A spartium junceum ferment, a preparation method thereof and application of the spartium junceum ferment in preparation of anti-wrinkle firming and soothing care products

Abstract

The present application belongs to the technical field of biological fermentation, and relates to a spirodelated sophora ferments, a preparation method thereof and application of the spirodelated sophora ferments in preparation of anti-wrinkle firming and soothing care products. The spirodelated sophora, glucose, dipotassium hydrogen phosphate and water are mixed, and after enzyme inactivation and sterilization, a fermentation culture medium is prepared; Lactobacillus plantarum and Lactobacillus rhamnosus are mixed and inoculated into the fermentation culture medium, and then fermentation is carried out in an anaerobic environment. The present application utilizes two strains to ferment spirodelated sophora, which not only has mild conditions, can avoid high-temperature damage to active ingredients such as total phenol, polysaccharide, flavone and amino acid, avoids the addition of organic solvents, and significantly improves the content of active ingredients such as total phenol, polysaccharide, flavone and amino acid, which is conducive to industrial production. At the same time, the fermentation liquor has the effects of anti-wrinkle firming, antioxidant, soothing and care.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, and relates to a fermentation product of *Broomia linearis*, its preparation method, and its application in the preparation of anti-wrinkle, firming, and soothing repair products. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] With economic development, people's awareness of skincare is constantly increasing, and the concept of healthy skin is gradually taking shape. As living conditions gradually improve, people's requirements for the performance of cosmetic products are gradually increasing. The application of general soothing, anti-allergic, and repairing ingredients in cosmetics is limited. The emergence of natural plant-based anti-allergic active ingredients has filled this gap and has been favored by the market and consumers.

[0004] Rooibos (scientific name: Aspalathus linearis), a rare herb unique to South Africa, shows broad application prospects in the cosmetics field due to its unique geographical scarcity and excellent bioactive components. Rooibos contains aspalathin, a unique and potent antioxidant that can directly neutralize free radicals and block the oxidative stress chain reaction. The SOD (superoxide dismutase) it contains works synergistically to form a multi-layered antioxidant defense system. This system not only eliminates existing free radicals but also reduces the generation of reactive oxygen species (ROS) at the source, thereby protecting skin cells from oxidative damage and delaying the cellular aging process. Meanwhile, the polyphenols and flavonoids in *Broomia lineare* can regulate the skin's inflammatory response pathways, reduce the release of pro-inflammatory cytokines, and alleviate skin redness, itching, and sensitivity caused by external stimuli, environmental pollution, or internal factors. This anti-inflammatory property makes it significantly effective in relieving inflammatory skin problems such as eczema and diaper rash, while also providing a gentle yet effective care solution for sensitive skin. Its mineral content, such as zinc, further supports skin repair functions and participates in cell regeneration and immune regulation. Furthermore, *Broomia lineare* enhances the physical structure and function of the skin barrier by promoting the orderly arrangement of lipids in the stratum corneum and the synthesis of ceramides, reducing transepidermal water loss, and improving the skin's moisturizing ability and resistance to external stimuli. AHA (alpha hydroxy acids) gently promote keratinocyte metabolism, accelerate the shedding of aged keratinocytes, stimulate cell renewal, and make the skin surface smoother and more delicate, while also creating favorable conditions for the penetration of subsequent active ingredients. The skincare mechanism of *Gnaphalium affine* is a comprehensive regulatory process involving multiple targets and pathways. Its core lies in blocking the source of aging and damage through powerful antioxidant capabilities, maintaining skin's internal environment stability through anti-inflammatory effects, and strengthening the skin's self-protective function through barrier repair, ultimately achieving a comprehensive improvement in skin health. This natural, gentle, and multifaceted nature makes it an ideal active ingredient in modern cosmetic formulations, bridging traditional botanical wisdom with modern dermatological science.

[0005] However, *Broomia linearis* cannot be used directly to make products; its active ingredients usually need to be extracted. Extraction processes for *Broomia linearis* mainly include steam distillation, solvent extraction (including volatile solvent extraction and ethanol reflux extraction), and supercritical fluid extraction. Steam distillation can easily damage heat-sensitive components (such as total phenols, polysaccharides, flavonoids, and amino acids), leading to reduced or even lost skincare efficacy. Solvent extraction carries the risk of solvent residue, posing safety concerns such as skin irritation, inflammation, and even barrier function damage. Supercritical fluid extraction requires large equipment investments and advanced technology, and its industrial application is not yet widespread. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a fermented product of *Broomia linearis*, its preparation method, and its application in the preparation of anti-wrinkle, firming, and soothing repair products. This invention utilizes two bacterial strains to ferment *Broomia linearis*, which not only provides mild conditions, avoiding the destruction of active ingredients such as total phenols, polysaccharides, flavonoids, and amino acids by high temperatures and avoiding the addition of organic solvents, but also significantly increases the content of active ingredients such as total phenols, polysaccharides, flavonoids, and amino acids, thus facilitating industrial production. Simultaneously, the fermented broth possesses anti-wrinkle, firming, antioxidant, and soothing repair effects.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, a method for preparing a fermented product of *Broomia linearifolia* includes the following steps: The enzymes of the linear-leaved broom flower were inactivated. Glucose and dipotassium hydrogen phosphate were mixed with water and sterilized. Then, enzyme-inactivated broom flowers were added to prepare a fermentation medium. Lactobacillus plantarum and Lactobacillus rhamnosus were activated to OD values ​​respectively. 600 The concentration is 0.8-1.0. Then, the activated solutions of Lactobacillus plantarum and Lactobacillus rhamnosus are mixed at a volume ratio of 1:0.9-1.1 and inoculated into the fermentation medium. Fermentation is then carried out in an anaerobic environment to obtain the final product.

[0008] *Lactobacillus plantarum* possesses highly efficient β-glucosidase activity, specifically hydrolyzing the glycosyl moiety of C-glycoside flavonoids (such as asparaghin and nothofagin) in *Broomria filamentosa*, converting bound polyphenols into free aglycones, significantly improving their bioavailability and antioxidant activity, while avoiding polyphenol oxidase-mediated oxidative degradation in traditional fermentation. This strain grows well in a low-temperature range of 25-30℃, exhibiting strong acid production capacity and controllable metabolic pathways. Furthermore, the lactic acid and short-chain fatty acids produced by *Lactobacillus plantarum* fermentation can form organic acid salts with minerals in *Broomria filamentosa*, enhancing bioavailability. Its metabolites also possess clear intestinal probiotic functions and immunomodulatory activities, synergistically complementing the anti-inflammatory properties of *Broomria filamentosa* itself. *Lactobacillus rhamnosus* exhibits unique polyphenol tolerance and metabolic adaptability, maintaining high activity in polyphenol-rich *Broomria filamentosa* substrates. Its extracellular polysaccharide production capacity can form protective biofilms, reducing oxidative damage to polyphenols from reactive oxygen species during fermentation. Lactobacillus rhamnosus can secrete various hydrolytic enzymes to selectively hydrolyze and structurally modify the complex polyphenols in *Broomia lineare*, producing derivatives with higher free radical scavenging activity. Simultaneously, this strain lacks amino acid decarboxylase activity, avoiding the formation of harmful biogenic amines. The combination of these two strains achieves functional complementarity and synergy: *Lactobacillus plantarum* dominates rapid acid production and the biotransformation of core polyphenols, establishing a stable acidic fermentation environment; *Lactobacillus rhamnosus* is responsible for deep metabolism and antioxidant protection, extending the stability of active ingredients throughout the fermentation cycle. Both are GRAS (Generally Recognized As Safe) strains, producing no ethanol. Their combined fermentation products can be directly applied to high-end cosmetics, simplifying the process while maximizing product functionality.

[0009] Secondly, a fermented product of *Broomia linearis* is obtained by the preparation method described in the first aspect of this invention.

[0010] Thirdly, the application of the *Broom's Threadleaf* ferment as described in the second aspect of the present invention in the preparation of anti-wrinkle, firming, and soothing repair products.

[0011] The beneficial effects of this invention are as follows: 1. Experiments show that the combined fermentation method of Lactobacillus plantarum and Lactobacillus rhamnosus in this invention significantly increases the content of key active ingredients such as total phenols, polysaccharides, flavonoids and amino acids in the fermentation broth, thus providing a material basis for the skin care efficacy of the fermentation broth.

[0012] 2. The fermentation broth obtained by the co-fermentation of Lactobacillus plantarum and Lactobacillus rhamnosus in this invention has multiple skin care effects such as firming, soothing, repairing and anti-oxidation, which can meet multiple skin care needs and avoid the complexity and compatibility problems caused by the compounding of multiple functional ingredients in traditional formulas.

[0013] 3. In the preparation method of this invention, the fermentation conditions are mild, effectively avoiding the destruction of heat-sensitive active ingredients such as total phenols, polysaccharides, flavonoids, and amino acids caused by high temperatures in traditional steam distillation methods. At the same time, this method does not add any organic solvents, avoiding the safety hazards caused by organic solvent residues in solvent extraction methods. It has advantages such as being green, environmentally friendly, and sustainable, and also ensures the purity and mildness of the final product.

[0014] 4. The *Lactobacillus plantarum* and *Lactobacillus rhamnosus* strains used in this invention are both GRAS strains. The fermentation process does not introduce any exogenous harmful substances, making the fermentation broth extremely safe for use in cosmetics or pharmaceuticals.

[0015] In summary, this invention not only achieves synergistic effects of multiple skin care benefits, but also has advantages such as high yield of active ingredients, mild processing conditions, environmental friendliness, and safe use, and has good prospects for industrial application and commercial value. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The following graphs show the results of the active ingredient content of the fermentation broth prepared in Examples 1-3 of this invention: (a) is the polyphenol content, (b) is the flavonoid content, (c) is the polysaccharide content, and (d) is the amino acid content.

[0018] Figure 2 The following figures show the evaluation results of the repair efficacy of the plant fermentation liquid in Example 5 of the present invention: (a) shows the CCK-8 test results, (b) shows the scratch test results, (c) shows the scratch test results, (d) shows the zebrafish tail fin repair test results, and (e) shows the zebrafish tail fin repair test results.

[0019] Figure 3 The following is a graph showing the evaluation results of the soothing effect of the plant fermentation liquid in Example 6 of the present invention: (a) is the hyaluronidase inhibition rate, (b) is the TNF-α content, (c) is the IL-6 content, and (d) is the IL-1 content.

[0020] Figure 4 The graphs show the antioxidant effects of the plant fermentation broth in Example 7 of this invention at the biochemical and cellular levels; (a) shows the scavenging efficiency of DPPH free radicals, and (b) shows the scavenging efficiency of O2. 2- (c) shows the scavenging efficiency of ·OH, and (d) shows the cellular level detection results.

[0021] Figure 5 This is a diagram showing the results of the safety test of the plant fermentation broth in Example 7 of the present invention.

[0022] Figure 6 The following is a graph showing the anti-wrinkle effect of the plant fermentation liquid in Example 7 of the present invention; (a) shows the content of type I collagen, and (b) shows the content of type III collagen. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] A typical embodiment of the present invention provides a method for preparing a fermented product of *Broomia linearis*, comprising the following steps: The enzymes of the linear-leaved broom flower were inactivated. Glucose and dipotassium hydrogen phosphate were mixed with water and sterilized. Then, enzyme-inactivated broom flowers were added to prepare a fermentation medium. Lactobacillus plantarum and Lactobacillus rhamnosus were activated to OD values ​​respectively. 600 The concentration is 0.8-1.0. Then, the activated solutions of Lactobacillus plantarum and Lactobacillus rhamnosus are mixed at a volume ratio of 1:0.9-1.1 and inoculated into the fermentation medium. Fermentation is then carried out in an anaerobic environment to obtain the final product.

[0026] In some embodiments, *Broomia linearis* is in powder form with a particle size of 40-60 mesh. Using powdered *Broomia linearis* is beneficial for increasing the content of active ingredients such as total phenols, polysaccharides, flavonoids, and amino acids in the fermentation broth.

[0027] In some embodiments, the ratio of *Broomia linearis* to water is 4.6-5.4:200, g / mL. Studies have shown that fermentation is more effective under these concentration conditions.

[0028] In some embodiments, the enzyme inactivation treatment conditions are: a temperature of 167-173°C and a time of 27-33 min.

[0029] In some embodiments, the sterilization conditions are: a temperature of 120-125°C and a time of 18-22 minutes. This treatment effectively kills bacterial strains and prevents other strains from affecting subsequent fermentation.

[0030] Lactobacillus plantarum and Lactobacillus rhamnosus can be co-fermented in any ratio. To control the ratio of Lactobacillus plantarum to Lactobacillus rhamnosus, adjustments are typically made using OD600 values ​​and volume. In some embodiments, the ratio of Lactobacillus plantarum to Lactobacillus rhamnosus is 1:0.9-1.1. Studies have shown that fermentation under these conditions yields better results.

[0031] In some embodiments, the total inoculum of Lactobacillus plantarum and Lactobacillus rhamnosus is 4.8–5.2% (v / v).

[0032] In some embodiments, the activation medium used is MRS medium.

[0033] The fermentation process of this invention is carried out in an anaerobic environment, which can be an oxygen-free aerobic environment or a micro-anaerobic environment containing trace amounts of oxygen. Since the *Lactobacillus plantarum* and *Lactobacillus rhamnosus* used in this invention are both facultative anaerobic bacteria, the operation is relatively simple, and the micro-anaerobic environment can be created by covering the liquid surface with liquid paraffin or sealing it with nitrogen gas.

[0034] In some embodiments, the fermentation temperature is 28-30℃ and the fermentation time is 18-24 h.

[0035] In some embodiments, the pH of the fermentation broth is 4.2-4.5 at the end of fermentation.

[0036] In some embodiments, pasteurization is performed after fermentation. Pasteurization conditions are relatively mild, which is beneficial for preserving heat-sensitive active ingredients such as total phenols, polysaccharides, flavonoids, and amino acids. Specifically, the pasteurization temperature is 62-68°C, and the time is 27-33 minutes.

[0037] In some embodiments, obtaining the fermentation broth after fermentation includes a process of removing precipitate. Specifically, the process of removing precipitate includes centrifugation and filtering the supernatant after centrifugation. When pasteurization is required after fermentation, the preferred process is to perform pasteurization first, followed by precipitate removal. If precipitate removal is performed first, followed by pasteurization, microbial debris will be generated, requiring further precipitate removal. Therefore, performing pasteurization first, followed by precipitate removal, simplifies the precipitation process.

[0038] In some embodiments, obtaining the fermentation broth after fermentation includes a decolorization process. Specifically, the decolorization process uses activated carbon for adsorption decolorization.

[0039] Another embodiment of the present invention provides a fermented product of *Broomia linearis*, obtained by the above preparation method.

[0040] A third embodiment of the present invention provides an application of the above-mentioned *Broomia linearis* ferment in the preparation of anti-wrinkle, firming, and soothing repair products.

[0041] The products described in this invention can be cosmetics or pharmaceuticals.

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0043] Example 1: Lactic acid bacteria fermentation broth from the plant *Broomia lineare* (1) Take 5 g of unfermented bromeliads that have passed through a 40-60 mesh sieve. Treat the dried raw material in a 170 ℃ oven for 30 min to inactivate endogenous oxidases. After taking it out, cool it to room temperature in a desiccator.

[0044] (2) Add 1 g of glucose and 0.4 g of dipotassium hydrogen phosphate and 200 mL of deionized water to the fermentation vessel, stir well and sterilize at 121°C for 20 min. After cooling, combine with the treated broom flower raw material to obtain the fermentation culture medium.

[0045] (3) Lactobacillus plantarum (ATCC 14917) and Lactobacillus rhamnosus (ATCC 53103) were activated separately in MRS medium until OD 600 When the pH is 1.0, it is inoculated into the above fermentation medium at a volume ratio of 1:1 and a total inoculum of 5% (v / v). Nitrogen gas is used to create a micro-anaerobic environment. Fermentation is carried out at 28.5℃ and 50 rpm for 24 h, with an endpoint pH of 4.35.

[0046] (4) After fermentation, pasteurize at 65°C for 30 min and cool rapidly to room temperature.

[0047] (5) Place it in a centrifuge and centrifuge at 12,000 rpm for 10 min. Take the supernatant and place it in a vacuum filter to filter it to obtain the unbleached fermentation broth.

[0048] (6) The obtained plant fermentation broth was decolorized using activated carbon. After decolorization, the broth was centrifuged to obtain the supernatant. The supernatant was then filtered through a 0.22 μM filter membrane to obtain a clear and transparent plant lactic acid bacteria fermentation broth.

[0049] Example 2: Fermentation of Lactobacillus plantarum with Broom's filamentosa fermentation broth (1) Take 5 g of unfermented bromeliads that have passed through a 40-60 mesh sieve. Treat the dried raw material in a 170 ℃ oven for 30 min to inactivate endogenous oxidases. After taking it out, cool it to room temperature in a desiccator.

[0050] (2) Add 1 g of glucose and 0.4 g of dipotassium hydrogen phosphate and 200 mL of deionized water to the fermentation vessel, stir well and sterilize at 121°C for 20 min. After cooling, combine with the treated broom flower raw material to obtain the fermentation culture medium.

[0051] (3) Inoculate Lactobacillus plantarum (ATCC 14917) into MRS broth medium and incubate at 30°C until the late logarithmic growth phase. OD 600 The pH reached 1.0. The activated bacterial solution was inoculated into the above fermentation medium at an inoculum rate of 5% (v / v), and a micro-anaerobic environment was created by sealing with nitrogen gas. Fermentation was carried out at 29°C and 50 rpm for 24 h, with the final pH being 4.25.

[0052] (4) After fermentation, pasteurize at 65°C for 30 min and cool rapidly to room temperature.

[0053] (5) Place it in a centrifuge and centrifuge at 12,000 rpm for 10 min. Take the supernatant and place it in a vacuum filter to filter it to obtain the unbleached fermentation broth.

[0054] (6) The obtained plant fermentation broth was decolorized using activated carbon. After decolorization, the supernatant was obtained by centrifugation. The supernatant was then filtered through a 0.22 μM filter membrane to obtain a clear and transparent Lactobacillus plantarum fermentation broth.

[0055] Example 3: Fermentation of Lactobacillus rhamnosus-Broomia linearis fermentation broth (1) Take 5 g of unfermented bromeliads that have passed through a 40-60 mesh sieve. Treat the dried raw material in a 170 ℃ oven for 30 min to inactivate endogenous oxidases. After taking it out, cool it to room temperature in a desiccator.

[0056] (2) Add 1 g of glucose and 0.4 g of dipotassium hydrogen phosphate and 200 mL of deionized water to the fermentation vessel, stir well and sterilize at 121°C for 20 min. After cooling, combine with the treated broom flower raw material to obtain the fermentation culture medium.

[0057] (3) Lactobacillus rhamnosus (ATCC 53103) was inoculated into MRS broth medium and cultured anaerobically at 37°C in the late logarithmic growth phase until the OD600 reached 1.0. The activated bacterial solution was inoculated into the above fermentation medium at an inoculation rate of 5% (v / v), and a strictly anaerobic environment was created by purging with nitrogen and sealing. Fermentation was carried out at 37°C and 50 rpm for 24 h, with the final pH being 4.4.

[0058] (4) After fermentation, pasteurize at 65°C for 30 min and cool rapidly to room temperature.

[0059] (5) Place it in a centrifuge and centrifuge at 12,000 rpm for 10 min. Take the supernatant and place it in a vacuum filter to filter it to obtain the unbleached fermentation broth.

[0060] (6) The obtained plant fermentation broth was decolorized using activated carbon. After decolorization, the broth was centrifuged to obtain the supernatant. The supernatant was then filtered through a 0.22 μM filter membrane to obtain a clear and transparent Lactobacillus rhamnosus fermentation broth.

[0061] Example 4: Determination of active ingredient content and determination of extraction process for plants using different extraction methods 1. Detection of total phenol content (1) Prepare a 60% ethanol solution and a 5 mg / mL gallic acid solution.

[0062] (2) Preparation of standard: Gallic acid standard at different concentrations of 0.625, 0.15625, 0.078125, 0.0391, 0.0191, 0.0098 and 0.0049 mg / mL was prepared by gradient dilution.

[0063] (3) Four experimental groups were set up and labeled as control group, test group, standard group and blank group respectively. Reagents were added according to the instructions of the total plant phenol (TP) content detection kit (catalog number: BC1345, Beijing Solarbio Science & Technology Co., Ltd.). The sample test solution was the solution obtained in Examples 1 to 3.

[0064] (4) The reaction was carried out according to the instructions of the Total Plant Phenolic (TP) Content Detection Kit (Catalog No.: BC1345, Beijing Solarbio Science & Technology Co., Ltd.).

[0065] (5) Vortex the above experimental groups, let them stand at room temperature for 10 min, and measure the absorbance at 760 nm in a 96-well plate.

[0066] (6) Plot the standard curve and calculate the total phenol content in each sample based on the standard curve: total phenol content (mg / mL) = x / W; x is the sample concentration obtained from the standard curve, mg / mL; W is the sample mass in g.

[0067] 2. Detection of crude polysaccharide content (1) Prepare a glucose solution of 10 mg / mL.

[0068] (2) Prepare glucose standards of 0.2, 0.16, 0.08, 0.04, 0.02, and 0.01 mg / mL using a gradient dilution method. Ensure thorough mixing during each dilution.

[0069] (3) The reaction was carried out according to the instructions of the Plant Crude Polysaccharide Content Detection Kit (Catalog No.: BC6165, Beijing Solarbio Science & Technology Co., Ltd.); the sample test solution was the solution obtained in Examples 1 to 3.

[0070] (4) Vortex the above experimental group, react in a boiling water bath for 20 min, take it out and cool it to room temperature immediately, and measure the absorbance at 490 nm after mixing.

[0071] (5) Plot the standard curve and calculate the crude polysaccharide content in each sample based on the standard curve: crude polysaccharide content (mg / mL) = x / W*F; x is the sample concentration obtained from the standard curve, mg / mL; W is the sample mass in g; F is the dilution factor.

[0072] 3. Detection of flavonoid content (1) Prepare a 60% ethanol solution and a 10 mg / mL rutin solution.

[0073] (2) Prepare rutin standards at concentrations of 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, 0.039, and 0.02 mg / mL using a gradient dilution method. Ensure thorough mixing during each dilution.

[0074] (3) The reaction was carried out according to the instructions of the flavonoid content detection kit (catalog number: BC1335, Beijing Solarbio Science & Technology Co., Ltd.); the sample test solution was the solution obtained in Examples 1 to 3.

[0075] (4) Vortex the above experimental groups, place them in a 37°C water bath for 45 min, centrifuge and take the supernatant to measure the absorbance at 470 nm in a 96-well plate.

[0076] (5) Plot the standard curve and calculate the flavonoid content in each sample based on the standard curve: flavonoid content (mg / mL) = x / W*F; x is the sample concentration obtained from the standard curve, mg / mL; W is the sample mass in g; F: dilution factor.

[0077] 4. Detection of amino acid content (1) Prepare 1.25 μmol / mL glutamic acid standard, mix thoroughly, and prepare immediately before use.

[0078] (2) The reaction was carried out according to the instructions of the amino acid content detection kit (catalog number: BC1575, Beijing Solarbio Science & Technology Co., Ltd.); the sample solution to be tested was the solution obtained in Examples 1 to 3.

[0079] (3) Vortex the above experimental groups, place them in a boiling water bath for 15 min, cool them, invert them repeatedly, centrifuge them, and take the supernatant to measure the absorbance at 570 nm in a 96-well plate.

[0080] (4) Calculate the amino acid content, (μmol / mL) = 2.5 * ΔA_determined / ΔA_standard * F, ΔA_determined = A_determined tube - A_blank tube, ΔA_standard = A_standard tube - A_blank tube; F: dilution factor.

[0081] Figure 1 Images (a), (b), (c), and (d) show the determination of the main components (total phenols, polysaccharides, flavonoids, and amino acids) in *Broomria filamentosa* after fermentation with *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and their combined fermentation. The results showed that compared with *Lactobacillus plantarum* alone, the combined fermentation increased the contents of total phenols, polysaccharides, flavonoids, and amino acids by 56%, 44%, 75%, and 50%, respectively; compared with *Lactobacillus rhamnosus* alone, the combined fermentation increased the contents of total phenols, polysaccharides, flavonoids, and amino acids by 42%, 67%, 44%, and 39%, respectively. Therefore, the dual-strain fermentation process can significantly increase the active ingredients in *Broomria filamentosa*. Subsequent studies will use the fermentation broth from the combined dual-strain fermentation of *Broomria filamentosa* to verify its efficacy.

[0082] Example 5: Evaluation of the restorative effects of plant fermentation broth Cell viability is the foundation for maintaining cell proliferation, repair and metabolic functions, and is crucial for skin damage repair. Cell scratch assays, to a certain extent, reflect the cell's repair ability.

[0083] The caudal fin is an important organ for locomotion and protection in zebrafish. Its damage repair process can directly reflect the body's ability to heal. The regeneration and repair model after caudal fin amputation in zebrafish is a classic model for evaluating the repair activity of test substances.

[0084] In this embodiment, the effects of plant fermentation liquid on the viability and repair ability of keratinocytes were detected at the cellular level, and the effects of plant fermentation liquid on the repair ability of zebrafish tail fins were further detected at the individual level, so as to comprehensively evaluate its repair efficacy.

[0085] 1. Effects of plant fermentation broth on cell viability (1) Select human skin fibroblasts in good logarithmic growth phase, digest them with trypsin, prepare cell suspensions, and count the cells. Use 1×10⁻⁶ cells per cell suspension. 6 Seeds were placed in 96-well plates at a density of 10 cells / well (marked on the back of the plates with a pencil for easy identification), shaken to mix, and placed in an incubator.

[0086] (2) The next day, observe the cell adhesion and growth status. After confirming that the cells adhered evenly, remove the original culture medium in each well. Add cell culture medium containing 1% serum (blank control group) or different concentrations of plant fermentation broth treatment solution (experimental group) prepared with cell culture medium containing 1% serum to each well. Set up 3 replicates for each group and continue to culture in the incubator for 24 h.

[0087] (3) After the culture is completed, add 0.5 mg / mL MTT solution to each well (usually 5 mg / mL, diluted 10 times with culture medium, 200 μL per well), shake gently to mix, and continue to incubate in the incubator for 2 h.

[0088] (4) After incubation, carefully discard the supernatant of each well, add 180 μL of DMSO solution to each well (usually 150 μL per well), and place on a shaker to shake at low speed for 10-15 min.

[0089] (5) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 570 nm and record the detection data.

[0090] (6) Calculate the cell proliferation rate based on the OD value. Cell proliferation rate = (OD value of experimental group - OD value of blank control group) / OD value of blank control group × 100%.

[0091] 2. The effect of plant fermentation broth on cell repair ability (1) Select cells in good growth condition, digest them with trypsin, prepare a cell suspension, and count the cells. Use 1×10⁻⁶ cells per cell. 5 Seeds were placed in 24-well plates at a density of 10 cells / well (marked with a pencil on the back of the 24-well plates for easy photography), shaken to mix, and placed in an incubator.

[0092] (2) The next day, when the cell growth density reaches 80%~90%, remove the culture medium, scratch each well with a 200 μL sterile pipette tip, so that the scratch area and area in each well are as consistent as possible, and wash the cells three times with PBS buffer to remove floating cells.

[0093] (3) Randomly select the scratch area of ​​each hole to take a picture, that is, the scratch width of 0 h, and record the photo position.

[0094] (4) Add cell culture medium containing 1% serum (control group) or plant fermentation broth treatment solution (experimental group) prepared with cell culture medium containing 1% serum to each well, and set up 3 replicates for each group.

[0095] (5) After 20 h of treatment, take a picture at the original position and calculate the migration rate = (0 h scratch width - 20 h scratch width) / 0 h scratch width × 100%.

[0096] 3. Experiment on zebrafish's resistance to tail fin repair (1) Select normal 2 dpf zebrafish, randomly select at least 10 zebrafish using zebrafish sampling forceps, put them into a container containing 3% methylcellulose solution, ensure that the zebrafish are completely submerged, let them stand for a period of time to allow the methylcellulose solution to fully contact the zebrafish, and complete the fixation of the zebrafish.

[0097] (2) Place the fixed zebrafish under a microscope, cut off the caudal fin of the zebrafish with a scalpel (avoiding the main vein and ensuring that the damage is consistent), observe and take pictures. All zebrafish should be photographed within 2 hours under the same instrument parameters and environmental conditions, and the corresponding photograph information of each group should be recorded.

[0098] (3) After gently rinsing the wound with sterile saline, place it in the culture medium to observe the phenotype and behavior. After confirming that there are no abnormalities, allocate it to a six-well plate, add 3 mL of the corresponding solution to each well (add standard dilution water to the blank group, add 1 mg / mL plant fermentation broth to the sample group), 10 tails per well, and place them in a (28.5±1.0)℃ biochemical incubator to be cultured in the dark for 72 h.

[0099] (4) After incubation, use zebrafish sampling forceps to randomly select at least 10 zebrafish and place them in a container containing 3% methylcellulose solution. Ensure that the zebrafish are completely submerged and let them stand for a period of time to allow the methylcellulose solution to fully contact the zebrafish and complete the fixation of the zebrafish.

[0100] (5) Place the fixed zebrafish under a microscope, observe and take pictures. All zebrafish should be photographed within 2 hours under the same instrument parameters and environmental conditions. Record the corresponding photograph information for each group. (6) Compare the before and after images of each group to evaluate the repair effect (the regenerated caudal fin area of ​​the sample group was significantly larger than that of the blank group and P <0.05, meaning it has a repair-promoting effect.

[0101] Figure 2 The results of the plant fermentation broth's repair efficacy evaluation are presented. The CCK-8 experimental results showed that, compared to the control group (CTR), the plant fermentation broth promoted cell viability in a concentration-dependent manner. Specifically, 1 mg / mL of plant fermentation broth significantly promoted cell viability; therefore, this concentration was chosen for subsequent experiments. Figure 2 As shown in (a) above. The scratch assay results showed that 1 mg / mL plant fermentation broth promoted cell repair with a healing rate of approximately 60%, as... Figure 2 As shown in (b) and (c) above. The results of the zebrafish tail fin repair experiment showed that 1 mg / mL plant fermentation broth promoted tail fin repair at a rate of 45% over 72 h, as... Figure 2 As shown in (d) and (e) above, the above experiments demonstrate that plant fermentation broth has excellent restorative effects.

[0102] Example 6: Evaluation of the soothing effects of plant fermentation liquid Hyaluronidase regulates the extracellular matrix by degrading hyaluronic acid (HA), and exhibits multiple functions in skin repair, such as accelerating healing, soothing, and promoting collagen production.

[0103] TNF-α, IL-6, and IL-1 are all important pro-inflammatory cytokines closely related to skin sensitivity. When the skin comes into contact with irritants or allergens, immune cells are activated and release various inflammatory mediators, including TNF-α. Inflammatory factors increase vascular permeability, attracting more immune cells to the site of inflammation, further exacerbating the inflammatory response and leading to increased skin sensitivity. LPS-induced RAW264.7 is a classic cell model for studying inflammatory factors. By comparing the differences in TNF-α, IL-6, and IL-1 secretion levels in RAW264.7 cells after administration of the test substance and those in the negative control, we can evaluate the soothing effect of the raw material on cells.

[0104] 1. Effects of plant fermentation broth on hyaluronidase (1) Add hyaluronidase, sodium hyaluronate and buffer solution to the reaction system to ensure that the pH and temperature of the reaction system are suitable. At the same time, set up a blank control group (without hyaluronidase) and a negative control group (without sample).

[0105] (2) Add the solution of the cosmetic raw material to be tested to the reaction system to ensure that the sample is in full contact with hyaluronidase. Incubate the reaction system under suitable conditions (e.g., 37°C) for a period of time (e.g., 30 min) to allow hyaluronidase to fully react with hyaluronic acid.

[0106] (3) Add acetylacetone and p-dimethylaminobenzaldehyde to carry out a color reaction, which is usually carried out under alkaline conditions.

[0107] (4) Use an ultraviolet spectrophotometer to measure the absorbance of the reaction system at 530 nm.

[0108] (5) Calculate the hyaluronidase inhibition rate: Hyaluronidase inhibition rate (%) = {1-(B1-B2) / (A1-A2)}*100, where: A1 is the absorbance of the reaction solution without the sample; A2 is the absorbance of the reaction solution without the sample and enzyme; B1 is the absorbance of the reaction solution containing the sample and enzyme; B2 is the absorbance of the reaction solution containing the sample and without the enzyme.

[0109] (6) Data analysis: The anti-allergic activity of the sample was determined by comparing the hyaluronidase inhibition rate of the sample group and the negative control group. If the hyaluronidase inhibition rate of the sample was significantly higher than that of the negative control group (P<0.05), the sample was considered to have anti-allergic activity.

[0110] 2. Effects of plant fermentation broth on inflammatory factors (1) Select RAW264.7 cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension.

[0111] (2) After thoroughly mixing the cell suspension, take 10 μL and count the cells using a hemocytometer. Dilute the cell suspension to a concentration of 4 × 10⁻⁶. 4 per mL.

[0112] (3) Mix the cell suspension thoroughly and then seed it into a six-well plate. Set up three replicates for each concentration gradient. Note that after adding a few wells, you need to resuspend the cells by pipetting.

[0113] (4) The next day, discard the old culture medium, add the pre-prepared working solution of different concentrations to the sample group, replace the LPS group and CTR group with fresh culture medium, and put them into the cell culture incubator to continue culturing for 24 h.

[0114] (5) After 24 h of treatment, discard the original culture medium and add 10-20 μL (20 μg / mL) of LPS to the sample group and LPS group, and incubate at 37℃ for 2 h in an incubator.

[0115] (6) After incubation, the ELISA kits for TNF-α, IL-6 and IL-1 were used to perform the experimental determination.

[0116] (7) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 min, and seal the remaining strips in a self-sealing bag and put them back at 4℃.

[0117] (8) Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well.

[0118] (9) Add 10 μL of the sample to be tested to the sample well first, and then add 40 μL of sample diluent (i.e., dilute the sample 5 times); do not add to the blank well.

[0119] (10) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate in a water bath or incubator at 37°C for 60 min.

[0120] (11) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0121] (12) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0122] (13) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0123] Figure 3 The results of the evaluation of the soothing effects of the plant fermentation liquid are presented. Figure 3 The results in (a) show that the plant fermentation broth inhibits hyaluronidase activity in a concentration-dependent manner, with 5 mg / mL of plant fermentation broth reaching the half-maximal inhibitory concentration, while 10 mg / mL of plant fermentation broth inhibits hyaluronidase activity by about 80%. Figure 3 The results in (b), (c), and (d) showed that, compared with the blank control group, the addition of LPS significantly promoted the release of TNF-α, IL-6, and IL-1 in RAW264.7 cells, while the addition of 1 mg / mL plant fermentation broth significantly reversed the release of TNF-α, IL-6, and IL-1 stimulated by LPS. The inhibitory effect of 2 mg / mL plant fermentation broth on TNF-α, IL-6, and IL-1 was not significantly different from that of 1 mg / mL plant fermentation broth. The above experiments indicate that 1 mg / mL plant fermentation broth has a good soothing effect.

[0124] Example 7: Evaluation of the antioxidant efficacy of plant fermentation broth at the biochemical level Extrinsic skin aging is primarily caused by photoaging, which leads to the excessive accumulation of free radicals within cells, inducing oxidative stress and ultimately resulting in skin cell aging. ROS (reactive oxygen species) is a collective term for the "reactive oxygen species family," which includes both true free radicals (such as O2) and... 2- ·, ·OH and RO2·), and also some non-free radical but highly reactive oxygen-containing molecules (such as H2O2, O 2- ONOO - Continuous skin exposure to ultraviolet radiation (especially UVA / UVB) and pollution induces significant ROS / free radical generation. The effects of this raw material on DPPH and O2 were first analyzed at the biochemical level. 2- The ability of the raw material to scavenge ·OH free radicals was then assessed, and its ability to scavenge ROS was further tested at the cellular level.

[0125] 1. DPPH scavenging rate of plant fermentation broth: (1) Prepare 0.1 mM DPPH solution and plant fermentation broth solutions of 1, 5, 10, 20, 50 and 100 mg / mL respectively; (2) Sample group, control group and blank group were set up respectively; the sample group was 180 µL + 90 µL DPPH of different concentrations of plant fermentation broth, the control group was 180 µL DMSO + 90 µL DPPH, and the blank group was 180 µL + 90 µL anhydrous ethanol of different concentrations of plant fermentation broth. (3) Shake in the dark for 10 minutes, and zero the container with anhydrous ethanol; (4) Test absorbance at 520 nm.

[0126] (5) Record and analyze the data. DPPH clearance rate % = A control group - (A sample group - A blank group) / A control group * 100%.

[0127] 2. Scavenging rate of ·OH by plant fermentation broth: (1) Prepare plant fermentation broth solutions of 9 mmol / L ethanol-salicylic acid, 9 mmol / L ferrous sulfate, 8.8 mmol / L H2O2 and different concentrations of 1, 5, 10, 20, 50 and 100 mg / ml respectively.

[0128] (2) The reaction system was carried out in a 1.5 mL colorimetric tube, and the order of sample addition is shown in Table 1 below; (3) Shake well, incubate in a 37℃ water bath for 15 min, and measure its absorbance; the absorbance of the sample is measured as A. X The blank control is A0, and the sample background is A. X0 ; (4) Record and analyze the data. ·OH removal rate % = [A0 - (A X -A X0 )] / A0*100%.

[0129] Table 1. Sample addition sequence for determining hydroxyl radical scavenging ability using the ethanol-salicylic acid method.

[0130] 3. The effect of plant fermentation broth on O 2- Sweep rate: (1) Prepare plant fermentation broth solutions of 5 mmol / L pyrogallol, 0.1 mol / L Tris-HCl and 1, 5, 10, 20, 50 and 100 mg / ml respectively.

[0131] (2) This reaction can be carried out in a cuvette, and the order of sample addition is shown in Table 2.

[0132] (3) After rapid mixing and shaking, the absorbance of the solution is measured at 325 nm as the first absorbance value. The absorbance is measured every 1 min for 4 min. The increase of absorbance per minute within the linear range is calculated. △A0 is the auto-oxidation rate of pyrogallol; △A is the auto-oxidation rate of pyrogallol after the sample solution is added.

[0133] (4) Record and analyze the data, O 2- Clearance rate (%) = (△A0 - △A) / △A0 × 100%.

[0134] Table 2. Sampling order for the determination of superoxide anion scavenging using the pyrogallol method

[0135] 4. The effect of plant fermentation broth on the scavenging capacity of ROS: (1) Seed cells at a suitable density in a six-well plate and process them after they adhere to the plate.

[0136] (2) After the experimental group was treated with 200 µM H2O2 for 2-3 h, it was washed with PBS 3 times. The control group was replaced with fresh culture medium. The experimental group was treated with 1 mg / mL of bromeliad fermentation solution for 24 h, and then ROS was detected.

[0137] (3) Dilute the DCFH-DA probe with serum-free medium at a ratio of 1:1000 to a final concentration of 10 μM; (4) Discard the original culture medium, add an appropriate volume of the diluted DCFH-DA probe, and incubate at 37°C for 20 min.

[0138] (5) Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA probes that have not entered the cells.

[0139] (6) Observe under a fluorescence microscope.

[0140] Figure 4 This study demonstrates the antioxidant effects of plant fermentation broth at both the biochemical and cellular levels. Figure 4 The results in (a), (b), and (c) show that the plant fermentation broth inhibited DPPH and O2 in a dose-dependent manner at the biochemical level. 2- The generation of ·OH free radicals, in which the plant fermentation broth reaches its effect on DPPH and O at 10 mg / mL. 2- The half-inhibitory concentration of ·OH free radicals; the effect of plant fermentation broth at 50 mg / mL on DPPH and O2. 2- The scavenging rates of ·OH radicals reached 85%, 80%, and 78%, respectively.

[0141] The antioxidant effects of plant fermentation broth were detected at the cellular level, and the results were as follows: Figure 4 As shown in (d), compared with the control group, H2O2 treatment of L929 cells significantly promoted ROS accumulation, while the addition of 1 mg / mL plant fermentation broth significantly reversed the ROS accumulation caused by H2O2 stimulation. These results indicate that the plant fermentation broth has a good antioxidant effect.

[0142] 5. Safety verification of plant fermentation broth: To further verify the safety of the plant fermentation broth, the safety of plant fermentation broth at low (5 mg / mL), medium (10 mg / mL), and high (50 mg / mL) concentrations was verified by taking advantage of the intact, clear, and transparent vascular system of the chorioallantoic membrane of hatched chicken embryos.

[0143] (1) CAM preparation: Select 7-day-old chicken embryos, check by candling, place the air cell end of the chicken embryo upwards, and draw the outline of the air cell of the chicken embryo with a pencil. (2) Drill a hole directly above the air cell of the chicken embryo with pointed tweezers, carefully peel off the eggshell above the air cell along the outline of the air cell with tweezers (be careful not to get too close to the outline of the air cell), blow off the eggshell that has fallen on the air cell membrane with a bulb blower, add 1 mL of physiological saline to moisten the eggshell membrane and discard the residual physiological saline, disinfect the pointed tweezers again with an alcohol lamp and carefully tear off the eggshell membrane (do not puncture the allantoic membrane and blood vessels during this process), exposing the allantoic membrane with a diameter of 2-3 cm; (3) At this point, observe the structure of the vascular system again and make a judgment on its integrity and suitability for the experiment; (4) Endpoint assessment method: Since the sample was a transparent liquid, the time assessment method was selected for detection. That is, 0.3 mL of the above transparent liquid was directly dropped onto the CAM surface, the CAM reaction was observed, and the time of occurrence of each toxic effect within 5 min was recorded; (5) The irritation score (IS) is shown in Table 5: (6) The reaction time method is used to conduct the test. The stimulus score (IS) is calculated using the following formula and the result is kept to two decimal places. According to the IS value, the eye irritation of the test substance is classified according to the following table.

[0144] IS=(301-sec H) 5 / 300+(301-sec L) x7 / 300+(301-sec C) x9 / 300; Note: sec H (bleeding time) - the average time observed on the CAM membrane for the onset of bleeding, in seconds (s); sec L (vascular dissolution time) - the average time observed on the CAM membrane for the onset of vascular dissolution, in seconds (s); sec C (clotting time) - the average time observed on the CAM membrane for the onset of clotting, in seconds (s).

[0145] Table 3. Evaluation of Results from the Reaction Time Method

[0146] Figure 5 The results showed the IS scores for the negative control group (CTR), positive control group (0.1 mol NaOH), low concentration group (5 mg / mL), medium concentration group (10 mg / mL), and high concentration group (50 mg / mL). Since the IS score of the positive control group was between 10 and 19, this experiment is considered reliable. Furthermore, the IS values ​​of all sample groups at different concentrations were less than 1; therefore, the plant fermentation broth at low, medium, and high concentrations is safe and non-irritating.

[0147] 6. Verification of the anti-wrinkle efficacy of plant fermentation liquid Collagen is the main structural protein of the skin. With age, collagen synthesis slows down while degradation accelerates, leading to decreased skin elasticity and firmness, resulting in wrinkles and sagging. To assess its anti-wrinkle effect by detecting whether plant fermentation broth can promote collagen regeneration, changes in type I and type III collagen content were analyzed using ELISA.

[0148] 1. Plant fermentation broth promotes the production of type I and III collagen: (1) Select HSF cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin and centrifuge them, discard the supernatant, and gently pipette the cell pellet with 2 mL of fresh culture medium to obtain a cell suspension. (2) After thoroughly mixing the cell suspension, take 10 µL and count the cells using a hemocytometer. Dilute the cell suspension to a concentration of 4 × 10⁻⁶. 4 cells / mL; (3) Mix the cell suspension thoroughly and then seed it into a six-well plate. Set up three replicates for each concentration gradient. Note that after adding a few wells, you need to resuspend the cells by pipetting. (4) The next day, the old culture medium was discarded, the blank control group was replaced with fresh culture medium, and the sample group was added with 1 mg / mL of plant fermentation broth and placed in a cell culture incubator to continue culturing for 24 h. (5) After 24 h of treatment, discard the original culture medium, wash the cells twice with PBS, add cell lysis buffer and lyse for 30 min, sonicate for 5-10 min, centrifuge to obtain supernatant for subsequent experiments. (6) After incubation, the corresponding type I and type III collagen ELISA kits were used for experimental determination; (7) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 min, and seal the remaining strips in a self-sealing bag and return them to 4℃; (8) Set up standard wells and sample wells, and add 50 µL of standard at different concentrations to each standard well; (9) Add 50 µL of the sample to be tested to the sample well first; do not add any to the blank well; (10) Except for the blank wells, add 100 µL of horseradish peroxidase (HRP) labeled antibody to each well of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate in a water bath or incubator at 37 ℃ for 60 min. (11) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 5 times. (12) Add 50 µL of substrate A and B to each well and incubate at 37°C in the dark for 15 min; (13) Add 50 µL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min. (14) Standard curve plotting and sample concentration calculation: Establish a standard curve based on the concentration (x, ng / mL) of the standard tube and the absorbance ΔA standard (y, ΔA standard). Based on the standard curve, substitute the ΔA measurement (y, ΔA measurement) into the formula to calculate the sample concentration (x, ng / mL).

[0149] Figure 6 The anti-wrinkle effect of the plant fermentation liquid was demonstrated. For example... Figure 6 As shown in (a) and (b), compared with the control group (CTR), the addition of plant fermentation broth significantly promoted the synthesis of type I and type III collagen. Specifically, the plant fermentation broth promoted the synthesis of type I and type III collagen at growth rates of 45% and 38%, respectively. These results indicate that plant fermentation broth has a good anti-wrinkle effect by promoting collagen regeneration.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a fermented product of *Broomia linearifolia*, characterized in that, Includes the following steps: The enzymes of the linear-leaved broom flower were inactivated. Glucose and dipotassium hydrogen phosphate were mixed with water and sterilized. Then, enzyme-inactivated broom flowers were added to prepare a fermentation medium. Lactobacillus plantarum and Lactobacillus rhamnosus were activated to OD values ​​respectively. 600 The concentration is 0.8-1.

0. Then, the activated solutions of Lactobacillus plantarum and Lactobacillus rhamnosus are mixed at a volume ratio of 1:0.9-1.1 and inoculated into the fermentation medium. Fermentation is then carried out in an anaerobic environment to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The linear-leaved broom flower is in powder form, with a particle size of 40-60 mesh.

3. The preparation method according to claim 1, characterized in that, The ratio of linear broom to water is 4.6-5.4: 200, g / mL.

4. The preparation method according to claim 1, characterized in that, The conditions for enzyme inactivation treatment were: temperature 167-173℃ and time 27-33 min.

5. The preparation method according to claim 1, characterized in that, The sterilization conditions are: temperature 120-125℃, time 18-22 min.

6. The preparation method according to claim 1, characterized in that, The ratio of Lactobacillus plantarum to Lactobacillus rhamnosus was 1:0.9-1.

1.

7. The preparation method according to claim 1, characterized in that, During fermentation, the fermentation temperature is 28-30℃, and the fermentation time is 18-24 hours. Alternatively, at the end of fermentation, the pH of the fermentation broth is 4.2-4.

5.

8. The preparation method according to claim 1, characterized in that, After fermentation, pasteurization is carried out; Alternatively, obtaining the fermentation broth after fermentation may include the process of removing the precipitate; Alternatively, obtaining the fermentation broth after fermentation may include a decolorization process.

9. A fermented product of *Broomia linearifolia*, characterized in that, Obtained by the preparation method according to any one of claims 1-8.

10. The use of the *Broomia linearis* ferment as described in claim 9 in the preparation of anti-wrinkle, firming, and soothing repair products.

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