A soothing, repairing and antioxidant filtrate of matsutake mycelium fermentation, and its preparation method and application

Abstract

The present invention relates to a fermentation filtrate of pine mushroom mycelium with soothing, repairing and antioxidant effects, as well as a preparation method and application thereof. The preparation method comprises the following steps: inoculating pine mushroom liquid seeds into a sterilized fermentation medium for a first-stage culture to obtain a first fermentation liquid; the first-stage culture is continued at 25-28°C for 5-10 days; then the first fermentation liquid is cultured in a second stage to obtain a second fermentation liquid; the second-stage culture is continued at 20-24°C for 5-8 days; and the second fermentation liquid is filtered to separate the pine mushroom mycelium and the fermentation filtrate. The present invention creatively performs a two-stage fermentation of pine mushroom liquid seeds. By controlling the fermentation time and fermentation temperature of each stage, it is possible to well balance the rapid growth of pine mushrooms and the rapid production of active ingredients such as pine mushroom polysaccharides. The final fermentation product filtrate has excellent soothing, moisturizing, repairing and antioxidant effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fungal fermentation, and relates to a matsutake mycelium fermentation filtrate with soothing, repairing and antioxidant effects, as well as a preparation method and application thereof. Background Art

[0002] Matsutake is a natural medicinal mushroom with high nutritional and medicinal value. Used as a medicine since ancient times, the Shennong Herbal Classic states, "Matsutake is sweet, mild, and non-toxic. It primarily treats urinary dysfunction, nourishes the five internal organs, replenishes qi and blood, strengthens the body and promotes salivation, and calms the mind. Long-term use can help with weight loss and strength. It is also known as the Immortal Mushroom." Matsutake contains active ingredients such as protein, amino acids, vitamins, carbohydrates, and minerals, and has numerous physiological functions. It strengthens the body, benefits the digestive system, enhances immunity, promotes the production of essence and qi, regulates qi and resolves phlegm, and delays aging.

[0003] Currently, the application of Matsutake in existing technologies mainly focuses on the development and application of Matsutake extracts, and there is less research on Matsutake mycelium fermentation products. Functional Matsutake mycelium fermentation products are a direction worth exploring, which will help the development and utilization of Matsutake resources.

[0004] Skin sensitivity is becoming increasingly common. Harmful substances in the environment, such as dust, chemicals, and heavy metals, constantly attack people's skin, causing damage to the skin barrier. At the same time, bad lifestyle habits such as staying up late, unhealthy diet, and lack of exercise also exacerbate the occurrence of skin sensitivity. Skin sensitivity symptoms are often doubly distressing. Even the slightest stimulation or touch can trigger skin discomfort such as stinging, redness, dryness, and itching. To address this problem, the market is constantly researching and developing cosmetic raw materials for sensitive skin care. Therefore, medicinal and edible plants that are both safe and have soothing and repairing effects are widely used in sensitive skin care products to help sensitive skin return to a healthy state and relieve discomfort.

[0005] At present, there are few reports on the use of matsutake mycelium fermentation products for soothing and repairing the skin. It would be very meaningful to develop a matsutake mycelium fermentation product that can be used for soothing and repairing. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a matsutake mycelium fermentation filtrate with soothing, repairing and antioxidant effects, as well as a preparation method and application thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a fermentation filtrate of Tricholoma matsutake mycelium having soothing, repairing and antioxidant effects, the preparation method comprising the following steps:

[0009] (1) inoculating the liquid seeds of Matsutake mushroom into a sterilized fermentation medium for a first stage of culture to obtain a first fermentation liquid; the first stage of culture is carried out at 25-28°C (e.g., 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, etc.) for 5-10 days (e.g., 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days, etc.);

[0010] (2) then subjecting the first fermentation broth to a second stage culture to obtain a second fermentation broth; the second stage culture is performed at 20-24°C (e.g., 20°C, 20.5°C, 21°C, 21.5°C, 22°C, 22.5°C, 23°C, 23.5°C, 24°C, etc.) for 5-8 days (e.g., 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, or 8.5 days, etc.);

[0011] (3) The second fermentation liquid is filtered to separate the matsutake mycelium and the fermentation filtrate, thereby obtaining the matsutake mycelium fermentation filtrate having soothing, repairing and antioxidant effects.

[0012] The present invention creatively conducts a two-stage fermentation of matsutake liquid seeds. By controlling the fermentation time and temperature of each stage, it can well take into account the rapid growth of matsutake and the rapid production of active ingredients such as matsutake polysaccharides. The final fermentation product filtrate has excellent soothing, moisturizing, repairing and antioxidant effects.

[0013] Preferably, the ventilation volumes in the first and second stages of culture are 80-150 L (e.g., 80 L, 90 L, 100 L, 110 L, 120 L, 130 L, 140 L, 150 L, etc.) and 50-120 L (e.g., 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 110 L, 120 L, etc.), respectively. Furthermore, the ventilation volume in the first stage of culture is greater than that in the second stage of culture.

[0014] By controlling the ventilation volume in each stage of the dual-stage fermentation, the soothing, moisturizing, repairing and antioxidant effects of the fermentation product filtrate are further improved.

[0015] Preferably, the stirring rates in the first and second stages of culture are 120-180 rpm (e.g., 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, etc.) and 50-120 rpm (e.g., 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, etc.), respectively. Furthermore, the stirring rate in the first stage of culture is greater than the stirring rate in the second stage of culture.

[0016] By controlling the stirring rate of each stage in the double-stage fermentation, the soothing, moisturizing, repairing and antioxidant effects of the fermentation product filtrate are further improved.

[0017] Preferably, in the first stage culture and the second stage culture, the pressure conditions are 0.1-0.15 MPa (e.g., 0.1 MPa, 0.11 MPa, 0.12 MPa, 0.13 MPa, 0.14 MPa, 0.15 MPa, etc.) and 0.1-0.15 MPa (e.g., 0.1 MPa, 0.11 MPa, 0.12 MPa, 0.13 MPa, 0.14 MPa, 0.15 MPa, etc.), respectively.

[0018] Preferably, the components of the fermentation medium contain, by concentration, 15-25% potato juice, 1-5% glucose, 1-5% malt extract, and 0.5-2% pine root extract.

[0019] The added concentration of the potato juice can be selected from 15%, 17%, 18%, 19%, 20%, 22%, 23%, 24%, 25%, etc.; the added concentration of the glucose can be selected from 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.; the added concentration of the malt extract can be selected from 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.; the added concentration of the pine root extract can be selected from 0.5%, 0.8%, 1%, 1.2%, 1.5%, 2%, etc.

[0020] The present invention also developed a culture medium formula that is more conducive to the fermentation of matsutake liquid seeds, that is, potato juice, glucose, malt extract and pine root extract are used as the carbon source and nitrogen source required for fermentation. Compared with other conventional carbon sources and nitrogen sources, the former has a better fermentation effect, which further improves the soothing, moisturizing, repairing and antioxidant effects of the fermentation product filtrate.

[0021] Preferably, the components of the fermentation medium further contain, in terms of concentration, 0.1-0.5% phosphate, 0.1-0.5% magnesium salt, and 0.0001-0.001% thiamine.

[0022] The added concentration of the phosphate can be selected from 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc.; the added concentration of the magnesium salt can be selected from 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc.; the added concentration of the thiamine can be selected from 0.0001%, 0.0002%, 0.0005%, 0.0007%, 0.0008%, 0.0009%, 0.001%, etc.

[0023] The phosphate includes any one of KH2PO4, K2HPO4, Na2HPO4 or NaH2PO4, or a combination of at least two thereof.

[0024] The magnesium salt includes any one of MgSO4·7H2O, MgSO4·H2O, MgSO4 or MgCl2, or a combination of at least two thereof.

[0025] The malt extract and pine root extract used in the fermentation medium of the present invention are prepared by conventional methods in the art, as follows:

[0026] Preferably, the malt extract is prepared by a method comprising the following steps: mixing ground barley malt with water to prepare a slurry; mixing the slurry with α-amylase to perform an enzymatic hydrolysis reaction; mixing the enzymatic hydrolyzate with trypsin to perform an enzymatic hydrolysis reaction; inactivating the enzyme in the enzymatic hydrolyzate, filtering the enzymatic hydrolyzate, and concentrating and drying the filtrate to obtain the malt extract.

[0027] Preferably, the malt extract is prepared by a method comprising the following steps: mixing ground barley malt with 5-10 times (e.g., 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.) the mass of water to prepare a slurry; mixing the slurry with 0.1-0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc.) of α-amylase, and performing an enzymatic hydrolysis reaction at 45-55°C (e.g., 45°C, 48°C, 50°C, 53°C, 55°C, etc.) for 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.); and then mixing the enzymatic hydrolysis solution with 0.1-0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc.) of trypsin, and performing an enzymatic hydrolysis reaction at 35-45°C (e.g., 35°C, 38°C, 40°C, 43°C, 45°C, etc.) for 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.). h, 1.5 h, 2 h, 2.5 h, 3 h, etc.); the enzymatic hydrolyzate was inactivated and filtered, and the filtrate was concentrated and dried to obtain the malt extract.

[0028] Preferably, the pine root extract is prepared by a method comprising the following steps: mixing crushed pine root with water and subjecting the mixture to reflux extraction, concentrating the extract under reduced pressure and drying to obtain the pine root extract.

[0029] Preferably, the pine root extract is prepared by a method comprising the following steps: mixing the crushed pine root with 10-15 times (for example, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, etc.) the mass of water, performing reflux extraction 1-3 times (for example, 1 time, 2 times or 3 times), each time for 0.5-2 hours (for example, 0.5 hours, 1 hours, 1.5 hours, 2 hours, etc.), combining the extracts, concentrating under reduced pressure and drying to obtain the pine root extract.

[0030] Preferably, the inoculation amount of the Matsutake liquid seeds is 5-15%, for example, 5%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0031] The preparation of the liquid seeds of Tricholoma matsutake can be obtained by using existing technical methods in the field. For example:

[0032] Under sterile conditions, inoculate the mushroom block into the seed activation solution and culture it in a shaking incubator at 20-30°C and 100-500 rpm for 20-40 days to obtain the first-grade seed liquid of Matsutake. Inoculate the first-grade seed liquid of Matsutake into the seed activation solution at a 2-10% inoculation rate and culture it in a shaking incubator at 15-25°C and 50-300 rpm for 10-25 days to obtain the liquid seeds of Matsutake.

[0033] Preferably, the filtration is performed using a plate and frame filter press.

[0034] Preferably, after separating the Tricholoma matsutake mycelium and the fermentation filtrate, the fermentation filtrate is further centrifuged and filtered through a 0.22 μm filter membrane.

[0035] In a second aspect, the present invention provides a fermentation filtrate of Tricholoma matsutake mycelium obtained according to the preparation method described in the first aspect.

[0036] In a third aspect, the present invention provides a use of the matsutake mycelium fermentation filtrate according to the second aspect in preparing a product with soothing, moisturizing, repairing or antioxidant effects.

[0037] All other specific point values ​​not listed in the numerical range of the present invention are within the protection scope of the present invention. In consideration of space and brevity, they will not be described one by one here.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention creatively conducts a two-stage fermentation of matsutake liquid seeds. By controlling the fermentation time and temperature of each stage, it can well take into account the rapid growth of matsutake and the rapid production of active ingredients such as matsutake polysaccharides. The final fermentation product filtrate has excellent soothing, moisturizing, repairing and antioxidant effects.

[0040] By controlling the aeration volume and stirring rate of each stage in the dual-stage fermentation, the soothing, moisturizing, repairing and antioxidant effects of the fermentation product filtrate are further improved.

[0041] By optimizing the carbon and nitrogen sources of the fermentation medium, the soothing, moisturizing, repairing and antioxidant effects of the fermentation product filtrate are further improved. DETAILED DESCRIPTION

[0042] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0043] The malt extracts involved in the following examples and comparative examples were prepared by the following method:

[0044] The crushed barley malt was mixed with 8 times the mass of water to prepare a slurry; the slurry was mixed with 0.3% α-amylase and subjected to enzymatic hydrolysis at 50°C for 2 hours; the enzymatic hydrolyzate was then mixed with 0.5% trypsin and subjected to enzymatic hydrolysis at 40°C for 2 hours; the enzymatic hydrolyzate was inactivated and filtered, and the filtrate was concentrated and dried to obtain.

[0045] The pine root extracts involved in the following examples and comparative examples were prepared by the following method:

[0046] The ground pine root was mixed with 12 times the mass of water, and reflux extraction was performed twice, each time for 1.5 hours. The two extracts were combined, concentrated under reduced pressure and dried to obtain the pine root extract.

[0047] The potato juices involved in the following examples and comparative examples were prepared by the following method:

[0048] The potato chunks were mixed with an equal amount of water to extract the juice, filtered, and the filtrate was collected.

[0049] The liquid seeds of Tricholoma matsutake involved in the following examples and comparative examples were prepared by the following method:

[0050] (a) Prepare culture medium: 20% potato juice, 2% glucose, 0.3% KH2PO4, 0.2% MgSO4·7H2O, and the balance water. After preparation, sterilize at 115°C for 30 minutes and set aside. Under sterile conditions, inoculate appropriate-sized mushroom blocks from the slant into a shake flask containing seed culture medium. Cultivate at 25°C, 200 rpm, and shake for 30 days to obtain the first-grade seed medium for Matsutake.

[0051] (b) Prepare culture medium: 20% potato juice, 2% glucose, 0.3% KH2PO4, 0.2% MgSO4·7H2O, and the balance water. After preparation, sterilize at 115°C for 30 min and set aside. Under sterile conditions, add 0.0001% thiamine, shake well, and then add 5% first-grade Matsutake seed liquid. Incubate at 20°C, 50 rpm, and shake for 20 days to obtain Matsutake liquid seeds.

[0052] Example 1

[0053] This embodiment provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is as follows:

[0054] (1) Prepare fermentation medium: potato juice 20%, glucose 2%, malt extract 2%, pine root extract 1%, KH2PO4 0.3%, MgSO4·7H2O 0.2%, and water as the balance. After preparation, sterilize at 115°C and 0.1 MPa for 50 min, cool, and set aside.

[0055] (2) Thiamine was pumped into the fermentation medium at a rate of 0.001% using a peristaltic pump, and Matsutake liquid seeds were inoculated into the fermentation medium at a rate of 10%. The culture was carried out at 27°C for 7 days with an aeration volume of 100 L, a stirring rate of 150 rpm, and a pressure of 0.15 MPa to obtain the first fermentation liquid.

[0056] (3) The first fermentation liquid was cooled to 20°C, the ventilation volume was 60 L, the stirring rate was 80 rpm, the pressure was 0.1 MPa, and the culture was continued for 8 days to obtain the second fermentation liquid.

[0057] (4) The second fermentation liquid was filtered using a plate and frame filter press to separate the mycelia of Tricholoma matsutake, and the fermentation filtrate was collected. The collected filtrate was centrifuged again (4°C, 3000 rpm, 10 min) and filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate of Tricholoma matsutake mycelia.

[0058] Example 2

[0059] This embodiment provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is as follows:

[0060] (1) Prepare fermentation medium: 15% potato juice, 5% glucose, 3% malt extract, 2% pine root extract, 0.5% KH2PO4, 0.1% MgSO4·7H2O, and the balance water. After preparation, sterilize at 115°C and 0.1 MPa for 50 min, cool, and set aside.

[0061] (2) Thiamine was pumped into the fermentation medium at an addition rate of 0.0007% using a peristaltic pump, and liquid Matsutake seeds were inoculated into the fermentation medium at an inoculum rate of 10%. The culture was carried out at 25°C for 9 days with an aeration volume of 90 L, a stirring rate of 120 rpm, and a pressure of 0.15 MPa to obtain the first fermentation liquid.

[0062] (3) The first fermentation liquid was cooled to 22°C, the ventilation volume was 50 L, the stirring rate was 60 rpm, the pressure was 0.1 MPa, and the culture was continued for 6 days to obtain the second fermentation liquid.

[0063] (4) The second fermentation liquid was filtered using a plate and frame filter press to separate the mycelia of Tricholoma matsutake, and the fermentation filtrate was collected. The collected filtrate was centrifuged again (4°C, 3000 rpm, 10 min) and filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate of Tricholoma matsutake mycelia.

[0064] Example 3

[0065] This embodiment provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is as follows:

[0066] (1) Prepare fermentation medium: 23% potato juice, 1% glucose, 1% malt extract, 0.5% pine root extract, 0.2% KH2PO4, 0.4% MgSO4·7H2O, and the balance water. After preparation, sterilize at 115°C and 0.1 MPa for 50 min, cool, and set aside.

[0067] (2) Thiamine was pumped into the fermentation medium at a rate of 0.0005% using a peristaltic pump, and liquid Matsutake seeds were inoculated into the fermentation medium at a rate of 10%. The culture was carried out at 28°C for 10 days with an aeration volume of 140 L, a stirring rate of 170 rpm, and a pressure of 0.15 MPa to obtain the first fermentation liquid.

[0068] (3) The first fermentation liquid was cooled to 24°C, the ventilation volume was 100 L, the stirring rate was 100 rpm, and the pressure was 0.1 MPa. The culture was continued for 5 days to obtain the second fermentation liquid.

[0069] (4) The second fermentation liquid was filtered using a plate and frame filter press to separate the mycelia of Tricholoma matsutake, and the fermentation filtrate was collected. The collected filtrate was centrifuged again (4°C, 3000 rpm, 10 min) and filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate of Tricholoma matsutake mycelia.

[0070] Example 4

[0071] This embodiment provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the formula of the culture medium in step (1) is different: "2% malt extract and 1% pine root extract" is replaced with "2% peptone and 1% yeast powder." Other conditions remain unchanged.

[0072] Example 5

[0073] This embodiment provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the formula of the culture medium in step (1) is different: "2% malt extract and 1% pine root extract" is replaced with "3% malt extract." Other conditions remain unchanged.

[0074] Example 6

[0075] This embodiment provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the formula of the culture medium in step (1) is different: "2% malt extract and 1% pine root extract" is replaced with "3% pine root extract." Other conditions remain unchanged.

[0076] Example 7

[0077] This example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in the difference in the aeration volume during the culture: the aeration volume during the first and second stages is 60 L. Other conditions remain unchanged.

[0078] Example 8

[0079] This example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in the difference in the stirring rate during the culture: the stirring rate during the first and second stages of the culture is 80 rpm. Other conditions remain unchanged.

[0080] Comparative Example 1

[0081] This comparative example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the first stage of culture is carried out at 27° C. for 12 days, and the second stage of culture is carried out at 20° C. for 3 days. Other conditions remain unchanged.

[0082] Comparative Example 2

[0083] This comparative example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the first stage of culture is cultured at 27° C. for 3 days, and the second stage of culture is cultured at 20° C. for 12 days. Other conditions remain unchanged.

[0084] Comparative Example 3

[0085] This comparative example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the first stage of culture is carried out at 30° C. for 7 days, and the second stage of culture is carried out at 18° C. for 8 days. Other conditions remain unchanged.

[0086] Comparative Example 4

[0087] This comparative example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the double-stage culture is replaced by a single-stage culture method:

[0088] (1) Prepare fermentation medium: potato juice 20%, glucose 2%, malt extract 2%, pine root extract 1%, KH2PO4 0.3%, MgSO4·7H2O 0.2%, thiamine 0.001%, and water as the balance. After preparation, sterilize at 115°C and 0.1 MPa for 50 min, cool, and set aside.

[0089] (2) The liquid seeds of Matsutake mushroom were inoculated into the fermentation medium at an inoculum rate of 10%, and cultured at 27°C for 15 days with an aeration volume of 100 L, a stirring rate of 150 rpm, and a pressure of 0.15 MPa to obtain the fermentation liquid.

[0090] (3) The fermentation liquid was filtered using a plate and frame filter press to separate the mycelia of Tricholoma matsutake, and the fermentation filtrate was collected. The collected filtrate was centrifuged again (4°C, 3000 rpm, 10 min) and filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate of Tricholoma matsutake mycelia.

[0091] Comparative Example 5

[0092] This comparative example provides a fermentation filtrate of Tricholoma matsutake mycelium. The preparation method thereof differs from that of Example 1 only in that the double-stage culture is replaced by a single-stage culture method:

[0093] (1) Prepare fermentation medium: potato juice 20%, glucose 2%, malt extract 2%, pine root extract 1%, KH2PO4 0.3%, MgSO4·7H2O 0.2%, thiamine 0.001%, and water as the balance. After preparation, sterilize at 115°C and 0.1 MPa for 50 min, cool, and set aside.

[0094] (2) The liquid seeds of Matsutake mushroom were inoculated into the fermentation medium at a 10% inoculum rate. The culture temperature was 20°C, the ventilation volume was 60 L, the stirring rate was 80 rpm, and the pressure was 0.1 MPa. The culture was carried out for 15 days to obtain the fermentation liquid.

[0095] (3) The fermentation liquid was filtered using a plate and frame filter press to separate the mycelia of Tricholoma matsutake, and the fermentation filtrate was collected. The collected filtrate was centrifuged again (4°C, 3000 rpm, 10 min) and filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate of Tricholoma matsutake mycelia.

[0096] Test Example 1

[0097] Safety performance assessment:

[0098] The safety performance of the products of Examples 1-8 and Comparative Examples 1-5 was evaluated. Each group of products was diluted with PBS to a 30% solution of the test sample, and PBS solution was used as a blank control. 30 social volunteers aged 20-45 years who met the requirements were selected and randomly divided into 3 groups. Each group tried 4 or 5 different products. Place the tester in a small chamber, and do not perform any treatment on the control well. Apply the tester with the test substance to the inner side of the subject's forearm with hypoallergenic tape, and gently press it with the palm of your hand to evenly apply it to the skin for 24 hours. Observe the skin reaction according to the standards in Table 1 30 minutes and 24 hours after removing the test substance tester.

[0099] Table 1

[0100]

[0101] The results showed that the products of Examples 1-8 of the present invention and Comparative Examples 1-5 all gave negative reactions after trial use, indicating that the Tricholoma matsutake mycelium fermentation filtrate of the present invention is safe and mild, and will not cause adverse reactions such as skin irritation and sensitization.

[0102] Test Example 2

[0103] Antioxidant effect evaluation test:

[0104] (1) Preparation of test samples: The products prepared in Examples 1-8 and Comparative Examples 1-5 were prepared into test samples with a concentration of 10% for later use.

[0105] (2) DPPH free radical scavenging experiment:

[0106] (2.1) Preparation of DPPH solution: Weigh 5.0 mg of DPPH and dissolve it in an appropriate amount of anhydrous ethanol. Sonicate in the dark to fully dissolve the DPPH. Then, dilute the volume to 100 mL with anhydrous ethanol to prepare a 50 μg / mL DPPH solution. This solution should be prepared immediately before use.

[0107] (2.2) Set up a sample group: 100 μL of sample solution mixed with 100 μL of DPPH solution (50.0 μg / mL); a blank group: 100 μL of sample solution mixed with 100 μL of solvent; and a control group: 100 μL of DPPH solution (50.0 μg / mL) mixed with 100 μL of solvent. Incubate at room temperature in the dark for 0.5 h. Measure the absorbance at 517 nm. Calculate the clearance rate.

[0108] (2.3) DPPH free radical scavenging rate calculation formula:

[0109] Clearance rate (%) = [1-(Ai-Aj) / Ac] × 100%

[0110] Among them, Aj is the absorbance value of the sample blank group; Ai is the absorbance value of the sample group; Ac is the absorbance value of the control group.

[0111] (3) ABTS free radical scavenging experiment:

[0112] (3.1) Preparation of ABTS stock solution: Dissolve ABTS in water to a 7 mmol / L solution and potassium persulfate in water to a 2.45 mmol / L solution. Mix the two solutions in a 1:1 volume ratio and allow to react at 25°C in the dark for 14 hours.

[0113] Preparation of ABTS solution: Dilute the ABTS stock solution with phosphate buffered saline (PBS, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm.

[0114] (3.2) Set up a sample group: 100 μL of sample solution mixed with 100 μL of ABTS working solution; a blank group: 100 μL of sample solution mixed with 100 μL of solvent; and a control group: 100 μL of ABTS working solution mixed with 100 μL of solvent. Incubate at 25°C in the dark for 10 min. Measure the absorbance at 734 nm. Calculate the clearance rate.

[0115] (3.3) Calculation formula for ABTS free radical scavenging rate:

[0116] Clearance rate (%) = [1-(Ai-Aj) / Ac] × 100%

[0117] Among them, Aj is the absorbance value of the sample blank group; Ai is the absorbance value of the sample group; Ac is the absorbance value of the control group.

[0118] (4) Hydroxyl free radical scavenging experiment:

[0119] (4.1) 6mmol / L FeSO4: Dissolve FeSO4 in water to prepare 6mmol / L FeSO4;

[0120] 6 mmol / L H2O2: Use 30% H2O2 to prepare 6 mmol / L H2O2;

[0121] 6 mmol / L salicylic acid solution: Prepare a 6 mmol / L salicylic acid solution in anhydrous ethanol.

[0122] (4.2) Set up the sample group: 100 μL of sample solution mixed with 100 μL of FeSO₄, 100 μL of H₂O₂, and 100 μL of salicylic acid solution; the blank group: 100 μL of sample solution mixed with 100 μL of solvent; and the control group: 100 μL of FeSO₄, 100 μL of H₂O₂, 100 μL of salicylic acid solution mixed with 100 μL of solvent. Incubate the mixture in a 37°C water bath in the dark for 30 min. Measure the absorbance at 510 nm. Calculate the clearance rate.

[0123] (4.3) Calculation formula for ABTS free radical scavenging rate:

[0124] Clearance rate (%) = [1-(Ai-Aj) / Ac] × 100%

[0125] Among them, Aj is the absorbance value of the sample blank group; Ai is the absorbance value of the sample group; Ac is the absorbance value of the control group.

[0126] (5) The results are shown in Table 2:

[0127] Table 2

[0128]

[0129] As shown in Table 2, the fermentation filtrate of Tricholoma matsutake mycelium obtained using the preparation method of the present invention exhibits significantly superior antioxidant efficacy compared to Comparative Examples 1-5. This demonstrates that the present invention enhances the antioxidant efficacy of the product by employing a dual-stage fermentation method for Tricholoma matsutake liquid seeds and controlling the fermentation time and temperature at each stage. Furthermore, a comparison of the results of Example 1 with those of Examples 7-8 shows that controlling the aeration rate and stirring rate at each stage during the fermentation process also affects the antioxidant efficacy of the product to a certain extent. A comparison of the results of Example 1 with those of Examples 4-6 shows that the fermentation medium formulation also affects the antioxidant efficacy of the product to a certain extent.

[0130] Test Example 3

[0131] Soothing effect evaluation test:

[0132] (1) Preparation of test samples: The products prepared in Examples 1-8 and Comparative Examples 1-5 were prepared into test samples with a concentration of 2.5% for later use.

[0133] (2) Experimental procedure: Test tubes A and B (0.5 mL of sample solution and 0.5 mL of hyaluronidase (500 U / mL)), test tubes C and D (0.5 mL of distilled water and 0.5 mL of acetic acid buffer solution), incubated at 37°C for 20 min; added 0.1 mL (2.5 mmol / L) CaCl2 solution, incubated at 37°C for 20 min; added 0.5 mL of sodium hyaluronate (0.5 mg / mL) to test tubes A and C, added 0.5 mL of acetic acid buffer solution to test tubes B and D, incubated at 37°C for 40 min, added 0.5 mL of acetylacetone solution, 0.1 mL of NaOH solution (5 mol / L) and 0.5 mL of distilled water, boiled in a water bath for 15 min, immediately cooled in ice water for 5 min, added 1 mL of P-DAB colorimetric reagent, shaken thoroughly, added 3.5 mL of anhydrous ethanol, left for 30 min, color developed, and then heated at 530°C. Measure the OD values ​​of the four test tubes A, B, C, and D at nm respectively.

[0134] The calculation formula for the hyaluronidase activity inhibition rate is:

[0135] Hyaluronidase inhibition rate (%) = [(C'-D')-(A'-B')] / (C'-D')×100

[0136] Where A' is the OD value of test tube A; B' is the OD value of test tube B; C' is the OD value of test tube C; and D' is the OD value of test tube D.

[0137] (3) The results are shown in Table 3:

[0138] Table 3

[0139]

[0140] As shown in Table 3, the fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method of the present invention exhibits significantly superior hyaluronidase inhibition compared to Comparative Examples 1-5. This indicates that the present invention enhances the soothing effect of the product by adopting a dual-stage fermentation method for Tricholoma matsutake liquid seeds and controlling the fermentation time and temperature of each stage. Furthermore, by comparing the results of Example 1 with those of Examples 7-8, it can be seen that controlling the ventilation volume and stirring rate at each stage during the fermentation process also affects the soothing effect of the product to a certain extent. By comparing the results of Example 1 with those of Examples 4-6, it can be seen that the fermentation medium formulation also affects the soothing effect of the product to a certain extent.

[0141] Test Example 4

[0142] Moisturizing and repairing effect evaluation test:

[0143] (1) Preparation of test samples: The products prepared in Examples 1-8 and Comparative Examples 1-5 were prepared into test samples with a concentration of 10% for later use.

[0144] (2) Test process:

[0145] (2.1) Subjects (140 participants, 14 groups, 10 participants each) were required to refrain from using any products (cosmetics or topical medications) on the test area for 3 days prior to the visit and avoid contact with water for 3 hours. They were required to clean the inner forearms of both hands (by wiping with dry tissue) and then equilibrate in the test environment for 20 minutes.

[0146] (2.2) Establishment of skin barrier impairment: Inject 5% sodium lauryl sulfate solution into the patch test chamber to soak the filter paper; apply the patch test chamber to the curved side of the subject's left and right forearms, 3 on each side, with the distance between the edges of adjacent patch test chambers being no less than 1 cm. Use the palm of your hand to gently press the patch test chamber evenly on the skin for 24 hours.

[0147] (2.3) Initial measurement value: After removing the patch tester, the subject equilibrates in the test environment for 30 minutes. The water content of the center of each patch area is measured using a stratum corneum hydration meter. Each area is measured in parallel three times. The measurement result is expressed as the average value of the patch area detection value, which is used as the initial value.

[0148] (2.4) After the initial value measurement is completed, take each group of samples (2 mg / cm 2 ) was applied to the test area. Two hours after application of the sample, the test area was measured using a stratum corneum hydration meter. The change rate (%) of the water content of the stratum corneum of each group was calculated, as shown in Table 4:

[0149] Table 4

[0150]

[0151] As shown in Table 4, the fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method of the present invention has significantly superior skin barrier repair efficacy compared to Comparative Examples 1-5. This indicates that the present invention enhances the moisturizing and repairing efficacy of the product by adopting a two-stage fermentation method for Tricholoma matsutake liquid seeds and controlling the fermentation time and temperature of each stage. Furthermore, by comparing the results of Example 1 with those of Examples 7-8, it can be seen that the control of the ventilation volume and stirring rate at each stage during the fermentation process also affects the moisturizing and repairing efficacy of the product to a certain extent. By comparing the results of Example 1 with those of Examples 4-6, it can be seen that the formula of the fermentation medium also affects the moisturizing and repairing efficacy of the product to a certain extent.

[0152] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0153] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within 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 scope of protection of the present invention.

[0154] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a fermentation filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects, characterized in that: The preparation method comprises the following steps: (1) Inoculating the liquid seeds of Matsutake mushroom into the sterilized fermentation medium for the first stage of culture to obtain the first fermentation liquid; the first stage of culture lasts for 5-10 days at 25-28°C; (2) The first fermentation broth is then subjected to a second stage of culturing to obtain a second fermentation broth; the second stage of culturing is continued at 20-24°C for 5-8 days; (3) filtering the second fermentation liquid to separate the mycelium of Tricholoma matsutake and the fermentation filtrate, thereby obtaining the fermentation filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects; The components of the fermentation medium include, by concentration, 15-25% potato juice, 1-5% glucose, 1-5% malt extract, 0.5-2% pine root extract, 0.1-0.5% phosphate, 0.1-0.5% magnesium salt, and 0.0001-0.001% thiamine; The malt extract is prepared by a method comprising the following steps: mixing ground barley malt with water to prepare a slurry; mixing the slurry with α-amylase to carry out an enzymatic hydrolysis reaction; mixing the enzymatic hydrolysis solution with trypsin to carry out an enzymatic hydrolysis reaction; inactivating the enzyme in the enzymatic hydrolysis solution, filtering the solution, concentrating the filtrate, and drying the solution to obtain the malt extract; The pine root extract is prepared by a method comprising the following steps: mixing crushed pine root with water and performing reflux extraction, concentrating the extract under reduced pressure and drying to obtain the pine root extract.

2. The method for preparing the fermentation filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 1, characterized in that: In the first stage culture and the second stage culture, the ventilation volumes are 80-150 L and 50-120 L, respectively.

3. The method for preparing the fermentation filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 1, characterized in that: The stirring rates in the first stage culture and the second stage culture were 120-180 rpm and 50-120 rpm, respectively.

4. The method for preparing the fermentation filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 1, characterized in that: In the first stage culture and the second stage culture, the pressure conditions are 0.1-0.15 MPa and 0.1-0.15 MPa, respectively.

5. The method for preparing the fermentation filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 1, characterized in that: The inoculation amount of the Matsutake liquid seeds is 5-15%; The filtration is carried out using a plate and frame filter press; After the separation of the Tricholoma matsutake mycelia and the fermentation filtrate, the fermentation filtrate was centrifuged again and filtered through a 0.22 μm filter membrane.

6. The fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the matsutake mycelium fermentation filtrate according to claim 6 in preparing products with soothing, moisturizing, repairing or antioxidant effects.

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