Preparation method of saffron crocus fermentation liquor and application of fermentation liquor in cosmetics

Abstract

The present invention discloses a saffron crocus petal fermentation broth, which is characterized in that the saffron crocus petal fermentation broth is obtained by fermenting saffron crocus petals by using plant lactobacillus (Lactiplantarum), and the saffron crocus petal fermentation broth is obtained by using the plant lactobacillus (Lactiplantarum) to ferment the saffron crocus petals. The fermentation method is simple and rapid, and the obtained saffron crocus petal fermentation liquor has skin care effects on skin whitening, anti-aging and the like.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a method for preparing saffron petal fermentation liquid and application of the fermentation liquid in cosmetics. Background Art

[0002] Saffron (Crocus sativus L.), also known as saffron or crocus, originated in Greece, Iran and India. It is a precious spice and medicinal plant. Saffron was first recorded in the "Essentials of Materia Medica" in my country: "It can relieve depression and regulate blood, relieve chest tightness, stimulate appetite, nourish the lower abdomen, improve complexion, and treat typhoid fever and madness after long-term use." Saffron contains rich active ingredients, such as crocin, crocin and crocetin, which also makes it have great potential in the fields of cosmetics and medicine.

[0003] At present, there is little research on the active ingredients of saffron, which mainly focuses on the saffron stigma with high active ingredients, and basically adopts the traditional organic solvent extraction method. However, the use of organic solvents has certain safety risks, environmental pollution and loss of active ingredients. Invention patent CN114146042B uses Saccharomyces cerevisiae and Monascus to ferment saffron stigma. The fermentation process does not use organic solvents, and the fermented product has good antioxidant activity and anti-glycation activity. In addition, invention patent CN116270386B selects Lactobacillus helveticus and Bifidobacterium adolescentis as fermentation strains, and uses supercritical CO 2 Through the combined process of extraction, enzymolysis and fermentation, we have developed saffron petal and saffron stigma extracts with soothing, firming and wrinkle-reducing effects.

[0004] However, the above patents all use dual-strain fermentation technology. Although this technology has advantages in improving product diversity, it also faces risks such as high difficulty in regulating fermentation conditions and large uncertainty in product composition. At the same time, there are many challenges in the preparation of fermented products by combined processes, such as complex extraction processes, difficult to control ingredients, and increased production costs. In addition, the yield of saffron stigma is extremely low, which is difficult to meet actual market demand, and further increases the difficulty of technology development and commercial promotion.

[0005] Saffron petals are a byproduct with abundant production, but are often neglected due to their low economic value, resulting in serious waste of resources. Literature shows that saffron petals also contain active ingredients, so they have great potential for development and utilization. At present, there are few reports on the research of fermenting saffron petals using fermentation technology.

[0006] Therefore, the present invention provides a simple and rapid fermentation method, and adopts plant lactobacillus (Lactiplantibacillusplantarum) to ferment saffron petals.Through the application of this innovative strain, the biological activity of active ingredients in fermented liquid is improved, and high-end cosmetic raw materials with multiple functions such as whitening and anti-aging are developed simultaneously.In addition, the present invention is in order to make up for the resource vacancy brought by saffron stigma raw material, and has huge market value and application potential. Summary of the invention

[0007] The invention provides a saffron petal fermentation liquid, which is obtained by fermenting the saffron petals with Lactobacillus plantarum.

[0008] According to another aspect of the present invention, there is provided a method for preparing the above fermentation broth, the method comprising the following steps:

[0009] (1) Weighing an appropriate amount of saffron petals, crushing, and sieving to obtain saffron petal powder;

[0010] (2) adding the culture medium into a container, adding the saffron petal powder and seed liquid, and fermenting to obtain a crude fermentation liquid; and

[0011] (3) inactivating and purifying the crude fermentation broth to obtain the saffron petal fermentation broth.

[0012] Furthermore, the sieve is a 10-mesh sieve.

[0013] Furthermore, the mass ratio of the saffron petal powder to the culture medium is 0.005-0.1, such as 0.01-0.08, such as 0.03-0.08.

[0014] Furthermore, the volume ratio of the seed solution to the culture medium is 0.03 to 0.2, such as 0.04 to 0.15, such as about 0.06 to 0.1.

[0015] Furthermore, the culture medium is a liquid culture medium.

[0016] Furthermore, the container is a fermentation tank.

[0017] Further, the fermentation temperature is 20-45°C, such as 20-40°C, such as 25-40°C.

[0018] Furthermore, the fermentation pH is 2-7, such as 3-7, such as 4-7.

[0019] Furthermore, the fermentation rotation speed is 10 to 1000 r / min, such as 50 to 800 r / min, such as 100 to 600 r / min.

[0020] Furthermore, the fermentation time is 12 to 72 hours, such as 18 to 48 hours.

[0021] Furthermore, the inactivation temperature is 115-121°C.

[0022] Furthermore, the inactivation time is 10 to 30 minutes, for example, about 20 minutes.

[0023] Further, the purification includes filtering and sterilizing the crude fermentation broth.

[0024] Furthermore, the seed solution is obtained by culturing the Lactobacillus plantarum in a seed culture medium.

[0025] Furthermore, the Lactobacillus plantarum is activated Lactobacillus plantarum.

[0026] Furthermore, the seed culture medium is a liquid seed culture medium.

[0027] Furthermore, the Lactobacillus plantarum is isolated from fermentation slurry.

[0028] Furthermore, the Lactobacillus plantarum is deposited in the General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures, and its deposit number is CGMCC No.28500.

[0029] According to another aspect of the present invention, there is provided a use of the fermentation broth in preparing cosmetics having whitening and / or anti-aging effects.

[0030] According to another aspect of the present invention, there is provided a use of a composition comprising the fermentation broth in preparing cosmetics having whitening and / or anti-aging effects.

[0031] Furthermore, the whitening is to inhibit cellular melanin synthesis.

[0032] Furthermore, the anti-aging effect is achieved through one or more of the following ways: protecting type I collagen, inhibiting the production of MMP-1 and inhibiting the production of MMP-9.

[0033] Furthermore, the composition further comprises one or more auxiliary materials.

[0034] Furthermore, the auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler.

[0035] Furthermore, the composition further comprises one or more whitening substances.

[0036] Beneficial effects of the present invention:

[0037] The invention utilizes Lactiplantibacillus plantarum separated from fermented slurry to ferment saffron petals. The method is simple and rapid. The obtained saffron petal fermentation liquid has skin care effects in whitening, anti-aging and the like on the skin, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0039] Figure 1 The result of the saffron petal fermentation liquid of the present invention inhibiting the synthesis of melanin in A375 cells is shown in the figure, wherein BC: blank control (A375 cells), PC: positive control (0.5% w / w 377), compared with BC, * represents P < 0.05, ** represents P < 0.01.

[0040] Figure 2 The saffron petal fermentation liquid of the present invention protects type I collagen under UVA stimulation. BC: blank control (human skin fibroblasts), NC: negative control (9J / cm 2 UVA stimulation), PC: positive control (100 μg / mL VC + 7 μg / mL VE); compared with BC, # represents P < 0.05, ## represents P < 0.01; compared with NC, * represents P < 0.05, ** represents P < 0.01.

[0041] Figure 3The result of the saffron petal fermentation liquid of the present invention inhibiting the synthesis of MMP-1 under UVA stimulation is shown in the figure. BC: blank control (human skin fibroblasts), NC: negative control (9J / cm2 UVA stimulation), PC: positive control (100μg / mL VC+7μg / mL VE); compared with BC, # represents P<0.05, ## represents P<0.01; compared with NC, * represents P<0.05, ** represents P<0.01.

[0042] Figure 4 The result of the saffron petal fermentation liquid of the present invention inhibiting the synthesis of MMP-9 under UVA stimulation is shown in the figure. BC: blank control (human skin fibroblasts), NC: negative control (9J / cm2 UVA stimulation), PC: positive control (100μg / mL VC+7μg / mL VE); compared with BC, # represents P<0.05, ## represents P<0.01; compared with NC, * represents P<0.05, ** represents P<0.01. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0045] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0046] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0048] As described in the background technology section, there is no related report in the prior art that the saffron petal fermentation liquid obtained by fermenting saffron petals with Lactiplantibacillus plantarum has skin care effects such as whitening and anti-aging. In order to solve the above problems, the present invention provides a saffron petal fermentation liquid, which is obtained by fermenting saffron petals with Lactiplantibacillus plantarum.

[0049] According to another aspect of the present invention, there is provided a method for preparing the above fermentation broth, the method comprising the following steps:

[0050] (1) Weighing an appropriate amount of saffron petals, crushing, and sieving to obtain saffron petal powder;

[0051] (2) adding the culture medium into a container, adding the saffron petal powder and seed liquid, and fermenting to obtain a crude fermentation liquid; and

[0052] (3) inactivating and purifying the crude fermentation broth to obtain the saffron petal fermentation broth.

[0053] In a preferred embodiment, the sieve is a 10 mesh sieve.

[0054] In the present invention, when a ratio, temperature, pH, rotation speed, time, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "0.01-0.08" is disclosed, the described range should be interpreted as including the range "0.01-0.08", "0.01-0.06", "0.01-0.04", "0.01-0.02", "0.02-0.08", "0.02-0.06", "0.02-0.04", "0.04-0.08", "0.04-0.06", "0.06-0.08" and the like. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0055] The present invention utilizes the metabolic activity of microorganisms to convert natural ingredients into more stable and more active compounds. Compared with traditional extraction methods, the fermentation process can not only improve the purity and stability of active ingredients, but also reduce dependence on raw materials and reduce the impact on the environment. Therefore, it is of great significance in the production of cosmetics.

[0056] In a preferred embodiment, the mass ratio of the saffron petal powder to the culture medium is 0.005-0.1, such as 0.01-0.08, such as 0.03-0.08.

[0057] In a preferred embodiment, the volume ratio of the seed solution to the culture medium is 0.03-0.2, such as 0.04-0.15, such as about 0.06-0.1.

[0058] In a preferred embodiment, the culture medium is a liquid culture medium.

[0059] In a preferred embodiment, the container is a fermenter.

[0060] In a preferred embodiment, the fermentation temperature is 20-45°C, such as 20-40°C, such as 25-40°C.

[0061] In a preferred embodiment, the pH of the fermentation is 2-7, such as 3-7, such as 4-7.

[0062] In a preferred embodiment, the fermentation rotation speed is 10-1000 r / min, such as 50-800 r / min, such as 100-600 r / min.

[0063] In a preferred embodiment, the fermentation time is 12 to 72 hours, such as 18 to 48 hours.

[0064] In a preferred embodiment, the inactivation temperature is 115-121°C.

[0065] In a preferred embodiment, the inactivation time is 10 to 30 minutes, such as about 20 minutes.

[0066] In a preferred embodiment, the purification comprises filtering and sterilizing the crude fermentation broth.

[0067] In a preferred embodiment, the seed liquid is obtained by culturing the Lactobacillus plantarum in a seed culture medium.

[0068] In a preferred embodiment, the Lactobacillus plantarum is activated Lactobacillus plantarum.

[0069] In a preferred embodiment, the seed culture medium is a liquid seed culture medium.

[0070] In a preferred embodiment, the Lactiplantibacillus plantarum is isolated from fermentation slurry.

[0071] In a preferred embodiment, the Lactobacillus plantarum is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and its deposit number is CGMCC No.28500.

[0072] For other information about the above-mentioned strains used in the present invention, please refer to the information disclosed in application number 202410502170.3 and publication number CN118165889A.

[0073] According to another aspect of the present invention, there is provided a use of the fermentation broth in preparing cosmetics having whitening and / or anti-aging effects.

[0074] According to another aspect of the present invention, there is provided a use of a composition comprising the fermentation broth in preparing cosmetics having whitening and / or anti-aging effects.

[0075] In a preferred embodiment, the whitening is to inhibit cellular melanin synthesis.

[0076] In a preferred embodiment, the anti-aging effect is achieved through one or more of the following ways: protecting type I collagen, inhibiting the production of MMP-1, and inhibiting the production of MMP-9.

[0077] In a preferred embodiment, the composition further comprises one or more auxiliary materials.

[0078] In a preferred embodiment, the auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler.

[0079] In a preferred embodiment, the composition further comprises one or more whitening substances.

[0080] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0081] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0082] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0083] Example

[0084] Preparation Example

[0085] Experimental instruments and materials

[0086] Biochemical incubator (LRH-150, Shanghai Yiheng), autoclave (SYQ-DSX-280B, Shanghai Shen'an), clean bench (SW-CJ-2F, Suzhou Purification), centrifuge (Hermle, Z216M), fully automatic double water purifier (Shanghai Jinke Industry, SZ-93), anaerobic box (ANAEROPACK, Mitsubishi Gas), rotary evaporator (R-206, Shanghai Shensheng), circulating water vacuum pump (SHZ-D, Shanghai Yuhua), MRS broth culture medium (Beijing Luqiao), MRS agar culture medium (Oxoid).

[0087] The saffron used in this example was purchased from Shanghai Jiangqiao Wholesale Market, a dried saffron sample produced in Tibet, China, and was identified as the petals of Crocus sativus L., a plant of the genus Crocus in the family Iridaceae. It was stored in a ventilated and dry place for later use. The white plum blossom used in this example was purchased from Shanghai Jiangqiao Wholesale Market, a dried white plum blossom sample produced in Anhui, and was identified as the buds of Prunus mumef.viridicalyx (Makino) TYChen, a plant of the family Rosaceae. It was stored in a ventilated and dry place for later use.

[0088] Example 1

[0089] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain saffron petal fermentation liquid A.

[0090] Example 2

[0091] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added according to 8% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added according to 10% inoculum of the volume percentage of the fermentation medium. The fermentation temperature was 40°C, the pH was 7.0, the speed was 600r / min, and the fermentation time was 48h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified several times to obtain saffron petal fermentation liquid B.

[0092] Comparative Example 1

[0093] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 15% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain saffron petal fermentation liquid C.

[0094] Comparative Example 2

[0095] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 2% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the saffron petal fermentation liquid D.

[0096] Comparative Example 3

[0097] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 65°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain saffron petal fermentation liquid E.

[0098] Comparative Example 4

[0099] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 9.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the saffron petal fermentation liquid F.

[0100] Comparative Example 5

[0101] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 1200r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the saffron petal fermentation liquid G.

[0102] Comparative Example 6

[0103] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 96h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the saffron petal fermentation liquid H.

[0104] Comparative Example 7

[0105] The saffron petals were crushed by a pulverizer and passed through a 10-mesh screen. The crushed saffron petals were then added at 3% of the mass fraction of the fermentation medium, and the sterile seed liquid culture medium system was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain saffron petal fermentation liquid I.

[0106] Comparative Example 8

[0107] The white plum blossoms were crushed by a grinder and passed through a 10-mesh screen. The crushed white plum blossoms were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the white plum blossom fermentation liquid J.

[0108] Efficacy Example

[0109] Efficacy Example 1: Determination of the inhibition rate of melanin synthesis in A375 cells (whitening)

[0110] 1.1 Experimental instruments and reagents

[0111] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), vortex oscillator (Shanghai Hanno, XH-D), CO 2 Incubator (Thermo Scientific), microplate reader (Thermo Scientific), 24-well plate (Coming Incorporated), 96-well plate (Coming Incorporated), 6 cm culture dish (Coming Incorporated), DMSO (Sigma), NaOH (Sinopharm), cell lysis buffer (Gibco), DMEM medium (Gibco), double antibody Pen Strep (Gibco), fetal bovine serum (Gibco), 0.05% Trypsin-EDTA (Gibco), PBS (Vivacell), MTT (Sigma).

[0112] 1.2 Experimental Methods

[0113] Cell seeding: 1×10 5 A375 cells (Stem Cell Bank, Chinese Academy of Sciences) were seeded into 24-well plates at a seeding density of 10 cells / well and incubated in an incubator (37°C, 5% CO 2) and incubate overnight.

[0114] Liquid preparation: Prepare the working solution of the test substance according to the experimental design (Table 1). Take 50 mg of 377, nonapeptide-1, and glabridin powder, respectively, and add 10 mL of DMEM culture medium (containing 10% fetal bovine serum) to obtain a cell culture solution with a concentration of 0.5% (w / w) of 377, nonapeptide-1, and glabridin; take 50 μL of saffron petal fermentation liquid AI and white plum blossom fermentation liquid J, respectively, and add 10 mL of DMEM culture medium (containing 10% fetal bovine serum) to obtain a cell culture solution with a concentration of 0.5% (w / w) of saffron petal fermentation liquid AI and white plum blossom fermentation liquid J; take 100 μL of saffron petal fermentation liquid AI and white plum blossom fermentation liquid J, respectively, and add 10 mL of DMEM culture medium (containing 10% fetal bovine serum) to obtain a cell culture solution with a concentration of 1% (w / w) of saffron petal fermentation liquid AI and white plum blossom fermentation liquid J;

[0115] Table 1 Pharmacodynamics Example 1 Experimental Design

[0116]

[0117]

[0118] Drug administration: After 24 hours of culture, the group administration was carried out according to the experimental design in Table 1. 1 mL of cell culture medium was added to each well of the blank control group; 1 mL of culture medium containing 0.5% (w / w) 377 was added to each well of the positive control group; 1 mL of culture medium containing the test substance at the corresponding concentration was added to each well of the sample group. After the administration was completed, the 24-well plate was placed in an incubator (37°C, 5% CO 2 ) for 24 h.

[0119] Detection: 72 hours after the third medium change, the melanin content of the cells was determined. The cells were washed twice with 1×PBS, the PBS was removed, 200 μL of 0.25% trypsin was added to each well to digest the cells, and then placed in CO 2 Digest in the incubator for 5 minutes. Add 2 mL of PBS twice to blow the cells, collect the cells into a centrifuge tube, and centrifuge at 4000 rpm for 5 minutes. Try to aspirate the residual PBS in the centrifuge tube, add 200 μL of melanin extract to each tube, shake evenly, and heat in an 80°C water bath for 1 hour. After cooling, centrifuge the droplets on the tube wall moderately, blow evenly, aspirate 150 μL of solution in each centrifuge tube, transfer to a 96-well plate, and detect the absorbance (OD) of each well at 405 nm with an enzyme reader.

[0120] The relative inhibition rate of melanin was calculated.

[0121] Melanin inhibition rate (%) = [1-(OD of drug well-OD of blank well) / (OD of cell control well-OD of blank well)] × 100

[0122] 1.3 Experimental Results

[0123] like Figure 1 As shown, compared with the BC group, the 0.5% (w / w) concentration of 377 in the PC group significantly increased the melanin inhibition rate, indicating that the experimental system is effective. Compared with the BC group, the common whitening monomer raw materials nonapeptide-1 and glabridin in the test group significantly improved the melanin inhibition rate at a concentration of 0.5%; compared with the BC group, the saffron petal fermentation liquid A and B described in Examples 1 and 2 improved the melanin inhibition rate at a concentration of 0.5%, and significantly improved the melanin inhibition rate at a concentration of 1%, which was close to the inhibition rate of nonapeptide-1 and glabridin at a concentration of 0.5%, indicating that the saffron petal fermentation liquid A and saffron petal fermentation liquid B have the ability to inhibit melanocytes from synthesizing melanin and have whitening effect; while in Comparative Examples 1-8, the differences between the groups of saffron petal fermentation liquid CI and white plum blossom fermentation liquid J at concentrations of 0.5% and 1% were not statistically significant (P>0.05) compared with the BC group, and therefore did not show the ability to inhibit melanocytes from synthesizing melanin; it proves that only within the scope of the preparation method described in the claims, the prepared saffron petal fermentation liquid has a whitening effect.

[0124] Efficacy Example 2: Protection of Type I Collagen (UVA) (Anti-aging) --- Determination of Type I Collagen Content in Fibroblasts Stimulated by UVA

[0125] 2.1 Experimental instruments and reagents

[0126] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), CO 2 Incubator (Thermo Scientific), microvibrator (Qilin Bell, TS-92), ultra-low temperature refrigerator (-80℃, Thermo), low-speed centrifuge (bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (ThermoScientific), adjustable pipette (eppendorf), PBS (Viva cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), Human Collagen I alpha 1 ELISA detection kit (Thermo), type I collagen primary antibody, secondary antibody (Thermo), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0127] 2.2 Experimental Methods

[0128] Cell inoculation: Human skin fibroblasts (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density of 45% to 60% confluence 24 hours after inoculation. 2 ) and incubate overnight.

[0129] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 2).

[0130] Table 2 Pharmacodynamics Example 2 Experimental Design

[0131]

[0132]

[0133] Drug administration: According to the experimental design in Table 2, when the cell plating rate in the 24-well plate reaches 40% to 60%, wash with PBS, and then culture and expose to 9J / cm 2 After exposure, the treated cells were washed with PBS and then divided into groups for drug administration. The sample group was added with culture medium containing the sample, and the positive control group was added with culture medium containing the positive control. They were exposed to different test substances for 24 hours. The dosage per well was 1.0 mL, and 3 replicates were set for each group. The blank control group BC group was added with cell culture medium and treated in the same way, but covered with aluminum foil to avoid ultraviolet exposure.

[0134] Detection: Cell supernatant collection: After the incubation, collect 1 mL of cell culture supernatant in a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator. Determine the type I collagen content according to the operating instructions of the ELISA detection kit.

[0135] Result analysis: Calculation of CollagenⅠ content: After subtracting the OD450 value of the blank well from the OD450 value of the standard and sample, the average value of the three parallel wells was calculated. Using the professional curve making software Curve Expert, the concentration of the standard was used as the ordinate and the OD450 value was used as the abscissa to draw the standard curve, and the regression equation was obtained. The OD450 value measured by the sample group was substituted into the equation to calculate the CollagenⅠ protein content of each group.

[0136] 2.3 Experimental Results

[0137] like Figure 2As shown in the data, compared with the BC group, the type I collagen concentration in the NC group was significantly decreased (P<0.01). Compared with the NC group, 100μg / mL VC+7μg / mL VE in the PC group significantly increased the type I collagen concentration (P<0.01), indicating that the experimental system was effective. Compared with the NC group, the saffron petal fermented liquid A and B described in Example 1 and Example 2 increased the concentration of type I collagen at a concentration of 0.5%, and significantly increased the concentration of type I collagen at a concentration of 1%, indicating that the saffron petal fermented liquid A and B have the ability to protect type I collagen of fibroblasts under UVA stimulation, and have anti-aging effect; while in Comparative Examples 1-8, the saffron petal fermented liquid CI and white plum blossom fermented liquid J were compared with the NC group at concentrations of 0.5% and 1%, and the differences between the groups were not statistically significant (P>0.05), so neither showed the ability to protect type I collagen; it is proved that only within the scope of the preparation method described in the claims, the prepared saffron petal fermented liquid has anti-aging effect.

[0138] Efficacy Example 3: Inhibition of MMP-1 Synthesis (UVA) (Anti-aging) --- Determination of MMP-1 Content in Fibroblasts Stimulated by UVA

[0139] 3.1 Experimental instruments and reagents

[0140] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), CO 2 Incubator (Thermo Scientific), microvibrator (Qilin Bell, TS-92), ultra-low temperature refrigerator (-80℃, Thermo), low-speed centrifuge (bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (ThermoScientific), adjustable pipette (eppendorf), PBS (Viva cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), human MMP-1 ELISA kit (Sigma), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0141] 3.2 Experimental Methods

[0142] Cell inoculation: Human skin fibroblasts (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density of 45% to 60% confluence 24 hours after inoculation. 2 ) and incubate overnight.

[0143] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 3).

[0144] Table 3 Pharmacodynamics Example 3 Experimental Design

[0145]

[0146]

[0147] Drug administration: According to the experimental design in Table 3, when the cell plating rate in the 24-well plate reaches 40% to 60%, wash with PBS, and then culture and expose to 9J / cm 2 After exposure, the treated cells were washed with PBS and then divided into groups for drug administration. The sample group was added with culture medium containing the sample, and the positive control group was added with culture medium containing the positive control. They were exposed to different test substances for 24 hours. The dosage per well was 1.0 mL, and 3 replicates were set for each group. The blank control group BC group was added with cell culture medium and treated in the same way, but covered with aluminum foil to avoid ultraviolet exposure.

[0148] Detection: Cell supernatant collection: After the incubation, collect 1 mL of cell culture supernatant in a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator. Determine the MMP-1 protein content according to the operating instructions of the ELISA detection kit.

[0149] Result analysis: The MMP-1 protein content was calculated according to the operating instructions of the ELISA detection kit.

[0150] 3.3 Experimental Results

[0151] like Figure 3 As shown, compared with the BC group, the MMP-1 concentration of the NC group was significantly increased (P<0.01), and compared with the NC group, 100μg / mL VC+7μg / mL VE in the PC group significantly reduced the MMP-1 concentration (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the saffron petal fermentation liquid A and B described in Example 1 and Example 2 reduced the MMP-1 concentration at a concentration of 0.5%, and significantly reduced the MMP-1 concentration at a concentration of 1%, indicating that the saffron petal fermentation liquid A and B have the ability to inhibit the synthesis of MMP-1 protein by fibroblasts under UVA stimulation, and have anti-aging effects; while in Comparative Examples 1-8, the saffron petal fermentation liquid CI and the white plum blossom fermentation liquid J were compared with the NC group at concentrations of 0.5% and 1%, and the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to inhibit the synthesis of MMP-1; it proves that only within the scope of the preparation method described in the claims, the prepared saffron petal fermentation liquid has anti-aging effects.

[0152] Efficacy Example 4: Inhibition of MMP-9 Synthesis (UVA) (Anti-aging) --- Determination of MMP-9 Content in Fibroblasts Stimulated by UVA

[0153] 4.1 Experimental instruments and reagents

[0154] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), CO 2 Incubator (Thermo Scientific), microvibrator (Qilin Bell, TS-92), ultra-low temperature refrigerator (-80℃, Thermo), low-speed centrifuge (bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (ThermoScientific), adjustable pipette (eppendorf), PBS (Viva cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), human MMP9 ELISA kit (Sigma), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0155] 4.2 Experimental Methods

[0156] Cell inoculation: Human skin fibroblasts (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density of 45% to 60% confluency 24 hours after inoculation and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0157] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 4).

[0158] Table 4 Pharmacodynamics Example 4 Experimental Design

[0159]

[0160] Drug administration: According to the experimental design in Table 4, when the cell plating rate in the 24-well plate reaches 40% to 60%, wash with PBS, and then culture and expose to 9J / cm 2 After exposure, the treated cells were washed with PBS and then divided into groups for drug administration. The sample group was added with culture medium containing the sample, and the positive control group was added with culture medium containing the positive control. They were exposed to different test substances for 24 hours. The dosage per well was 1.0 mL, and 3 replicates were set for each group. The blank control group BC group was added with cell culture medium and treated in the same way, but covered with aluminum foil to avoid ultraviolet exposure.

[0161] Detection: Cell supernatant collection: After the incubation, collect 1 mL of cell culture supernatant in a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator. Determine the MMP-9 protein content according to the operating instructions of the ELISA detection kit.

[0162] Result analysis: The MMP-9 protein content was calculated according to the operating instructions of the ELISA detection kit.

[0163] 4.3 Experimental Results

[0164] like Figure 4 As shown, compared with the BC group, the MMP-9 concentration of the NC group was significantly increased (P<0.01), and compared with the NC group, 100μg / mL VC+7μg / mL VE in the PC group significantly reduced the MMP-9 concentration (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the saffron petal fermentation liquid A and B described in Example 1 and Example 2 reduced the MMP-9 concentration at a concentration of 0.5%, and significantly reduced the MMP-9 concentration at a concentration of 1%, indicating that the saffron petal fermentation liquid A and B have the ability to inhibit the synthesis of MMP-9 protein by fibroblasts under UVA stimulation, and have anti-aging effects; while in Comparative Examples 1-8, the saffron petal fermentation liquid CI and the white plum blossom fermentation liquid J were compared with the NC group at concentrations of 0.5% and 1%, and the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to inhibit the synthesis of MMP-9; it proves that only within the scope of the preparation method described in the claims, the prepared saffron petal fermentation liquid has anti-aging effects.

[0165] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A saffron petal fermentation liquid, characterized in that: The fermented liquid is obtained by fermenting saffron petals with Lactiplantibacillus plantarum.

2. A method for preparing the fermentation broth according to claim 1, characterized in that: The method comprises the following steps: (1) Weighing an appropriate amount of saffron petals, crushing, and sieving to obtain saffron petal powder; (2) adding the culture medium into a container, adding the saffron petal powder and seed liquid, and fermenting to obtain a crude fermentation liquid; and (3) inactivating and purifying the crude fermentation broth to obtain the saffron petal fermentation broth.

3. The method according to claim 2, characterized in that The sieve is a 10-mesh sieve; Preferably, the mass ratio of the saffron petal powder to the culture medium is 0.005 to 0.1, such as 0.01 to 0.08, such as 0.03 to 0.08; More preferably, the volume ratio of the seed solution to the culture medium is 0.03 to 0.2, such as 0.04 to 0.15, such as about 0.06 to 0.1; Also preferably, the culture medium is a liquid culture medium.

4. The method according to claim 2, characterized in that: The container is a fermentation tank; Preferably, the fermentation temperature is 20-45°C, such as 20-40°C, such as 25-40°C; Preferably, the pH of the fermentation is 2 to 7, such as 3 to 7, such as 4 to 7; More preferably, the fermentation speed is 10 to 1000 r / min, such as 50 to 800 r / min, such as 100 to 600 r / min; More preferably, the fermentation time is 12 to 72 hours, such as 18 to 48 hours.

5. The method according to claim 2, characterized in that: The inactivation temperature is 115-121°C; Preferably, the inactivation time is 10 to 30 minutes, for example 20 minutes; More preferably, the purification comprises filtering and sterilizing the crude fermentation broth.

6. The method according to claim 2, characterized in that The seed solution is obtained by culturing the Lactobacillus plantarum in a seed culture medium; Preferably, the Lactobacillus plantarum is an activated Lactobacillus plantarum; More preferably, the seed culture medium is a liquid seed culture medium; Preferably, the Lactobacillus plantarum is isolated from fermentation slurry; Particularly preferably, the Lactobacillus plantarum is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, and its deposit number is CGMCC No.28500.

7. Use of the fermentation broth according to claim 1 in preparing cosmetics having whitening and / or anti-aging effects.

8. Use of a composition comprising the fermentation broth according to claim 1 in preparing cosmetics having whitening and / or anti-aging effects.

9. The use according to claim 7 or 8, characterized in that The whitening is to inhibit the synthesis of cellular melanin; Preferably, the anti-aging effect is achieved through one or more of the following ways: protecting type I collagen, inhibiting the production of MMP-1 and inhibiting the production of MMP-9.

10. The use according to claim 8, characterized in that The composition further comprises one or more excipients; Preferably, the excipient is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler; Also preferably, the composition further comprises one or more whitening substances.

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