An anti-wrinkle and anti-aging macrofungus extract, its preparation method and application

Abstract

This invention proposes an anti-wrinkle and anti-aging macrofungus extract, its preparation method, and its applications, belonging to the cosmetics field. The macrofungus extract is obtained by using macrofungus fruiting bodies as fermentation raw materials, employing aerobic-facultative anaerobic staged fermentation with Bacillus subtilis, followed by extraction and purification. This invention provides a method for preparing the macrofungus extract. Using macrofungus fruiting bodies as the main fermentation substrate, precise-controlled anaerobic solid-state fermentation with Bacillus subtilis is carried out using specific inducers. This process can specifically stimulate and enrich secondary metabolites. The fermentation products are finally extracted using a water / ethanol mixed extraction technique to efficiently obtain an extract rich in highly active ingredients. This macrofungus extract exhibits significant efficacy in anti-wrinkle and anti-aging skin, providing a high-quality natural raw material solution for the development of related cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics, and in particular relates to an anti-wrinkle and anti-aging macrofungus extract, its preparation method, and its application. Background Technology

[0002] The application of fungi and their fermentation products in the skincare field stems from in-depth exploration of their unique bioactivity, particularly their immense potential in skin anti-aging. With the continuous advancement of research into the mechanisms of skin aging, fungal-derived active ingredients have attracted significant attention due to their multi-target and high biocompatibility characteristics. Skin aging is a complex biological process, primarily characterized by collagen loss, elastin degradation, decreased antioxidant defense system function, and weakened cell regeneration capacity. The active ingredients in fungi and their fermentation products can precisely target these key aspects.

[0003] In anti-aging skincare applications, various fungi have been proven to have significant efficacy. The polysaccharides in the fermentation products of *Schizophyllum commune* effectively enhance the skin barrier function, increase the hydration of the stratum corneum, and delay skin cell aging by regulating autophagy. The pigments produced by the fermentation of *Monascus purpureus*, such as ankaflavin and monascin, not only neutralize free radicals but also inhibit the activity of matrix metalloproteinases MMP-1 and MMP-3, two key enzymes that degrade collagen and elastin. The acidic polysaccharides in *Tremella fuciformis* extract possess moisturizing capabilities surpassing hyaluronic acid; their unique three-dimensional network structure forms a highly effective moisturizing film while promoting collagen synthesis by activating fibroblasts. Studies have shown that *Tremella fuciformis* polysaccharides can significantly enhance the gene expression of type I and type III collagen, both crucial for maintaining skin elasticity and firmness.

[0004] At the molecular level, the anti-aging effects of fungal active ingredients are mainly achieved through multiple signaling pathways. In addition to the aforementioned TGF-β / Smad pathway, some fungal extracts can also activate the Nrf2 / ARE antioxidant pathway, enhancing the activity of intracellular antioxidant enzymes and reducing oxidative stress damage to skin cells. Furthermore, some active peptides in fungal fermentation products can mimic the function of Sirtuin family proteins, regulating cell metabolism and DNA repair through deacetylation, thereby prolonging skin cell lifespan. Notably, certain fungal extracts can also inhibit the production of the aging-associated secretory phenotype (SASP), reduce the release of inflammatory factors, and block the negative impact of senescent cells on surrounding tissues.

[0005] In terms of formulation technology, the application forms of fungal anti-aging ingredients are constantly being innovated. Microencapsulation technology can improve the stability and transdermal absorption rate of active ingredients; for example, encapsulating Monascus ferment products in liposomes can both protect their activity and promote skin penetration. Biotransformation technology, through specific enzyme treatment, can optimize the molecular weight of fungal polysaccharides, obtaining fragments that are more easily absorbed by the skin while retaining their biological activity. Triple formulation technology scientifically combines active ingredients from different fungal sources to produce synergistic effects in antioxidation, collagen synthesis promotion, and cell repair.

[0006] Despite the promising prospects of fungal ingredients in the anti-aging field, several technical challenges remain. The stability, standardization, and transdermal absorption efficiency of active ingredients are key issues requiring continuous optimization. Furthermore, different strains, culture conditions, and extraction processes can all affect the content and efficacy of active ingredients in the final product, necessitating a more comprehensive quality control system. Future research needs to further elucidate the interaction mechanisms between fungal active ingredients and skin aging-related signaling pathways, providing a theoretical foundation for developing more efficient and targeted anti-aging skincare products. With advancements in synthetic biology and metabolic engineering, targeted modification of fungal metabolic pathways holds promise for obtaining more potent and purer anti-aging ingredients, bringing new breakthroughs to the skincare industry. Summary of the Invention

[0007] The first objective of this invention is to provide a macrofungus extract and its preparation method, wherein the preparation method involves using macrofungus entities as fermentation raw materials, fermenting them in aerobic-facultative anaerobic stages using Bacillus subtilis, and then extracting and purifying them to obtain the extract. The fermentation substrate used for Bacillus subtilis fermentation comprises the following components, by weight percentage: Large fungal fruiting bodies, ≥90%; Pine bark extract, 1-3%; The Bacillus subtilis is a pre-induced Bacillus subtilis cell suspension; the pre-induced Bacillus subtilis cell suspension contains at least 1 wt% inactivated competitive microbial cell walls; The large fungal fruiting body is at least one of matsutake, black truffle, and white truffle. Preferably, the pine bark extract is a hydro-alcoholic mixed extract of pine bark, wherein the condensed tannin content is ≥4wt%.

[0008] Preferably, the inactivated competitive microbial cell wall is derived from the cell wall components of Bacillus amyloliquefaciens after inactivation, followed by crushing and purification.

[0009] Preferably, the fermentation substrate further contains the following components: a carbon source, a nitrogen source, and inorganic salts; the initial water content of the fermentation substrate is 55-65%. The fermentation substrate also includes the following components by mass percentage: Carbon source, ≥2%; Nitrogen source, ≥3%; Complex inorganic salts, ≥1%; The composite inorganic salt includes at least phosphate, magnesium salt and manganese salt.

[0010] Preferably, the fermentation conditions for the aerobic-facultative anaerobic staged fermentation using Bacillus subtilis are as follows: Active oxygen supply was provided during the first 0-24 hours of fermentation; the fermentation temperature was 37±1℃. After 24 hours of fermentation, stop the active oxygen supply and adjust the fermentation temperature to 31±1℃. Fermentation time is at least 48 hours; initial pH ≤ 6.5; anaerobic solid-state fermentation; fermentation endpoint is reached when pH drops to ≤ 4.5 and changes ≤ 0.1 within 4 hours.

[0011] Preferably, the large fungal fruiting bodies are pretreated before fermentation. The pretreatment includes the following steps: taking dried large fungal fruiting bodies, cleaning the surface and chopping them; freeze-drying the chopped fruiting bodies to constant weight; then ultra-fine pulverizing them to ≥300 mesh to obtain large fungal ultra-fine powder; and sterilizing the large fungal ultra-fine powder.

[0012] The enzyme used in the enzymatic hydrolysis is at least one of papain, cellulase, and pectinase.

[0013] Preferably, the extraction and purification process involves: sterilization followed by freeze-drying and pulverization after reaching the fermentation endpoint; then, ethanol / water extraction of the macrofungi ferment; the extract is a macrofungi extract, which is usually freeze-dried again to a solid state for ease of transportation and storage.

[0014] A second objective of this invention is to provide the application of the aforementioned macrofungus extract in products with anti-aging effects; more specifically, in cosmetics; such as serums, lotions, creams, and essential oils.

[0015] The method for preparing macrofungi extract provided by this invention achieves efficient biotransformation of matsutake mushrooms or other macrofungi fruiting bodies through specific fermentation substrate composition, inducer addition, and optimized fermentation process. The resulting macrofungi extract exhibits significant beneficial effects on skin anti-wrinkle and anti-aging. Specifically, the beneficial effects of this invention include: (1) Highly efficient stimulation of secondary metabolite synthesis: By adding inactivated competitive microbial cell walls and pine bark extracts as inducers, natural stress was simulated, effectively stimulating the production of defensive secondary metabolites by Bacillus subtilis and matsutake matrix. This process significantly promoted the biosynthesis and accumulation of terpenoids (such as sterols and triterpenes unique to matsutake), unsaturated fatty acids, polyphenols and fat-soluble antioxidants (such as tocopherols), as well as Bacillus subtilis-derived metabolites.

[0016] (2) Enrichment of highly bioactive components: The fermentation process not only enriches fat-soluble active substances but also retains water-soluble active components. The final extract obtained by water / ethanol mixed extraction is rich in matsutake mushroom-derived terpenes, sterols, fermentation-derived unsaturated fatty acids, polyphenols, flavonoids, and polysaccharides. These components exert their effects through both water-soluble and fat-soluble pathways, effectively neutralizing free radicals and inhibiting the activity of elastase and collagenase, thereby playing a synergistic role in anti-wrinkle, improving skin elasticity, and delaying photoaging.

[0017] (3) Fermentation process optimization ensures efficient conversion of active precursor substances: Anaerobic solid-state fermentation is adopted, combined with strict control of temperature, pH and fermentation endpoint, to create an optimal environment for the metabolic activities of Bacillus subtilis. This process ensures that the active precursor substances in the cell walls of macrofungi (such as matsutake, black truffle and white truffle) are fully released and converted, which significantly improves the yield and diversity of the final target active substances.

[0018] (4) Pretreatment and broad-spectrum extraction technology ensure component integrity: Ultrafine grinding and enzymatic hydrolysis pretreatment of large fungal fruiting bodies effectively disrupts the cell wall structure, releasing intracellular lipid-soluble and water-soluble components, laying the foundation for subsequent fermentation and extraction. A water / ethanol mixed solvent is specifically used for targeted extraction. By adjusting the ethanol ratio, efficient and compatible extraction of lipid-soluble active components (such as terpenes and sterols) and water-soluble active components (such as polysaccharides and polyphenols) is achieved. This process is also mild, perfectly preserving the chemical structure and biological activity of heat-sensitive components, resulting in a comprehensive range of active substances and synergistic effects in the final product.

[0019] In summary, this invention provides a method for preparing a macrofungus extract. Using matsutake mushrooms or other macrofungi fruiting bodies as the main fermentation substrate, precise anaerobic solid-state fermentation is conducted using Bacillus subtilis with specific inducers. This process specifically stimulates and enriches secondary metabolites. The fermentation product is then subjected to broad-spectrum extraction using a water / ethanol mixed extraction technique, thereby efficiently obtaining an extract rich in various highly active ingredients. This macrofungus ferment exhibits significant efficacy in anti-wrinkle and anti-aging skincare, providing a high-quality natural raw material solution for the development of related cosmetics. Detailed Implementation

[0020] To better understand the present invention, the present invention will be further described below with reference to specific serial numbers. The terminology used in the serial numbers is for describing specific embodiments and does not constitute a limitation on the scope of protection of the present invention.

[0021] In the specific implementation methods, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0022] Unless otherwise specified, percentages, % and so on in the specific implementation method are assumed to be mass percentages.

[0023] The raw materials used in this invention are as follows: Matsutake: Commercially available dried matsutake fruiting bodies, identified as *Tricholoma matsutake* (S. Ito & S. Imai) Singer.

[0024] Black truffle: Fresh Yunnan truffles sold in the market, identified as Tuber melanosporum.

[0025] White truffles: Fresh white truffles imported from Italy and sold in the market are identified as Tuber Magnatum.

[0026] Bacillus amyloliquefaciens: Bacillus amyloliquefaciens CICC11035 Bacillus subtilis: Bacillus subtilis CGMCC3825 Lactobacillus bulgaricus: Lactobacillus delbrueckii subsp. bulgaricus CICC6045 Pine bark extract: Commercially available. The content of proanthocyanidins, the main component of condensed tannins, was determined by vanillin-hydrochloric acid spectrophotometry, and the content of proanthocyanidins was found to be 60%.

[0027] Example 1: Preparation of inactivated competitive microbial cell walls (1) Method for preparing Bacillus amyloliquefaciens cell walls Cell culture and collection: Bacillus amyloliquefaciens was inoculated into LB liquid medium and cultured at 37°C and 220 rpm for 24 hours with shaking. The fermentation broth was centrifuged at 4°C and 8000 g for 10 minutes, the supernatant was discarded, and the cell pellet was collected. The cells were washed twice with pre-cooled sterile deionized water to remove any residual culture medium.

[0028] Cell disruption: The washed bacterial pellet was resuspended in an appropriate amount of pre-cooled phosphate-buffered saline (PBS, pH 7.4). The cells were homogenized using a high-pressure homogenizer under ice bath conditions until most cells were observed to be disrupted under a microscope.

[0029] Impurity removal: The lysate was centrifuged at 4°C and 13000 g for 15 minutes, and the precipitate (mainly cell wall fragments) was collected. This precipitate was boiled in a solution containing 2% sodium dodecyl sulfate (SDS) for 30 minutes to thoroughly remove proteins and lipids. Subsequently, it was repeatedly washed by centrifugation with hot sterile deionized water until no white precipitate was detected in the supernatant using barium chloride solution, ensuring that SDS was completely removed.

[0030] Enzymatic purification: The SDS-treated precipitate was sequentially treated with α-amylase (to remove glycogen) and trypsin (to remove residual protein) in suitable buffers and at appropriate temperatures. After enzymatic hydrolysis, the precipitate was collected by centrifugation again and thoroughly washed with water.

[0031] Inactivation and preservation: The purified cell walls were suspended in an appropriate amount of physiological saline and inactivated by autoclaving at 121 degrees Celsius for 30 minutes. The inactivated cell wall suspension was then freeze-dried to prepare a dry powder and stored at -20 degrees Celsius for later use.

[0032] (2) Method for preparing Lactobacillus bulgaricus cell walls Bacterial culture and collection: Purchased Lactobacillus bulgaricus strain was inoculated into MRS liquid medium and cultured with gentle shaking at 37°C for 16 hours. The culture was centrifuged at 4°C and 8000 g for 10 minutes to collect the bacterial cells, which were then washed twice with pre-cooled 0.1 mol / L phosphate buffer (pH 6.2).

[0033] Protoplast preparation: The washed bacterial cells were resuspended in an osmotic stabilizer solution (0.5 mol / L sucrose solution) containing a high concentration of lysozyme (10 mg / mL). The cells were gently shaken in a 36°C water bath for 30 minutes for enzymatic digestion. Samples were taken periodically for microscopic examination. When the vast majority of cells transformed into spherical protoplasts, it indicated that the cell wall had been effectively removed.

[0034] Cell wall separation: The protoplast suspension was centrifuged at 4°C and 8000 g for 15 minutes. The precipitate contained cell wall fragments dissociated by lysozyme. The precipitate was carefully washed several times with an osmotic stabilizer to remove cytoplasmic residues.

[0035] Inactivation and preservation: The obtained cell wall fragments were resuspended in physiological saline and inactivated by autoclaving at 121°C for 30 minutes. The cells were then freeze-dried into powder and stored at -20°C.

[0036] (3) Method for preparing Bacillus subtilis cell walls Cell preparation: Bacillus subtilis was cultured in LB liquid medium at 37°C with shaking until the late logarithmic phase. The cells were collected by centrifugation and washed with pre-cooled PBS buffer.

[0037] Lysozyme treatment: The bacterial cells were resuspended in a hypertonic Tris-HCl buffer containing 0.5 M sucrose. Lysozyme was added to a final concentration of 5 mg / mL, and the mixture was incubated in a water bath at 37°C for 60 minutes. During this period, microscopic observation was performed. When most rod-shaped bacterial cells transformed into spherical protoplasts, it indicated that the cell wall had been successfully removed.

[0038] Ultrasonic disruption: Collect the enzymatically hydrolyzed protoplasts by centrifugation and resuspend them in pre-cooled PBS. Disrupt the cells using an ultrasonic cell disruptor under ice bath conditions (300W power, 3 seconds on, 5 seconds off, total duration 20 minutes) to fully release cell wall fragments.

[0039] Purification and inactivation: Centrifuge the lysate and collect the precipitate. Boil in a solution containing 2% SDS for 30 minutes to remove contaminating proteins, then repeatedly centrifuge and wash with hot water until no SDS residue remains. Finally, suspend the purified cell walls in physiological saline and autoclave at 121°C for 30 minutes to complete inactivation, obtaining the final product. Freeze-dry into powder and store at -20°C.

[0040] Example 2 Preparation of Matsutake Fermented Product Includes the following steps: S1 Fermentation Raw Material Pretreatment: Take dry, mold-free matsutake fruiting bodies and clean surface impurities. Chop them and freeze-dry them to constant weight. Use an ultrafine pulverizer to pulverize the dried matsutake to 300 mesh to obtain matsutake ultrafine powder.

[0041] Mix matsutake mushroom ultrafine powder with warm water at 40-45℃ to form a slurry (material-to-water ratio 1:10), add a compound enzyme (papain:cellulase:pectinase = 2:3:1, total enzyme activity ≥50000 U / g), and enzymatically hydrolyze for 3 hours at pH 5.5-6.0 and 50℃. After enzymatic hydrolysis, autoclave at 121℃ for 20 minutes to obtain matsutake mushroom enzymatically hydrolyzed powder for later use.

[0042] Bacillus subtilis seed culture was inoculated into the corresponding culture medium, and an additional 1% of different inactivated cell walls prepared in Example 1 was added; after culturing for a period of time, a pre-induced microbial suspension was obtained, with a cell concentration ≥1× CFU / mL.

[0043] Prepared separately: Bacillus subtilis suspension (a suspension that breaks down the cell walls of Bacillus amyloliquefaciens); also known simply as Bacillus subtilis suspension (a suspension that breaks down the amyloliquefaciens cell walls). Bacillus subtilis suspension (Bacillus subtilis cell wall); abbreviated as Bacillus subtilis suspension (Bacillus subtilis). Bacillus subtilis suspension (cell wall of Lactobacillus bulgaricus); abbreviated as Bacillus subtilis suspension (Lactobacillus).

[0044] Bacillus subtilis suspension (without inactivated cell walls); abbreviated as Bacillus subtilis suspension (without).

[0045] S2 Fermentation Substrate Preparation and Fermentation Control: The fermentation substrate was prepared based on dry weight, as shown in Table 1. Perform aerobic-facultative anaerobic staged fermentation according to the following parameters: Aerobic fermentation stage (0-24 hours): The prepared fermentation substrate is placed into a shallow fermentation tank, with a thickness not exceeding 15cm.

[0046] Inoculate the pre-induced Bacillus subtilis cell suspension according to the inoculation amount in Table 2, and mix thoroughly.

[0047] The fermentation temperature is controlled at 37±1℃, and active oxygen supply is provided through forced ventilation and stirring to maintain air circulation inside the tank.

[0048] Facultative anaerobic fermentation stage (from 24 hours later to the end): After 24 hours of fermentation, stop active oxygen supply and stirring, seal the fermentation tank, and switch to anaerobic solid-state fermentation mode. At the same time, adjust the fermentation temperature to 31±1℃.

[0049] Continuously monitor the pH value of the fermentation system. When the pH value drops to ≤4.5, and the pH value fluctuates by ≤0.1 within 4 hours thereafter, the fermentation can be considered to have reached its endpoint.

[0050] The specific inoculum suspensions used for fermentation are shown in Table 2. Table 1. Parameters of Matsutake Fermentation Substrate

[0051] Table 2 Fermentation strains Inoculation amount of hay suspension (starch desiccant) 5% 5% 5% 5% 0 0 0 0 0 Dried hay suspension (dried hay) inoculation amount 0 0 0 0 5% 0 0 0 0 Inoculation amount of Bacillus subtilis suspension (Lactobacillus) 0 0 0 0 0 5% 0 0 0 Hay suspension (no) inoculation amount 0 0 0 0 0 0 5% 0 0 Bacillus amyloliquefaciens suspension inoculation volume 0 0 0 0 0 0 0 5% 0 Lactobacillus bulgaricus suspension inoculation volume 0 0 0 0 0 0 0 0 5% In Table 1, except for pine bark powder, the components of the fermentation substrate are all calculated by dry weight. Pine bark powder is commercially available pine bark extract powder, which is dissolved in 5 times its volume of water to obtain a solution of pine bark extract with a proanthocyanidin content of 10%.

[0052] S3 fermentation product extraction and purification: Upon arrival at the destination, all fermentation materials are immediately subjected to high-pressure steam sterilization at 121°C for 20 minutes to terminate all biological reactions.

[0053] The sterilized material is freeze-dried until its weight is constant to completely remove moisture; the dried block material is then crushed to 80 mesh to obtain uniform matsutake fermented powder.

[0054] The dried powder of matsutake fermentation was transferred to an extraction tank as the material to be extracted.

[0055] Perform water / ethanol mixed solvent extraction according to the following parameters: Extraction solvent: a mixed solution of ethanol and pure water, wherein the ethanol volume concentration is 50%; Material-to-liquid ratio: 1:8 (w / v), the specific ratio should be optimized and adjusted according to the characteristics of the material; Extraction temperature: 70±5℃; Extraction method: Hot reflux extraction is used to reduce solvent evaporation and improve extraction efficiency; Extraction times and time: Under the above conditions, extract twice, each time for 1.5 to 2 hours.

[0056] After extraction, the mixture was separated into solid and liquid components using a filtration device, and all the filtrate was collected.

[0057] The combined filtrate is transferred to a concentration tank and subjected to vacuum distillation at a temperature below 60°C to recover the ethanol solvent.

[0058] The remaining aqueous concentrate was further concentrated and then freeze-dried to obtain the matsutake mushroom extract.

[0059] The contents of ergosterol and total phenols in the matsutake extracts prepared according to each serial number in this embodiment were tested. The test method for ergosterol is the method described in "GH / T 1475-2024 Determination of Free Ergosterol in Edible Fungi by High Performance Liquid Chromatography".

[0060] The total phenol content was determined using the Folin-Ciocalteu reagent colorimetric method, referring to "GB / T 44349-2024 Determination of Total Polyphenols in Bee Pollen - Folin-Ciocalteu Reagent Colorimetric Method"; details are as follows: Preparation of S1 standard curve: Accurately weigh gallic acid standard and prepare a series of standard solutions of different concentrations.

[0061] Take standard solutions of various concentrations, add Folin-Ciocalteu reagent, mix well, and let stand for a period of time.

[0062] Add sodium carbonate solution, mix well, and let stand in the dark for 60 minutes.

[0063] The absorbance was measured at a wavelength of 760 nm using a spectrophotometer.

[0064] A standard curve was plotted with gallic acid concentration on the x-axis and absorbance on the y-axis.

[0065] S2 sample determination: Extraction: Accurately weigh an appropriate amount of sample and extract it using 60% ethanol solution via ultrasonication or oscillation.

[0066] Color development: Take an appropriate amount of sample extract and add Folin-Ciocalteu reagent and sodium carbonate solution according to the same steps as in the "Preparation of Standard Curve" above for color development.

[0067] Measurement: The absorbance of the sample tube was measured at a wavelength of 760 nm.

[0068] Calculation: Based on the absorbance value of the sample, find the corresponding total phenol concentration from the gallic acid standard curve, and then calculate the total phenol content in the sample.

[0069] The results are shown in Table 3 below.

[0070] Table 3. Content of marker active ingredients at different serial numbers 1 54.87 209.17 2 56.28 213.11 3 65.91 267.78 4 68.12 287.91 5 45.24 181.40 6 46.91 178.47 7 45.81 180.41 8 38.19 145.19 9 39.04 140.21 The component content analysis of each sample in Table 3 is as follows: Serial number 1 served as the baseline control group, using Bacillus subtilis pre-induced by Bacillus amyloliquefaciens cell wall for fermentation, but without the addition of pine bark extract. Its ergosterol and total phenol content were at moderate levels, confirming that the pre-induction treatment itself could activate the metabolic activity of Bacillus subtilis, promoting the decomposition and transformation of the matsutake matrix. However, due to the lack of abundant phenolic precursors provided by pine bark extract, the synthetic potential of the active ingredients was not fully realized.

[0071] Sample 2, based on sample 1, added a small amount of pine bark extract, with little change in the content of the two active ingredients. This indicates that this concentration of pine bark extract did not achieve an effective stimulating effect.

[0072] Further increasing the amount of pine bark extract in step 3 resulted in a significant increase in the content of both components. This demonstrates that an appropriate amount of pine bark extract provides the optimal concentration of phenolic precursors for the fermentation system and produces a strong synergistic effect with pre-induced Bacillus subtilis (amylopectin-degrading cell wall), greatly promoting the biochemical process by which the fungus converts the matsutake substrate into ergosterol and total phenols.

[0073] While maintaining the optimal amount of pine bark extract, combination 4 appropriately increased the substrate moisture content and optimized the pH value. Its component content reached the peak among all combinations, indicating that the more relaxed moisture conditions and more suitable acid-base environment further improved the growth status of the microorganisms and the substrate mass transfer efficiency, allowing the metabolic synthesis reactions to proceed more fully and thoroughly.

[0074] In sequence 5, the pre-induction was performed using the cell wall of Bacillus subtilis itself. Compared with sequence 3, the content of its components decreased significantly. This reveals that the induction effect of the heterologous microorganism (Bacillus amyloliquefaciens) cell wall is better than that of the homologous cell wall, possibly because the heterologous cell wall can induce a stronger or more specific stress and defense response in Bacillus subtilis, thereby activating a more efficient secondary metabolic pathway.

[0075] In sequence 6, an attempt was made to pre-induce the bacteria using the cell wall of *Lactobacillus bulgaricus*, but the effect was even slightly lower than that of sequence 5. This indicates that *Bacillus subtilis*, as a Gram-positive bacterium, has a higher efficiency in signal recognition and response to cell walls from Gram-positive bacteria (*Bacillus subtilis*, *Bacillus amyloliquefaciens*) than to cell walls from Gram-positive lactic acid bacteria that differ in morphology and composition, and that different cell wall components are specific to the induction effect.

[0076] Number 7 served as a negative control for Bacillus subtilis fermentation, without any cell wall pre-induction. Its component content was significantly lower than all pre-induction groups, directly demonstrating that the pre-induction step is a key innovation of this process. It can pre-train and "activate" the production strain, enabling it to exhibit stronger product synthesis capabilities in subsequent fermentation.

[0077] Group 8 was fermented directly using Bacillus amyloliquefaciens. Its component content was significantly lower than that of the group using pre-induced Bacillus subtilis. This indicates that although the Bacillus amyloliquefaciens cell wall is an effective inducer, its overall ability as a fermentation strain in decomposing the matsutake substrate and synthesizing the target product is inferior to that of specially induced Bacillus subtilis.

[0078] Fermentation using *Lactobacillus bulgaricus* in sequence 9 yielded results similar to those in sequence 8, but with the lowest content. This clearly indicates that *Lactobacillus bulgaricus* is not suitable for this specific matsutake solid-state fermentation system, as its ability to decompose the fibrous matsutake substrate and its synthesis pathways for related target products are weak, thus highlighting the irreplaceable role of *Bacillus subtilis* in this process.

[0079] Example 3 Performance testing of matsutake mushroom extract The performance of each matsutake ferment prepared in Example 2 was tested, including: its ability to scavenge free radicals and its ability to promote type I collagen production.

[0080] (1) DPPH scavenging experiment, Step 1: Reagent and Sample Preparation DPPH working solution (0.1 mM): Accurately weigh approximately 3.94 mg of DPPH powder, dissolve it in anhydrous ethanol, and dilute to the mark in a 100 mL brown volumetric flask. Shake well. This solution should be stored in the dark and refrigerated, and it is recommended to prepare it immediately before use.

[0081] 1% (w / v) Sample stock solution: Accurately weigh 50.0 mg of matsutake fermentation material from each serial number in Example 2, dissolve it in acetone and transfer it to a 5 mL volumetric flask, dilute to the mark with acetone, and shake well to obtain a sample stock solution with a concentration of 10 mg / mL (i.e., 1%).

[0082] Negative control solvent: Prepare sufficient pure acetone.

[0083] Blank control: No separate preparation is required; it is prepared directly during the testing process.

[0084] Step 2: Establishing the testing system and adding samples It is recommended to establish the following four reaction systems, with three parallel tubes for each: Sample tube (S): Used to test the true activity of the sample. First add 100 μL of 1% sample stock solution, then add 1500 μL of DPPH working solution.

[0085] Sample background tube (SB): Used to correct the sample's own color. First add 100 μL of 1% sample stock solution, then add 1500 μL of anhydrous ethanol (Note: Do not add DPPH working solution here).

[0086] Blank tube (B): Represents the initial concentration of DPPH. First add 100 μL of pure acetone, then add 1500 μL of DPPH working solution.

[0087] Solvent control tube (SC): Used to correct for potential effects of the solvent system. First add 100 μL of pure acetone, then add 1500 μL of anhydrous ethanol (Note: Do not add DPPH working solution here).

[0088] Step 3: Reaction and Measurement Mixing: Use a vortex mixer to thoroughly mix all test tubes to ensure the solution is homogeneous.

[0089] Light-protected reaction: Wrap all test tubes with aluminum foil and let stand at room temperature in the dark for 60 minutes to allow the reaction to proceed fully.

[0090] Absorbance measurement: Using a spectrophotometer or microplate reader, the absorbance values ​​of all test tubes were measured sequentially at a wavelength of 517 nm and recorded as AS (sample tube), ASB (sample background tube), AB (blank tube), and ASC (solvent control tube).

[0091] Step 4: Calculate the DPPH free radical scavenging rate Use the following fully corrected formula for calculation: Sweep rate (%) = { 1 - [ (AS - ASB) / (AB - ASC) ]} × 100%; the results are shown in Table 4.

[0092] Table 4. Antioxidant Test Results 1 89.01 2 90.16 3 93.45 4 96.61 5 86.84 6 87.01 7 86.33 8 77.04 9 76.12 The serial numbers in Table 4 follow those in Example 2.

[0093] (2) Test for inhibiting porcine pancreatic elastase activity Elastase degrades elastin, leading to skin aging. This experiment evaluated the skin-firming and anti-wrinkle effects of an oil-soluble matsutake mushroom ferment broth by simulating its inhibitory effect on porcine pancreatic elastase activity. The yellow product generated by the enzyme hydrolysis of the substrate AAAPVN showed characteristic absorption at 420 nm, and the inhibition rate was calculated by measuring changes in absorbance.

[0094] 2. Solution preparation First, prepare the Tris-HCl buffer solution: Weigh 2.42g of Tris, add 200mL of ultrapure water to dissolve it, and adjust the pH to 8.0 with concentrated hydrochloric acid.

[0095] Preparation of sample solution: Accurately weigh 10 mg of matsutake fermentation extract, add 50 μL of DMSO and vortex to dissolve. After ensuring complete dissolution of the sample, bring the volume to 1 mL with Tris-HCl buffer to obtain a 1% sample stock solution.

[0096] Preparation of positive control: Weigh 5 mg of tea polyphenols and dissolve them in 5 mL of Tris-HCl buffer to prepare a 1 mg / mL tea polyphenol solution.

[0097] Preparation of substrate solution: Accurately weigh 4.51 mg AAAPVN, dissolve it in 5 mL Tris-HCl buffer to prepare a 2 mM substrate solution.

[0098] Preparation of enzyme working solution: Take 280 μL of porcine pancreatic elastase stock solution, dilute with 10 mL of Tris-HCl buffer, and prepare an enzyme working solution of 0.171 U / mL.

[0099] Preparation of solvent control: Take 50 μL of DMSO and dilute it with 950 μL of Tris-HCl buffer to obtain a 5% DMSO solution as a solvent control.

[0100] 3. Experimental grouping and sample addition The experiment consisted of five groups, with four replicates in each group. The specific sample loading plan is as follows: Model control group: Add 25 μL of enzyme working solution, 50 μL of substrate solution and 25 μL of Tris-HCl buffer to form a complete reaction system.

[0101] Blank control group: 50 μL of substrate solution and 50 μL of Tris-HCl buffer were added to determine the background value of non-enzymatic reactions.

[0102] Positive control group: 25 μL of tea polyphenol solution, 25 μL of enzyme working solution and 50 μL of substrate solution were added to verify the reliability of the experimental system.

[0103] Sample group: Add 25 μL of sample solution, 25 μL of enzyme working solution and 50 μL of substrate solution to test the inhibitory effect of the sample.

[0104] Solvent control group: 25 μL of solvent control solution, 25 μL of enzyme working solution and 50 μL of substrate solution were added to eliminate the potential influence of DMSO solvent on enzyme activity.

[0105] 4. Measurement and Calculation All reaction systems were reacted at room temperature in the dark for 15 minutes. The absorbance of each well was measured at a wavelength of 420 nm using an ELISA reader. The results are shown in Table 5.

[0106] The formula for calculating the inhibition rate is: Inhibition rate (%) = [1 - (absorbance of sample group - absorbance of blank group) / (absorbance of model group - absorbance of blank group)] × 100%.

[0107] Table 5 Results of detection of porcine pancreatic elastase inhibition rate Model comparison 0.00 Blank control / Positive control tea polyphenols 71.03 1 79.21 2 80.14 3 86.77 4 87.18 5 76.81 6 76.45 7 76.12 8 71.68 9 72.03 Analysis of the results in Tables 4 and 5: Group 1, serving as the basic experimental group, exhibited good levels of DPPH scavenging and elastase inhibition, primarily due to the enhancement of Bacillus subtilis metabolic activity induced by Bacillus amyloliquefaciens cell wall pre-induction. However, the lack of pine bark extract resulted in insufficient polyphenolic antioxidants in the fermentation products, leaving significant room for improvement in both antioxidant and enzyme-inhibiting capabilities.

[0108] Although a small amount of pine bark extract was added in sample 2, the concentration did not reach the effective threshold, resulting in only a slight increase in DPPH scavenging and elastase inhibition rates compared to sample 1. This difference is within the normal experimental fluctuation range. This indicates that excessively low concentrations of pine bark extract cannot provide sufficient polyphenol precursors for the fermentation system, making it difficult to produce a significant synergistic effect.

[0109] In step 3, both indicators were significantly improved after reaching the optimal amount of pine bark extract. Sufficient polyphenol precursors not only directly contribute to antioxidant capacity but are also converted into more active molecules during fermentation. These molecules can effectively scavenge free radicals and exert an inhibitory effect by binding to the active site of elastase.

[0110] In step 4, further optimization of fermentation conditions resulted in both indicators reaching their peak values. Suitable moisture content and pH created an ideal fermentation environment, leading to high concentrations of antioxidants in the metabolites and the production of more small-molecule active substances that specifically inhibit elastase.

[0111] After replacing item 5 with pre-induction using Bacillus subtilis' own cell wall, both indicators showed a significant decrease. This indicates that the metabolic profile produced by homologous cell wall induction is fundamentally different from that produced by heterologous induction, and the resulting active substances are inferior in both structure and efficacy, demonstrating that heterologous cell wall induction has a unique advantage in activating specific metabolic pathways.

[0112] Pre-induction using Lactobacillus bulgaricus cell wall in sequence 6 was less effective. Significant differences in cell wall components among different microorganisms resulted in weak or nonspecific induction signals, failing to effectively activate the expression of key genes required for Bacillus subtilis to produce highly active metabolites.

[0113] Number 7, serving as the uninduced control group, showed only slightly better performance than numbers 5 and 6. This further confirms that pre-induction treatment can indeed significantly alter the metabolic pathways of Bacillus subtilis and is a key process step in enhancing product activity.

[0114] The fermentation effect of Bacillus amyloliquefaciens directly using No. 8 was significantly poor, with its activity level at the same low level as No. 9. This indicates that although the cell wall of Bacillus amyloliquefaciens is an effective inducer, the adaptability and transformation ability of this strain in the matsutake solid-state fermentation system are limited.

[0115] The fermentation effect of Lactobacillus bulgaricus in item 9 was comparable to that in item 8, and the slight difference between the two is normal experimental fluctuation. This fully demonstrates that neither Lactobacillus bulgaricus nor Bacillus amyloliquefaciens is suitable for this matsutake solid-state fermentation system, and their metabolic characteristics cannot effectively decompose the matsutake substrate and produce abundant active metabolites.

[0116] Example 4: Experiments with different bacterial strains To further investigate whether the extraction and fermentation method of the present invention is applicable to different strains, two large edible fungi with growth environments similar to matsutake mushrooms were selected for experiments. Fresh black and white truffles were taken, pre-dried, and chopped; then freeze-dried to constant weight; and then fermented under the same conditions using three different strains of bacteria, namely, Bacillus amyloliquefaciens suspension, Bacillus amyloliquefaciens, and Lactobacillus bulgaricus, according to the preparation method in Example 2; to obtain the corresponding black truffle ferment and white truffle ferment.

[0117] The same test for inhibiting porcine pancreatic elastase activity was performed on both black truffle and white truffle ferments as in Example 3; the results are as follows: Black truffle fermentation: Bitter hay suspension (starch-degrading): elastase inhibition rate (%): 81.23% Bacillus amyloliquefaciens: elastase inhibition rate (%): 67.23% Lactobacillus bulgaricus: elastase inhibition rate (%): 65.78% White truffle fermentation: Bitter hay suspension (starch decomposition): elastase inhibition rate (%): 84.45%.

[0118] Bacillus amyloliquefaciens: elastase inhibition rate (%): 68.11% Lactobacillus bulgaricus: elastase inhibition rate (%): 67.49% Experimental results show that black and white truffles and white truffles, prepared using the same method, have the same beneficial effects as matsutake mushroom extract.

[0119] Example 5: Preparation of Oil-Based Anti-Aging Skincare Products This embodiment provides a skin anti-aging essence oil containing the matsutake fermentation product prepared in Example 2 above, which contains the following components by mass percentage: Matsutake mushroom fermentation product of Example 2, No. 4, 1.0 wt%. Camellia seed oil, 45.0 wt% Jojoba oil, 25wt% Macadamia seed oil, 15wt% bis-ethylhexyloxyphenol methoxyphenyl triazine, 3wt% Cetearyl glucoside, 0.2 wt% Vitamin E, 1.0 wt% Ethylhexylglycerin, 0.8 wt% Bisabolol, 0.2 wt%.

[0120] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing an extract from macrofungi, characterized in that, The preparation method involves using large fungal fruiting bodies as fermentation raw materials, fermenting them in aerobic-facultative anaerobic stages using Bacillus subtilis, and then extracting and purifying them to obtain the final product. The fermentation substrate used for Bacillus subtilis fermentation comprises the following components, by weight percentage: Large fungal fruiting bodies, ≥90%; Pine bark extract, 1-3%; The Bacillus subtilis is a pre-induced Bacillus subtilis cell suspension; the pre-induced Bacillus subtilis cell suspension is a pre-induced microbial suspension obtained by inoculating Bacillus subtilis seed culture into a culture medium with an additional 1 wt% inactivated competitive microbial cell wall and culturing it for a period of time, with a cell concentration of... ; The large fungal fruiting body is at least one of matsutake, black truffle, and white truffle; The pine bark extract is a water-alcohol mixture extract of pine bark, wherein the condensed tannin content is ≥4wt%; The inactivated competitive microbial cell wall is derived from the cell wall components of Bacillus amyloliquefaciens after inactivation, followed by disruption and purification. The fermentation conditions for aerobic-facultative anaerobic staged fermentation using Bacillus subtilis are as follows: Active oxygen supply was provided during the first 0-24 hours of fermentation; the fermentation temperature was 37±1℃. After 24 hours of fermentation, active oxygen supply was stopped, and the fermentation temperature was adjusted to 31±1℃. Fermentation time is at least 48 hours; initial pH ≤ 6.5; anaerobic solid-state fermentation; fermentation endpoint is reached when pH drops to ≤ 4.5 and changes ≤ 0.1 within 4 hours. The extraction and purification process involves: sterilization followed by freeze-drying and pulverization after reaching the fermentation endpoint; then, ethanol / water extraction of the macrofungi ferment; the extract is a macrofungi extract. The ethanol / water has a volume concentration of 50%.

2. The method for preparing macrofungi extract according to claim 1, characterized in that, The fermentation substrate also contains components: carbon source, nitrogen source and inorganic salt; the initial water content of the fermentation substrate is 55-65 wt%.

3. The method for preparing macrofungi extract according to claim 1, characterized in that, The fermentation substrate also includes the following components by mass percentage: Carbon source, ≥2%; Nitrogen source, ≥3%; Complex inorganic salts, ≥1%; The composite inorganic salt includes at least phosphate, magnesium salt and manganese salt.

4. The method for preparing macrofungi extract according to claim 1, characterized in that, Before fermentation, the fruiting bodies of large fungi are pretreated. The pretreatment includes the following steps: taking dried large fungal fruiting bodies, cleaning the surface and chopping them; freeze-drying the chopped fruiting bodies to constant weight; then ultra-fine pulverizing them to ≥300 mesh to obtain large fungal ultra-fine powder; and sterilizing the large fungal ultra-fine powder.

5. The macrofungus extract prepared by any one of claims 1-4.

6. The application of the macrofungus extract according to claim 5 in the preparation of cosmetics with anti-aging effects.

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