-15 in Table 10 and 400 μmol / L hydrogen peroxide; the model group was treated with 200 μL of serum-free low-glucose DMEM medium containing 400 μmol / L hydrogen peroxide every day at 37°C for 2 h, and the human skin fibroblasts were washed with PBS for 3 times, and then replaced with low-glucose DMEM medium containing serum, and then cultured for 22 h, a total of 3 days of treatment; the control group was serum-free low-glucose DMEM medium without hydrogen peroxide.

[0257] (2) After 72 h of treatment in each group, the cells were collected, RNA was extracted, and after reverse transcription, GAPDH was used as an internal reference to detect the expression of type I collagen-related genes (COL1A1 and COL1A2), elastin gene (ELN) in the sample group, model group and control group by qPCR, respectively. The primer sequences are shown in Table 11. GAPDH COL1A1 COL1A2 ELNA

[0258] Table 11 Primer sequences

[0259]

[0260] The growth rate of gene expression (%) = (M-T) ÷ M × 100%; and whether there is a statistical difference is analyzed by using software SPSS. COL1A1 COL1A2 ELNA

[0261] In the formula: T represents the average value of the relative expression of the genes in the sample group; M represents the average value of the relative expression of the genes in the model group.

[0262]

[0263] 3.2 Experimental results: as shown in​​​​​​​​Figure 7 As shown in the figure, compared with the blank control group, after hydrogen peroxide treatment, the type I collagen gene in the model group COL1A1 and COL1A2、 Elastin gene ELNA The contents of β-actin and β-actin in the human fibroblasts were significantly decreased, indicating that the human fibroblast aging model has been successfully established.

[0264] Depend on Figure 7 Further calculation of type I collagen gene ( COL1A1 and COL1A2 ), elastin gene ( ELNA ) expression growth rate. The results showed that:

[0265] Compared with the model group, the Asparagus cochinchinensis fermentation extract groups (No. 1-5) had no significant effect on the gene expression. COL1A1 The growth rates of expression were 66.7%, 56.0%, 48.3%, 40.3% and 34.7% respectively; COL1A2 The growth rates of expression were 66.3%, 54.3%, 45.1%, 36.8% and 31.6% respectively; ELNA The growth rates of expression were 47.3%, 40.0%, 24.0%, 19.7% and 16.3% respectively. This indicates that 10-30% concentration of Asparagus cochinchinensis fermentation extract can significantly promote the expression of type I collagen gene. COL1A1 and COL1A2、 Elastin gene ELNA The expression ( P <0.001), and the promoting effect was dose-dependent with the fermented extract of Asparagus cochinchinensis.

[0266] Compared with the model group, the magnesium ascorbyl phosphate group (No. 6-10) had a negative effect on the gene COL1A1 The growth rates of expression were 30.3%, 21.7%, 20.0%, 9.0% and 4.3%; COL1A2 The growth rates of expression were 27.4%, 18.2%, 16.7%, 9.7% and 6.1% respectively; ELNA The growth rates of expression were 21.0%, 16.0%, 14.7%, 11.7% and 9.3% respectively. This indicates that 0.2-1.5% concentration of magnesium ascorbyl phosphate can promote the expression of type I collagen related genes ( COL1A1 and COL1A2 ), elastin gene ( ELNA ) expression.

[0267] Compared with the model group, the compound group (No. 11-15) had COL1A1 The growth rates of expression were 100.7%, 95.0%, 83.0%, 83.0% and 55.7%; COL1A2The growth rates of expression amounts were 106.9%, 94.3%, 82.7%, 73.3% and 51.9%, respectively; genes ELNA The growth rates of expression amounts were 68.0%, 57.0%, 48.7%, 41.3% and 30.7%, respectively. And compared with the asparagus fermentation extract group (Nos. 1-5), the compound group (Nos. 11-15) showed a significant improvement in promoting collagen type I and elastin, and the anti-wrinkle and firming effects were the best.

[0268] Test Example 5 Evaluation of soothing and anti-aging effects of cosmetics

[0269] 1. Evaluation of soothing effects of cosmetics prepared in Examples 17-21 and Comparative Examples 6-10

[0270] A copper sulfate-induced zebrafish embryo lateral line region neural crest cell injury-induced neutrophil aggregation model was used to test the soothing effect.

[0271] 1.1 Experimental method:

[0272] (1) Set up experimental groups: sample group, positive control group, model group and blank group. Among them, the sample group is 10 µM anhydrous copper sulfate + 0.1% by mass concentration of the cosmetic solution prepared in Examples 4-8 and Comparative Examples 5-9 (dissolve the cosmetic in zebrafish embryo culture solution); the positive control group is 10 µM anhydrous copper sulfate + 10 µM indomethacin; the model group is 10 µM anhydrous copper sulfate; and the blank group is zebrafish embryo culture solution.

[0273] (2) Divide wild-type AB strain zebrafish embryos into 13 groups on average, 15 tails per group, expose them to the sample group, the positive control group, the model group and the blank control group, and after incubation in a constant temperature incubator at 28±1°C for 40 min, fix the fish embryos and perform Sudan black staining, observe under a body microscope and record the number of neutrophils in the lateral line region. The neutrophil aggregation inhibition rate of each treatment group was calculated according to the following formula.

[0274] Neutrophil aggregation inhibition rate (%) = (M-S) ÷ M x 100%

[0275] In the formula, S represents the average number of neutrophils in zebrafish embryos in each treatment group; M represents the average number of neutrophils in zebrafish embryos in the model group.

[0276] 1.2 Experimental results: As shown in Tables 12 and Figure 6 Compared with the blank control group, the number of neutrophils on the surface of the zebrafish skin in the model group increased significantly, indicating that the zebrafish soothing model was successfully established.

[0277] Compared with the model group, the inhibition rates of the comparative examples 6-10 on neutrophil aggregation in zebrafish embryos were 24.22%, 23.32%, 21.75%, 18.39% and 27.58%, respectively; while the inhibition rates of the examples 17-21 were 44.39%, 42.60%, 38.12%, 37.00% and 34.53%, respectively, and the examples 17-21 had significant differences compared with the comparative examples 6-10, respectively P <0.001).

[0278] Table 12 Number of centrosomes in zebrafish embryos

[0279]

[0280] 2. Evaluation of the anti-aging efficacy of the cosmetics prepared from the examples 17-21 and the comparative examples 6-10

[0281] The anti-aging efficacy test was performed by using Caenorhabditis elegans as a model system for aging research.

[0282] 2.1 Experimental method:

[0283] (1) Set up experimental groups: sample group and blank group, wherein the sample group was 1 g of the cosmetics prepared from the examples 17-21 and the comparative examples 6-10 + NGM medium containing E. coli OP50 bacterial liquid, and the blank group was M9 buffer + NGM medium containing E. coli OP50 bacterial liquid.

[0284] (2) After synchronization, the N2 wild-type Caenorhabditis elegans was picked into the medium of the sample group and the blank group, and cultured at 20°C, with not less than 50 Caenorhabditis elegans per plate. The growth of Caenorhabditis elegans was observed every day, and the escaped, dead and prolapsed Caenorhabditis elegans were removed, and the live Caenorhabditis elegans was picked to a new plate, and the average lifespan and the maximum lifespan (days) of Caenorhabditis elegans were recorded. If Caenorhabditis elegans did not respond to stimulation twice, it was judged to be dead, wherein the death time of the last Caenorhabditis elegans in each group was determined as the maximum lifespan of Caenorhabditis elegans. Each group of experiments was repeated 3 times.

[0285] 2.2 Experimental results: as Figure 8- Figure 9As shown, compared with the blank group, the average lifespan and the maximum lifespan of C. elegans were significantly prolonged in Examples 17-21. The average lifespan of Examples 17-21 was 14.23 d, 12.93 d, 12.05 d, 11.36 d and 11.12 d, respectively, and the maximum lifespan was 24.32 d, 22.16 d, 21.06 d, 20.13 d and 19.69 d, respectively; while the average lifespan of Comparative Examples 6-10 was 10.32 d, 10.16 d, 10.06 d, 10.35 d and 10.33 d, respectively, and the maximum lifespan was 15.34 d, 15.16 d, 14.96 d, 15.69 d and 16.03 d, respectively. There was a significant difference between Examples 17-21 and Comparative Examples 6-10 (P<0.001). P <0.001).

[0286] In Examples 17-21, the longer lifespan effect of C. elegans was more obvious with the increase of the concentration of the asparagus fermentation extract and the magnesium ascorbyl phosphate composition. Among them, Example 19 (the content of asparagus fermentation extract and magnesium ascorbyl phosphate was 30% and 1.5%, respectively) had the best effect of prolonging lifespan. Compared with the blank group, the average lifespan was increased by 29.2%, and the maximum lifespan was increased by 37.9%; compared with Comparative Example 6, the average lifespan was prolonged by 27.5%, and the maximum lifespan was increased by 36.9%.

[0287] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing an asparagus fermentation extract, characterized in that: The method comprises the following steps: preparing a saccharomyces cerevisiae strain with a deposit number of CCTCC KY 2008613 ( Saccharomyces cerevisiae ) of the bacterial liquid, and fermented it with the asparagus extract as the raw material to obtain, The preparation process of the asparagus asparagus extract comprises: first placing the asparagus asparagus in water, adding pectinase and cellulase for enzymolysis, then centrifuging and filtering, adding anhydrous glucose to the filtrate, and sterilizing to obtain the asparagus asparagus extract; the enzymolysis temperature is 50° C., and the enzymolysis time is 2 hours; the total amount of the pectinase and cellulase added is 1.5% of the mass of the asparagus asparagus, and the amount of the cellulase added is 1% of the mass of the asparagus asparagus; The fermentation process includes: inoculating a culture of saccharomyces cerevisiae into an asparagus extract, adjusting the initial pH to 7, and then fermenting at a rotation speed of 150 rpm and 30° C. for 40 hours.

2. The preparation method according to claim 1, characterized in that The preparation process of the bacterial liquid comprises the following steps: inoculating saccharomyces cerevisiae into a YPD liquid culture medium, and activating the culture medium at 25-35° C. for 10-14 hours.

3. The preparation method according to claim 1, characterized in that The material-liquid ratio of the asparagus and water is 1 g:8-12 mL; the enzymatic hydrolysis needs to be stirred at 800-1200 rpm; the centrifugal speed is 1800-2200 rpm, and the centrifugation time is 12-18 min; the mass concentration of the anhydrous glucose in the filtrate is 0.8-1.2%.

4. The preparation method according to claim 1, characterized in that The volume concentration of the bacterial liquid in the asparagus extract is 0.5-2%. After the fermentation is completed, post-treatment is performed, which includes sterilization at 120-122° C. for 28-32 minutes, centrifugation at 1500-2500 rpm for 10-20 minutes, and filtration.

5. An Asparagus cochinchinensis fermentation extract prepared by the preparation method according to any one of claims 1 to 4.

6. A soothing and anti-aging cosmetic composition, characterized in that: The invention comprises magnesium ascorbyl phosphate and the asparagus cochinchinensis fermentation extract according to claim 5.

7. The cosmetic composition according to claim 6, characterized in that The mass percentage of the magnesium ascorbyl phosphate in the cosmetic composition is 0.2-1.5%, and the mass percentage of the asparagus fermentation extract in the cosmetic composition is 10-30%.

8. A soothing and anti-aging cosmetic, characterized in that: A cosmetic composition comprising the composition of any one of claims 6 to 7.

9. Use of the fermented asparagus extract prepared by the preparation method according to any one of claims 1 to 4 in the preparation of cosmetics with antioxidant, anti-inflammatory, soothing and anti-aging effects.

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