A plant fermentation composition for enhancing scalp immune barrier and melanocyte vitality, its preparation method and application

Abstract

The present invention discloses a plant fermentation composition for enhancing the scalp immune barrier and the vitality of melanocytes, and its preparation method and application, belonging to the technical fields of microbial fermentation and skin immunity. The preparation method of the plant fermentation composition comprises the following steps: using the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea as substrates, and fermenting with Wickerhamomyces anomalus and Saccharomyces cerevisiae; the mass ratio of Wickerhamomyces anomalus to Saccharomyces cerevisiae is 3-10:1; the weight ratio of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea is 10-30:1-10:1. The present invention utilizes microbial fermentation technology to provide a cosmetic raw material that promotes melanogenesis by enhancing the innate immune response of melanocytes, and solves the technical problem of black hair turning white. Applying the cosmetics of the present invention can improve the health of hair and scalp, and has a good effect of blackening hair, effectively preventing hair from turning grayish-white.

Description

Technical Field

[0001] The present invention belongs to the technical fields of microbial fermentation and skin immunity. Specifically, it relates to a preparation method and application of a plant fermentation composition for enhancing the scalp immune barrier and melanocyte viability. Background Art

[0002] Hair whitening is a common hair disease in the middle-aged and elderly population, which is caused by factors such as the decline of immunity, the decrease of melanocyte viability, and the reduction of tyrosinase activity caused by aging, resulting in the obstruction of melanin synthesis. At present, there is no effective clinical treatment for this disease. Most people use hair dyeing to cover it, but hair dyeing has a great impact on physical health. Hair dyes cannot fundamentally change the hair quality and may induce various diseases, such as contact dermatitis, leukemia, osteoporosis, and even canceration. Therefore, developing a safe and effective treatment method is crucial for solving the problem of hair whitening in the middle-aged and elderly population.

[0003] Studies have found that melanocytes have dual functions of immune defense and melanin synthesis. Like other epidermal resident cells, melanocytes play a role in enhancing the innate immune inflammatory response: melanocytes sense environmental stimuli by expressing Toll-like receptors and participate in the innate immune response. Subsequently, the activation of Toll-like receptors (such as TLR4) can enhance melanin synthesis. In addition, melanocytes promote the expression of antimicrobial peptides (such as β-defensin) in keratinocytes through paracrine cytokines such as IL-1 / TNF-α and interaction with keratinocytes. Therefore, developing a cosmetic raw material that promotes melanogenesis by enhancing the innate immune response of melanocytes is of great significance in the treatment of hair whitening.

[0004] A variety of plant extracts have extensive applications in promoting the conversion of white hair to black hair. For example, Patent CN104042752A discloses a composition for treating white hair, which is prepared from plant extracts in the following parts by weight: 25 - 35 parts by weight of Polygonum multiflorum Thunb., 20 - 30 parts by weight of black sesame, 15 - 25 parts by weight of ginseng, 25 - 35 parts by weight of Paeonia suffruticosa Andr., 20 - 30 parts by weight of Chinese date, 20 - 30 parts by weight of Rehmannia glutinosa Libosch., and 5 - 10 parts by weight of liquorice. Patent CN109939061A discloses a safe and non - irritating essence for converting white hair to black hair, which includes the following raw materials: water, flaxseed extract, angelica extract, aloe extract, tea polyphenols, ethanol, sodium benzoate, potassium sorbate, citric acid, ginseng extract, polygonum multiflorum extract, γ - linolenic acid, glucomannan, and crude cuttlefish ink extract. In addition, improving the efficacy of plant extracts by fermentation technology has become a research hotspot in the current cosmetics field. For example, the research on the proliferation effect of compound - strain - fermented ginseng extract on mouse hair follicle cells [J] (Wang Jingxia et al., Central South Pharmacy, 2024, 22(10), 2674 - 2679) found that when ginseng was fermented with a combination of three strains, Bacillus subtilis, Lactobacillus rhamnosus, and Lactobacillus casei, and the compound - strain - fermented ginseng extract was obtained after extraction, the proliferation rate of mouse hair follicle cells was 59.93%, and the proliferation effect was better than that of non - fermented ginseng extract. Patent CN119033641A discloses a method for preparing plant fermentation products by multi - strain co - fermentation, using a mixture of Bacillus velezensis and Bacillus subtilis as the fermentation broth; obtaining an extract from the plant raw material tea bran by water extraction; inoculating and fermenting the water - extracted product with the mixed broth of Bacillus velezensis and Bacillus subtilis; separating and purifying the fermentation broth to obtain the plant combined fermentation broth. This method can effectively increase the active substances in the fermentation broth and improve its antioxidant and other effects. However, none of the above - mentioned published patents or non - patent literatures mention that plant extracts increase melanin production by enhancing the immune function of melanocytes.

[0005] In the production of fruit wine, yeast plays an important role and can be divided into two categories: Saccharomyces cerevisiae and non-Saccharomyces cerevisiae (such as Wickerhamomyces anomalus). Saccharomyces cerevisiae often dominates in the initial stage of fermentation due to its high ethanol production ability and high tolerance. In contrast, Wickerhamomyces anomalus has the ability to produce diverse flavor components and extracellular enzymes and other metabolites, and it can more effectively decompose raw materials and convert these components into aroma-rich substances such as higher alcohols and esters. The synergistic interaction between these two yeasts can greatly improve the overall quality of fruit wine. However, Zhao Jianlei et al. found that in the mixed fermentation of Wickerhamomyces anomalus and Saccharomyces cerevisiae, a high concentration of Saccharomyces cerevisiae will cause growth stagnation and death of Wickerhamomyces anomalus in the initial stage of fermentation through a cell-cell contact mechanism. Therefore, in the mixed fermentation, the influence of the inoculation ratio of these two strains on the fermentation process and the product still needs to be further studied.

[0006] Currently, there is no research report on the use of a plant fermentation composition (Asparagus cochinchinensis, Polygonum multiflorum, black tea) prepared by the combined fermentation of Wickerhamomyces anomalus and Saccharomyces cerevisiae in improving white hair. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a plant fermentation composition for enhancing the scalp immune barrier and the vitality of melanocytes, and its preparation and application. The invention uses microbial fermentation technology to provide a cosmetic raw material that promotes melanogenesis by enhancing the innate immune response of melanocytes, and solves the technical problem of black hair turning white.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a plant fermentation composition for enhancing the scalp immune barrier and the vitality of melanocytes, which is prepared by fermenting the water extracts of Polygonum multiflorum, Asparagus cochinchinensis and black tea with Wickerhamomyces anomalus and Saccharomyces cerevisiae.

[0010] Asparagus, black tea, and Polygonum multiflorum all have various pharmacological activities and biological effects. Asparagus polysaccharide activates immune responses and innate immune memory through the TLR4-JNK / p38 / ERK signaling pathway, and is accompanied by the production of inflammatory factors, which may cause skin adverse reactions. Black tea can stimulate the proliferation of hair matrix keratinocytes by promoting the expression of insulin-like growth factor, prolong the hair growth cycle, and is beneficial to the generation of melanin in hair follicles during the growth phase (III–VI). This may be the effect of caffeine contained in black tea, but different production processes affect the yield of caffeine. Polygonum multiflorum promotes the generation of melanin in B16 cells by promoting the gene expression and protein synthesis of tyrosinase and microphthalmia-associated transcription factor and activating the activity of tyrosinase. However, Polygonum multiflorum has certain toxicity, especially serious harm to the liver. For example, the 95% ethanol extract of Polygonum multiflorum can significantly inhibit the growth of human liver L-02 cells.

[0011] The fermentation method provided by the present invention utilizes the metabolic activities of microorganisms and the advantages of the enzyme catalytic system to enrich the active ingredients in raw materials, and can significantly improve the efficacy and safety of plant extracts. In addition to producing secondary metabolites through fermentation to enhance the biological activity of plant extracts, microorganisms can also decompose and transform certain toxic components in plant extracts, converting toxic components into low-toxic or non-toxic substances, thereby reducing the damage to the body. At the same time, microorganisms can secrete various extracellular enzymes to decompose the tightly structured plant cell wall, making the gaps between cells larger, providing channels for the diffusion of substances inside and outside the cells, and thus increasing the extraction rate and absorption and utilization rate of active ingredients.

[0012] In some embodiments, the preparation method of the aqueous extract is as follows:

[0013] Step 1: Dry Polygonum multiflorum, Asparagus, and black tea, and collect the powder after crushing treatment;

[0014] Step 2: Add water to the powders of Polygonum multiflorum, Asparagus, and black tea obtained in Step 1, extract with ultrasonic assistance, separate the solid and liquid, and combine the filtrates to obtain the aqueous extract.

[0015] Preferably, the weight ratio of Polygonum multiflorum, Asparagus, and black tea in Step 1 is 10-30:1-10:1; more preferably 15-25:3-8:1.

[0016] Preferably, the drying in Step 1 is: drying at 45°C - 60°C for 3h - 8h; more preferably drying at 50°C - 55°C for 4h - 6h.

[0017] Preferably, the crushing in Step 1 is crushing through a 20-mesh - 120-mesh sieve; more preferably through a 40-mesh - 100-mesh sieve; even more preferably through a 40-mesh - 80-mesh sieve.

[0018] Preferably, the amount of water added in step 2 is 3 to 12 times the total weight of polygonum multiflorum, asparagus and black tea powder; more preferably 5 to 10 times.

[0019] Preferably, the power of ultrasonic-assisted extraction in step 2 is 200W - 300W, the time is 20min - 30min, and the number of extractions is at least 1 time; more preferably, the ultrasonic power is 250W - 280W and the time is 25min - 30min.

[0020] In some more specific embodiments, the preparation method of the aqueous extract is as follows:

[0021] Step 1: Weigh 10 to 30 parts of polygonum multiflorum, 1 to 10 parts of asparagus and 1 part of black tea respectively, dry them in an oven at 50°C - 55°C for 4h - 6h, pulverize them through a 20-mesh to 120-mesh sieve, and collect the powder.

[0022] Step 2: According to the solid-liquid weight ratio of 1:3, add water to the polygonum multiflorum, asparagus and black tea powder obtained in step 1, perform ultrasonic-assisted extraction at 200W - 300W for 20min - 30min, separate the solid and liquid, and combine the filtrates.

[0023] In some embodiments, the ratio of Wickerhamomyces anomalus to Saccharomyces cerevisiae is 3 - 10:1; more preferably 5 - 8:1.

[0024] In some more specific embodiments, the plant fermentation composition is prepared by fermenting the aqueous extracts of polygonum multiflorum, asparagus and black tea with Wickerhamomyces anomalus and Saccharomyces cerevisiae under the condition of 26°C - 32°C.

[0025] In a second aspect, the present invention provides a method for preparing the above plant fermentation composition, comprising the following steps:

[0026] S1. Substrate preparation: Sterilize the aqueous extracts of polygonum multiflorum, asparagus and black tea to obtain the substrate.

[0027] S2. Fermentation: Inoculate Wickerhamomyces anomalus and Saccharomyces cerevisiae into the substrate for fermentation to obtain the fermentation broth.

[0028] S3. Separate the solid and liquid of the fermentation broth, filter, and take the filtrate to obtain the product.

[0029] In some embodiments, the inoculation amount of Wickerhamomyces anomalus and Saccharomyces cerevisiae in step S2 is 3% - 10% of the weight of the substrate.

[0030] In some embodiments, the fermentation conditions in step S2 are: fermentation temperature 26°C - 32°C, fermentation time 60h - 120h.

[0031] In a third aspect, the present invention provides the use of the above preparation method or the above fermentation composition in the preparation of cosmetics.

[0032] In some embodiments, the cosmetics are scalp care and / or hair care products.

[0033] In a fourth aspect, the present invention provides a cosmetic for improving hair and scalp health, comprising the above plant fermentation composition.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) The plant fermentation composition of the present invention is prepared by fermenting the water extracts of Polygonum multiflorum, Asparagus cochinchinensis, and black tea with Wickerhamomyces anomalus and Saccharomyces cerevisiae. It is beneficial to the complementary and synergistic system of the active components of Polygonum multiflorum, Asparagus cochinchinensis, and black tea, and jointly exerts multiple effects such as inhibiting the growth of harmful bacteria, scavenging free radicals, enhancing the vitality of melanocytes and melanin synthesis. At the same time, the synergistic effect of Wickerhamomyces anomalus and Saccharomyces cerevisiae is used to convert the macromolecular active ingredients in the water extracts of Polygonum multiflorum, Asparagus cochinchinensis, and black tea into small molecular active ingredients that are easy to absorb, further improving the efficacy of the plant extracts.

[0036] (2) The plant fermentation composition of the present invention promotes the expression of β-defensin in keratinocytes and simultaneously inhibits the production of the inflammatory factor IL-6, and has the effect of enhancing the scalp immune barrier.

[0037] (3) The cosmetics applying the plant fermentation composition of the present invention can inhibit Propionibacterium acnes and Staphylococcus aureus, maintain the stability of the microbiota, regulate the microecological balance of the skin surface to enhance the scalp immune barrier, improve hair and scalp health, have a good effect of blackening hair, and effectively prevent hair from turning gray.

[0038] (4) On the one hand, the plant fermentation composition of the present invention has antioxidant properties and can reduce the damage caused by free radicals to follicular melanocytes. On the other hand, it can increase the expression of Toll-like receptor 4, tyrosinase activity of melanocytes, and promote melanogenesis, and jointly achieve a good effect of blackening hair from two aspects of enhancing antioxidant capacity and promoting melanogenesis.

[0039] (5) The preparation method of the present invention is simple, has good safety, low cost, and is suitable for large-scale popularization and application. Detailed embodiments

[0040] The following description of the embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The following description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but can be applied to a wider range consistent with the principles and novel features disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0041] In addition to the above basic components (plant fermentation composition), the optional components of the cosmetics described in the present invention may also contain one or more known or other effective optional components used in scalp care, hair care or personal care products, as long as these optional components are compatible with the physical and chemical properties of the basic components described herein, or do not overly affect the stability, aesthetics or performance of the product. Non-limiting examples of such optional components are described in the "Technical Specifications for Cosmetics Safety, 2015 Edition", which are incorporated herein by reference.

[0042] In the solid-liquid separation involved in the present invention, the purpose is to achieve the separation of the solid phase and the liquid phase. Therefore, conventional technical means such as centrifugation, filtration or pressure filtration can be used.

[0043] Unless otherwise specified, all operations herein are carried out at room temperature, and the solvents used are all water.

[0044] In the present invention, "melanin" and "melanochrome" have the same meaning and can be replaced with each other.

[0045] Next, the embodiments and comparative examples of the present invention are shown to describe the present invention in more detail, but the present invention is not limited thereto. The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products in this technical field. In the embodiments, Wickerhamomyces anomalus, with the Latin name Wickerhamomyces anomalus, is purchased from the Guangdong Provincial Microbial Culture Collection Center, with the number ZKCC12538, and Saccharomyces cerevisiae is purchased from the China Center for Type Culture Collection, with the preservation numbers CCTCC KY 2008613 respectively.

[0046] [Preparation of Wickerhamomyces anomalus liquid and Saccharomyces cerevisiae liquid]

[0047] Streak Wickerhamomyces anomalus and Saccharomyces cerevisiae on YPD solid medium respectively, and culture at 28±2°C for 36 h to restore the activity of the strains. Pick single colonies on the plate into 100 mL of sterilized YPD liquid medium, and culture at 28±2°C and 150 rpm / min for 36 hours to obtain Wickerhamomyces anomalus seed liquid and Saccharomyces cerevisiae seed liquid respectively. Take an appropriate amount of the bacterial liquid, dilute it with sterile normal saline, and adjust the OD 600 value to the same range (OD 600 =1.0) to ensure that the initial concentrations of the two yeast strains are the same.

[0048] Example 1 Preparation of plant fermentation composition

[0049] S1. By weight, take 10 parts of Polygonum multiflorum, 1 part of Asparagus cochinchinensis, and 1 part of black tea respectively, dry them in an oven at 52±2°C for 5 hours, pulverize them, and pass through an 80-mesh sieve. According to the material-liquid weight ratio of 1:3, add distilled water to the powder, place it in an ultrasonic extractor, set the ultrasonic power to 200W and the time to 30min. After the ultrasonic treatment, centrifuge at 2500 rpm for 20 min to obtain an aqueous extract. Sterilize the aqueous extract at 121°C under high temperature and high pressure for 20 min, and cool to obtain a substrate.

[0050] S2. Take 3 parts of Wickerhamomyces anomalus liquid and 1 part of Saccharomyces cerevisiae liquid and mix them according to a weight ratio of 3:1 to prepare a mixed bacterial liquid. Inoculate the mixed bacterial liquid (inoculation amount is 10% of the weight of the substrate) into the substrate obtained in step S1, and ferment at a stirring speed of 150 rpm / min and a temperature of 30±2°C for 60 h to obtain a fermentation product.

[0051] S3. Centrifuge the fermentation product obtained in step S2 at 2500 rpm / min for 20 min, and filter it with a 0.22μm ultrafiltration membrane to obtain a plant fermentation composition.

[0052] Example 2 Preparation of plant fermentation composition

[0053] S1. By weight, take 30 parts of Polygonum multiflorum, 10 parts of Asparagus cochinchinensis, and 1 part of black tea respectively, dry them in an oven at 52±2°C for 5 hours, pulverize them, and pass through an 80-mesh sieve. According to the material-liquid weight ratio of 12:1, add distilled water to the powder, place it in an ultrasonic extractor, set the ultrasonic power to 300W and the time to 20min. After the ultrasonic treatment, centrifuge at 2500 rpm for 20 min to obtain an aqueous extract. Sterilize the aqueous extract at 121°C under high temperature and high pressure for 30 min, and cool to obtain a substrate.

[0054] S2. Take 10 parts of Wickerhamomyces anomalus yeast liquid and 1 part of Saccharomyces cerevisiae yeast liquid, mix them according to a weight ratio of 10:1 to prepare a mixed yeast liquid. Inoculate the mixed yeast liquid into the substrate obtained in step S1 (inoculation amount is 3% of the substrate weight), and ferment for 120 h under the conditions of a stirring speed of 150 rpm / min and a temperature of 28 ± 2 °C to obtain a fermentation product.

[0055] S3. Centrifuge the fermentation product obtained in step S2 at 2500 rpm for 20 min, and filter it with a 0.22 μm ultrafiltration membrane to obtain a plant fermentation composition.

[0056] Example 3 Preparation of plant fermentation composition

[0057] S1. Take 20 parts of Polygonum multiflorum, 5 parts of Asparagus cochinchinensis, and 1 part of black tea by weight, dry them in an oven at 52 ± 2 °C for 5 h, pulverize them, and then pass through an 80-mesh sieve. Add distilled water to the powder according to a material-liquid weight ratio of 8:1, place it in an ultrasonic extractor, set the ultrasonic power to 250 W and the time to 25 min. After the ultrasonic treatment, centrifuge at 2500 rpm for 20 min to obtain an aqueous extract. Sterilize the aqueous extract at 121 °C under high temperature and high pressure for 20 min, and cool to obtain a substrate.

[0058] S2. Take 6 parts of Wickerhamomyces anomalus yeast liquid and 1 part of Saccharomyces cerevisiae yeast liquid, mix them according to a weight ratio of 6:1 to prepare a mixed yeast liquid. Inoculate the mixed yeast liquid into the substrate obtained in step S1 (inoculation amount is 5% of the substrate weight), and ferment for 90 h under the conditions of a stirring speed of 150 rpm / min and a temperature of 28 ± 2 °C to obtain a fermentation product. [[ID=!4]]

[0059] S3. Centrifuge the fermentation product obtained in step S2 at 2500 rpm for 20 min, and filter it with a 0.22 μm ultrafiltration membrane to obtain a plant fermentation composition.

[0060] Comparative Example 1 Preparation of plant fermentation composition

[0061] The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the yeast is only Wickerhamomyces anomalus.

[0062] Comparative Example 2 Preparation of plant fermentation composition

[0063] The preparation steps and parameters of this comparative example are basically the same as those of Example 3, except that the yeast is only Saccharomyces cerevisiae.

[0064] Comparative Example 3 Preparation of plant fermentation composition

[0065] This comparative example has the same preparation steps and parameters as Example 3, except that the ratio of Wickerhamomyces anomalus to Saccharomyces cerevisiae in the mixed bacterial solution is different.

[0066] Specifically: According to a weight ratio of 2:1, take 2 parts of Wickerhamomyces anomalus bacterial solution and 1 part of Saccharomyces cerevisiae bacterial solution and mix them to prepare a mixed bacterial solution.

[0067] Preparation of the plant fermentation composition of Comparative Example 4

[0068] This comparative example has the same preparation steps and parameters as Example 3, except that the ratio of Wickerhamomyces anomalus to Saccharomyces cerevisiae in the mixed bacterial solution is different.

[0069] Specifically: According to a weight ratio of 12:1, take 12 parts of Wickerhamomyces anomalus bacterial solution and 1 part of Saccharomyces cerevisiae bacterial solution and mix them to prepare a mixed bacterial solution.

[0070] Preparation of the plant fermentation composition of Comparative Example 5

[0071] This comparative example has the same preparation steps and parameters as Example 3, except that Schizosaccharomyces pombe is used to replace Wickerhamomyces anomalus.

[0072] Preparation of the plant fermentation composition of Comparative Example 6

[0073] This comparative example has the same preparation steps and parameters as Example 3, except that Schizosaccharomyces pombe is used to replace Saccharomyces cerevisiae in step (1).

[0074] Preparation of the plant fermentation composition of Comparative Example 7

[0075] This comparative example has the same preparation steps and parameters as Example 3, except that the weight ratio of Polygonum multiflorum, Asparagus cochinchinensis and black tea in the substrate is different;

[0076] Specifically: Weigh 35 parts of Polygonum multiflorum, 0.5 part of Asparagus cochinchinensis and 1 part of black tea respectively.

[0077] Preparation of the plant fermentation composition of Comparative Example 8

[0078] This comparative example has the same preparation steps and parameters as Example 3, except that the weight ratio of Polygonum multiflorum, Asparagus cochinchinensis and black tea in the substrate is different.

[0079] Specifically: Weigh 8 parts of Polygonum multiflorum, 12 parts of Asparagus cochinchinensis and 1 part of black tea respectively.

[0080] Preparation of the plant composition of Comparative Example 9

[0081] By weight, take 20 parts of Polygonum multiflorum, 5 parts of Asparagus cochinchinensis, and 1 part of black tea respectively, dry them in an oven at 52 ± 2 °C for 5 hours, and after pulverization, pass through an 80-mesh sieve. Add distilled water to make the solid-liquid ratio 5 - 10:1, place it in an ultrasonic extractor, set the ultrasonic power to 250 W and the time to 25 min. After ultrasonic extraction, centrifuge at 2500 rpm for 20 min to obtain an aqueous extract, and sterilize the aqueous extract at 121 °C under high temperature and high pressure for 20 min, then cool to obtain the product.

[0082] Effect Example 1 Enhancement of scalp immunity

[0083] 1. Detection of the viability of keratinocytes (HaCaT)

[0084] In this experiment, a blank group (DMEM medium), a cell control group (DMEM medium + HaCaT cells), and a sample group (DMEM medium + HaCaT cells + test samples provided by Examples 1 - 3 and Comparative Examples 1 - 9) were set up for cell viability testing, and each group had 3 parallel wells.

[0085] Seed HaCaT cells in the logarithmic growth phase at a density of 1×10 5 cells / mL into a 96-well plate. Incubate at 37 °C and 5% CO2 for 24 hours. Remove the culture medium, add 100 μL of sample solution to each well respectively, and continue to incubate at 37 °C and 5% CO2 for 24 hours. Add 20 μL of MTT solution to each well and continue to culture for 2 hours. Discard the supernatant, and add 100 μL of isopropanol to each well. Under dark conditions at room temperature, shake at a rate of 100 r / min for 30 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each well at a wavelength of 570 nm.

[0086] Calculate the cell viability according to the following formula, screen the sample mass concentration with a cell viability greater than 90% and no visible morphological changes, and carry out subsequent efficacy evaluation research.

[0087]

[0088] Among them, OD (sample group) represents the absorbance of the sample group, OD (blank group) represents the absorbance of the blank group, and OD (cell control group) represents the absorbance of the cell control group.

[0089] The results are shown in Table 1.

[0090] Table 1

[0091]

[0092] The results showed that when the concentration was 5 mg / mL, the cell viability of each group of samples was maintained above 90%, indicating that the plant fermentation composition of the present invention had no obvious cytotoxic effect, so it could be used for the determination of the contents of IL-6 and HBD-2.

[0093] 2. Determination of the contents of interleukin-6 (IL-6) and human β-defensin-2 (HBD-2)

[0094] Grouping for the IL-6 content determination test: A control group (DMEM), a model group (1 μg / mL LPS + cells + DMEM), a positive control group (1 μg / mL LPS + 35 μM quercetin + cells + DMEM), and a sample group (1 μg / mL LPS + 5 mg / mL test samples provided by Examples 1-3 and Comparative Examples 1-9 + cells + DMEM) were set up to determine the content of IL-6. Each group had 3 parallel wells.

[0095] Grouping for the HBD-2 content determination test: A control group (DMEM), a sample group (5 mg / mL test samples provided by Examples 1-3 and Comparative Examples 1-9 + HaCaT cells + DMEM), and a positive control group (100 mg / mL L-arginine) were set up to determine the content of HBD-2. Each group had 3 parallel wells.

[0096] HaCaT cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 1×10 4 cells / mL. They were incubated at 37°C and 5% CO2 for 24 hours. The culture medium was removed, and 500 μL of sample solution diluted with DMEM as the culture medium was added to each well. Incubation was continued at 37°C and 5% CO2 for 24 hours. The supernatant was centrifuged for 3 minutes (4°C, 12,000 r / min).

[0097] According to the operation instructions of the kits for interleukin-6 (IL-6) and human β-defensin-2 (HBD-2) respectively, after adding samples, incubation, washing the plates, adding enzyme reagents, incubating and washing the plates again, color reaction was carried out, and the reaction was terminated. The contents of IL-6 and HBD-2 in the supernatant were detected by machine respectively: Standard curves were drawn, and the concentrations of IL-6 and HBD-2 (in units of pg / mL) in the cell culture supernatants of each group were calculated respectively.

[0098] The inhibition rate of IL-6 and the promotion rate of HBD-2 were calculated according to the following formulas respectively. The higher the inhibition rate of IL-6, the stronger the ability of the plant fermentation composition to inhibit the inflammatory reaction of HaCaT cells; the higher the promotion rate of HBD-2, the stronger the ability of the plant fermentation composition to enhance the innate immune response of HaCaT cells, and the immune function of the scalp can be enhanced.

[0099]

[0100] The results are shown in Table 2.

[0101] Table 2

[0102]

[0103] The results show that the plant fermentation composition provided by the present invention can enhance the scalp immune function by enhancing the expression of β - defensin - 2 (HBD - 2); at the same time, it can inhibit the secretion of IL - 6 in HaCaT cells to avoid excessive inflammatory reactions, and has the effect of enhancing the scalp immune barrier.

[0104] Effect Example 2: Regulating the microecological balance of the scalp surface

[0105] Antibacterial evaluation: The Oxford cup punching method was used to analyze the antibacterial effects of the plant fermentation composition against Propionibacterium acnes (ATCC11827) and Staphylococcus aureus (ATCC6538).

[0106] The above strains were made into a bacterial suspension with a concentration of 2×10 5 CFU / mL using PBS. The bacterial suspension and the culture medium were added to the petri dish and mixed well. Sterile Oxford cups were placed on the surface of the culture medium to punch holes. After solidification, 100 μg of the test sample (the test samples provided in Examples 1 - 3 and Comparative Examples 1 - 9 at 5 mg / mL) was added. Each group had 3 parallel holes. Staphylococcus aureus was cultured under aerobic conditions at 37 ± 2°C for 2 days, and Propionibacterium acnes was cultured under anaerobic conditions at 37 ± 2°C for 2 days. After cultivation, the diameter of the antibacterial zone was measured with a vernier caliper, and the antibacterial effect was evaluated by the diameter of the antibacterial zone. Among them, if the diameter of the antibacterial zone of the test sample > 8 mm, it has an antibacterial effect; if the diameter of the antibacterial zone of the test sample ≤ 8 mm, it has no antibacterial effect.

[0107] The results are shown in Table 3.

[0108] Table 3

[0109]

[0110] The results show that the plant fermentation compositions prepared in Examples 1 - 3 of the present invention can simultaneously inhibit Propionibacterium acnes and Staphylococcus aureus, maintain the stability of the microbiota, and have the effect of regulating the microecological balance of the skin surface.

[0111] Comparing the groups of Comparative Examples 1-9 and Example 3, it can be seen that the plant fermentation compositions prepared in Comparative Examples 1-9 cannot effectively inhibit Propionibacterium acnes and Staphylococcus aureus, indicating that the co-fermentation of the abnormal strains Wickerhamomyces anomalus and Saccharomyces cerevisiae during the fermentation process and the dosage of plant components (Asparagus cochinchinensis, Polygonum multiflorum, and black tea) have a greater impact on the antibacterial effect of the obtained plant fermentation composition.

[0112] Effect Example 3 Enhancing the viability of melanoma B16 cells

[0113] Dilute melanoma B16 cells with the culture medium and seed them into a 96-well plate at a cell density of 0.8×10 4 cells per well. After culturing for 24 h, add 100 μL of the sample solution, blank control group (DMEM medium), cell control group (melanoma B16 cells + DMEM medium), and sample group (melanoma B16 cells + test samples provided by Examples 1-3 and Comparative Examples 1-9 + DMEM medium) in sequence, and continue to culture for 24 h. Add 10 μL of CCK-8 solution to each well, incubate in a CO2 incubator at 37 °C for 3 h, and measure the absorbance (OD) at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0114] According to the following formula, calculate the cell viability (%) of melanoma B16 cells under the treatment of the test samples provided by Examples 1-3 and Comparative Examples 1-9.

[0115]

[0116] Among them, OD (sample group) represents the absorbance of the test sample group, OD (blank control group) represents the absorbance of the blank control group, and OD (cell control group) represents the absorbance of the cell control group.

[0117] The results are shown in Table 4.

[0118] Table 4

[0119]

[0120] The results show that the samples provided by Examples 1-3 of the plant fermentation composition have a cell viability greater than 100% in the concentration range of 1.25-5 mg / mL, which can promote the proliferation of mouse melanoma B16 cells. And as the concentration of the test sample increases, the viability of B16 cells gradually increases. However, the test samples provided by Comparative Examples 1-9 failed to enhance the viability of B16 cells. Among them, for the test samples provided by Examples 1-3 and Comparative Examples 1-9, at a treatment concentration of 5 mg / mL, the viability of B16 cells is the highest, greater than 90%, and no morphological changes are observed, which can be used for subsequent efficacy evaluation studies.

[0121] Effect Example 4 Enhances the Antioxidant Capacity of Melanoma B16 Cells

[0122] In this experiment, a blank control group (melanoma B16 cells + DMEM medium), a model group (melanoma B16 cells + DMEM medium), a positive control group (melanoma B16 cells + 5 mg / mL vitamin C + DMEM medium), and a sample group (melanoma B16 cells + test samples provided by Examples 1 - 3 and Comparative Examples 1 - 9 + DMEM medium) were set up for ROS testing, with 6 parallel wells in each group. Melanoma B16 cells were diluted with DMEM medium, and 100 μL of cell suspension (the number of cells per well was 1×10 4 cells) was added dropwise to a 96-well plate. When the cell density reached 70% - 80%, the sample group and the model group were treated with 150 μmol / L H2O2 for 10 min. After the treatment, the test samples were added to the experimental groups, and serum-free DMEM medium was added to the blank control group and the model group. Then, they were incubated in a CO2 incubator for 30 min. Subsequently, DCFH-DA (2',7'-dichlorofluorescein diacetate fluorescence probe) was diluted with serum-free medium to a final concentration of 5 μmol / L and incubated in a 37 °C incubator for 30 min. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. The absorbance value was detected using an enzyme-linked immunosorbent assay (ELISA) reader with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

[0123] According to the following formula, the cell viability (%) of melanoma B16 cells treated with the test samples provided by Examples 1 - 3 and Comparative Examples 1 - 9 was calculated. Compared with the model group, a higher ROS inhibition rate indicates stronger antioxidant capacity, which is more beneficial for reducing the damage caused by oxidative stress to melanocytes.

[0124]

[0125] The results are shown in Table 5.

[0126] Table 5

[0127]

[0128] The results showed that the plant fermentation compositions provided in Examples 1 - 3 of the present invention could effectively reduce the ROS level, had strong antioxidant capacity, could effectively reduce the oxidative damage caused by free radicals to follicular melanocytes, prevent the occurrence of problems such as white hair, and the effect was significantly better than that of the positive drug.

[0129] Comparing the groups of Comparative Examples 1-2 and Example 3, it can be seen that in the present invention, when only Wickerhamomyces anomalus or Saccharomyces cerevisiae is used to ferment the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea, the ROS inhibition rate of the prepared plant fermentation composition is only 58.7%-66.2%. However, in the group of Example 3, when Wickerhamomyces anomalus and Saccharomyces cerevisiae are used in combination to ferment the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea, the ROS inhibition rate of the prepared plant fermentation composition reaches 94.8%, indicating that the combination of Wickerhamomyces anomalus and Saccharomyces cerevisiae in the present invention has a synergistic effect in terms of antioxidant capacity.

[0130] Comparing the groups of Comparative Examples 3-6 and Example 3, it can be seen that in the present invention, when the combination of Wickerhamomyces anomalus and Saccharomyces cerevisiae in conventional dosages or the combination of conventional yeasts is used to ferment the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea, the ROS inhibition rate of the prepared plant fermentation composition is only 60.9%-70.8%, which is much lower than that of the group of Example 3. It shows that the plant fermentation composition prepared by the combination of Wickerhamomyces anomalus and Saccharomyces cerevisiae in conventional dosages or the combination of conventional yeasts cannot produce a synergistic effect in terms of antioxidant capacity.

[0131] Comparing the groups of Comparative Examples 7-8 and Example 3, it can be seen that in the present invention, the dosages of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea have a great influence on the antioxidant capacity of the plant fermentation composition, and the water extracts prepared with conventional dosages of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea cannot achieve the technical effects of the invention.

[0132] Effect Example 5 Promotion of the expression of Toll-like receptor 4 (TLR4) in melanoma B16 cells

[0133] The upstream and downstream sequences of GAPDH (internal reference) and TLR4 were purchased from Beijing Tsingke Biotechnology Co., Ltd. In this experiment, mouse melanoma B16 cells were treated with a blank control group (DMEM medium) and a sample group (5 mg / mL plant fermentation compositions provided in Examples 1-3 and Comparative Examples 1-9 + DMEM medium) for 48 h. RNA of the above samples was extracted using an RNA extraction kit. The RNA concentration and purity were detected by a NANO-400A ultra-micro nucleic acid analyzer. According to the instructions of the reverse transcription kit, a reverse transcription reaction system was prepared on ice. After reverse transcribing RNA into cDNA on a PCR instrument according to the instructions on the reverse transcription kit, a real-time fluorescence quantitative PCR (RT-PCR) reaction system was prepared on ice: 5 μL of RT-PCR SYBR Green was taken on a 96-well plate dedicated to fluorescence quantitative PCR, 0.2 μL of each upstream and downstream primer, 1 μL of the cDNA of the sample, and 3.6 μL of double-distilled water; the RT-PCR reaction conditions of Bio-rad: pre-denaturation at 95 °C for 2 min, denaturation at 95 °C for 10 s, annealing / extension at 60 °C for 30 s, 40 cycles. According to the obtained Ct values, analysis was performed using 2-△△Ct. After repeating the experiment three times, the activation rate of TLR4 was calculated according to the following formula.

[0134]

[0135] The higher the activation rate, the stronger the ability of the plant fermentation composition to enhance the response of melanocytes to innate immune stimulation.

[0136] The results are shown in Table 6.

[0137] Table 6

[0138]

[0139] The results showed that: the plant fermentation compositions provided in Examples 1-3 of the present invention all had an activating effect, which was significantly better than that of Comparative Examples 1-9. It was shown that the plant fermentation composition provided by the present invention could promote the expression of Toll-like receptor 4 (TLR4) in melanoma B16 cells and play a role in promoting melanogenesis by participating in the innate immune response.

[0140] Effect Example 6 Enhancement of tyrosinase activity

[0141] Cell culture and experimental treatment were the same as in Effect Example 4. An enzyme kinetics experiment grouping was set up: a control group (DMEM medium + B16 cells) and a sample group (DMEM medium + B16 cells + test samples provided in Examples 1 - 3 and Comparative Examples 1 - 9). A 200 μL reaction system was used, with 5 replicates set for each concentration, and cultured for 48 h. The supernatant was discarded, and the cells were washed twice with PBS. 80 μL of PBS buffer containing 1% TritonX - 100 was added to each well, and the plate was quickly placed in a -80°C freezer for 1 h. It was taken out and thawed at room temperature, incubated at 37°C for 5 min, 20 μL of levodopa with a mass fraction of 0.5% was added, and the reaction was carried out at 37°C for 6 h. The absorbance value at 490 nm was measured using an enzyme - linked immunosorbent assay (ELISA) reader. [[ID=②]]

[0142] The enzyme activity was calculated according to the following formula: Tyrosinase activity = OD value of the experimental group / OD value of the control group × 100%.

[0143]

[0144] The higher the activation rate of tyrosinase, the stronger the ability of the plant fermentation composition to promote melanin production, and the better the hair - blackening effect.

[0145] The results are shown in Table 7.

[0146] Table 7

[0147]

[0148] The results showed that the plant fermentation compositions provided in Examples 1 - 3 all had the effect of activating tyrosinase, and were significantly superior to Comparative Examples 1 - 9.

[0149] At the same time, by comparing the groups of Comparative Examples 1 - 2 and Example 3, it can be seen that in the present invention, when only Wickerhamomyces anomalus or Saccharomyces cerevisiae is used to ferment the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr., and black tea, the plant fermentation composition prepared therefrom has a weak effect on activating tyrosinase, and the activation rate is only 35.2% - 37.8%. While in Example 3, Wickerhamomyces anomalus and Saccharomyces cerevisiae are used in combination to ferment the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr., and black tea, and the activation rate of the plant fermentation composition prepared therefrom reaches 89.6%, indicating that the combination of Wickerhamomyces anomalus and Saccharomyces cerevisiae in the present invention has a synergistic effect in activating tyrosinase. [[ID=②⑤]] [[ID=②⑥]]

[0150] Comparison of Comparative Examples 3-6 with Example 3 shows that in the present invention, the plant fermentation compositions prepared by fermenting the aqueous extracts of Polygonum multiflorum, Asparagus cochinchinensis, and black tea using conventional amounts of a combination of abnormal Wickham's yeast and Saccharomyces cerevisiae, or a combination of conventional yeasts, exhibited tyrosinase activation rates of no more than 40%, significantly lower than that of Example 3. This indicates that conventional amounts of a combination of abnormal Wickham's yeast and Saccharomyces cerevisiae, or a combination of conventional yeasts, do not produce a synergistic effect in activating tyrosinase.

[0151] Effect Example 7: Increase the melanin content of melanocytes

[0152] Cell culture and experimental grouping were the same as in Example 4. 100 μL of B16 melanoma cell suspension was added to a 96-well plate to a cell density of 0.5×10 4 Each well was plated with 5 replicates and cultured in an incubator (37°C, 5% carbon dioxide) for 12 h. 1 mL of culture medium without the test sample was added to the blank control group and the negative control group, and 1 mL of culture medium containing the corresponding test sample (concentration 5 mg / mL) was added to the sample group. Five replicates were set up for each group and the cells were cultured in an incubator (37°C, 5% carbon dioxide) for 56 h.

[0153] After the incubation period, the culture medium was aspirated, and the cells were digested with 0.25% trypsin and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded to obtain the precipitate. 200 μL of 1 mol / L NaOH solution containing 10% DMSO was added to the B16 cells in each treatment group and shaken for 5 minutes to completely dissolve the intracellular melanin granules. The absorbance at 490 nm was measured using a spectrophotometer, and the melanin content was calculated. Three measurements were performed for each group, and the average value was calculated.

[0154]

[0155] Among them, OD1 is the absorbance value of the sample group, OD2 is the absorbance value of the negative control group, and OD3 is the absorbance value of the blank control group.

[0156] The results are shown in Table 8.

[0157] Table 8

[0158]

[0159] The results showed that compared with the negative control group, the melanin content in the B16 melanoma cells of Examples 1 to 3 groups was significantly increased (P < 0.05), indicating that the plant fermentation compositions prepared in Examples 1 to 3 have the effect of promoting melanin production.

[0160] Compared with the negative control group, there was no significant change in the melanin content in B16 melanoma cells in Comparative Examples 1-9 (P>0.05), indicating that the plant fermentation composition prepared in Comparative Examples 1-9 does not have the effect of promoting melanogenesis.

[0161] The above is a further description of the present invention in combination with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

Claims

1. A preparation method of a plant fermentation composition for enhancing the scalp immune barrier and the vitality of melanocytes, characterized in that, It includes the following steps: Using the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea as substrates, fermenting with Wickerhamomyces anomalus and Saccharomyces cerevisiae; The Wickerhamomyces anomalus has the number ZKCC12538, and the Saccharomyces cerevisiae has the deposit number CCTCC KY 2008613; The weight ratio of the Wickerhamomyces anomalus to the Saccharomyces cerevisiae is 3-10:1; The preparation method of the water extract is as follows: Step 1: Dry Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea, and perform comminution treatment to collect the powder; Step 2: Add water to the powders of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea obtained in Step 1, perform ultrasonic-assisted extraction, separate the solid and liquid, and combine the filtrates; wherein, the weight ratio of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea is 10-30:1-10:1; In Step 2, the addition amount of water is 3 to 12 times the total amount of the powders of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea. In Step 2, the power of the ultrasonic-assisted extraction is 200W-300W, and the time is 20min-30min.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the Wickerhamomyces anomalus to the Saccharomyces cerevisiae is 5-8:1; the weight ratio of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea is 15-25:3-8:

1.

3. The preparation method according to claim 1, wherein, The inoculation amount of the Wickerhamomyces anomalus and the Saccharomyces cerevisiae is 3%-10% of the weight of the substrate.

4. The preparation method according to claim 1, wherein In Step 2, the addition amount of water is 5 to 10 times the total amount of the powders of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea. In Step 2, the power of the ultrasonic-assisted extraction is 250W-280W, and the time is 25min-30min.

5. The preparation method according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Substrate preparation: Sterilize the water extracts of Polygonum multiflorum Thunb., Asparagus cochinchinensis (Lour.) Merr. and black tea to obtain the substrate; S2. Fermentation: Inoculate the Wickerhamomyces anomalus and the Saccharomyces cerevisiae into the substrate for fermentation to obtain the fermentation broth; S3. Perform solid-liquid separation on the fermentation broth, filter, and take the filtrate.

6. The preparation method according to claim 5, wherein The conditions of the fermentation in Step S2 are: the fermentation temperature is from 26°C to 32°C, and the fermentation time is from 60h to 120h.

7. A plant fermentation composition prepared by the preparation method according to any one of claims 1-6.

8. Use of the plant fermentation composition according to claim 7 in the preparation of cosmetics.

9. The application according to claim 8, wherein The cosmetics are at least one of scalp care products and hair care products.

10. A cosmetic for improving hair and scalp health, characterized in that, It includes the plant fermentation composition according to claim 7.

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