Tricholoma matsutake endophytic fungus fermented oil and preparation method and application thereof

Abstract

The present application relates to a tricholoma matsutake endophytic fungus fermentation oil and a preparation method and application thereof. The preparation method comprises the following steps: (1) inoculating tricholoma matsutake endophytic fungus seed liquid into a fermentation medium to carry out fermentation culture, and obtaining a primary fermentation liquid; (2) mixing the primary fermentation liquid with vegetable oil and a linoleic acid-based deep eutectic solvent to continue fermentation culture, and centrifuging to collect an upper oil phase, thereby obtaining the tricholoma matsutake endophytic fungus fermentation oil. In the present application, the linoleic acid in the vegetable oil and the linoleic acid-based deep eutectic solvent is used as a precursor of tricholoma matsutake alcohol, so that the content of tricholoma matsutake alcohol in the fermentation product is increased. Meanwhile, the deep eutectic solvent is added in the fermentation process to regulate metabolism, and the fermentation product obtained by the above method has more excellent antioxidant level and soothing and repairing capacity.

Description

Technical Field

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

[0002] Matsutake alcohol, a characteristic fat-soluble active ingredient unique to matsutake mushrooms, has significant antioxidant, anti-inflammatory, and immune-regulating physiological functions. It can effectively scavenge free radicals and delay skin aging, demonstrating important application value in the fields of cosmetics, food, and medicine.

[0003] Currently, matsutake alcohol is mainly obtained by extraction from matsutake fruiting bodies. However, matsutake has extremely demanding requirements for its growth environment, making large-scale artificial cultivation impossible. Furthermore, matsutake fruiting bodies have low oil synthesis efficiency due to their slow growth and metabolism, which is biased towards structural substances and secondary metabolism. In addition, traditional extraction techniques (such as solvent extraction and supercritical fluid extraction) not only suffer from low extraction rates and high energy consumption, but may also lead to the destruction of matsutake alcohol structure due to process limitations, greatly reducing the utilization rate of matsutake.

[0004] The growth and development of fungal fruiting bodies are closely related to endophytic fungi. These endophytic fungi form a symbiotic relationship with fungal fruiting bodies and do not cause significant harm to host cells. At the same time, they are also a rich source of novel bioactive metabolites. Endophytic fungi can not only produce metabolites with medicinal value similar to those of the host plant, but also directly synthesize bioactive secondary metabolites.

[0005] Current matsutake endophytic fungal fermentation technology still has significant shortcomings: the synthesis efficiency of matsutake alcohol during fermentation is low, making it impossible to achieve targeted enrichment, and the purity of matsutake alcohol in the product is difficult to meet the needs of high-end applications; at the same time, the fermentation products have poor compatibility with oily application scenarios, making it difficult to directly apply them to skin care essential oils, functional oils and other products, thus limiting the further application of matsutake endophytic fungal fermentation products.

[0006] Therefore, optimizing the fermentation process of matsutake endophytic fungi to achieve targeted and efficient enrichment of matsutake alcohol, while solving problems such as difficulty in extracting fermentation products, insufficient safety, and poor adaptability to oily environments, has become one of the urgent technical problems to be solved. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a fermented oil of matsutake endophytic fungi, its preparation method, and its application.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing fermented oil from matsutake mushroom endophytic fungi, the method comprising:

[0010] (1) The seed liquid of matsutake endophytic fungi was inoculated into a fermentation medium for fermentation culture to obtain a primary fermentation broth;

[0011] (2) The primary fermentation broth was mixed with vegetable oil and linoleic acid-based eutectic solvent (linoleic acid-based DES), and fermentation was continued. The upper oil phase was collected by centrifugation to obtain the fermented oil of matsutake endophytic fungi.

[0012] This invention uses matsutake endophytic fungi as a highly efficient substitute for matsutake, and creatively employs vegetable oil and linoleic acid-based eutectic solvent to ferment the primary fermentation broth of matsutake endophytic fungi. By utilizing linoleic acid in vegetable oil and linoleic acid-based eutectic solvent as a precursor of matsutake alcohol, the content of matsutake alcohol in the fermentation product is increased.

[0013] Meanwhile, linoleic acid-based eutectic solvents are added during the fermentation process to regulate metabolism. Compared with traditional fermentation methods, the fermentation products obtained by the above method have a significantly increased content of the active ingredient matsutake alcohol, and the fermented oil of the product has better antioxidant, soothing and repairing capabilities.

[0014] Meanwhile, this invention utilizes the self-assembly of linoleic acid-based eutectic solvents to form water-in-oil vesicles. On the one hand, this increases the solubility of plant oils and linoleic acid, enabling better extraction of oil-soluble active ingredients (matsutake alcohol) from the fermentation broth. On the other hand, it encapsulates water-soluble active ingredients (such as matsutake polysaccharides) in the fermentation broth, enhancing the skin penetration of water-soluble active ingredients and giving the fermented oil product superior antioxidant, soothing, and repairing abilities.

[0015] Preferably, the vegetable oil in step (2) includes any one or a combination of at least two of the following: babassu seed oil, meadowfoam seed oil, grape seed oil, flaxseed oil, camellia oil, safflower seed oil, or perilla seed oil.

[0016] Preferably, the vegetable oil in step (2) comprises a combination of babassu seed oil and grape seed oil.

[0017] This invention creatively discovers that the combination of babassu seed oil and grape seed oil in the preparation of matsutake endophytic fungal fermentation oil can better improve the content of matsutake alcohol and the skin penetration ability of matsutake polysaccharides in the product. Compared with babassu seed oil or grape seed oil alone, the matsutake endophytic fungal fermentation oil prepared by the compound plant oil (the combination of babassu seed oil and grape seed oil) has a higher content of matsutake alcohol and better skin penetration ability of matsutake polysaccharides, indicating that babassu seed oil and grape seed oil have a significant synergistic effect in improving the antioxidant and soothing repair capabilities of the product.

[0018] Preferably, the mass ratio of babassu seed oil to grape seed oil is 1:0.5-1:5, for example, it can be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0019] Preferably, the linoleic acid-based eutectic solvent in step (2) is obtained by reacting hydrogen bond acceptors and hydrogen bond donors at 50-90°C (e.g., 50°C, 60°C, 70°C, 80°C, 90°C, etc.) and then reacting it with linoleic acid.

[0020] Preferably, the molar ratio of the hydrogen bond acceptor, hydrogen bond donor and linoleic acid is (1-5):(2-6):(1-5).

[0021] Among them, the specific point values ​​for 1-5 can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc., and the specific point values ​​for 2-6 can be 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0022] Preferably, the hydrogen bond acceptor of the linoleic acid-based eutectic solvent includes any one or a combination of at least two of proline, lauric acid, and decanoic acid.

[0023] Preferably, the hydrogen bond donor of the linoleic acid-based eutectic solvent includes any one or a combination of at least two of ethyl lactate, lauric acid, and decanoic acid.

[0024] Preferably, the linoleic acid-based eutectic solvent is prepared from any one of the following two raw materials:

[0025] (i) Linoleic acid, proline, ethyl lactate;

[0026] (ii) Linoleic acid, lauric acid, and decanoic acid.

[0027] In raw material (i), proline acts as a hydrogen bond acceptor, and ethyl lactate acts as a hydrogen bond donor; in raw material (ii), lauric acid and decanoic acid both serve as hydrogen bond acceptors and donors. The eutectic solvent prepared from the above raw materials can form a more stable water-in-oil vesicle structure, increasing the content of both oil-soluble and water-soluble active ingredients, thereby enhancing the antioxidant level and soothing repair capabilities of the fermented oil.

[0028] Preferably, the linoleic acid-based eutectic solvent is prepared from linoleic acid, proline, and ethyl lactate.

[0029] This invention creatively discovers that linoleic acid-based DES prepared from linoleic acid, proline, and ethyl lactate can further enhance the antioxidant level and soothing repair ability of the fermented oil.

[0030] Preferably, the mass ratio of the primary fermentation broth to vegetable oil and linoleic acid-based eutectic solvent in step (2) is (30-60):(40-60):(1-10).

[0031] Among them, the specific point values ​​in the range of 30-60 can be 30, 35, 40, 45, 50, 55, 60, etc.; the specific point values ​​in the range of 40-60 can be 40, 45, 50, 55, 60, etc.; and the specific point values ​​in the range of 1-10 can be 1, 3, 5, 7, 9, 10, etc.

[0032] Preferably, the matsutake endophytic fungus in the matsutake endophytic fungal seed liquid in step (1) is the matsutake endophytic fungus strain XWJ0001.

[0033] 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.

[0034] 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.

[0035] Preferably, the fermentation medium in step (1) comprises: yeast powder, peptone, glycerol, potassium dihydrogen phosphate, magnesium sulfate and water.

[0036] 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.

[0037] 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.

[0038] 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.).

[0039] Preferably, the fermentation culture in step (1) is carried out under agitation conditions with an aeration rate of 40-80 L (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.).

[0040] 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:

[0041] The endophytic fungal strain of matsutake mushroom was inoculated into YPD medium and cultured to the logarithmic growth phase to obtain seed culture.

[0042] 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.).

[0043] Secondly, the present invention provides a matsutake mushroom endophytic fungal fermentation oil prepared by the preparation method described in the first aspect.

[0044] Thirdly, the present invention provides an application of the matsutake endophytic fungal fermentation oil as described in the second aspect in the preparation of products having any one or at least a combination of the following effects:

[0045] (I) Antioxidant effects;

[0046] (II) Soothing and repairing effects.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] This invention uses matsutake endophytic fungi as a highly efficient substitute for matsutake, and creatively employs vegetable oil and linoleic acid-based eutectic solvent to ferment the primary fermentation broth of matsutake endophytic fungi. By utilizing linoleic acid in vegetable oil and linoleic acid-based eutectic solvent as a precursor of matsutake alcohol, the content of matsutake alcohol in the fermentation product is increased.

[0049] Meanwhile, linoleic acid-based eutectic solvents are added during the fermentation process to regulate metabolism. Compared with traditional fermentation methods, the fermentation products obtained by the above method have a significantly increased content of the active ingredient matsutake alcohol, and the fermented oil of the product has better antioxidant, soothing and repairing capabilities.

[0050] Meanwhile, this invention utilizes the self-assembly of linoleic acid-based eutectic solvents to form water-in-oil vesicles. On the one hand, this increases the solubility of plant oils and linoleic acid, enabling better extraction of oil-soluble active ingredients (matsutake alcohol) from the fermentation broth. On the other hand, it encapsulates water-soluble active ingredients (such as matsutake polysaccharides) in the fermentation broth, enhancing the skin penetration of water-soluble active ingredients and giving the fermented oil product superior antioxidant, soothing, and repairing abilities.

[0051] 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

[0052] 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.

[0053] The method for preparing the Matsutake endophytic fungal seed liquid described in the following specific embodiments is as follows:

[0054] 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.

[0055] Preparation Example 1

[0056] This preparation example provides a linoleic acid-based DES, prepared by the following method:

[0057] Proline and ethyl lactate were reacted at 60°C for 2 h and then mixed with linoleic acid for 30 min (the molar ratio of proline, ethyl lactate, and linoleic acid was 3:4:3) to obtain the final product.

[0058] Preparation Example 2

[0059] This preparation example provides a linoleic acid-based DES, prepared by the following method:

[0060] Proline and ethyl lactate were reacted at 50°C for 4 h and then mixed with linoleic acid for 30 min (the molar ratio of proline, ethyl lactate, and linoleic acid was 1:2:2) to obtain the final product.

[0061] Preparation Example 3

[0062] This preparation example provides a linoleic acid-based DES, prepared by the following method:

[0063] Proline and ethyl lactate were reacted at 70°C for 2 h and then mixed with linoleic acid for 30 min (the molar ratio of proline, ethyl lactate, and linoleic acid was 5:5:4) to obtain the final product.

[0064] Preparation Example 4

[0065] This preparation example provides a linoleic acid-based DES, prepared by the following method:

[0066] Lauric acid and decanoic acid were reacted at 60℃ for 2 h and then mixed with linoleic acid for 30 min (the molar ratio of lauric acid, decanoic acid and linoleic acid was 2:5.5:5) to obtain the product.

[0067] Preparation Example 5

[0068] This preparation example provides a linoleic acid-based DES, prepared by the following method:

[0069] Choline chloride and glycerol were reacted at 60°C for 2 h, and then mixed with linoleic acid for 30 min (the molar ratio of choline chloride, glycerol, and linoleic acid was 3:4:3) to obtain the product.

[0070] Comparative Preparation Example 1

[0071] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0072] Proline and ethyl lactate were mixed in a molar ratio of 3:4 and reacted at 60°C for 2 h to obtain the final product.

[0073] Example 1

[0074] This embodiment provides a fermented oil made from matsutake endophytic fungi, prepared by the following method:

[0075] (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 0.1 MPa, and cultured for 18 h to obtain the primary fermentation liquid.

[0076] The fermentation medium is formulated as follows: 1% yeast extract, 3% peptone, 2% glycerol, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, and the remainder water.

[0077] (2) Mix 45% of the primary fermentation broth with 50% of vegetable oil (Babassu seed oil and grape seed oil in a mass ratio of 1:3) and 5% of the linoleic acid-based DES prepared in Preparation Example 1, and continue to culture at 30°C for 18 h. After centrifugation, take the upper oil phase to obtain the fermented oil of matsutake endophytic fungi.

[0078] Example 2

[0079] This embodiment provides a fermented oil made from matsutake endophytic fungi, prepared by the following method:

[0080] (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 0.2 MPa, and cultured for 24 h to obtain the primary fermentation liquid.

[0081] The fermentation medium is formulated as follows: 2% yeast extract, 1% peptone, 5% glycerol, 0.3% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and the remainder water.

[0082] (2) Mix 50% of the primary fermentation broth with 40% of vegetable oil (Babassu seed oil and grape seed oil in a mass ratio of 1:5) and 10% of the linoleic acid-based DES prepared in Preparation Example 2, and continue to culture at 25°C for 6 h. After centrifugation, take the upper oil phase to obtain the fermented oil of matsutake endophytic fungi.

[0083] Example 3

[0084] This embodiment provides a fermented oil made from matsutake endophytic fungi, prepared by the following method:

[0085] (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 35℃, an aeration rate of 40 L, a stirring speed of 50 rpm, a pressure of 0.05 MPa, and cultured for 18 h to obtain the primary fermentation liquid.

[0086] The fermentation medium is formulated as follows: 0.5% yeast extract, 5% peptone, 1% glycerol, 0.1% potassium dihydrogen phosphate, 0.3% magnesium sulfate, and the remainder water.

[0087] (2) Mix 42% of the primary fermentation broth with 54% of vegetable oil (Babassu seed oil and grape seed oil in a mass ratio of 1:0.5) and 4% of the linoleic acid-based DES prepared in Preparation Example 3, and continue to culture at 35°C for 24 h. After centrifugation, take the upper oil phase to obtain the fermented oil of matsutake endophytic fungi.

[0088] Example 4

[0089] This embodiment provides a fermented oil of matsutake endophytic fungi, which differs from Example 1 only in that the linoleic acid-based DES prepared in Preparation Example 1 is replaced with the linoleic acid-based DES prepared in Preparation Example 4, while the other steps are consistent with Example 1.

[0090] Example 5

[0091] This embodiment provides a fermented oil of matsutake endophytic fungi, which differs from Example 1 only in that the linoleic acid-based DES prepared in Preparation Example 1 is replaced with the linoleic acid-based DES prepared in Preparation Example 5, while the other steps are consistent with Example 1.

[0092] Example 6

[0093] This embodiment provides a matsutake endophytic fungal fermented oil, which differs from Embodiment 1 only in that the amount of vegetable oil added remains unchanged at 50%, and the vegetable oil is adjusted to be a single babassu seed oil. All other steps are consistent with Embodiment 1.

[0094] Example 7

[0095] This embodiment provides a matsutake endophytic fungal fermented oil, which differs from Embodiment 1 only in that the amount of vegetable oil added remains unchanged at 50%, and the vegetable oil is adjusted to be a single grape seed oil. All other steps are consistent with Embodiment 1.

[0096] Comparative Example 1

[0097] This comparative example provides a fermented oil of matsutake endophytic fungi, which differs from Example 1 only in that the linoleic acid-based DES prepared in Preparation Example 1 is replaced with the eutectic solvent prepared in Comparative Preparation Example 1, while the other steps are consistent with Example 1.

[0098] Comparative Example 2

[0099] This comparative example provides a fermented oil of matsutake endophytic fungi, which differs from Example 1 only in that the linoleic acid-based DES prepared in Example 1 is replaced with an equal mass of a mixture of proline, ethyl lactate, and linoleic acid (proline, ethyl lactate, and linoleic acid are mixed in a molar ratio of 3:4:3). All other steps are the same as in Example 1.

[0100] Comparative Example 3

[0101] This comparative example provides a matsutake endophytic fungal fermented oil, which differs from Example 1 only in that linoleic acid DES is not added in step (2), that is, 45% primary fermentation liquid is mixed with 55% vegetable oil for fermentation, and the remaining steps are consistent with Example 1.

[0102] Comparative Example 4

[0103] This comparative example provides a fermented oil of matsutake endophytic fungi, which differs from Example 1 only in that the vegetable oil is replaced with synthetic GTCC (caprylic / capric triglyceride) in step (2), that is, 45% primary fermentation broth, 50% GTCC and 5% linoleic acid-based DES prepared in Example 1 are mixed and fermented. The remaining steps are consistent with Example 1.

[0104] Comparative Example 5

[0105] This comparative example provides a matsutake mushroom endophytic bacteria fermentation broth, prepared by the following method:

[0106] Seed culture of the endophytic fungus strain XWJ0001 of matsutake mushroom was inoculated into 10 mL of YPD medium at a 10% inoculation rate and cultured at 30℃ for 5 days. The culture was then centrifuged and filtered to obtain the final product.

[0107] Comparative Example 6

[0108] This comparative example provides a direct-extract oil from matsutake mushrooms, prepared by the following method:

[0109] Wipe fresh matsutake mushrooms clean with a damp cloth, slice them thinly, and soak them in twice their weight of vegetable oil (babassu seed oil and grape seed oil in a 1:3 weight ratio). Simmer over low heat for 60 minutes, then filter and collect the filtrate.

[0110] Test Example 1

[0111] Matsutake alcohol content test:

[0112] (1) Test method:

[0113] S1. The matsutake fermented oil prepared in Examples 1-7 and the products obtained in Comparative Examples 1-6 were added to acetonitrile at a volume ratio of 1:1 into a 50 mL centrifuge tube. The mixture was shaken at 200 r / min for 5 min. 25 mg of C18 adsorbent was added to remove excess oil and obtain purified solution. 1 mL of purified solution was filtered through a 0.22 μm filter membrane into a sample vial for GC-MS / MS determination.

[0114] S2. Preparation of matsutake alcohol standard solution: Weigh 0.01 g of matsutake alcohol standard (accurate to 0.0001 g), dissolve in methanol and dilute to 10 mL, store at -18℃. Take a certain amount of matsutake alcohol standard solution and dilute it stepwise with acetonitrile to obtain a series of standard solutions, which should be prepared and used immediately.

[0115] S3. Instrument and mass spectrometry conditions: DB-WAXX capillary column (30m×250μm×0.25μm); injection port temperature 210℃; carrier gas He (purity ≥99.999%), flow rate 1.0mL / min; temperature program conditions: initial temperature 50℃, hold for 2min, increase to 170℃ at 15℃ / min, continue to increase to 210℃ at 30℃ / min, hold for 3min; injection volume 1.0μL, splitless injection.

[0116] (2) Test results:

[0117] The results of the test for the content of matsutake alcohol in the products obtained in each embodiment and comparative example are shown in Table 1.

[0118] Table 1

[0119]

[0120]

[0121] As shown in Table 1, the fermented oil of this invention contains more than 20 ppm of matsutake alcohol, which is significantly higher than that of traditional extraction technology for direct extraction of oil from live matsutake mushrooms.

[0122] A comparison of the data from Examples 1 and 4-5 shows that the linoleic acid-based DES formed by proline, ethyl lactate, and linoleic acid has a superior ability to enrich matsutake alcohol compared to other formulations, while the linoleic acid-based DES formed by lauric acid, decanoic acid, and linoleic acid has a better effect on increasing the matsutake alcohol content in the product.

[0123] Comparison of data from Examples 1, 6-7, Comparative Examples 1, and 4 shows that linoleic acid and vegetable oil, as precursors for matsutake synthesis, can significantly increase matsutake alcohol content when added. In addition, babassu seed oil and grape seed oil have a significant synergistic effect in increasing matsutake alcohol content.

[0124] A comparison of the data from Example 1 with Comparative Examples 2 and 3 shows that by forming linoleic acid DES, the enrichment effect of matsutake alcohol can be significantly improved, thereby increasing the content of matsutake alcohol in the product.

[0125] Test Example 2

[0126] Polysaccharide skin cumulative transdermal rate test:

[0127] (1) Test method:

[0128] According to the Franz diffusion cell transdermal absorption assay, porcine ear skin was used for the experiment. Before the experiment, the porcine skin was cut into small circular pieces the size of the receiving cell and placed in physiological saline. Physiological saline was used as the receiving medium in this experiment. During the experiment, the skin model was fixed between the release cell and the receiving cell, with the stratum corneum side facing the release cell and the dermis side facing the receiving cell, ensuring that the skin and the receiving solution were in close contact and that no air bubbles were present between them.

[0129] 2g of samples from Examples 1-7 and Comparative Examples 1-6 were added to each release tank. The temperature of the receiving tank was maintained at 32℃. A magnetic ball was placed in the receiving tank, and the rotation speed was maintained at 300 rpm throughout the experiment. After 24 hours, the skin was removed, and the skin surface was cleaned with cotton soaked in physiological saline, then dried with filter paper. Pigskin was cut into small pieces with a particle diameter of less than 0.2cm, placed in a 10mL grinding tube, and 1mL of physiological saline was added. The mixture was ground using a rapid biological sample preparation system at 6.5m / s for 2 minutes, 10 cycles, followed by sonication for 20 minutes. The grinding solution was removed, and 1mL of physiological saline was added to the grinding tube. This process was repeated once. The two grinding solutions were combined, centrifuged at 12000rpm for 10 minutes, and the supernatant was collected. The concentration of matsutake polysaccharide in the supernatant, the sample tank, and the receiving tank was determined using the phenol-sulfuric acid method. The cumulative transdermal permeability of matsutake polysaccharide was calculated.

[0130] Cumulative transdermal rate calculation formula:

[0131] Cumulative transdermal penetration rate (%) = (polysaccharide concentration in supernatant + polysaccharide concentration in receiving cell) / initial polysaccharide concentration in sample × 100%;

[0132] (2) Test results:

[0133] The cumulative skin transdermal permeability test results of polysaccharides in the products obtained from each embodiment and comparative example are shown in Table 2.

[0134] Table 2

[0135]

[0136] As shown in Table 2, the fermented oil involved in this invention can significantly improve the skin permeability of polysaccharides, effectively solving the problem of polysaccharide penetration.

[0137] A comparison of the data from Examples 1 and 4 and 5 shows that the linoleic acid-based DES formed by proline, ethyl lactate, and linoleic acid, compared with the linoleic acid-based DES formed by lauric acid, decanoic acid, and linoleic acid, has a better transport capacity for water-soluble polysaccharides by self-assembling water-in-oil vesicles during fermentation, compared with other formulations. In contrast, the linoleic acid-based DES formed by proline, ethyl lactate, and linoleic acid has a better effect on increasing polysaccharide content and transdermal absorption of polysaccharides.

[0138] Test Example 3

[0139] DPPH free radical scavenging ability test:

[0140] (1) Test method:

[0141] Preparation of DPPH solution: Weigh 5.0 mg of DPPH, dissolve it in an appropriate amount of anhydrous ethanol, sonicate it in the dark until fully dissolved, and then dilute to 100 mL with anhydrous ethanol to prepare a 50 μg / mL DPPH solution. This solution should be prepared and used immediately.

[0142] The sample group consisted of 100 μL of sample solution mixed with 100 μL of DPPH solution (50.0 μg / mL); the blank group consisted of 100 μL of sample solution mixed with 100 μL of anhydrous ethanol; and the control group consisted of 100 μL of DPPH solution (50.0 μg / mL) mixed with 100 μL of anhydrous ethanol. The mixture was allowed to react at room temperature in the dark for 0.5 h, and the absorbance was measured at 517 nm. The scavenging rate was calculated.

[0143] Formula for calculating DPPH free radical scavenging rate:

[0144] Clearance rate (%) = [1 - (Ai - Aj) / Ac] × 100%;

[0145] Where Aj is the absorbance value of the blank sample group; Ai is the absorbance value of the sample group; and Ac is the absorbance value of the control group.

[0146] (2) Test results:

[0147] The DPPH free radical scavenging rate test results of the matsutake endophytic fungal fermented oil / matsutake extract / matsutake direct extract oil obtained in each embodiment and comparative example are shown in Table 3.

[0148] Table 3

[0149]

[0150] As shown in Table 4, compared with Comparative Examples 1-6, the matsutake endophytic fungal fermentation oil prepared by the method of this invention has excellent DPPH free radical scavenging ability, that is, excellent antioxidant properties. Furthermore, a comparison of the data from Example 1 and Examples 4-7 shows that the selection of linoleic acid DES and the formulation of the vegetable oil also affect the antioxidant capacity of the matsutake endophytic fungal fermentation oil to some extent.

[0151] Test Example 4

[0152] Soothing and repairing efficacy test:

[0153] (1) Test method:

[0154] Healthy zebrafish embryos, 3 days post-fertilization, were selected and divided into a model group, a positive control group, and 13 test substance groups. In the model group, 6 embryos were randomly selected and placed into 3 cm culture dishes, with 5 mL of 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 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 embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate and 5 mg / mL of the test substance (the product obtained in each example and comparative example).

[0155] 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.

[0156] 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.

[0157] Formula for calculating neutrophil aggregation inhibition rate:

[0158] Inhibition rate (%) = [(MS) / M] × 100%;

[0159] 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; the average values ​​of each test group were calculated, and the results are shown in Table 4.

[0160] Table 4

[0161]

[0162]

[0163] As shown in Table 4, compared with the comparative example, the matsutake endophytic fungal fermented oil prepared by the method of this invention has excellent zebrafish embryo neutrophil aggregation effect, reflecting its potential in soothing and repairing effects. Furthermore, a comparison of the data from Example 1 and Examples 4-7 shows that the selection of linoleic acid DES and the formulation of the vegetable oil also affect the soothing and repairing effects of the matsutake endophytic fungal fermented oil to some extent.

[0164] 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.

[0165] 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.

[0166] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing fermented oil from matsutake mushroom endophytic fungi, characterized in that, The preparation method includes: (1) The seed liquid of matsutake endophytic fungi was inoculated into a fermentation medium for fermentation culture to obtain a primary fermentation broth; (2) The primary fermentation broth was mixed with vegetable oil and linoleic acid-based eutectic solvent, and fermentation was continued. The upper oil phase was collected by centrifugation to obtain the fermented oil of matsutake endophytic fungi. The vegetable oil mentioned in step (2) is a combination of babassu seed oil and grape seed oil in a mass ratio of 1:(0.5-1:5); The linoleic acid-based eutectic solvent in step (2) is obtained by reacting proline and ethyl lactate at 50-90°C, followed by reaction with linoleic acid. The molar ratio of proline, ethyl lactate and linoleic acid is (1-5):(2-6):(1-5); The endophytic fungus (Umbelopsis sp.) mentioned is strain XWJ0001 with preservation number CGMCC No.42463.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the primary fermentation broth to vegetable oil and linoleic acid-based eutectic solvent in step (2) is (30-60):(40-60):(1-10).

3. The preparation method according to claim 1, characterized in that, In step (1), the inoculation volume of the matsutake endophytic fungal seed solution is 1-10% of the fermentation medium volume; The fermentation culture temperature in step (1) is 25-35℃, and the fermentation culture time is 6-24 h; The fermentation temperature in step (2) is 25-35℃, and the fermentation time is 6-24 h.

4. A fermented oil of matsutake endophytic fungi prepared by any one of claims 1-3.

5. The use of the matsutake endophytic fungal fermented oil according to claim 4 in the preparation of products with antioxidant and / or soothing and repairing effects.

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