Preparation method of jasmine flower fermentation liquor and application of the fermentation liquor in cosmetics

Abstract

The application discloses a jasmine fermentation liquor, characterized in that the fermentation liquor is obtained by fermenting jasmine flowers by using Lactiplantibacillus plantarum. The fermentation method is simple and rapid, and the obtained jasmine fermentation liquor has skin care effects such as skin whitening, moisturizing, soothing and anti-aging.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a preparation method of jasmine flower fermentation liquor and application of the fermentation liquor in cosmetics. BACKGROUND

[0002] At present, whitening products are popular in the market. Common whitening raw materials include salicylic acid, fruit acid, vitamin C, 377, etc. However, these raw materials may have certain safety problems in some cases. For example, salicylic acid may cause skin allergic reactions, and fruit acid may cause skin irritation and be easily irritated after sun exposure. In addition, vitamin C has poor stability in some formulations and is easily oxidized, which affects its whitening effect. Studies have also shown that 377 may cause skin allergic reactions and may cause irritation or adverse reactions in some specific skin types. Therefore, there is an urgent need for a safe and effective whitening raw material in the current market to meet the needs of consumers for whitening products.

[0003] Jasmine (Jasminum sambac (L.) Aiton) is a plant of the Oleaceae family. It was introduced from India to China during the Han Dynasty and was initially cultivated in the southeastern coastal areas of Fujian and Guangzhou. According to the Compendium of Materia Medica, jasmine has the properties of "acrid, hot, sweet, and warm, and can harmonize the center, lower qi, dispel turbidity, and treat dysentery and abdominal pain." It is commonly used to treat external heat, red and swollen eyes, and other conditions. The main producing areas of jasmine in China are Guangxi, Fujian, Sichuan, and Yunnan. Jasmine has a rich and pleasant fragrance and is often used for fragrance blending, making jasmine tea, and making perfumes. Its fragrance is fresh and elegant, not only improving people's mood, but also helping to relieve stress and improve sleep quality. However, the petal structure of jasmine is relatively fragile, making it difficult to extract effective components, so its application in skincare products is relatively limited, and it is more commonly used for fragrance blending, flower viewing, and making jasmine tea. The industrial chain has not been effectively extended, and the application of resources is relatively limited. SUMMARY

[0004] Therefore, the present application provides a jasmine fermentation liquor, which is obtained by fermenting jasmine with Lactiplantibacillus plantarum.

[0005] According to another aspect of the present application, a method for preparing the above-mentioned fermentation liquor is provided, which comprises the following steps:

[0006] (1) A suitable amount of jasmine is weighed, crushed, and sieved to obtain jasmine powder;

[0007] (2) The culture medium is added to a container, the jasmine powder and seed liquid are added, and fermentation is carried out to obtain a crude fermentation liquor; and

[0008] (3) The crude fermentation liquid was inactivated and purified to obtain the jasmine fermentation liquid.

[0009] Furthermore, the sieve is a 10-mesh sieve.

[0010] Furthermore, the mass ratio of the jasmine powder to the culture medium is 0.005 to 0.1, for example 0.01 to 0.08, for example 0.03 to 0.08.

[0011] Furthermore, the volume ratio of the seed culture to the culture medium is 0.03 to 0.2, for example 0.04 to 0.15, for example 0.06 to 0.1.

[0012] Furthermore, the culture medium is a liquid culture medium.

[0013] Furthermore, the container is a fermentation tank.

[0014] Furthermore, the fermentation temperature is 20–45°C, for example 20–40°C, or for example 25–40°C.

[0015] Furthermore, the pH of this fermentation is 2–7, for example 3–7, for example 4–7.

[0016] Furthermore, the fermentation speed is 10–1000 r / min, for example 50–800 r / min, for example 100–600 r / min.

[0017] Furthermore, the fermentation time is 12–72 hours, for example, 18–48 hours.

[0018] Furthermore, the inactivation temperature is 115–121°C.

[0019] Furthermore, the inactivation time is 10–30 minutes, for example, about 20 minutes.

[0020] Furthermore, the purification process includes filtering and sterilizing the crude fermentation broth.

[0021] Furthermore, the seed culture was obtained by culturing Lactiplantibacillus plantarum in a seed culture medium.

[0022] Furthermore, this *Lactiplantibacillus plantarum* is an activated *Lactiplantibacillus plantarum*.

[0023] Furthermore, the seed culture medium is a liquid seed culture medium.

[0024] Furthermore, the plant lactobacillus plantarum was isolated from the fermentation slurry.

[0025] Furthermore, this *Lactiplantibacillus plantarum* is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28500.

[0026] According to another aspect of the present invention, the use of the above-mentioned fermentation broth in the preparation of cosmetics having whitening, moisturizing, soothing, anti-inflammatory and / or anti-aging properties is provided.

[0027] According to another aspect of the invention, there is provided the use of a composition comprising the above-described fermentation broth in the preparation of cosmetics having whitening, moisturizing, soothing, anti-inflammatory, and / or anti-aging properties.

[0028] Furthermore, this whitening effect works by inhibiting melanin synthesis in cells.

[0029] Furthermore, this moisturizing effect promotes the synthesis of aquaporin 3.

[0030] Furthermore, this anti-aging mechanism works through one or more of the following methods: protecting type I collagen, inhibiting MMP-1 production, and inhibiting MMP-9 production.

[0031] Furthermore, this soothing and anti-inflammatory effect is achieved by inhibiting the production of TNF-α.

[0032] Furthermore, the composition further comprises one or more excipients.

[0033] Furthermore, the excipient is selected from one or more of the following: thickeners, surfactants, co-surfactants, skin conditioners, preservatives, fragrances, lubricants, disintegrants, humectants, binders, and fillers.

[0034] Furthermore, the composition further comprises one or more skin-whitening substances.

[0035] The beneficial effects of this invention are:

[0036] This invention utilizes a plant lactobacillus isolated from fermented slurry to ferment jasmine flowers. This method is simple and rapid, and the resulting jasmine fermentation liquid has skin care effects such as whitening, moisturizing, soothing, and anti-aging, making it suitable for widespread application. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0038] Figure 1 This diagram illustrates the results of the jasmine fermentation broth inhibiting melanin synthesis in A375 cells according to the present invention. In the diagram, BC represents the blank control (A375 cells), PC represents the positive control (0.5% w / w 377), and compared to BC, * represents P < 0.05, ** represents P < 0.01.

[0039] Figure 2 This diagram illustrates the results of promoting aquaporin 3 (AQP3) synthesis with jasmine fermentation broth according to the present invention. In the diagram, BC represents the blank control (HaCaT cells), PC represents the positive control (0.5% w / w CaCl2), and compared to BC, * represents P < 0.05, ** represents P < 0.01.

[0040] Figure 3 This diagram illustrates the results of the jasmine fermentation broth protecting type I collagen under UVA stimulation, as described in this invention. Wherein, BC: blank control (human skin fibroblasts), NC: negative control (9 J / cm²). 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 4 This diagram illustrates the results of inhibiting MMP-1 synthesis in the jasmine fermentation broth under UVA stimulation according to the present invention. Wherein, BC: blank control (human skin fibroblasts), NC: negative control (UVA stimulation of 9 J / cm²), 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.

[0042] Figure 5This diagram illustrates the results of the jasmine fermentation broth inhibiting MMP-9 synthesis under UVA stimulation, as described in this invention. In the diagram, BC: blank control (human skin fibroblasts), NC: negative control (UVA stimulation at 9 J / cm²), PC: positive control (100 μg / mL VC + 7 μg / mL VE); compared to BC, # represents P < 0.05, ## represents P < 0.01; compared to NC, * represents P < 0.05, ** represents P < 0.01.

[0043] Figure 6 This diagram illustrates the results of the jasmine fermentation broth inhibiting TNF-α production under LPS stimulation, as described in this invention. In the diagram, BC represents the blank control (RAW264.7 cells), NC represents the negative control (0.00001% LPS), and PC represents the positive control (0.01% dexamethasone). Compared to BC, # represents P < 0.05, and ## represents P < 0.01; compared to NC, * represents P < 0.05, and ** represents P < 0.01. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0046] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0047] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0048] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0049] As described in the background section, there are no existing reports on the skincare effects of jasmine fermentation liquid obtained by fermenting jasmine flowers with Lactiplantibacillus plantarum, such as whitening, moisturizing, soothing, and anti-aging. To address these issues, this invention provides a jasmine fermentation liquid obtained by fermenting jasmine flowers with Lactiplantibacillus plantarum.

[0050] According to another aspect of the present invention, a method for preparing the above-mentioned fermentation broth is provided, the method comprising the following steps:

[0051] (1) Weigh an appropriate amount of jasmine flowers, crush them, and sieve them to obtain jasmine flower powder;

[0052] (2) Add the culture medium to a container, add the jasmine flower powder and seed liquid, and ferment to obtain a crude fermentation liquid; and

[0053] (3) The crude fermentation liquid was inactivated and purified to obtain the jasmine fermentation liquid.

[0054] In a preferred embodiment, the sieve is a 10-mesh sieve.

[0055] In this invention, when ratios, temperatures, pH, rotational speeds, times, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “0.01 to 0.08” is disclosed, the described range should be interpreted as including ranges “0.01 to 0.08”, “0.01 to 0.06”, “0.01 to 0.04”, “0.01 to 0.02”, “0.02 to 0.08”, “0.02 to 0.06”, “0.02 to 0.04”, “0.04 to 0.08”, “0.04 to 0.06”, “0.06 to 0.08”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0056] This invention utilizes the metabolic activities of microorganisms to transform natural ingredients into more stable and highly active compounds. Compared to traditional extraction methods, fermentation not only improves the purity and stability of active ingredients but also reduces dependence on raw materials and minimizes environmental impact, thus holding significant importance in cosmetic production.

[0057] In a preferred embodiment, the mass ratio of the jasmine powder to the culture medium is 0.005 to 0.1, for example 0.01 to 0.08, for example 0.03 to 0.08.

[0058] In a preferred embodiment, the volume ratio of the seed culture to the culture medium is 0.03 to 0.2, for example 0.04 to 0.15, for example 0.06 to 0.1.

[0059] In a preferred embodiment, the culture medium is a liquid culture medium.

[0060] In a preferred embodiment, the container is a fermentation tank.

[0061] In a preferred embodiment, the fermentation temperature is 20–45°C, for example 20–40°C, for example 25–40°C.

[0062] In a preferred embodiment, the pH of the fermentation is 2 to 7, for example 3 to 7, or for example 4 to 7.

[0063] In a preferred embodiment, the fermentation speed is 10 to 1000 r / min, for example 50 to 800 r / min, for example 100 to 600 r / min.

[0064] In a preferred embodiment, the fermentation time is 12 to 72 hours, for example, 18 to 48 hours.

[0065] In a preferred embodiment, the inactivation temperature is 115–121°C.

[0066] In a preferred embodiment, the inactivation time is 10 to 30 minutes, for example, about 20 minutes.

[0067] In a preferred embodiment, the purification includes filtering and sterilizing the crude fermentation broth.

[0068] In a preferred embodiment, the seed culture is obtained by culturing Lactiplantibacillus plantarum in a seed culture medium.

[0069] In a preferred embodiment, the Lactiplantibacillus plantarum is an activated Lactiplantibacillus plantarum.

[0070] In a preferred embodiment, the seed culture medium is a liquid seed culture medium.

[0071] In a preferred embodiment, the Lactiplantibacillus plantarum is isolated from fermented slurry.

[0072] In a preferred embodiment, the Lactiplantibacillus plantarum is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28500 and deposit date of September 20, 2023.

[0073] Other information about the strains used in this invention can be found in the information disclosed in application number 202410502170.3 and publication number CN118165889A.

[0074] According to another aspect of the present invention, the use of the above-mentioned fermentation broth in the preparation of cosmetics having whitening, moisturizing, soothing, anti-inflammatory and / or anti-aging properties is provided.

[0075] According to another aspect of the invention, there is provided the use of a composition comprising the above-described fermentation broth in the preparation of cosmetics having whitening, moisturizing, soothing, anti-inflammatory, and / or anti-aging properties.

[0076] In a preferred embodiment, the whitening effect is achieved by inhibiting melanin synthesis in cells.

[0077] In a preferred embodiment, the moisturizing effect promotes the synthesis of aquaporin 3.

[0078] In a preferred embodiment, the anti-aging is achieved through one or more of the following mechanisms: protecting type I collagen, inhibiting MMP-1 production, and inhibiting MMP-9 production.

[0079] In a preferred embodiment, the soothing and anti-inflammatory effect is the inhibition of TNF-α production.

[0080] In a preferred embodiment, the composition further comprises one or more excipients.

[0081] In a preferred embodiment, the excipient is selected from one or more of the following: thickeners, surfactants, co-surfactants, skin conditioners, preservatives, fragrances, lubricants, disintegrants, humectants, binders, and fillers.

[0082] In a preferred embodiment, the composition further comprises one or more skin-whitening substances.

[0083] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0085] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0086] Example

[0087] Preparation Examples

[0088] Experimental instruments and materials

[0089] Biochemical incubator (Shanghai Yiheng, LRH-150), autoclave (Shanghai Shenan, SYQ-DSX-280B), clean bench (Suzhou Purification, SW-CJ-2F), centrifuge (Hermle, Z216M), fully automatic double pure water system (Shanghai Jinke Industrial, SZ-93), anaerobic chamber (Mitsubishi Gas, ANAEROPACK), rotary evaporator (Shanghai Shensheng, R-206), circulating water vacuum pump (Shanghai Yuhua, SHZ-D), MRS broth medium (Beijing Luqiao), MRS agar medium (Oxoid).

[0090] The jasmine flowers used in this embodiment were purchased from the Shanghai Jiangqiao Wholesale Market. They were dried jasmine flowers from Guangxi and were identified as flower buds of *Jasminum sambac* (L.) Aiton, a plant in the Oleaceae family. They were stored in a well-ventilated and dry place for later use. The white plum blossoms used in this embodiment were also purchased from the Shanghai Jiangqiao Wholesale Market. They were dried white plum blossoms from Anhui and were identified as flower buds of *Prunus mumef. viralyx* (Makino) TYChen, a plant in the Rosaceae family. They were stored in a well-ventilated and dry place for later use.

[0091] Example 1

[0092] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added at 3% of the fermentation medium's mass fraction, followed by 6% of activated *Lactobacillus lactis* seed culture at a volume percentage of the fermentation medium. The fermentation was carried out at 25℃, pH 4.0, 100 rpm, and for 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth A.

[0093] Example 2

[0094] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added to the fermentation medium at 8% by weight, followed by activated *Lactobacillus lactis* seed culture at 10% by volume. The fermentation was carried out at 40℃, pH 7.0, 600 rpm, and for 48 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth B.

[0095] Comparative Example 1

[0096] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added to the fermentation medium at 15% by weight, followed by activated *Lactobacillus lactis* seed culture at 6% by volume. The fermentation was carried out at 25℃, pH 4.0, 100 rpm, and for 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth C.

[0097] Comparative Example 2

[0098] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added at 3% of the fermentation medium's mass fraction, followed by 2% of activated *Lactobacillus lactis* seed culture at 2% of the fermentation medium's volume fraction. The fermentation temperature was 25℃, pH 4.0, rotation speed 100 rpm, and fermentation time 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth D.

[0099] Comparative Example 3

[0100] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added at 3% of the fermentation medium's mass fraction, followed by 6% of activated *Lactobacillus lactis* seed culture at a volume percentage of the fermentation medium. The fermentation was carried out at 65℃, pH 4.0, 100 rpm, and for 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth E.

[0101] Comparative Example 4

[0102] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added to the fermentation medium at 3% by mass, followed by activated *Lactobacillus lactis* seed culture at 6% by volume. The fermentation was carried out at 25℃, pH 9.0, 100 rpm, and for 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth F.

[0103] Comparative Example 5

[0104] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added at 3% of the fermentation medium's mass fraction, followed by 6% of activated *Lactobacillus lactis* seed culture at 6% of the fermentation medium's volume fraction. The fermentation was carried out at 25℃, pH 4.0, 1200 rpm, and for 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth G.

[0105] Comparative Example 6

[0106] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added at 3% of the fermentation medium's mass fraction, followed by 6% of activated *Lactobacillus lactis* seed culture at a volume percentage of the fermentation medium. The fermentation was carried out at 25℃, pH 4.0, 100 rpm, and for 96 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth H.

[0107] Comparative Example 7

[0108] Jasmine flowers were crushed using a grinder and passed through a 10-mesh sieve. The crushed jasmine flowers were then added at 3% of the fermentation medium's mass fraction, followed by an inoculum of sterile seed culture medium at 6% of the fermentation medium's volume percentage. The fermentation was carried out at 25℃, pH 4.0, 100 rpm, and for 18 hours. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine flower fermentation broth I.

[0109] Comparative Example 8

[0110] White plum blossoms were crushed using a pulverizer and passed through a 10-mesh sieve. Then, pulverized jasmine blossoms were added at 3% of the fermentation medium's mass fraction, followed by activated *Lactobacillus lactis* seed culture at 6% of the fermentation medium's volume percentage. The fermentation temperature was 25℃, pH 4.0, rotation speed 100 r / min, and fermentation time 18 h. After fermentation, the resulting fermentation broth was inactivated and then purified multiple times to obtain jasmine blossom fermentation broth J.

[0111] Pharmacological Examples

[0112] Example 1: Determination of the inhibition rate of melanin synthesis in A375 cells (whitening)

[0113] 1.1 Experimental Instruments and Reagents

[0114] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), vortex mixer (Shanghai Hanno, XH-D), CO2 incubator (Thermo Scientific), microplate reader (Thermo Scientific), 24-well plate (Coming Incorporated), 96-well plate (Coming Incorporated), 6cm culture dish (Coming Incorporated), DMSO (Sigma), NaOH (Sinopharm), cell lysis buffer (Gibco), DMEM medium (Gibco), pen Strep (Gibco), fetal bovine serum (Gibco), 0.05% Trypsin-EDTA (Gibco), PBS (Vivacell), MTT (Sigma).

[0115] 1.2 Experimental Methods

[0116] Cell seeding: 1×10 5 A375 cells (Chinese Academy of Sciences Stem Cell Bank) were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0117] Preparation of solutions: Working solutions for the test substances were prepared according to the experimental design (Table 1). 50 mg each of 377, nonapeptide-1, and glycyrrhizin powder were added to 10 mL of DMEM medium (containing 10% fetal bovine serum) to obtain cell culture solutions with a concentration of 0.5% (w / w) for 377, nonapeptide-1, and glycyrrhizin. 50 μL each of jasmine fermentation broth AI and white plum fermentation broth J were added to 10 mL of DMEM medium (containing 10% fetal bovine serum) to obtain cell culture solutions with a concentration of 0.5% (w / w) for jasmine fermentation broth AI and white plum fermentation broth J. 100 μL each of jasmine fermentation broth AI and white plum fermentation broth J were added to 10 mL of DMEM medium (containing 10% fetal bovine serum) to obtain cell culture solutions with a concentration of 1% (w / w) for jasmine fermentation broth AI and white plum fermentation broth J.

[0118] Table 1. Pharmacological Examples 1. Experimental Design

[0119]

[0120] Drug administration: After 24 h of culture, according to the experimental design in Table 1, the drugs were administered to the groups. 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; and 1 mL of culture medium containing the corresponding concentration of the test substance was added to each well of the sample group. After drug administration, the 24-well plate was placed in an incubator (37℃, 5% CO2) and cultured for 24 h.

[0121] Detection: 72 hours after the third medium change, the melanin content of the cells was measured. Cells were washed twice with 1×PBS, the PBS was removed, and 200 μL of 0.25% trypsin was added to each well to digest the cells. The cells were then incubated in a CO2 incubator for 5 min. 2 mL of PBS was added twice, and the cells were collected by pipetting and centrifuging into centrifuge tubes. The tubes were centrifuged at 4000 rpm for 5 min. Any remaining PBS in the centrifuge tubes was removed as much as possible. 200 μL of melanin extraction solution was added to each tube, and the mixture was shaken well. The tubes were then heated in an 80℃ water bath for 1 h. After cooling, the droplets on the tube wall were centrifuged gently, and the mixture was pipetted and resuspended. 150 μL of the solution from each centrifuge tube was transferred to a 96-well plate, and the absorbance (OD) at 405 nm was measured using a microplate reader.

[0122] Calculate the relative inhibition rate of melanin in cells.

[0123] Cellular melanin inhibition rate (%) = [1 - (OD of drug administration well - OD of blank well) / (OD of cell control well - OD of blank well)] × 100

[0124] 1.3 Experimental Results

[0125] like Figure 1As shown, compared with the BC group, the 0.5% (w / w) concentration of 377 in the PC group significantly improved the melanin inhibition rate, indicating that the experimental system was effective. Compared with groups BC, the common whitening monomer raw materials nonapeptide-1 and glycyrrhizin in the experimental group significantly increased the melanin inhibition rate at a concentration of 0.5%. Compared with groups BC, the jasmine fermentation liquids A and B described in Examples 1 and 2 increased the melanin inhibition rate at a concentration of 0.5% and significantly increased it at a concentration of 1%, approaching the inhibition rate of nonapeptide-1 and glycyrrhizin at a concentration of 0.5%. This indicates that jasmine fermentation liquids A and B have the ability to inhibit melanin synthesis by melanocytes and have whitening effects. However, in comparative examples 1-8, the differences between jasmine fermentation liquid CI and white plum fermentation liquid J at concentrations of 0.5% and 1% and groups BC were not statistically significant (P>0.05), therefore neither showed the ability to inhibit melanin synthesis by melanocytes. This proves that only the jasmine fermentation liquid prepared within the scope of the preparation method described in the claims has whitening effects.

[0126] Example 2 of efficacy: Promoting the synthesis of aquaporin 3 (AQP3) (moisturizing) --- Determination of aquaporin 3 content

[0127] 2.1 Experimental Instruments and Reagents

[0128] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), microoscillator (Qilinbeier, TS-92). High-glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), trypsin (Gibco).

[0129] 2.2 Experimental Methods

[0130] Cell seeding: 1×10 5 Keratinocytes (HaCaT, stem cell bank of Chinese Academy of Sciences) were seeded into 24-well plates at a seeding density of 1 cell per well and incubated overnight in an incubator (37°C, 5% CO2).

[0131] Solution preparation: Following the solution preparation method in Example 1 of the efficacy study, the working solution of the test substance was prepared according to the experimental design (Table 2).

[0132] Table 2. Pharmacological Examples 2. Experimental Design

[0133]

[0134] Drug administration: According to the experimental design in Table 2, when the cell deposition rate in the 24-well plate reached 40% to 60%, the cells were administered to groups, with 1.0 mL administered to each well. Each group had 3 replicates, and the cells were cultured in an incubator (37℃, 5% CO2) for 24 h.

[0135] Detection: Collect samples, discard the supernatant, and wash cells three times with PBS. Perform routine immunofluorescence staining. Fix cells with 100% methanol, wash three times with PBS, and block each well with 1 mL of 1% BSA for 1 h. Discard the blocking solution, add 5 μg / mL primary antibody to each well, and incubate overnight at 4°C. Discard the primary antibody and wash three times with PBS. Add secondary antibody to each well and incubate for 2 h. Discard the secondary antibody and wash three times with PBS. Add 1.43 μM DAPI to each well for nuclear staining, incubate for 1 h, discard the DAPI, wash three times with PBS, and then photograph using a fluorescence microscope.

[0136] Results analysis: The fluorescence intensity of AQP3 was quantitatively analyzed using Image Pro Plus software.

[0137] 2.3 Experimental Results

[0138] like Figure 2 As shown, compared with group BC, the 0.5% (w / w) CaCl2 concentration in group PC significantly increased the fluorescence intensity (IOD) of AQP3 protein, indicating that the experimental system was effective. Compared with group BC, the jasmine fermentation broths A and B described in Examples 1 and 2 significantly increased the IOD at concentrations of 0.5% and 1%, indicating that jasmine fermentation broths A and B have the ability to promote the synthesis of aquaporin 3 (AQP3) and have moisturizing effects. However, in comparative examples 1-8, the differences between jasmine fermentation broth CI and white plum fermentation broth J at concentrations of 0.5% and 1% and group BC were not statistically significant (P>0.05), therefore neither showed the ability to promote AQP3 synthesis. This proves that only jasmine fermentation broths prepared within the scope of the preparation method described in the claims have moisturizing effects.

[0139] Example 3 of efficacy: Protection of type I collagen (UVA) (anti-aging) --- Determination of type I collagen content in UVA-stimulated fibroblasts

[0140] 3.1 Experimental Instruments and Reagents

[0141] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), CO2 incubator (Thermo Scientific), micro-shaker (Qilinbeier, TS-92), ultra-low temperature freezer (-80℃, Thermo), low-speed centrifuge (Bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), ELISA reader (Thermo Scientific), 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 kit (Thermo), type I collagen primary antibody and secondary antibody (Thermo), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0142] 3.2 Experimental Methods

[0143] Cell seeding: Human skin fibroblasts (Chinese Academy of Sciences Stem Cell Bank) were seeded into 24-well plates at a seeding density of 45%–60% confluence 24 hours after seeding and incubated overnight in an incubator (37°C, 5% CO2).

[0144] Solution preparation: Following the solution preparation method in Example 1 of the efficacy study, the working solution of the test substance was prepared according to the experimental design (Table 3).

[0145] Table 3. Pharmacological Examples 3. Experimental Design

[0146]

[0147]

[0148] Drug administration: According to the experimental design in Table 3, when the cell deposition rate in the 24-well plate reached 40%–60%, the cells were washed with PBS, and then cultured in PBS exposed to 9J / cm². 2 Under ultraviolet light. 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, while the positive control group was added with culture medium containing the positive control. Different test substances were used for 24 hours of exposure, with 1.0 mL administered per well, and each group had 3 replicates. The blank control group (groups BC) was added with cell culture medium and treated in the same way, but fully covered with aluminum foil to avoid ultraviolet exposure.

[0149] Detection: Cell supernatant collection: After incubation, collect 1 mL of cell culture supernatant into a 1.5 mL sterile centrifuge tube and store it at -80°C. Determine the type I collagen content according to the ELISA kit instructions.

[0150] Results Analysis: Collagen I Content Calculation: The OD450 values ​​of the standard and sample wells were subtracted from the OD450 values ​​of the blank wells, and the average value of the three parallel wells was taken. A standard curve was plotted using Curve Expert software, with the concentration of the standard on the ordinate and the OD450 value on the abscissa. A regression equation was derived, and the OD450 values ​​of the sample groups were substituted into the equation to calculate the Collagen I protein content of each group.

[0151] 3.3 Experimental Results

[0152] like Figure 3 As shown, compared with group BC, the concentration of type I collagen in group NC was significantly lower (P<0.01). Compared with group NC, the concentration of type I collagen in group PC was significantly increased by 100 μg / mL VC + 7 μg / mL VE (P<0.01), indicating that the experimental system was effective. Compared with group NC, the jasmine fermentation broths A and B described in Examples 1 and 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 jasmine fermentation broths A and B have the ability to protect type I collagen in fibroblasts under UVA stimulation and have anti-aging effects. However, in comparative examples 1-8, the differences between jasmine fermentation broth CI and white plum fermentation broth J at concentrations of 0.5% and 1% and group NC were not statistically significant (P>0.05), therefore neither showed the ability to protect type I collagen. This proves that only the jasmine fermentation broth prepared within the scope of the preparation method described in the claims has anti-aging effects.

[0153] Example 4 of pharmacological efficacy: Inhibition of MMP-1 synthesis (UVA) (anti-aging) --- Measurement of MMP-1 content in UVA-stimulated fibroblasts

[0154] 4.1 Experimental Instruments and Reagents

[0155] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), CO2 incubator (Thermo Scientific), micro-shaker (Qilinbeier, TS-92), ultra-low temperature freezer (-80℃, Thermo), low-speed centrifuge (Bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (Thermo Scientific), adjustable pipette (Eppendorf), PBS (Viva Cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), human MMP1 ELISA kit (Sigma), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0156] 4.2 Experimental Methods

[0157] Cell seeding: Human skin fibroblasts (Chinese Academy of Sciences Stem Cell Bank) were seeded into 24-well plates at a seeding density of 45%–60% confluence 24 hours after seeding and incubated overnight in an incubator (37°C, 5% CO2).

[0158] Solution preparation: Following the solution preparation method in Example 1 of the efficacy study, the working solution of the test substance was prepared according to the experimental design (Table 4).

[0159] Table 4. Pharmacological Examples 4. Experimental Design

[0160]

[0161]

[0162] Drug administration: According to the experimental design in Table 4, when the cell deposition rate in the 24-well plates reached 40%–60%, the cells were washed with PBS, and then cultured in PBS exposed to 9 J / cm². 2 Under ultraviolet light. 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, while the positive control group was added with culture medium containing the positive control. Different test substances were used for 24 hours of exposure, with 1.0 mL administered per well, and each group had 3 replicates. The blank control group (groups BC) was added with cell culture medium and treated in the same way, but fully covered with aluminum foil to avoid ultraviolet exposure.

[0163] Detection: Cell supernatant collection: After incubation, collect 1 mL of cell culture supernatant into a 1.5 mL sterile centrifuge tube and store it at -80°C. Determine the MMP-1 protein content according to the ELISA kit instructions.

[0164] Results analysis: MMP-1 protein content was calculated according to the operating instructions of the ELISA detection kit.

[0165] 4.3 Experimental Results

[0166] like Figure 4 As shown, compared with group BC, the MMP-1 concentration in group NC was significantly increased (P<0.01). Compared with group NC, the MMP-1 concentration in group PC was significantly decreased by 100 μg / mL VC + 7 μg / mL VE (P<0.01), indicating that the experimental system was effective. Compared with group NC, the jasmine fermentation broths A and B described in Examples 1 and 2 decreased the MMP-1 concentration at 0.5% concentration and significantly decreased the MMP-1 concentration at 1% concentration, indicating that jasmine fermentation broths A and B have the ability to inhibit the synthesis of MMP-1 protein by fibroblasts under UVA stimulation and have anti-aging effects. However, in comparative examples 1-8, the differences between jasmine fermentation broth CI and white plum fermentation broth J at 0.5% and 1% concentrations and group NC were not statistically significant (P>0.05), therefore neither showed the ability to inhibit MMP-1 synthesis. This proves that only the jasmine fermentation broth prepared within the scope of the preparation method described in the claims has anti-aging effects.

[0167] Example 5 of pharmacological efficacy: Inhibition of MMP-9 synthesis (UVA) (anti-aging) --- Determination of MMP-9 content in UVA-stimulated fibroblasts

[0168] 5.1 Experimental Instruments and Reagents

[0169] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), CO2 incubator (Thermo Scientific), micro-shaker (Qilinbeier, TS-92), ultra-low temperature freezer (-80℃, Thermo), low-speed centrifuge (Bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (Thermo Scientific), 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.

[0170] 5.2 Experimental Methods

[0171] Cell seeding: Human skin fibroblasts (Chinese Academy of Sciences Stem Cell Bank) were seeded into 24-well plates at a seeding density of 45%–60% confluence 24 hours after seeding and incubated overnight in an incubator (37°C, 5% CO2).

[0172] Solution preparation: Following the solution preparation method in Example 1 of the efficacy study, the working solution of the test substance was prepared according to the experimental design (Table 5).

[0173] Table 5. Pharmacological Examples 5. Experimental Design

[0174]

[0175]

[0176] Drug administration: According to the experimental design in Table 5, when the cell deposition rate in the 24-well plates reached 40%–60%, the cells were washed with PBS, and then cultured in PBS exposed to 9J / cm². 2 Under ultraviolet light. 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, while the positive control group was added with culture medium containing the positive control. Different test substances were used for 24 hours of exposure, with 1.0 mL administered per well, and each group had 3 replicates. The blank control group (groups BC) was added with cell culture medium and treated in the same way, but fully covered with aluminum foil to avoid ultraviolet exposure.

[0177] Detection: Cell supernatant collection: After incubation, collect 1 mL of cell culture supernatant into a 1.5 mL sterile centrifuge tube and store it at -80°C. Determine the MMP-9 protein content according to the ELISA kit instructions.

[0178] Results analysis: MMP-9 protein content was calculated according to the operating instructions of the ELISA detection kit.

[0179] 5.3 Experimental Results

[0180] like Figure 5As shown, compared with group BC, the MMP-9 concentration in group NC was significantly increased (P<0.01). Compared with group NC, the MMP-9 concentration in group PC was significantly decreased by 100 μg / mL VC + 7 μg / mL VE (P<0.01), indicating that the experimental system was effective. Compared with group NC, the jasmine fermentation broths A and B described in Examples 1 and 2 decreased the MMP-9 concentration at 0.5% concentration and significantly decreased the MMP-9 concentration at 1% concentration, indicating that jasmine fermentation broths A and B have the ability to inhibit the synthesis of MMP-9 protein by fibroblasts under UVA stimulation and have anti-aging effects. However, in comparative examples 1-8, the differences between jasmine fermentation broth CI and white plum fermentation broth J at 0.5% and 1% concentrations and group NC were not statistically significant (P>0.05), therefore neither showed the ability to inhibit MMP-9 synthesis. This proves that only the jasmine fermentation broth prepared within the scope of the preparation method described in the claims has anti-aging effects.

[0181] Example 6: Determination of TNF-α content (Shumin)

[0182] 6.1 Experimental Instruments and Reagents

[0183] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), microoscillator (Qilinbeier, TS-92). High-glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), trypsin (Gibco), bacterial lipopolysaccharide (LPS) (AbMole). Human TNF alpha ELISA kit (Thermo).

[0184] 6.2 Experimental Methods

[0185] Cell seeding: 1×10 5 RAW264.7 cells (Chinese Academy of Sciences Stem Cell Bank) were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0186] Solution preparation: Following the solution preparation method in Example 1 of the efficacy study, the working solution of the test substance was prepared according to the experimental design (Table 6).

[0187] Table 6. Pharmacological Examples 6. Experimental Design

[0188]

[0189] Drug administration: According to the experimental design in Table 6, when the cell deposition rate in the 24-well plate reached 40% to 60%, culture medium containing the sample was added to each group, with 3 replicates per group. The 24-well plate was then placed in an incubator (37℃, 5% CO2) and cultured for 24 hours.

[0190] Detection: After culturing for 24 hours, collect the cell supernatant and determine the TNF-α content according to the instructions of the ELISA kit.

[0191] Results analysis: TNF-α levels were calculated according to the instructions of the ELISA kit.

[0192] 6.3 Experimental Results

[0193] like Figure 6 As shown, compared with group BC, the TNF-α concentration in group NC was significantly increased (P<0.01). Compared with group NC, the TNF-α concentration in group PC was significantly decreased by 0.01% dexamethasone (P<0.01), indicating that the experimental system was effective. Compared with group NC, the jasmine fermentation broths A and B described in Examples 1 and 2 significantly reduced the TNF-α concentration at concentrations of 0.5% and 1%, respectively, indicating that jasmine fermentation broths A and B have the ability to inhibit TNF-α production under LPS stimulation and have a soothing effect. However, in comparative examples 1-8, the differences between jasmine fermentation broth CI and white plum fermentation broth J at concentrations of 0.5% and 1% and group NC were not statistically significant (P>0.05), therefore neither showed the ability to inhibit TNF-α production. This proves that only jasmine fermentation broths prepared within the scope of the preparation method described in the claims have a soothing effect.

[0194] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The use of jasmine ferment broth as the sole active ingredient in the preparation of soothing and anti-inflammatory cosmetics, characterized in that, The fermentation broth was obtained by fermenting jasmine flowers with Lactiplantibacillus plantarum, wherein Lactiplantibacillus plantarum is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28500. The method for preparing the fermentation broth includes the following steps: (1) Weigh an appropriate amount of jasmine flowers, crush them, and sieve them to obtain jasmine flower powder; (2) Add the culture medium to a container, add the jasmine powder and seed liquid, and ferment to obtain a crude fermentation liquid; and (3) The crude fermentation broth is inactivated and purified to obtain the jasmine fermentation broth; Wherein, the mass ratio of jasmine powder to culture medium is 0.03, the volume ratio of seed liquid to culture medium is 0.06, the fermentation temperature is 25℃, the fermentation pH is 4, the fermentation speed is 100 r / min, and the fermentation time is 18 h; or the mass ratio of jasmine powder to culture medium is 0.08, the volume ratio of seed liquid to culture medium is 0.1, the fermentation temperature is 40℃, the fermentation pH is 7, the fermentation speed is 600 r / min, and the fermentation time is 48 h.

2. The use of a composition containing jasmine ferment broth as the sole active ingredient in the preparation of a soothing and anti-inflammatory cosmetic, characterized in that, The fermentation broth was obtained by fermenting jasmine flowers with Lactiplantibacillus plantarum, wherein Lactiplantibacillus plantarum is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28500. The method includes the following steps: (1) Weigh an appropriate amount of jasmine flowers, crush them, and sieve them to obtain jasmine flower powder; (2) Add the culture medium to a container, add the jasmine powder and seed liquid, and ferment to obtain a crude fermentation liquid; and (3) The crude fermentation broth is inactivated and purified to obtain the jasmine fermentation broth; Wherein, the mass ratio of jasmine powder to culture medium is 0.03, the volume ratio of seed liquid to culture medium is 0.06, the fermentation temperature is 25℃, the fermentation pH is 4, the fermentation speed is 100 r / min, and the fermentation time is 18 h; or the mass ratio of jasmine powder to culture medium is 0.08, the volume ratio of seed liquid to culture medium is 0.1, the fermentation temperature is 40℃, the fermentation pH is 7, the fermentation speed is 600 r / min, and the fermentation time is 48 h.

3. The use according to claim 1 or 2, characterized in that, The sieve is a 10-mesh sieve.

4. The use according to claim 1 or 2, characterized in that, The culture medium is a liquid culture medium.

5. The use according to claim 1 or 2, characterized in that, The container is a fermentation tank.

6. The use according to claim 1 or 2, characterized in that, The inactivation temperature is 115–121°C.

7. The use according to claim 1 or 2, characterized in that, The inactivation time is 10-30 minutes.

8. The use according to claim 7, characterized in that, The inactivation time is 20 minutes.

9. The use according to claim 1 or 2, characterized in that, The purification process includes filtering and sterilizing the crude fermentation broth.

10. The use according to claim 1 or 2, characterized in that, The seed culture was obtained by culturing *Lactiplantibacillus plantarum* in a seed culture medium.

11. The use according to claim 10, characterized in that, The *Lactiplantibacillus plantarum* mentioned is an activated *Lactiplantibacillus plantarum*.

12. The use according to claim 10, characterized in that, The seed culture medium is a liquid seed culture medium.

13. The use according to claim 10, characterized in that, The plant lactobacillus plantarum was isolated from fermentation slurry.

14. The use according to claim 2, characterized in that, The composition further comprises one or more excipients.

15. The use according to claim 14, characterized in that, The excipients are selected from one or more of the following: thickeners, surfactants, co-surfactants, skin conditioners, preservatives, fragrances, lubricants, disintegrants, humectants, binders, and fillers.

Images