Ganoderma lucidum probiotic fermented whitening composition as well as preparation method and application thereof

Abstract

The invention provides a ganoderma lucidum probiotic fermented whitening composition as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. Comprising the following steps: S1, fermentation of ganoderma lucidum; preparing a bacteria dissolving product; s3, preparation of a camptothecin / quercetin eutectic crystal; s4, preparation of the bornyl gallate; and S5, preparing the ganoderma lucidum probiotic fermented whitening composition. The ganoderma lucidum probiotic fermented whitening composition prepared by the invention has good beautifying effects of whitening, moisturizing, relieving, resisting oxidation, removing freckles, removing wrinkles and the like, increases the wettability of the skin, is good in stability, mild and non-irritating to the skin, has a good transdermal absorption promoting effect, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a probiotic fermentation whitening composition of Tai Sui bacteria and a preparation method and application thereof. Background Art

[0002] With the improvement of modern living standards, people pay more and more attention to their external image, and use a good image to increase their self-confidence in various activities and occasions. At the same time, under a good appearance, they pursue skin health more, and use healthy skin and a good image to show people's pursuit of high-end life. Whitening cosmetics have evolved from physical covering whitening to physical and chemical peeling, and then to interfering with and blocking the normal metabolism of melanin, and then to "synergistic effect" whitening. However, the existing whitening methods will cause skin damage to consumers and have poor whitening effects, so new drugs need to be developed.

[0003] The Compendium of Materia Medica records that the Tai Sui is "a meat-like ganoderma. The red ones are like coral, the white ones are like fat, and the black ones are like lacquer... all are bright and clear like ice." Because the living meat balls found in various places have similar characteristics to the Tai Sui recorded in the book, the name has been adopted. The main active ingredients in Tai Sui are pyrroloquinoline quinone, amino acids, vitamins, and trace elements, which have pharmacological effects such as replenishing qi, eliminating fatigue, and delaying aging.

[0004] At present, some bacteria, yeasts, molds and a small amount of slime molds have been isolated from Gastrodia elata. However, the microorganisms isolated by traditional methods are far from representing all types of microorganisms in Gastrodia elata. Continuously developing new methods to separate and recognize the unidentified types of microorganisms in Gastrodia elata is of great significance for its large-scale utilization. Summary of the invention

[0005] The purpose of the present invention is to propose a Tai Sui probiotic fermented whitening composition and its preparation method and application, which has good whitening, moisturizing, soothing, anti-oxidation, freckle removal, wrinkle removal and other beauty effects, increases the moisture content of the skin, has good stability, is mild and non-irritating to the skin, has good transdermal absorption promotion effect, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way: The present invention provides a method for preparing a probiotic fermented whitening composition of Tai Sui bacteria, comprising the following steps: S1. Fermentation of Tai Sui: The Tai Sui was dried, crushed, added to sterile water, inoculated with Aspergillus niger and Aspergillus oryzae activation solution, fermented, filtered, washed with solids, the culture solution was collected, freeze-dried, and a composite bacteria was obtained; the filtrate was freeze-dried to obtain Tai Sui fermentation broth; S2. Preparation of lysate: adding the composite bacteria to water, adding composite enzyme for enzymolysis, inactivating the enzyme, filtering, and freeze-drying the filtrate to obtain a lysate; S3. Preparation of camptothecin / quercetin cocrystal: camptothecin was dissolved in ethanol to obtain solution 1; quercetin was dissolved in ethyl acetate to obtain solution 2, solution 1 and solution 2 were mixed uniformly, the solvent was evaporated, filtered, washed, dried, and ball-milled to obtain camptothecin / quercetin cocrystal; S4. Preparation of gallic acid bornyl alcohol ester: Gallic acid and bornyl alcohol were added to toluene, concentrated sulfuric acid was added, the reaction was heated to reflux, cooled to room temperature, filtered, washed, and dried to obtain gallic acid bornyl alcohol ester; S5. Preparation of Tai Sui probiotic fermentation whitening composition: Tai Sui fermentation broth, lysate, camptothecin / quercetin eutectic, menthol and bornyl gallate are uniformly mixed to prepare Tai Sui probiotic fermentation whitening composition.

[0007] As a further improvement of the present invention, the bacterial content of the Aspergillus niger and Aspergillus oryzae activated solution in step S1 is 10 8 -10 9 cfu / mL, the inoculation amounts were 1-2v / v% and 0.5-1.5v / v%, respectively, and the fermentation culture conditions were 25-30°C, 100-200r / min, and the fermentation culture was 24-36h.

[0008] As a further improvement of the present invention, the mass ratio of the composite bacteria and the composite enzyme in step S2 is 10:1-2, the composite enzyme is a mixture of modified lysozyme, snail enzyme and laccase in a mass ratio of 10:3-5:2-3, the temperature of the enzymatic hydrolysis is 35-45°C, and the time is 2-4h.

[0009] As a further improvement of the present invention, the preparation method of the modified lysozyme is as follows: T1. Stearic acid and oleic acid were mixed and added to water, NHS and EDC were added to activate the reaction, lysozyme was added, the reaction was stirred, dialyzed, and freeze-dried to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to react and obtain modified lysozyme.

[0010] As a further improvement of the present invention, the mass ratio of stearic acid, oleic acid, NHS, EDC and lysozyme is 3-5:2-3:0.5-1:0.7-1.2:10, the activation reaction time is 20-40 min, the stirring reaction time is 4-6 h, the heating reaction temperature is 55-65° C., and the time is 30-40 min.

[0011] As a further improvement of the present invention, the mass ratio of camptothecin to quercetin in step S3 is 3-5:2-3.

[0012] As a further improvement of the present invention, the molar ratio of gallic acid to borneol in step S4 is 1-1.1:1, and the heating reflux reaction time is 5-7h.

[0013] As a further improvement of the present invention, the mass ratio of the Tai Sui fermentation broth, the lysate, the camptothecin / quercetin cocrystal, the menthol and the gallic acid bornyl alcohol ester in step S5 is 15-20:3-5:1-2:0.2-0.4:0.1-0.2.

[0014] The present invention further protects a probiotic fermentation whitening composition prepared by the above-mentioned preparation method.

[0015] The present invention further protects the use of the above-mentioned Tai Sui probiotic fermentation whitening composition in cosmetics.

[0016] The present invention has the following beneficial effects: Aspergillus oryzae fermentation can change the chemical composition of the matrix, help the active compounds to be converted into different metabolites, thereby exerting other biological activities, and is also beneficial to increase the content of biologically active compounds in the extract, produce components that have an inhibitory effect on tyrosinase, and whiten the skin. Aspergillus niger fermentation can increase the concentration of active components in the fermented product, produce new compounds of polyphenols and flavonoids, improve the antioxidant effect of the fermented product, and reduce the production of melanin, which has the effect of whitening the skin. The present invention ferments the ginseng by composite Aspergillus, greatly improving the output of active components, rich in organic germanium, nucleic acids, polysaccharides, vitamins and amino acids, lecithin, coenzymes and other nutrients, and has good whitening, moisturizing, soothing skin and other effects.

[0017] At the same time, the lysate obtained by enzymatic hydrolysis of fungal mud has high safety and can also be used as a moisturizing ingredient. The neutral glycosphingolipids present in its cell membrane can increase the moisture content of the skin. At the same time, the fungal cell wall is one of the main natural sources of β-glucan and has good antioxidant and moisturizing effects.

[0018] Lysozyme achieves antibacterial effect by destroying the β-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in the bacterial peptidoglycan layer. Since the peptidoglycan content in Gram-positive bacteria reaches 80%, while there is only a small amount of peptidoglycan in Gram-negative bacteria, this affects its antibacterial effect on negative bacteria to a certain extent, and the bacteriolytic effect on fungi is not good. The present invention uses stearic acid and oleic acid to mix and modify lysozyme to obtain an amphiphilic enzyme molecule. At the same time, the three-dimensional structure of the enzyme molecule is changed by heating, exposing more hydrophobic groups, and greatly improving the fungal cell wall breaking effect. By cooperating with snail enzyme and laccase for enzymolysis, the fungal cell wall can be broken to obtain a lysate.

[0019] Camptothecin can inhibit human skin pigmentation and inhibit tyrosinase activity in B16 cells induced by α-melanocyte stimulating hormone. Quercetin exhibits good tyrosinase inhibition, so the synthesis of melanin is limited. However, their water solubility is poor, which greatly limits their bioavailability. The present invention greatly improves the water solubility of the product by preparing cocrystals, thereby facilitating skin penetration and absorption and improving the whitening effect.

[0020] Gallic acid inhibits melanin synthesis and tyrosinase activity, reduces the gene expression of proteins related to melanin synthesis, and inhibits melanin synthesis by phosphorylating and activating melanin production inhibitory proteins, and can also inhibit melanin synthesis by inhibiting the downstream signal transduction pathway of melanin synthesis. However, it has a certain irritation to the skin and is prone to cause problems such as redness, swelling, and allergies. Borneol has a good effect of promoting transdermal absorption, but it is volatile and has poor storage stability. The gallic acid borneol ester prepared by the present invention greatly improves the molecular weight of the product, thereby improving its stability, is not easy to volatilize, and is mild and non-irritating to the skin, has a good effect of promoting transdermal absorption, and has a synergistic effect with menthol.

[0021] The Tai Sui probiotic fermented whitening composition prepared by the present invention has good whitening, moisturizing, soothing, anti-oxidation, freckle removal, wrinkle removal and other cosmetic effects, increases the moisture content of the skin, has good stability, is mild and non-irritating to the skin, has good transdermal absorption promotion effect, and has broad application prospects. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The preparation method of Aspergillus niger and Aspergillus oryzae activation liquid is as follows: Aspergillus niger and Aspergillus oryzae are inoculated into Goulburn medium respectively, and activated and cultured for 18-24 hours at 25°C and 100r / min.

[0024] Aspergillus niger, 20 billion cfu / mL, was purchased from Shandong Changtai Biotechnology Co., Ltd.; Aspergillus oryzae, 20 billion cfu / mL, was purchased from Hubei Xingdongcheng Chemical Co., Ltd.

[0025] Lysozyme, 100 U / mg, was purchased from Shandong Gushuo Biotechnology Co., Ltd.; snail enzyme, with a cell wall breaking rate of 90%, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; laccase, 10,000 U / g, was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.

[0026] Preparation Example 1 Preparation of modified lysozyme Here’s how: T1. 3g of stearic acid and 2g of oleic acid were mixed and added to water, 0.5g of NHS and 0.7g of EDC were added, the activation reaction was carried out for 20min, 10g of lysozyme was added, the reaction was stirred for 4h, dialyzed, and freeze-dried to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to 55°C and react for 30 minutes to obtain modified lysozyme.

[0027] Preparation Example 2 Preparation of modified lysozyme Here’s how: T1. 5 g of stearic acid and 3 g of oleic acid were mixed and added to water, 1 g of NHS and 1.2 g of EDC were added, the activation reaction was carried out for 40 min, 10 g of lysozyme was added, the reaction was stirred for 6 h, dialyzed, and freeze-dried to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to 65°C and react for 40 minutes to obtain modified lysozyme.

[0028] Preparation Example 3 Preparation of modified lysozyme Here’s how: T1. 4 g of stearic acid and 2.5 g of oleic acid were mixed and added to water, 0.7 g of NHS and 1 g of EDC were added, the reaction was activated for 30 min, 10 g of lysozyme was added, the reaction was stirred for 5 h, dialyzed, and freeze-dried to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to 60°C and react for 35 minutes to obtain modified lysozyme.

[0029] Comparative Preparation Example 1 Compared with Preparation Example 3, the difference is that stearic acid is not added in step T1.

[0030] The details are as follows: T1. 6.5 g of oleic acid was mixed and added to water, 0.7 g of NHS and 1 g of EDC were added, the activation reaction was carried out for 30 min, 10 g of lysozyme was added, the reaction was stirred for 5 h, dialyzed, and freeze-dried to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to 60°C and react for 35 minutes to obtain modified lysozyme.

[0031] Comparative Preparation Example 2 Compared with Preparation Example 3, the difference is that oleic acid is not added in step T1.

[0032] The details are as follows: T1. 6.5 g of stearic acid was mixed with water, 0.7 g of NHS and 1 g of EDC were added, the activation reaction was carried out for 30 min, 10 g of lysozyme was added, the reaction was stirred for 5 h, dialyzed, and freeze-dried to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to 60°C and react for 35 minutes to obtain modified lysozyme.

[0033] Comparative Preparation Example 3 Compared with Preparation Example 3, the difference is that step T2 is not performed.

[0034] The details are as follows: T1. Mix 4g stearic acid and 2.5g oleic acid and add to water, add 0.7g NHS and 1g EDC, activate reaction for 30min, add 10g lysozyme, stir reaction for 5h, dialyze, freeze-dry to obtain modified lysozyme.

[0035] Example 1

[0036] This embodiment provides a method for preparing a probiotic fermented whitening composition of Tai Sui, comprising the following steps: S1. Fermentation of Ganoderma lucidum: 10 g of Ganoderma lucidum was dried, crushed, and added to 200 mL of sterile water. The inoculation amount was 10 8 -10 9 cfu / mL of Aspergillus niger and Aspergillus oryzae activated liquid, inoculated at 1v / v% and 0.5v / v%, respectively, at 25°C, 100r / min, fermented for 24h, filtered, washed with solid, collected the bacterial liquid, freeze-dried to obtain composite bacteria; the filtrate was freeze-dried to obtain Tai Sui fermented liquid; S2. Preparation of lysate: 10 g of composite bacteria was added to 300 mL of water, 1 g of composite enzyme was added, enzymolysis was carried out at 35°C for 2 h, the enzyme was inactivated, the filtrate was filtered, and the filtrate was freeze-dried to obtain a lysate; The complex enzyme is a mixture of modified lysozyme, snail enzyme and laccase, with a mass ratio of 10:3:2; S3. Preparation of camptothecin / quercetin cocrystal: 3 g of camptothecin was dissolved in 200 mL of ethanol to obtain solution 1; 2 g of quercetin was dissolved in 200 mL of ethyl acetate to obtain solution 2, solution 1 and solution 2 were mixed for 10 min, the solvent was evaporated, filtered, washed, dried, and ball-milled for 1 h to obtain camptothecin / quercetin cocrystal; S4. Preparation of gallic acid bornyl alcohol ester: 0.1 mol of gallic acid and 0.1 mol of bornyl alcohol were added to 500 mL of toluene, 1 mL of concentrated sulfuric acid was added, and the reaction was heated under reflux for 5 h, cooled to room temperature, filtered, washed, and dried to obtain gallic acid bornyl alcohol ester; S5. Preparation of Tai Sui probiotic fermentation whitening composition: 15 g Tai Sui fermentation broth, 3 g lysate, 1 g camptothecin / quercetin eutectic, 0.2 g menthol and 0.1 g bornyl gallate were stirred and mixed for 15 min to prepare Tai Sui probiotic fermentation whitening composition.

[0037] Example 2

[0038] This embodiment provides a method for preparing a probiotic fermented whitening composition of Tai Sui, comprising the following steps: S1. Fermentation of Ganoderma lucidum: 10 g of Ganoderma lucidum was dried, crushed, and added to 200 mL of sterile water. The inoculation amount was 10 8 -10 9 cfu / mL of Aspergillus niger and Aspergillus oryzae activated liquid, inoculated at 2v / v% and 1.5v / v% respectively, at 30°C, 200r / min, fermented for 36h, filtered, washed with solid, collected the bacterial liquid, freeze-dried to obtain composite bacteria; the filtrate was freeze-dried to obtain Tai Sui fermented liquid; S2. Preparation of lysate: 10 g of the composite bacteria was added to 300 mL of water, 2 g of the composite enzyme was added, enzymatic hydrolysis was performed at 45°C for 4 h, the enzyme was inactivated, the filtrate was filtered, and the filtrate was freeze-dried to obtain a lysate; The complex enzyme is a mixture of modified lysozyme, snail enzyme and laccase, with a mass ratio of 10:5:3; S3. Preparation of camptothecin / quercetin cocrystal: 5 g of camptothecin was dissolved in 200 mL of ethanol to obtain solution 1; 3 g of quercetin was dissolved in 200 mL of ethyl acetate to obtain solution 2, solution 1 and solution 2 were mixed for 10 min, the solvent was evaporated, filtered, washed, dried, and ball-milled for 1 h to obtain camptothecin / quercetin cocrystal; S4. Preparation of gallic acid bornyl alcohol ester: 0.11 mol of gallic acid and 0.1 mol of bornyl alcohol were added to 500 mL of toluene, 2 mL of concentrated sulfuric acid was added, and the reaction was heated under reflux for 7 h, cooled to room temperature, filtered, washed, and dried to obtain gallic acid bornyl alcohol ester; S5. Preparation of Tai Sui probiotic fermentation whitening composition: 20 g Tai Sui fermentation broth, 5 g lysate, 2 g camptothecin / quercetin eutectic, 0.4 g menthol and 0.2 g bornyl gallate were stirred and mixed for 15 min to prepare Tai Sui probiotic fermentation whitening composition.

[0039] Example 3

[0040] This embodiment provides a method for preparing a probiotic fermented whitening composition of Tai Sui, comprising the following steps: S1. Fermentation of Ganoderma lucidum: 10 g of Ganoderma lucidum was dried, crushed, and added to 200 mL of sterile water. The inoculation amount was 10 8 -10 9cfu / mL of Aspergillus niger and Aspergillus oryzae activated liquid, inoculated at 1.5 v / v% and 1 v / v%, respectively, at 27°C, 150 r / min, fermented for 30 h, filtered, washed with solids, collected the bacterial liquid, freeze-dried to obtain a composite bacteria; the filtrate was freeze-dried to obtain a Tai Sui fermentation liquid; S2. Preparation of lysate: 10 g of composite bacteria was added to 300 mL of water, 1.5 g of composite enzyme was added, enzymolysis was performed at 40°C for 3 h, the enzyme was inactivated, the filtrate was filtered, and the filtrate was freeze-dried to obtain a lysate; The complex enzyme is a mixture of modified lysozyme, snail enzyme and laccase, with a mass ratio of 10:4:2.5; S3. Preparation of camptothecin / quercetin cocrystal: 4 g of camptothecin was dissolved in 200 mL of ethanol to obtain solution 1; 2.5 g of quercetin was dissolved in 200 mL of ethyl acetate to obtain solution 2, solution 1 and solution 2 were mixed for 10 min, the solvent was evaporated, filtered, washed, dried, and ball-milled for 1 h to obtain camptothecin / quercetin cocrystal; S4. Preparation of gallic acid bornyl alcohol ester: 0.105 mol of gallic acid and 0.1 mol of bornyl alcohol were added to 500 mL of toluene, 1.5 mL of concentrated sulfuric acid was added, and the reaction was heated under reflux for 6 h, cooled to room temperature, filtered, washed, and dried to obtain gallic acid bornyl alcohol ester; S5. Preparation of Tai Sui probiotic fermentation whitening composition: 17 g Tai Sui fermentation broth, 4 g lysate, 1.5 g camptothecin / quercetin eutectic, 0.3 g menthol and 0.15 g bornyl gallate were stirred and mixed for 15 min to prepare Tai Sui probiotic fermentation whitening composition.

[0041] Comparative Example 1 Compared with Example 3, the difference is that the Aspergillus niger activation liquid is not inoculated in step S1.

[0042] The details are as follows: S1. Fermentation of Ganoderma lucidum: 10 g of Ganoderma lucidum was dried, crushed, and added to 200 mL of sterile water. The inoculation amount was 10 8 -10 9 cfu / mL of Aspergillus oryzae activated liquid, inoculation amount of 2.5v / v%, 27℃, 150r / min, fermentation culture for 30h, filtration, solid washing, collection of bacterial liquid, freeze-drying to obtain composite bacteria; filtrate freeze-dried to obtain Tai Sui fermentation liquid.

[0043] Comparative Example 2 Compared with Example 3, the difference is that the Aspergillus oryzae activation liquid is not inoculated in step S1.

[0044] The details are as follows: S1. Fermentation of Ganoderma lucidum: 10 g of Ganoderma lucidum was dried, crushed, and added to 200 mL of sterile water. The inoculation amount was 10 8 -10 9 cfu / mL of Aspergillus niger activated liquid, inoculation amount of 2.5v / v%, 27℃, 150r / min, fermentation culture for 30h, filtration, solid washing, collection of bacterial liquid, freeze-drying to obtain composite bacteria; filtrate freeze-dried to obtain Tai Sui fermentation liquid.

[0045] Comparative Example 3 Compared with Example 3, the difference is that the modified lysozyme in step S2 is prepared by Comparative Preparation Example 1.

[0046] Comparative Example 4 Compared with Example 3, the difference is that the modified lysozyme in step S2 is prepared by Comparative Preparation Example 2.

[0047] Comparative Example 5 Compared with Example 3, the difference is that the modified lysozyme in step S2 is prepared by Comparative Preparation Example 3.

[0048] Comparative Example 6 Compared with Example 3, the difference is that no modified lysozyme is added in step S2.

[0049] The details are as follows: S2. Preparation of lysate: 10 g of composite bacteria was added to 300 mL of water, 1.5 g of composite enzyme was added, enzymolysis was performed at 40°C for 3 h, the enzyme was inactivated, the filtrate was filtered, and the filtrate was freeze-dried to obtain a lysate; The complex enzyme is a mixture of snail enzyme and laccase, with a mass ratio of 4:2.5.

[0050] Comparative Example 7 Compared with Example 3, the difference is that in step S3, the camptothecin / quercetin co-crystal is replaced by a mixture of camptothecin and quercetin.

[0051] The details are as follows: S3. Preparation of a mixture of camptothecin and quercetin: 4 g of camptothecin and 2.5 g of quercetin were uniformly mixed to prepare a mixture.

[0052] Comparative Example 8 Compared with Example 3, the difference is that menthol is not added in step S5.

[0053] The details are as follows: S5. Preparation of Tai Sui probiotic fermentation whitening composition: 17 g Tai Sui fermentation broth, 4 g lysate, 1.5 g camptothecin / quercetin eutectic, 0.3 g menthol and 0.15 g bornyl gallate were stirred and mixed for 15 min to prepare Tai Sui probiotic fermentation whitening composition.

[0054] Comparative Example 9 Compared with Example 3, the difference is that no bornyl gallate is added in step S5.

[0055] The details are as follows: S5. Preparation of Tai Sui probiotic fermentation whitening composition: 17 g Tai Sui fermentation broth, 4 g lysate, 1.5 g camptothecin / quercetin eutectic, 0.3 g menthol and 0.15 g bornyl gallate were stirred and mixed for 15 min to prepare Tai Sui probiotic fermentation whitening composition.

[0056] Comparative Example 10 Compared with Example 3, the difference is that bornyl gallate and menthol are not added in step S5.

[0057] The details are as follows: S5. Preparation of Tai Sui probiotic fermentation whitening composition: 17 g Tai Sui fermentation broth, 4 g lysate, 1.5 g camptothecin / quercetin eutectic, 0.3 g menthol and 0.15 g bornyl gallate were stirred and mixed for 15 min to prepare Tai Sui probiotic fermentation whitening composition.

[0058] Test Example 1 The probiotic fermented whitening composition solution (1 wt%) prepared in Examples 1-3 or Comparative Examples 1-7 was prepared with PBS at pH = 7.4. The addition amounts of PBS, tyrosinase solution and L-tyrosine are shown in Table 1. Each colorimetric tube was divided into a sample group, a sample blank group, a positive group (arbutin, 0.05 wt%), a positive blank group, a negative control group and a negative blank group. After being kept in a water bath at 37°C for 10 minutes, the mixture was reacted for another 10 minutes at pH = 7.4, and then added. L -The concentration of tyrosine was 0.6 mmol / mL, and the enzyme volume was 1.0 mL (97 IU / mL). After the reaction was completed, the absorbance at 475 nm was measured, and the parallel experiment was repeated 3 times to obtain the average value. The tyrosinase activity inhibition rate was calculated according to the following formula ( IS , %): IS =[( A A - A B )-( A C - A D)] / ( A A - A B )×100% Where: A A : absorbance value of negative control group; A B : absorbance value of negative blank group; A C : Absorbance value of sample group / positive group; A D : Blank absorbance value of sample group / positive group.

[0059] Table 1

[0060] The results are shown in Table 2.

[0061] Table 2

[0062] It can be seen from the above table that the Tai Sui probiotic fermentation whitening composition prepared in Examples 1-3 of the present invention has good tyrosinase inhibition activity.

[0063] Test Example 2 Determination of DPPH free radical scavenging rate Prepare a 0.2mmol / L DPPH solution and store it in the dark. Prepare the Tai Sui probiotic fermented whitening composition solution (1wt%) prepared in Example 1-3 or Comparative Example 1-7 with PBS at pH=7.4. Prepare a vitamin C solution with a mass concentration of 7.5μg / mL with PBS at pH=7.4, prepare the reaction system according to the amount of each reagent added in Table 3, react for 30min at 25℃ in the dark, centrifuge, and measure its absorbance at a wavelength of 517nm. Each sample is measured in parallel 3 times and the average value is taken.

[0064] Clearance rate (%) = (A2 + A3 - A1) / A2 × 100% Wherein, A2: absorbance of negative control group; A3: absorbance of blank control group; A1: absorbance of sample group / positive control group.

[0065] Table 3

[0066] The results are shown in Table 4.

[0067] Table 4

[0068] It can be seen from the above table that the Tai Sui probiotic fermentation whitening composition prepared in Examples 1-3 of the present invention has good antioxidant activity.

[0069] Test Example 3 Melanin inhibition assay According to 2×10 5 Inoculate mouse melanoma B16-F10 cells into 6-well plates at 37°C and 5% CO2 incubator for 24 hours. Prepare each reaction system according to Table 5. When the cell plating rate in the 6-well plate reaches 60%, group administration is carried out. Each group has 3 replicate wells. Incubate for 3 days at 37°C and 5% CO2 incubator, centrifuge and discard the supernatant. Add 1 mL of 2.5 g / L trypsin digestion solution to each well and digest for 5 minutes. Add 4 mL of RPMI-1640 complete medium to stop digestion and blow into a single cell suspension. Take 0.5 mL for cell counting, centrifuge the remaining cell suspension and discard the supernatant, add 1 mL of 1 mol / L NaOH solution, incubate in a 90°C water bath for 2 hours, and measure its absorbance at a wavelength of 490 nm.

[0070] Inhibition rate (%) = (1-absorbance of sample well / cell density of sample well / absorbance of blank control well / cell density of blank control well) × 100% Table 5

[0071] The results are shown in Table 6.

[0072] Table 6

[0073] It can be seen from the above table that the Tai Sui probiotic fermentation whitening composition prepared in Examples 1-3 of the present invention has good melanin inhibition activity.

[0074] Test Example 4 Skin barrier function test 1 part by weight of glyceryl monostearate and 3 parts by weight of jojoba oil were mixed, 3 parts by weight of the probiotic fermented whitening composition prepared in Examples 1-3 and Comparative Examples 1-10 were added, and the mixture was stirred and mixed evenly, 30 parts by weight of deionized water, 1 part by weight of carbomer, and 2 parts by weight of glycerol were added, and the mixture was stirred and mixed evenly to prepare a skin cream.

[0075] 130 people aged 22-54 years old were selected, including 62 males and 68 females, and randomly divided into 24 groups, namely a skin care cream base group (without adding Tai Sui probiotic fermentation whitening composition), Example 1-3 groups, and Comparative Example 1-10 groups, with 10 people in each group.

[0076] Usage: Apply the corresponding prepared skin cream, 1g / person, once in the morning and evening; apply the cream without the Tai Sui probiotic fermentation whitening composition, 1g / person, once in the morning and evening for the skin cream matrix group. Use continuously for 1 week.

[0077] The skin moisture content tester was used to test the moisture content of the stratum corneum of each group of subjects; the average value was taken after 3 measurements. The moisture content of the stratum corneum of normal skin is 20%-30%.

[0078] The results are shown in Table 7.

[0079] Table 7

[0080] It can be seen from the above table that the Tai Sui probiotic fermented whitening compositions prepared in Examples 1-3 of the present invention have good moisturizing activity.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a probiotic fermented whitening composition, characterized in that: The following steps are involved: S1. Fermentation of Ganoderma lucidum: drying and crushing Ganoderma lucidum, adding it into sterile water, inoculating the activation solution of Aspergillus niger and Aspergillus oryzae, fermenting and culturing, filtering, washing the solid, collecting the bacterial solution, freeze-drying, and obtaining the composite bacteria; freeze-drying the filtrate to obtain the fermentation solution of Ganoderma lucidum; S2. Preparation of lysate: adding the composite bacteria into water, adding composite enzyme for enzymolysis, inactivating the enzyme, filtering, freeze-drying the filtrate, and obtaining the lysate; S3. Preparation of camptothecin / quercetin eutectic: dissolving camptothecin in ethanol to obtain solution 1; dissolving quercetin in ethyl acetate to obtain solution 2, mixing solution 1 and solution 2 evenly, volatilizing the solvent, filtering, washing, drying, and ball milling to obtain camptothecin / quercetin eutectic; S4. Preparation of gallic acid borneol ester: add gallic acid and borneol to toluene, add concentrated sulfuric acid, heat to reflux reaction, cool to room temperature, filter, wash, and dry to obtain gallic acid borneol ester; S5. Preparation of Tai Sui probiotic fermentation whitening composition: mix Tai Sui fermentation broth, lysate, camptothecin / quercetin eutectic, menthol and gallic acid borneol ester evenly to obtain Tai Sui probiotic fermentation whitening composition.

2. The preparation method according to claim 1, characterized in that: The bacterial content of the Aspergillus niger and Aspergillus oryzae activated solution in step S1 is 10 8 -10 9 cfu / mL, the inoculation amounts were 1-2v / v% and 0.5-1.5v / v%, respectively, and the fermentation culture conditions were 25-30°C, 100-200r / min, and the fermentation culture was 24-36h.

3. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of the composite bacteria to the composite enzyme is 10:1-2, the composite enzyme is a mixture of modified lysozyme, snail enzyme and laccase in a mass ratio of 10:3-5:2-3, the temperature of the enzymolysis is 35-45°C, and the time is 2-4h.

4. The preparation method according to claim 3, characterized in that: The preparation method of the modified lysozyme is as follows: T1. Stearic acid and oleic acid are mixed and added to water, NHS and EDC are added, activation reaction is carried out, lysozyme is added, stirring reaction is carried out, dialysis is carried out, and freeze-drying is carried out to obtain a preliminary modified lysozyme; T2. Heat the preliminary modified lysozyme to react and obtain modified lysozyme.

5. The preparation method according to claim 4, characterized in that: The mass ratio of stearic acid, oleic acid, NHS, EDC and lysozyme is 3-5:2-3:0.5-1:0.7-1.2:10, the activation reaction time is 20-40 minutes, the stirring reaction time is 4-6 hours, the heating reaction temperature is 55-65°C, and the time is 30-40 minutes.

6. The preparation method according to claim 1, characterized in that: The mass ratio of camptothecin to quercetin in step S3 is 3-5:2-3.

7. The preparation method according to claim 1, characterized in that: In step S4, the molar ratio of gallic acid to borneol is 1-1.1:1, and the heating reflux reaction time is 5-7h.

8. The preparation method according to claim 1, characterized in that: The mass ratio of the fermentation broth, lysate, camptothecin / quercetin eutectic, menthol and bornyl gallate in step S5 is 15-20:3-5:1-2:0.2-0.4:0.1-0.

2.

9. A probiotic fermentation whitening composition made by the preparation method according to any one of claims 1 to 8.

10. Use of the Tai Sui probiotic fermentation whitening composition as claimed in claim 9 in cosmetics.

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