Tricholoma matsutake endophytic fungus fermentation self-microemulsion as well as preparation method and application thereof

Abstract

The invention relates to tricholoma matsutake endophytic fungus fermentation self-microemulsion as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) inoculating a tricholoma matsutake endophytic fungus seed solution into a fermentation culture medium for fermentation culture to obtain a primary fermentation solution; and (2) mixing the primary fermentation liquor, vegetable oil, an amphiphilic deep-eutectic solvent and polyol, continuing fermentation culture, filtering and collecting a liquid phase to obtain the tricholoma matsutake endophytic fungus fermentation self-microemulsion. According to the tricholoma matsutake endophytic fungus fermentation self-microemulsion prepared by the preparation method disclosed by the invention, the utilization rate of tricholoma matsutake endophytic fungi is increased to the maximum efficiency, oil-soluble and water-soluble functional components in a tricholoma matsutake endophytic fungus fermentation process can be directionally enriched, and a nano oil-in-water microcapsule structure with a uniform particle size is formed; the stability and permeability of active ingredients are remarkably improved, and the prepared tricholoma matsutake endophytic fungus fermented self-microemulsion has excellent soothing and repairing effects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a fermentation microemulsion of matsutake endophytic fungi, its preparation method, and its application. Background Technology

[0002] Matsutake mushrooms, also known as pine mushrooms, are fungi belonging to the genus *Tricholoma* in the family Tricholomataceae. They are well-known wild edible and medicinal fungi, typically growing at high altitudes in the roots of pine and oak forests in temperate and cold-temperate zones. As one of the most famous higher fungi, matsutake mushrooms contain dozens of active nutrients and are rich in protein and carbohydrates.

[0003] Currently, the main method for obtaining the active ingredients in matsutake mushrooms is through direct extraction from the fruiting body.

[0004] CN118078698A discloses a matsutake mushroom extract with soothing and anti-aging effects. The preparation method includes the following steps: mixing matsutake raw material with an ethanol-water solution and refluxing to obtain an extract; filtering the extract to obtain an ethanol extract filtrate and a residue; purifying the ethanol extract filtrate using a macroporous resin and collecting the eluent; mixing the residue with water for water extraction to obtain an aqueous extract, and filtering to obtain the aqueous extract filtrate; combining the eluent and the aqueous extract filtrate to obtain the matsutake mushroom extract. This invention combines the aqueous extract of the ethanol extract filtrate and the ethanol extract residue, not only achieving more efficient utilization of the matsutake raw material but also significantly improving the safety of the final product and its efficacy in anti-inflammatory, soothing, antioxidant, anti-aging, and anti-photoaging effects, providing a new strategy for preparing anti-aging and soothing cosmetics.

[0005] CN118384070A discloses a matsutake mushroom extract with soothing and repairing effects. The preparation method includes the following steps: mixing matsutake ultrafine powder with a eutectic solvent and a complex enzyme for extraction to obtain an extract; centrifuging the extract to collect the supernatant; eluting the supernatant through a macroporous resin to obtain the matsutake extract. The preparation method involved in this invention uses matsutake ultrafine powder as the extraction raw material, employs a eutectic solvent and complex enzyme co-extraction method, and combines it with subsequent purification operations to obtain a matsutake extract product with excellent soothing and repairing effects, specifically manifested in its ability to significantly inhibit hyaluronidase activity and the aggregation of neutrophils in zebrafish embryos.

[0006] Endophytic fungi of matsutake mushrooms are a group of microorganisms that have long coexisted with matsutake mushrooms. Through long-term co-evolution, their metabolic pathways are somewhat similar to those of matsutake mushrooms, and they possess the potential to synthesize the characteristic active ingredients of matsutake mushrooms. Compared with natural matsutake fruiting bodies, matsutake endophytic fungi have advantages such as faster growth rate, milder cultivation conditions, and the ability to achieve large-scale production through fermentation engineering. Furthermore, by optimizing fermentation process parameters, their metabolic flow can be directionally regulated.

[0007] Therefore, developing a fermentation process for matsutake mushroom endophytic fungi to achieve targeted and efficient enrichment of active ingredients and improve the compatibility of fermentation products in different cosmetics has become one of the urgent technical problems to be solved. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing and applying a microemulsion fermented by endophytic fungi of matsutake mushrooms.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing a microemulsion by fermentation of matsutake endophytic fungi, the method comprising the following steps:

[0011] (1) The seed liquid of matsutake endophytic fungi was inoculated into the fermentation medium for fermentation culture to obtain the primary fermentation liquid;

[0012] (2) Mix the primary fermentation broth, vegetable oil, amphiphilic eutectic solvent and polyol, continue fermentation and culture, filter and collect the liquid phase to obtain the fermentation microemulsion of matsutake endophytic fungi.

[0013] This invention uses endophytic fungi from matsutake mushrooms as a highly efficient substitute for matsutake mushrooms. First, a primary fermentation broth is obtained through fermentation. Next, a self-emulsion with an amphiphilic eutectic solvent (DES) is formed during co-fermentation and self-assembly, exhibiting an oil-in-water vesicle structure. Simultaneously, vegetable oil (as the oil phase core of the self-emulsion system) and polyols (possessing both emulsification stability and moisturizing activity) are added. The primary fermentation broth serves as the continuous phase (aqueous phase) of the self-emulsion, ultimately forming an oil-in-water nanovesicle self-emulsion with uniform particle size.

[0014] The microemulsion prepared by this invention, derived from the fermentation of matsutake endophytic fungi, can efficiently extract water-soluble components from matsutake mushrooms, solving the problem of water-soluble component loss due to centrifugation to extract the upper oil phase in conventional fermentation oil processes, and improving the utilization rate of matsutake endophytic fungi. This method can directionally enrich oil-soluble and water-soluble components during the fermentation process of matsutake endophytic fungi and form a nano-microcapsule structure with uniform particle size, significantly improving the stability and permeability of active ingredients. Furthermore, the prepared microemulsion derived from the fermentation of matsutake endophytic fungi has excellent soothing and repairing effects.

[0015] Furthermore, the matsutake endophytic fungal fermentation microemulsion involved in this invention does not use organic solvents during the preparation process and does not require the addition of additional emulsifiers, which has the advantages of being green and environmentally friendly. It also has the compatibility of an amphiphilic microenvironment and surpasses traditional formulations in terms of complex ingredient loading, precise delivery, and adaptation to special scenarios. At the same time, it improves its soothing and repairing effects and the feel on the skin, opening up innovative application avenues for matsutake skin care.

[0016] Preferably, the raw materials for preparing the amphiphilic eutectic solvent in step (2) are selected from any one or at least two of the following combinations:

[0017] (i) Proline and xylitol;

[0018] (ii) Glucose and decanoic acid;

[0019] (iii) Glucose and choline;

[0020] (iv) Matrine and coconut oil acid.

[0021] Studies have found that eutectic solvents obtained by any of the above combinations can form relatively stable oil-in-water vesicle structures during the fermentation of matsutake endophytic fungi. However, the stability of the microemulsion structure and the content of active ingredients in the product vary. The following specific types of eutectic solvents are preferred, as they can further enhance the repair efficacy and stability of fermented microemulsions.

[0022] Preferably, the amphiphilic eutectic solvent in step (2) is a mixture of a first eutectic solvent and a second eutectic solvent.

[0023] The raw materials for preparing the first eutectic solvent include proline and xylitol (proline is used as a hydrogen bond acceptor and xylitol is used as a hydrogen bond donor to obtain the reaction).

[0024] The raw materials for preparing the second eutectic solvent include matrine and coconut oil acid (matrine is reacted as a hydrogen bond acceptor and coconut oil acid is reacted as a hydrogen bond donor).

[0025] This invention creatively prepares a first eutectic solvent and a second eutectic solvent using different raw materials. Studies have found that, compared with a single first eutectic solvent or a single second eutectic solvent, the fermented oil produced has superior stability and soothing and repairing effects.

[0026] Preferably, the molar ratio of proline to xylitol is 1:1 to 1:4, for example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, etc.

[0027] Preferably, the molar ratio of matrine to coconut oil acid is 1:1 to 1:6, for example, it can be 1:1, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0028] Preferably, the volume ratio of the first eutectic solvent to the second eutectic solvent is 1:3 to 3:1, for example, it can be 1:3, 1.5:3, 2:3, 2.5:3, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, etc.

[0029] Preferably, the polyol includes any one or a combination of at least two of glycerol, butylene glycol, or propylene glycol.

[0030] Preferably, the polyol comprises a combination of butanediol and propylene glycol.

[0031] Preferably, the mass ratio of butanediol to propylene glycol is 1:3 to 3:1, for example, it can be 1:3, 1.5:3, 2:3, 2.5:3, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, etc.

[0032] This invention creatively discovers that the combination of butanediol and propylene glycol in the preparation of fermented self-microemulsions has a significant synergistic effect in improving the stability of fermented self-microemulsions.

[0033] Preferably, the vegetable oil includes any one or a combination of at least two of the following: sea buckthorn fruit oil, meadowfoam seed oil, grape seed oil, flaxseed oil, camellia oil, sunflower seed oil, or perilla seed oil.

[0034] Preferably, the mass ratio of the primary fermentation broth, vegetable oil, amphiphilic eutectic solvent, and polyol is (10-60):(30-50):(10-30):(5-10).

[0035] Among them, the specific point values ​​in 10-60 can be 10, 20, 30, 40, 50, 60, etc.; the specific point values ​​in 30-50 can be 30, 35, 40, 45, 50, etc.; the specific point values ​​in 10-30 can be 10, 15, 20, 25, 30, etc.; and the specific point values ​​in 5-10 can be 5, 6, 7, 8, 9, 10, etc.

[0036] Preferably, the matsutake endophytic fungus in the matsutake endophytic fungal seed liquid in step (1) is the matsutake endophytic fungus strain XWJ0001.

[0037] The endophytic fungus strain XWJ0001 of the matsutake mushroom is classified as Umbelopsis sp., with accession number CGMCCNo.42463, accession date of December 4, 2025, and deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0038] Preferably, the inoculation volume of the matsutake endophytic fungal seed liquid in step (1) is 1-10% of the fermentation medium volume, for example, it can be 1%, 3%, 5%, 7%, 9%, 10%, etc.

[0039] Preferably, the fermentation medium in step (1) comprises: yeast powder, peptone, glycerol, potassium dihydrogen phosphate, magnesium sulfate and water.

[0040] Preferably, the fermentation culture medium in step (1) comprises, by mass percentage: 0.5-2% yeast powder, 1-5% peptone, 1-5% glycerol, 0.1-0.3% potassium dihydrogen phosphate, 0.1-0.3% magnesium sulfate, and the remainder water.

[0041] Among them, the specific point values ​​in the 0.5-2% range can be 0.5%, 1%, 1.5%, 2%, etc.; the specific point values ​​in the 1-5% range can be 1%, 2%, 3%, 4%, 5%, etc.; and the specific point values ​​in the 0.1-0.3% range can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc.

[0042] Preferably, the fermentation temperature in step (1) is 25-35℃ (e.g., 25℃, 27℃, 29℃, 31℃, 33℃, 35℃, etc.), and the fermentation time is 6-24 h (e.g., 6 h, 10 h, 14 h, 18 h, 22 h, 24 h, etc.).

[0043] Preferably, the fermentation culture in step (1) is carried out under agitation conditions with an aeration rate (e.g., 40 L, 50 L, 60 L, 70 L, 80 L, etc.), a pressure of 0.05-0.3 MPa (e.g., 0.05 MPa, 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, etc.), and a speed of 50-150 rpm (e.g., 50 rpm, 80 rpm, 110 rpm, 130 rpm, 150 rpm, etc.).

[0044] Preferably, the preparation of the matsutake endophytic fungal seed liquid in step (1) can be obtained using existing techniques in the art, for example, by the following process:

[0045] The endophytic fungal strain of matsutake mushroom was inoculated into YPD medium and cultured to the logarithmic growth phase to obtain seed culture.

[0046] Preferably, the fermentation temperature in step (2) is 25-35℃ (e.g., 25℃, 27℃, 29℃, 31℃, 33℃, 35℃, etc.), and the fermentation time is 6-24 h (e.g., 6 h, 10 h, 14 h, 18 h, 22 h, 24 h, etc.).

[0047] In a second aspect, the present invention provides a self-fermented microemulsion of matsutake endophytic fungi prepared by the preparation method described in the first aspect.

[0048] Thirdly, the present invention provides an application of the fermentation microemulsion of matsutake endophytic fungi as described in the second aspect in cosmetics.

[0049] Preferably, the cosmetic product has repairing and soothing effects.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This invention uses endophytic fungi from matsutake mushrooms as a highly efficient substitute for matsutake mushrooms. First, a primary fermentation broth is obtained through fermentation. Next, a self-emulsion with an amphiphilic eutectic solvent (DES) is formed during co-fermentation and self-assembly, exhibiting an oil-in-water vesicle structure. Simultaneously, vegetable oil (as the oil phase core of the self-emulsion system) and polyols (possessing both emulsification stability and moisturizing activity) are added. The primary fermentation broth serves as the continuous phase (aqueous phase) of the self-emulsion, ultimately forming an oil-in-water nanovesicle self-emulsion with uniform particle size.

[0052] The microemulsion prepared by this invention, derived from the fermentation of matsutake endophytic fungi, can efficiently extract water-soluble components from matsutake mushrooms, solving the problem of water-soluble component loss due to centrifugation to extract the upper oil phase in conventional fermentation oil processes, and improving the utilization rate of matsutake endophytic fungi. This method can directionally enrich oil-soluble and water-soluble components during the fermentation process of matsutake endophytic fungi and form a nano-microcapsule structure with uniform particle size, significantly improving the stability and permeability of active ingredients. Furthermore, the prepared microemulsion derived from the fermentation of matsutake endophytic fungi has excellent soothing and repairing effects.

[0053] Furthermore, the matsutake endophytic fungal fermentation microemulsion involved in this invention does not use organic solvents during the preparation process and does not require the addition of additional emulsifiers, which has the advantages of being green and environmentally friendly. It also has the compatibility of an amphiphilic microenvironment and surpasses traditional formulations in terms of complex ingredient loading, precise delivery, and adaptation to special scenarios. At the same time, it improves its soothing and repairing effects and the feel on the skin, opening up innovative application avenues for matsutake skin care.

[0054] The matsutake endophytic fungus strain XWJ0001 involved in this invention is classified as Umbelopsis sp., with accession number CGMCC No.42463, accession date of December 4, 2025, and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0055] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0056] The method for preparing the Matsutake endophytic fungal seed liquid described in the following specific embodiments is as follows:

[0057] The endophytic fungus strain XWJ0001 from matsutake mushrooms was added to 10 mL of YPD medium and cultured at 30℃ and 180 rpm for 14 h until mid-log phase to obtain seed culture. The YPD medium formula was: 1% yeast extract, 2% peptone, and 2% glucose, with natural pH.

[0058] Preparation Example 1

[0059] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0060] (1) Proline and xylitol were mixed in a molar ratio of 1:3 and reacted at 70°C for 2 h to obtain the first eutectic solvent;

[0061] (2) Matrine and coconut oil acid were mixed in a 1:1 molar ratio and reacted at 70°C for 2 h to obtain a second eutectic solvent;

[0062] (3) The first eutectic solvent and the second eutectic solvent are mixed in a volume ratio of 1:1 to obtain a composite eutectic solvent.

[0063] Preparation Example 2

[0064] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0065] (1) Proline and xylitol were mixed in a 1:1 molar ratio and reacted at 50°C for 4 h to obtain the first eutectic solvent;

[0066] (2) Matrine and coconut oil acid were mixed in a molar ratio of 1:2 and reacted at 50°C for 4 h to obtain a second eutectic solvent;

[0067] (3) The first eutectic solvent and the second eutectic solvent are mixed in a volume ratio of 1:3 to obtain a composite eutectic solvent.

[0068] Preparation Example 3

[0069] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0070] (1) Proline and xylitol were mixed in a molar ratio of 1:4 and reacted at 70°C for 2 h to obtain the first eutectic solvent;

[0071] (2) Matrine and coconut oil acid were mixed in a molar ratio of 1:3 and reacted at 70°C for 2 h to obtain a second eutectic solvent;

[0072] (3) The first eutectic solvent and the second eutectic solvent are mixed at a volume ratio of 3:1 to obtain a composite eutectic solvent.

[0073] Preparation Example 4

[0074] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0075] (1) Glucose and decanoic acid were mixed in a molar ratio of 1:3 and reacted at 70°C for 2 h to obtain the first eutectic solvent;

[0076] (2) Glucose and choline were mixed in a 1:1 molar ratio and reacted at 70°C for 2 h to obtain a second eutectic solvent;

[0077] (3) The first eutectic solvent and the second eutectic solvent are mixed in a volume ratio of 1:1 to obtain a composite eutectic solvent.

[0078] Preparation Example 5

[0079] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0080] Proline and xylitol are mixed in a molar ratio of 1:3 and reacted at 70°C for 2 h to obtain the final product.

[0081] Preparation Example 6

[0082] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0083] Matrine and coconut oil acid are mixed in a 1:1 molar ratio and reacted at 70°C for 2 h to obtain the product.

[0084] Comparative Preparation Example 1

[0085] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0086] Choline chloride and urea are mixed in a molar ratio of 1:4 and reacted at 70°C for 2 hours to obtain the product.

[0087] Example 1

[0088] This embodiment provides a method for preparing a microemulsion fermented from matsutake endophytic fungi, as follows:

[0089] (1) The endophytic fungal seed liquid of matsutake mushroom was fermented in a fermentation medium at an inoculation rate of 10% at a fermentation temperature of 30℃, an aeration rate of 60 L, a stirring speed of 100 rpm, a pressure of 0.2 MPa, and cultured for 18 h to obtain the first-stage fermentation liquid.

[0090] The fermentation medium, by mass, is formulated as follows: 1% yeast extract, 3% peptone, 3% glycerol, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and the remainder water.

[0091] (2) Mix 32% of the primary fermentation broth with 40% of vegetable oil (sea buckthorn fruit oil), 20% of the composite eutectic solvent prepared in Preparation Example 1, and 8% of polyol (butanediol and propylene glycol in a mass ratio of 1:1), and continue to culture at 30°C for 18 h. Filter and collect the liquid phase to obtain the fermentation microemulsion of matsutake endophytic fungi.

[0092] Example 2

[0093] This embodiment provides a method for preparing a microemulsion fermented from matsutake endophytic fungi, as follows:

[0094] (1) The endophytic fungal seed liquid of matsutake mushroom was fermented in a fermentation medium at an inoculation rate of 10% at a fermentation temperature of 25℃, an aeration rate of 80 L, a stirring speed of 150 rpm, a pressure of 0.05 MPa, and cultured for 24 h to obtain the first-stage fermentation liquid.

[0095] The fermentation medium, by mass, is formulated as follows: 0.5% yeast extract, 5% peptone, 1% glycerol, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and the remainder water.

[0096] (2) Mix 35% of the primary fermentation broth with 35% of vegetable oil (meadowfoam seed oil), 25% of the composite eutectic solvent prepared in Preparation Example 1, and 5% of polyol (butanediol and propylene glycol in a mass ratio of 1:3), and continue to culture at 25°C for 6 h. Filter and collect the liquid phase to obtain the fermentation microemulsion of matsutake endophytic fungi.

[0097] Example 3

[0098] This embodiment provides a method for preparing a microemulsion fermented from matsutake endophytic fungi, as follows:

[0099] (1) The endophytic fungal seed liquid of matsutake mushroom was fermented in a fermentation medium at an inoculation rate of 7% at a fermentation temperature of 35℃, an aeration rate of 40 L, a stirring speed of 50 rpm, a pressure of 0.3 MPa, and cultured for 6 h to obtain the first-stage fermentation liquid.

[0100] The fermentation medium, by mass, is formulated as follows: 2% yeast extract, 1% peptone, 5% glycerol, 0.1% potassium dihydrogen phosphate, 0.3% magnesium sulfate, and the remainder water.

[0101] (2) Mix 30% of the primary fermentation broth with 45% of vegetable oil (sunflower seed oil), 15% of the composite eutectic solvent prepared in Preparation Example 1, and 10% of polyol (butanediol and propylene glycol in a mass ratio of 3:1), and continue to culture at 35°C for 24 h. Filter and collect the liquid phase to obtain the fermentation microemulsion of matsutake endophytic fungi.

[0102] Examples 4-6

[0103] Examples 4-6 each provide a microemulsion fermented by endophytic fungi of matsutake mushrooms. The only difference between them and Example 1 is that the composite eutectic solvent prepared in Preparation Example 1 is replaced with the composite eutectic solvent or eutectic solvent prepared in Preparation Examples 4-6 respectively. All other steps are the same as in Example 1.

[0104] Example 7

[0105] This embodiment provides a microemulsion fermented by endophytic fungi of matsutake mushrooms. The only difference between this embodiment and Example 1 is that the amount of polyol added remains unchanged at 8%, but the polyol is adjusted to a single butylene glycol. All other steps are consistent with Example 1.

[0106] Example 8

[0107] This embodiment provides a method for fermenting matsutake endophytic fungi into microemulsions. The only difference between this embodiment and Example 1 is that the amount of polyol added remains unchanged at 8%, but the polyol is adjusted to a single propylene glycol. All other steps are consistent with Example 1.

[0108] Comparative Example 1

[0109] This comparative example provides a microemulsion fermented by endophytic fungi of matsutake mushrooms. The only difference between this example and Example 1 is that the composite eutectic solvent prepared in Example 1 is replaced with the eutectic solvent prepared in Comparative Example 1. All other steps are the same as in Example 1.

[0110] Comparative Example 2

[0111] This comparative example provides a microemulsion fermentation of matsutake endophytic fungi. The only difference between this example and Example 1 is that polyols are not added in step (2). Instead, 40% of the primary fermentation broth is mixed with 40% of vegetable oil and 20% of the composite eutectic solvent prepared in Example 1 for fermentation. All other steps are consistent with Example 1.

[0112] Comparative Example 3

[0113] This comparative example provides a microemulsion fermented by endophytic fungi of matsutake mushrooms. The only difference between this example and Example 1 is that in step (2), the composite eutectic solvent is adjusted to an equal mass of emulsifier (polyglycerol-3 diisostearate). All other steps are consistent with Example 1.

[0114] Test Example 1

[0115] Particle size analysis:

[0116] (1) Test method:

[0117] The particle size and PDI of each sample (the self-fermented microemulsion of matsutake endophytic fungi provided in each example and comparative example) were detected by a laser particle size analyzer.

[0118] (2) Test results:

[0119] The particle size test results for each sample are shown in Table 1.

[0120] Table 1

[0121]

[0122]

[0123] Test Example 2

[0124] Stability test:

[0125] (1) Test method: Each sample to be tested (the Matsutake endophytic fungus fermentation microemulsion provided in each example and comparative example) was placed at 25°C for 3 months. After 3 months, the samples to be tested were observed to see if discoloration, oil seepage or precipitation occurred, so as to evaluate the stability of fermentation microemulsion.

[0126] (2) Test results:

[0127] The stability test results of each sample are shown in Table 2.

[0128] Table 2

[0129]

[0130] As can be seen from the comparison of the data in Tables 1 and 2, the fermentation microemulsion of matsutake endophytic fungi involved in this invention is a nano-sized microemulsion with uniform particle size distribution and excellent storage stability.

[0131] Comparison of the data from Example 1 with Comparative Examples 1 and 3 shows that the self-microemulsion of matsutake endophytic fungi obtained by co-fermentation with an amphiphilic eutectic solvent and self-assembly to form a microemulsion has better stability.

[0132] A comparison of the data from Examples 1 and 4-6 shows that the first eutectic solvent prepared from proline and xylitol and the second eutectic solvent prepared from matrine and coconut oil acid have a better effect on improving the uniformity and stability of self-microemulsions compared to eutectic solvents in other formulations, and the two have a significant synergistic effect in improving the uniformity and stability of self-microemulsions.

[0133] A comparison of the data from Example 1 with Examples 7-8 and Comparative Example 2 shows that the addition of polyols can improve particle size uniformity and product stability during the preparation of fermented self-microemulsions. In addition, butanediol and propylene glycol have significant synergistic effects in improving particle size uniformity and self-microemulsion stability.

[0134] Test Example 3

[0135] Solubility test:

[0136] (1) Test method:

[0137] (1.1) Water solubility test: Accurately weigh 1 mg of the sample to be tested and put it into a 1.5 mL centrifuge tube; add 1 mL of distilled water and tighten the cap; vortex for 10 minutes (2000 rpm) to ensure that the sample and solvent are in full contact; let stand for 30 minutes and observe the state inside the centrifuge tube.

[0138] (1.2) Lipid solubility test: Accurately weigh 1 mg of the sample to be tested and put it into a 1.5 mL centrifuge tube; add 1 mL of n-octanol and tighten the cap; vortex for 10 minutes (2000 rpm) to ensure that the sample and solvent are in full contact; let stand for 30 minutes and observe the state inside the centrifuge tube.

[0139] (1.3) Judgment:

[0140] Easily soluble: The solution is clear and transparent, without precipitation, turbidity, or layering;

[0141] Soluble: The solution is translucent, without precipitate, turbidity, or separation;

[0142] Slightly soluble: The solution is turbid, or shows slight separation.

[0143] Sparingly soluble: Completely separates into layers, the solution remains clear.

[0144] (2) Test results:

[0145] Table 3

[0146]

[0147]

[0148] As can be seen from the data in Table 3, the microemulsion fermented by the matsutake endophytic fungi involved in this invention has the compatibility of an amphiphilic microenvironment, excellent water solubility, and can be well dissolved in oils.

[0149] Comparison of the data from Example 1 with Comparative Examples 1 and 3 shows that the self-assembly of the Matsutake endophytic fungus fermentation self-microemulsion obtained by co-fermentation with an amphiphilic eutectic solvent and self-assembly to form a self-microemulsion has better compatibility with the amphiphilic microenvironment.

[0150] A comparison of the data from Examples 1 and 4-6 shows that the first eutectic solvent prepared from proline and xylitol and the second eutectic solvent prepared from matrine and coconut oil acid have a better effect on improving the self-microemulsion dissolution compatibility compared to eutectic solvents in other formulations, and the two have a significant synergistic effect in improving the self-microemulsion dissolution compatibility.

[0151] A comparison of the data from Example 1 with Examples 7-8 and Comparative Example 2 shows that the addition of polyols can further improve the solubility of the self-microemulsion during the preparation of fermentation self-microemulsion. In addition, butanediol and propylene glycol have significant synergistic effects in improving the solubility of the self-microemulsion.

[0152] Test Example 4

[0153] Active ingredient content test:

[0154] (1) Test method:

[0155] (1.1) Total polyphenol content:

[0156] (1.1.1) Detection using the Folin-Ciocalteu method. Prepare a series of gallic acid standard solutions of different concentrations. Take 100 µL of the gallic acid working solution, add 500 µL of 10% Folin-Ciocalteu reagent, shake well, and let stand for 5 min. Add 400 µL of 7.5% sodium carbonate solution, shake well, incubate in a water bath at 40℃ for 60 min, and then let cool for 20 min. Measure the absorbance at 760 nm. Plot a standard curve with gallic acid concentration on the x-axis and absorbance on the y-axis.

[0157] (1.1.2) Accurately weigh 250 mg of the products obtained in Examples 1-8 and Comparative Examples 1-3, add 5 mL of 80% ethanol, sonicate for 30 min, centrifuge at 10000 r / min for 10 min, and collect the supernatant for later use. Accurately pipette 100 µL of the sample, add 500 µL of 10% Folin-Ciocalteu reagent, shake well, and let stand for 5 min. Add 400 µL of 7.5% sodium carbonate solution, shake well, incubate in a water bath at 40℃ for 60 min, and let stand and cool for 20 min. Measure the absorbance at 760 nm, and calculate the total polyphenol content in the sample according to the standard curve.

[0158] (1.2) Total polysaccharide content:

[0159] (1.2.1) Detection using the Benfen-sulfuric acid method. Prepare a series of glucose standard solutions of different concentrations. Take 1 mL of each glucose standard solution to a 10 mL stoppered test tube, add 1.0 mL of 5% phenol solution, and then quickly add 5.0 mL of concentrated sulfuric acid (add perpendicularly to the liquid surface, avoiding contact with the test tube wall to ensure thorough mixing). Use a vortex mixer to mix the reaction solution and let it stand for 10 min. Accurately heat in a boiling water bath for 15 min, then rapidly cool to room temperature and let it stand for 10 min. Take an appropriate amount of the reaction solution and measure the absorbance at 490 nm. Develop a standard curve with glucose mass concentration as the x-axis and absorbance as the y-axis.

[0160] (1.2.2) Accurately pipette 1 mL of the sample test solution into a 10 mL stoppered test tube, follow the steps in 1.2.1, measure the absorbance, and calculate the total sugar content using the standard curve.

[0161] (2) Test results:

[0162] The test results of total polyphenols and total polysaccharides in each sample are shown in Table 4.

[0163] Table 4

[0164]

[0165] As shown in Table 4, compared with the methods of Comparative Examples 1-3, the Matsutake endophytic fungal fermentation microemulsion prepared by the method of this invention has a high content of total polyphenols and total polysaccharides. Furthermore, the formulation composition of the amphiphilic eutectic solvent and the type of polyol selected in the method also affect the levels of total polyphenols and total polysaccharides in the product to some extent.

[0166] Test Example 5

[0167] Soothing and repairing effect test:

[0168] (1) Test method:

[0169] Healthy zebrafish embryos, 3 days post-fertilization, were selected and divided into a model group, a positive control group, and 11 test substance groups. In the model group, 6 embryos were randomly selected and placed into 3 cm culture dishes, with 5 mL of fish embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate. In the positive control group, 6 embryos were randomly selected and placed into 3 cm culture dishes, with 5 mL of fish embryo culture medium containing 0.1 mg / L copper sulfate pentahydrate and 0.0036 mg / L indomethacin. In each test substance group, 6 embryos were randomly selected and placed into 3 cm culture dishes, with 5 mL of fish embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate and 5 mg / mL of the test substance (fermented from microemulsions of the matsutake endophytic fungi obtained in each example and comparative example).

[0170] All groups were incubated at 28℃ for 45 min. Then, the fish embryos in each group were fixed in paraformaldehyde for 1 h, treated with PBST three times for 5 min each time, followed by treatment with 50% ethanol for 3 min. For the second group, the fish embryos were stained with Sudan Black solution for 1 h, washed four times with 70% ethanol for 5 min each time, and then treated with PBST twice for 5 min each time. For the third group, the fish embryos were treated with bleaching solution for 10 min, followed by treatment with 70% ethanol solution for 5 min, PBST for 1 min, clearing solution 1 for 15 min, clearing solution 2 for 10 min, and then PBST for 3 min.

[0171] The fish embryos were placed on their sides and observed under a stereomicroscope at the tail. The number of neutrophils in the three-quarters of the tail region from the anus was counted for each embryo, and the neutrophil aggregation inhibition rate was calculated.

[0172] Formula for calculating neutrophil aggregation inhibition rate:

[0173] Inhibition rate (%) = [(MS) / M] × 100%;

[0174] Wherein, S is the average number of neutrophils in the embryos of fish in the test group, in units of neutrophils per fish; M is the average number of neutrophils in the embryos of fish in the model group, in units of neutrophils per fish.

[0175] (2) Test results:

[0176] The results of the inhibition test on zebrafish neutrophils by each sample are shown in Table 5.

[0177] Table 5

[0178]

[0179] As can be seen from the data comparison in Table 5, the microemulsion of matsutake endophytic fungi fermented using the method of the present invention has excellent zebrafish embryo neutrophil aggregation effect, reflecting its potential in soothing and repairing effects.

[0180] Comparison of the data from Example 1 with Comparative Examples 1 and 3 shows that the self-microemulsion fermented by Matsutake endophytic fungi obtained by co-fermentation with an amphiphilic eutectic solvent and self-assembly to form a microemulsion has a better soothing and repairing effect.

[0181] A comparison of the data from Examples 1 and 4-6 shows that the first eutectic solvent prepared from proline and xylitol and the second eutectic solvent prepared from matrine and coconut oil acid are more effective in improving the soothing and repairing effects of self-microemulsions compared to eutectic solvents in other formulations, and the two have a synergistic effect in this regard.

[0182] A comparison of the data from Example 1 with Examples 7-8 and Comparative Example 2 shows that the addition of polyols during the preparation of fermented microemulsions can improve the stability of the microemulsions, thereby enhancing their soothing and repairing effects. Furthermore, butylene glycol and propylene glycol have a significant synergistic effect in improving the soothing and repairing effects of microemulsions.

[0183] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0184] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0185] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A preparation method of Tricholoma matsutake endophytic fungus fermented self-microemulsion, characterized in that, The preparation method comprises the following steps: (1) inoculating the seed liquid of the Tricholoma matsutake endophytic fungus into a fermentation medium for fermentation culture to obtain a primary fermentation liquid; (2) mixing the primary fermentation liquid, plant oil, amphiphilic eutectic solvent and polyhydric alcohol, continuing the fermentation culture, filtering and collecting the liquid phase to obtain the Tricholoma matsutake endophytic fungus fermentation self-microemulsion.

2. The production method according to claim 1, characterized by, The raw materials for preparing the amphiphilic eutectic solvent in step (2) are selected from any one or a combination of at least two of the following combinations: (i) proline and xylitol; (ii) glucose and decanoic acid; (iii) glucose and choline; (iv) matrine and coconut oil acid.

3. The preparation method according to claim 1, characterized in that, The amphiphilic eutectic solvent in step (2) is a mixture of a first eutectic solvent and a second eutectic solvent; The raw materials for preparing the first eutectic solvent include proline and xylitol; The raw materials for preparing the second eutectic solvent include matrine and coconut oil acid.

4. The production method according to claim 3, characterized by, The molar ratio of the proline and xylitol is 1:1-1:4; The molar ratio of the matrine and coconut oil acid is 1:1-1:6; The volume ratio of the first eutectic solvent to the second eutectic solvent is 1:3-3:

1.

5. The preparation method according to claim 1, characterized in that, The polyhydric alcohol includes any one or a combination of at least two of glycerol, butanediol or propylene glycol.

6. The production method according to claim 5, wherein The polyhydric alcohol includes a combination of butanediol and propylene glycol; The mass ratio of the butanediol to the propylene glycol is 1:3-3:

1.

7. The preparation method according to claim 1, characterized in that, The plant oil includes any one or a combination of at least two of sea buckthorn fruit oil, white pool flower seed oil, grape seed oil, flaxseed oil, camellia oil, sunflower seed oil or perilla seed oil.

8. The method of claim 1, wherein, The mass ratio of the primary fermentation liquid, plant oil, amphiphilic eutectic solvent and polyhydric alcohol is (10-60):(30-50):(10-30):(5-10).

9. A Tricholoma matsutake endophytic fungus fermentation self-microemulsion prepared by the preparation method in any one of claims 1-8.

10. Use of the Tricholoma matsutake endophytic fungus fermentation self-microemulsion in claim 9 in cosmetics.

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