Asparagus cochinchinensis fermentation extracting solution, preparation method and application thereof, soothing and anti-aging cosmetic composition and cosmetic

Abstract

The invention belongs to the technical field of cosmetic preparation, and particularly relates to a radix asparagi fermentation extracting solution, a preparation method and application thereof, a soothing and anti-aging cosmetic composition and a cosmetic. The preparation method comprises the following steps: fermenting a bacterial liquid of saccharomyces cerevisiae with the preservation number of CCTCC KY 2008613 by taking a radix asparagi extracting solution as a raw material, thereby obtaining the saccharomyces cerevisiae. The radix asparagi fermentation extracting solution provided by the invention not only can retain the natural characteristics of radix asparagi to the greatest extent, but also further enhances the efficacy of radix asparagi in cosmetics through the metabolic reaction of saccharomyces cerevisiae, so that the cosmetics have remarkable antioxidant, anti-aging and skin-soothing capabilities. The preparation method of the radix asparagi fermentation extracting solution is easy to operate and environmentally friendly, the whole fermentation and enzymolysis process is carried out at low temperature, the stability of active ingredients is protected, energy is saved, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetic preparation, and particularly relates to an asparagus 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 a variety of active ingredients, including saponins, polysaccharides, amino acids and various antioxidants. Due to its significant moisturizing, antioxidant and anti-inflammatory properties, it has a wide range of application potential in the cosmetics field. Asparagus cochinchinensis extract can help improve skin hydration, reduce free radical damage and delay skin aging. With the increasing demand of consumers for natural and safe cosmetics, cosmetic manufacturers have a growing demand for ingredients derived from natural plants. Asparagus cochinchinensis, as a traditional Chinese herbal medicine, has attracted the attention of the cosmetics industry due to its mild and natural properties. However, the absorption and bioavailability of active ingredients may be limited when Asparagus cochinchinensis extract is used directly in cosmetics.

[0003] Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) is widely used in the food industry due to its strong fermentation ability, short growth cycle, potential low cost and easy large-scale cultivation. The metabolites of Saccharomyces cerevisiae have good affinity to the skin, which can not only improve the vitality of skin cells and promote metabolism, but also have significant effects on repairing skin, moisturizing, whitening and delaying skin aging. Existing studies have shown that extracts prepared from plants fermented with Saccharomyces cerevisiae can effectively improve the activity of plant ingredients and enhance their antioxidant, moisturizing and anti-inflammatory effects.

[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 enzymes for enzymolysis, enzymolysis to the end point, and then heating to inactivate the enzymes 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 for fermentation to obtain a fermentation solution; removing impurities and sterilizing the fermentation solution to obtain the Rhodiola rosea fermentation extract. The Rhodiola rosea fermentation extract prepared by the method provided by the invention has good whitening and freckle-removing, anti-oxidation and anti-inflammatory effects.

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

[0006] In view of this, there is an urgent need in the art to provide a natural, low-irritation cosmetic raw material with multiple functions such as moisturizing, anti-aging and soothing, so as to provide a new raw material preparation method and application scheme for the cosmetics industry. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides an asparagus fermentation extract and a preparation method and application thereof, a soothing and anti-aging cosmetic composition and cosmetics, which can improve the low stability of effective ingredients in conventional asparagus extracts and the serious loss of effective ingredients, thereby improving the comprehensive efficacy of asparagus cosmetic raw materials.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing an asparagus fermentation extract comprises the following steps: preparing a saccharomyces cerevisiae fermented with a deposit number of CCTCC KY2008613 ( Saccharomyces cerevisiae )'s bacterial liquid is fermented with asparagus extract as raw material to obtain the product.

[0009] Preferably, the preparation process of the bacterial solution comprises inoculating Saccharomyces cerevisiae into a YPD liquid culture medium and activating the culture medium at 25-35° C. for 10-14 h.

[0010] Preferably, the preparation process of the asparagus extract comprises: firstly placing the 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 extract.

[0011] Preferably, the solid-liquid ratio of the asparagus to water is 1 g:8-12 mL.

[0012] More preferably, the solid-liquid ratio of the asparagus to water is 1 g:9-10 mL.

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

[0014] Preferably, the total added amount of the pectinase and cellulase is 1-2% of the mass of the asparagus.

[0015] Preferably, the added amount of the cellulase is 0.5-1.5% of the mass of the asparagus.

[0016] More preferably, the added amount of the cellulase is 0.8-1.2% of the mass of the asparagus.

[0017] Further preferably, the added amount of the cellulase is 1% of the mass of the asparagus.

[0018] Preferably, the enzymatic hydrolysis temperature is 40-60° C., the enzymatic hydrolysis time is 1.5-3 h, and the enzymatic hydrolysis needs to be stirred at 800-1200 rpm.

[0019] More preferably, the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis time is 2 h, and the enzymatic hydrolysis needs to be stirred at 1000 rpm.

[0020] Preferably, the centrifugal speed is 1800-2200 rpm, and the centrifugal time is 12-18 min.

[0021] More preferably, the centrifugal speed is 2000 rpm and the centrifugal time is 15 min.

[0022] Preferably, the mass concentration of the anhydrous glucose in the filtrate is 0.8-1.2%.

[0023] More preferably, the mass concentration of the anhydrous glucose in the filtrate is 1%.

[0024] Further preferably, the mass concentration of anhydrous glucose in the filtrate is 1.0%.

[0025] Preferably, the volume concentration of the yeast liquid of Saccharomyces cerevisiae in the asparagus extract is 0.5-2%.

[0026] More preferably, the volume concentration of the Saccharomyces cerevisiae culture solution in the Asparagus cochinchinensis extract is 1.5%.

[0027] Preferably, the fermentation process comprises inoculating the bacterial liquid of Saccharomyces cerevisiae into the asparagus extract, adjusting the measured initial pH to 6-7, and then fermenting at a rotation speed of 120-180 rpm and 25-35° C. for 36-48 h.

[0028] More preferably, the fermentation process comprises inoculating the bacterial liquid of Saccharomyces cerevisiae into the asparagus extract, adjusting the measured initial pH to 7, and then fermenting at a rotation speed of 150 rpm and 30° C. for 40 h.

[0029] Preferably, the treatment comprises sterilization at 120-122°C for 28-32 min, centrifugation at 1500-2500 rpm for 10-20 min and filtration.

[0030] More preferably, the treatment comprises sterilization at 121° C. for 30 min, centrifugation at 2000 rpm for 15 min, and filtration.

[0031] The present invention also provides the asparagus fermentation extract prepared by the above preparation method.

[0032] The present invention also provides a soothing and anti-aging cosmetic composition, comprising magnesium ascorbyl phosphate and the above-mentioned asparagus fermentation extract.

[0033] Preferably, the mass percentage of the magnesium ascorbyl phosphate in the cosmetic composition is 0.2-1.5%.

[0034] 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%.

[0035] Further preferably, the mass percentage of the magnesium ascorbyl phosphate in the cosmetic composition is 1.5%.

[0036] Preferably, the mass percentage of the Asparagus cochinchinensis fermentation extract in the cosmetic composition is 10-30%.

[0037] More preferably, the mass percentage of the Asparagus cochinchinensis fermentation extract in the cosmetic composition is 10%, 15%, 20%, 25% or 30%.

[0038] Further preferably, the mass percentage of the Asparagus cochinchinensis fermentation extract in the cosmetic composition is 30%.

[0039] The present invention also provides a soothing and anti-aging cosmetic comprising the above-mentioned cosmetic composition.

[0040] Preferably, the mass percentage of the cosmetic composition in the cosmetic is 10-50%.

[0041] More preferably, the mass percentage of the cosmetic composition in the cosmetic is 10.2%, 15.3%, 20.4%, 25.5% or 31.5%.

[0042] Further preferably, the mass percentage of the cosmetic composition in the cosmetics is 31.5%.

[0043] Preferably, the cosmetics include any one of toner, essence, eye cream, lotion, face cream, facial mask, scalp essence, shampoo, body lotion, sunscreen and shower gel.

[0044] The present invention also provides the use of the asparagus fermentation extract prepared by the above preparation method in the preparation of cosmetics with anti-oxidation, anti-inflammatory, soothing and anti-aging effects.

[0045] The present invention utilizes saccharomyces cerevisiae to ferment asparagus, and combines the metabolic advantages of yeast to further decompose and transform the active ingredients in asparagus, thereby obtaining a novel asparagus fermented cosmetic raw material. Yeast fermentation can release a variety of metabolites, including amino acids, polysaccharides and polyphenols, etc. These components are combined with the unique plant active ingredients of asparagus, so that the prepared cosmetic raw material has significant effects in anti-oxidation, anti-aging, anti-inflammatory and skin soothing. The small molecules generated during the fermentation process can be more easily absorbed by the skin, thereby improving the bioavailability of the active ingredients of asparagus and making the product more effective.

[0046] Using asparagus fermented extract as a cosmetic raw material in cosmetics can not only bring out the natural activity of asparagus, but also make use of the additional effects produced by yeast fermentation to make the ingredients in skin care products interact with each other and achieve the effect of 1+1>2. When asparagus fermented extract is used as a raw material in skin care products, it not only makes the skin moisturized and smooth, but also effectively resists the damage of external pollution to the skin, delays skin aging, provides soothing and anti-inflammatory effects, and has the effect of improving skin quality and skin health.

[0047] Compared with the prior art, the present invention has the following beneficial effects: (1) The fermented extract of Asparagus cochinchinensis provided by the present invention can not only retain the natural characteristics of Asparagus cochinchinensis to the greatest extent, but also further enhance the efficacy of Asparagus cochinchinensis in cosmetics through the metabolic reaction of Saccharomyces cerevisiae, so that the cosmetics have significant antioxidant, anti-aging and skin soothing capabilities.

[0048] (2) The method for preparing the fermented extract of Asparagus cochinchinensis provided by the present invention is simple to operate, green and environmentally friendly, avoids the addition of exogenous chemicals, and uses natural enzymes in the basic culture medium for biotransformation. The entire fermentation and enzymatic hydrolysis process is carried out at a low temperature (below 40°C), which helps to protect the stability of the active ingredients, save energy and reduce production costs. In addition, the fermented extract of Asparagus cochinchinensis is light yellow-green in color, which meets the natural and pure requirements of cosmetic raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The toxic effects of the Asparagus cochinchinensis fermentation extracts prepared in Example 1 and Comparative Example 1 on RAW264.7 cells at different dilution multiples.

[0050] Figure 2 The fermented extracts of Asparagus cochinchinensis prepared in Example 1 and Comparative Example 1 were used to respectively treat inflammatory factors IL-6 The impact of expression.

[0051] Figure 3 The antioxidant capacity test results of the Asparagus cochinchinensis fermentation extract prepared in Example 1 and Comparative Example 1 are shown.

[0052] Figure 4 The toxic effects of the cosmetic compositions No. 1-15 on RAW264.7 cells.

[0053] Figure 5 The cosmetic compositions No. 1-15 are effective against inflammatory factors IL-6 The impact of expression.

[0054] Figure 6 Effects of the cosmetics prepared in Examples 17-21 and Comparative Examples 6-10 on the aggregation of zebrafish neutrophils.

[0055] Figure 7 The cosmetic composition No. 1-15 is used to treat the type I collagen gene of human fibroblasts COL1A1 and COL1A2 and elastin gene ELNA The effect on gene expression (where A is the COL1A1 The effect on gene expression, B is COL1A2 The effect on gene expression, C is ELNA expression).

[0056] Figure 8 Effects of the cosmetics prepared in Examples 17-21 and Comparative Examples 6-10 on the average lifespan of Caenorhabditis elegans.

[0057] Fig. 9 Effects of the cosmetics prepared in Examples 17-21 and Comparative Examples 6-10 on the maximum lifespan of Caenorhabditis elegans.

[0058] Significance analysis: Figure 1 , Figure 4 and Figure 7 Compared with the control group, ns indicates P >0.05.

[0059] Figure 2 , Figure 5 and Figure 7 Compared with the model group, ns means P >0.05,*denotes P <0.05, ** indicates P <0.01, *** indicates P <0.001; ### indicates that compared with the control group, P <0.001.

[0060] Figure 6 , Figure 8 and Fig. 9 In the figure, a represents that Example 17 is compared with 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

[0061] 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 (preservation number is CCTCC KY 2008613) and Pichia pastoris (preservation number is 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 Inc.; 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 (RAW264.7 cells), purchased from Shanghai Chuanqiu Biotechnology Co., Ltd.; SOD activity detection kit, purchased from Beijing Solebow Technology Co., Ltd.; human dermal fibroblast primary cells (P2 generation), purchased from Guangdong Boxi Biotechnology Co., Ltd.; wild-type AB strain zebrafish, purchased from China National Zebrafish Resource Center; N2 wild-type Caenorhabditis elegans, purchased from Caenorhabditis elegans Genetics Center; nematode growth medium, Shandong Top Biotechnology Co., Ltd.; Escherichia coli OP50, purchased from Hubei Apti Biotechnology Co., Ltd.

[0062] Example 1 A method for preparing an asparagus fermentation extract comprises the following steps: (1) First, the brewer's yeast with the deposit number of CCTCC KY 2008613 ( Saccharomyces cerevisiae ) was inoculated into YPD liquid culture medium and activated at 30℃ for 12 h to obtain the bacterial liquid of Saccharomyces cerevisiae.

[0063] (2) Cut off the two ends of the asparagus and take the middle, cut it into small pieces evenly, and put 50 g of asparagus into 500 mL of deionized water according to the solid-liquid ratio of 1:10, add 0.5% pectinase and 1% cellulase of asparagus for enzymolysis, stir at 1000 rpm, the temperature of enzymolysis is 50℃, and the time is 2 h. After the enzymolysis is completed, filter with gauze, centrifuge at 2000 rpm for 15 min, remove the supernatant, cool it, and filter it, add 1% anhydrous glucose of the solution to the filtrate, sterilize it at high temperature, and obtain the asparagus extract.

[0064] (3) Finally, the yeast culture liquid of brewer's yeast was inoculated into the asparagus extract at a volume concentration of 1.5%, and the initial pH was adjusted to 7. The mixture was 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 to obtain the asparagus fermentation extract.

[0065] Example 2 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the added amount of pectinase is 0.5% of the mass of the asparagus, and the added amount of cellulase is 0.5% of the mass of the asparagus.

[0066] Example 3 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the added amount of pectinase is 0.5% of the mass of the asparagus, and the added amount of cellulase is 1.5% of the mass of the asparagus.

[0067] Example 4 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the enzymatic hydrolysis temperature in step (2) is 40°C.

[0068] Example 5 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the enzymatic hydrolysis temperature in step (2) is 60°C.

[0069] Example 6 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the enzymatic hydrolysis time in step (2) is 1.5 h.

[0070] Example 7 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the enzymatic hydrolysis time in step (2) is 3 h.

[0071] Example 8 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the rotation speed during enzymatic hydrolysis in step (2) is 800 rpm.

[0072] Example 9 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the rotation speed during the enzymatic hydrolysis in step (2) is 1200 rpm.

[0073] Example 10 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that in step (3), the volume concentration of the saccharomyces cerevisiae fermentation liquid in the asparagus extract is 0.5%.

[0074] Embodiment 11 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that in step (3), the volume concentration of the saccharomyces cerevisiae fermentation liquid in the asparagus extract is 2%.

[0075] Example 12 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the initial pH of the fermentation in step (3) is 6.

[0076] Embodiment 13 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the rotation speed of the shaking table in step (3) is 120 rpm.

[0077] Embodiment 14 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the rotation speed of the shaking table in step (3) is 180 rpm.

[0078] Embodiment 15 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the fermentation time in step (3) is 36 hours.

[0079] Example 16 A method for preparing an asparagus fermentation extract, compared with Example 1, the only difference is that the fermentation time in step (3) is 48 hours.

[0080] Embodiment 17 A soothing and anti-aging essence, the contents of which are shown in Table 1 by mass percentage.

[0081] Table 1 A soothing and anti-aging essence formula

[0082] The preparation process of the essence is as follows: First, mix glycerin, sodium hyaluronate, panthenol and purified water; then add the asparagus fermentation extract prepared in Example 1, niacinamide, magnesium ascorbyl phosphate and vitamin E in sequence, and stir well to ensure that the ingredients are evenly dispersed; then slowly add recombinant type III human collagen and stir until completely dissolved; finally, add phenoxyethanol and mix well to obtain.

[0083] Embodiment 18 A soothing and anti-aging eye cream, the contents of which are shown in Table 2 by mass percentage.

[0084] Table 2 A soothing and anti-aging eye cream formula

[0085] The preparation process of the eye cream is as follows: First, jojoba oil and polysorbate-60 are placed in a 60° C. water bath and heated until completely melted to obtain an oil phase; then glycerin, sodium hyaluronate, caffeine and purified water are stirred evenly, and heated in a water bath to 60° C. to obtain an aqueous phase; the oil phase is gradually added to the aqueous phase, and stirring is continued to form a uniform emulsion; after cooling to 40° C., the asparagus fermentation extract, magnesium ascorbyl phosphate, avocado extract and ceramide prepared in Example 1 are added and stirred evenly; finally, phenoxyethanol is added and mixed evenly to obtain the product.

[0086] Embodiment 19 A soothing and anti-aging moisturizing lotion, the contents of which are shown in Table 3 by mass percentage.

[0087] Table 3 A soothing and anti-aging moisturizing lotion formula

[0088] The preparation process of the moisturizing emulsion is as follows: first, jojoba oil, squalane, ceramide, sorbitan stearate and cetearyl alcohol are heated to 85°C, and stirred until completely dissolved to obtain an oil phase; then, glycerin, carbomer, panthenol and purified water are stirred evenly, and heated to 85°C in a water bath to obtain an aqueous phase; the oil phase is gradually added to the aqueous phase, and stirred homogeneously to form a uniform emulsion, and after cooling to 40°C, the asparagus fermentation extract prepared in Example 1, magnesium ascorbyl phosphate and triethanolamine are added, and stirred evenly; finally, phenoxyethanol and ethylhexylglycerin are added, and mixed evenly to obtain the emulsion.

[0089] Embodiment 20 A soothing and anti-aging sunscreen, the contents of its ingredients are shown in Table 4 by mass percentage.

[0090] Table 4 A sunscreen formula for soothing and anti-aging

[0091] The preparation process of the sunscreen is as follows: First, titanium dioxide, zinc oxide, jojoba oil, polysorbate 80 and cetearyl alcohol are heated to 80° C. and ground evenly to obtain an oil phase; then glycerin, niacinamide, purified water and carbomer are stirred evenly, and heated to 85° C. in a water bath to obtain an aqueous phase; the oil phase is gradually added to the aqueous phase, and homogenized and continuously stirred to form a uniform emulsion; after cooling to 40° C., the asparagus fermentation extract prepared in Example 1 and magnesium ascorbyl phosphate are added and stirred evenly; finally, phenoxyethanol is added and mixed evenly to obtain the product.

[0092] Embodiment 21 A soothing and anti-aging repairing facial mask, the contents of its ingredients, measured by mass percentage, are shown in Table 5.

[0093] Table 5 A soothing and anti-aging repair mask formula

[0094] The preparation process of the repair mask is as follows: First, glycerin, panthenol and purified water are stirred evenly, and heated in a water bath to 60° C. to obtain an aqueous phase; vitamin E in the oil phase is cooled to 40° C., and then the asparagus fermentation extract, magnesium ascorbyl phosphate, Centella asiatica extract, green tea extract and dipotassium glycyrrhizinate prepared in Example 1 are added in sequence and stirred evenly; finally, phenoxyethanol as a preservative is added and mixed evenly to obtain the product.

[0095] Comparative Example 1 Compared with Example 1, the only difference is that the bacterial solution does not contain brewer's yeast.

[0096] A method for preparing an asparagus extract comprises the following steps: Cut off the two ends of the asparagus and take the middle, cut it into small pieces evenly, and put 50 g of asparagus in 500 mL of deionized water according to the solid-liquid ratio of 1:10, add 2% pectinase and 1% cellulase of asparagus for enzymolysis, stir at 1000 rpm, the enzymolysis temperature is 50℃, and the time is 2 h. After the enzymolysis, filter with gauze, centrifuge at 2000 rpm for 15 min, take out the supernatant, cool it, and filter it, add 1% anhydrous glucose of the solution mass to the filtrate, sterilize it at high temperature, and obtain the asparagus extract.

[0097] Comparative Example 2 Compared with Example 1, the only difference is that the fermentation medium does not contain the asparagus extract.

[0098] A bacterial liquid of brewer's yeast, the preparation steps of which are as follows: The cerevisiae yeast ( Saccharomyces cerevisiae) was inoculated into YPD liquid culture medium and activated at 30℃ for 12 h to obtain the bacterial liquid of Saccharomyces cerevisiae.

[0099] Then, the yeast culture liquid of brewer's yeast was inoculated into YPD liquid medium at a volume concentration of 1.5%, and fermented 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.

[0100] Comparative Example 3 Compared with Example 1, the only difference is that Saccharomyces cerevisiae is replaced by Pichia pastoris.

[0101] A method for preparing an asparagus fermentation extract comprises the following steps: (1) First, the Pichia pastoris with the deposit number of CCTCC KY 2008612 ( Pichia pastoris ) was inoculated into YPD liquid culture medium and activated at 30℃ for 12 h to obtain the bacterial liquid of Pichia pastoris.

[0102] (2) Cut off the two ends of the asparagus and take the middle, cut it into small pieces evenly, and put 50 g of asparagus into 500 mL of deionized water according to the solid-liquid ratio of 1:10, add 0.5% pectinase and 1% cellulase of asparagus for enzymolysis, stir at 1000 rpm, the temperature of enzymolysis is 50℃, and the time is 2 h. After the enzymolysis is completed, filter with gauze, centrifuge at 2000 rpm for 15 min, remove the supernatant, cool it, and filter it, add 1% anhydrous glucose of the solution to the filtrate, sterilize it at high temperature, and obtain the asparagus extract.

[0103] (3) Finally, the bacterial suspension of Pichia pastoris was inoculated into the asparagus extract at a volume concentration of 1.5%, and the initial pH was adjusted to 7. The mixture was 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.

[0104] Comparative Example 4 Compared with Example 1, the only difference is that the initial pH of the fermentation is 5.5.

[0105] A method for preparing an asparagus fermentation extract comprises the following steps: (1) First, the brewer's yeast with the deposit number of CCTCC KY 2008613 ( Saccharomyces cerevisiae) was inoculated into YPD liquid culture medium and activated at 30℃ for 12 h to obtain the bacterial liquid of Saccharomyces cerevisiae.

[0106] (2) Cut off the two ends of the asparagus and take the middle, cut it into small pieces evenly, and put 50 g of asparagus into 500 mL of deionized water according to the solid-liquid ratio of 1:10, add 0.5% pectinase and 1% cellulase of asparagus for enzymolysis, stir at 1000 rpm, the temperature of enzymolysis is 50℃, and the time is 2 h. After the enzymolysis is completed, filter with gauze, centrifuge at 2000 rpm for 15 min, remove the supernatant, cool it, and filter it, add 1% anhydrous glucose of the solution to the filtrate, sterilize it at high temperature, and obtain the asparagus extract.

[0107] (3) Finally, the yeast culture liquid of brewer's yeast was inoculated into the asparagus extract at a volume concentration of 1.5%, and the initial pH was adjusted to 5.5. The fermentation was carried out 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.

[0108] Comparative Example 5 Compared with Example 1, the only difference is that the enzymolysis temperature is 30°C.

[0109] A method for preparing an asparagus fermentation extract comprises the following steps: (1) First, the brewer's yeast with the deposit number of CCTCC KY 2008613 ( Saccharomyces cerevisiae ) was inoculated into YPD liquid culture medium and activated at 30℃ for 12 h to obtain the bacterial liquid of Saccharomyces cerevisiae.

[0110] (2) Cut off the two ends of the asparagus and take the middle, cut it into small pieces evenly, and put 50 g of asparagus into 500 mL of deionized water according to the solid-liquid ratio of 1:10, add 0.5% pectinase and 1% cellulase of asparagus for enzymolysis, stir at 1000 rpm, the temperature of enzymolysis is 30℃, and the time is 2 h. After the enzymolysis is completed, filter with gauze, centrifuge at 2000 rpm for 15 min, remove the supernatant, cool it, and filter it, add 1% anhydrous glucose of the solution to the filtrate, sterilize it at high temperature, and obtain the asparagus extract.

[0111] (3) Finally, the yeast culture liquid of brewer's yeast was inoculated into the asparagus extract at a volume concentration of 1.5%, and the initial pH was adjusted to 7. The fermentation was carried out 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.

[0112] Comparative Example 6 A soothing and anti-aging essence, compared with Example 17, the only difference is that the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1 are replaced by an equal amount of purified water.

[0113] Comparative Example 7 A soothing and anti-aging eye cream, compared with Example 18, the only difference is that the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1 are replaced by an equal amount of purified water.

[0114] Comparative Example 8 A soothing and anti-aging moisturizing lotion, compared with Example 19, the only difference is that the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1 are replaced by an equal amount of purified water.

[0115] Comparative Example 9 A soothing and anti-aging sunscreen formula, compared with Example 20, the only difference is that the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1 are replaced with an equal amount of purified water.

[0116] Comparative Example 10 A soothing and anti-aging repairing facial mask, compared with Example 21, the only difference is that the asparagus fermentation extract and magnesium ascorbyl phosphate prepared in Example 1 are replaced by an equal amount of purified water.

[0117] Test Example 1 Evaluation of the soothing effect of Asparagus cochinchinensis fermented extract 1. Effects on RAW264.7 cell activity 1.1 Experimental methods: (1) Mouse monocytes / macrophages (RAW264.7 cells) were first inoculated in DMEM medium containing 10% fetal bovine serum at a cell density of 1×10 4 The cells were cultured in a 37°C incubator containing 5% CO2 for 24 h.

[0118] (2) Experimental groups were set up: Example 1 group, Comparative Example 1 group and control group, wherein, Example 1 group was added with 200 μL of the Asparagus cochinchinensis fermentation extract prepared in Example 1 diluted 1000 times, 800 times, 600 times, 400 times, 300 times, 200 times, 100 times, 50 times and 25 times with cell culture medium; Comparative Example 1 group was added with 200 μL of the Asparagus cochinchinensis extract prepared in Comparative Example 1 diluted 1000 times, 800 times, 600 times, 400 times, 300 times, 200 times, 100 times, 50 times and 25 times with cell culture medium; and the control group was added with 200 μL of cell culture medium.

[0119] (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.

[0120] 1.2 Experimental results: Figure 1 As shown, the cell viability of each group diluted from 1000 times to 25 times is maintained above 80%. With the increase of the dilution multiple, the cell activity of Example 1 shows a trend of increase → decrease → increase → decrease; while the cell activity of Comparative Example 1 shows a trend of increase → decrease → increase. Among them, the cell viability of the 25-fold and 50-fold dilution groups in Example 1 and Comparative Example 1 is still above 80%, indicating that the fermented extract of Asparagus cochinchinensis and the extract of Asparagus cochinchinensis have no obvious toxicity to cells, have good biosafety, and can be used as cosmetic raw materials to test their efficacy.

[0121] 2. Cellular inflammatory factors IL-6 The impact of expression 2.1 Experimental methods: (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 The inoculated cells were placed in a 37°C, 5% CO2 incubator for 24 h to allow them to adhere to the wall and grow.

[0122] (2) Experimental groups were set up: sample group, model group and control group. The control group was used as the baseline, and the model group was treated with the pro-inflammatory agent LPS to induce an inflammatory response. IL-6 increased expression levels.

[0123] Among them, the sample groups were 194 μL cell culture medium + 2 μL LPS (1 mg / mL) + 4 μL asparagus fermentation extract prepared in Example 1 (diluted 50 times), 190 μL cell culture medium + 2 μL LPS (1 mg / mL) + 8 μL asparagus fermentation extract prepared in Example 1 (diluted 25 times), 194 μL cell culture medium + 2 μL LPS (1 mg / mL) + 4 μL asparagus 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 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.

[0124] (3) After treatment, each group was cultured at 37℃ with 5% CO2 for 48 h. GAPDH As an internal reference, qPCR experiments were performed to detect IL-6 The primer sequences are shown in Table 6 to evaluate the effects of different concentrations of extract on IL-6 The inhibitory effect.

[0125] Table 6 Primer sequences

[0126] 2.2 Experimental results: Figure 2 As shown, in comparative example 1, when diluted 25 and 50 times, the cellular inflammatory factors IL-6 The expression of the asparagus extract under this concentration condition was similar to that of the model group. IL-6 The level was similar to that of the model group, indicating that the inflammatory response was not effectively inhibited at this concentration; while under higher concentration conditions, the 25-fold dilution group significantly reduced IL-6 The inhibition rate was 32.8%, indicating that at this concentration, the active ingredients in the fermented extract of Asparagus cochinchinensis effectively inhibited IL-6 , showing anti-inflammatory and soothing effects.

[0127] Test Example 2 Antioxidant Capacity Detection of Asparagus Fermentation Extract 1. Superoxide dismutase (SOD) activity assay 1.1 Experimental method: The supernatant of the fermented extract of Asparagus cochinchinensis prepared in Example 1 and the extract of Asparagus cochinchinensis prepared in Comparative Example 1 was diluted 25 times with distilled water as the test sample. The superoxide dismutase (SOD) activity was determined using a SOD activity detection kit in accordance with the instructions of the kit.

[0128] 1.2 Experimental results: Figure 3 As shown, the SOD activity of the asparagus fermented extract of Example 1 is 70.9 U / mg, and the SOD activity of the asparagus extract of Comparative Example 1 is 55 U / mg. This indicates that the fermentation treatment significantly increases the SOD activity in the asparagus extract, and its ability to scavenge superoxide free radicals is enhanced.

[0129] 2.ABTS + Free radical scavenging ability assay 2.1 Experimental methods: (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.

[0130] (2) Prepare 7 mM ABTS solution and 2.45 mM potassium persulfate solution, mix them, and place them away from light for 12-16 hours to generate ABTS radical cations. Before use, dilute the ABTS radical solution to an absorbance of 0.70±0.02 at 734 nm.

[0131] (3) Take 0.2 mL of the sample to be tested and add it to 2.8 mL of ABTS free radical solution in the sample tube. Place it in the dark at room temperature for 6 min and measure the absorbance at 734 nm using a spectrophotometer. Replace the sample solution with distilled water in the blank tube and replace ABTS in the control tube. + Working fluid, respectively denoted as A 样品 , A 空白 , A 对照 ABTS is calculated according to the following formula: + Free radical scavenging rate.

[0132] ABTS + Free radical scavenging rate (%) = [[A 空白 - (A 样品 -A 对照 )]÷A 空白 ]×100% Where: A 对照 Represents the absorbance of ABTS free radical solution; A 样品 It indicates the absorbance after adding the sample to be tested.

[0133] 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.

[0134] 3. DPPH free radical scavenging ability determination 3.1 Experimental methods: (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.

[0135] (2) Preheat the spectrophotometer for more than 30 min, adjust the wavelength to 515 nm, and set the wavelength to zero with anhydrous ethanol. Dilute the 1 mg / mL DPPH anhydrous ethanol solution with anhydrous ethanol until the absorbance at 515 nm is 0.8-1.0.

[0136] (3) Vortex mix, and leave to 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%.

[0137] 3.2 Experimental results: Figure 3 As shown, the DPPH free radical scavenging rate of the asparagus fermented extract of Example 1 is 83.4%, while that of Comparative Example 1 is 63.1%. This indicates that the fermented asparagus extract has a stronger free radical scavenging ability in the DPPH free radical scavenging experiment, and has a higher antioxidant activity, which helps to resist skin aging.

[0138] In summary, fermentation treatment significantly improved the performance of Asparagus cochinchinensis extract in multiple antioxidant indicators, especially in superoxide dismutase activity, ABTS + The fermentation process promotes the release and transformation of antioxidant components in the asparagus extract, improves its antioxidant activity, and the fermented asparagus extract can more effectively scavenge free radicals, delay cell aging, reduce inflammatory responses, and enhance its value as a cosmetic raw material in applications such as antioxidants and anti-inflammatory.

[0139] Test Example 3 Detection of active ingredients in fermented extract of Asparagus cochinchinensis 1. Determination of total polyphenol content As a key ingredient in cosmetics, polyphenols have powerful antioxidant and anti-inflammatory capabilities, which can effectively scavenge free radicals, reduce inflammation and delay skin aging.

[0140] 1.1 The total polyphenol content in the sample was determined by the Folin-phenol reagent method. The experimental method is: (1) The fermented asparagus extracts prepared in Examples 1-16, the fermented asparagus extracts prepared in Comparative Examples 3-5, the asparagus extract prepared in Comparative Example 1, and the saccharomyces cerevisiae culture solution prepared in Comparative Example 2 were diluted 25 times with distilled water as test samples.

[0141] (2) Prepare gallic acid standard solutions with concentrations of 0, 20, 40, 60, 80 and 100 µg / mL. Add 2.5 mL of Folin-phenol reagent to each solution and dilute the two solutions in a volume ratio of 1:10. After standing for 5 min, add 2 mL of 7.5% sodium carbonate solution. After reacting at room temperature in the dark for 30 min, use a spectrophotometer to measure the absorbance at a wavelength of 760 nm and draw a standard curve.

[0142] (3) Take 0.5 mL of the sample to be tested and place it in a 25 mL volumetric flask. Add 5 mL of distilled water to dilute and shake well. Add 2.5 mL of Folin-phenol reagent. After standing for 5 minutes, add 2 mL of 7.5% sodium carbonate solution. After reacting in the dark for 30 minutes, measure the sample absorbance at a wavelength of 760 nm. Calculate the total polyphenol content in the sample using the standard curve according to the following formula: Total polyphenol content (mg / mL) = (C×V1)÷V2.

[0143] Wherein: 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 the asparagus extract, mL; V2 represents the total volume of the asparagus fermentation extract, mL.

[0144] 1.2 Experimental results: As shown in Table 7, the polyphenol content in the asparagus fermentation extract is as high as 48.5 mg / mL, indicating that the fermentation treatment increases the polyphenol content in the asparagus extract, while the saccharomyces cerevisiae in the YPD liquid medium without the asparagus extract (Comparative Example 2) does not produce polyphenols during the fermentation process. The metabolic action of saccharomyces cerevisiae during the fermentation process of Example 1 promotes the release and activation of polyphenols in the asparagus fermentation extract, which has the potential to enhance the anti-inflammatory, antioxidant and anti-aging functions of the asparagus extract in cosmetics. In addition, the asparagus fermentation extract may also increase the bioavailability of polyphenols during the fermentation process, making it easier for the human body to absorb and utilize.

[0145] Table 7 Total polyphenol content

[0146] Note: Compared with Example 1, *** express P <0.001.

[0147] 2. Determination of saponin content 2.1 The saponin content in the sample was determined by the vanillin-sulfuric acid method. Experimental method: (1) The fermented asparagus extracts prepared in Examples 1-16, the fermented asparagus extracts prepared in Comparative Examples 3-5, the asparagus extract prepared in Comparative Example 1, and the saccharomyces cerevisiae culture liquid prepared in Comparative Example 2 were subjected to saponin extraction using ethanol as a solvent at a solid-liquid ratio of 1 g:10 mL. The extraction was carried out in a 50°C water bath for 2 h. After filtration, the ethanol was removed by rotary evaporation. The supernatant was concentrated and diluted 25 times with distilled water as a sample to be tested.

[0148] (2) Diosgenin was prepared into saponin standard solutions with concentrations of 0, 10, 20, 40, 60, 80 and 100 µg / mL. To 1 mL of the saponin standard solution, 1 mL of 1% vanillin solution (vanillin dissolved in glacial acetic acid) was added first, and then 5 mL of concentrated sulfuric acid was quickly added. The solution was allowed to stand at room temperature for 15 min. The absorbance was measured at a wavelength of 560 nm using a spectrophotometer and a standard curve was drawn.

[0149] (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 let stand at room temperature for 15 min to generate red compounds. Detect the absorbance at a wavelength of 560 nm. Calculate the total saponin content in the sample using the standard curve according to the following formula.

[0150] Total saponin content (mg / mL) = (C×V1) ÷ V2 Wherein: C represents the concentration of saponin in the sample to be tested obtained according to the standard curve, mg / mL; V1 represents the total volume of the asparagus extract, mL; V2 represents the total volume of the asparagus fermentation extract, mL.

[0151] 2.2 Experimental results: As shown in Table 8, the saponin content of the asparagus fermentation extract was 19.5 mg / mL at the highest, which was higher than 10.3 mg / mL in Comparative Example 1, indicating that the fermentation treatment increased the saponin content in the asparagus extract, while the saccharomyces cerevisiae in the YPD liquid medium without the asparagus extract (Comparative Example 2) did not produce saponins during the fermentation process. This indicates that the metabolism of saccharomyces cerevisiae during the fermentation process promoted the release and activation of saponins in the asparagus fermentation extract, making it have stronger anti-inflammatory and anti-aging potential in cosmetics.

[0152] Table 8 Total saponin content

[0153] Note: Compared with Example 1, *** express P<0.001.

[0154] 3. Determination of polysaccharide content 3.1 Experimental methods: (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 bacterial solution prepared in Comparative Example 2 were subjected to polysaccharide extraction using distilled water as a solvent at a solid-liquid ratio of 1 g:10 mL. The extraction was carried out in a 50°C water bath for 2 h, filtered, and centrifuged at 4000 rpm for 10 min. The supernatant was concentrated and diluted 25 times with distilled water as a sample to be tested.

[0155] (2) Prepare glucose standard solutions with concentrations of 0, 10, 20, 40, 60, 80 and 100 µg / mL. Take 1 mL of glucose standard solution, first add 1 mL of 5% phenol solution, then quickly add 5 mL of concentrated sulfuric acid. 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.

[0156] (4) Take 1 mL of the sample to be tested, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid, mix well and let stand at room temperature for 15-30 min to generate yellow to orange compounds. Detect the absorbance at 490 nm. Calculate the polysaccharide content in the sample using the standard curve according to the following formula.

[0157] Polysaccharide content (mg / mL) = (C×V1) ÷ V2 Wherein: C represents the concentration of glucose in the sample to be tested obtained according to the standard curve, mg / mL; V1 represents the total volume of the asparagus extract, mL; V2 represents the total volume of the asparagus fermentation extract, mL.

[0158] 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 the fermentation treatment significantly increased the polysaccharide content in the asparagus extract, while the cerevisiae yeast in the YPD liquid medium without the asparagus extract (Comparative Example 2) only metabolized and produced a small amount of polysaccharides during the fermentation process. This indicates that the metabolism of cerevisiae yeast during the fermentation process promoted the release and activation of polysaccharides in the asparagus fermentation extract, which has the potential effects of moisturizing, anti-oxidation, immunomodulation and promoting collagen production.

[0159] Table 9 Polysaccharide content

[0160] Note: Compared with Example 1, *** express P <0.001.

[0161] Test Example 4 Evaluation of the soothing and anti-aging effects of cosmetic compositions 1. Effects on RAW264.7 cell activity 1.1 Experimental methods: (1) RAW264.7 cells were inoculated in DMEM medium containing 10% fetal bovine serum at a cell density of 1×10 4 The cells were cultured in a 37°C incubator containing 5% CO2 for 24 h.

[0162] (2) Experimental groups were set up: sample group and control group. The sample group was added with 200 μL of cell culture medium containing the cosmetic composition numbered 1-15 in Table 10; the control group was added with 200 μL of cell culture medium containing PBS. When the cell confluence reached 60%, the drug treatment was given according to the above grouping.

[0163] (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.

[0164] Table 10 Composition of cosmetic composition

[0165] 1.2 Experimental results: Figure 4 As shown, no cell morphology changes and no obvious toxicity were observed in each sample group within the test range.

[0166] 2. Cellular inflammatory factors IL-6 The impact of expression 2.1 Experimental methods: (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 The seeded cells were placed in a 37°C, 5% CO2 incubator for 24 h to allow them to adapt to the environment and adhere to the wall.

[0167] (2) Experimental groups were set up: sample group, model group and control group. The sample group was added with 200 μL of cell culture medium containing the cosmetic composition numbered 1-15 in Table 10; the model group was added with 200 μL of cell culture medium containing 10 μg / mL LPS; and the control group was added with 200 μL of cell culture medium containing PBS. When the cell fusion degree reached 60%, the drug treatment was carried out according to the above grouping. Taking the control group as the baseline, the model group was treated with the pro-inflammatory agent LPS to induce an inflammatory response, so that IL-6 increased expression levels.

[0168] (3) After treatment, each group was cultured at 37℃ with 5% CO2 for 48 h. GAPDH As an internal reference, qPCR experiments were performed to detect IL-6 The expression level, primer sequences are shown in Table 6, and the expression was calculated according to the following formula IL-6 inhibition rate.

[0169] Inhibition rate (%) = (1- IL-6 Expression level ÷ IL-6 expression level in the model group) × 100% 2.2 Experimental results: Inflammatory factors in each group IL-6 The relative expression level Figure 5 As shown in the figure, compared with the control group, the inflammatory factors in the model group IL-6 The content of was significantly increased, indicating that the RAW264.7 cell inflammation model has been successfully established.

[0170] Compared with the model group, the fermented extract of Asparagus cochinchinensis (No. 1-5) had an effect on the expression of 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 fermentation extract could significantly inhibit the inflammatory factors. IL-6 The expression ( P <0.001), and the inhibitory effect was dose-dependent with Asparagus cochinchinensis fermentation extract; 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 group 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.

[0171] 3. Evaluation of anti-aging efficacy 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 helix 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 helix structure. The human type I collagen gene mainly includes COL1A1 and COL1A2 . COL1A1The gene encodes the α1 chain of type I collagen, which is an important component of the triple helix structure of type I collagen; COL1A2 Gene encoding the α2 chain of type I collagen COL1A1 The α1 chain encoded by the gene is synthesized in a ratio of 2:1 to form the triple helix structure of type I collagen. Type I collagen and elastin are distributed in the dermis. After being affected by factors such as hydrogen peroxide, the content of collagen and elastin in the extracellular matrix decreases, showing degeneration of the skin's appearance and structure and decreased function. The senescence of human skin fibroblasts (HSF) was induced by H2O2 to verify the anti-aging effect of the cosmetic compositions numbered 1-15 in Table 10.

[0172] 3.1 Experimental methods: (1) Culture primary human dermal fibroblasts (P2) to 2×10 5 After culturing for 24 hours, when the cell confluence reached 60%, the experimental groups were set up: sample group, model group and control group. Among them, the sample group was 200 μL of serum-free low-sugar DMEM medium containing the cosmetic composition numbered 1-15 in Table 10 and 400 μmol / L hydrogen peroxide; the model group was treated with 200 μL of serum-free low-sugar DMEM medium containing 400 μmol / L hydrogen peroxide at 37°C for 2 hours every day, and the human skin fibroblasts were washed 3 times with PBS, replaced with low-sugar DMEM medium containing serum, and then continued to be cultured for 22 hours, for a total of 3 days; the control group was serum-free low-sugar DMEM medium without hydrogen peroxide.

[0173] (2) After 72 h of treatment, cells were collected and RNA was extracted. GAPDH As an internal reference, qPCR was used to detect the type I collagen-related genes in the sample group, model group and control group ( COL1A1 and COL1A2 ), elastin gene ( ELNA ), and the primer sequences are shown in Table 11.

[0174] Table 11 Primer sequences

[0175] The gene is calculated according to the following formula COL1A1 , COL1A2 and ELNA The growth rate of expression was analyzed using SPSS software to see if there was any statistical difference.

[0176] Growth rate of gene expression (%) = (M-T) ÷ M × 100%; Where: T represents the average relative gene expression level of the sample group; M represents the average relative gene expression level of the model group.

[0177] 3.2 Experimental results: 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.

[0178] Depend on Figure 7 The type I collagen gene ( COL1A1 and COL1A2 ), elastin gene ( ELNA ) expression growth rate. The results show that: 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 extract of Asparagus cochinchinensis fermentation.

[0179] Compared with the model group, the magnesium ascorbyl phosphate group (No. 6-10) had a negative effect on 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.

[0180] Compared with the model group, the combined groups (No. 11-15) had COL1A1 The growth rates of expression were 100.7%, 95.0%, 83.0%, 83.0% and 55.7%; COL1A2 The growth rates of expression were 106.9%, 94.3%, 82.7%, 73.3% and 51.9% respectively; ELNA The growth rates of expression were 68.0%, 57.0%, 48.7%, 41.3% and 30.7% respectively. Compared with the Asparagus cochinchinensis fermented extract group (No. 1-5), the compound group (No. 11-15) showed a significant improvement in promoting type I collagen and elastin, and its anti-wrinkle and firming effects were the best.

[0181] Test Example 5 Evaluation of the soothing and anti-aging effects of cosmetics 1. Evaluation of the soothing effects of the cosmetics prepared in Examples 17-21 and Comparative Examples 6-10 A copper sulfate-induced neutrophil aggregation model was used to test the soothing efficacy.

[0182] 1.1 Experimental methods: (1) Experimental groups were set up: sample group, positive control group, model group and blank group. The sample groups were 10 µM anhydrous copper sulfate + 0.1% mass concentration of cosmetic solutions prepared in Examples 4-8 and Comparative Examples 5-9 (the cosmetics were dissolved in zebrafish embryo culture medium); the positive control group was 10 µM anhydrous copper sulfate + 10 µM indomethacin; the model group was 10 µM anhydrous copper sulfate; and the blank group was zebrafish embryo culture medium.

[0183] (2) Wild-type AB strain zebrafish embryos were divided into 13 groups, 15 in each group, and exposed to the sample group, positive control group, model group, and blank control group. After being cultured in a constant temperature incubator at 28±1℃ for 40 min, the fish embryos were fixed and stained with Sudan black, and the number of neutrophils in the lateral line area was observed and recorded under a stereo microscope. The neutrophil aggregation inhibition rate of each treatment group was calculated according to the following formula.

[0184] Neutrophil aggregation inhibition rate (%) = (M-S) ÷ M × 100% Where: S represents the average number of neutrophils in zebrafish embryos of each treatment group; M represents the average number of neutrophils in zebrafish embryos of the model group.

[0185] 1.2 Experimental results: As shown in Table 12 and Figure 6 As shown, compared with the blank control group, the number of neutrophils on the skin surface of zebrafish in the model group was significantly increased, indicating that the zebrafish soothing model was successfully established.

[0186] Compared with the model group, the inhibition rates of comparative examples 6-10 groups on zebrafish embryo neutrophil aggregation were 24.22%, 23.32%, 21.75%, 18.39% and 27.58%, respectively; while the inhibition rates of examples 17-21 groups were 44.39%, 42.60%, 38.12%, 37.00% and 34.53%, respectively, and the examples 17-21 groups were significantly different from the comparative examples 6-10 groups ( P <0.001).

[0187] Table 12 Number of centrioles in zebrafish embryos

[0188] 2. Evaluation of the anti-aging effects of the cosmetics prepared in Examples 17-21 and Comparative Examples 6-10 Using Caenorhabditis elegans as a model system for aging research, anti-aging efficacy was tested.

[0189] 2.1 Experimental methods: (1) Experimental groups were set up: a sample group and a blank group, wherein the sample groups were 1 g of the cosmetics prepared in Examples 17-21 and Comparative Examples 6-10 respectively + a nematode growth medium containing Escherichia coli OP50 liquid, and the blank group was M9 buffer + NGM medium containing Escherichia coli OP50 liquid.

[0190] (2) The synchronized N2 wild-type C. elegans were placed in the culture medium of the sample group and the blank group and cultured at 20°C, with no less than 50 C. elegans per plate. The growth of C. elegans was observed every day, and the nematodes that escaped, died from drying, or had their reproductive tract everted were removed. The living C. elegans were placed on a new plate, and the average lifespan and maximum lifespan (in days) of the C. elegans were recorded. If the C. elegans did not respond to stimulation twice, it was considered dead. The death time of the last nematode in each group was determined as the maximum lifespan of the nematode. Each group of experiments was repeated 3 times.

[0191] 2.2 Experimental results: Figure 8-Figure 9As shown, compared with the blank group, Examples 17-21 can significantly prolong the average lifespan and maximum lifespan of Caenorhabditis elegans. The average lifespans of Examples 17-21 are 14.23 d, 12.93 d, 12.05 d, 11.36 d and 11.12 d, respectively, and the maximum lifespans are 24.32 d, 22.16 d, 21.06 d, 20.13 d and 19.69 d, respectively; while the average lifespans of Comparative Examples 6-10 are 10.32 d, 10.16 d, 10.06 d, 10.35 d and 10.33 d, respectively, and the maximum lifespans are 15.34 d, 15.16 d, 14.96 d, 15.69 d and 16.03 d, respectively. Compared with Comparative Examples 6-10, Examples 17-21 have extremely significant differences ( P <0.001).

[0192] In Examples 17-21, as the concentration of the asparagus fermentation extract and magnesium ascorbyl phosphate composition increases, the effect of extending the lifespan of Caenorhabditis elegans becomes more obvious. Among them, Example 19 (the content of asparagus fermentation extract and magnesium ascorbyl phosphate is 30% and 1.5%, respectively) has the best effect of extending the lifespan. Compared with the blank group, the average lifespan increased by 29.2% and the maximum lifespan increased by 37.9%; compared with Comparative Example 6, the average lifespan increased by 27.5% and the maximum lifespan increased by 36.9%.

[0193] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

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 )'s bacterial liquid is fermented with asparagus extract as raw material to obtain the product.

2. The preparation method according to claim 1, characterized in that: The preparation process of the bacterial liquid includes inoculating brewer's yeast into a YPD liquid culture medium and activating it at 25-35° C. for 10-14 h.

3. The preparation method according to claim 1, characterized in that: The preparation process of the asparagus extract comprises: firstly placing the 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 extract.

4. The preparation method according to claim 3, characterized in that: The solid-liquid ratio of the asparagus and water is 1 g:8-12 mL; the total amount of the pectinase and the cellulase added is 1-2% of the mass of the asparagus, and the amount of the cellulase added is 0.5-1.5% of the mass of the asparagus; the temperature of the enzymatic hydrolysis is 40-60° C., the time of the enzymatic hydrolysis is 1.5-3 h, and the enzymatic hydrolysis needs to be stirred at 800-1200 rpm; the speed of the centrifugation is 1800-2200 rpm, and the time of the centrifugation is 12-18 min; the mass concentration of the anhydrous glucose in the filtrate is 0.8-1.2%.

5. 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%; the fermentation process includes inoculating the bacterial liquid of brewer's yeast into the asparagus extract, adjusting the measured initial pH to 6-7, and then fermenting for 36-48 hours at a rotation speed of 120-180 rpm and 25-35°C; after the fermentation is completed, post-treatment is also performed, and the post-treatment includes sterilization at 120-122°C for 28-32 minutes, centrifugation at 1500-2500 rpm for 10-20 minutes and filtration.

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

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

8. The cosmetic composition according to claim 7, 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%.

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

10. Use of the fermented asparagus extract prepared by the preparation method according to any one of claims 1 to 5 in the preparation of cosmetics with antioxidant, anti-inflammatory, soothing and anti-aging effects.

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