A preparation method of a pine mushroom mycelium fermentation product and its product and application

Abstract

The present invention relates to a preparation method of a pine mushroom mycelium fermentation product, and its products and applications, including: inoculating pine mushroom liquid seeds into a culture medium for culturing, solid-liquid separation, obtaining pine mushroom mycelium and fermentation filtrate; subjecting the fermentation filtrate to alcohol precipitation treatment to obtain pine mushroom mycelium crude polysaccharide; inoculating fermentation bacteria seed liquid into a fermentation medium added with pine mushroom mycelium and pine mushroom mycelium crude polysaccharide for culturing to obtain a fermentation liquid; centrifuging the fermentation liquid, filtering the supernatant and sterilizing to obtain the pine mushroom mycelium fermentation product. The present invention develops a new fermentation product with pine mushroom mycelium as a fermentation substrate, the fermentation product has excellent soothing and antioxidant effects, and compared with the method of fermenting a single pine mushroom mycelium or a single pine mushroom mycelium crude polysaccharide, the fermentation product of the present invention has better soothing and antioxidant effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant fermentation, and relates to a method for preparing a pine twig mycelium fermentation product, a product thereof and an application thereof, and in particular to a method for preparing a pine twig mycelium fermentation product with a soothing effect, a product thereof and an application thereof. Background Art

[0002] 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. These symptoms are not only related to people's appearance, but also have a significant impact on their daily life and mood. 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.

[0003] Matsutake is one of the natural medicinal mushrooms. Due to its high nutritional value and medicinal value, it is known as the "king of edible mushrooms". 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 mainly treats urinary problems, replenishes the five internal organs, nourishes qi and blood, consolidates and produces body fluids, and calms the mind. 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 qi, regulate qi and resolve phlegm, and delay aging.

[0004] However, the current application of matsutake in the prior art mainly focuses on the development and application of matsutake extracts, and there is little research on fermentation products made from matsutake mycelium as a fermentation substrate. Therefore, functional matsutake mycelium fermentation products are a direction worth exploring, which is conducive to the development and utilization of matsutake resources. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a Tricholoma matsutake mycelium fermentation product, and its products and applications, and in particular to provide a method for preparing a Tricholoma matsutake mycelium fermentation product with soothing effect, and its products and applications.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a Tricholoma matsutake mycelium fermentation product, the preparation method comprising the following steps:

[0008] (1) inoculating Tricholoma matsutake liquid seeds into a culture medium for culturing, separating the solid and the liquid to obtain Tricholoma matsutake mycelium and a fermentation filtrate; subjecting the fermentation filtrate to alcohol precipitation to obtain Tricholoma matsutake mycelium crude polysaccharide;

[0009] (2) inoculating a fermentation culture medium supplemented with pine twig mycelium and pine twig mycelium crude polysaccharide with a fermentation culture medium to obtain a fermentation liquid;

[0010] The fermentation bacteria are selected from any one or a combination of at least two of Saccharomyces cerevisiae, Pichia pastoris, Acetobacter xylinum, Sphingomonas or Alcaligenes;

[0011] (3) centrifuging the fermentation liquid, filtering the supernatant liquid through a membrane and sterilizing the supernatant liquid to obtain the Tricholoma matsutake mycelium fermentation product.

[0012] The present invention develops a new fermentation product using pine mushroom mycelium as a fermentation substrate, specifically, pine mushroom liquid seeds are fermented to harvest pine mushroom mycelium, and the harvested fermentation filtrate is further treated with alcohol precipitation to harvest another fermentation product, crude polysaccharide; pine mushroom mycelium and pine mushroom mycelium crude polysaccharide are used as fermentation substrates and specific strains are selected to ferment them to obtain a fermentation product. The fermentation product has excellent soothing and antioxidant effects, and compared with the fermentation method of single pine mushroom mycelium or single pine mushroom mycelium crude polysaccharide, the fermentation product of the present invention has better soothing and antioxidant effects.

[0013] Preferably, the total dry weight of the Tricholoma matsutake mycelium and crude polysaccharides from the Tricholoma matsutake mycelium in step (2) is 0.5-5% of the weight of the fermentation medium, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0014] The most suitable amount of the Tricholoma matsutake mycelium and Tricholoma matsutake mycelium crude polysaccharide added to the fermentation medium is 0.5-5%. Too much or too little addition will affect the soothing and anti-oxidation effects.

[0015] Preferably, the mass ratio of the dry weight of Tricholoma matsutake mycelium to the dry weight of crude polysaccharide from Tricholoma matsutake mycelium in the fermentation medium of step (2) is (10-20):1, for example, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, etc.

[0016] Based on the synergistic effect of the dry weight of Matsutake mycelium and the crude polysaccharides of Matsutake mycelium as fermentation substrates in promoting the efficacy of fermentation products, when the two meet the above-mentioned specific mass ratio, the soothing and antioxidant effects of the fermentation products are better.

[0017] Preferably, the fermentation medium in step (2) further contains, by mass percentage, any one of 1-10% (e.g., 1%, 2%, 3%, 5%, 7%, 8%, 10%, etc.) nitrogen source, 1-10% (e.g., 1%, 2%, 3%, 5%, 7%, 8%, 10%, etc.) carbon source, 0.5-3% (e.g., 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc.) inorganic salt or 0.5-3% (e.g., 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc.) pH adjuster or a combination of at least two thereof.

[0018] Preferably, the inoculation amount of the fermentation bacteria seed liquid in step (2) is 1-10%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 10%, etc.

[0019] Preferably, the culturing in step (2) is carried out at 28-34°C (e.g., 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, etc.) for 16-30h (e.g., 16h, 18h, 20h, 24h, 26h, 28h, 30h, etc.).

[0020] Preferably, the fermentation bacteria in step (2) are selected from a combination of Acetobacter xylinum and Sphingomonas.

[0021] Preferably, the fermentation bacteria in step (2) are selected from a combination of Saccharomyces cerevisiae, Acetobacter xylinum and Sphingomonas.

[0022] Any one or a combination of at least two of Saccharomyces cerevisiae, Pichia pastoris, Acetobacter xylinum, Sphingomonas or Alcaligenes selected in the preparation method of the present invention can yield a fermentation product with excellent soothing and antioxidant effects. When a combination of Acetobacter xylinum and Sphingomonas or a combination of Saccharomyces cerevisiae, Acetobacter xylinum and Sphingomonas is selected for joint fermentation, the product has better soothing and antioxidant effects. The specific joint fermentation adopts the following method:

[0023] Preferably, when the fermentation strain is selected from a combination of Acetobacter xylinum and Sphingomonas, the process of inoculating the fermentation strain seed solution for culturing in step (2) comprises:

[0024] The fermentation medium to which the mycelium of Tricholoma matsutake and the crude polysaccharide of the mycelium of Tricholoma matsutake were added was inoculated with the seed liquid of Acetobacter xylinum and the seed liquid of Sphingomonas sp. to carry out fermentation and obtain the fermentation liquid.

[0025] Preferably, the inoculation amounts of the Acetobacter xylinum seed solution and the Sphingomonas seed solution are independently 1-5%, such as 1%, 2%, 3%, 4%, 5%, etc.

[0026] Preferably, the fermentation is carried out at 28-34°C (e.g., 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, etc.) for 16-30h (e.g., 16h, 17h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, etc.).

[0027] Preferably, when the fermentation strain is selected from a combination of Saccharomyces cerevisiae, Acetobacter xylinum and Sphingomonas, the process of inoculating the fermentation strain seed solution for culturing in step (2) comprises:

[0028] Inoculating Acetobacter xylinum seed solution and Sphingomonas seed solution into a fermentation medium added with Tricholoma matsutake mycelia and Tricholoma matsutake mycelia crude polysaccharide, performing a primary fermentation to obtain a one-step fermentation liquid;

[0029] The first-stage fermentation liquid is then inoculated with brewer's yeast seed liquid to carry out secondary fermentation to obtain a fermentation liquid.

[0030] Preferably, the inoculation amount of the Acetobacter xylinum seed solution and the Sphingomonas seed solution are independently 1-3%, such as 1%, 1.5%, 2%, 2.5%, 3%, etc.; the inoculation amount of the Saccharomyces cerevisiae seed solution is 1-5%, such as 1%, 2%, 3%, 4%, 5%, etc.

[0031] Preferably, the primary fermentation is carried out at 28-34°C (e.g., 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, etc.) for 12-18h (e.g., 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc.); the secondary fermentation is carried out at 28-34°C (e.g., 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, etc.) for 6-12h (e.g., 6h, 7h, 8h, 9h, 10h, 11h, 12h, etc.).

[0032] Preferably, the inoculation amount of the Tricholoma matsutake liquid seeds in step (1) is 7-15%, for example, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0033] The preparation of the Tricholoma matsutake liquid seeds can be obtained by using the existing technical methods in the field.

[0034] Preferably, the culture temperature in step (1) is 15-30°C (e.g., 15°C, 17°C, 20°C, 22°C, 25°C, 28°C, 30°C, etc.), and the culture time is 10-30 days (e.g., 10 days, 13 days, 15 days, 18 days, 20 days, 22 days, 25 days, 27 days, 30 days, etc.).

[0035] The culture medium in step (1) can be any culture medium formula that can be used to produce Tricholoma matsutake mycelium, for example: 10-30% potato juice, 1-5% glucose, 1-5g KH2PO4, 1-3g MgSO4.7H20, 0.01-0.1g thiamine, and the balance water; after preparation, sterilize at 115-121° C. and 0.05-0.3MPa pressure for 15-30min.

[0036] Preferably, after obtaining the mycelium in step (1), it is also washed and / or freeze-dried.

[0037] Preferably, the alcohol precipitation treatment in step (1) is carried out using 90-98% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, etc.) ethanol aqueous solution; the volume ratio of the fermentation filtrate to the ethanol aqueous solution is 1:(3-8), for example, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, etc.

[0038] Preferably, after obtaining the crude polysaccharide from Tricholoma matsutake mycelium in step (1), it is freeze-dried.

[0039] Preferably, the centrifugation in step (3) is performed at 12000-18000 rpm (e.g., 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm, 16000 rpm, 18000 rpm, etc.) for 20-40 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, etc.).

[0040] Preferably, the membrane in step (3) is a 0.22 μm membrane.

[0041] Preferably, freeze-drying is further performed after membrane or sterilization in step (3).

[0042] In a second aspect, the present invention provides a Tricholoma matsutake mycelium fermentation product obtained according to the preparation method described in the first aspect.

[0043] In a third aspect, the present invention provides a use of the Tricholoma matsutake mycelium fermentation product described in the second aspect in the preparation of a product with soothing and / or antioxidant effects.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention develops a new fermentation product using pine mushroom mycelium as a fermentation substrate, specifically, pine mushroom liquid seeds are fermented to harvest pine mushroom mycelium, and the harvested fermentation filtrate is further treated with alcohol precipitation to harvest another fermentation product, crude polysaccharide; pine mushroom mycelium and pine mushroom mycelium crude polysaccharide are used as fermentation substrates and specific strains are selected to ferment them to obtain a fermentation product. The fermentation product has excellent soothing and antioxidant effects, and compared with the fermentation method of single pine mushroom mycelium or single pine mushroom mycelium crude polysaccharide, the fermentation product of the present invention has better soothing and antioxidant effects. DETAILED DESCRIPTION

[0046] 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.

[0047] The strains used in the following implementation cases are commercial strains purchased from the Guangdong Provincial Microbiological Strain Collection Center, namely Saccharomyces cerevisiae strain (GDMCC 2.207), Pichia pastoris strain (GDMCC 2.248), Acetobacter xylinum strain (GDMCC1.423), Sphingomonas strain (GDMCC 1.696); and commercial strains purchased from the China Industrial Microbiological Culture Collection Center, namely Alcaligenes strain (CICC 24302).

[0048] The liquid seeds of Tricholoma matsutake used in the following implementation cases were prepared by the following method:

[0049] Prepare the culture medium: 20% potato juice, 20g glucose, 3g KH2PO4, 1.5g MgSO4.7H20, and the rest water. After the preparation is completed, sterilize at 121℃ for 20min, take out the culture medium, take appropriate size mushroom blocks from the slope under sterile conditions, inoculate them into a shaking bottle, and culture them in a shaking table at 28℃ and 200rpm for 10 days to obtain the Matsutake seed liquid.

[0050] Example 1

[0051] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0052] (1) 10% of Matsutake liquid seeds were inoculated into a culture medium (20% potato juice, 2% glucose, 3 g KH2PO4, 1.5 g MgSO4.7H20, 0.1% thiamine, the balance being water; sterilized at 121°C and 0.1 MPa for 20 min and then cooled to room temperature) for culturing at 28°C for 20 days.

[0053] (2) Solid-liquid separation to obtain Tricholoma matsutake mycelium and fermentation filtrate; the mycelium is washed with pure water for 3 times and centrifuged again to remove excess water. The mycelium is quickly frozen to -20°C, maintained for 2 hours, and placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%. It is crushed using a grinder to obtain powdered Tricholoma matsutake mycelium powder; the fermentation filtrate is centrifuged again, filtered through a 0.22 μm filter membrane, and the filtrate is mixed with 4 times the volume of 95% ethanol aqueous solution, allowed to stand for 12 hours for alcohol precipitation treatment, the bottom precipitate is collected, the excess liquid is removed by centrifugation, and the collected precipitate is placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%, which is crude polysaccharide from Tricholoma matsutake mycelium.

[0054] (3) Preparation of brewer's yeast seed solution: Under sterile conditions, the brewer's yeast strain was inoculated into a shake flask at a rate of 4% (2% peptone, 1% yeast powder, 2% glucose, the remainder water, pH natural; sterilized at 115°C for 20 min), and cultured in a shaker at 30°C and 200 rpm for 24 h to obtain the brewer's yeast seed solution.

[0055] (4) Prepare fermentation medium: 2% peptone, 2% yeast powder, 2% glucose, 2% matsutake mycelium powder obtained in step (2), 0.15% mycelium crude polysaccharide, the balance is water, and the pH is natural. After preparation, sterilize at 115° C. and 0.1 MPa pressure for 30 min.

[0056] (5) Inoculate 4% of the brewer's yeast seed liquid into the fermentation medium prepared in step (4) for cultivation at a temperature of 30° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 24 h, and harvest the fermentation liquid in a tank.

[0057] (6) The fermentation liquid was centrifuged at 15000 r / min for 30 min, the supernatant was passed through a 0.22 μm organic membrane, sterilized at 115° C. for 30 min, and freeze-dried to obtain the Tricholoma matsutake mycelium fermentation product.

[0058] Example 2

[0059] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0060] (1) 7% of Matsutake liquid seeds were inoculated into a culture medium (20% potato juice, 2% glucose, 3 g KH2PO4, 1.5 g MgSO4.7H20, 0.1% thiamine, the balance being water; sterilized at 121°C and 0.1 MPa for 20 min and then cooled to room temperature) for 30 days at 20°C.

[0061] (2) Solid-liquid separation to obtain Tricholoma matsutake mycelium and fermentation filtrate; the mycelium is washed with pure water for 3 times and centrifuged again to remove excess water. The mycelium is quickly frozen to -20°C, maintained for 2 hours, and placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%. It is crushed using a grinder to obtain powdered Tricholoma matsutake mycelium powder; the fermentation filtrate is centrifuged again, filtered through a 0.22 μm filter membrane, and the filtrate is mixed with 8 times the volume of 92% ethanol aqueous solution, allowed to stand for 12 hours for alcohol precipitation treatment, the bottom precipitate is collected, the excess liquid is removed by centrifugation, and the collected precipitate is placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%, which is crude polysaccharide from Tricholoma matsutake mycelium.

[0062] (3) Preparation of Pichia pastoris seed solution: Under sterile conditions, Pichia pastoris was inoculated into a shake flask at a 4% inoculum (1% tryptone, 0.5% yeast powder, 1% sodium chloride, 2% glucose, the balance being water, pH 7; sterilized at 115°C for 20 min), and cultured in a shaker at 25°C and 200 rpm for 24 h to obtain the Pichia pastoris seed solution.

[0063] (4) Prepare fermentation medium: 1% tryptone, 0.5% yeast powder, 1% sodium chloride, 2% matsutake mycelium powder obtained in step (2), 0.1% mycelium crude polysaccharide, the balance is water, and the pH is natural. After preparation, sterilize at 121° C. and 0.1 MPa pressure for 20 min.

[0064] (5) Inoculate 4% of Pichia pastoris seed liquid into the fermentation medium prepared in step (4) for cultivation at a temperature of 30° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 24 h, and harvest the fermentation liquid in a tank.

[0065] (6) The fermentation liquid was centrifuged at 12000 r / min for 40 min, the supernatant was passed through a 0.22 μm organic membrane, sterilized at 115° C. for 30 min, and freeze-dried to obtain the Tricholoma matsutake mycelium fermentation product.

[0066] Example 3

[0067] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0068] (1) 15% of Matsutake liquid seeds were inoculated into a culture medium (20% potato juice, 2% glucose, 3 g KH2PO4, 1.5 g MgSO4.7H20, 0.1% thiamine, the balance being water; sterilized at 121°C and 0.1 MPa for 20 min and then cooled to room temperature) for 10 days at 30°C.

[0069] (2) Solid-liquid separation to obtain Tricholoma matsutake mycelium and fermentation filtrate; the mycelium is washed with pure water for 3 times and centrifuged again to remove excess water. The mycelium is quickly frozen to -20°C, maintained for 2 hours, and placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%. It is crushed using a grinder to obtain powdered Tricholoma matsutake mycelium powder; the fermentation filtrate is centrifuged again, filtered through a 0.22 μm filter membrane, and the filtrate is mixed with 3 volumes of 97% ethanol aqueous solution, allowed to stand for 12 hours for alcohol precipitation treatment, the bottom precipitate is collected, the excess liquid is removed by centrifugation, and the collected precipitate is placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%, which is crude polysaccharide from Tricholoma matsutake mycelium.

[0070] (3) Preparation of Acetobacter xylinum seed solution: Under sterile conditions, Acetobacter xylinum strains were inoculated at a 4% inoculum into a shake flask (2% glucose, 0.5% peptone, 0.5% yeast powder, 1% citric acid, pH 6, balance water; sterilized at 115° C. for 20 min), and cultured in a shaker at 30° C. and 160 rpm for 24 h to obtain Acetobacter xylinum seed solution.

[0071] (4) Prepare fermentation medium: 2% glucose, 0.5% peptone, 0.5% yeast powder, 1% citric acid, 2% matsutake mycelium powder, 0.2% mycelium crude polysaccharide, pH 6, and the balance water. After preparation, sterilize at 121°C and 0.1 MPa for 20 min.

[0072] (5) Inoculate 3% of the seed solution of Acetobacter xylinum into the fermentation medium prepared in step (4) for cultivation at a temperature of 30° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 24 h, and harvest the fermentation liquid.

[0073] (6) The fermentation liquid was centrifuged at 18000 r / min for 20 min, the supernatant was passed through a 0.22 μm organic membrane, sterilized at 115° C. for 30 min, and freeze-dried to obtain the Tricholoma matsutake mycelium fermentation product.

[0074] Example 4

[0075] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0076] (1) 10% of Matsutake liquid seeds were inoculated into a culture medium (20% potato juice, 2% glucose, 3 g KH2PO4, 1.5 g MgSO4.7H20, 0.1% thiamine, the balance being water; sterilized at 121°C and 0.1 MPa for 20 min and then cooled to room temperature) for culturing at 28°C for 15 days.

[0077] (2) Solid-liquid separation to obtain Tricholoma matsutake mycelium and fermentation filtrate; the mycelium is washed with pure water for 3 times and centrifuged again to remove excess water. The mycelium is quickly frozen to -20°C, maintained for 2 hours, and placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%. It is crushed using a grinder to obtain powdered Tricholoma matsutake mycelium powder; the fermentation filtrate is centrifuged again, filtered through a 0.22 μm filter membrane, and the filtrate is mixed with 5 times the volume of 95% ethanol aqueous solution, allowed to stand for 12 hours for alcohol precipitation treatment, the bottom precipitate is collected, the excess liquid is removed by centrifugation, and the collected precipitate is placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%, which is crude polysaccharide from Tricholoma matsutake mycelium.

[0078] (3) Preparation of Sphingomonas seed solution: Under sterile conditions, the Sphingomonas strain was inoculated into a shake flask at a 5% inoculation rate (sucrose 2%, yeast extract 2.5%, NaCl 1%, KH2PO4 0.2%, pH 6.0, balance water; sterilized at 115°C for 20 min), and cultured in a shaker at 30°C and 200 rpm for 24 h to obtain the Sphingomonas seed solution.

[0079] (4) Prepare fermentation medium: 2% sucrose, 2.5% yeast extract powder, 1% NaCl, 0.2% KH2PO4, 4% matsutake mycelium powder, 0.2% mycelium crude polysaccharide, pH 6, and the balance water. After preparation, sterilize at 121°C and 0.1 MPa for 20 min.

[0080] (5) Inoculate 6% of the Sphingomonas seed solution into the fermentation medium prepared in step (4) for cultivation at a temperature of 30° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 24 h, and harvest the fermentation liquid.

[0081] (6) The fermentation liquid was centrifuged at 15000 r / min for 30 min, the supernatant was passed through a 0.22 μm organic membrane, sterilized at 115° C. for 30 min, and freeze-dried to obtain the Tricholoma matsutake mycelium fermentation product.

[0082] Example 5

[0083] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0084] (1) 7% of Matsutake liquid seeds were inoculated into a culture medium (20% potato juice, 2% glucose, 3 g KH2PO4, 1.5 g MgSO4.7H20, 0.1% thiamine, the balance being water; sterilized at 121°C and 0.1 MPa for 20 min and then cooled to room temperature) for culturing at 28°C for 15 days.

[0085] (2) Solid-liquid separation to obtain Tricholoma matsutake mycelium and fermentation filtrate; the mycelium is washed with pure water for 3 times and centrifuged again to remove excess water. The mycelium is quickly frozen to -20°C, maintained for 2 hours, and placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%. It is crushed using a grinder to obtain powdered Tricholoma matsutake mycelium powder; the fermentation filtrate is centrifuged again, filtered through a 0.22 μm filter membrane, and the filtrate is mixed with 5 times the volume of 95% ethanol aqueous solution, allowed to stand for 12 hours for alcohol precipitation treatment, the bottom precipitate is collected, the excess liquid is removed by centrifugation, and the collected precipitate is placed in a freeze drying oven for drying. The resulting product has a moisture content of ≤5%, which is crude polysaccharide from Tricholoma matsutake mycelium.

[0086] (3) Preparation of Alcaligenes seed solution: Under sterile conditions, the Alcaligenes strain was inoculated into a shake flask at a 5% inoculum (1.5% tryptone, 0.5% soytone, 0.5% NaCl, pH 7.3, balance water; sterilized at 115°C for 20 min), and cultured in a shaker at 30°C and 200 rpm for 24 h to obtain the Alcaligenes seed solution.

[0087] (4) Prepare fermentation medium: 1.5% tryptone, 0.5% soytone, 0.5% NaCl, 1% matsutake mycelium powder, 0.1% mycelium crude polysaccharide, pH 7.3, and the balance water. After preparation, sterilize at 121°C and 0.1 MPa for 20 min.

[0088] (5) Inoculate 4% of the Alcaligenes seed solution into the fermentation medium prepared in step (4) for cultivation at a temperature of 28° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 24 h, and harvest the fermentation liquid in a tank.

[0089] (6) The fermentation liquid was centrifuged at 15000 r / min for 30 min, the supernatant was passed through a 0.22 μm organic membrane, sterilized at 115° C. for 30 min, and freeze-dried to obtain the Tricholoma matsutake mycelium fermentation product.

[0090] Example 6

[0091] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0092] (1) Same as Example 1.

[0093] (2) Same as Example 1.

[0094] (3) Preparation of brewer's yeast seed solution: Under sterile conditions, the brewer's yeast strain was inoculated into a shake flask at a rate of 4% (2% peptone, 1% yeast powder, 2% glucose, the remainder water, pH natural; sterilized at 115°C for 20 min), and cultured in a shaker at 30°C and 200 rpm for 24 h to obtain the brewer's yeast seed solution.

[0095] Preparation of Acetobacter xylinum seed solution: Under sterile conditions, inoculate Acetobacter xylinum with a 4% inoculum amount into a shake flask (2% glucose, 0.5% peptone, 0.5% yeast powder, 1% citric acid, pH 6, balance water; sterilize at 115°C for 20 min), and culture in a shaker at 30°C and 160 rpm for 24 h to obtain Acetobacter xylinum seed solution.

[0096] Preparation of Sphingomonas seed solution: Under sterile conditions, the Sphingomonas strain was inoculated into a shake flask at a 5% inoculation rate (sucrose 2%, yeast extract 2.5%, NaCl 1%, KH2PO4 0.2%, pH 6.0, balance water; sterilized at 115°C for 20 min), and cultured in a shaker at 30°C and 200 rpm for 24 h to obtain the Sphingomonas seed solution.

[0097] (4) Same as Example 1.

[0098] (5) inoculating 2% of Acetobacter xylinum seed solution and 2% of Sphingomonas seed solution into the fermentation medium prepared in step (4) for culturing at a temperature of 30° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, and a culturing time of 16 h to obtain a one-step fermentation liquid;

[0099] 4% of the brewer's yeast seed solution was inoculated into the one-step fermentation liquid for cultivation at a temperature of 30°C, a ventilation volume of 50 L, a stirring of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 8 h, and the fermentation liquid was harvested in a tank.

[0100] (6) Same as Example 1.

[0101] Example 7

[0102] This embodiment provides a Tricholoma matsutake mycelium fermentation product, which is prepared by the following preparation method:

[0103] (1) Same as Example 1.

[0104] (2) Same as Example 1.

[0105] (3) Preparation of Acetobacter xylinum seed solution: Under sterile conditions, Acetobacter xylinum strains were inoculated at a 4% inoculum into a shake flask (2% glucose, 0.5% peptone, 0.5% yeast powder, 1% citric acid, pH 6, balance water; sterilized at 115° C. for 20 min), and cultured in a shaker at 30° C. and 160 rpm for 24 h to obtain Acetobacter xylinum seed solution.

[0106] Preparation of Sphingomonas seed solution: Under sterile conditions, the Sphingomonas strain was inoculated into a shake flask at a 5% inoculation rate (sucrose 2%, yeast extract 2.5%, NaCl 1%, KH2PO4 0.2%, pH 6.0, balance water; sterilized at 115°C for 20 min), and cultured in a shaker at 30°C and 200 rpm for 24 h to obtain the Sphingomonas seed solution.

[0107] (4) Same as Example 1.

[0108] (5) Inoculate 2% of Acetobacter xylinum seed solution and 2% of Sphingomonas seed solution into the fermentation medium prepared in step (4) for cultivation at a temperature of 30° C., a ventilation volume of 50 L, a stirring speed of 100 r / min, a pressure of 0.1 MPa, a cultivation time of 24 h, and harvest the fermentation liquid in a tank.

[0109] (6) Same as Example 1.

[0110] Comparative Examples 1-5

[0111] This comparative example provides five kinds of Tricholoma matsutake mycelium fermentation products, and the preparation methods thereof are different from those of Examples 1-5 respectively in that the mycelium crude polysaccharide is absent in step (4), and the reduced amount thereof is distributed to the Tricholoma matsutake mycelium powder in the corresponding examples. Other conditions remain unchanged.

[0112] Comparative Examples 6-10

[0113] This comparative example provides five kinds of Tricholoma matsutake mycelium fermentation products, and the preparation methods thereof are different from those of Examples 1-5 respectively in that Tricholoma matsutake mycelium powder is absent in step (4), and its reduced amount is allocated to the mycelium crude polysaccharide in the corresponding example. Other conditions remain unchanged.

[0114] Test Example 1

[0115] Safety performance evaluation:

[0116] The safety performance of the products of Examples 1-7 and Comparative Examples 1-10 was evaluated. Each group of products was diluted with PBS to a 10% sample solution to be tested. 40 social volunteers aged 20-45 years old who met the requirements were selected and randomly divided into 4 groups. Each group tried 4 or 5 different products. Put it into the small chamber of the spot tester, and the control well was not treated. 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 at 30 minutes, 24 hours and 48 hours after removing the test substance spot tester.

[0117] Table 1

[0118]

[0119] The results showed that the products of Examples 1-7 of the present invention and Comparative Examples 1-10 all had negative reactions after trial use, indicating that the Tricholoma matsutake mycelium fermentation products of the present invention are safe and mild, and will not cause adverse reactions such as skin irritation and sensitization.

[0120] Test Example 2

[0121] Antioxidant effect evaluation test:

[0122] (1) Preparation of test samples: The products prepared in Examples 1-7 and Comparative Examples 1-10 were prepared into test samples with a concentration of 5% for later use.

[0123] (2) DPPH free radical scavenging experiment:

[0124] (2.1) Preparation of DPPH solution: Weigh 5.0 mg of DPPH and dissolve it in an appropriate amount of anhydrous ethanol. Protect from light and sonicate until it is fully dissolved. Then use anhydrous ethanol to make the volume to 100 mL to prepare a 50 μg / mL DPPH solution. This solution should be prepared and used immediately.

[0125] (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.

[0126] (2.3) DPPH free radical scavenging rate calculation formula:

[0127] Clearance (%) = [1-(Ai-Aj) / Ac] × 100%

[0128] 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.

[0129] (3) ABTS free radical scavenging experiment:

[0130] (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;

[0131] 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.

[0132] (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. React at 25℃ in the dark for 10 minutes, and measure the absorbance at 734 nm. Calculate the clearance rate.

[0133] (3.3) ABTS free radical scavenging rate calculation formula:

[0134] Clearance (%) = [1-(Ai-Aj) / Ac] × 100%

[0135] 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.

[0136] (4) Hydroxyl free radical scavenging experiment:

[0137] (4.1) 6mmol / L FeSO4: Dissolve FeSO4 in water to prepare 6mmol / L FeSO4;

[0138] 6mmol / L H2O2: Use 30% H2O2 to prepare 6mmol / L H2O2;

[0139] 6mmol / L salicylic acid solution: Use anhydrous ethanol to prepare 6mmol / L salicylic acid solution.

[0140] (4.2) Set up sample group: 100μL sample solution mixed with 100μL FeSO4, 100μL H2O2, 100μL salicylic acid solution; sample blank group: 100μL sample solution mixed with 100μL solvent; control group: 100μL FeSO4, 100μL H2O2, 100μL salicylic acid solution mixed with 100μL solvent. React in a 37℃ water bath in the dark for 30min, and measure the absorbance at 510nm. Calculate the clearance rate.

[0141] (4.3) ABTS free radical scavenging rate calculation formula:

[0142] Clearance (%) = [1-(Ai-Aj) / Ac] × 100%

[0143] 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.

[0144] (5) The results are shown in Table 2:

[0145] Table 2

[0146]

[0147]

[0148] As shown in Table 2, compared with Comparative Examples 1-10, the fermented product of Tricholoma matsutake mycelium used Tricholoma matsutake mycelium and Tricholoma matsutake mycelium crude polysaccharide as fermentation substrates and selected specific strains for fermentation, and the obtained fermented product has more prominent antioxidant effect. The difference in fermentation strains also affects the antioxidant effect of the product to a certain extent.

[0149] Test Example 3

[0150] Soothing effect evaluation test:

[0151] (1) Preparation of test samples: The products prepared in Examples 1-5 and Comparative Examples 1-7 were prepared into test samples with a concentration of 5% for later use.

[0152] (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 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 NaOH solution (5 mol / L) and 0.5 mL of distilled water were placed in a boiling water bath for 15 min, and then immediately cooled with ice water for 5 min. 1 mL of P-DAB colorimetric reagent was added, and after sufficient shaking, 3.5 mL of anhydrous ethanol was added. The mixture was allowed to stand for 30 min for color development, and the OD values ​​of the four test tubes A, B, C, and D were measured at 530 nm, respectively.

[0153] The calculation formula of hyaluronidase activity inhibition rate is:

[0154] Hyaluronidase inhibition rate (%) = [(CD) - (AB)] / (CD) × 100

[0155] (3) The results are shown in Table 3:

[0156] Table 3

[0157]

[0158]

[0159] It can be seen from the data results in Table 3 that, compared with Comparative Examples 1-10, the pine mushroom mycelium fermentation product of the present invention uses pine mushroom mycelium and pine mushroom mycelium crude polysaccharide as fermentation substrates and selects specific strains for fermentation treatment, and the obtained fermentation product has a more prominent soothing effect. And the difference in fermentation strains also affects the soothing effect of the product to a certain extent.

[0160] 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.

[0161] 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.

[0162] 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 product of Tricholoma matsutake mycelium fermentation, characterized in that: The preparation method comprises the following steps: (1) inoculating Tricholoma matsutake liquid seeds into a culture medium for culturing, separating the solid and the liquid to obtain Tricholoma matsutake mycelium and a fermentation filtrate; subjecting the fermentation filtrate to alcohol precipitation to obtain Tricholoma matsutake mycelium crude polysaccharide; (2) inoculating a fermentation culture medium supplemented with pine twig mycelium and pine twig mycelium crude polysaccharide with a fermentation culture medium to obtain a fermentation liquid; The fermentation bacteria are selected from any one or a combination of at least two of Saccharomyces cerevisiae, Pichia pastoris, Acetobacter xylinum, Sphingomonas or Alcaligenes; (3) centrifuging the fermentation liquid, filtering the supernatant liquid through a membrane and sterilizing the supernatant liquid to obtain the Tricholoma matsutake mycelium fermentation product.

2. The method for preparing a Tricholoma matsutake mycelium fermentation product according to claim 1, characterized in that: The total dry weight of the Tricholoma matsutake mycelium and Tricholoma matsutake mycelium crude polysaccharide in step (2) is 0.5-5% of the weight of the fermentation medium; Preferably, the mass ratio of the dry weight of Tricholoma matsutake mycelium to the dry weight of crude polysaccharide from Tricholoma matsutake mycelium in the fermentation medium of step (2) is (10-20):

1.

3. The method for preparing a Tricholoma matsutake mycelium fermentation product according to claim 1 or 2, characterized in that: The fermentation medium in step (2) further contains, by weight percentage, any one of 1-10% nitrogen source, 1-10% carbon source, 0.5-3% inorganic salt or 0.5-3% pH regulator or a combination of at least two thereof; Preferably, the inoculation amount of the fermentation bacteria seed liquid in step (2) is 1-10%; Preferably, the culturing in step (2) is carried out at 28-34°C for 16-30 hours.

4. The method for preparing a Tricholoma matsutake mycelium fermentation product according to any one of claims 1 to 3, characterized in that: The fermentation bacteria in step (2) are selected from a combination of Acetobacter xylinum and Sphingomonas; Preferably, the fermentation bacteria in step (2) are selected from a combination of Saccharomyces cerevisiae, Acetobacter xylinum and Sphingomonas.

5. The method for preparing a Tricholoma matsutake mycelium fermentation product according to claim 4, characterized in that: When the fermentation strain is selected from a combination of Saccharomyces cerevisiae, Acetobacter xylinum and Sphingomonas, the process of inoculating the fermentation strain seed solution for culturing in step (2) comprises: Inoculating Acetobacter xylinum seed solution and Sphingomonas seed solution into a fermentation medium added with Tricholoma matsutake mycelia and Tricholoma matsutake mycelia crude polysaccharide, performing a primary fermentation to obtain a one-step fermentation solution; The first-stage fermentation liquid is then inoculated with brewer's yeast seed liquid to carry out secondary fermentation to obtain a fermentation liquid.

6. The method for preparing a Tricholoma matsutake mycelium fermentation product according to claim 5, characterized in that: The inoculation amount of the Acetobacter xylinum seed solution and the Sphingomonas seed solution are independently 1-3%; the inoculation amount of the Saccharomyces cerevisiae seed solution is 1-5%; Preferably, the primary fermentation is carried out at 28-34°C for 12-18 hours; and the secondary fermentation is carried out at 28-34°C for 6-12 hours.

7. The method for preparing a Tricholoma matsutake mycelium fermentation product according to any one of claims 1 to 6, characterized in that: The inoculation amount of the Tricholoma matsutake liquid seeds in step (1) is 7-15%; Preferably, the culture temperature in step (1) is 15-30°C and the culture time is 10-30 days; Preferably, after obtaining the mycelium in step (1), the mycelium is washed and / or freeze-dried; Preferably, the alcohol precipitation treatment in step (1) is carried out using a 90-98% ethanol aqueous solution; the volume ratio of the fermentation filtrate to the ethanol aqueous solution is 1:(3-8); Preferably, after obtaining the crude polysaccharide from Tricholoma matsutake mycelium in step (1), it is freeze-dried.

8. The method for preparing a Tricholoma matsutake mycelium fermentation product according to any one of claims 1 to 7, characterized in that: The centrifugation in step (3) is performed at 12000-18000 rpm for 20-40 min; Preferably, the membrane in step (3) is a 0.22 μm membrane; Preferably, freeze-drying is further performed after membrane or sterilization in step (3).

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

10. Use of the fermented product of Tricholoma matsutake mycelium according to claim 9 in the preparation of a product having soothing and / or antioxidant effects.

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