Preparation method, product and application of matsutake polysaccharide with soothing effect

Abstract

The present invention relates to a preparation method, product and application of Tricholoma matsutake polysaccharide with a soothing effect. The preparation method includes: mixing Tricholoma matsutake dry powder with distilled water and a composite enzyme, and performing ultrasonic-assisted extraction to obtain a Tricholoma matsutake extraction product; centrifuging the extraction product to take the supernatant, mixing the concentrated supernatant with a fermentation medium and inoculating a bacterial solution, and performing fermentation culture to obtain a Tricholoma matsutake fermentation filtrate; mixing the Tricholoma matsutake fermentation filtrate with water, performing water extraction treatment, centrifuging to take the supernatant, and filtering to obtain a water extraction filtrate; performing alcohol precipitation treatment on the water extraction filtrate, centrifuging to take the supernatant, and filtering to obtain a crude polysaccharide; redissolving the crude polysaccharide in water, and then performing deproteinization treatment to obtain a deproteinized product; sequentially performing elution on the deproteinized product through an anion exchange chromatography column and a Sephadex column, collecting the eluate and concentrating and drying it. The prepared product has excellent soothing effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and relates to a preparation method, product and application of matsutake polysaccharide with a soothing effect. Background Art

[0002] Skin sensitivity is becoming increasingly common among people. Harmful substances in the environment, such as dust, chemicals and heavy metals, continuously attack people's skin, resulting in damaged skin barriers. At the same time, bad living habits such as staying up late, unhealthy diet and lack of exercise also exacerbate the occurrence of skin sensitivity problems. Skin sensitivity symptoms are often very disturbing. Slight irritation or touch may trigger uncomfortable skin reactions, such as stinging, flushing and dry itching. These symptoms not only affect people's appearance, but also have a certain impact on daily life and mood. In order to address this problem, continuous research and development are being carried out on cosmetic raw materials for sensitive skin care in the market. Medicinal and edible homologous plants with both safety and soothing and repairing effects are thus widely used in sensitive skin care products, aiming to help sensitive skin recover to a healthy state and relieve uncomfortable reactions.

[0003] Matsutake is one of the natural medicinal fungi, which has high nutritional and medicinal values. According to modern scientific research, the extract of matsutake can inhibit the expression of inflammatory factors and inflammation-related proteins by blocking the upstream signaling pathway of mitogen-activated protein kinase (MAPK) and further inhibiting the activation of nuclear factor κB (NF-κB) pathway, and can play a role in relieving skin sensitivity. At present, the preparation processes of matsutake cosmetic raw materials are generally water extraction method, alcohol extraction method, etc. For example, a preparation method of matsutake extract with anti-aging, moisturizing and penetration-promoting effects disclosed in Patent CN102895172B uses boiling water for extraction for 2-5 hours, but long-term high temperature is likely to damage the structure of active ingredients in matsutake, resulting in a decline in efficacy. There is also research on extracting total triterpenoids from matsutake by refluxing with 80% ethanol solution in a water bath (Liu Junhong, Chu Jiahao, Gong Xiaoqing, etc. Research on the extraction process of total triterpenoids from matsutake and its whitening effect [J]. Contemporary Chemical Industry, 2022, 51 (09): 2110-2113.), and this extraction process has problems of relatively low extraction efficiency and less enrichment of active substances.

[0004] At present, there are few reports in the existing technology on how to extract and enrich matsutake polysaccharide. Therefore, developing a preparation process that can efficiently enrich matsutake polysaccharide and applying the obtained product is a direction worthy of exploration. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a preparation method, product and application of matsutake polysaccharide with a soothing effect.

[0006] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing Tricholoma matsutake polysaccharide with a soothing effect, and the preparation method includes:

[0008] (1) Mix the dried Tricholoma matsutake powder with distilled water and a composite enzyme, and perform ultrasonic-assisted extraction to obtain a Tricholoma matsutake extraction product;

[0009] (2) Centrifuge the extraction product to obtain the supernatant, mix the concentrated supernatant with a fermentation medium and inoculate the bacterial liquid, and perform fermentation culture to obtain a Tricholoma matsutake fermentation filtrate;

[0010] (3) Mix the Tricholoma matsutake fermentation filtrate with water, perform water extraction treatment, centrifuge to obtain the supernatant, and filter to obtain a water extraction filtrate;

[0011] (4) Perform alcohol precipitation treatment on the water extraction filtrate, centrifuge to obtain the precipitate, and obtain crude polysaccharide;

[0012] (5) Redissolve the crude polysaccharide in water, and then perform deproteinization treatment to obtain a deproteinized product;

[0013] (6) Elute the deproteinized product successively through an anion exchange chromatography column and a Sephadex column, collect the eluate and concentrate and dry it to obtain the Tricholoma matsutake polysaccharide.

[0014] The preparation method involved in the present invention creatively adopts the method of extracting the dried Tricholoma matsutake powder with a composite enzyme, and ultrasonic-assisted extraction is added, which can extract the target product more efficiently, and make the performance of the target product stable, with excellent soothing effect; further, using microorganisms to ferment the extraction supernatant can decompose part of the macromolecular polysaccharide into medium-molecular polysaccharide and small-molecular polysaccharide, and the specific polysaccharide microstructure makes the product have a more excellent soothing effect; further, the successive water extraction treatment, alcohol precipitation treatment and deproteinization treatment remove non-target components, which is beneficial to enriching polysaccharide components; performing anion exchange chromatography column elution and Sephadex column elution is to further remove non-target impurities, so that the target product is further enriched, and finally the product has a high concentration of polysaccharide components and excellent anti-inflammatory and soothing effects.

[0015] Preferably, the material-liquid ratio of the dried Tricholoma matsutake powder to distilled water is 1 g:(50 - 80) mL, such as 1 g:50 mL, 1 g:55 mL, 1 g:60 mL, 1 g:65 mL, 1 g:70 mL, 1 g:75 mL, 1 g:80 mL, etc.

[0016] Preferably, the composite enzyme is composed of cellulase and pectinase.

[0017] The present invention selects a specific composite enzyme formula, which can make the dissolution efficiency of matsutake polysaccharide higher, and finally the content of enriched matsutake polysaccharide is more.

[0018] Preferably, the total addition amount of the composite enzyme in the extraction system is 0.3 - 0.6 mg / mL, such as 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, etc.; the mass ratio of cellulase to pectinase is (1 - 5):1, such as 1:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc.

[0019] Preferably, the enzyme activity of the cellulase is 20000 - 40000 U / g, such as 20000 U / g, 25000 U / g, 30000 U / g, 35000 U / g, 40000 U / g, etc.; the enzyme activity of the pectinase is 10000 - 20000 U / g, such as 10000 U / g, 12000 U / g, 15000 U / g, 17000 U / g, 20000 U / g, etc.

[0020] Preferably, the power of the ultrasonic wave is 250 - 450 W, such as 250 W, 300 W, 350 W, 400 W, 450 W, etc.

[0021] Preferably, the temperature of the extraction is 25 - 45 °C, such as 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, etc.; the time is 30 - 60 min, such as 30 min, 40 min, 50 min, 60 min, etc.

[0022] Preferably, the supernatant in step (2) is concentrated to 1 / 5 - 1 / 2 of the original volume of the supernatant, such as 1 / 5, 2 / 5, 3 / 10, 4 / 10, 1 / 2, etc.

[0023] Preferably, the volume ratio of the fermentation medium to the concentrated supernatant is (200 - 300):1, such as 200:1, 230:1, 250:1, 280:1, 300:1, etc.

[0024] Preferably, the bacterial liquid is a combination of yeast bacterial liquid and trichoderma bacterial liquid.

[0025] The present invention selects a specific composite fermentation bacterium, and the two bacteria cooperate with each other and ferment together, so that the final product has a more excellent soothing effect.

[0026] Preferably, the ratio of the viable cell count of the yeast to that of the Trichoderma is (1 - 10):1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0027] Preferably, the initial inoculation concentration of the yeast solution and the Trichoderma solution is independently OD600 = 2 - 6, such as 2, 2.5, 3, 3.5, 4, 5, 6, etc., and the inoculation amount is independently 1 - 5%, such as 1%, 2%, 3%, 4%, 5%, etc.

[0028] Preferably, the temperature of the fermentation culture is 25 - 35 °C, such as 25 °C, 28 °C, 30 °C, 32 °C, 35 °C, etc., and the time is 12 - 24 h, such as 12 h, 15 h, 18 h, 20 h, 22 h, 23 h, 24 h, etc.

[0029] Preferably, the volume ratio of the Tricholoma matsutake fermentation filtrate to water in step (3) is 1:(1 - 3), such as 1:1, 1:1.5, 1:2, 1:2.5, 1:3, etc.

[0030] Preferably, the temperature of the water extraction treatment is 25 - 40 °C, such as 25 °C, 28 °C, 30 °C, 32 °C, 35 °C, 38 °C, 40 °C, etc.; the time is 10 - 40 min, such as 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, etc.

[0031] Preferably, the centrifugation in step (3) is carried out at 4 - 10 °C (such as 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, etc.) and 6000 - 8000 rpm (such as 6000 rpm, 7000 rpm, 8000 rpm, etc.) for 15 - 20 min (such as 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, etc.).

[0032] Preferably, the alcohol precipitation treatment uses absolute ethanol or an ethanol aqueous solution, and its final concentration in the system is 85 - 95%, such as 85%, 87%, 88%, 90%, 92%, 93%, 94%, 95%, etc.

[0033] In the present invention, the protein removal treatment uses Sevag reagent, and its volume ratio to the water extraction filtrate is 1:(1 - 5), such as 1:1, 1:2, 1:3, 1:4, 1:5, etc.; the treatment method is to shake for 20 - 40 min first, such as 20 min, 25 min, 30 min, 35 min, 40 min, etc.; then let it stand for 1 - 3 h, such as 1 h, 2 h, 3 h, etc.; discard the bottom white protein, and repeat the above operation until there is no protein at the two - phase interface.

[0034] In the present invention, the anion exchange chromatography column is selected from a DEAE-52 cellulose anion exchange chromatography column.

[0035] Compared with other types of anion exchange chromatography columns, the end product obtained by elution with the DEAE-52 cellulose anion exchange chromatography column has a more excellent soothing effect.

[0036] Preferably, the elution method of the anion exchange chromatography column is to elute with a 0.1 - 0.3 mol / L (such as 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, etc.) NaCl solution, and collect the eluate of 5 - 10 BV (such as 5, 6, 7, 8, 9 BV, etc.).

[0037] Preferably, the dextran gel column is selected from a Sephadex-50 dextran gel column.

[0038] Preferably, the elution method of the dextran gel column is to elute with a 0.05 - 0.15 mol / L (such as 0.08 mol / L, 0.1 mol / L, 0.12 mol / L, etc.) NaCl solution, and collect the eluate of 5 - 10 BV (such as 5, 6, 7, 8, 9 BV, etc.).

[0039] All the point values listed after the numerical ranges involved in the present invention are examples, and other specific point values not listed within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0040] In a second aspect, the present invention provides a product prepared by the method for preparing the matsutake polysaccharide having a soothing effect according to the first aspect.

[0041] In a third aspect, the present invention provides the application of the product according to the second aspect in the preparation of a product having a soothing effect;

[0042] Preferably, the product includes cosmetics.

[0043] In a fourth aspect, the present invention provides the application of the prepared product according to the second aspect in the preparation of an inflammatory factor inhibitor.

[0044] According to the research results of the present invention, the extraction product involved in the present invention can inhibit the expression of inflammatory factors at the in vitro level, that is, the prepared product involved in the present invention can also be made into a simple test preparation. The above-mentioned inflammatory factor inhibitor is not used for skin soothing, but is used alone for the inhibition of inflammatory factors.

[0045] In a fifth aspect, the present invention provides the application of the prepared product according to the second aspect in the preparation of a neutrophil aggregation inhibitor in zebrafish embryos.

[0046] According to the research results of the present invention, the extraction product involved in the present invention can inhibit the aggregation of neutrophils in zebrafish embryos, that is, the extraction product involved in the present invention can also be made into a simple test preparation. The inhibitor mentioned above is not used for skin soothing and repair, but is used alone for the study of the behavior of neutrophils in zebrafish embryos.

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

[0048] The preparation method involved in the present invention creatively adopts the co-extraction method of alcohol solvent and complex enzyme for the dried powder of Tricholoma matsutake, and ultrasonic-assisted extraction is added, which can extract the target product more efficiently, and make the performance of the target product stable, with excellent soothing effect; further, the supernatant of the extraction is fermented by microorganisms, which can decompose part of the macromolecular polysaccharides into medium-molecular polysaccharides and small-molecular polysaccharides, and the specific polysaccharide microstructure makes the product have a more excellent soothing effect; further, the water extraction treatment, alcohol precipitation treatment and protein removal treatment carried out in sequence remove non-target components, which is beneficial to enrich polysaccharide components; anion exchange chromatography column elution and Sephadex column elution are carried out to further remove non-target impurities, so that the target product is further enriched, and finally the product has a high concentration of polysaccharide components and excellent anti-inflammatory and soothing effects at the same time. Detailed implementation mode

[0049] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solutions of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0050] The sources of some reagents or raw materials involved in the following examples or comparative examples are as follows:

[0051] Preparation of YPD fermentation medium: Weigh 18 parts of peptone, 15 parts of yeast extract, 20 parts of anhydrous glucose, 2.0 parts of magnesium sulfate, 3.0 parts of potassium dihydrogen phosphate, and 0.03 parts of vitamin B1, add 1000 mL of distilled water, stir evenly, adjust to pH 6.0, autoclave at 120 °C for 20 min, and after cooling to room temperature, filter impurities with a 0.22 μm filter membrane to obtain.

[0052] The yeast is Pichia pastoris (CICC 1688), and the Trichoderma is Trichoderma koningii LZ51.

[0053] Methods for obtaining yeast cell suspension and Trichoderma cell suspension: Inoculate yeast or Trichoderma into YPD medium, place it on a shaking incubator at a temperature of 30 °C and a rotation speed of 250 rpm, and shake and culture for 24 h to obtain.

[0054] The Sevag reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1.

[0055] Example 1

[0056] This example provides a method for preparing matsutake polysaccharide, which is as follows:

[0057] (1) Take the matsutake fruit bodies, wash them clean, slice and dry them, then crush them and pass through a 60-mesh sieve;

[0058] (2) Mix the dried matsutake powder with distilled water and a complex enzyme, and perform ultrasonic-assisted extraction at 350 W for 30 min at 30 °C. The material-liquid ratio of the dried matsutake powder to distilled water is 1 g:70 mL. The complex enzyme is composed of cellulase and pectinase with a mass ratio of 3:1. The total addition amount of the complex enzyme in the extraction system is 0.4 mg / mL. The enzyme activity of cellulase is 30000 U / g, and the enzyme activity of pectinase is 20000 U / g, to obtain the matsutake extraction product;

[0059] (3) Centrifuge the extraction product to obtain the supernatant. After concentrating the supernatant to 1 / 5 of the original volume, mix it with a YPD fermentation medium 250 times its volume and simultaneously inoculate 4% yeast cell suspension (OD600 = 0.20) and 1% Trichoderma cell suspension (OD600 = 0.20) (the viable cell number ratio is 4:1), and perform fermentation culture at 35 °C for 18 h to obtain the matsutake fermentation filtrate;

[0060] (4) Mix the matsutake fermentation filtrate with 2 volumes of water, perform water extraction treatment in a water bath at 35 °C for 20 min, then centrifuge at 10 °C and 7000 rpm for 15 min, take the supernatant, and filter it through a 0.22-μm filter membrane to obtain the water extraction filtrate;

[0061] (5) Add 95% ethanol to the water extraction filtrate to make the final concentration 90%, perform alcohol precipitation treatment, centrifuge, and take the precipitate to obtain the crude polysaccharide;

[0062] (6) Redissolve the crude polysaccharide in water, then mix it with 1 / 2 volume of Sevag reagent, shake vigorously for 30 min, let it stand for 2 h, then discard the bottom white protein emulsion, and repeat the operation 10 times until there is no denatured protein at the two-phase interface to obtain the protein-depleted product;

[0063] (7) Concentrate the protein-depleted product and load it onto a DEAE-52 cellulose anion exchange chromatography column, and elute it with 0.2 mol / L NaCl solution, and collect 8 BV of eluate;

[0064] (8) Concentrate the eluate and load it onto a Sephadex-50 dextran gel column (1.6 cm * 80 cm), and elute it with 0.1 mol / L NaCl solution, and collect 8 BV of eluate;

[0065] (9) Concentrate and dry the eluate to obtain the product.

[0066] Example 2

[0067] This example provides a method for preparing matsutake polysaccharide, which is as follows:

[0068] (1) Take the fruiting body of matsutake, wash it clean, slice and air-dry, then pulverize it and pass through a 60-mesh sieve;

[0069] (2) Mix the dried matsutake powder with 55% distilled water and a complex enzyme, and perform ultrasonic-assisted extraction at 40 °C for 15 min with 450 W. The material-liquid ratio of the dried matsutake powder to distilled water is 1 g:80 mL. The complex enzyme is composed of cellulase and pectinase with a mass ratio of 2:1. The total addition amount of the complex enzyme in the extraction system is 0.5 mg / mL. The enzyme activity of cellulase is 20000 U / g, and the enzyme activity of pectinase is 10000 U / g to obtain the matsutake extraction product;

[0070] (3) Centrifuge the extraction product to obtain the supernatant. Concentrate the supernatant to 1 / 4 of the original volume, then mix it with a YPD fermentation medium 300 times its volume and simultaneously inoculate 3% yeast culture solution (OD600 = 0.20) and 1% Trichoderma culture solution (OD600 = 0.20) (the viable cell number ratio is 3:1), and perform fermentation culture at 32 °C for 20 h to obtain the matsutake fermentation filtrate;

[0071] (4) Mix the matsutake fermentation filtrate with 3 times its volume of water, perform water extraction treatment in a 40 °C water bath for 15 min, then centrifuge at 4 °C and 6000 rpm for 15 min, take the supernatant, and filter it through a 0.22-μm filter membrane to obtain the water extraction filtrate;

[0072] (5) Add 95% ethanol to the water extraction filtrate to make the final concentration 88%, perform alcohol precipitation treatment, centrifuge, and take the precipitate to obtain the crude polysaccharide;

[0073] (6) Redissolve the crude polysaccharide in water, then mix it with 1 / 2 volume of Sevag reagent, shake it vigorously for 40 min, let it stand for 1 h, then discard the bottom white protein emulsion, and repeat the operation 8 times until there is no denatured protein at the two-phase interface to obtain the protein-depleted product;

[0074] (7) Concentrate the protein-depleted product and load it onto a DEAE-52 cellulose anion exchange chromatography column, elute it with 0.2 mol / L NaCl solution, and collect 7 BV of the eluate;

[0075] (8) After concentrating the eluate, load it onto a Sephadex-50 dextran gel column (1.6 cm * 80 cm), elute with 0.1 mol / L NaCl solution, and collect 6 BV of eluate;

[0076] (9) Concentrate and dry the eluate to obtain the product.

[0077] Example 3

[0078] This example provides a method for preparing matsutake polysaccharide, which is as follows:

[0079] (1) Take matsutake fruit bodies, wash them clean, slice and air-dry, then crush and pass through a 60-mesh sieve;

[0080] (2) Mix the dried matsutake powder with 75% distilled water and a complex enzyme, perform ultrasonic-assisted extraction at 300 W for 30 min at 30 °C. The material-liquid ratio of the dried matsutake powder to distilled water is 1 g:60 mL. The complex enzyme is composed of cellulase and pectinase with a mass ratio of 4:1. The total addition amount of the complex enzyme in the extraction system is 0.3 mg / mL. The enzyme activity of cellulase is 40000 U / g, and the enzyme activity of pectinase is 20000 U / g to obtain the matsutake extraction product;

[0081] (3) Centrifuge the extraction product to obtain the supernatant. After concentrating the supernatant to 1 / 3 of the original volume, mix it with 200 times the volume of YPD fermentation medium and simultaneously inoculate 5% yeast cell suspension (OD600 = 0.20) and 1% Trichoderma cell suspension (OD600 = 0.20) (the viable cell number ratio is 5:1), and perform fermentation culture at 35 °C for 24 h to obtain the matsutake fermentation filtrate;

[0082] (4) Mix the matsutake fermentation filtrate with 3 times the volume of water, perform water extraction treatment in a 30 °C water bath for 35 min, then centrifuge at 4 °C and 6000 rpm for 15 min, take the supernatant, and filter it through a 0.22-μm filter membrane to obtain the water extraction filtrate;

[0083] (5) Add 95% ethanol to the water extraction filtrate to make the final concentration 88%, perform alcohol precipitation treatment, centrifuge, and take the precipitate to obtain the crude polysaccharide;

[0084] (6) Re-dissolve the crude polysaccharide in water, then mix the water extraction filtrate with 1 / 2 volume of Sevag reagent, shake vigorously for 40 min, let it stand for 1 h, and discard the bottom white protein emulsion. Repeat the operation 8 times until there is no denatured protein at the two-phase interface to obtain the protein-depleted product;

[0085] (7) After concentrating the protein-depleted product, load it onto a DEAE-52 cellulose anion exchange chromatography column, elute with 0.2 mol / L NaCl solution, and collect 10 BV of eluate;

[0086] (8) The concentrated eluate was loaded onto a Sephadex-50 dextran gel column (1.6 cm * 80 cm), eluted with 0.1 mol / L NaCl solution, and 8 BV of eluate was collected;

[0087] (9) The eluate was concentrated and dried to obtain the product.

[0088] Example 4

[0089] This example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (3), only yeast cell suspension was inoculated: the supernatant was obtained by centrifuging the extraction product, and after concentrating the supernatant to 1 / 5 of the original volume, it was mixed with YPD fermentation medium at 250 times the volume and inoculated with 5% yeast cell suspension (OD600 = 0.20), and fermented at 35 °C for 18 h to obtain matsutake fermentation filtrate. Other steps remained unchanged.

[0090] Example 5

[0091] This example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (3), only Trichoderma cell suspension was inoculated: the supernatant was obtained by centrifuging the extraction product, and after concentrating the supernatant to 1 / 5 of the original volume, it was mixed with YPD fermentation medium at 250 times the volume and inoculated with 5% Trichoderma cell suspension (OD600 = 0.20), and fermented at 35 °C for 18 h to obtain matsutake fermentation filtrate. Other steps remained unchanged.

[0092] Example 6

[0093] This example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (7), the DEAE-52 cellulose anion exchange chromatography column was replaced with a Q-Sepharose Fast Flow anion exchange chromatography column, and other conditions remained unchanged. Other steps remained unchanged.

[0094] Comparative Example 1

[0095] This comparative example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (2), no complex enzyme was added: the dried matsutake powder was mixed with distilled water, and ultrasonic-assisted extraction was carried out at 350 W for 30 min at 30 °C, where the material-liquid ratio of the dried matsutake powder to the ethanol aqueous solution was 1 g:70 mL to obtain the matsutake extraction product. Other steps remained unchanged.

[0096] Comparative Example 2

[0097] This comparative example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (2), an equal amount of a single enzyme, cellulase, is added: The dried matsutake powder is mixed with distilled water and cellulase, and ultrasonic-assisted extraction is carried out at 350 W for 30 min at 30°C. The solid-liquid ratio of the dried matsutake powder to the ethanol aqueous solution is 1 g:70 mL. The total addition amount of cellulase in the extraction system is 0.4 mg / mL, and the enzyme activity of cellulase is 30000 U / g, obtaining the matsutake extraction product. Other steps remain unchanged.

[0098] Comparative Example 3

[0099] This comparative example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (2), an equal amount of a single enzyme, pectinase, is added: The dried matsutake powder is mixed with distilled water and pectinase, and ultrasonic-assisted extraction is carried out at 350 W for 30 min at 30°C. The solid-liquid ratio of the dried matsutake powder to the ethanol aqueous solution is 1 g:70 mL. The total addition amount of pectinase in the extraction system is 0.4 mg / mL, and the enzyme activity of pectinase is 20000 U / g, obtaining the matsutake extraction product. Other steps remain unchanged.

[0100] Comparative Example 4

[0101] This comparative example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that in step (2), ultrasonic assistance is not carried out: The dried matsutake powder is mixed with distilled water and complex enzyme, and water bath extraction is carried out at 30°C for 30 min. The solid-liquid ratio of the dried matsutake powder to the ethanol aqueous solution is 1 g:70 mL. The complex enzyme is composed of cellulase and pectinase with a mass ratio of 3:1. The total addition amount of the complex enzyme in the extraction system is 0.4 mg / mL, the enzyme activity of cellulase is 30000 U / g, and the enzyme activity of pectinase is 20000 U / g, obtaining the matsutake extraction product. Other steps remain unchanged.

[0102] Comparative Example 5

[0103] This comparative example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that the fermentation treatment in step (3) is not carried out, that is, the matsutake extraction product obtained in step (2) is directly subjected to the water extraction treatment in step (4). Other steps remain unchanged.

[0104] Comparative Example 6

[0105] This comparative example provides a method for preparing matsutake polysaccharide. The difference in operation from Example 1 is only that the column chromatography treatment in step (7) is not carried out, and the product in step (6) is directly concentrated and then subjected to step (8). Other steps remain unchanged.

[0106] Comparative Example 7

[0107] This comparative example provides a method for preparing matsutake polysaccharide. The only difference in operation from Example 1 is that the column chromatography treatment in step (8) is not carried out, and the product of step (6) is directly dried after concentration. Other steps remain unchanged.

[0108] Test Example 1

[0109] The total polysaccharide content of the prepared products obtained in Examples 1-6 and Comparative Examples 1-7 was determined. The steps of the total polysaccharide content detection method (sulfuric acid-phenol method) are as follows:

[0110] (1) Preparation of standard solution: Weigh accurately 100 mg of reference substance glucose, place it in a 100 mL volumetric flask, dissolve it with purified water to the scale, accurately pipette 10 mL and place it in another 100 mL volumetric flask, and make up the volume with pure water to prepare a standard glucose stock solution with a concentration of 0.1 mg / mL, and dilute it to gradient concentrations.

[0111] Preparation of sample solution: Weigh accurately 100 mg of the prepared product of each group in a 100 mL volumetric flask, dissolve it completely with purified water to the scale, accurately pipette 10 mL and place it in another 100 mL volumetric flask, and make up the volume with pure water to prepare a sample solution with a concentration of 0.1 mg / ml.

[0112] Preparation of 5% phenol solution: Weigh accurately 5.0 g of phenol, dissolve it with an appropriate amount of purified water, quantitatively transfer it to a 100 mL brown volumetric flask, slowly add water to the scale, stir well, and prepare a 5% aqueous phenol solution. Store it in the dark.

[0113] (2) Reaction of standard solution and sample solution:

[0114] Sequentially pipette 1 mL of the prepared series of standard solutions into test tubes, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution for reaction, shake gently, use ddH 2 O as the blank control, measure its absorbance at 490 nm with a microplate reader, and plot a standard curve with the concentration as the abscissa and the absorbance as the ordinate. Pipette 1 mL of the prepared sample solutions of each group into test tubes, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution for reaction, shake gently, use ddH 2 O as the blank control, measure its absorbance at 490 nm with a microplate reader, substitute the absorbance of the sample solution into the standard curve for calculation. The results are shown in Table 1.

[0115] Table 1

[0116]

[0117] As can be seen from the data results in Table 1: Compared with the comparative examples, the preparation method involved in the present invention uses a composite enzyme to extract the dried matsutake powder, adds ultrasonic-assisted extraction, and subsequent microbial fermentation and purification treatments, which can efficiently extract the target product and make the prepared matsutake polysaccharide product have a high content of polysaccharide components.

[0118] Test Example 2

[0119] The inhibitory effects of the preparation products obtained in Examples 1-6 and Comparative Examples 1-7 on inflammatory factors were evaluated:

[0120] In this experiment, the inhibitory effects of the samples on TNF-α and IL-6 were determined through an LPS-induced inflammatory model of mouse macrophages (RAW264.7).

[0121] (1) Determination of the maximum safe concentration:

[0122] (1.1) The MTT method was used to determine the effect of the sample solution diluted from the product of Example 1 at different concentrations on the cell viability of mouse macrophages (RAW264.7). According to the test results, when the concentration of the sample solution ≤ 1.25%, the cell viability was greater than 90%. Therefore, 1.00% was selected as the safe concentration for subsequent experiments.

[0123] (1.2) RAW264.7 cells were inoculated. When the cell density ≥ 70%, the culture medium was discarded, 5 mL of PBS was added to wash the cells, and then 4 mL of DMEM basal medium was added again. The cells were pipetted and resuspended, transferred to a 2 mL centrifuge tube, centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded; 1 mL of complete medium was added again, pipetted and mixed evenly, diluted by an appropriate multiple and counted. According to the counting results, the cell density was adjusted to 8×10 4 cell / mL, and the cells were inoculated into a 96-well plate at a volume of 200 μL per well and returned to the incubator for incubation (37 °C, 5% CO 2 )

[0124] (1.3) There were a total of 16 groups, namely a blank control group (without LPS stimulation and without sample intervention, 180 μL of basal medium was given), a negative control group (stimulated with 1 μg / mL of LPS but without sample intervention), a positive control group (stimulated with 1 μg / mL of LPS and simultaneously given 180 μL of 100 μg / mL of dexamethasone intervention), experimental groups 1-13 (stimulated with 1 μg / mL of LPS and simultaneously given 180 μL of 1% sample solutions of Examples 1-6 and Comparative Examples 1-7). In addition, 3 cell-free wells were set as zero adjustment groups, and the plates were arranged with 4 replicates in each group.

[0125] (1.4)Intervention method: First, add the basal medium or sample solution to each group, and then place it back in the incubator for incubation (37 °C, 5% CO 2 ); After 1 h, take out the 96-well plate. Add 20 μL of basal medium to each well of the zero-adjustment well and the blank control group, and add 20 μL of LPS (1 μg / mL) to each well of the negative control group, positive control group, and experimental groups 1-13. Then place it back in the incubator for continued incubation.

[0126] (1.5)After 24 h, take out the 96-well plate, collect the cell supernatants corresponding to each sample group into centrifuge tubes, centrifuge at 1000 rpm for 10 min, collect the supernatants and place them in 1.5 mL centrifuge tubes, and use an ELISA kit to measure the concentrations of TNF-α and IL-6 in the cell supernatants. The results are shown in Table 2.

[0127] Table 2

[0128]

[0129] It can be seen from the data results in Table 2 that compared with the comparative example, the preparation method involved in the present invention uses a complex enzyme to extract the dried matsutake powder, adds ultrasonic-assisted extraction, and subsequent microbial fermentation and purification treatment, which can efficiently extract the target product, making the prepared matsutake polysaccharide product have a high inhibitory effect on inflammatory factors. Moreover, the selection of the fermenting microorganism and the selection of the purification chromatography column also affect this inhibitory activity. Although the polysaccharide concentrations of the products in Examples 4 and 5 are close to that of the product in Example 1, their inhibitory effects on inflammatory factors are far less than that of the product in Example 1, indicating that the selection of the fermenting microorganism affects the polysaccharide structure composition method, and thus affects the inhibitory effect on inflammatory factors.

[0130] Test Example 3

[0131] Inhibitory test of neutrophil aggregation in zebrafish embryos:

[0132] Select healthy zebrafish embryos at 3 days post-fertilization, and divide them into a model control group, a positive control group, and a test substance group. Among them, randomly select 24 fish embryos in the model control group into a 3 cm culture dish, and add mL of fish embryo culture solution containing 0.16 mg / L of copper sulfate pentahydrate; randomly select 24 fish embryos in the positive control group into a 3 cm culture dish, and add mL of fish embryo culture solution containing 0.1 mg / L of copper sulfate pentahydrate and 0.0036 mg / L of indomethacin; randomly select 24 fish embryos in the test substance group into a 3 cm culture dish, and add 5 mL of fish embryo culture solution containing 0.16 mg / L of copper sulfate pentahydrate and 5 mg / mL of each test substance (the extraction products prepared in Examples 1-6 and Comparative Examples 1-7).

[0133] Each group was placed in an incubator at 28 °C for 45 min. Then, the fish embryos in each group were fixed in paraformaldehyde for 1 h, and the fish embryos were treated with PBST three times, 5 min each time. Then, they were treated with 50% ethanol for 3 min. After staining the fish embryos with Sudan black staining solution for 1 h, they were washed with 70% ethanol four times, 5 min each time. Then, they were treated with PBST twice, 5 min each time. The fish embryos were treated with bleaching solution for 10 min, then treated with 70% ethanol solution for 5 min, PBST for 1 min, cleared with clearing solution 1 for 15 min, cleared with clearing solution 2 for 10 min, and then treated with PBST for 3 min.

[0134] Place the fish embryos on their sides and then observe the tails of the fish embryos under a stereomicroscope. Count the number of neutrophils in the three-quarter tail region starting from the anus of each fish embryo, and calculate the neutrophil aggregation inhibition rate. Inhibition rate (%) = [(M - S) / M] × 100%, where S represents the average number of neutrophils in the fish embryos of the test substance treatment group, in units of grains per fish (grains / fish); M represents the average number of neutrophils in the fish embryos of the model control group, in units of grains per fish (grains / fish); the average values of each test group are calculated and shown in Table 3.

[0135] Table 3

[0136]

[0137] From the data results in Table 3, it can be seen that compared with the comparative example, the preparation method involved in the present invention uses a composite enzyme to extract the dried matsutake powder, adds ultrasonic-assisted extraction, and subsequent microbial fermentation and purification treatment, which can efficiently extract the target product, making the prepared matsutake polysaccharide product have a high inhibitory effect on neutrophil aggregation in zebrafish embryos, that is, a soothing effect. Moreover, the selection of fermentation microorganisms and the selection of purification chromatography columns also affect this inhibitory activity. Although the polysaccharide concentrations of the products in Examples 4 and 5 are close to that of the product in Example 1, their inhibitory effects on inflammatory factors are far less than that of the product in Example 1, indicating that the selection of fermentation microorganisms affects the structural composition mode of polysaccharides, thereby affecting the inhibitory effect on inflammatory factors.

[0138] The applicant declares that the present invention uses the above embodiments to illustrate the technical solution of the present invention, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and public scope of the present invention.

[0139] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0140] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for preparing Tricholoma matsutake polysaccharide with soothing effect, characterized in that: The preparation method comprises: (1) Mixing Tricholoma matsutake dried powder with distilled water and a composite enzyme, and performing ultrasonic-assisted extraction to obtain a Tricholoma matsutake extraction product; the composite enzyme is composed of cellulase and pectinase; the total addition amount of the composite enzyme in the extraction system is 0.3-0.6 mg / mL, the mass ratio of the cellulase to the pectinase is (1-5):1; the enzymatic activity of the cellulase is 20000-40000 U / g, and the enzymatic activity of the pectinase is 10000-20000 U / g; (2) The extracted product is centrifuged to obtain a supernatant, the supernatant is concentrated and then mixed with a fermentation medium and inoculated with a bacterial solution for fermentation to obtain a matsutake fermentation filtrate; the bacterial solution is a combination of a yeast bacterial solution and a Trichoderma bacterial solution; the ratio of the number of live bacteria of the yeast to that of the Trichoderma is (3-10):1; the yeast is Pichia pastoris CICC 1688, and the Trichoderma is Trichoderma koningii LZ51; (3) mixing the matsutake fermentation filtrate with water, performing water extraction, centrifuging to obtain the supernatant, filtering, and obtaining a water extraction filtrate; (4) subjecting the water extract filtrate to alcohol precipitation treatment, and centrifuging to obtain the precipitate to obtain crude polysaccharide; (5) re-dissolving the crude polysaccharide in water, and then performing a deproteinization treatment to obtain a deproteinized product; (6) The deproteinized product is sequentially eluted by anion exchange chromatography column and dextran gel column, and the eluate is collected and concentrated to obtain the Tricholoma matsutake polysaccharide.

2. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The solid-liquid ratio of the dry matsutake powder to distilled water is 1g:(50-80)mL.

3. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The power of the ultrasound is 250-450 W.

4. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The extraction temperature is 25-45°C and the extraction time is 30-60 min.

5. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The supernatant in step (2) is concentrated to 1 / 5-1 / 2 of the original volume of the supernatant.

6. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The volume ratio of the fermentation medium to the concentrated supernatant is (200-300):

1.

7. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The initial inoculation concentrations of the yeast solution and the Trichoderma solution are independently OD600=2-6, and the inoculation amounts are independently 1-5%.

8. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The fermentation culture temperature is 25-35°C and the fermentation time is 12-24 hours.

9. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: In step (3), the volume ratio of the matsutake fermentation filtrate to water is 1:(1-3).

10. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The water extraction treatment is carried out at a temperature of 25-40°C and for a time of 10-40 min.

11. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The centrifugation in step (3) is carried out at 4-10°C and 6000-8000 rpm for 15-20 min.

12. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The alcohol precipitation treatment uses anhydrous ethanol or ethanol aqueous solution to make its final concentration in the system 85-95%.

13. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The deproteinization treatment uses Sevag reagent, and the volume ratio of Sevag reagent to the water filtrate is 1:(1-5); the treatment method is to first oscillate for 20-40 minutes, then stand for 1-3 hours, discard the bottom white protein, and repeat the above operation until there is no protein at the interface between the two phases.

14. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The anion exchange chromatography column is selected from a DEAE-52 cellulose anion exchange chromatography column.

15. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The anion exchange chromatography column is eluted with a 0.1-0.3 mol / L NaCl solution, and 5-10 BV of the eluate is collected.

16. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The dextran gel column is selected from Sephadex-50 dextran gel column.

17. The method for preparing Tricholoma matsutake polysaccharide with soothing effect according to claim 1, characterized in that: The dextran gel column is eluted with a 0.05-0.15 mol / L NaCl solution, and 5-10 BV of the eluate is collected.

18. A product obtained by the method for preparing Tricholoma matsutake polysaccharide with soothing effect according to any one of claims 1 to 17.

19. Use of the product of claim 18 in the preparation of a product with soothing effect.

20. The use according to claim 19, characterized in that The products include cosmetics.

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