Tricholoma matsutake mycelium fermentation filtrate with soothing, repairing and anti-oxidation effects as well as preparation method and application of tricholoma matsutake mycelium fermentation filtrate

Abstract

The invention relates to tricholoma matsutake mycelium fermentation filtrate with soothing, repairing and anti-oxidation effects and a preparation method and application thereof, the preparation method comprises the following steps: inoculating tricholoma matsutake liquid seeds into a sterilized fermentation culture medium for first-stage culture to obtain first fermentation liquor; the first-stage culture lasts for 5-10 days at the temperature of 25-28 DEG C; performing second-stage culture on the first fermentation liquor to obtain second fermentation liquor; the second-stage culture lasts for 5-8 days at the temperature of 20-24 DEG C; and carrying out filter pressing on the second fermentation liquor, and separating tricholoma matsutake mycelia and fermentation filtrate. According to the method, the tricholoma matsutake liquid seeds are creatively subjected to two-stage fermentation, and the fermentation time and the fermentation temperature of each stage are controlled, so that the rapid growth of tricholoma matsutake and the rapid generation of active ingredients such as tricholoma matsutake polysaccharides can be well considered, and the final fermentation product filtrate has excellent soothing, moisturizing, repairing and antioxidant effects.

Description

Technical Field

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

[0002] Matsutake is one of the natural medicinal mushrooms with high nutritional and medicinal value. It has been used as medicine since ancient times. It is recorded in Shennong's Herbal Classic that "Matsutake is sweet, flat and non-toxic. It is mainly used to treat urinary problems, nourish the five internal organs, replenish qi and nourish blood, consolidate and promote the production of body fluids, and calm the mind and nerves. Long-term use can make the body lighter and increase qi and strength. It is also called fairy mushroom." Matsutake contains effective ingredients such as protein, amino acids, multiple vitamins, carbohydrates and minerals, and has multiple physiological functions. It can strengthen the body, benefit the stomach and intestines, enhance human immunity, produce essence and replenish qi, regulate qi and resolve phlegm, and delay aging.

[0003] At present, the application of Matsutake in the existing technology is mainly focused 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 more and more common among the population. 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 living habits such as staying up late, unhealthy diet and lack of exercise also aggravate the occurrence of skin sensitivity problems. Skin sensitivity symptoms are often more troublesome. Slight stimulation or touch may cause skin discomfort, such as tingling, redness, dryness and itching. In order to cope with this problem, cosmetic raw materials for sensitive skin care are also being continuously researched and developed on the market. Medicinal and edible plants that are both safe and soothing and repairing are therefore widely used in sensitive skin care products, aiming 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 fermented products for soothing and repairing the skin. It would be very meaningful if a Matsutake mycelium fermented product that can be used for soothing and repairing can be developed. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a pine mushroom mycelium fermentation filtrate with soothing, repairing and antioxidant effects, and 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 pine mushroom mycelium fermentation filtrate having soothing, repairing and antioxidant effects, the preparation method comprising the following steps:

[0009] (1) inoculating the liquid seeds of Matsutake into the sterilized fermentation medium for the first stage of culture to obtain the 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 continued 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 pine mushroom mycelium and the fermentation filtrate, thereby obtaining the pine mushroom 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 fermentation 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, in the first stage culture and the second stage culture, the ventilation volumes 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. And the ventilation volume of the first stage culture is greater than the ventilation volume of the second stage culture.

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

[0015] Preferably, in the first stage culture and the second stage culture, the stirring rates 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. And the stirring rate of the first stage culture is greater than the stirring rate of the second stage 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% and the like; the added concentration of the glucose can be selected from 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% and the like; the added concentration of the malt extract can be selected from 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% and the like; the added concentration of the pine root extract can be selected from 0.5%, 0.8%, 1%, 1.2%, 1.5%, 2% and the like.

[0020] The present invention has 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% and the like; 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% and the like; the added concentration of the thiamine can be selected from 0.0001%, 0.0002%, 0.0005%, 0.0007%, 0.0008%, 0.0009%, 0.001% and the like.

[0023] The phosphate includes KH 2 PO 4 , K 2 HPO 4 、Na 2 HPO 4 or NaH 2 PO 4 Any one or a combination of at least two of the following.

[0024] The magnesium salt includes MgSO 4 7H 2 O、MgSO 4 ·H 2 O、MgSO 4 or MgCl 2 Any one or a combination of at least two of the following.

[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 for enzymatic hydrolysis; mixing the enzymatic hydrolyzate with trypsin for enzymatic hydrolysis; filtering the enzymatic hydrolyzate after enzyme inactivation treatment, 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.) 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 mixing the enzymatic hydrolyzate with 0.1-0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc.) 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.); 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 pine root crushed material with water for 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 pine root crushed material with 10-15 times (e.g., 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, etc.) the mass of water, performing reflux extraction 1-3 times (e.g., 1 time, 2 times or 3 times), each time for 0.5-2 h (e.g., 0.5 h, 1 h, 1.5 h, 2 h, etc.), combining the extracts, concentrating under reduced pressure and drying to obtain the pine root extract.

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

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

[0032] Under sterile conditions, the mushroom block is inoculated into the seed activation liquid, and cultured in a shaking incubator at 20-30°C and 100-500 rpm for 20-40 days to obtain the first-grade seed liquid of Tricholoma matsutake. The first-grade seed liquid of Tricholoma matsutake is inoculated into the seed activation liquid at a rate of 2-10%, and cultured in a shaking incubator at 15-25°C and 50-300 rpm for 10-25 days to obtain the liquid seeds of Tricholoma 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 centrifuged again and filtered through a 0.22 μm filter membrane.

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

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

[0037] All other unlisted specific point values ​​within the numerical range involved in 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 fermentation 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 in each stage of the double-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 explain the technical means and effects adopted by the present invention, the technical solution of the present invention is 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 obtain a slurry; the slurry was mixed with 0.3% α-amylase and subjected to enzymolysis at 50°C for 2 h; the enzymolysis solution was then mixed with 0.5% trypsin and subjected to enzymolysis at 40°C for 2 h; the enzymolysis solution 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 refluxed for extraction twice, each time for 1.5 h. The two extracts were combined, concentrated under reduced pressure and dried to obtain the pine root extract.

[0047] The potato juice involved in the following examples and comparative examples was 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 are prepared by the following method:

[0050] (a) Preparation of culture medium: 20% potato juice, 2% glucose, KH 2 PO 4 0.3%, MgSO 4 7H 2 O 0.2%, the balance is water. After the preparation is completed, sterilize at 115℃ for 30min and set aside; under sterile conditions, take appropriate size mushroom blocks from the slant, inoculate them into a shaking bottle filled with seed culture medium, and culture them in a shaking table at 25℃ and 200rpm for 30 days to obtain the first-level seed liquid of Tricholoma matsutake;

[0051] (b) Preparation of culture medium: 20% potato juice, 2% glucose, KH 2 PO 4 0.3%, MgSO 4 7H 2 O 0.2%, the rest is water. After the preparation is completed, sterilize at 115℃ for 30min and set aside; under sterile conditions, add 0.0001% thiamine, shake well, add 5% first-grade seed liquid of Tricholoma matsutake, and culture on a shaker at 20℃ and 50 rpm for 20 days to obtain liquid seeds of Tricholoma matsutake.

[0052] Example 1

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

[0054] (1) Preparation of fermentation medium: 20% potato juice, 2% glucose, 2% malt extract, 1% pine root extract, KH 2 PO 4 0.3%, MgSO 4 7H 2 O 0.2%, water for the remainder. After preparation, sterilize at 115℃ 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 liquid Matsutake 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 mycelium 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 mycelium.

[0058] Example 2

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

[0060] (1) Preparation of fermentation medium: 15% potato juice, 5% glucose, 3% malt extract, 2% pine root extract, KH 2 PO 4 0.5%, MgSO 4 7H 2 O 0.1%, water for the remainder. After preparation, sterilize at 115℃ and 0.1 MPa for 50 min, cool and set aside.

[0061] (2) Thiamine was pumped into the fermentation medium at a rate of 0.0007% 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 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 mycelium 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 mycelium.

[0064] Example 3

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

[0066] (1) Preparation of fermentation medium: 23% potato juice, 1% glucose, 1% malt extract, 0.5% pine root extract, KH 2 PO 4 0.2%, MgSO 4 7H 2 O 0.4%, water for the remainder. After preparation, sterilize at 115℃ 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, the pressure was 0.1 MPa, and 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 mycelium 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 mycelium.

[0070] Example 4

[0071] This embodiment provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is different from that of Embodiment 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 by "2% peptone and 1% yeast powder". Other conditions remain unchanged.

[0072] Example 5

[0073] This embodiment provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is different from that of Embodiment 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 filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is different from that of Embodiment 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 embodiment provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is different from that of Example 1 only in that the ventilation volume of the culture is different: the ventilation volume of the first stage and the second stage culture is 60 L. Other conditions remain unchanged.

[0078] Example 8

[0079] This embodiment provides a fermentation filtrate of Tricholoma matsutake mycelium, and the preparation method thereof is different from that of Example 1 only in that the stirring rate of the culture is different: the stirring rate of the culture in the first stage and the second stage is 80 rpm. Other conditions remain unchanged.

[0080] Comparative Example 1

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

[0082] Comparative Example 2

[0083] This comparative example provides a filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is different from that of Example 1 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 filtrate of Tricholoma matsutake mycelium fermentation, and the preparation method thereof is different from that of Example 1 in that the first stage of culture is cultured at 30° C. for 7 days, and the second stage of culture is cultured at 18° C. for 8 days. Other conditions remain unchanged.

[0086] Comparative Example 4

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

[0088] (1) Preparation of fermentation medium: 20% potato juice, 2% glucose, 2% malt extract, 1% pine root extract, KH 2 PO 4 0.3%, MgSO 4 7H 2 O 0.2%, thiamine 0.001%, water for the rest. After preparation, sterilize at 115℃ and 0.1 MPa for 50 min, cool and set aside.

[0089] (2) The liquid seeds of Tricholoma matsutake 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 a fermentation liquid.

[0090] (3) The fermentation liquid was filtered using a plate and frame filter press to separate the mycelium 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 mycelium.

[0091] Comparative Example 5

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

[0093] (1) Preparation of fermentation medium: 20% potato juice, 2% glucose, 2% malt extract, 1% pine root extract, KH 2 PO 4 0.3%, MgSO 4 7H 2 O 0.2%, thiamine 0.001%, water for the rest. After preparation, sterilize at 115℃ and 0.1 MPa for 50 min, cool and set aside.

[0094] (2) The liquid seeds of Tricholoma matsutake were inoculated into the fermentation medium at a rate of 10%. The culture temperature was 20°C, the ventilation volume was 60 L, the stirring rate was 80 rpm, the pressure was 0.1 MPa, and 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 mycelium 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 mycelium.

[0096] Test Example 1

[0097] Safety performance evaluation:

[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 sample to be tested, and PBS solution was used as a blank control. 30 social volunteers who met the requirements, aged 20-45 years old, were selected and randomly divided into 3 groups, and each group tried 4 or 5 different products. Placed in a small chamber of the spot tester, the control well was not treated in any way. The spot tester with the test substance was applied to the inner side of the forearm of the subject with a low-allergenic tape, and gently pressed with the palm of the 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 spot 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 had 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. Protect it from light and sonicate it to make it fully dissolved. Then use anhydrous ethanol to make up to 100 mL to prepare a 50 μg / mL DPPH solution. This solution should be prepared and used immediately.

[0107] (2.2) Set up the sample group: 100 μL sample solution mixed with 100 μL DPPH solution (50.0 μg / mL); sample blank group: 100 μL sample solution mixed with 100 μL solvent; control group: 100 μL DPPH solution (50.0 μg / mL) mixed with 100 μL solvent. React at room temperature in the dark for 0.5 h, and 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 prepare a 7 mmol / L solution, and potassium persulfate to prepare a 2.45 mmol / L solution. Then mix the two solutions in a volume ratio of 1:1 and let them 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 a value of 0.70 ± 0.02 at a wavelength of 734 nm.

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

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

[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 FeSO 4 : Convert FeSO 4 Dissolve in water to prepare 6mmol / L FeSO 4 ;

[0120] 6 mmol / LH 2 O 2 : Use 30% H 2 O 2 Formulated to 6 mmol / LH 2 O 2 ;

[0121] 6 mmol / L salicylic acid solution: Use anhydrous ethanol to prepare 6 mmol / L salicylic acid solution.

[0122] (4.2) Set up sample set: 100 μL sample solution and 100 μL FeSO 4 , 100 μL HO 2 O 2 , 100 μL salicylic acid solution; sample blank group: 100 μL sample solution mixed with 100 μL solvent; control group: 100 μL FeSO 4 , 100 μL HO 2 O 2 , 100 μL salicylic acid solution was mixed with 100 μL solvent. The mixture was reacted in a 37°C water bath in the dark for 30 min, and the absorbance was measured at 510 nm. The clearance rate was calculated.

[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 can be seen from the data results in Table 2, compared with Comparative Examples 1-5, the fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method of the present invention has significantly excellent antioxidant effect, indicating that the present invention improves the antioxidant effect of the product by adopting a two-stage fermentation method for Tricholoma matsutake liquid seeds and controlling the fermentation time and fermentation temperature of each stage. Further, by comparing the results of Example 1 with 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 antioxidant effect of the product to a certain extent; by comparing the results of Example 1 with Examples 4-6, it can be seen that the culture medium formula used for fermentation also affects the antioxidant effect 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 process: 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 acetate buffer solution), incubated at 37°C for 20 min; 0.1 mL (2.5 mmol / L) CaCl was added 2 solution, and incubate at 37°C for 20 min; add 0.5 mL of sodium hyaluronate (0.5 mg / mL) to test tubes A and C, and add 0.5 mL of acetate buffer solution to test tubes B and D, and keep warm at 37°C for 40 min, add 0.5 mL of acetylacetone solution, 0.1 mL of NaOH solution (5 mol / L) and 0.5 mL of distilled water, and boil in a water bath for 15 min, immediately cool with ice water for 5 min, add 1 mL of P-DAB colorimetric reagent, shake well, add 3.5 mL of anhydrous ethanol, and let stand for 30 min for color development, and measure the OD values ​​of the four test tubes A, B, C, and D at 530 nm, respectively.

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

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

[0136] Among them, 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; D' is the OD value of test tube D.

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

[0138] Table 3

[0139]

[0140] From the data results in Table 3, it can be seen that compared with Comparative Examples 1-5, the fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method of the present invention has a significantly excellent effect of inhibiting hyaluronidase, indicating that the present invention improves the soothing effect of the product by adopting a two-stage fermentation method for Tricholoma matsutake liquid seeds and controlling the fermentation time and fermentation temperature of each stage. Further, by comparing the results of Example 1 and 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 soothing effect of the product to a certain extent; by comparing the results of Example 1 and Examples 4-6, it can be seen that the culture medium formula used for fermentation 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) The subjects (140 people, 14 groups, 10 people in each group) did not use any products (cosmetics or topical drugs) on the test area 3 days before the visit, and did not come into contact with water within 3 hours. They cleaned the inner forearms of both hands (cleaning method was to wipe them with dry tissue paper), and after cleaning their arms, they balanced in the test environment for 20 minutes.

[0146] (2.2) Establishment of skin barrier damage: Inject 5% sodium dodecyl sulfate solution into the patch test chamber to soak the filter paper. Apply the patch test chamber to the curved side of the left and right forearms of the subject, 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 to evenly adhere to the skin for 24 hours.

[0147] (2.3) Initial measurement value: After removing the patch tester, the subjects were allowed to balance in the test environment for 30 minutes. A stratum corneum hydration meter was used to measure the water content of the center of each patch area. Each area was measured three times in parallel. The measurement result was expressed as the average value of the patch area detection value, which was 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, and the test area was measured with a stratum corneum hydration meter 2 hours after the sample was used, and 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 can be seen from the data results in Table 4, compared with Comparative Examples 1-5, the fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method of the present invention has a significantly excellent effect of repairing the skin barrier, indicating that the present invention improves the moisturizing and repairing effect of the product by adopting a two-stage fermentation method for Tricholoma matsutake liquid seeds and controlling the fermentation time and fermentation temperature of each stage. Further, by comparing the results of Example 1 and 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 effect of the product to a certain extent; by comparing the results of Example 1 and Examples 4-6, it can be seen that the culture medium formula used for fermentation also affects the moisturizing and repairing effect of the product to a certain extent.

[0152] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope 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 suitable 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 filtrate of Tricholoma matsutake mycelium fermentation having soothing, repairing and antioxidant effects, characterized in that: The preparation method comprises the following steps: (1) inoculating the liquid seeds of Tricholoma matsutake 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) then subjecting the first fermentation broth to a second stage of culturing to obtain a second fermentation broth; the second stage of culturing lasts for 5 to 8 days at 20 to 24° C.; (3) The second fermentation liquid is filtered to separate the pine mushroom mycelium and the fermentation filtrate, thereby obtaining the pine mushroom mycelium fermentation filtrate having soothing, repairing and antioxidant effects.

2. The method for preparing the fermented 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 fermented 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 stirring rates are 120-180 rpm and 50-120 rpm, respectively.

4. The method for preparing the fermented 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 fermented filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 1, characterized in that: The components of the fermentation medium include, by concentration, 15-25% potato juice, 1-5% glucose, 1-5% malt extract, and 0.5-2% pine root extract.

6. The method for preparing the fermented filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 5, characterized in that: 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.

7. The method for preparing the fermented filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 5, characterized in that: The malt extract is prepared by a method comprising the following steps: mixing crushed barley malt with water to obtain 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; filtering the enzymatic hydrolyzate after inactivating the enzyme, and concentrating and drying the filtrate to obtain the malt extract; The pine root extract is prepared by a method comprising the following steps: mixing pine root crushed material with water for reflux extraction, and concentrating the extract under reduced pressure and drying to obtain the pine root extract.

8. The method for preparing the fermented filtrate of Tricholoma matsutake mycelium with soothing, repairing and antioxidant effects according to claim 1, characterized in that: The inoculation amount of the Tricholoma 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 mycelium and the fermentation filtrate, the fermentation filtrate was centrifuged again and filtered through a 0.22 μm filter membrane.

9. The fermentation filtrate of Tricholoma matsutake mycelium obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the fermentation filtrate of Tricholoma matsutake mycelium according to claim 9 in preparing products with soothing, moisturizing, repairing or anti-oxidation effects.

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