Process for preparing tremella extract by fermentation and composition comprising tremella extract

Abstract

The application discloses a kind of fermentation process for preparing tremella extract, comprising: S1.tremella fruiting body is crushed, add water, boil 10~30min;After cooling, dissolve by adding sugar source, obtain mixed liquor;S2.to mixed liquor Add compound lactobacillus, after sealing, warm fermentation 12h or more, obtain fermentation liquor;S3.to fermentation liquor Add water, normal pressure boiling extraction 2~3h after, remove precipitate by centrifugation, obtain tremella extract.The application also discloses a composition comprising the above-mentioned tremella extract, including tremella extract, sargassum polycystum extract, artemisia flower extract and mussel extract.The fermentation process for preparing tremella extract of the application, the energy consumption of the extraction process is low, and the components such as tremella polysaccharide will not be damaged, the yield of tremella polysaccharide and polypeptide is higher, the quality of product is more stable, and the batch uniformity is good.

Description

Technical Field

[0001] This invention relates to the field of biological extract technology, and in particular to a process for preparing Tremella fuciformis extract by fermentation and a composition containing Tremella fuciformis extract. Background Technology

[0002] Tremella fuciformis, a traditional Chinese edible fungus used both as food and medicine, is prized for its nutritious, gelatinous fruiting body and has long been considered a superior tonic for beauty and health. Traditional Chinese medicine believes that tremella has various health benefits, including strengthening the kidneys, moisturizing the intestines, replenishing qi, harmonizing blood, nourishing the brain, beautifying the skin, and softening the complexion. Tremella is rich in polysaccharides, proteins, fats, inorganic salts, and B vitamins. It contains 17 amino acids, with proline being the most abundant. Minerals include potassium, calcium, magnesium, and iron. Tremella polysaccharides are the most abundant and widely studied active ingredient in the fruiting body, possessing various physiological functions such as immune regulation, anti-aging, anti-tumor activity, and anti-neurodegenerative diseases. While other components have been studied less extensively, their beneficial effects on human health are also undeniable.

[0003] Currently, methods for extracting active ingredients from Tremella fuciformis include water extraction, alkali extraction, and acid extraction. To improve the yield of effective components, auxiliary techniques are often added, such as enzymatic hydrolysis, ultrasound, microwave, and high / ultra-high pressure extraction. However, the use of acidic and alkaline solvents often disrupts the glycosidic bonds of Tremella fuciformis polysaccharides, causing random degradation of large-molecule polysaccharides. This makes it difficult to control product quality, resulting in poor uniformity between different batches and greatly limiting its efficacy and application. Summary of the Invention

[0004] The primary objective of this invention is to provide a fermentation process for preparing Tremella fuciformis extract. This extraction process has low energy consumption, does not damage components such as Tremella fuciformis polysaccharides, has a higher yield of Tremella fuciformis polysaccharides and polypeptides, and results in more stable product quality and good batch-to-batch uniformity.

[0005] The second objective of this invention is to provide an anti-inflammatory and repairing composition in which all components are natural, safe and non-irritating, effectively inhibiting skin oil production, while also possessing good anti-inflammatory and repairing effects.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a process for preparing Tremella fuciformis extract by fermentation, comprising the following steps:

[0008] S1. Crush the fruiting body of Tremella fuciformis to 60-80 mesh, add water equivalent to 20-40 times the weight of the fruiting body, boil for 10-30 minutes; after cooling, add sugar source, dissolve to obtain a mixture;

[0009] S2. Add compound lactobacillus to the mixture, seal it, and ferment at room temperature for more than 12 hours to obtain fermentation broth;

[0010] S3. Add water equivalent to 20 to 40 times the weight of the Tremella fruiting body to the fermentation broth, and extract by boiling at normal pressure for 2 to 3 hours to obtain an extract; then, centrifuge the extract to remove the precipitate and obtain the Tremella extract.

[0011] In some embodiments of the present invention, in step S1, the sugar source can be any existing sugar source, including but not limited to at least one of sucrose and glucose. The weight ratio of the sugar source to the fruiting body of the Tremella fuciformis is preferably (0.6-1.3):1, for example, it can be 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, etc., and more preferably 1:1.

[0012] In some embodiments of the present invention, in step S2, the compound lactobacillus includes three types of lactobacillus: *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Lactobacillus bulgaricus*. The present invention selects three types of lactobacillus as the fermentation bacteria because the combined fermentation of multiple probiotics can produce a rich enzyme system, which can fully enzymatically break down the cell walls of *Tremella fuciformis*, thereby promoting the dissolution of effective components in the *Tremella fuciformis* cells and thus improving the product yield.

[0013] In some embodiments of the present invention, the amount of the compound lactobacillus added per gram of the above mixture is (1-5) × 10⁻⁶. 3 The concentration of the mixed solution is preferably 3 × 10⁻⁶ g / g. 3 =1×10⁶ Lactobacillus / g mixture, that is, 1×10⁶ Lactobacillus per group 3 The mixture contains [number] lactobacilli per g. The preferred ratio of the three types of lactobacilli in the compound lactobacillus formulation is 1:1:1.

[0014] Secondly, the present invention provides an anti-inflammatory and repairing composition comprising Tremella fuciformis extract, Alternaria latifolia extract, Artemisia capillaris flower extract, and mussel extract. Preferably, the Tremella fuciformis extract is the Tremella fuciformis extract prepared by the above-mentioned fermentation method.

[0015] Tremella fuciformis, a traditional Chinese edible fungus used both as food and medicine, is prized for its nutritious, gelatinous fruiting body, and has been considered a superior tonic and beauty product since ancient times. It is rich in polysaccharides, protein, fat, inorganic salts, and B vitamins. Polysaccharides, the most abundant component in the fruiting body, possess strong antioxidant properties, effectively scavenging free radicals, slowing cell aging, and preventing various diseases. They also promote collagen synthesis, increasing skin elasticity and maintaining smooth, delicate skin. Furthermore, polysaccharides can regulate endocrine function, improve skin pigmentation and age spots, and have a certain whitening effect.

[0016] Sargassum pallidum is a plant belonging to the Sargassum family and the Sargassum genus. It grows in shallow sea areas below the low tide line. Extracts from Sargassum pallidum are rich in alginate, vitamins, and amino acids. Alginate is a natural moisturizing and emollient that promotes cell regeneration, strengthens skin metabolism, and helps renew the skin, preventing wrinkles.

[0017] Artemisia capillaris Thunb., a semi-shrubby herbaceous plant belonging to the genus Artemisia in the family Asteraceae, grows along roadsides, riverbanks, sandy beaches, and damp low-lying slopes. It is distributed in China, Korea, Japan, and Southeast Asian countries. Artemisia capillaris is rich in vitamin C and vitamin B, and contains various trace elements and more than 20 amino acids essential for the human body, offering excellent health benefits. Artemisia capillaris flower extract can inhibit the expression level of hyaluronic acid (HA) lyase in cells, thus inhibiting the degradation of HA in the skin and helping to improve sagging skin and wrinkles.

[0018] Mussels are molluscs that live along the coast of China, inhabiting coastal rocks and primarily in areas with strong currents and clear water. Mussel extract is rich in collagen and amino acids, which can promote skin elasticity and firmness, reducing wrinkles and fine lines. Secondly, mussel extract contains abundant minerals and vitamins, nourishing and replenishing the skin's nutritional needs and improving its overall health. Mussel extract also possesses antioxidant properties, combating free radical damage and slowing down the skin aging process. Furthermore, mussel extract has soothing and calming effects, relieving sensitivity and inflammation, and promoting skin repair and healing. In this invention, the inventors, through extensive experimentation, surprisingly discovered that combining Tremella fuciformis extract with extracts of large-leaved seaweed, Artemisia capillaris flower, and mussel extract in a specific ratio produces a synergistic effect, giving the composition a good effect in inhibiting oil production. In cell experiments, the inventors found that, compared to the control group, the composition of this invention can significantly inhibit lipid synthesis in SZ95 cells, and its use in skincare products is expected to reduce oil production. In addition, the composition has a significant inhibitory effect on lipopolysaccharide-induced inflammatory factors in RAW264.7 cells, and can also promote the synthesis of collagen and hyaluronic acid in oxidatively damaged HSF cells. Compared with the control group, this efficacy is also improved.

[0019] In some embodiments of the present invention, the anti-inflammatory and repairing composition comprises, by weight, 5-30 parts of Tremella fuciformis extract, 1-5 parts of Alternaria latifolia extract, 1-5 parts of Artemisia capillaris flower extract, and 1-20 parts of mussel extract. For example, the anti-inflammatory and repairing composition may include 5, 10, 15, 20, 25, or 30 parts of Tremella fuciformis extract, 1, 2, 3, 4, or 5 parts of Alternaria latifolia extract, 1, 2, 3, 4, or 5 parts of Artemisia capillaris flower extract, and 1, 2, 3, 5, 6, 8, 10, 12, 15, 16, 18, or 20 parts of mussel extract.

[0020] In some embodiments of the present invention, the anti-inflammatory and repairing composition comprises, by weight, 15-25 parts of Tremella fuciformis extract, 2-4 parts of Sargassum fusiforme extract, 1-3 parts of Artemisia capillaris flower extract, and 8-20 parts of mussel extract. For example, the anti-inflammatory and repairing composition may include 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 parts of Tremella fuciformis extract, 2, 3, or 4 parts of Sargassum fusiforme extract, 1, 2, or 3 parts of Artemisia capillaris flower extract, and 8, 10, 12, 15, 18, or 20 parts of mussel extract.

[0021] In some embodiments of the present invention, the anti-inflammatory and repairing composition further includes nonapeptide-1. Nonapeptide-1 is a small-molecule whitening biomimetic peptide composed of nine amino acid molecules. It is non-irritating and non-toxic to the skin. Nonapeptide-1 has excellent compatibility with the MC1 receptor on melanocytes, thus competitively binding to the MC1 receptor and preventing further activation of tyrosinase to produce melanin. This inhibits the activation of tyrosinase by melanocytes, thereby reducing melanin production and achieving a whitening and spot-fading effect. Secondly, nonapeptide-1 can stimulate the body's own secretion and metabolic functions, promoting the secretion of deficient collagen and elastin, inhibiting muscle movement, and reducing wrinkles. Thirdly, nonapeptide-1 has strong skin penetration, allowing it to penetrate the stratum corneum and bind to the skin epithelial tissue, participating in and improving skin cell metabolism, enhancing collagen activity in the skin, maintaining the integrity of the stratum corneum's moisture and fiber structure, enhancing blood circulation, and providing nutrients to cells.

[0022] In some embodiments of the present invention, the weight ratio of the nonapeptide-1 to the tremella extract is (0.01 to 0.1):1. For example, the weight ratio of the nonapeptide-1 to the tremella extract can be 0.01:1, 0.05:1, 0.1:1, etc.

[0023] In some embodiments of the present invention, the anti-inflammatory and repairing composition comprises, by weight, 15-25 parts of Tremella fuciformis extract, 2-4 parts of Alternaria latifolia extract, 1-3 parts of Artemisia capillaris flower extract, 8-20 parts of mussel extract, and 0.5-1 parts of nonapeptide-1. For example, the anti-inflammatory and repairing composition may include 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 parts of Tremella fuciformis extract, 2, 3, or 4 parts of Alternaria latifolia extract, 1, 2, or 3 parts of Artemisia capillaris flower extract, 8, 10, 12, 15, 18, or 20 parts of mussel extract, and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 part of nonapeptide-1.

[0024] In some embodiments of the present invention, the concentration of soluble solids in the Tremella fuciformis extract is 2.0-2.5%, for example, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, etc.; the concentration of Tremella fuciformis polysaccharide is 1.3%-1.8%, for example, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, etc.; and the concentration of polypeptide is 3.6-4.0 mg / mL, for example, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 mg / mL, 4.0 mg / mL, etc.

[0025] Secondly, the present invention also provides the application of the aforementioned anti-inflammatory and repairing composition in the preparation of cosmetics.

[0026] The aforementioned cosmetics use the aforementioned anti-inflammatory and repairing composition as an active ingredient and include a cosmetically acceptable matrix. It is understood that such cosmetics therefore possess anti-inflammatory, oil-controlling, and skin-repairing effects.

[0027] In some embodiments, the amount of the anti-inflammatory and repairing composition added to the cosmetic can be from 1 wt% to 50 wt%, for example, it can be any value or a range of any two values ​​from 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 8 wt%, 10 wt%, 12 wt%, 15 wt%, 18 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%.

[0028] In this invention, the aforementioned cosmetic can be prepared in various forms. For example, the cosmetic can be prepared into any dosage form conventionally prepared in this field, including but not limited to solutions, suspensions, ointments, gels, creams, lotions, loose powders, facial cleansers, essential oils, foundations, lotion foundations, wax foundations, sprays, etc. Furthermore, specifically, it can be selected from various types within the group consisting of, but not limited to, toners, lotions, softening lotions, astringents, skin lotions, emulsions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, moisturizing creams, hand creams, serums, face masks, soaps, shampoos, facial foams, cleansing milks, cleansing creams, body lotions, shower gels, emulsions, lipsticks, sunscreens, liquid foundations, pressed powders, and loose powders.

[0029] This invention can achieve at least the following technical effects:

[0030] 1. This invention provides a process for preparing Tremella fuciformis extract by fermentation. This extraction process has low energy consumption and does not damage components such as Tremella fuciformis polysaccharides. The yield of Tremella fuciformis polysaccharides and polypeptides is higher, the product quality is more stable, and the batch-to-batch uniformity is good.

[0031] 2. This invention combines Tremella fuciformis extract, Alternaria latifolia extract, Artemisia capillaris flower extract, and mussel extract in a certain proportion to produce a synergistic effect. The resulting composition can effectively inhibit skin oil production and also has anti-inflammatory and skin repair-promoting effects. Attached Figure Description

[0032] Figure 1 This is a process flow diagram of the fermentation method for preparing Tremella fuciformis extract in this invention;

[0033] Figure 2 This is a standard curve for glucose solution;

[0034] Figure 3 The standard curve for reduced glutathione. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] I. Main Reagents and Instruments

[0037] (1) Reagents

[0038] Glucose (98%, Shanghai Yuanye Biotechnology Co., Ltd.); concentrated sulfuric acid, hydrochloric acid (analytical grade, Zhuhai Huachengda Chemical Co., Ltd.); phenol, 95% ethanol, methanol, reduced glutathione, trichloroacetic acid, copper sulfate pentahydrate, sodium hydroxide, linoleic acid, nitrocellulose, vitamin C (analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.); lipopolysaccharide (99%, Shanghai Maclean's Biochemical Technology Co., Ltd.); cellulase (50U / mg), pectinase (500U / mg), Shanghai Yuanye Biotechnology Co., Ltd.; isotretinoin (99%, Shanghai Maclean's Biochemical Technology Co., Ltd.); hydrogen peroxide (3%, Guangdong Hengjian Pharmaceutical Co., Ltd.); sodium chloride (Hangzhou Microbiology). Reagent Co., Ltd.; DMEM high glucose culture medium, fetal bovine serum (Gibco, USA); DMSO, 100 U / mL penicillin-100 μg / mL streptomycin solution (Beijing Lanjieke Technology Co., Ltd.), dihydrotestosterone (10 mM, Shanghai Aladdin Biochemical Technology Co., Ltd.), PBS (Wuhan Pronosei Life Science Co., Ltd.); hydroxyproline kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.); mouse TNF-α ELISA kit, mouse IL-1β ELISA kit (Wuhan Huamei Bioengineering Co., Ltd.); human hyaluronic acid (HA) ELISA kit (Shanghai Hengda Biotechnology Co., Ltd.); all other reagents were of analytical grade.

[0039] (2) Probiotic strains

[0040] Lactobacillus plantarum and Lactobacillus rhamnosus were purchased from Koto Biotechnology; Lactobacillus bulgaricus was purchased from Runying Biotechnology (Shanghai) Co., Ltd.

[0041] (3) Cells

[0042] SZ95 cells were obtained from Shanghai BLUEFBIO, while RAW264.7 and HSF cells were obtained from Shenzhen Yongpo Biotechnology Co., Ltd.

[0043] (4) Instruments

[0044] UV-Vis spectrophotometer (752N, Shanghai Jingke Industrial Co., Ltd.), constant temperature water bath (HH-2, Changzhou Aohua Instrument Co., Ltd.), vortex mixer (MX-S, Beijing Dalong Instrument Co., Ltd.), automatic amino acid analyzer (L-8900, Hitachi, Japan), constant temperature incubator (ZXDP-B2270, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), cell culture incubator (HERACELL 150i, Thermo Fisher Scientific, USA), autoclave (SQ510C, Chongqing Yamato Technology Co., Ltd.), clean bench (SW-CJ-2FD, Suzhou Antai Air Technology Co., Ltd.), centrifuge (KA-1000, Shanghai Anting Scientific Instrument Factory), microbalance (BSA224S, Sartorius Scientific Instruments (Beijing) Co., Ltd.), inverted microscope (BDS400, Chongqing Aote Optical Instrument Co., Ltd.), microplate reader (SpectraMax i3x, Meigu Molecular Instruments (Shanghai) Co., Ltd.).

[0045] II. Examples and Comparative Examples

[0046] Preparation Example 1: Preparation of Tremella fuciformis extract by fermentation

[0047] Crush the fruiting bodies of the white fungus to 80 mesh, add 30 times its weight of water, and boil for 30 minutes. After cooling, add sucrose in a 1:1 ratio with the weight of the white fungus and dissolve completely. Then, add 1×10 3 Equal amounts of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Lactobacillus bulgaricus* were added to the mixture, which was then sealed and fermented at room temperature overnight (≥12 h). Then, water equivalent to 30 times the weight of the *Tremella fuciformis* was added, and the mixture was boiled at normal pressure for 3 h. The extract was then centrifuged at high speed to remove the precipitate, yielding the fermentation-assisted *Tremella fuciformis* extract.

[0048] Refractometer analysis revealed that the soluble solids content in the Tremella fuciformis extract was 2.2%.

[0049] Preparation Example 2: Conventional Method for Preparing Tremella Extract

[0050] The fruiting bodies of Tremella fuciformis were pulverized to 80 mesh, and 60 times their weight of water were added. The mixture was boiled at normal pressure for 3 hours. The extract was centrifuged at high speed to remove the precipitate. The clear liquid after centrifugation was concentrated under vacuum (temperature 60℃~70℃, vacuum degree -0.05~-0.07Mpa) to a soluble solids content of 2.5%, thus obtaining Tremella fuciformis extract.

[0051] Preparation Example 3: Enzymatic Hydrolysis-Assisted Preparation of Tremella Extract Referring to the method in the reference [Peng Yunfei, Enzymatic Extraction and Bioactivity Study of Tremella Polysaccharides (D), Fujian Agriculture and Forestry University, 2017], the Tremella fruiting body was pulverized to 80 mesh, and 60 times its weight of water was added. Cellulase 1% and pectinase 0.8% were added according to the substrate weight, respectively. The enzymatic hydrolysis temperature was controlled at 55℃ for 4 hours, and the mixture was stirred and boiled at normal pressure for 3 hours. The extract was centrifuged at high speed to remove the precipitate. The supernatant was then concentrated under vacuum (temperature 60℃~70℃, vacuum degree -0.05~-0.07Mpa) to a soluble solids content of 2.5%, yielding the enzymatically extracted Tremella extract, referred to as the enzymatically extracted Tremella extract.

[0052] Preparation Example 4: Preparation of Tremella polysaccharide

[0053] According to the literature [Wang Zhaojing et al., study on physicochemical characteristics, microstructure and antioxidant and anti-inflammatory effects of four kinds of Tremella polysaccharides. Chinese Pharmaceutical Journal, 2019, 54(21):1788-1793.], the polysaccharides of Tremella fruiting bodies were extracted, and after alcohol precipitation to obtain crude Tremella polysaccharide, the protein was removed by Sevag method and freeze-dried to obtain pure Tremella polysaccharide (hereinafter referred to as Tremella polysaccharide).

[0054] Detection of Tremella fuciformis extract

[0055] (1) Polysaccharide content detection

[0056] The polysaccharide content was determined by visible spectrophotometry using the concentrated sulfuric acid-phenol method for color development.

[0057] Construction of the glucose standard curve: Accurately pipette 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of glucose standard solution into 15 mL reaction tubes, respectively. Add distilled water to each tube to a final volume of 2.0 mL. Add 1.0 mL of 6% phenol solution to each tube and mix well. At room temperature, vertically add 6.0 mL of concentrated sulfuric acid while the tube is suspended in the air, and let stand for 10 min. Mix thoroughly using a vortex mixer. Incubate in a boiling water bath for 30 min. After the reaction, incubate in an ice-water bath for 1 min, then cool to room temperature. Measure the absorbance at 490 nm. Use 2.0 mL of water as a blank control, following the same colorimetric procedure. Plot the standard curve with absorbance (A)y as the ordinate and glucose concentration (μg)x as the abscissa. Sample determination: Add 8 mL of 95% ethanol to a centrifuge tube, accurately transfer 1 mL of sample and weigh it (m), slowly add it dropwise to the ethanol while shaking, let it stand for 2 hours, then transfer it to a centrifuge for centrifugation (3000 r / min, 10 min), remove the supernatant, dissolve the precipitate in 5 mL of 2 mol / L sulfuric acid, transfer it to a 25 mL (V1) volumetric flask, and dilute to the mark with water. Shake well to obtain the test solution. Accurately pipette 1 mL (V2) of the test solution, add water to 2.0 mL, and perform 3 parallel tests for each sample. Use 2 mL of water as a blank control, following the same colorimetric procedure. Measure the absorbance at 490 nm using the same method as the standard curve preparation. Measure the absorbance at 490 nm according to the standard curve procedure, and determine the sugar content based on the standard curve.

[0058] Total polysaccharide content calculation method: (Note the dilution factor of sample weight / volume)

[0059]

[0060] In the formula:

[0061] X — Total sugar content, %;

[0062] Y—The absorbance of the sample measured;

[0063] a—Standard curve intercept;

[0064] V1—Sample dilution volume, in milliliters (mL);

[0065] V2—Volume of the test sample, in milliliters (mL);

[0066] b—Slope of the standard curve;

[0067] m — Sample mass, in grams (g);

[0068] f—The conversion factor for polysaccharides to glucose is 0.9;

[0069] n—Dilution factor of the test sample.

[0070] (2) Detection of peptide content

[0071] Reduced glutathione was used as a standard, and the biuret method was employed for colorimetric development. The peptide content was determined by visible spectrophotometry. 10% trichloroacetic acid can precipitate large protein molecules in the sample, thus removing their influence on peptide determination.

[0072] Construction of the reduced glutathione standard curve: Prepare a 4 mg / mL glutathione standard solution using deionized water. Take 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mL of the glutathione standard solution sequentially, and dilute to 6.0 mL with deionized water. Add 4.0 mL of biuret reagent (solution A:solution B = 3:1, V / V), mix thoroughly using a vortex mixer, let stand for 10 min, centrifuge at 2000 rpm for 10 min, and measure the absorbance of the supernatant at 540 nm (using the first tube as a blank control). Plot the standard curve with absorbance (A)y as the ordinate and peptide concentration (mg / mL)x as the abscissa.

[0073] Sample determination: Take 2.5 mL of sample solution, add 2.5 mL of 10% (w / v) trichloroacetic acid, mix thoroughly on a vortex mixer, let stand for 10 min, then centrifuge at 4000 r / min for 15 min. Transfer the supernatant to a 50 mL volumetric flask and dilute to the mark with 5% trichloroacetic acid, then shake well. Then take 6.0 mL of the above solution and place it in another test tube, add 4.0 mL of biuret reagent (sample solution: biuret reagent = 3:2, v / v), mix thoroughly on a vortex mixer, let stand for 10 min, centrifuge at 2000 r / min for 10 min, take the supernatant and measure the absorbance value (A) at 540 nm. Calculate the peptide concentration (mg / mL) in the sample solution by referring to the standard curve, and then determine the peptide content in the sample.

[0074] (3) Results Analysis

[0075] Please see Figure 2 Plotting glucose content on the x-axis and absorbance on the y-axis, the linear regression equation between glucose concentration and absorbance is: y = 0.008x - 0.0177, with a linear correlation coefficient R0. 2 =0.9977.

[0076] According to the absorbance values, the polysaccharide contents of the samples in Preparation Examples 1-4 were as follows: fermented Tremella fuciformis extract 1.5%, Tremella fuciformis extract 1.1%, enzymatic Tremella fuciformis extract 1.2%, and Tremella fuciformis polysaccharide (dry powder) 97%. Therefore, compared with other extraction methods of Tremella fuciformis extract, the fermented extract had the highest polysaccharide content.

[0077] Please see Figure 3A standard curve was plotted with reduced glutathione concentration on the x-axis and the corresponding absorbance value on the y-axis. The regression equation between reduced glutathione concentration and absorbance was obtained as: y = 0.1207x + 0.0015, R0 2 =0.9989.

[0078] According to the absorbance values, the polypeptide contents of the samples in Preparation Examples 1-4 were as follows: fermented Tremella fuciformis extract: 3.8 mg / mL; Tremella fuciformis extract: 0.9 mg / mL; enzymatic Tremella fuciformis extract: 1.5 mg / mL; Tremella fuciformis polysaccharide (dried powder) was not detected. Therefore, the polypeptide content in the fermented extract was significantly higher than that in the other Tremella fuciformis extracts.

[0079] Table 1

[0080]

[0081] As can be seen from the data in Table 1, the yields of soluble solids, polysaccharides, and polypeptides were the lowest among the extracts obtained by conventional water extraction. Enzymatic extraction can improve the yields of relevant indicators, indicating that the added cellulase and pectinase can disrupt the integrity of the cell wall to a certain extent, thereby promoting the dissolution of active ingredients. The product obtained by fermentation has the highest yield, which may be related to the rich enzyme system produced by various probiotics.

[0082] Examples and Comparative Examples

[0083] The formulations of the compositions in Examples 1-4 and Comparative Examples 1-10 are shown in Table 2, where each component is expressed in parts by weight. The components are weighed and mixed thoroughly to obtain each composition.

[0084] Table 2

[0085]

[0086]

[0087] Note: The extract of large-leaved seaweed was purchased from Ningxia Xiangcao Biotechnology Co., Ltd.; the extract of Artemisia capillaris flower was purchased from Shaanxi Sinote Biotechnology Co., Ltd.; and the extract of mussel was purchased from Shaanxi Lonier Biotechnology Co., Ltd.

[0088] Test

[0089] (1) Study on the inhibition of lipid secretion in SZ95 cells by samples

[0090] Cell Culture: SZ95 cells were cultured in DMEM high-glucose medium containing 1% penicillin-drug antibiotics and 10% fetal bovine serum, and incubated at 37°C in a 5% CO2 saturated humidity incubator, with the medium changed every two days. Experimental Methods: SZ95 sebaceous gland cells in the logarithmic growth phase and in good growth were digested and seeded at 100 μL into 96-well plates with a black transparent bottom, at a density of 5 × 10⁶ cells per well. 3 The experiment included a blank control group, a model group, a positive control group, and an experimental group. The blank control group received only 100 μM linoleic acid, while the model group, positive control group, and experimental group received 10 μM dihydrotestosterone and 100 μM linoleic acid, with three replicates per group. After overnight incubation, the blank control group received an equal volume of blank culture medium, the positive control group received 0.01 mM isotretinoin, and the experimental group received the composition of the example or comparative example at a final concentration of 500 μg / mL for 48 h.

[0091] After 48 hours of treatment, the culture medium was discarded and the cells were washed with PBS. The cells were then stained with 10 μg / mL Nile Red diluted in PBS and incubated at 37°C in the dark for 10 minutes. After washing with PBS, the Nile Red intensity was measured using a multi-mode microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 565 nm. The results are expressed as Nile Red absorbance, and the percentages in the experimental and control groups reflect the relative intracellular lipid content.

[0092] (2) Immunomodulatory effects of the sample on RAW264.7 cells

[0093] Cell culture: RAW264.7 cells were cultured in DMEM high glucose medium containing 1% penicillin antibiotics and 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO2 saturated humidity, with the medium changed every two days.

[0094] Experimental methods: RAW264.7 cells in the logarithmic growth phase and with good growth were passaged into 96-well plates, with 5 × 10⁶ cells per well. 3 Three wells were set up: a model group, a blank group, and a positive group, with three replicates for each group. The positive group was treated with 100 μg / mL yeast dextran, the model group was treated with 500 μg / mL of Tremella fuciformis extract from the examples or comparative studies, and the blank group was treated with an equal volume of culture medium. All treatments were carried out for 24 hours.

[0095] TNF-α and IL-1β levels were detected by ELISA. The procedure was followed according to the instructions in the ELISA kit. 100 μL of cell supernatant was taken from each well and added to the well plate of the ELISA kit. Each group was divided into two wells. The TNF-α and IL-1β levels were measured according to the instructions.

[0096] (3) Inhibitory effect of the sample on lipopolysaccharide-induced inflammatory factors in RAW264.7 cells: RAW264.7 cells were cultured in DMEM high glucose medium containing 1% penicillin and 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO2 saturated humidity, with the medium changed every two days.

[0097] Experimental methods: RAW264.7 cells in the logarithmic growth phase and with good growth were passaged into 96-well plates, with 5 × 10⁶ cells per well. 3 The experiment included a control group, a model group, a blank group, and an experimental group, with three replicates per group. The model group was first treated with 2 mg / L LPS for 2 h. The control group, blank group, and experimental group were added with an equal volume of culture medium. After stimulation, the positive group was added with 100 μg / mL yeast dextran, and the experimental group was added with 500 μg / mL Tremella fuciformis extract from the example or comparative example. The control group, model group, and blank group were added with an equal volume of culture medium, and the mixture was cultured for another 24 h.

[0098] TNF-α and IL-1β levels were detected using ELISA. The procedure was followed according to the instructions in the ELISA kit. 100 μL of cell supernatant was taken from each well and added to the ELISA kit plate. Each group was divided into two wells. The cells were incubated in multiple steps as instructed in the kit. The TNF-α and IL-1β levels in each group were calculated based on the standard curve.

[0099] (4) Effect of the sample on the secretion of collagen and hyaluronic acid by HSF cells induced by hydrogen peroxide. Hydroxyproline is a unique amino acid in collagen, accounting for about 13% of the amino acids in collagen. By measuring the content of hydroxyproline, the collagen content can be indirectly measured.

[0100] Cell culture: HSF cells were cultured in DMEM high-glucose medium containing 1% penicillin-drug antibiotics and 10% fetal bovine serum, and incubated at 37°C in a 5% CO2 saturated humidity incubator, with the medium changed every two days. Determination of HSF cell concentration damaged by hydrogen peroxide: HSF cells in the logarithmic growth phase were cultured at a concentration of 1×10⁻⁶ cells / cell. 5 100 μL of HSF cells were seeded into each well of a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 10 μL of DMEM high-glucose medium solution containing hydrogen peroxide was added to each well until the hydrogen peroxide concentration reached 25–400 μmol / L (freshly prepared before use). A control well was also included. Each group had three replicates, and the incubation period was 4 h. Finally, the MTT assay was performed according to the manufacturer's instructions, using the dose corresponding to the half-maximal survival rate of HSF cells as the lesion concentration.

[0101] The effect of the sample on promoting the secretion of collagen and hyaluronic acid in HSF cells damaged by hydrogen peroxide: Logarithmic growth phase cells in good growth condition were taken, digested, and diluted to 1×10⁻⁶. 5 / mL, take 500μL and inoculate into 24-well culture plates, 5×10⁶ per well.4 Cells were cultured for 24 hours. Except for the control group, 10 μL of DMEM high glucose medium solution containing hydrogen peroxide was added to each cell until the final hydrogen peroxide concentration was the damage concentration (400 μM). After incubation for 4 hours, the original medium was removed and replaced with 500 μL of each of the control group, model group, vitamin C positive group, and test samples. Each group was divided into 3 replicates. The cells were cultured for another 48 hours and the supernatant was collected.

[0102] Hydroxyproline content was detected using a colorimetric method. 250 μL of cell supernatant was taken from each well of a 24-well plate. Following the instructions of the hydroxyproline assay kit, the supernatant was used to measure the absorbance (A) of hydroxyproline at 550 nm using a microplate reader. The hydroxyproline content and collagen content were then calculated. The calculation formula is as follows:

[0103]

[0104] Collagen content = hydroxyproline content × 7.69

[0105] C standard: standard solution concentration, 5 μg / mL; N: sample dilution factor before testing. The hyaluronic acid level in the cell culture supernatant was detected by ELISA. 100 μL of sample was taken from each well of a 24-well plate and added to the ELISA plate. The plate was set up for double-well measurement and incubated at 37℃ for 2 h. Then, reagents were added sequentially according to the instructions. Finally, the absorbance (A) was measured at 450 nm using a microplate reader, and the hyaluronic acid content was calculated.

[0106] (5) Statistical Analysis

[0107] Data are expressed as mean ± standard deviation. ANOVA one-way ANOVA was used for comparisons among multiple groups, and LSD was used for post-hoc comparisons. P < 0.05 was considered statistically significant.

[0108] Results Analysis

[0109] 1. Effects of the sample on lipid synthesis in SZ95 cells

[0110] After treating sebaceous gland cells with different samples, Nile Red staining was used to detect the amount of lipid secretion in sebaceous gland cells. The results are shown in Table 3.

[0111] Table 3. Effects of each sample on lipid synthesis in SZ95 cells.

[0112] Group Absorbance ratio (%) Blank group <![CDATA[44±3.2 a ]]> Dihydrotestosterone + linoleic acid group (model group) <![CDATA[100 b ]]> Isotretinoin group <![CDATA[51±3.3 c <!-- 10 -->]]> Example 1 <![CDATA[69±2.7 d ]]> Example 2 <![CDATA[65±1.4 d ]]> Example 3 <![CDATA[67±5.1 d ]]> Example 4 <![CDATA[62±3.7 d ]]> Comparative Example 1 <![CDATA[84±6.5 e ]]> Comparative Example 2 <![CDATA[97±5.4 b ]]> Comparative Example 3 <![CDATA[95±4.8 b ]]> Comparative Example 4 <![CDATA[96±6.7 b ]]> Comparative Example 5 <![CDATA[88±5.1 e ]]> Comparative Example 6 <![CDATA[86±3.4 e ]]> Comparative Example 7 <![CDATA[87±2.3 e ]]> Comparative Example 8 <![CDATA[96±5.8 b ]]> Comparative Example 9 <![CDATA[85±3.6 e ]]> Comparative Example 10 <![CDATA[85±4.9 e ]]>

[0113] Note: Different letters in intergroup comparisons represent P < 0.05. Sample concentrations are all calculated based on dry matter (the same applies below).

[0114] As can be seen from the table, dihydrotestosterone plus linoleic acid can significantly promote lipid synthesis in cells; fermented tremella extract has a certain inhibitory effect on lipid synthesis in cells (P<0.05), while seaweed extract, artemisia capillaris flower extract and mussel extract have no inhibitory effect on lipid synthesis in SZ95 cells (P>0.05).

[0115] When fermented Tremella fuciformis extract was combined with several other components, all showed some inhibitory effect on cellular lipid synthesis (P<0.05). Surprisingly, when fermented Tremella fuciformis extract, seaweed extract, Artemisia capillaris flower extract, and mussel extract were combined, they showed a significant inhibitory effect on lipid synthesis in SZ95 cells, with a several-fold increase compared to the other control groups.

[0116] Furthermore, compared with conventional and enzymatically prepared Tremella fuciformis extracts, the combination of fermented Tremella fuciformis extract with extracts of large-leaved seaweed, Artemisia capillaris flower, and mussel extract showed better inhibitory effects on lipid synthesis in Z95 cells. This may be because the exogenous addition of lactic acid bacteria during fermentation resulted in a different composition of the extract compared to water-extracted and enzymatically hydrolyzed Tremella fuciformis extracts. 2. Immunomodulatory effects of the samples on RAW264.7 cells

[0117] After treating macrophages with different samples, the concentrations of TNF-α and IL-1β in the macrophage culture supernatant were determined by ELISA. The results are shown in Table 4.

[0118] Table 4. Effects of the samples on the secretion of TNF-α and IL-1β by RAW264.7 cells.

[0119]

[0120]

[0121] Please refer to Table 4. Compared with the blank group, all sample groups showed the effect of promoting the secretion of TNF-α and IL-1β (P<0.05). Among them, compared with comparative examples 1-8, the compositions of examples 1-4 were able to better promote the secretion of TNF-α and IL-1β. This may be because the four components—fermented Tremella fuciformis extract, Alternanthera philoxeroides extract, Artemisia capillaris flower extract, and mussel extract—all have immunomodulatory effects and are of different components. Therefore, when combined, they can better promote the activity of immune cells, enabling them to secrete more TNF-α and IL-1β.

[0122] 3. Inhibitory effect of the sample on lipopolysaccharide-induced inflammatory factors in RAW264.7 cells

[0123] An inflammation model was established by treating RAW264.7 macrophages with LPS. After treating cells with different samples for 24 hours, the concentrations of TNF-α and IL-1β in the culture supernatant were measured by ELISA. The results are shown in Table 5.

[0124] Table 5. Effects of the samples on TNF-α and IL-1β in RAW264.7 cells.

[0125] Group TNF-α (pg / mL) IL-1β (pg / mL) normal group <![CDATA[20.1±1.4 a ]]> <![CDATA[85±3.5 a ]]> LPS Group <![CDATA[80.5±2.9 b ]]> <![CDATA[214±8.8 b ]]> Example 1 <![CDATA[56.8±5.5 c ]]> <![CDATA[152±3.7 c ]]> Example 2 <![CDATA[56.5±4.3 c ]]> <![CDATA[155±3.1 c ]]> Example 3 <![CDATA[57.3±6.8 c ]]> <![CDATA[152±2.5 c ]]> Example 4 <![CDATA[55.7±2.3 c ]]> <![CDATA[151±4.4 c ]]> Comparative Example 1 <![CDATA[64.8±5.5 d ]]> <![CDATA[162±6.4 d ]]> Comparative Example 2 <![CDATA[68.3±3.2 d ]]> <![CDATA[170±1.7 e ]]> Comparative Example 3 <![CDATA[71.8±3.4 d ]]> <![CDATA[185±4.7 f ]]> Comparative Example 4 <![CDATA[70.1±6.1 d ]]> <![CDATA[181±2.3 f ]]> Comparative Example 5 <![CDATA[65.1±3.3 d ]]> <![CDATA[168±6.7 d <!-- 12 -->]]> Comparative Example 6 <![CDATA[67.4±4.8 d ]]> <![CDATA[174±7.1 e ]]> Comparative Example 7 <![CDATA[66.8±4.6 d ]]> <![CDATA[169±4.9 e ]]> Comparative Example 8 <![CDATA[76.7±2.4 b ]]> <![CDATA[177±3.8 e ]]> Comparative Example 9 <![CDATA[67.5±1.9 d ]]> <![CDATA[179±4.8 e ]]> Comparative Example 10 <![CDATA[68.1±2.5 d ]]> <![CDATA[172±4.4 e ]]>

[0126] As shown in Table 5, compared with the normal group, the model group exhibited significantly increased levels of TNF-α and IL-1β, indicating the successful establishment of the cellular inflammation model. Compared with the model group, all samples showed varying degrees of inhibition of TNF-α and IL-1β secretion. Among them, compared with Comparative Examples 1-10, the compositions of Examples 1-4 showed stronger inhibition of TNF-α and IL-1β secretion, indicating that the four components—fermented Tremella fuciformis extract, Alternanthera philoxeroides extract, Artemisia capillaris flower extract, and mussel extract—produced a synergistic effect, thereby exhibiting better anti-inflammatory activity.

[0127] 4. Effects of the sample on collagen and hyaluronic acid synthesis in hydrogen peroxide-induced damaged HSF cells.

[0128] An oxidative damage model was successfully established by treating HSF cells with hydrogen peroxide solution. After treating cells with different samples for 48 hours, the culture supernatant was collected, and the collagen content was detected by colorimetric method, while the hyaluronic acid content was determined by ELISA. The results are shown in Table 6.

[0129] Table 6. Effects of samples on collagen and hyaluronic acid synthesis in HSF cells.

[0130] Group Collagen (μg / mL) Hyaluronic acid (μg / mL) normal group <![CDATA[4.48±0.21 a ]]> <![CDATA[3.42±0.22 a ]]> Model group <![CDATA[1.42±0.12 b ]]> <![CDATA[0.64±0.24 b ]]> Vitamin C group <![CDATA[2.41±0.43 c ]]> <![CDATA[1.82±0.31 c ]]> Example 1 <![CDATA[2.65±0.32 c ]]> <![CDATA[2.35±0.16 c ]]> Example 2 <![CDATA[2.70±0.40 c ]]> <![CDATA[2.36±0.33 c ]]> Example 3 <![CDATA[2.78±0.34 c ]]> <![CDATA[2.41±0.27 c ]]> Example 4 <![CDATA[2.88±0.23 c ]]> <![CDATA[2.49±0.17 c ]]> Comparative Example 1 <![CDATA[2.23±0.34 d ]]> <![CDATA[1.95±0.36 d ]]> Comparative Example 2 <![CDATA[2.17±0.45 d ]]> <![CDATA[1.91±0.16 d ]]> Comparative Example 3 <![CDATA[2.10±0.32 d ]]> <![CDATA[2.04±0.18 d ]]> Comparative Example 4 <![CDATA[2.21±0.17 d ]]> <![CDATA[1.88±0.41 d ]]> Comparative Example 5 <![CDATA[2.30±0.24 d ]]> <![CDATA[2.07±0.11 d ]]> Comparative Example 6 <![CDATA[2.20±0.15 d ]]> <![CDATA[2.16±0.31 d ]]> Comparative Example 7 <![CDATA[2.22±0.41 d ]]> <![CDATA[2.07±0.40 d ]]> Comparative Example 8 <![CDATA[2.18±0.16 d ]]> <![CDATA[1.99±0.15 d ]]> Comparative Example 9 <![CDATA[2.34±0.11 d ]]> <![CDATA[2.04±0.32 d ]]> Comparative Example 10 <![CDATA[2.40±0.15 d ]]> <![CDATA[2.11±0.31 d ]]>

[0131] Please refer to Table 6. Compared with the model group, the secretion of collagen and hyaluronic acid in each sample group was significantly increased, indicating that each sample has a certain protective effect against oxidatively damaged cells. Compared with Comparative Examples 1-8, the compositions obtained by combining fermented Tremella fuciformis extract, seaweed extract, Artemisia capillaris flower extract, and mussel extract in Examples 1-4 produced a synergistic effect, thus significantly increasing the secretion of collagen and hyaluronic acid. In addition, comparing Examples 1-4 with Comparative Examples 9-10, it can be seen that fermented Tremella fuciformis extract is superior to conventional Tremella fuciformis extract and enzymatic Tremella fuciformis extract in increasing the secretion of collagen and hyaluronic acid, indicating that fermented Tremella fuciformis extract can better promote the synthesis of collagen and hyaluronic acid in oxidatively damaged HSF cells.

[0132] In summary, although the present invention has been disclosed above with reference to preferred embodiments, the above preferred embodiments are not intended to limit the present invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope defined in the claims.

Claims

1. A process for preparing Tremella fuciformis extract by fermentation, characterized in that, Includes the following steps: S1. Crush the fruiting body of Tremella fuciformis to 60-80 mesh, add water equivalent to 20-40 times the weight of the fruiting body, boil for 10-30 minutes; after cooling, add sugar source, dissolve to obtain a mixture; the sugar source includes at least one of sucrose and glucose; the weight ratio of the sugar source to the fruiting body of Tremella fuciformis is (0.6-1.3):1; S2. Add the compound lactobacillus to the mixture, seal it, and ferment at room temperature for more than 12 hours to obtain the fermentation broth; the compound lactobacillus includes Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus bulgaricus in a ratio of 1:1:1; the amount of the compound lactobacillus added is (1~5)×10 3 pcs / g of mixed solution; S3. Add water equivalent to 20-40 times the weight of the Tremella fruiting body to the fermentation broth, and extract by boiling at normal pressure for 2-3 hours to obtain an extract; then, centrifuge the extract to remove the precipitate and obtain the Tremella extract.

2. An anti-inflammatory and repairing composition, characterized in that, The composition includes Tremella fuciformis extract, Alternaria macrocarpa extract, Artemisia capillaris flower extract, and mussel extract; wherein the Tremella fuciformis extract is prepared by the process described in claim 1; by weight, the anti-inflammatory and repairing composition includes 5-30 parts Tremella fuciformis extract, 1-5 parts Alternaria macrocarpa extract, 1-5 parts Artemisia capillaris flower extract, and 1-20 parts mussel extract.

3. The anti-inflammatory and repairing composition as described in claim 2, characterized in that, The anti-inflammatory and repairing composition comprises, by weight, 15-25 parts of Tremella fuciformis extract, 2-4 parts of Sargassum fusiforme extract, 1-3 parts of Artemisia capillaris flower extract, and 8-20 parts of mussel extract.

4. The anti-inflammatory and repairing composition as described in claim 2, characterized in that, The anti-inflammatory and repairing composition also includes nonapeptide-1; The weight ratio of the nonapeptide-1 to the tremella extract is (0.01~0.1):

1.

5. The anti-inflammatory and repairing composition as described in claim 4, characterized in that, The anti-inflammatory and repairing composition comprises, by weight, 15-25 parts of Tremella fuciformis extract, 2-4 parts of Sargassum fusiforme extract, 1-3 parts of Artemisia capillaris flower extract, 8-20 parts of mussel extract, and 0.5-1 parts of nonapeptide-1.

6. The anti-inflammatory and repairing composition as described in claim 2, characterized in that, The concentration of soluble solids in the Tremella fuciformis extract is 2.0-2.5%, the concentration of Tremella fuciformis polysaccharide is 1.3%-1.8%, and the concentration of polypeptide is 3.6-4.0 mg / mL.

7. The use of the anti-inflammatory and repairing composition according to any one of claims 2 to 6 in the preparation of cosmetics.

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