A method for preparing a black truffle extract and a composition containing the extract and applications thereof

Abstract

The present application relates to a kind of preparation methods of black truffle extract and the composition containing the extract and its application, the preparation method includes: after steam explosion, black truffle is dried, and the black truffle dry product is obtained;Black truffle dry product is mixed with water, ultrasonic extraction is filtered by ceramic membrane, the filtrate is concentrated, dried, and it is obtained immediately.The extraction method used in the present application can complete the process of breaking wall extraction in a shorter time, improve the active material yield and production efficiency.The solvent used in the extraction process is water, no organic solvent is involved, improve the safety of product, and reduce the pollution to environment, can be applied to anti-saccharification, anti-inflammatory, antioxidant product.

Description

Technical Field

[0001] This invention belongs to the field of material extraction technology, and relates to a method for preparing black truffle extract, a composition containing the extract, and its application. Background Technology

[0002] The global market for medicinal mushroom extracts is experiencing rapid growth, driven primarily by increased consumer health awareness and technological advancements. Yunnan, a core global mushroom-producing region, cultivates black truffles with significant advantages in polysaccharide and androstenol content. However, Yunnan black truffles have thick cell walls (microscopic analysis shows a thickness >2 μm), which, while forming a natural protective barrier, increases the difficulty of extracting active ingredients. Existing research indicates that compound enzymatic hydrolysis technology can specifically disrupt the cell walls of black truffles, improving extraction efficiency; however, this technology suffers from high cost and low efficiency, limiting its large-scale application. CN117598941A discloses a method for mixed fermentation of truffles using Saccharomyces cerevisiae and Lactobacillus mucosa. The fermentation process is gentle, and the resulting prebiotics can increase product activity, exhibiting good DPPH free radical scavenging ability and anti-glycation effects, contributing to improved skin radiance and elasticity. CN118415924A discloses a hydroxypropyl-β-cyclodextrin-assisted water extraction method that significantly increases the water solubility of fat-soluble components in black truffles through molecular adsorption and inclusion. Combined with pressure filtration and sterilization, a complete black truffle extract is obtained and can be applied in cosmetics to achieve skin conditioning effects. However, these methods still have certain limitations in terms of extraction efficiency, cost, and industrial application.

[0003] In the development of glycation-related chronic diseases, skin aging, and metabolic disorders, the abnormal formation of advanced glycation end products (AGEs) is considered a key driving factor. AGEs are stable end products formed by the complex Maillard reaction between reducing sugars and proteins, lipids, or nucleic acids under non-enzymatic conditions. Their formation is accompanied by the accumulation of reactive carbonyl compounds (such as methylglyoxal and acetylacetaldehyde), and is significantly accelerated under hyperglycemic and hyperlipidemic conditions. Once formed, AGEs can bind to their specific receptor RAGE, activating downstream signaling pathways such as NF-κB and MAPK, inducing the expression of inflammatory factors, and promoting the generation of reactive oxygen species (ROS), triggering oxidative stress. Oxidative stress, in turn, can accelerate glycation and exacerbate inflammation, forming a positive feedback loop of "glycation-oxidation-inflammation," further promoting tissue damage and functional decline. Therefore, intervention strategies surrounding AGEs have gradually formed a multi-pathway parallel prevention and control mechanism: on the one hand, antioxidant effects can be used to remove ROS generated during the formation of AGEs, thus slowing down the glycation reaction chain; on the other hand, the inflammatory response amplification effect after AGEs-RAGE binding can be reduced by inhibiting inflammatory pathways; in addition, by lowering blood sugar or inhibiting α-glucosidase activity, the supply of available glycosides can be reduced from the source, thereby slowing down the synthesis of AGEs; at the same time, various active ingredients in plant extracts can also chelate with metal ions and react with active carbonyl intermediates or directly inhibit the formation of AGEs, constituting a direct intervention on the formation of advanced glycation end products.

[0004] CN118844628A and CN118615222A both disclose anti-glycation-related compositions. Although they differ in their composition and application, their formulation designs share certain commonalities, characterized by diverse components and complex formulations, but with relatively simple mechanisms of action, primarily focusing on intervention through direct inhibition of AGEs formation pathways. Furthermore, there are few reports on the anti-glycation activity of black truffle extracts, which limits the application of Yunnan black truffles.

[0005] Based on the shortcomings of existing technologies, the extracts and compositions proposed in this patent achieve sugar suppression from the source, possessing stronger scientific rigor, systematicity, and product development feasibility, demonstrating significant innovative advantages. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing black truffle extract, a composition containing the extract, and its application.

[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 black truffle extract, the method comprising:

[0009] (1) Black truffles are dried by steam explosion to obtain dried black truffle products;

[0010] (2) Mix the dried black truffle product with water, extract by ultrasonication, filter through a ceramic membrane, concentrate and dry the filtrate to obtain the product.

[0011] This invention provides an innovative method for extracting black truffles, combining steam explosion, ultrasonic extraction, and membrane separation technologies. The aim is to improve extraction efficiency and product activity, reduce production costs, and realize the high-value-added industrial application of black truffle extracts. This method effectively disrupts the cell walls of black truffles in a short time through steam explosion technology, promoting the release of active ingredients and significantly improving extraction efficiency and the yield of active substances. Water is used as the solvent during the extraction process, avoiding the use of organic solvents, improving product safety, and reducing environmental pollution. Furthermore, the combination of ultrasonic extraction and membrane separation technologies further optimizes the extraction process and improves production efficiency. The black truffle extract obtained under these process conditions shows significantly enhanced efficacy in inhibiting AGEs formation, scavenging free radicals, chelating metal ions, and reducing inflammatory responses. It possesses multiple biological activities including anti-glycation, hypoglycemic, antioxidant, and anti-inflammatory properties, and can be widely used in anti-glycation, antioxidant, and anti-inflammatory products, broadening the application scope of black truffles and demonstrating a promising market prospect.

[0012] Preferably, the black truffle in step (1) has a water content of 15-25 wt%, a steam explosion pressure of 0.4-1.2 MPa, and a time of 30-60 s.

[0013] The moisture content can be selected from 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, etc.; the pressure can be selected from 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, 1 MPa, 1.1 MPa, 1.2 MPa, etc.; the time can be selected from 30 s, 32 s, 34 s, 36 s, 38 s, 40 s, 42 s, 44 s, 46 s, 48 ​​s, 50 s, 52 s, 54 s, 56 s, 58 s, 60 s, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0014] Preferably, the temperature of the steam explosion is 160-210 ℃, such as 160 ℃, 165 ℃, 170 ℃, 175 ℃, 180 ℃, 185 ℃, 190 ℃, 195 ℃, 200 ℃, 205 ℃, 210 ℃, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0015] Preferably, the moisture content of the dried black truffle product is 3-6 wt%, such as 3 wt%, 3.2 wt%, 3.5 wt%, 3.8 wt%, 4 wt%, 4.2 wt%, 4.5 wt%, 4.8 wt%, 5 wt%, 5.2 wt%, 5.5 wt%, 5.8 wt%, 6 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0016] Preferably, the ratio of the dried black truffle 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, and will not be elaborated here.

[0017] Preferably, the ultrasonic extraction temperature is 60-80 ℃, the time is 30-60 min, and the power is 200-400 W.

[0018] Temperatures can be selected from 60℃, 65℃, 70℃, 75℃, 80℃, etc.; time can be selected from 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc.; power can be selected from 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, 320 W, 340 W, 360 W, 380 W, 400 W, etc. Other specific values ​​within the above ranges can also be selected, which will not be elaborated here.

[0019] Preferably, the pore size of the ceramic membrane is 100-200 nm, such as 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0020] Preferably, the solid content of the concentrated solution obtained by concentration is 8-12%, such as 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, etc. Other specific 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 4-5, such as 4, 4.2, 4.5, 4.8, 5, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] In a second aspect, the present invention provides a black truffle extract prepared according to the method for preparing black truffle extract described in the first aspect.

[0023] Thirdly, the present invention provides a composition with anti-glycation effects, wherein the composition comprises, by weight, 5-20 parts of black truffle extract, 10-30 parts of raspberry extract and 20-50 parts of rose extract as described in the second aspect.

[0024] The composition prepared in this invention, through the selection of natural extracts with synergistic mechanisms and high activity, achieves multi-pathway intervention from the source, including inhibiting sugar production, scavenging free radicals, chelating metal ions, directly inhibiting AGEs formation, and reducing inflammatory responses. Furthermore, our composition features a concise composition but a closed-loop mechanism, clearly defined indicators, and strong dosage form adaptability. Therefore, this patent utilizes a plant-based active composition with a multi-target combined strategy, from anti-oxidation and anti-inflammation to lowering blood sugar and directly inhibiting glycation, to prevent AGEs formation and delay the related tissue damage caused by them. Moreover, black truffle extract, raspberry extract, and rose extract exhibit certain synergistic effects in the aforementioned efficacy, suggesting a potential synergistic effect.

[0025] The mass fractions of black truffle extract can be selected from 5, 8, 10, 12, 15, 18, 20, etc.; the mass fractions of raspberry extract can be selected from 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, etc.; and the mass fractions of rose extract can be selected from 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, etc. Other specific values ​​within the above ranges can also be selected, which will not be elaborated here.

[0026] Preferably, the raspberry extract is prepared by a method comprising the following steps:

[0027] (1) Raspberries are dried after being steam-exploded to obtain dried raspberry product;

[0028] (2) The dried raspberry product was mixed with water and extracted by microjet to obtain the first extract;

[0029] (3) The first extract was heated and refluxed to obtain the second extract;

[0030] (4) Filter the second extract, concentrate it, refrigerate it to flocculate, centrifuge it and dry it to obtain the final product.

[0031] Preferably, the water content of the raspberries in step (1) is 10-20 wt%, the steam explosion pressure is 0.2-0.6 MPa, and the time is 0.5-2 min.

[0032] The moisture content can be selected from 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, etc. The pressure can be selected from 0.2 MPa, 0.22 MPa, 0.25 MPa, 0.28 MPa, 0.3 MPa, 0.32 MPa, 0.35 MPa, 0.38 MPa, 0.4 MPa, 0.42 MPa, 0.45 MPa, 0.48 MPa, 0.5 MPa, 0.52 MPa, 0.55 MPa, 0.58 MPa, 0.6 MPa, etc. The time can be selected from 0.5 min, 0.8 min, 1 min, 1.2 min, 1.5 min, 1.8 min, 2 min, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0033] Preferably, the moisture content of the dried raspberry product is 4-6 wt%, such as 4 wt%, 4.2 wt%, 4.5 wt%, 4.8 wt%, 5 wt%, 5.2 wt%, 5.5 wt%, 5.8 wt%, 6 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0034] Preferably, the temperature of the steam explosion is 140-170 ℃, such as 140 ℃, 145 ℃, 150 ℃, 155 ℃, 160 ℃, 165 ℃, 170 ℃, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0035] Preferably, the ratio of dried raspberry product to water is 1 g:(10-15) mL, where the specific values ​​in (10-15) can be selected from 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 4, 14.5, 15, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0036] Preferably, the microjet extraction is performed at a temperature of 60-80 ℃, a pressure of 100-200 MPa, and 2-3 times.

[0037] Temperatures can be selected from 60℃, 62℃, 65℃, 68℃, 70℃, 72℃, 75℃, 78℃, 80℃, etc., and pressures can be selected from 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200 MPa, etc. Other specific values ​​within the above ranges can also be selected, which will not be elaborated here.

[0038] Preferably, the heating and reflux extraction time is 60-90 min, such as 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0039] Preferably, the solid content of the concentrate obtained by concentration is 10-15 wt%, such as 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0040] Preferably, the refrigerated flocculation temperature is 2-6 ℃ and the time is 8-12 h.

[0041] Temperatures can be selected from 2 ℃, 2.5 ℃, 3 ℃, 3.5 ℃, 4 ℃, 4.5 ℃, 5 ℃, 5.5 ℃, 6 ℃, etc., and time can be selected from 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0042] Preferably, the rose extract is prepared by a method comprising the following steps: mixing rose with water for extraction, filtering, concentrating, and drying.

[0043] Preferably, the ratio of rose to water is 1 g:(30-50) mL, where the specific values ​​in (30-50) can be selected from 30, 32, 35, 38, 40, 42, 45, 48, 50, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0044] Preferably, the extraction is carried out under stirring.

[0045] Preferably, the extraction temperature is 60-70 ℃ and the extraction time is 60-90 min.

[0046] Temperatures can be selected from 60 ℃, 62 ℃, 64 ℃, 66 ℃, 68 ℃, 70 ℃, etc., and time can be selected from 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0047] Fourthly, the present invention provides the use of the black truffle extract according to the second aspect or the composition according to the third aspect in the preparation of products having anti-glycation, antioxidant or anti-inflammatory effects.

[0048] Preferably, the product includes cosmetics.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The extraction method employed in this invention can complete the cell-wall breaking extraction process in a short time, tailored to the characteristics of Yunnan black truffles, thus improving the yield of active ingredients and production efficiency. The solvent used in the extraction process is water, without the involvement of organic solvents, improving product safety and reducing environmental pollution. Furthermore, this invention selects natural extracts with synergistic mechanisms and high activity characteristics to achieve multi-pathway intervention, including inhibiting sugar production at the source, chelating metal ions, directly inhibiting AGEs formation, scavenging free radicals, and regulating inflammatory responses. Black truffle extract, raspberry extract, and rose extract exhibit certain synergistic effects in the aforementioned efficacy, demonstrating potential synergistic properties. Attached Figure Description

[0051] Figure 1 Examples 1, 8, Comparative Example 1, and Application Example 1 are examples of Fe... 2+ Chelation capacity results. * indicates comparison with Example 1, P < 0.05; *** indicates comparison with Example 1, P < 0.001.

[0052] Figure 2 This refers to the effect of Example 1 on the ROS clearance of HaCaT cells induced by UVA. ### indicates P < 0.001 compared with the blank control group (NC); * indicates P < 0.05 compared with the model control group (MC); ** indicates P < 0.01 compared with the model control group; *** indicates P < 0.001 compared with the model control group. Detailed Implementation

[0053] 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.

[0054] The black truffles used in the following examples are from Yunnan.

[0055] Preparation Example 1-1

[0056] This preparation example provides a method for preparing raspberry extract, the method comprising:

[0057] (1) The moisture content of raspberry raw material was adjusted to 15 wt%, and steam explosion treatment was carried out at 0.4 MPa and 150 ℃ for 1 min, with an explosion rate of <0.01 s. The explosion was repeated once, and the product was dried to a moisture content of 5 wt% to obtain dried raspberry product.

[0058] (2) The dried raspberry product was pulverized and mixed with water at a ratio of 1 g:12 mL. Microfluidic extraction was performed at a temperature of 70 °C and a pressure of 150 MPa, and the mixture was circulated twice to obtain the first extract. The first extract was then heated and refluxed for 70 min to obtain the second extract. The second extract was cooled and filtered through a 600-mesh, 5 μm filter bag and concentrated under vacuum to obtain a concentrate. The solid content of the concentrate was 12 wt%.

[0059] (3) The concentrate was refrigerated at 4 ℃ for 10 h to flocculate, then sterilized by centrifugation and dried to obtain the final product.

[0060] Preparation Examples 1-2

[0061] This preparation example provides a method for preparing raspberry extract, the method comprising:

[0062] (1) The moisture content of raspberry raw material was adjusted to 10 wt%, and steam explosion treatment was carried out at 0.2 MPa and 140 ℃ for 2 min, with an explosion rate of <0.01 s. The explosion was repeated once, and the product was dried to a moisture content of 4 wt% to obtain dried raspberry product.

[0063] (2) The dried raspberry product was pulverized and mixed with water at a ratio of 1 g:10 mL. Microfluidic extraction was performed at a temperature of 60 °C and a pressure of 100 MPa, and the mixture was circulated twice to obtain the first extract. The first extract was then heated and refluxed for 90 min to obtain the second extract. The second extract was cooled and filtered through a 600-mesh, 5 μm filter bag and concentrated under vacuum to obtain a concentrated solution with a solid content of 15 wt%.

[0064] (3) The concentrate was refrigerated at 2 ℃ for 8 h to flocculate, then sterilized by centrifugation and dried to obtain the final product.

[0065] Preparation Examples 1-3

[0066] This preparation example provides a method for preparing raspberry extract, the method comprising:

[0067] (1) The moisture content of raspberry raw material was adjusted to 20 wt%, and steam explosion treatment was carried out at 0.6 MPa and 170 ℃ for 0.5 min, with an explosion rate of <0.01 s. The explosion was repeated once, and the product was dried to a moisture content of 6 wt% to obtain dried raspberry product.

[0068] (2) The dried raspberry product was pulverized and mixed with water at a material-to-liquid ratio of 1 g:15 mL. Microfluidic extraction was performed at a temperature of 80 ℃ and a pressure of 200 MPa, and the mixture was circulated twice to obtain the first extract. The first extract was then heated and refluxed for 60 min to obtain the second extract. The second extract was cooled and filtered through a 600-mesh, 5 μm filter bag, and concentrated under vacuum to obtain a concentrated solution with a solid content of 10 wt%.

[0069] (3) The concentrate was refrigerated at 6 ℃ for 12 h to flocculate, then sterilized by centrifugation and dried to obtain the final product.

[0070] Preparation Examples 1-4

[0071] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (1) is "adjusting the moisture content of raspberry raw material to 5 wt%, performing steam explosion treatment at 0.4 MPa and 150 ℃ for 1 min, with an explosion rate of <0.01 s, cyclic explosion once, and drying to a moisture content of 5 wt% to obtain dried raspberry product", while other operations remain unchanged.

[0072] Preparation Examples 1-5

[0073] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (1) is "adjusting the moisture content of raspberry raw material to 25 wt%, performing steam explosion treatment at 0.4 MPa and 150 ℃ for 1 min, with an explosion rate of <0.01 s, cyclic explosion once, and drying to a moisture content of 5 wt% to obtain dried raspberry product", while other operations remain unchanged.

[0074] Preparation Examples 1-6

[0075] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (1) is "adjusting the moisture content of raspberry raw material to 15 wt%, performing steam explosion treatment at 0.1 MPa and 120 ℃ for 3 min, with an explosion rate of <0.01 s, cyclic explosion once, and drying to a moisture content of 5 wt% to obtain dried raspberry product", while other operations remain unchanged.

[0076] Preparation Examples 1-7

[0077] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (1) is "adjusting the moisture content of raspberry raw material to 15 wt%, performing steam explosion treatment at 0.8 MPa and 175 ℃ for 15 s, with an explosion rate of <0.01 s, cyclic explosion once, and drying to a moisture content of 5 wt% to obtain dried raspberry product", while other operations remain unchanged.

[0078] Preparation Examples 1-8

[0079] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (1) is "adjusting the moisture content of raspberry raw material to 15 wt%, keeping it heated and pressure-treated at 0.4 MPa and 150 ℃ for 1 min, and drying it to a moisture content of 5 wt% to obtain dried raspberry product", while other operations remain unchanged.

[0080] Preparation Examples 1-9

[0081] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (2) is "to crush the dried raspberry product and mix it with water at a ratio of 1 g: 12 mL, and perform micro-jet extraction at a temperature of 70 ℃ and a pressure of 150 MPa, and to cycle 3 times to obtain the first extract; after cooling the first extract, filter it through a 600-mesh, 5 μm filter bag and concentrate it under vacuum to obtain a concentrate, the solid content of which is 12 wt%", and other operations remain unchanged.

[0082] Preparation Examples 1-10

[0083] This preparation example provides a method for preparing raspberry extract, which differs from preparation example 1-1 only in that step (2) is "to crush the dried raspberry product and mix it with water at a ratio of 1 g: 12 mL, and extract it by heating and reflux for 90 min to obtain the first extract. After cooling the first extract, filter it through a 600-mesh, 5 μm filter bag and concentrate it under vacuum to obtain a concentrated solution with a solid content of 12 wt%". Other operations remain unchanged.

[0084] Preparation Example 2-1

[0085] This preparation example provides a method for preparing rose extract, the method comprising:

[0086] The rose powder was mixed with water at a ratio of 1 g to 40 mL. The mixture was stirred and extracted at 65 °C for 70 min. The mixture was then filtered through 600 mesh and 5 μm filter bags, concentrated under reduced pressure, sterilized, and dried to obtain the final product.

[0087] Preparation Example 2-2

[0088] This preparation example provides a method for preparing rose extract, the method comprising:

[0089] The rose powder was mixed with water at a ratio of 1 g to 30 mL. The mixture was stirred and extracted at 60 °C for 90 min. The mixture was then filtered through 600 mesh and 5 μm filter bags, concentrated under reduced pressure, sterilized, and dried to obtain the final product.

[0090] Preparation Examples 2-3

[0091] This preparation example provides a method for preparing rose extract, the method comprising:

[0092] The rose powder was mixed with water at a ratio of 1 g to 50 mL. The mixture was stirred and extracted at 70 °C for 60 min. The mixture was then filtered through 600 mesh and 5 μm filter bags, concentrated under reduced pressure, sterilized, and dried to obtain the final product.

[0093] Example 1

[0094] This embodiment provides a method for preparing black truffle extract, the preparation method comprising:

[0095] (1) Cut the black truffle raw material into 2-5cm flaky particles, adjust the moisture content to 20 wt%, and carry out steam explosion treatment at 0.8 MPa and 175℃ for 45 s, with an explosion rate of <0.01 s. Repeat the explosion once and dry to a moisture content of 4 wt% to obtain the dried black truffle product.

[0096] (2) Pulverize the dried black truffle product, pass it through a 40-mesh sieve, mix it with water at a ratio of 1 g: 15 mL, extract it at 80 ℃ and 300 W for 45 min, pass it through a 600-mesh filter cloth and then through a 140 nm ceramic membrane, concentrate it under reduced pressure to a solid content of 10 wt%, adjust the pH to 4.5, sterilize and dry it to obtain the product.

[0097] Example 2

[0098] This embodiment provides a method for preparing black truffle extract, the preparation method comprising:

[0099] (1) Cut the black truffle raw material into 2-5 cm flaky particles, adjust the moisture content to 15 wt%, and carry out steam explosion treatment at 0.4 MPa and 160 ℃ for 60 s, with an explosion rate of <0.01 s. Repeat the explosion twice and dry to a moisture content of 6 wt% to obtain the dried black truffle product.

[0100] (2) Pulverize the dried black truffle product, pass it through a 40-mesh sieve, mix it with water at a ratio of 1 g: 10 mL, extract it at 60 ℃ and 200 W for 60 min using ultrasonic extraction, pass it through a 600-mesh filter cloth and then through a 200 nm ceramic membrane, concentrate it under reduced pressure to a solid content of 8 wt%, adjust the pH to 4, sterilize and dry it to obtain the product.

[0101] Example 3

[0102] This embodiment provides a method for preparing black truffle extract, the preparation method comprising:

[0103] (1) Cut the black truffle raw material into 2-5 cm flaky particles, adjust the moisture content to 25 wt%, and carry out steam explosion treatment at 1.2 MPa and 195 ℃ for 30 s, with an explosion rate of <0.01 s. Repeat the explosion once and dry to a moisture content of 3 wt% to obtain the dried black truffle product.

[0104] (2) Pulverize the dried black truffle product, pass it through a 40-mesh sieve, mix it with water at a ratio of 1 g: 20 mL, extract it at 70 ℃ and 400 W for 30 min, pass it through a 600-mesh filter cloth and then through a 100 nm ceramic membrane, concentrate it under reduced pressure until the solid content is 12 wt%, adjust the pH to 5, sterilize and dry it to obtain the product.

[0105] Example 4

[0106] This embodiment provides a method for preparing black truffle extract. The only difference between this method and Example 1 is that step (1) is to "cut the black truffle raw material into 2-5 cm flake particles, adjust the moisture content to 10 wt%, perform steam explosion treatment at 0.8 MPa and 175 ℃ for 45 s, with an explosion rate of <0.01 s, perform one cycle of explosion, and dry to a moisture content of 4 wt% to obtain the dried black truffle product". Other operations remain unchanged.

[0107] Example 5

[0108] This embodiment provides a method for preparing black truffle extract. The only difference between this method and Example 1 is that step (1) is to "cut the black truffle raw material into 2-5 cm flaky particles, adjust the moisture content to 30 wt%, perform steam explosion treatment at 0.8 MPa and 175 ℃ for 45 s, with an explosion rate of <0.01 s, repeat the explosion once, and dry to a moisture content of 4 wt% to obtain the dried black truffle product". Other operations remain unchanged.

[0109] Example 6

[0110] This embodiment provides a method for preparing black truffle extract. The only difference between this method and Example 1 is that step (1) is to "cut the black truffle raw material into 2-5 cm flaky particles, adjust the moisture content to 20 wt%, perform steam explosion treatment at 0.2 MPa and 135 ℃ for 70 s with an explosion rate of <0.01 s, repeat the explosion once, and dry to a moisture content of 4 wt% to obtain the dried black truffle product". Other operations remain unchanged.

[0111] Example 7

[0112] This embodiment provides a method for preparing black truffle extract. The only difference between this method and Example 1 is that step (1) is to "cut the black truffle raw material into 3 cm flakes, adjust the moisture content to 20 wt%, perform steam explosion treatment at 1.45 MPa and 200 ℃ for 20 s, with an explosion rate of <0.01 s, perform one cycle of explosion, and dry to a moisture content of 4 wt% to obtain the dried black truffle product". Other operations remain unchanged.

[0113] Example 8

[0114] This embodiment provides a method for preparing black truffle extract. The only difference between this method and Example 1 is that step (1) is to "cut the black truffle raw material into 3 cm flakes, adjust the moisture content to 20 wt%, and hold it under pressure at 0.8 MPa and 175 ℃ for 45 s, and dry it to a moisture content of 4 wt% to obtain the dried black truffle product". Other operations remain unchanged.

[0115] Comparative Example 1

[0116] This comparative example provides a method for preparing black truffle extract, which differs from Example 1 only in that step (1) is "drying black truffles to a water content of 4 wt% to obtain dried black truffle product", while other operations remain unchanged.

[0117] Application Example 1

[0118] This application example provides a composition comprising, by mass parts, 30 parts of raspberry extract prepared in Preparation Example 1-1, 20 parts of black truffle extract prepared in Example 1, and 50 parts of rose extract prepared in Preparation Example 2-1.

[0119] The preparation method is as follows: the raw materials are physically mixed to obtain the product.

[0120] Application Example 2

[0121] This application example provides a composition comprising, by weight, 10 parts of raspberry extract prepared in Preparation Examples 1-2, 15 parts of black truffle extract prepared in Example 2, and 30 parts of rose extract prepared in Preparation Examples 2-2.

[0122] The preparation method is described in Application Example 1.

[0123] Application Example 3

[0124] This application example provides a composition comprising, by weight, 20 parts of raspberry extract prepared in Preparation Examples 1-3, 8 parts of black truffle extract prepared in Example 3, and 20 parts of rose extract prepared in Preparation Examples 2-3.

[0125] The preparation method is described in Application Example 1.

[0126] Application Example 4-10

[0127] This application example provides 7 compositions, which differ from Application Example 1 only in that the raspberry extract prepared in Preparation Example 1-1 is replaced in equal amounts with the raspberry extracts prepared in Preparation Examples 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, and 1-10 in sequence, while other components and contents remain unchanged.

[0128] The preparation method is described in Application Example 1.

[0129] Comparative Application Example 1

[0130] This comparative application example provides a composition that differs from Application Example 1 only in that it does not contain the raspberry extract prepared in Preparation Example 1-1, and the reduced mass of the raspberry extract is proportionally allocated to the mass of the black truffle extract prepared in Example 1 and the rose extract prepared in Preparation Example 2-1.

[0131] The preparation method is described in Application Example 1.

[0132] Comparative Application Example 2

[0133] This comparative application example provides a composition that differs from Application Example 1 only in that it does not contain the black truffle extract prepared in Example 1, and the reduced mass of the black truffle extract is proportionally allocated to the mass of the raspberry extract prepared in Preparation Example 1-1 and the rose extract prepared in Preparation Example 2-1.

[0134] The preparation method is described in Application Example 1.

[0135] Comparative Application Example 3

[0136] This comparative application example provides a composition that differs from application example 1 only in that it does not contain the rose extract prepared in preparation example 2-1, and the reduced mass of the rose extract is proportionally allocated to the mass of the black truffle extract prepared in example 1 and the raspberry extract prepared in preparation example 1-1.

[0137] The preparation method is described in Application Example 1.

[0138] Test Example 1

[0139] Antioxidant effect test

[0140] DPPH solution was dissolved in anhydrous ethanol to a concentration of 0.1 mg / mL. Vitamin C (positive control) and sample extract solution (concentration: 250 μg / mL) were prepared simultaneously. 150 μL of DPPH ethanol solution and 150 μL of extract 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:

[0141]

[0142] Sample blank group: 150 μL DPPH solution + 150 μL sample solvent

[0143] Sample group: 150 μL sample solution + 150 μL DPPH alcohol solution

[0144] Control group: 150 μL sample solution + 150 μL anhydrous ethanol

[0145] Control group: 150 μL anhydrous ethanol + 150 μL sample solvent

[0146] The DPPH free radical scavenging rate (%) results are shown in Table 1.

[0147] Table 1

[0148]

[0149] As shown in Table 1, both the black truffle extract and the anti-glycation composition prepared in this invention exhibit DPPH free radical scavenging effects. Adjustments to the sample's moisture content, pressure, and temperature during steam explosion can affect the antioxidant effect. Failure to perform steam explosion or the use of high-temperature, high-pressure methods instead will also negatively impact the antioxidant effect. The black truffle extract, raspberry extract, and rose extract demonstrate a certain synergistic effect in enhancing the antioxidant efficacy.

[0150] Test Example 2

[0151] Anti-glycation test

[0152] A 1 mg / mL sample solution was prepared using phosphate buffer (50 mmol / L, pH 7.4). 1 mL of the sample, 0.8 mg / mL bovine serum albumin (BSA) solution, 200 mM glucose solution, and phosphate buffer (PBS) were added to separate test tubes, and the tubes were heated at 60 °C for 24 h. Aminoguanidine hydrochloride (AG) was used 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 fluorescence intensity (AU) of the AGEs was expressed as a fluorescence intensity.

[0153] Table 2

[0154]

[0155] The inhibition rate of fluorescent AGEs formation by the sample is calculated as follows:

[0156]

[0157] 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.

[0158] Table 3

[0159]

[0160] As shown in Table 3, both the black truffle extract and the anti-glycation composition prepared in this invention exhibit AGEs inhibition effects. Adjustments to the sample's moisture content, pressure, and temperature during steam explosion all affect the AGEs inhibition effect. Furthermore, omitting steam explosion or using high-temperature, high-pressure methods instead will also negatively impact the AGEs inhibition effect. The black truffle extract, raspberry extract, and rose extract demonstrate a certain synergistic effect in enhancing these effects.

[0161] Test Example 3

[0162] Fe 2+ Chelation capacity test

[0163] The Fenton reaction involves the reaction of iron ions with hydrogen peroxide under acidic conditions to generate hydroxyl radicals. These radicals accelerate the decomposition of polysaccharides during saccharification. If plant extracts possess metal chelating capabilities, they can bind to iron ions, inhibiting the generation of hydroxyl radicals and thus mitigating the accelerating effect of the Fenton reaction on saccharification.

[0164] Principle: Based on phenanthridine and Fe 2+ The colorimetric reaction is used to determine the chelating ability of iron ions; phenanthridine can chelate with Fe. 2+ A specific reaction occurs, forming a complex with a strong absorption peak at 562 nm, and the absorbance is similar to that of Fe. 2+ Concentration is directly proportional. When there is no chelating agent in the system, phenanthridine reacts with Fe... 2+ The reaction was fully completed. The chelation rate was calculated by comparing the absorbance of the sample (containing the potential chelating agent) with that of the control group (blank group) without the chelating agent. A higher chelation rate indicates a greater affinity of the sample for Fe. 2+ The stronger the chelating ability, the better. Citric acid, which is known to have chelating ability, was used as a positive control.

[0165] Two mL of 10% citric acid and 10 mg / mL sample solutions from Examples 1, 8, Comparative Example 1, and Application Example 1 were mixed with 2.7 mL of distilled water and 0.1 mL of FeCl₂·4H₂O solution (2 mmol / L). Then, 0.2 mL of phenoxylate (5 mmol / L) was added, and the mixture was reacted at room temperature for 10 min. The absorbance was then measured at 562 nm. Simultaneously, distilled water was used as a control group instead of the sample, and ultrapure water was used as a blank group instead of phenoxylate. 2+ The chelation rate is calculated using the following formula, and the result is as follows: Figure 1 As shown.

[0166]

[0167] In the formula:

[0168] A sample : Sample group absorbance measurement

[0169] A sampleblank : Absorbance determination of blank sample group

[0170] A control : Control group absorbance measured

[0171] A controlblank : Control blank group for absorbance measurement

[0172] Fe 2+ Fe is a highly reactive transition-state metal ion that can catalyze and accelerate protein glycosylation. Therefore, by chelating Fe... 2+This is an important pathway to inhibit the formation of AGEs. For example... Figure 1 As shown, Examples 1, 8, Comparative Example 1, and Application Example 1 all involve Fe 2+ It has chelating ability, and the Fe in Example 1 2+ The chelating ability was significantly better than that of Example 8 and Comparative Example 1 (P < 0.001), indicating that the black truffle extract of Example 1 has a stronger activity advantage in inhibiting AGEs formation. The above data show that the black truffle extract and its composition of the present invention can effectively chelate Fe... 2+ It significantly reduces protein glycosylation levels and effectively inhibits AGEs formation. Black truffle extract, rose extract, and raspberry extract have a certain synergistic effect on the above-mentioned effects.

[0173] Test Example 4

[0174] Anti-inflammatory effect test

[0175] 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 hours.

[0176] Drug administration and LPS: A blank control group, an induction stimulation group, a positive control group, and a sample group were set up.

[0177] Blank control group: Add complete culture medium to each well.

[0178] Positive control group: 5 μM dexamethasone acetate (DEX) diluted with complete culture medium was added to each well.

[0179] Negative control group: LPS at a final concentration of 1 μg / mL

[0180] Add the test sample (concentration of 500 μg / mL) diluted with complete culture medium to each well of the sample group.

[0181] 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 in a 37 ℃, 5% CO2 incubator for 24 h.

[0182] Detection of inflammatory factors in cell supernatant: Cell supernatant was collected by centrifugation at 4 ℃, 1000 g, for 10 min. The levels of NO and IL-6 in the collected cell supernatant were detected according to the instructions of the NO detection kit and ELISA kit. The experimental results were analyzed using data processing software, and the results are shown in Table 4.

[0183] Table 4

[0184]

[0185] Note: ### * indicates P < 0.001 compared to the blank control group; * indicates P < 0.05 compared to the model control group; ** indicates P < 0.01 compared to the model control group. *** This indicates a comparison with the model control group, where P < 0.001.

[0186] As shown in Table 4, LPS stimulation significantly increased the relative expression levels of NO and IL-6 (P<0.001), indicating the successful establishment 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 Example 1 and Application Example 1 were at a high level. These results indicate that black truffle extract and its composition have strong anti-inflammatory effects.

[0187] Test Example 5

[0188] α-glucosidase inhibition effect test

[0189] Using phosphate buffer (pH 6.8) as the solvent, prepare a 5 mmol / L PNPG solution, a 0.5 μg / mL α-glucosidase solution, a 1 mol / L Na₂CO₃ solution, and an acarbose solution (positive control) at a concentration of 100 μg / mL, along with a sample solution. Mix 50 μL of the α-glucosidase solution with 50 μL of the 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 mixture to stop the reaction. Measure the absorbance at 405 nm. A blank control group was also included.

[0190] Table 5

[0191]

[0192] Calculate the inhibition rate of the sample against α-glucosidase using the following formula:

[0193]

[0194] The results are shown in Table 6.

[0195] Table 6

[0196]

[0197] As shown in Table 6, the composition described in this application has a good inhibitory effect on α-glucosidase. The preparation method of raspberry extract also affects the effect of the composition. Furthermore, black truffle extract, raspberry extract and rose extract have a certain synergistic effect on the above-mentioned effects. They complement each other and are indispensable.

[0198] Test Example 6

[0199] ROS Cleanup Effectiveness Test

[0200] Healthy cells in the exponential growth phase were selected, digested with trypsin, and counted before being seeded into 12-well plates, approximately 2.5 × 10⁶ cells per well. 5 Cells were cultured in an incubator for 24 h. 1 mL of complete culture medium was added to each well of the control and model groups, and 1 mL of complete culture medium containing 62.5, 125, and 250 μg / mL of the composition from Example 1 was added to each well of the sample groups, respectively. After incubation for 2 h, the culture medium was discarded. Subsequently, 1 mL of PBS was added to each well of the control and model groups, and 1 mL of PBS containing 62.5, 125, and 250 μg / mL of the composition from Example 1 was added to each well of the sample groups, respectively. The model and sample groups were then exposed to 15 J / cm². 2 Under ultraviolet radiation, a 2-hour UVA irradiation treatment was performed (UVA irradiation was performed using a 3T3 NRU phototoxic UV radiometer, UVA wavelength was 350-400 nm, peak emission λ=365 nm, and dose was 2 mW / cm²). 2 Meanwhile, the control group was placed in the same environment but without UVA irradiation. After irradiation, all groups were placed in an incubator for 1 h to stabilize. ROS were detected using flow cytometry. Results are as follows. Figure 2 As shown.

[0201] During glycation, ROS and free radicals promote the formation of AGEs. Natural plant extracts have a certain free radical scavenging ability and can indirectly inhibit the accumulation of AGEs. For example... Figure 2 As shown, after UVA radiation, HaCaT cells in the model control group (MC) produced a large amount of ROS, and the fluorescence intensity was significantly increased compared with the blank control group (NC) (P<0.001), indicating significant oxidative stress damage. Compared with MC, the intracellular ROS fluorescence intensity in the application group (Example 1) was significantly reduced (P<0.001). The results indicate that the application group (Example 1) can effectively reduce UVA-induced ROS levels and alleviate cell damage caused by oxidative stress.

[0202] The applicant declares that this invention illustrates a method for preparing a black truffle extract, a composition containing the extract, 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, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0203] 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.

[0204] 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 describe the various possible combinations separately.

Claims

1. A composition having an anti-glycemic effect, characterized by comprising, The composition comprises black truffle extract 5-20 parts by mass, raspberry extract 10-30 parts by mass, and rose extract 20-50 parts by mass; The black truffle extract is prepared by a method comprising the following steps: (1) drying the black truffle after steam explosion to obtain a black truffle dried product; (2) mixing the black truffle dried product with water, ultrasonic extraction, ceramic membrane filtration, concentration, drying, and obtaining the extract; The water content of the black truffle is 15-25 wt%, the pressure of steam explosion is 0.4-1.2 MPa, and the time is 30-60 s; The raspberry extract is prepared by a method comprising the following steps: (1) drying the raspberry after steam explosion to obtain a raspberry dried product; (2) mixing the raspberry dried product with water, micro-jet extraction, and obtaining a first extract; (3) heating reflux extraction of the first extract to obtain a second extract; (4) filtering, concentrating, cold storage flocculation, centrifugation, and drying, and obtaining the extract; The water content of the raspberry is 10-20 wt%, the pressure of steam explosion is 0.2-0.6 MPa, and the time is 0.5-2 min.

2. The composition having anti-glycative efficacy according to claim 1, characterized in that, The ultrasonic extraction temperature is 60-80 ℃, the time is 30-60 min, and the power is 200-400 W.

3. The composition having anti-glycative efficacy according to claim 1, characterized in that, The micro-jet extraction temperature is 60-80 ℃, the pressure is 100-200 MPa, and the number of times is 2-3.

4. The composition having anti-glycative efficacy according to claim 1, characterized in that, The rose extract is prepared by a method comprising the following steps: mixing the rose with water, extraction, filtration, concentration, and drying.

5. Use of the composition according to any one of claims 1-4 in the preparation of a cosmetic product having anti-glycation, antioxidant or anti-inflammatory efficacy.

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