Natural plant fermentation liquor, preparation method and application thereof

Abstract

The present application belongs to the technical field of biological fermentation, and discloses a natural plant fermentation liquor, which is prepared from acanthopanax, angelica sinensis, rosa davurica, phellinus baumii, tea leaves, water, mixed enzymes and mixed bacterial agents; wherein the mixed enzymes include cellulase, pectinase and tannase, and the mixed bacterial agents include lactobacillus plantarum, schizosaccharomyces and lactobacillus acidophilus.The natural plant fermentation liquor has high safety, good clarity after adjusting the concentration of crude drugs, and can be added into a cosmetic matrix.The preparation method is simple, and the effective active ingredients of the natural plants and tea leaves are extracted by the synergistic effect of bacteria and enzymes to the maximum extent.In addition, the present application also discloses the application of the natural plant fermentation liquor in preparing cosmetics for treating oily sensitive skin.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to natural plant fermentation broth, its preparation method, and its application. Background Technology

[0002] Oily sensitive skin is a special type of skin characterized by significantly increased sebum secretion and heightened sensitivity to environmental or physical stimuli. Oily sensitive skin manifests differently in different areas of the face. The T-zone, including the forehead, nose, and chin, is prone to oiliness, with a shiny surface, large pores, and a tendency to develop blackheads and acne. The U-zone, the sides of the cheeks, often feels dry and sensitive, potentially experiencing erythema, flaking, itching, burning, stinging, dryness, or tightness. Under physiological or pathological conditions, oily sensitive skin exhibits high reactivity. When exposed to physical, chemical, or psychological stimuli, it easily experiences discomfort such as burning, stinging, itching, and tightness, and may even develop objective signs such as erythema, scaling, and telangiectasia. Improvement can be achieved by repairing the skin barrier, regulating sebum secretion, and soothing skin inflammation.

[0003] In recent years, the proportion of people with oily and sensitive skin has been rising steadily, reaching as high as 48.5% in 2022. Developing new cosmetic ingredients for treating oily and sensitive skin has become a current hot topic. my country has abundant plant resources. With the development of enzymatic extraction and bio-fermentation technologies, the synergistic effect between microorganisms can be leveraged through the screening and combination of natural plants, enzymes, and microbial strains, thereby improving the efficiency of extracting effective components from natural plants. This has a positive effect on discovering a natural plant fermentation liquid that can be used to treat oily and sensitive skin. Summary of the Invention

[0004] The first objective of this invention is to provide a natural plant fermentation liquid to solve at least one of the above-mentioned technical problems.

[0005] A second objective of this invention is to provide a method for preparing natural plant fermentation liquid to solve at least one of the above-mentioned technical problems.

[0006] A third objective of this invention is to provide the application of natural plant fermentation liquid in the preparation of cosmetics, thereby solving at least one of the aforementioned technical problems.

[0007] According to a first aspect of the present invention, a natural plant fermentation liquid is provided, which is prepared from Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fertilizer, tea, water, mixed enzymes and mixed microbial agents as raw materials; wherein the mixed enzymes include cellulase, pectinase and tanninase, and the mixed microbial agents include Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus.

[0008] In some embodiments, the tea leaves may be selected from at least one of green tea, black tea, white tea, yellow tea, oolong tea, and dark tea.

[0009] Among them, Acanthopanax senticosus (Rupr. et Maxim.) Harms, a plant of the Araliaceae family, is the dried root and rhizome or stem; Angelica dahurica (Siebet Zucc.) Kitag, a plant of the Apiaceae family, is the dried root; Rosa laevigata (Rosa laevigata Michx.), a plant of the Rosaceae family, is the dried mature fruit; and Polyporaceae fungus is the fungus *Polyporaceae*. Fomes officinalis The dried fruiting body of (Vill. ex Fr.)Ames., green tea is the tea plant of the Theaceae family. Camellia senensis (L.) O. Kuntze: Fresh leaves that have undergone blanching treatment.

[0010] This invention utilizes natural plants, medicinal pores (Polyporus flocculationus), tea, mixed enzymes, and mixed microbial agents as raw materials, ensuring high safety. Among these, Acanthopanax senticosus and Angelica sinensis are used medicinally from their rhizomes and are rich in cellulose; Rosa laevigata is used medicinally from its fruit; Polyporus flocculationus is used medicinally from its fruiting body; and tea is high in tannins. The mixed enzyme, composed of cellulase, pectinase, and tanninase, can disrupt the dense structure of natural plant cell walls, enhancing the permeability of cell walls and intercellular barriers, and promoting the diffusion of active ingredients from the intracellular to the extracellular medium. The mixed microbial agent, composed of Lactobacillus plantarum, Bifida fragrans, and Lactobacillus acidophilus, degrades the macromolecular active ingredients of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Polyporus flocculationus, and tea into smaller molecule active ingredients, significantly increasing the absorption rate of active ingredients from these ingredients. The synergistic fermentation of these microorganisms and enzymes maximizes the extraction of effective active ingredients from natural plants, thereby enhancing the medicinal efficacy of the natural plant fermentation liquid.

[0011] In some embodiments, by weight parts, Acanthopanax senticosus is 1-5 parts, Angelica sinensis is 1-5 parts, Rosa laevigata is 1-5 parts, Phytotrichum candida is 1-5 parts, tea is 1-5 parts, and water is 6-8 parts.

[0012] In some embodiments, by mass parts, Acanthopanax senticosus is 1 part, Angelica sinensis is 1 part, Rosa laevigata is 1 part, Phytotrichum candida is 1 part, tea is 1 part, and water is 6 parts.

[0013] In some embodiments, the mass of the mixed enzyme can be 0.3% to 0.5% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Flos, tea, and water; wherein the mass ratio of cellulase, pectinase, and tanninase in the mixed enzyme can be 1:1:2.

[0014] In other embodiments, the mass of the mixed enzyme can be 0.4% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Medicinal Fomitopsis, tea, and water.

[0015] In some embodiments, the amount of the mixed microbial agent can be 0.5% to 0.7% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Flos medicata, tea and water; preferably, the amount of the mixed microbial agent can be 0.5% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Flos medicata, tea and water.

[0016] In some embodiments, the mass ratio of *Lactobacillus plantarum*, *Bifida ferment lysate*, and *Lactobacillus acidophilus* can be 1:1:1; *Lactobacillus plantarum* can be *Lactobacillus plantarum* Lp-G18, *Bifida ferment lysate* can be *Bifida ferment lysate* BB536, and *Lactobacillus acidophilus* can be *Lactobacillus acidophilus* LA-G80.

[0017] In some embodiments, the raw materials for the natural plant fermentation liquid also include sucrose and rice flour. Specifically, sucrose and rice flour provide energy for *Lactobacillus plantarum*, *Bifida fumica*, and *Lactobacillus acidophilus*.

[0018] In some embodiments, the amount of sucrose used can be 7% to 8% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Phytotrichum candida, tea and water, and the amount of rice flour used can be 3% to 5% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Phytotrichum candida, tea and water.

[0019] In some embodiments, the amount of sucrose used can be 7.5% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Phytotrichum candida, tea and water, and the amount of rice flour used can be 3% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Phytotrichum candida, tea and water.

[0020] According to a second aspect of the present invention, a method for preparing a natural plant fermentation broth is provided, comprising the following steps:

[0021] (1) Mix Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis, tea leaves and water and grind them to obtain a paste;

[0022] (2) Add mixed enzymes to the slurry at 50~60℃ to hydrolyze it and obtain the hydrolysate;

[0023] (3) Dissolve sucrose and rice flour in the enzymatic hydrolysate to obtain a mixture;

[0024] (4) Filter the mixture and seal the filtrate. Sterilize the sealed filtrate with high pressure steam and cool it.

[0025] (5) Activate Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus. Mix the activated Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus to obtain a mixed bacterial agent.

[0026] (6) Inoculate the mixed bacterial agent into the cooled filtrate;

[0027] (7) Ferment the filtrate after inoculation at 35~50℃ for 6~8 days to obtain the final product.

[0028] The method for preparing the natural plant fermentation liquid of the present invention first involves grinding to initially break down the cell walls of natural plants. Then, a mixed enzyme is added to carry out an enzymatic hydrolysis reaction to further break down the dense structure of the plant cell walls, promoting the diffusion of macromolecular active ingredients from natural plants such as Acanthopanax senticosus, Angelica sinensis, and Rosa laevigata, as well as medicinal Ferula meliflora and tea, from the cells into the enzymatic hydrolysate. Nutrients such as sucrose and rice flour are then added to provide nutrition for the mixed microbial agent. Finally, the mixed microbial agent is used for fermentation to degrade the macromolecular active ingredients into smaller molecule active ingredients. The preparation method is simple and not only improves the extraction efficiency of effective ingredients from natural plants, medicinal Ferula meliflora, and tea, but also increases the absorption rate of natural plant active ingredients by the human body, thereby enhancing the medicinal efficacy of the natural plant fermentation liquid.

[0029] In some implementations, the enzymatic hydrolysis time in step (2) can be 3 to 5 hours.

[0030] In some embodiments, in step (4), the filtration method may be to filter the mixture using clean gauze.

[0031] In some embodiments, in step (4), the method for sealing the filtrate can be to dispense the filtrate into 50 mL Erlenmeyer flasks and then seal the mouth of the Erlenmeyer flasks with a sterile culture container sealing film. Specifically, this can effectively prevent external microorganisms from contaminating the filtrate.

[0032] In some embodiments, in step (4), the high-pressure steam sterilization method can be sterilization for 10 to 15 minutes at a temperature of 100 to 110°C and a pressure of 102 to 105 kPa.

[0033] In some embodiments, the activation method in step (5) can be to remove the cryopreservation tubes containing Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus respectively from the cryopreservation environment, and then gently shake the cryopreservation tubes in a water bath at 36~37°C until the ice in the cryopreservation tubes is completely dissolved.

[0034] According to a third aspect of the present invention, the application of natural plant fermentation liquid in the preparation of cosmetics is provided. The cosmetic is one that possesses at least one of the following functions:

[0035] (1) Improves the skin's moisture-locking function;

[0036] (2) Improves skin barrier function;

[0037] (3) Improves skin roughness and luster;

[0038] (4) Antihistamine, anti-allergic and soothing effects;

[0039] (5) Moisturizes and controls oil.

[0040] The microbial indicators of the natural plant fermentation liquid of the present invention meet the hygiene standards of cosmetics, have high safety, and the natural plant fermentation liquid after adjusting the concentration of raw materials has a light color and high clarity, making it suitable for addition to cosmetic bases to prepare cosmetics.

[0041] In some embodiments, the application can be the use of natural plant fermentation liquid in the preparation of cosmetics for treating oily and sensitive skin. Specifically, when natural plant fermentation liquid is added to cosmetics as a raw material, the cosmetics are safe and non-irritating, and the easily absorbed properties and medicinal efficacy of the natural plant fermentation liquid can be utilized.

[0042] According to a fourth aspect of the present invention, a cosmetic containing the above-mentioned natural plant fermentation liquid is provided, which is also a cosmetic having at least one of the following functions:

[0043] (1) Improves the skin's moisture-locking function;

[0044] (2) Improves skin barrier function;

[0045] (3) Improves skin roughness and luster;

[0046] (4) Antihistamine, anti-allergic and soothing effects;

[0047] (5) Moisturizes and controls oil.

[0048] In some embodiments, the cosmetic can be a serum, lotion, cream, mask, or liquid. Specifically, a serum containing natural plant fermentation liquid, prepared from natural plant fermentation liquid as a raw material, is non-irritating. Tests have shown that this serum not only has good antihistamine and anti-allergic skin-soothing effects, but also has significant moisturizing and oil-controlling effects on the skin, and exhibits good tolerance in the treatment of combination and oily skin.

[0049] The technical effects of this invention are as follows:

[0050] (1) The natural plant fermentation liquid of the present invention is made from Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal pore fungus, tea, mixed enzymes and mixed bacterial agents as raw materials. It has high effective active ingredients and good medicinal effects.

[0051] (2) The method for preparing the natural plant fermentation liquid of the present invention, by adding mixed enzymes and mixed bacterial agents in sequence, promotes the diffusion of macromolecular active ingredients of natural plants, medicinal fumonis and tea from the cell to the cell, and degrades macromolecular active ingredients into small molecule active ingredients, which not only improves the extraction efficiency of effective ingredients in natural plants, medicinal fumonis and tea, but also increases the absorption rate of natural plant active ingredients by the human body, thereby enhancing the medicinal efficacy of the natural plant fermentation liquid;

[0052] (3) The natural plant fermentation liquid of the present invention has high clarity after adjusting the concentration of raw medicinal materials, and is suitable for adding to cosmetic matrix to prepare cosmetics. The prepared cosmetics are safe and non-irritating. The essence made from natural plant fermentation liquid has significant moisturizing and oil-controlling effects on facial skin, and has good antihistamine and anti-allergic skin soothing effects. Attached Figure Description

[0053] Figure 1 Images showing the products of Example 1, Comparative Example 1, and Comparative Example 2 after adjusting the concentration of the raw medicinal material. Figure 1 (A) is an image of the natural plant extract of Comparative Example 1 after adjusting the concentration of the raw medicinal material. Figure 1 (B) is an image of the natural plant fermentation broth from Example 1 after adjusting the concentration of the medicinal materials. Figure 1 (C) is an image of the natural plant extract from Comparative Example 2 after adjusting the concentration of the raw medicinal material;

[0054] Figure 2 These are magnified images of the skin of the subjects in Experiment 2 before and after using the serum containing natural plant fermentation fluid. Figure 2 (A) A magnified image of the subject's skin before using an antihistamine serum containing natural plant fermentation fluid. Figure 2 (B) A magnified image of the subject's skin after using an antihistamine serum containing natural plant fermentation fluid;

[0055] Figure 3 This describes the changes in facial skin of the subjects before and after treatment in Experiment 3. Figure 3 (A) is a picture of the subject's skin before treatment with an essence containing natural plant fermentation fluid. Figure 3 (B) A picture of the subject's skin 14 days after treatment with an essence containing natural plant fermentation fluid. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0057] The Acanthopanax senticosus, Angelica sinensis, and Rosa laevigata were purchased from Kangmei Pharmaceutical Co., Ltd.; the medicinal Fomitopsis pilosula was purchased from Qingping Traditional Chinese Medicine Market in Guangzhou; and the green tea was purchased from Hanshi Tea Co., Ltd. in Guangshan County, Henan Province. All the above medicinal materials were identified as genuine by Chief Pharmacist Qiu Zhenwen of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Lactobacillus plantarum Lp-G18, Bifida ferment lysate BB536, and Lactobacillus acidophilus LA-G80 were purchased from the China Center for Type Culture Collection; and cellulase (200 U / mg), pectinase (100 U / mg), and tanninase (100 U / mg) were purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0058] It should be noted that green tea is only one way to implement tea varieties. Other tea varieties can be selected as needed, such as black tea, white tea, yellow tea, oolong tea, or dark tea.

[0059] The homogenizing emulsification equipment used in this invention is the FV-30L FISCO vacuum homogenizing emulsifier manufactured by Shanghai Fluke Fluid Machinery Manufacturing Co., Ltd., which has functions such as homogenization, stirring, and temperature control. Homogenizing emulsification equipment used in cosmetic preparation from other manufacturers can also be used in this invention, as long as the process parameters described in this invention are strictly followed, the effects described in this invention can be achieved.

[0060] Example 1

[0061] This embodiment provides a method for preparing natural plant fermentation liquid, including the following steps:

[0062] (1) Weigh out 50 grams each of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Flos flos and green tea, crush them, add 300 grams of water, mix and grind to obtain a paste;

[0063] (2) Mix cellulase, pectinase and tanninase in a mass ratio of 1:1:2 to obtain a mixed enzyme. Add 0.4% of the mixed enzyme to the slurry at 55℃ and hydrolyze for 4 hours to obtain the hydrolysate.

[0064] (3) Add 7.5% of the total mass of slurry sucrose and 3% of the total mass of slurry rice flour to the enzymatic hydrolysate, mix well until the sucrose and rice flour are completely dissolved in the enzymatic hydrolysate to obtain a mixture.

[0065] (4) Filter the mixture with clean gauze, and then dispense the filtrate into 50 mL Erlenmeyer flasks. Seal the flask openings with sterile culture container sealing film. Then sterilize the Erlenmeyer flasks at 105℃ and 103.4 kPa for 10 min, cool to room temperature, and then transfer the Erlenmeyer flasks to a clean bench.

[0066] (5) Take out the cryopreservation tubes containing Lactobacillus plantarum Lp-G18, Bifida ferment 200g BB536, and Lactobacillus acidophilus LA-G80 respectively from the cryopreservation environment, place them at 37°C and gently shake them continuously until the ice completely melts. Then, use a sterile micropipette to take out the activated Lactobacillus plantarum Lp-G18, Bifida ferment 200g, and Lactobacillus acidophilus LA-G80 bacterial solutions from the cryopreservation tubes respectively, and mix them in a mass ratio of 1:1:1 to obtain a mixed bacterial agent.

[0067] (6) Inoculate the mixture in the conical flask with a mixed microbial agent, the amount of which is 0.5% of the total mass of the slurry;

[0068] (7) Place the conical flask in a constant temperature and humidity incubator and carry out closed fermentation for 7 days at a temperature of 40℃ to obtain natural plant fermentation liquid.

[0069] Comparative Example 1

[0070] This comparative example provides a natural plant extract, the preparation method of which includes the following steps:

[0071] Weigh out 50 grams each of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis pinnatifida, and green tea, crush them, then add 300 grams of 70% ethanol, heat to 100℃ and decoct for 30 minutes, extract once with the filtrate, and you will get the product.

[0072] Comparative Example 2

[0073] This comparative example provides a natural plant extract, the preparation method of which includes the following steps:

[0074] Weigh out 50 grams each of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis pinnatifida, and green tea, crush them, add 300 grams of water, heat to 100℃ and decoct for 30 minutes, extract the filtrate once, and you will get the product.

[0075] The natural plant fermentation broth prepared in Example 1, the natural plant extract of Comparative Example 1, and the natural plant extract of Comparative Example 2 were adjusted to a crude drug concentration of 0.1 g crude drug / mL, respectively. Then, the color and clarity of the three were compared, and the results are as follows: Figure 1 As shown, where, Figure 1 (A) is an image of the natural plant extract of Comparative Example 1 after adjusting the concentration of the raw medicinal material. Figure 1 (B) is an image of the natural plant fermentation broth from Example 1 after adjusting the concentration of the medicinal materials. Figure 1(C) is an image of the natural plant extract of Comparative Example 2 after adjusting the concentration of the raw medicinal material. It can be observed that the natural plant fermentation liquid of Example 1 has the lightest color and good clarity after adjusting the concentration of the raw medicinal material, making it suitable for addition to cosmetic bases.

[0076] Application Example 1

[0077] This application example demonstrates the use of natural plant fermentation liquid in the preparation of serums. The formulation of the serum containing natural plant fermentation liquid is shown in Table 1.

[0078] Table 1. Formula of Essence Containing Natural Plant Fermentation Liquid

[0079]

[0080] The production process of this serum includes the following steps:

[0081] According to the formula in Table 1, low molecular weight hyaluronic acid and xanthan gum were first soaked in water and fully swollen for 12 hours. Then, the swollen low molecular weight hyaluronic acid and xanthan gum were added to water and stirred to disperse evenly to obtain a mixture. Then, the other raw materials in Table 1 except for polyhexamethylene biguanide and p-hydroxyacetophenone were added to the mixture in sequence. The temperature was raised to 85°C and stirred at 500 r / min for 30 minutes. Then, the temperature was lowered to 40°C, polyhexamethylene biguanide and p-hydroxyacetophenone were added, and emulsification was continued at 500 r / min for 15 minutes to obtain an essence containing natural plant fermentation liquid.

[0082] It should be noted that the raw materials and addition amounts selected in Table 1 represent the optimal choices for maximizing the efficacy of the natural plant fermentation liquid of this invention. However, the selection of cosmetic bases for the essence containing natural plant fermentation liquid of this invention is not limited to these. Commonly used cosmetic bases produced by other manufacturers and applicable to cosmetics can also be used as raw materials in the formulation of essences containing natural plant fermentation liquid, provided that the dosage complies with national standards for cosmetic additives. It should also be noted that the preparation of essences is only one implementation method, as the natural plant fermentation liquid exerts its medicinal effects in cosmetics to achieve corresponding skincare benefits. By selecting and adjusting the dosage of the cosmetic base, the natural plant fermentation liquid can also be used as a raw material to prepare lotions, creams, masks, and water-based cosmetics.

[0083] Experimental Example 1

[0084] This experiment tested the number of microorganisms in the natural plant fermentation broth of Example 1, and the results are shown in Table 2.

[0085] Table 2 Microbiological indicators of natural plant fermentation broth

[0086]

[0087] As shown in Table 2, in the natural plant fermentation liquid prepared in Example 1, the most probable number of microorganisms (MPN) per 100g of sample of coliform bacteria is less than 100, the total number of molds is less than 50 CFU / g, the total number of yeasts is less than 50 CFU / g, and no pathogenic bacteria were detected. This indicates that the microbial indicators of the natural plant fermentation liquid meet the hygiene standards of cosmetics, are suitable for use on the skin, and have high safety.

[0088] Experimental Example 2

[0089] This experimental example uses the essence containing natural plant fermentation liquid from Application Example 1 for clinical trials to study the effect of the essence containing natural plant fermentation liquid of the present invention on facial skin moisturizing and repairing.

[0090] 1.1 Subject Inclusion Criteria

[0091] (1) Healthy female subjects aged 20-55 years with dry skin, moisture content less than 60%, positive lactic acid sting test, and no clear systemic organic disease; (2) Subjects are willing to refrain from using other skin care products, cosmetics, and receiving other facial treatments (such as drugs, chemical peels, lasers, etc.) throughout the study period; (3) Subjects are willing to comply with the study requirements and complete the entire study period and be able to complete the follow-up as required; (4) Subjects have signed the informed consent form and are willing to complete the study process.

[0092] 1.2 Exclusion criteria for subjects

[0093] (1) Those who are allergic to cosmetic preparations and ingredients or have a history of severe skin allergies; (2) Those who cannot attend follow-up appointments on time during treatment or who use other hormone-containing drugs on their own; (3) Pregnant or lactating women.

[0094] 1.3 General Information of Subjects

[0095] We commissioned Dr. Xiao Wei, Deputy Chief Physician of Guangzhou Zihetang Xinghe Traditional Chinese Medicine Clinic, to recruit 82 healthy female subjects who met the criteria for this study. Their average age was (36.15±7.23) years.

[0096] 1.4 Test Methods

[0097] Referring to the method described in the literature (Lei Cuiting, Zheng Muchuang, Chen Chuwei. Discussion on the efficacy evaluation method of skin repair cosmetics [J]. Daily Chemical Science, 2021, 44(12): 28-30), this clinical trial adopted a self-controlled pre- and post-treatment approach. The right cheek of the subject was selected as the test area. After cleansing the skin, the product was applied in a circular motion, avoiding the corners of the eyes and other easily allergenic areas. The product was gently massaged until all the remaining essence was absorbed. The product was applied once in the morning, noon and evening, 5g each time, for 14 consecutive days. One course of treatment was required. During the treatment, other similar products should not be changed or used together.

[0098] Precautions: During the trial, participants are advised to change their lifestyle and behavior, such as ensuring sufficient sleep, quitting smoking and drinking, avoiding spicy, cold, and greasy foods, avoiding dust stimulation and strenuous exercise, and avoiding exposure to strong sunlight.

[0099] 1.5 Observation Indicators

[0100] 1.5.1 Detection of Skin Physiological Function Indicators

[0101] Transepidermal water loss (TEWL) and stratum corneum hydration of the subjects' facial skin were measured using a Courage Khazaka MPA9 multifunctional skin analyzer (Germany) before treatment, and at 14 and 28 days after treatment. Skin pH was measured using a Courage Khazaka Skin-pH meter (pH905, Germany). The testing environment was a dedicated skin function testing room with no ventilation, no direct sunlight, a room temperature of 22–24°C, and a relative humidity of 45%–55%. Specifically, on the day of testing, subjects did not use any topical facial medications or cosmetics, routinely cleansed their faces, and rested for 30 minutes in the skin function testing room to acclimatize before the test. Following the instrument operating instructions, all three indicators at the same test site were tested three times, with approximately 5 minutes between each test. The average value was taken as the result of this measurement.

[0102] 1.5.2 Lactic acid stinging score

[0103] Subjects cleansed their faces and dried them with lint-free tissues. After resting for 30 minutes in the skin function testing room to acclimatize, the test was conducted. 50 μL of a 10% lactic acid solution was applied to one cheek on either side of the nasolabial fold. Subjects assessed the degree of discomfort (itching, stinging, burning) at the test site at 30 seconds, 2.5 minutes, and 5 minutes. A 4-point scale was used for scoring (0 points: no sensation; 1 point: mild; 2 points: moderate; 3 points: severe). A cumulative stinging score of 3 or higher at 30 seconds, 2.5 minutes, and 5 minutes was considered a positive result for lactic acid stinging.

[0104] 1.5.3 Human Patch Test

[0105] Thirty female volunteers who met the inclusion criteria were selected, with a mean age of (30.56±5.63) years. Using qualified patch testing equipment, a closed patch test was conducted. Approximately 0.02–0.025 mL of the test substance was placed in the patch device, and a hypoallergenic adhesive tape was applied to the back of the subject. The test substance was removed after 24 hours, and skin reactions were observed at 0.5, 24, and 48 hours after removal. The results were recorded according to the skin reaction grading standards in the 2015 edition of the "Cosmetic Safety Technical Specifications".

[0106] 1.5.4 Subjects' self-satisfaction evaluation

[0107] Subjects evaluated their overall skin improvement based on their own skin type, including indicators such as skin dryness and skin radiance. Compared to baseline (pre-treatment skin condition), self-satisfaction was categorized into four levels: very satisfied (improvement ≥ 90%), satisfied (60% ≤ improvement < 90%), average (20% ≤ improvement < 60%), and dissatisfied (improvement < 20%). Satisfaction rate = (Number of very satisfied + number of satisfied cases) / Total number of cases × 100%.

[0108] 1.6 Adverse Reactions

[0109] The subjects were observed for any adverse reactions, such as facial allergies, before and after treatment through consultation or follow-up visits.

[0110] 1.7 Statistical Methods

[0111] The data were double-checked before being entered into SPSS 25.0 software and statistically analyzed. Quantitative data were expressed as mean ± standard deviation. The independent samples t-test was used for pairwise comparisons between groups, and the paired t-test was used for within-group comparisons. Clinical efficacy evaluations were ordinal data, expressed as frequencies and relative frequencies, and analyzed using the rank-sum test. A p-value < 0.05 was considered statistically significant.

[0112] 2 Results

[0113] 2.1 Comparison of skin physiological function indicators before and after treatment in subjects

[0114] Table 3 shows the skin physiological function indicators of the subjects before and after treatment. Data shows that compared with before treatment, 14 days after treatment, the stratum corneum water content of the subjects significantly increased, TEWL significantly decreased, with statistically significant differences (P < 0.01), while pH value showed no significant change. 28 days after treatment, the stratum corneum water content of the subjects significantly increased, TEWL significantly decreased, with statistically significant differences (P < 0.01), while pH value showed no significant change.

[0115] Table 3. Skin physiological function test indicators of subjects before and after treatment ( )

[0116]

[0117] 2.2 Lactic acid stinging score

[0118] Table 4 shows the lactic acid stinging scores of the subjects before and after treatment. It can be seen that, compared with before treatment, the lactic acid stinging scores of the subjects were significantly reduced at 14 days and 28 days after treatment, with statistical differences (P<0.01).

[0119] Table 4. Skin lactic acid stinging scores of subjects before and after treatment ( )

[0120]

[0121] 2.3 Results of Human Patch Test

[0122] Table 5 shows the results of patch tests on human skin using the essence containing natural plant fermentation fluid. The results showed that none of the 82 subjects experienced symptoms such as light redness, erythema, edematous erythema, or significant edema, indicating that under certain environmental conditions, usage methods, and dosages, the essence containing natural plant fermentation fluid does not affect the body's barrier function and has no significant irritation to human skin.

[0123] Table 5 Summary of Human Skin Patch Test Results

[0124]

[0125] 2.4 Comparison of self-satisfaction evaluation before and after treatment

[0126] Table 6 compares the self-satisfaction ratings of the subjects before and after treatment. The questionnaire results showed that the subjects found the product to be of good texture, easy to apply, and easily absorbed. After use, the skin was not sticky, moisturized, and dryness and tightness were relieved. Skin dehydration, fine lines, skin sensitivity, and redness also improved. As shown in Table 6, the subject satisfaction rate was 2.44% before treatment, 58.33% after 14 days of treatment, and 86.59% after 28 days of treatment, with statistically significant differences (P < 0.01). All 82 subjects were satisfied or relatively satisfied with both the product's suitability for their skin type and overall product satisfaction, and were willing to purchase the product if it was within their acceptable price range.

[0127] Table 6 Comparison of self-satisfaction ratings of subjects before and after treatment N (p / %)

[0128]

[0129] 2.5 Adverse Reactions

[0130] After 28 days of treatment and observation, no adverse reactions occurred, all enrolled subjects completed their respective treatment regimens, and compliance was good throughout the study period with no dropouts.

[0131] 3. Conclusion

[0132] The results of this experiment show that the essence containing natural plant fermentation fluid of the present invention can significantly improve the skin's moisture retention and barrier function, effectively improve the roughness and luster of the subjects' facial skin, and the skin lactic acid stinging score results show that after using the essence containing natural plant fermentation fluid of the present invention for 28 days, the subjects' skin's ability to respond to external stimuli was significantly enhanced, and their skin tolerance increased by 4.09 times. Human patch testing results show that the formula of the essence containing natural plant fermentation fluid of the present invention is safe and non-irritating or only slightly irritating to human skin.

[0133] Experimental Example 3

[0134] This experimental example uses the essence containing natural plant fermentation liquid from Application Example 1 to conduct a histamine test, and studies the antihistamine soothing effect of the essence containing natural plant fermentation liquid of the present invention.

[0135] 1.1 Test Methods

[0136] Following the method described in the literature (Cai Yiwen, Liu Yuan, Song Chuang, et al. Research progress on the safety evaluation model of cosmetic anti-allergy[J]. Daily Chemical Science, 2021, 44(3): 48-54), histamine was applied to the skin to simulate an acute allergic reaction. Then, the essence containing natural plant fermentation liquid from Application Example 1 was applied to the skin test area to test the soothing effect of the essence containing natural plant fermentation liquid of the present invention on allergic skin.

[0137] 1.2 Test conditions and test procedures

[0138] 1.2.1 Test Conditions

[0139] Testing instrument: German CK Cutometer dual MPA580 skin tester; Probe used: Skin red pigment test probe; Testing environment conditions: Indoors, without strong sunlight or artificial light; Testing environment temperature is 20~25℃, humidity is 40%~60%, and real-time dynamic monitoring is performed.

[0140] 1.2.2 Test Procedure

[0141] The subjects washed the inside of their elbows with water and dried them with paper towels. Two 2×2cm marks were made on the inside of each elbow during the experiment. 2In the test area, photographs were taken and the red pigment was tested using a CK instrument, and the results were recorded in the antihistamine test red pigment record sheet. 10 μL of 5% histamine solution was dropped onto the marked skin surface, massaged and rubbed to absorb the solution, and the application time was recorded. The peak time of redness, swelling, and itching was recorded within fifteen minutes, photographed, and the red pigment was tested using a CK instrument, and the results were recorded in the antihistamine test red pigment record sheet. The essence containing natural plant fermentation liquid of this invention was applied to one test skin, while deionized water was applied to another test skin as a blank control, and the application time was recorded. The red pigment changes were tested using a CK instrument at 15 min, 30 min, 60 min, and 90 min after applying the test product, and the results were recorded in the antihistamine test red pigment record sheet. The subject felt relief from itching after the test product was applied, and the time was recorded. Redness and papules were observed, and the area of ​​fading was greater than 1 / 2, and the time was recorded in the antihistamine relief effect test record sheet.

[0142] 2 Results

[0143] The subjects' skin before and after using the serum containing natural plant fermentation fluid was as follows: Figure 2 As shown, Figure 2 (A) A magnified image of the subject's skin before using an antihistamine serum containing natural plant fermentation fluid. Figure 2 (B) Enlarged images of the subjects' skin after using the serum containing natural plant fermentation extract for antihistamine treatment. It can be observed that the subjects' skin showed significant redness and swelling before using the serum, and the redness and swelling significantly subsided after use. The antihistamine soothing effect is shown in Table 7.

[0144] Table 7. Antihistamine soothing effect in subjects

[0145]

[0146] 3. Conclusion

[0147] The results of this experiment show that the essence containing natural plant fermentation fluid has a significant effect on reducing swelling and redness of the skin. Compared with the control area, the red pigment value of the skin tested with the essence containing natural plant fermentation fluid showed a more significant downward trend. After applying the essence containing natural plant fermentation fluid of this invention, the itching sensation subsided significantly after about 8 minutes, the swelling subsided significantly after 15 minutes, and the redness began to fade after 23 minutes. In conclusion, the essence containing natural plant fermentation fluid of this invention has a good antihistamine and anti-allergic skin soothing effect.

[0148] Test Example 4

[0149] This experimental example uses the essence containing natural plant fermentation liquid from Application Example 1 to conduct a clinical trial on facial skin, studying the clinical efficacy of the essence containing natural plant fermentation liquid of the present invention in moisturizing and oil control of facial skin.

[0150] 1.1 Subject Inclusion Criteria

[0151] (1) Healthy subjects aged 18-40 years with oily facial skin; (2) Able to cooperate well with the experimenters and maintain a regular lifestyle during the study period; (3) Able to read and understand all contents of the informed consent form and voluntarily sign the informed consent form; (4) Discontinue the use of skin care products during the trial period, and will not participate in any clinical trials at other research centers, agreeing not to use any cosmetics, drugs, or health products that may affect the results; (5) When testing the oil control effect, the subject's face must be of combination or oily skin and the initial amount of oil on the forehead on the day of the test must be greater than 150 μg / cm. 2 .

[0152] 1.2 Exclusion criteria for subjects

[0153] (1) Those with a history of severe skin allergies; (2) Those with infectious or inflammatory diseases on the face; (3) Those who have received any facial cosmetic treatment in the past six months; (4) Pregnant or breastfeeding women.

[0154] 1.3 General Information of Subjects

[0155] Forty volunteers, aged 20.25–30.72 (23.52±4.61) years, were treated at the Guangzhou Zihetang Xinghe Traditional Chinese Medicine Clinic by Dr. Xiao Wei, Deputy Chief Physician. All volunteers signed informed consent forms before the trial began.

[0156] 1.4 Test Methods

[0157] Referring to the method described in the literature (China Association for Industry-University-Research Collaboration. Test methods for seven efficacy items of cosmetics: anti-wrinkle, firming, moisturizing, oil control, repairing, nourishing and soothing [J]. Group Standard of China Association for Industry-University-Research Collaboration, T / CAB 0152-2022, released on 2022-06-24), this clinical trial adopted a self-controlled pre- and post-treatment approach. After cleansing their skin, the subjects applied the essence containing natural plant fermentation liquid of this invention to their right cheek, 3 mL / time, twice a day, for 14 consecutive days, for a total of 28 applications. During this period, they were not allowed to change or use other similar products.

[0158] Precautions: During the trial, researchers advised participants to change their lifestyle and behavior, such as ensuring sufficient sleep, quitting smoking and alcohol, avoiding spicy, cold, and greasy foods, avoiding dust stimulation and strenuous exercise, and avoiding exposure to strong sunlight.

[0159] 1.5 Detection of skin barrier function indicators in subjects

[0160] Researchers used a Courage Khazaka MPA9 multifunctional skin analyzer (Germany) to measure transepidermal water loss (TEWL), stratum corneum hydration, and sebum content of the subjects' facial skin before and 14 days after treatment. The testing environment was a dedicated skin function testing room with no ventilation, no direct sunlight, a room temperature of 22–24°C, and a relative humidity of 45%–55%. Specifically, on the day of testing, subjects did not use any topical facial medications or cosmetics, routinely moisturized their faces, and rested for 30 minutes in the skin function testing room to acclimatize before the test. Following the instrument's operating instructions, researchers tested the same area of ​​the subject three times, with approximately 5 minutes between each test, and took the average value as the result of that measurement.

[0161] 1.6 Subjective evaluations by test takers

[0162] A questionnaire survey was used to allow participants to self-assess the condition of their skin at the test site. 1 point was rated as unsatisfactory, 2 as somewhat unsatisfactory, 3 as neutral, 4 as satisfied, and 5 as completely satisfied. Scores greater than 3 were considered valid. Satisfaction rate was calculated as (valid number of participants / valid data) × 100%.

[0163] 1.7 Statistical Methods

[0164] The data were double-checked before being entered into SPSS 25.0 software and statistically analyzed. The measurement data adopted (…). The numbers indicate that paired t-tests were used to compare pre- and post-administration results within the same group. One-way ANOVA was used for comparisons among multiple groups, and independent samples t-tests were used for pairwise comparisons between groups. Count data were expressed as rates and analyzed using the chi-square test. P < 0.05 was considered statistically significant.

[0165] 2 Results

[0166] 2.1 Clinical efficacy

[0167] Changes in facial skin of subjects before and after treatment, such as Figure 3 As shown, Figure 3 (A) is a picture of the subject's skin before treatment with an essence containing natural plant fermentation fluid. Figure 3 (B) A picture of the subject's skin 14 days after treatment with an essence containing natural plant fermentation fluid.

[0168] According to statistics, among 40 subjects treated with the essence containing natural plant fermentation fluid for 14 days, 20 subjects (50.00%) showed significant improvement in skin oiliness and stickiness, and 15 subjects (37.50%) showed improvement, with a total effective rate of 87.50%. This indicates that the essence containing natural plant fermentation fluid of the present invention has good moisturizing and oil-controlling effects.

[0169] 2.2 Skin barrier function indicators of subjects before and after treatment

[0170] The skin barrier function indicators before and after treatment are shown in Table 8. The data in Table 8 show that, compared with before treatment, the stratum corneum moisture content of the subjects increased significantly after treatment, while TEWL and sebum content decreased significantly, with statistical differences (P < 0.01).

[0171] Table 8. Indicators of skin barrier function in subjects before and after treatment ( )

[0172]

[0173] 2.3 Statistical results of subjective evaluations by test takers

[0174] The subjects' self-assessment results of their facial skin condition are shown in Table 9. As can be seen from Table 9, after 14 days of treatment with the essence containing natural plant fermentation fluid of this invention, the overall skin satisfaction rate of the subjects was 90.00%.

[0175] Table 9. Subjects' self-assessment results of facial skin condition

[0176]

[0177] 2.5 Adverse Reactions

[0178] After 14 days of treatment and observation, no adverse reactions occurred, all enrolled subjects completed their respective treatment regimens, and compliance was good throughout the study period with no dropouts.

[0179] 3. Conclusion

[0180] The results of this study indicate that when the facial skin is combination or oily, the essence containing natural plant fermentation liquid of this invention has a significant moisturizing and oil-controlling effect on the facial skin, and is non-irritating. The subjects tolerated it well, with no side effects reported, and compliance was high.

[0181] In summary, the advantages of this invention over the prior art are as follows:

[0182] (1) The method for preparing the natural plant fermentation liquid of the present invention is simple. By adding mixed enzymes and mixed bacterial agents sequentially, the extraction efficiency of the effective components of natural plants is improved, thereby enhancing the medicinal efficacy of the natural plant fermentation liquid. Testing shows that the microbial indicators of the natural plant fermentation liquid prepared by the method of the present invention meet the hygiene standards for cosmetics, indicating high safety. Furthermore, compared to extracts obtained through other extraction methods, the natural plant fermentation liquid of the present invention has the lightest color and highest clarity after adjusting the concentration of the raw medicinal material, making it suitable as a raw material for addition to cosmetic bases.

[0183] (2) The natural plant fermentation liquid of the present invention can be added to the cosmetic matrix as a raw material to prepare cosmetics for treating oily and sensitive skin. The essence containing natural plant fermentation liquid obtained from the natural plant fermentation liquid is non-irritating. Experiments have shown that the essence can improve the skin's water-locking and barrier function, effectively improve the roughness and luster of the subject's facial skin, and reduce the skin's lactic acid stinging score. It not only has a good soothing effect on allergic skin, but also has a significant moisturizing and oil-controlling effect in the treatment of combination and oily facial skin. It is well tolerated, has no side effects, and has high compliance. It can treat oily and sensitive skin.

[0184] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A natural plant fermentation liquid, characterized in that, It is mainly prepared from Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis, tea, water, mixed enzymes, and mixed microbial agents as raw materials; wherein, the mixed enzymes are composed of cellulase, pectinase and tanninase, and the mixed microbial agents are composed of Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus. The ingredients, by weight, consist of 1-5 parts of Acanthopanax senticosus, 1-5 parts of Angelica sinensis, 1-5 parts of Rosa laevigata, 1-5 parts of Phytotrichum candida, 1-5 parts of tea leaves, and 6-8 parts of water.

2. The natural plant fermentation liquid according to claim 1, characterized in that, The mass of the mixed enzyme is 0.3% to 0.5% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis, tea, and water; wherein the mass ratio of cellulase, pectinase, and tanninase in the mixed enzyme is 1:1:

2.

3. The natural plant fermentation liquid according to claim 1, characterized in that, The dosage of the mixed microbial agent is 0.5% to 0.7% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis, tea, and water; the mass ratio of Lactobacillus plantarum, Bifida fumica, and Lactobacillus acidophilus in the mixed microbial agent is 1:1:

1.

4. The natural plant fermentation liquid according to any one of claims 1-3, characterized in that, The raw materials of the natural plant fermentation liquid also include sucrose and rice flour. The amount of sucrose used is 7% to 8% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Phytotrichum candida, tea and water. The amount of rice flour used is 3% to 5% of the total mass of Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, Phytotrichum candida, tea and water.

5. The method for preparing the natural plant fermentation liquid according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Mix Acanthopanax senticosus, Angelica sinensis, Rosa laevigata, medicinal Fomitopsis, tea leaves and water and grind them to obtain a paste; (2) Add mixed enzymes to the slurry at 50~60℃, and perform enzymatic hydrolysis to obtain the hydrolysate; (3) Dissolve sucrose and rice flour in the enzymatic hydrolysate to obtain a mixture; (4) Filter the mixture and seal the filtrate. Sterilize the sealed filtrate with high pressure steam and cool it. (5) Activate Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus. Mix the activated Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus to obtain a mixed bacterial agent. (6) Inoculate the mixed bacterial agent into the cooled filtrate; (7) Ferment the filtrate after inoculation at 35-50℃ for 6-8 days to obtain the final product.

6. The preparation method according to claim 5, characterized in that, In step (4), the filtration method is to filter the mixture using clean gauze; The method for sealing the filtrate involves dispensing the filtrate into conical flasks and then sealing the mouths of the conical flasks with a sterile culture container sealing film.

7. The preparation method according to claim 5, characterized in that, In step (5), the activation method is to remove the cryopreservation tubes containing Lactobacillus plantarum, Bifida ferment lysate and Lactobacillus acidophilus respectively from the cryopreservation environment, and then gently shake the cryopreservation tubes in a water bath environment of 36~37℃ until the ice in the cryopreservation tubes is completely dissolved.

8. The use of the natural plant fermentation liquid according to any one of claims 1-4 in the preparation of cosmetics, wherein the cosmetic is a cosmetic having at least one of the following functions: (1) Improves the skin's moisture-locking function; (2) Improves skin barrier function; (3) Improves skin roughness and luster; (4) Anti-allergic and soothing effects; (5) Control oil.

9. A cosmetic product, characterized in that, A cosmetic containing the natural plant fermentation liquid according to any one of claims 1-4, and possessing at least one of the following functions: (1) Improves the skin's moisture-locking function; (2) Improves skin barrier function; (3) Improves skin roughness and luster; (4) Anti-allergic and soothing effects; (5) Control oil.

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