Tricholoma matsutake endophytic fungus fermentation oil with whitening effect, and preparation method and application thereof

Abstract

The present application relates to a kind of pine mushroom endophytic fungi fermentation oil with whitening effect and its preparation method and application.The preparation method includes: (1) the pine mushroom endophytic fungi seed liquid is inoculated in fermentation medium and is carried out fermentation culture, and the first order fermentation liquor is obtained;(2) the first order fermentation liquor is mixed with vegetable oil, phosphatidylcholine base eutectic solvent, continues fermentation culture, and the upper oil phase is collected by centrifugation, and the pine mushroom endophytic fungi fermentation oil is obtained.The pine mushroom endophytic fungi fermentation oil prepared by the present application has the transdermal ability of high level oil-soluble efficacy component and water-soluble efficacy component, and has excellent whitening effect.

Description

Technical Field

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

[0002] Matsutake mushrooms, also known as pine mushrooms, are fungi belonging to the Tricholomataceae family and the Tricholomataceae genus. They are well-known wild edible and medicinal fungi, and typically grow at high altitudes in the roots of temperate and cold-temperate pine and oak forests.

[0003] As one of the renowned higher fungi, matsutake mushrooms contain dozens of active nutrients, rich in protein and carbohydrates. Studies have shown that matsutake contains a variety of active substances with important physiological functions, among which polysaccharides and polyphenols are two core functional components: matsutake polysaccharides, as natural high-molecular-weight compounds, possess various biological activities such as immunomodulation, antioxidation, and antitumor activity, and have broad application prospects in the fields of health products and medicine; matsutake polyphenols, on the other hand, are a class of secondary metabolites rich in phenolic hydroxyl groups, possessing significant free radical scavenging ability, anti-inflammatory and antibacterial activities, and have important development value in the fields of food preservation and cosmetic raw materials.

[0004] Currently, the acquisition of matsutake polysaccharides and polyphenols mainly relies on direct extraction from the matsutake fruiting body. However, matsutake has extremely demanding requirements for its growth environment, making large-scale artificial cultivation impossible. Furthermore, the slow growth and metabolic focus of matsutake fruiting bodies on structural substances and secondary metabolism result in low synthesis efficiency of polyphenols and polysaccharides. 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 structural damage to polyphenols and polysaccharides due to process limitations, significantly reducing the utilization rate of matsutake.

[0005] 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 culture 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.

[0006] Therefore, developing a fermentation process for matsutake mushroom endophytic fungi to achieve targeted and efficient enrichment of matsutake polyphenols and polysaccharides and improve the whitening effect of fermentation products has become one of the urgent technical problems to be solved. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a fermented oil of matsutake endophytic fungi with whitening effect, its preparation method and 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 of matsutake endophytic fungi with whitening effects, the preparation method comprising:

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

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

[0012] This invention uses matsutake endophytic fungi as a highly effective substitute for matsutake, and creatively utilizes vegetable oil and phosphatidylcholine eutectic solvent to ferment the primary fermentation broth of matsutake endophytic fungi. The eutectic solvent can regulate the metabolism of matsutake endophytic fungi, directionally enriching and synthesizing polyphenols and polysaccharides. Furthermore, the phosphatidylcholine eutectic solvent can self-assemble into water-in-oil vesicles, efficiently promoting the permeation of polyphenols and polysaccharides into the matsutake endophytic fungal fermentation broth, thus enhancing the whitening effect of the fermented oil. The vegetable oil further regulates the metabolism of matsutake endophytic fungi, enhancing the directional enrichment and synthesis of active ingredients, thereby improving the whitening effect of the product.

[0013] Preferably, the hydrogen bond donor of the phosphatidylcholine eutectic solvent in step (2) is selected from fatty acids.

[0014] Preferably, the fatty acid is selected from any one or a combination of at least two of palmitic acid, linolenic acid, myristic acid, or coconut acid.

[0015] Preferably, the hydrogen bond acceptor of the phosphatidylcholine eutectic solvent is selected from phosphatidylcholine.

[0016] Studies have found that the whitening effect of fermented oils prepared by phosphatidylcholine eutectic solvents obtained by any combination of the above-mentioned hydrogen bond acceptors and hydrogen bond donors varies. More preferably, the following specific eutectic solvent types can further enhance the whitening effect of fermented oils.

[0017] Preferably, the phosphatidylcholine eutectic solvent in step (2) is a mixture of a first eutectic solvent formed by the reaction of palmitoleic acid and phosphatidylcholine and a second eutectic solvent formed by the reaction of myristic acid and phosphatidylcholine.

[0018] This invention creatively uses different hydrogen bond donors as raw materials to prepare a first eutectic solvent and a second eutectic solvent, respectively. Studies have found that, compared with a single first eutectic solvent or a single second eutectic solvent, the fermented oil of the product has a better whitening effect.

[0019] Preferably, the molar ratio of palmitoleic acid to phosphatidylcholine in the first eutectic solvent is 3:1-5:1, for example, it can be 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc.

[0020] Preferably, the molar ratio of myristic acid and phosphatidylcholine in the second eutectic solvent is 1:1 to 3:1, for example, it can be 1:1, 1.5:1, 2:1, 2.5:1, 3:1, etc.

[0021] Preferably, the reaction temperature of palmitoleic acid and phosphatidylcholine is 50-90℃ (e.g., 50℃, 60℃, 70℃, 80℃, 90℃, etc.), and the reaction time is 1-4 h (e.g., 1 h, 2 h, 3 h, 4 h, etc.).

[0022] Preferably, the reaction temperature of myristic acid and phosphatidylcholine is 50-90℃ (e.g., 50℃, 60℃, 70℃, 80℃, 90℃, etc.), and the reaction time is 1-4 h (e.g., 1 h, 2 h, 3 h, 4 h, etc.).

[0023] Preferably, the volume ratio of the first eutectic solvent and 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.

[0024] Preferably, the vegetable oil in step (2) includes any one or a combination of at least two of grape seed oil, rosehip oil, sea buckthorn oil, olive oil, sweet almond oil, avocado oil, or camellia oil.

[0025] Preferably, the vegetable oil in step (2) comprises a combination of rosehip oil and sea buckthorn oil.

[0026] This invention creatively discovers that the combination of rosehip oil and sea buckthorn oil in the preparation of fermented oil from matsutake mushroom endophytic fungi can better enhance the whitening effect of the fermentation liquid. The content of matsutake polysaccharides and matsutake polyphenols in the product is higher than that of rosehip oil or sea buckthorn oil alone, indicating that rosehip oil and sea buckthorn oil have a significant synergistic effect in enhancing the whitening effect of the product.

[0027] Preferably, the mass ratio of rosehip oil to sea buckthorn oil 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.

[0028] Preferably, the mass ratio of the primary fermentation broth to vegetable oil and phosphatidylcholine eutectic solvent in step (2) is (30-60):(40-60):(1-10).

[0029] 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.

[0030] Preferably, the matsutake endophytic fungus in the matsutake endophytic fungal seed liquid in step (1) is the matsutake endophytic fungus strain XWJ0001.

[0031] 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.

[0032] 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.

[0033] Preferably, the fermentation medium in step (1) comprises: yeast powder, peptone, glycerol, potassium dihydrogen phosphate, magnesium sulfate and water.

[0034] 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.

[0035] 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.

[0036] 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.).

[0037] 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.).

[0038] 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:

[0039] The endophytic fungal strain of matsutake mushroom was inoculated into YPD medium and cultured to the logarithmic growth phase to obtain seed culture.

[0040] 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.).

[0041] Secondly, the present invention provides a matsutake mushroom endophytic fungal fermentation oil prepared by the preparation method described in the first aspect.

[0042] Thirdly, the present invention provides the application of matsutake endophytic fungal fermented oil as described in the second aspect in the preparation of products with whitening effects.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention uses matsutake endophytic fungi as a highly effective substitute for matsutake, and creatively utilizes vegetable oil and phosphatidylcholine eutectic solvent to ferment the primary fermentation broth of matsutake endophytic fungi. The eutectic solvent can regulate the metabolism of matsutake endophytic fungi, directionally enrich and synthesize polyphenols, and the phosphatidylcholine eutectic solvent can self-assemble into water-in-oil vesicles, efficiently promoting the permeation of polyphenols and polysaccharides in the matsutake endophytic fungal fermentation broth, thereby enhancing the whitening effect of the fermented oil. The vegetable oil can further regulate the metabolism of matsutake endophytic fungi, improve the directional enrichment and synthesis of active ingredients, and enhance the whitening effect of the product.

[0045] 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

[0046] 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.

[0047] The method for preparing the Matsutake endophytic fungal seed liquid described in the following specific embodiments is as follows:

[0048] 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.

[0049] Preparation Example 1

[0050] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0051] (1) Palmitoleic acid and phosphatidylcholine were mixed in a molar ratio of 4:1 and reacted at 70°C for 2 h to obtain the first eutectic solvent;

[0052] (2) Myristic acid and phosphatidylcholine were mixed in a molar ratio of 2:1 and reacted at 70°C for 2 h to obtain a second eutectic solvent;

[0053] (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.

[0054] Preparation Example 2

[0055] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0056] (1) Palmitoleic acid and phosphatidylcholine were mixed in a molar ratio of 3:1 and reacted at 50°C for 4 h to obtain the first eutectic solvent;

[0057] (2) Myristic acid and phosphatidylcholine were mixed in a 1:1 molar ratio and reacted at 50°C for 4 h to obtain a second eutectic solvent;

[0058] (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.

[0059] Preparation Example 3

[0060] This preparation example provides a composite eutectic solvent, and the preparation method is as follows:

[0061] (1) Palmitoleic acid and phosphatidylcholine were mixed in a molar ratio of 5:1 and reacted at 90°C for 1 h to obtain the first eutectic solvent;

[0062] (2) Myristic acid and phosphatidylcholine were mixed in a molar ratio of 3:1 and reacted at 90°C for 1 h to obtain a second eutectic solvent;

[0063] (3) The first eutectic solvent and the second eutectic solvent are mixed in a volume ratio of 3:1 to obtain a composite eutectic solvent.

[0064] Preparation Example 4

[0065] This preparation example provides a composite eutectic solvent, which differs from Preparation Example 1 only in that step (1) replaces palmitoleic acid with an equimolar amount of linolenic acid, while the rest of the preparation method is consistent with Preparation Example 1.

[0066] Preparation Example 5

[0067] This preparation example provides a composite eutectic solvent, which differs from Preparation Example 1 only in that in step (2), myristic acid is replaced with an equimolar amount of linolenic acid, while the rest of the preparation method is consistent with Preparation Example 1.

[0068] Preparation Example 6

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

[0070] Palmitoleic acid and phosphatidylcholine are mixed in a molar ratio of 4:1 and reacted at 70°C for 2 h to obtain the product.

[0071] Preparation Example 7

[0072] This preparation example provides a eutectic solvent, and the preparation method is as follows:

[0073] Myristic acid and phosphatidylcholine are mixed in a molar ratio of 2:1 and reacted at 70°C for 2 h to obtain the product.

[0074] Comparative Preparation Example 1

[0075] This preparation example provides a composite eutectic solvent, which differs from Preparation Example 1 only in that the phosphatidylcholine in steps (1) and (2) is replaced with an equimolar amount of betaine, while the rest of the preparation method is consistent with Preparation Example 1.

[0076] Example 1

[0077] This embodiment provides a fermented oil made from matsutake endophytic fungi, prepared by the following method:

[0078] (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.

[0079] 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.

[0080] (2) Mix 44% of the primary fermentation broth with 50% of vegetable oil (rosehip oil and sea buckthorn oil in a mass ratio of 1:1) and 6% of the composite eutectic solvent prepared in Preparation Example 1, and continue to culture at 30°C for 12 h. After centrifugation, take the upper oil phase to obtain the fermentation oil of matsutake endophytic fungi.

[0081] Example 2

[0082] This embodiment provides a fermented oil made from matsutake endophytic fungi, prepared by the following method:

[0083] (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.

[0084] 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.

[0085] (2) Mix 50% of the primary fermentation broth with 40% of vegetable oil (rosehip oil and sea buckthorn oil in a mass ratio of 1:3) and 10% of the composite eutectic solvent prepared in Example 2, and continue to culture at 25°C for 6 h. After centrifugation, take the upper oil phase to obtain the fermentation oil of matsutake endophytic fungi.

[0086] Example 3

[0087] This embodiment provides a fermented oil made from matsutake endophytic fungi, prepared by the following method:

[0088] (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 0.3 MPa, and cultured for 6 h to obtain the first-stage fermentation liquid.

[0089] 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.

[0090] (2) Mix 42% of the primary fermentation broth with 54% of vegetable oil (rosehip oil and sea buckthorn oil in a mass ratio of 3:1) and 4% of the composite eutectic solvent prepared in Example 3, and continue to culture at 35°C for 24 h. After centrifugation, take the upper oil phase to obtain the fermentation oil of matsutake endophytic fungi.

[0091] Examples 4-7

[0092] Examples 4-7 each provide a matsutake endophytic fungal fermentation oil, which differs from Example 1 only in that the composite eutectic solvent prepared in Example 1 is replaced with the composite eutectic solvent or eutectic solvent prepared in Examples 4-7 respectively. All other steps are the same as in Example 1.

[0093] Example 8

[0094] 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 rosehip oil. All other steps are consistent with Embodiment 1.

[0095] Example 9

[0096] This embodiment provides a fermented oil of matsutake endophytic fungi, 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 sea buckthorn fruit oil. All other steps are consistent with Embodiment 1.

[0097] Comparative Example 1

[0098] This comparative example provides a fermented oil of matsutake endophytic fungi, which differs from Example 1 only in that the composite eutectic solvent prepared in Preparation Example 1 is replaced with the composite eutectic solvent prepared in Comparative Preparation Example 1, while the other steps are consistent with Example 1.

[0099] Comparative Example 2

[0100] This comparative example provides a matsutake endophytic fungal fermentation oil, which differs from Example 1 only in that a composite eutectic solvent is not added in step (2), that is, 44% of the primary fermentation liquid is mixed with 56% of vegetable oil for fermentation, and the remaining steps are consistent with Example 1.

[0101] Comparative Example 3

[0102] 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, 44% of primary fermentation broth, 50% GTCC and 6% of the composite eutectic solvent prepared in Example 1 are mixed and fermented. The remaining steps are consistent with Example 1.

[0103] Comparative Example 4

[0104] This comparative example provides a matsutake mushroom fermentation liquid, prepared by the following method:

[0105] 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.

[0106] Comparative Example 5

[0107] This comparative example provides a direct extraction method for matsutake mushroom oil, and the preparation method is as follows:

[0108] Wipe fresh matsutake mushrooms clean with a damp cloth, slice them thinly, and soak them in twice the weight of vegetable oil (rosehip oil and sea buckthorn oil in a 1:1 weight ratio). Simmer over low heat for 60 minutes, then filter and collect the filtrate.

[0109] Test Example 1

[0110] Total polyphenol content test:

[0111] (1) Test method:

[0112] (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 40℃ water bath 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.

[0113] (1.2) Accurately weigh 250 mg of the products obtained in Examples 1-9 and Comparative Examples 1-5, 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 products obtained in Examples 1-9 and Comparative Examples 1-5, 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 to each, 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.

[0114] (2) Test results:

[0115] The total polyphenol content test results of the products obtained in each embodiment and comparative example are shown in Table 1.

[0116] Table 1

[0117]

[0118]

[0119] As shown in Table 1, the total polyphenol content in the fermented oil involved in this invention is above 1000 mg GA / kg, which is significantly higher than that in traditional extraction techniques.

[0120] A comparison of the data from Examples 1 and 4-7 shows that by using different hydrogen bond donors as raw materials to prepare the first eutectic solvent and the second eutectic solvent, the fermented oil produced has a higher total polyphenol content compared to either the first eutectic solvent or the second eutectic solvent alone. Furthermore, the combined use of the first eutectic solvent prepared from palmitoleic acid and phosphatidylcholine and the second eutectic solvent prepared from myristic acid and phosphatidylcholine can further increase the polyphenol content in the fermented oil.

[0121] Comparison of data from Examples 1, 8-9, and Comparative Example 3 shows that the addition of vegetable oil can further regulate the metabolism of matsutake endophytic fungi and improve the targeted enrichment and synthesis of total polyphenols. In addition, the combination of rosehip oil and sea buckthorn oil in the preparation of matsutake endophytic fungal fermentation oil has a significant synergistic effect in increasing the polyphenol content in the product.

[0122] A comparison of the data from Example 1 with Comparative Examples 1 and 2 shows that the addition of phosphatidylcholine eutectic solvents can enhance the enrichment capacity of polyphenols and increase the polyphenol content in the fermented oil of the product compared to other types of eutectic solvents.

[0123] Test Example 2

[0124] Total polyphenols and total polysaccharides skin cumulative transdermal rate test:

[0125] (1) Test method:

[0126] (1.1) According to the Franz diffusion cell transdermal absorption assay, pig ear skin was used for the experiment. Before the experiment, the pig skin was cut into small circular pieces the size of the receiving cell and placed in physiological saline for later use. 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, and ensuring that the skin and the receiving fluid were in close contact, with no air bubbles appearing between them.

[0127] (1.2) 2g of samples from Examples 1-9 and Comparative Examples 1-5 were added to the skin models of the control and experimental groups in the release cell, respectively. The temperature of the receiving cell was maintained at 32℃, and a magnetic ball was placed in the receiving cell. The rotation speed was maintained at 300rpm / min during the experiment.

[0128] (1.3) After 24 hours, remove the skin and clean the skin surface with cotton soaked in physiological saline. Then, dry the skin surface with filter paper. Cut the pigskin into small pieces with a particle diameter of less than 0.2 cm, put them into a 10 ml grinding tube, add 1 mL of physiological saline, and grind using a rapid biological sample preparation system at 6.5 m / s for 2 min, 10 cycles, followed by sonication for 20 min. Take out the grinding solution, add 1 mL of physiological saline to the grinding tube, and repeat the above operation once. Combine the two grinding solutions, centrifuge at 12000 rpm for 10 min, and collect the supernatant.

[0129] (1.4) Cumulative skin transdermal permeability of polysaccharides: The concentration of matsutake polysaccharides in the supernatant, sample tank and receiving tank was determined by phenol-sulfuric acid method, and the cumulative skin transdermal permeability of matsutake polysaccharides was calculated.

[0130] (1.5) Cumulative skin transdermal permeability of polyphenols: The concentration of matsutake polyphenols in the supernatant, sample cell and receiving cell was detected by the Folin-Ciocalteu method according to the procedure of Test Example 1, and the cumulative skin transdermal permeability of matsutake polyphenols was calculated.

[0131] Cumulative transdermal permeability calculation formula: Cumulative transdermal permeability (%) = (Supernatant component concentration + Receiving cell component concentration) / Initial sample component concentration × 100%;

[0132] (2) Test results:

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

[0134] Table 2

[0135]

[0136] As can be seen from the data in Table 2, the fermented oil involved in this invention can significantly improve the skin penetration of polysaccharide and polyphenol components, effectively solving the penetration problem of polysaccharide and polyphenol.

[0137] A comparison of the data from Examples 1 and 4-7 shows that by using different hydrogen bond donors as raw materials to prepare the first eutectic solvent and the second eutectic solvent, the fermented oil of the product has better polysaccharide and polyphenol permeability compared to either the first eutectic solvent or the second eutectic solvent alone. In addition, the combined use of the first eutectic solvent prepared from palmitoleic acid and phosphatidylcholine and the second eutectic solvent prepared from myristic acid and phosphatidylcholine yields even better results.

[0138] Comparison of data from Examples 1, 8-9, and Comparative Example 3 shows that the addition of vegetable oil can further regulate the metabolism of matsutake endophytic fungi, improve the directional enrichment capacity of polysaccharides and polyphenols, and thus increase the permeation rate. In addition, the combination of rosehip oil and sea buckthorn oil in the preparation of matsutake endophytic fungal fermentation oil has a significant synergistic effect on the above-mentioned effects.

[0139] A comparison of the data from Example 1 with Comparative Examples 1 and 2 shows that the addition of phosphatidylcholine-based eutectic solvents significantly improves the skin penetration of polysaccharides and polyphenols compared to other types of eutectic solvents.

[0140] Test Example 3

[0141] Whitening effect test:

[0142] Tyrosinase is a key enzyme in pigment biosynthesis. Inhibiting tyrosinase is a common method to reduce pigmentation spots. Therefore, the whitening effect can be evaluated by analyzing the ability of a sample to inhibit tyrosinase.

[0143] (1) Test method:

[0144] (1.1) Preparation of test samples: The matsutake endophytic fungal fermentation oil / matsutake extract / matsutake direct extract oil obtained in each example and comparative example were dissolved in DMSO and then added to DMEM medium to make the final concentration of each test sample 10 μg / mL.

[0145] (1.2) Mouse melanoma cells (B16F10) were inoculated into DMEM medium and cultured at 37°C and 5% CO2. The cultured B16F10 cells were digested with trypsin containing 0.25% EDTA and then popped into 96-well plates at a density of 5 × 10⁶ cells per well. 3Cells were seeded. Once the cells in each well of a 96-well plate reached 50% capacity, the test samples were added, with an equal volume of fresh culture medium added to the control group. After 24 h of culture, the cells were washed twice with PBS, and 50 μL / mL of 1% Triton X-100 solution was added. The plates were then rapidly frozen at -20°C for 1 h. This rapid freezing process caused cell rupture. After the cells were removed and allowed to return to room temperature, 10 μL of 0.1% L-DOPA solution was added, and the plates were incubated at 37°C for 4 h. The absorbance (A value) was measured at 492 nm. The tyrosinase inhibition rate was calculated.

[0146] Tyrosinase inhibition rate (%) = 1 - A1 / A2 × 100%.

[0147] In the formula, A1 is the absorbance value of each sample group at a wavelength of 492 nm, and A2 is the absorbance value of the blank group at a wavelength of 492 nm.

[0148] (2) Test results:

[0149] The inhibition rates of different test samples on tyrosinase are shown in Table 3.

[0150] Table 3

[0151]

[0152] As shown in Table 3, compared with the comparative example, the matsutake endophytic fungal fermented oil prepared by the method of this invention has excellent tyrosinase inhibition ability, that is, excellent whitening effect. Furthermore, a comparison of the data from Example 1 and Examples 4-8 shows that the selection of eutectic solvent raw materials and the formulation of vegetable oil also affect the whitening effect of the matsutake endophytic fungal fermented oil to a certain extent.

[0153] 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.

[0154] 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.

[0155] 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 with whitening effects, characterized in that, The preparation method includes: (1) The seed liquid of matsutake endophytic fungi was inoculated into the fermentation medium for fermentation culture to obtain the primary fermentation liquid; (2) The primary fermentation broth was mixed with vegetable oil and phosphatidylcholine eutectic solvent, and fermentation was continued. The upper oil phase was collected by centrifugation to obtain the fermentation oil of matsutake endophytic fungi. The phosphatidylcholine eutectic solvent in step (2) is a mixture of a first eutectic solvent formed by the reaction of palmitoleic acid and phosphatidylcholine and a second eutectic solvent formed by the reaction of myristic acid and phosphatidylcholine; the volume ratio of the first eutectic solvent to the second eutectic solvent is 1:3-3:

1. The vegetable oil mentioned in step (2) is a combination of rosehip oil and sea buckthorn oil in a mass ratio of 1:3 to 3:1; The matsutake endophytic fungus in the matsutake endophytic fungus seed liquid in step (1) is the matsutake endophytic fungus strain XWJ0001, which is classified as Umbelopsis sp. and has the preservation number CGMCC No.42463.

2. The preparation method according to claim 1, characterized in that, The molar ratio of palmitoleic acid to phosphatidylcholine in the first eutectic solvent is 3:1-5:1; The molar ratio of myristic acid to phosphatidylcholine in the second eutectic solvent is 1:1 to 3:

1.

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

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

5. The application of the matsutake endophytic fungal fermented oil according to claim 4 in the preparation of products with whitening effects.

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