Preparation method of bolete extract as well as product and application of bolete extract

Abstract

The invention relates to a preparation method of a bolete extract as well as a product and application thereof, the preparation method comprises the following steps: drying bolete after steam explosion to obtain a dried bolete product; mixing the dried bolete product with water, and performing reflux extraction to obtain an extracting solution; concentrating the extracting solution to obtain a concentrated solution, mixing the concentrated solution with a flocculating agent, flocculating, filtering, and drying to obtain a finished product. According to the invention, an extraction method combining steam explosion and heating reflux is adopted, so that the extraction efficiency and biological activity of active components in the extract are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of substance extraction, and relates to a preparation method of boletus extract, a product thereof and application. BACKGROUND

[0002] Yunnan is one of the richest regions of global biodiversity, and its diverse species and complex habitat conditions make it famous for its reputation as the "wild mushroom kingdom". Here is not only the natural distribution center of wild boletus in China, but also the area with the richest yield, accounting for more than 90% of the total yield in China. Wild edible boletus in Yunnan has become a hot spot for nutrition and health research due to its outstanding antioxidant, antitumor, antifatigue and immunomodulatory functions. However, the existing extraction technology generally has problems such as low extraction efficiency and limited application range, which seriously restricts the development and application of high value-added products of boletus. It is worth noting that although boletus has been studied in depth in terms of antioxidant and other aspects, the study of its anti-glycation activity is still blank, which limits the application range of boletus.

[0003] Under oxidative stress conditions, the glucose autoxidation reaction initiates the AGEs generation pathway, and the high activity carbonyl compounds generated can be effectively blocked by antioxidant components such as polyphenols through the DPPH free radical scavenging mechanism. The formed AGEs can activate the NF-κB signal transduction pathway, induce the release of pro-inflammatory factors such as NO and IL-6, and then up-regulate the expression of tyrosinase gene to promote melanin biosynthesis. Based on this mechanism, targeted inhibition of AGEs activity can achieve direct anti-glycation by blocking the formation of glycation end products, and can also produce whitening effect by interfering with the melanin synthesis pathway. This multi-target synergistic mode provides an important theoretical basis and application prospect for the development of functional ingredients with anti-glycation, whitening and other multiple effects.

[0004] CN108653342B discloses an ultrasonic extraction technology, which uses ethanol to extract boletus fruiting bodies, and the obtained product has significant microbial antibacterial activity. However, the ultrasonic extraction method may damage the structure of polysaccharides, thereby affecting their biological activity. CN102000120B discloses a method of using three groups of organic solvents with different polarities, namely non-polar petroleum ether, medium-polar ethyl acetate and polar ethanol, to extract boletus fruiting bodies step by step, and combined with chromatography technology to separate a single component with antioxidant activity. However, the extraction solvents used are all organic solvents, which affect the safety of the extract. In addition, the traditional complex enzymatic hydrolysis technology has problems such as high cost, low extraction efficiency and insufficient application in practical application.

[0005] Therefore, there is an urgent need to provide an efficient and safe preparation method of boletus extract. SUMMARY

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing Boletus extract, as well as its products and applications.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a boletus extract, the method comprising:

[0009] (1) Boletus edulis is dried after being steam-exploded to obtain dried Boletus edulis product;

[0010] (2) Mix the dried Boletus edulis product with water and reflux to extract the extract;

[0011] (3) Concentrate the extract to obtain a concentrate, mix the concentrate with a flocculant to flocculate, filter, and dry to obtain the final product.

[0012] This invention employs a combined steam explosion and reflux extraction method, significantly improving the extraction efficiency and bioactivity of active ingredients in the extract. This provides a valuable technical solution for the high-value utilization of *Boletus edulis*. The study uses water as the extraction solvent and pre-treats the fruiting bodies of *Boletus edulis* with steam explosion. By disrupting the cell wall structure, this effectively alters the intermolecular forces. Combined with reflux extraction and flocculation technology, this significantly enhances the anti-glycation and whitening bioactivity of *Boletus edulis*, providing high-value-added raw materials for the functional food and cosmetic industries, meeting market demand, and promoting the development of related industries.

[0013] This process not only shortens extraction time and reduces energy costs, but also significantly enhances the extract's multiple effects such as anti-glycation, whitening, and anti-inflammation. Furthermore, it does not use any organic reagents, conforming to green and environmentally friendly principles, and provides reliable technical support for its application in health foods.

[0014] Preferably, the steam explosion is performed at a pressure of 0.4-0.8 MPa, a temperature of 140-180 ℃, and a time of 30-60 s.

[0015] The pressure can be selected from 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, etc., the temperature can be selected from 140℃, 150℃, 160℃, 170℃, 180℃, etc., and the time can be selected from 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0016] Preferably, the water content of the bolete in step (1) is 20-30 wt%, such as 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0017] The moisture content of porcini mushrooms is crucial to the effectiveness of steam explosion; both excessively high and low moisture content will result in poor performance.

[0018] Preferably, the moisture content of the dried Boletus edulis product does not exceed 8 wt%, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0019] Preferably, the ratio of the dried boletus product to water is 1 g:(10-20) mL, where the specific values ​​in (10-20) can be selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0020] Preferably, the reflux extraction time is 0.5-2 h, such as 0.5 h, 0.6 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc. Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0021] Preferably, the concentration process further includes adjusting the pH to 5-6, such as 5, 5.2, 5.4, 5.6, 5.8, 6, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] Preferably, the solid content of the concentrate is 8-12 wt%, such as 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, 10.5 wt%, 11 wt%, 11.5 wt%, 12 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0023] Preferably, the flocculant includes gelatin, chitosan, and agar.

[0024] Preferably, the flocculant comprises, by weight, 3-10 parts gelatin, 3-20 parts chitosan, and 5-20 parts agar.

[0025] The mass fractions of gelatin can be selected from 3, 4, 5, 6, 7, 8, 9, 10, etc.; the mass fractions of chitosan can be selected from 3, 5, 8, 10, 12, 15, 18, 20, etc.; the mass fractions of agar can be selected from 5, 8, 10, 12, 15, 18, 20, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0026] Preferably, the amount of flocculant added is 0.2-0.6% of the mass of the concentrate, such as 0.2%, 0.22%, 0.25%, 0.28%, 0.3%, 0.32%, 0.35%, 0.38%, 0.4%, 0.42%, 0.45%, 0.48%, 0.5%, 0.52%, 0.55%, 0.58%, 0.6%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0027] Preferably, the flocculation time is 0.5-2 h, such as 0.5 h, 0.6 h, 0.7 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0028] In a second aspect, the present invention provides a boletus extract prepared according to the method for preparing boletus extract described in the first aspect.

[0029] Thirdly, the present invention provides the use of the Boletus extract according to the second aspect in the preparation of products with anti-glycation, anti-oxidation, whitening or anti-inflammatory effects.

[0030] Preferably, the product includes food or cosmetics.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) High efficiency: The extraction method used in this invention can complete the cell wall breaking extraction in a short time, which improves the yield of active ingredients and production efficiency, saves energy consumption, and improves efficiency.

[0033] (2) Environmental friendliness: The solvent used in the extraction process is water, without the involvement of organic solvents, which improves the safety of the product and reduces environmental pollution.

[0034] (3) Enhanced bioactivity: It has anti-glycation, antioxidant, whitening or anti-inflammatory effects.

[0035] (4) Wide range of applications: This invention is not only applicable to food, functional food and health products, but can also be applied to other anti-glycation, anti-oxidation and whitening products.

[0036] (5) Simple operation: The preparation method of the present invention has no organic reagents, the process is simple, energy-saving and efficient, which is conducive to industrial production. Attached Figure Description

[0037] Figure 1 This is a graph showing the relative content of AGEs in a zebrafish anti-glycation model. ### This indicates that compared with the normal control group, p < 0.001; *** This indicates that compared with the model control group, p < 0.001. + This indicates that compared with Example 1, p < 0.05. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0039] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0040] Chitosan was purchased from Sinopharm Chemical Reagent Co., Ltd. and its product is called chitosan.

[0041] The gelatin was purchased from Henan Zhongchen Biotechnology Co., Ltd. and is classified as edible gelatin.

[0042] The agar was purchased from Henan Zhongchen Biotechnology Co., Ltd. and its product is called agar powder.

[0043] Preparation Example 1

[0044] This preparation example provides a flocculant comprising, by weight, 3 parts gelatin, 3 parts chitosan, and 10 parts agar.

[0045] The preparation method is as follows: the raw materials are physically mixed to obtain the product.

[0046] Preparation Example 2

[0047] This preparation example provides a flocculant comprising, by weight, 3 parts gelatin, 7 parts chitosan, and 6 parts agar.

[0048] The preparation method is the same as in Preparation Example 1.

[0049] Preparation Example 3

[0050] This preparation example provides a flocculant comprising, by weight, 6 parts gelatin, 3 parts chitosan, and 7 parts agar.

[0051] The preparation method is the same as in Preparation Example 1.

[0052] Preparation Example 4

[0053] This preparation example provides a flocculant that differs from Preparation Example 1 only in that it does not contain chitosan, and the reduced mass of chitosan is proportionally allocated to the mass of gelatin and agar.

[0054] The preparation method is the same as in Preparation Example 1.

[0055] Preparation Example 5

[0056] This preparation example provides a flocculant that differs from Preparation Example 1 only in that it does not contain agar, and its reduced mass is proportionally allocated to the mass of gelatin and chitosan.

[0057] The preparation method is the same as in Preparation Example 1.

[0058] Preparation Example 6

[0059] This preparation example provides a flocculant that differs from Preparation Example 1 only in that it does not contain gelatin, and its reduced mass is proportionally allocated to the mass of agar and chitosan.

[0060] The preparation method is the same as in Preparation Example 1.

[0061] Example 1

[0062] This embodiment provides a method for preparing Boletus extract, the preparation method comprising:

[0063] (1) Cut the raw material of Boletus edulis into 2-5 cm particles, adjust the moisture content to 20 wt%, and carry out steam explosion treatment at 0.6 MPa and 165 ℃ for 40 s. The pressure release rate is <0.1s. The explosion is repeated once, and the product is dried to a moisture content of 4 wt% to obtain dried Boletus edulis product.

[0064] (2) The dried Boletus edulis product was pulverized, passed through a 40-mesh sieve, and mixed with water at a ratio of 1 g: 15 mL. The mixture was refluxed for 1 h, and the residue was separated by passing through a 600-mesh sieve. The pH was adjusted to 5.5, centrifuged, filtered, and concentrated under reduced pressure to a solid content of 10 wt% to obtain a concentrated solution.

[0065] (3) The flocculant prepared in Preparation Example 1 is mixed with the concentrate. The amount of flocculant added is 0.3% of the mass of the concentrate. After adsorption and flocculation for 1 h, the filtrate is sterilized and dried to obtain the final product.

[0066] Example 2

[0067] This embodiment provides a method for preparing Boletus extract, the preparation method comprising:

[0068] (1) Cut the raw material of Boletus edulis into 2-5 cm particles, adjust the moisture content to 30 wt%, and carry out steam explosion treatment at 0.4 MPa and 140 ℃ for 60 s, with a pressure release rate of <0.1s. Circulate the explosion once and dry to a moisture content of 2 wt% to obtain the dried product of Boletus edulis.

[0069] (2) The dried Boletus edulis product was pulverized, passed through a 40-mesh sieve, and mixed with water at a ratio of 1 g: 10 mL. The mixture was refluxed for 1.5 h, and the residue was separated by passing through a 600-mesh sieve. The pH was adjusted to 5.3, centrifuged, filtered, and concentrated under reduced pressure to a solid content of 8 wt% to obtain a concentrated solution.

[0070] (3) The flocculant prepared in Preparation Example 2 was mixed with the concentrate. The amount of flocculant added was 0.4% of the mass of the concentrate. After adsorption and flocculation for 1.5 h, the filtrate was sterilized and dried to obtain the final product.

[0071] Example 3

[0072] This embodiment provides a method for preparing Boletus extract, the preparation method comprising:

[0073] (1) Cut the raw material of Boletus edulis into 2-5 cm particles, adjust the moisture content to 25 wt%, and carry out steam explosion treatment at 0.8 MPa and 180 ℃ for 30 s, with a pressure release rate of <0.1s. Circulate the explosion once and dry to a moisture content of 8 wt% to obtain the dried product of Boletus edulis.

[0074] (2) The dried Boletus edulis product was pulverized, passed through a 40-mesh sieve, and mixed with water at a ratio of 1 g: 20 mL. The mixture was refluxed for 0.8 h, and the residue was separated by passing through a 600-mesh sieve. The pH was adjusted to 5.7, centrifuged, filtered, and concentrated under reduced pressure to a solid content of 12 wt% to obtain the concentrate.

[0075] (3) The flocculant prepared in Preparation Example 3 was mixed with the concentrate. The amount of flocculant added was 0.5% of the mass of the concentrate. After adsorption and flocculation for 0.8 h, the filtrate was sterilized and dried to obtain the final product.

[0076] Example 4

[0077] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (1) is "cutting Boletus raw material into 2-5 cm particles, adjusting the moisture content to 10 wt%, performing steam explosion treatment at 0.6 MPa and 165 ℃ for 40 s, with a pressure release rate of <0.1s, cyclic explosion once, and drying to a moisture content of 4 wt% to obtain dried Boletus product", while other operations remain unchanged.

[0078] Example 5

[0079] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (1) is "cutting Boletus raw material into 2-5 cm particles, adjusting the moisture content to 40 wt%, performing steam explosion treatment at 0.6 MPa and 165 ℃ for 40 s, with a pressure release rate of <0.1s, cyclic explosion once, and drying to a moisture content of 4 wt% to obtain dried Boletus product", while other operations remain unchanged.

[0080] Example 6

[0081] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (1) is "cutting Boletus raw material into 2-5 cm particles, adjusting the moisture content to 20 wt%, performing steam explosion treatment at 0.6 MPa and 165 ℃ for 20 s, with a pressure release rate of <0.1 s, cyclic explosion once, and drying to a moisture content of 4 wt% to obtain dried Boletus product", while other operations remain unchanged.

[0082] Example 7

[0083] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (1) is "cutting Boletus raw material into 2-5 cm particles, adjusting the moisture content to 20 wt%, performing steam explosion treatment at 0.6 MPa and 165 ℃ for 100 s, with a pressure release rate of <0.1 s, cyclic explosion once, and drying to a moisture content of 4 wt% to obtain dried Boletus product", with other operations remaining unchanged.

[0084] Example 8

[0085] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (1) is "cutting Boletus raw material into 2-5 cm particles, adjusting the moisture content to 20 wt%, performing steam explosion treatment at 0.2 MPa and 130 ℃ for 40 s, with a pressure release rate of <0.1 s, cyclic explosion once, and drying to a moisture content of 4 wt% to obtain dried Boletus product", while other operations remain unchanged.

[0086] Example 9

[0087] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (1) is "cutting Boletus raw material into 2-5 cm particles, adjusting the moisture content to 20 wt%, performing steam explosion treatment at 1.4 MPa and 210 ℃ for 40 s, with a pressure release rate of <0.1 s, cyclic explosion once, and drying to a moisture content of 4 wt% to obtain dried Boletus product", while other operations remain unchanged.

[0088] Example 10

[0089] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is "mixing the flocculant prepared in Preparation Example 4 with the concentrate, the amount of flocculant added is 0.3% of the mass of the concentrate, adsorbing and flocculating for 1 h, filtering and sterilizing the filtrate, drying, and obtaining the extract", while other operations remain unchanged.

[0090] Example 11

[0091] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is to "mix the flocculant prepared in Preparation Example 5 with the concentrate, the amount of flocculant added is 0.3% of the mass of the concentrate, adsorb and flocculate for 1 h, filter and sterilize the filtrate, and dry it to obtain the extract", while other operations remain unchanged.

[0092] Example 12

[0093] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is to "mix the flocculant prepared in Preparation Example 6 with the concentrate, the amount of flocculant added is 0.3% of the mass of the concentrate, adsorb and flocculate for 1 h, filter and sterilize the filtrate, dry it, and obtain the product", while other operations remain unchanged.

[0094] Example 13

[0095] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is to "mix the flocculant prepared in Example 1 with the concentrate, the amount of flocculant added is 0.3% of the mass of the concentrate, adsorb and flocculate for 15 min, filter and sterilize the filtrate, and dry it to obtain the extract", while other operations remain unchanged.

[0096] Example 14

[0097] This embodiment provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is to "mix the flocculant prepared in Example 1 with the concentrate, the amount of flocculant added is 0.3% of the mass of the concentrate, adsorb and flocculate for 5 h, filter and sterilize the filtrate, dry it, and obtain the product", while other operations remain unchanged.

[0098] Comparative Example 1

[0099] This comparative example provides a method for preparing a Boletus extract, which differs from Example 1 only in that step (1) is "cutting the Boletus raw material into 2-5 cm particles and drying them to a moisture content of 4 wt% to obtain the dried Boletus product", while other operations remain unchanged.

[0100] Comparative Example 2

[0101] This comparative example provides a method for preparing a Boletus extract, which differs from Example 1 only in that step (1) is "cutting the Boletus raw material into 2-5 cm particles, adjusting the moisture content to 20 wt%, subjecting it to high temperature and high pressure treatment at 0.2 MPa and 130 ℃ for 40 s, and drying it to a moisture content of 4 wt% to obtain the dried Boletus product", while other operations remain unchanged.

[0102] Comparative Example 3

[0103] This comparative example provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is "to flocculate the concentrate at 4 ℃ for 1 h, filter it, sterilize the filtrate, and dry it to obtain the extract", while other operations remain unchanged.

[0104] Comparative Example 4

[0105] This comparative example provides a method for preparing Boletus extract, which differs from Example 1 only in that step (3) is "filtering the concentrate, sterilizing the filtrate, drying it, and obtaining the extract", while other operations remain unchanged.

[0106] Comparative Example 5

[0107] This comparative example provides a method for preparing Boletus extract, which differs from Example 1 only in that step (2) is: “the dried Boletus product is pulverized, passed through a 40-mesh sieve, and mixed with water at a material-to-liquid ratio of 1 g:15 mL. Ultrasonic extraction is performed for 1 h at a temperature of 60 °C and a power of 640 W. The residue is separated by passing through a 600-mesh sieve, the pH is adjusted to 5.5, centrifuged, filtered, and concentrated under reduced pressure to a solid content of 10 wt% to obtain a concentrated solution.” Other operations remain unchanged.

[0108] Test Example 1

[0109] Anti-glycation test

[0110] Test method: Prepare a 1 mg / mL sample solution using phosphate buffer (50 mmol / L, pH 7.4). Add 1 mL of the sample, 0.8 mg / mL bovine serum albumin (BSA) solution, 200 mM glucose solution, and phosphate buffer (PBS) to separate test tubes and heat at 60 °C for 24 h. Use aminoguanidine hydrochloride (AG) as a positive control. Fluorescent AGEs were measured using a fluorescence microplate reader with an excitation wavelength of 370 nm and an emission wavelength of 420 nm. The content of fluorescent AGEs is expressed as fluorescence intensity (AU).

[0111] Table 1

[0112]

[0113] The inhibition rate of fluorescent AGEs formation by the sample is calculated as follows:

[0114]

[0115] Each group was measured an average of 3 times, and the average value was taken to calculate the AGEs formation inhibition rate. The results are shown in Table 2.

[0116] Table 2

[0117]

[0118] As shown in Table 2, the Boletus extract prepared in this application has a good inhibitory effect on AGEs and a significant anti-glycation effect. The selection of steam explosion parameters and flocculant has a significant impact on the anti-glycation effect of Boletus.

[0119] Test Example 2

[0120] Antioxidant effect test

[0121] Test method: DPPH was dissolved in anhydrous ethanol to a concentration of 0.1 mg / mL. Simultaneously, vitamin C (positive control) and a 0.5 mg / mL sample solution were prepared. 120 μL of the DPPH ethanol solution and 120 μL of the sample solution were mixed thoroughly. A blank control group was set up. The mixture was reacted at room temperature in the dark for 30 min, shaken well, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate of the sample was calculated using the following formula:

[0122]

[0123] Sample blank group: 150 μL DPPH solution + 150 μL sample solvent

[0124] Sample group: 150 μL sample solution + 150 μL DPPH alcohol solution

[0125] Control group: 150 μL sample solution + 150 μL anhydrous ethanol

[0126] Control group: 150 μL anhydrous ethanol + 150 μL sample solvent

[0127] The results of DPPH free radical scavenging are shown in Table 3.

[0128] Table 3

[0129]

[0130] As shown in Table 3, the Boletus extract prepared in this application has a good scavenging effect on DPPH free radicals and a significant antioxidant effect. The selection of steam explosion parameters and flocculant has a significant impact on the antioxidant effect of Boletus.

[0131] Test Example 3

[0132] Tyrosinase inhibition effect test

[0133] A 0.5 mg / mL L-tyrosine solution and a 100 μg / mL tyrosinase solution were prepared using phosphate buffer (pH 6.8) as the solvent. A 1 mg / mL arbutin (positive control) solution and the sample solution were prepared using water as the solvent. The tyrosine solution, tyrosinase solution, and sample solution were mixed thoroughly. A blank control group was set up. The mixture was incubated at 37 °C for 30 min, and the absorbance was measured at 475 nm. The inhibition rate of tyrosinase activity by the sample was calculated using the following formula:

[0134]

[0135] Sample group: 100 μL tyrosine solution + 50 μL sample solution + 50 μL tyrosinase solution

[0136] Sample blank group: 100 μL tyrosine solution + 50 μL sample solution + 50 μL buffer

[0137] Control group: 100 μL tyrosine solution + 50 μL tyrosinase + 50 μL buffer solution

[0138] Control group: 100 μL tyrosine solution + 100 μL buffer solution

[0139] Each group was measured an average of 3 times, and the average value was taken to calculate the tyrosinase inhibition rate. The results are shown in Table 4.

[0140] Table 4

[0141]

[0142] As shown in Table 4, the Boletus extract prepared in this application has a good inhibitory effect on tyrosinase, and the selection of steam explosion parameters and flocculant has a significant impact on the whitening effect of Boletus.

[0143] Test Example 4

[0144] α-glucosidase inhibition effect test

[0145] Using phosphate buffer (pH 6.8) as the solvent, prepare a 5 mmol / L PNPG solution, a 0.5 μg / mL α-glucosidase solution, and a 1 mol / L Na₂CO₃ solution. The initial concentrations are 5 μg / mL acarbose and 1 mg / mL sample solution. Mix 50 μL of α-glucosidase solution with 50 μL of sample solution thoroughly and incubate at 37 °C for 10 min. Then, add 50 μL of PNPG solution to the mixture to initiate the reaction. After mixing, incubate at 37 °C for 20 min. Finally, add 100 μL of sodium carbonate solution to the reaction solution to stop the reaction. Measure the absorbance at 405 nm. A blank control group is included.

[0146] Table 5

[0147]

[0148] Calculate the inhibition rate of the sample against α-glucosidase using the following formula:

[0149]

[0150] The results are shown in Table 6.

[0151] Table 6

[0152]

[0153] As shown in Table 6, the Boletus extract prepared in this application has a good inhibitory effect on α-glucosidase, and the selection of steam explosion parameters and flocculant has a significant impact on the hypoglycemic effect of Boletus.

[0154] Test Example 5

[0155] Anti-inflammatory effect test

[0156] RAW264.7 cells in logarithmic growth phase with good morphology were selected and prepared into 5×10⁶ cells / years using complete culture medium. 4 A cell suspension of 1000 μL / mL was seeded into each well of a 24-well plate, i.e., 5 × 10⁶ cells / mL. 4 Each sample was incubated at 37 °C in a 5% CO2 incubator for 24 h.

[0157] Drug administration and LPS: Blank control group, model control group, positive control group and sample group were set up.

[0158] Blank control group: Add complete culture medium to each well.

[0159] Positive control group: 5 μM dexamethasone acetate (DEX) diluted with complete culture medium was added to each well.

[0160] Negative control group: LPS with a final concentration of 1 μg / mL

[0161] Sample group: Add the test sample (concentration of 500 μg / mL) diluted with complete culture medium to each well.

[0162] Each group was set up with 3 replicates and incubated in a 37 ℃, 5% CO2 incubator for 2 h. Except for the blank control group, LPS was added to each well to a final concentration of 1 μg / mL and incubated again in the incubator for 24 h.

[0163] Inflammatory factor detection: The levels of NO and IL-6 in the collected cell supernatant were measured according to the instructions of the NO and ELISA detection kits. The experimental results were analyzed using data processing software, and the results are shown in Table 7.

[0164] Table 7

[0165]

[0166] Note: ### This indicates that compared with the blank control group, p < 0.001; *** This indicates a comparison with the model control group, p < 0.001.

[0167] As shown in Table 7, LPS stimulation significantly increased the relative expression levels of NO and IL-6 (p<0.001), indicating the successful construction of the inflammation model. Compared with the model control group, the test sample treatment significantly reduced the relative expression of NO and IL-6 (p<0.05), and the effects of Examples 1-3 were at a high level. These results indicate that Boletus extract has a strong anti-inflammatory effect.

[0168] Test Example 6

[0169] Zebrafish Anti-glycation Test

[0170] Four dpf wild-type AB strain zebrafish were randomly selected and placed in 24-well plates, with 10 zebrafish treated in each well. The embryo culture medium was removed from the 24-well plates without harming the juveniles. A normal control group, a model control group (3% glucose), and a positive control group were set up, and the experiment was repeated three times. Except for the normal control group, each experimental group was given 500 μL of the corresponding concentration of the test substance dilution (containing the modeling agent 3% glucose solution) to establish a zebrafish anti-glycation model. The concentration of the test substance was 500 μg / mL, and the concentration of the positive control (metformin) was 500 μM. The 24-well cell culture plates were wrapped with aluminum foil and incubated in a biochemical incubator at (28.5±1.0℃) in the dark for 24 h. After incubation, the 24-well plate was removed, shaken well, and 100 μL of liquid was pipetted from each well into an opaque 96-well plate. The AGEs content was determined by detecting the fluorescence intensity at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The anti-glycation efficacy of the samples was evaluated using statistical analysis of this indicator. Statistical results are expressed as mean ± SE. Results are as follows... Figure 1 As shown.

[0171] Exogenous glucose exposure significantly increased the content of fluorescent advanced glycation end products (AGEs) in zebrafish culture (p<0.001), confirming that a high-glucose environment promotes glycosylation in zebrafish, while natural plant extracts can reduce glycosylation levels and inhibit AGE formation in zebrafish. Figure 1 As shown, 3% glucose was used as the model group, resulting in the production of a large amount of AGEs. Compared with the model group, the AGEs content in Example 1 and Comparative Example 1 was significantly reduced (p<0.05), and Example 1 had even lower AGEs content, indicating better anti-glycation efficacy.

[0172] The applicant declares that this invention illustrates a method for preparing a Boletus extract, its product, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0173] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0174] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing a boletus extract, characterized by, The preparation method comprises: (1) drying the Boletus after steam explosion to obtain a dried Boletus product; (2) mixing the dried Boletus product with water, reflux extraction to obtain an extract; (3) concentrating the extract to obtain a concentrated liquid, mixing the concentrated liquid with a flocculating agent, filtering and drying to obtain the Boletus extract.

2. The method of preparing the boletus extract according to claim 1, characterized by, The pressure of the steam explosion is 0.4-0.8 MPa, the temperature is 140-180 ℃, and the time is 30-60 s.

3. The method of preparing the boletus extract according to claim 1, characterized by, The water content of the Boletus in step (1) is 20-30 wt%.

4. The method of preparing the boletus extract according to claim 1, characterized by, The time of the reflux extraction is 0.5-2 h.

5. The method of preparing the boletus extract according to claim 1, characterized by, The flocculating agent comprises gelatin, chitosan and agar.

6. The method of preparing Boletus extract according to claim 5, characterized by, The flocculating agent comprises 3-10 parts of gelatin, 3-20 parts of chitosan and 5-20 parts of agar by mass fraction.

7. The method of preparing the boletus extract according to claim 1, characterized by, The addition amount of the flocculating agent is 0.2-0.6% of the mass of the concentrated liquid.

8. The method of preparing the boletus extract according to claim 1, characterized by, The time of the flocculation is 0.5-2 h.

9. The Boletus extract prepared by the preparation method of the Boletus extract according to any one of claims 1-8.

10. The use of the Boletus extract according to claim 9 in the preparation of a product having the efficacy of antioxidation, whitening, anti-allergy, anti-sugar or anti-inflammation.

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