Tricholoma matsutake polyphenol extraction method and preparation

Abstract

The present application relates to the technical field of pine mushroom polyphenol extraction, in particular to a pine mushroom polyphenol extraction method and preparation. The method uses an ethanol solution to extract pine mushroom raw materials at least once under heating and ultrasonic conditions to obtain pine mushroom polyphenol extract; wherein the extraction temperature is 45-55 DEG C, the solid-liquid ratio of the pine mushroom raw materials and ethanol is 1:38-42, and the ultrasonic time is 55-65 min in the ultrasonic conditions. The cavitation effect, mechanical effect and thermal effect produced by the ultrasonic waves synergistically act on the special cell wall structure of pine mushrooms, effectively break the special cell wall structure of pine mushrooms, realize the efficient dissolution of polyphenols while avoiding thermal degradation and oxidative loss, and solve the technical problems existing in the application of conventional extraction methods to pine mushrooms.

Description

Technical Field

[0001] This invention relates to the field of matsutake polyphenol extraction technology, and more specifically, to a method and preparation for extracting matsutake polyphenols. Background Technology

[0002] Matsutake mushrooms ( Tricholoma Matsutake (S. Ito & S. Imai) Singer is one of the rarest, most precious and commercially valuable wild edible ectomycorrhizal mushrooms in the world. It belongs to the genus S. Ito in the family Tricholomataceae. It is an ectomycorrhizal fungus that grows on trees such as pine and oak. It is named for growing under pine trees and for the shape of its buds resembling deer antlers. It is also known as pine mushroom or pine fungus.

[0003] Matsutake mushrooms are rich in protein, amino acids, unsaturated fatty acids, vitamins, and essential trace elements, while being low in fat. They also contain various bioactive components such as matsutake polysaccharides, matsutake polypeptides, and matsutake alcohol, exhibiting a range of pharmacological effects including anti-inflammatory, antioxidant, anti-diabetic, anti-fatigue, neuroprotective, immunomodulatory, and wound-healing properties. However, current research on matsutake mushrooms largely focuses on polysaccharides and proteins, with limited research on the polyphenolic compounds present in lower concentrations.

[0004] Matsutake polyphenols are present in extremely low concentrations in matsutake mushrooms, with existing literature reporting levels typically ranging from 0.001% to 0.74%, far lower than common plant materials such as tea (18%-36%) and grape seeds (60%-70%). This extremely low concentration makes it difficult to achieve ideal yields using conventional extraction methods, placing stringent demands on the efficiency and selectivity of the extraction process. Furthermore, matsutake polyphenols coexist with other bioactive components such as polysaccharides, polypeptides, and volatile substances. These substances may compete with polyphenols for solvents or form complexes during extraction, increasing the difficulty of separation and purification. Conventional extraction methods often lack consideration for the selectivity of such complex matrices.

[0005] In addition, matsutake polyphenols are significantly unstable. High temperature and long-term extraction can cause matsutake polyphenols to decompose and oxidize, which reduces the antioxidant capacity of the extracted matsutake polyphenols and makes them lose their practical application value.

[0006] Specifically, the main drawbacks of existing extraction methods are as follows: organic solvent extraction is time-consuming and has a low extraction rate; ultrasonic extraction can easily denature or deactivate heat-sensitive substances; supercritical fluid extraction equipment is expensive and occupies a large space, and cannot achieve continuous extraction; enzyme-assisted extraction requires stringent conditions, making it difficult to industrialize, and the high price and large dosage of some enzyme preparations lead to high costs; dynamic ultra-high pressure microfluidic technology has complex extraction operations and high energy consumption.

[0007] Therefore, there is an urgent need to develop an efficient and optimized matsutake polyphenol extraction process to improve the comprehensive utilization of matsutake raw materials and provide technical support for the industrial application of matsutake polyphenols. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method and preparation for extracting matsutake polyphenols.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: Based on the above technical solution, the present invention can be further improved as follows.

[0010] This invention provides a method for extracting matsutake polyphenols, which involves extracting matsutake raw materials at least once with an ethanol solution under heating and ultrasonic conditions to obtain matsutake polyphenol extract; wherein the extraction temperature is 45℃-55℃, the ratio of matsutake raw materials to ethanol is 1:38-42, and the ultrasonic conditions are met for 55-65 minutes.

[0011] Furthermore, the ethanol solution has a mass percentage of 35%-45%.

[0012] Furthermore, in the ultrasonic conditions, the ultrasonic frequency is 35-45Hz.

[0013] Furthermore, the extraction temperature is 50℃, the ultrasonic time is 60 min, the material-to-liquid ratio is 1:40, the mass percentage of the ethanol solution is 40%, and the ultrasonic frequency is 40 Hz.

[0014] Furthermore, the matsutake mushroom raw material is freeze-dried matsutake mushroom powder.

[0015] Furthermore, each extraction includes the following steps: S1. Mix the matsutake mushroom raw material or the filter residue obtained from the previous extraction with the ethanol solution to obtain a mixture; S2. Extract the mixture under heating and ultrasonic conditions; S3. After cooling the extracted mixture, centrifuge and filter it to obtain filter residue and supernatant; S4. Concentrate the supernatant to obtain the matsutake polyphenol extract.

[0016] Furthermore, the centrifugation speed is 3500-4500 r / min, and the time is 5-15 min.

[0017] Furthermore, the temperature of the cooled mixture is 20℃-25℃.

[0018] Furthermore, the filtration method is vacuum filtration, and in step S4, the concentration method is rotary evaporation.

[0019] The present invention also provides a matsutake polyphenol preparation, wherein the matsutake polyphenol in the preparation is extracted by the method described above.

[0020] The beneficial effects of this invention are as follows: (1) The method for extracting matsutake polyphenols of the present invention significantly improves the extraction efficiency and polyphenol yield by using ethanol solution to extract matsutake raw materials under specific heating and ultrasonic conditions. The extraction temperature is controlled within the range of 45℃-55℃, which can effectively promote the dissolution of polyphenols while avoiding polyphenol decomposition and oxidation and ethanol volatilization caused by excessively high temperature. (2) The method for extracting matsutake polyphenols in this invention controls the material-liquid ratio within the range of 1:38-42, ensuring that the solvent can fully wet the matsutake raw material so that the polyphenols can be fully extracted, while avoiding the dilution effect caused by an excessively high material-liquid ratio, which leads to a decrease in yield, thus achieving an optimized balance between solvent usage and extraction effect. (3) The method for extracting polyphenols from matsutake mushrooms in this invention controls the ultrasonic time within the range of 55-65 min, which ensures that the matsutake mushrooms and solvent have sufficient contact time to fully extract the polyphenols, while avoiding the oxidation and decomposition of polyphenols caused by excessive extraction time, thereby obtaining a higher polyphenol yield. (4) In the method for extracting matsutake polyphenols of the present invention, the mass percentage of ethanol solution is controlled within the range of 35%-45%. This concentration range can effectively match the polarity characteristics of matsutake polyphenols, promote the dissolution of polyphenols into ethanol solution, and avoid the problem of protein denaturation and formation of dense cell wall structure due to excessively high ethanol concentration, which hinders the dissolution of polyphenols. (5) The method for extracting matsutake polyphenols of the present invention is simple to operate, saves time and cost, is suitable for the extraction of heat-sensitive substances, and the ethanol solvent is relatively environmentally friendly, less toxic and easy to recycle, which meets the requirements of green extraction process. (6) The matsutake polyphenol extraction method of the present invention yields matsutake polyphenols with excellent antioxidant activity, exhibiting scavenging rates of up to 79.40%, 97.33%, and 98.75% against DPPH free radicals, ABTS+ free radicals, and hydroxyl free radicals, respectively. 50 The values ​​were 3.92 mg / mL, 3.78 mg / mL and 4.89 mg / mL, respectively, indicating that it has potential antioxidant activity; (7) The matsutake polyphenols of the present invention provide important technical support for the intensive processing and comprehensive utilization of matsutake resources. They can be widely used in the development of functional foods, cosmetics and pharmaceutical products, and improve the comprehensive utilization level and added value of matsutake raw materials. Attached Figure Description

[0021] Figure 1 This is a standard curve diagram in Embodiment 1 of the present invention; Figure 2 This is a line graph showing the relationship between different ethanol concentrations and matsutake polyphenol yield in Example 1 of the present invention; Figure 3 This is a line graph showing the relationship between different temperatures and the yield of matsutake polyphenols in Example 1 of the present invention. Figure 4 This is a line graph showing the relationship between different extraction times and matsutake polyphenol yield in Example 1 of the present invention; Figure 5 This is a line graph showing the yield of matsutake polyphenols with different material-to-liquid ratios in Example 1 of the present invention. Figure 6 This is a graph showing the interaction results between pairs of factors affecting polyphenol yield in Example 2 of the present invention. Figure 6 In Figure 'a', the effect of the interaction between ultrasonic temperature and ultrasonic time on the yield of matsutake polyphenols is shown. Figure 6 In Figure b, the interaction between the material-liquid ratio and the ultrasonic time affects the yield of matsutake polyphenols. Figure 6 c represents the effect of the interaction between ethanol concentration and ultrasonic time on the yield of matsutake polyphenols; Figure 6 In the figure, d represents the effect of the interaction between ultrasonic temperature and the material-liquid ratio on the yield of matsutake polyphenols. Figure 6 In the figure, e represents the effect of the interaction between ultrasonic temperature and ethanol concentration on the yield of matsutake polyphenols. Figure 6 f represents the effect of the interaction between ethanol concentration and the material-to-liquid ratio on the yield of matsutake polyphenols; Figure 7 This is a comparison chart of the DPPH free radical scavenging rates of matsutake polyphenol solutions and vitamin C at different concentrations in Example 3 of the present invention. Figure 8 This is a comparison chart of the scavenging rates of hydroxyl radicals by matsutake polyphenol solutions and vitamin C at different concentrations in Example 3 of the present invention. Figure 9 This is a comparison chart of the scavenging rates of ABTS+ free radicals by matsutake polyphenol solutions and vitamin C at different concentrations in Example 3 of the present invention. Detailed Implementation

[0022] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] The method for extracting matsutake polyphenols of the present invention involves extracting matsutake raw materials at least once with an ethanol solution under heating and ultrasonic conditions to obtain matsutake polyphenol extract; wherein the extraction temperature is 45℃-55℃, the ratio of matsutake raw materials to ethanol is 1:38-42, and the ultrasonic conditions are for an ultrasonic time of 55-65 min.

[0024] The method for extracting matsutake polyphenols of the present invention significantly improves the extraction efficiency and polyphenol yield by using an ethanol solution to extract matsutake raw materials under specific heating and ultrasonic conditions.

[0025] The extraction temperature is controlled within the range of 45℃-55℃, which can effectively promote the dissolution of polyphenols while avoiding polyphenol decomposition and oxidation, as well as ethanol volatilization caused by excessively high temperatures. The material-to-liquid ratio is controlled within the range of 1:38-42 to ensure that the solvent can fully wet the matsutake mushroom raw material and fully extract the polyphenols, while avoiding the dilution effect caused by excessively high material-to-liquid ratio, which would lead to a decrease in yield. The ultrasonic time is controlled within the range of 55-65 minutes, which ensures that the matsutake mushroom and the solvent have sufficient contact time to fully extract the polyphenols, while avoiding polyphenol oxidation and decomposition caused by excessively long extraction time.

[0026] The ultrasound-assisted ethanol extraction method utilizes the cavitation, mechanical, and thermal effects of ultrasound to promote the disruption of matsutake cell walls, releasing more intracellular polyphenols. Simultaneously, the microfluids generated by ultrasound facilitate solvent penetration into the cells, significantly improving polyphenol diffusion and leaching efficiency. This method is simple to operate, saves time and costs, is suitable for the extraction of heat-sensitive substances, and uses relatively environmentally friendly, low-toxicity, and easily recyclable ethanol solvent. The resulting matsutake polyphenol extract has a high polyphenol content and exhibits excellent antioxidant activity, demonstrating good scavenging ability against DPPH, ABTS+, and hydroxyl radicals. It can be widely applied in the development of functional foods, cosmetics, and pharmaceutical products, providing effective technical support for the comprehensive utilization and industrialization of matsutake resources.

[0027] Preferably, the mass percentage of the ethanol solution is 35%-45%. This concentration range can effectively match the polarity characteristics of matsutake polyphenols, promoting the dissolution of polyphenols into the ethanol solution. When the ethanol concentration is within this range, the ethanol solution can effectively penetrate into the interior of matsutake cells, promoting better dissolution of polyphenols and thus increasing the polyphenol yield. Compared with excessively high concentrations of ethanol solution, this concentration range avoids the problem of protein denaturation and the formation of dense cell wall structures due to excessively high ethanol concentration, which hinders the dissolution of polyphenols. It also avoids the defects of insufficient polyphenol solubility and low extraction efficiency due to excessively low ethanol concentration.

[0028] Preferably, the ultrasonic frequency is 35-45 Hz. This frequency range can effectively generate ultrasonic cavitation, mechanical, and thermal effects, promoting the disruption of matsutake cell walls and allowing the full release of polyphenols from the cells. Simultaneously, ultrasound in this frequency range can form stable microfluids, enhancing the solvent's penetration into the matsutake raw material, making it easier for ethanol solutions to enter the cells, accelerating the diffusion and dissolution of polyphenols, thereby improving extraction efficiency and polyphenol yield. This frequency range ensures efficient extraction while avoiding the problems of excessively high frequencies that may lead to excessively high local temperatures that could damage polyphenol activity or denature heat-sensitive substances, and also avoids the defects of insufficient cavitation effect, incomplete cell wall disruption, and low extraction efficiency caused by excessively low frequencies.

[0029] In one embodiment of the present invention, the extraction temperature of the method is 50°C, the ultrasonic time is 60 min, the material-to-liquid ratio is 1:40, the mass percentage of the ethanol solution is 40%, and the ultrasonic frequency is 40 Hz.

[0030] The optimal combination of process parameters was determined based on single-factor experiments and Box-Behnken response surface methodology. The synergistic effect among the parameters was significant, enabling efficient extraction of matsutake polyphenols. Under these conditions, the yield of matsutake polyphenols reached 4.108 mg / g, which is basically consistent with the model prediction of 4.163 mg / g, with a relative error of only 1.32%. This indicates that the process conditions are stable, reliable, and have good reproducibility.

[0031] Preferably, the matsutake mushroom raw material is freeze-dried matsutake mushroom powder.

[0032] In the matsutake polyphenol extraction method of the present invention, each extraction includes the following steps: S1. Mix the matsutake mushroom raw material or the filter residue obtained from the previous extraction with the ethanol solution to obtain a mixture; S2. Extraction of the mixture under heating and ultrasonic conditions; S3. After cooling the extracted mixture, centrifuge and filter it to obtain filter residue and supernatant.

[0033] Preferably, the centrifugation speed is 3500-4500 r / min and the time is 5-15 min.

[0034] Preferably, the temperature of the cooled mixture is 20℃-25℃.

[0035] Preferably, the filtration method is pressure-reducing filtration.

[0036] S4. Concentrate the supernatant to obtain matsutake polyphenol extract. When performing multiple extractions, the supernatants obtained each time can be combined and then concentrated.

[0037] The preferred method of concentration is rotary evaporation.

[0038] Experimental verification shows that the yield of matsutake polyphenols extracted by the method described above in this invention can reach 0.4108% (4.108 mg / g). Therefore, in practical applications, the obtained matsutake polyphenols can be directly applied after extraction without calculating the yield. In other cases, if it is necessary to calculate the yield of the product, a standard curve can be established for calculation.

[0039] The matsutake polyphenol preparation of the present invention is wherein the matsutake polyphenol is extracted by the method described above.

[0040] The effects of the present invention will be illustrated by specific embodiments below.

[0041] The main materials and reagents used in each embodiment are shown in Table 1, and the main instruments and equipment used in each embodiment are shown in Table 2.

[0042] Table 1 Experimental Materials and Reagents Table 2 Experimental Instruments and Equipment Example 1: Single-factor experiment on the extraction of matsutake polyphenols This embodiment uses single-factor experiments to determine the effects of four factors—ethanol concentration, extraction temperature, ultrasonic time, and material-to-liquid ratio—on the polyphenol yield in the extraction method of the present invention.

[0043] Specifically, in this embodiment, each factor was set with five levels: ethanol concentration of 30%, 40%, 50%, 60%, and 70%; extraction temperature of 30, 40, 50, 60, and 70°C; ultrasonic time of 20, 40, 60, 80, and 100 min; and material-to-liquid ratio of 1:10, 1:20, 1:30, 1:40, and 1:50 g / mL. The effects of different levels of this factor on the yield of matsutake polyphenols were compared sequentially to determine the optimal process conditions. Each group was measured in triplicate, and the result was the average of the three measurements.

[0044] This embodiment uses a gallic acid standard curve for calculation. The standard curve is plotted as follows: using gallic acid as a standard, the total phenol content is determined by the Folin-Ciocalteu method. Accurately weigh 10 mg of gallic acid standard into a 50 mL volumetric flask, dissolve the standard in a small amount of distilled water, and then dilute to 50 mL with water to obtain a 0.2 mg / mL standard solution. Sequentially measure 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the above standard solution into 25 mL colorimetric tubes, add distilled water to each to 1 mL, then add 1 mL of 1 mol / L Folin-Ciocalteu reagent, shake well, and let stand in the dark for 6 min. Then add 5 mL of 5% (mass fraction) sodium carbonate solution, mix thoroughly, and dilute to 25 mL. Let stand in the dark for 1 h, and measure the absorbance at 760 nm. Plot the curve with gallic acid concentration on the x-axis and absorbance on the y-axis as shown below. Figure 1 The standard curve is shown. The equation of the standard curve is y = 0.1463x + 0.0125, R0. 2 =0.9994.

[0045] The determination of matsutake polyphenol content in this embodiment: 1.0000 g of matsutake powder was accurately weighed into a 50 mL colorimetric tube, dispersed in ethanol of a certain concentration at a solid-liquid ratio (g:mL), shaken well, and extracted by ultrasonication (40 kHz) for a certain time at a certain temperature. After extraction, the powder was cooled to room temperature, centrifuged at 4000 r / min for 10 min, and the residue was collected. A certain volume of ethanol was added, and the extraction was repeated once under the same conditions. The residue was cooled to room temperature, centrifuged at 4000 r / min for 10 min, and the supernatants from both extractions were combined and diluted to a 100 mL volumetric flask as the test solution. 1 mL of matsutake ethanol extract was accurately measured into a 25 mL volumetric flask, and the yield of the matsutake polyphenol solution was calculated according to formula (1).

[0046] In the formula: c is the concentration of matsutake polyphenols after ethanol extraction, mg / L; V is the solution volume, mL; n is the dilution factor; m is the mass of matsutake powder, g.

[0047] Line graphs showing the relationship between different ethanol concentrations and matsutake polyphenol yield are shown below. Figure 2 As shown. By Figure 2It can be seen that the yield of matsutake polyphenols first increases and then decreases with increasing ethanol concentration. Differences in ethanol concentration lead to variations in polarity, which in turn affects the dissolution of polyphenols. When the ethanol concentration is between 30% and 50%, the solubility of matsutake polyphenols gradually increases because within this concentration range, the ethanol solution can effectively penetrate cells, promoting better dissolution of polyphenols. However, when the ethanol concentration is between 50% and 70%, the yield gradually decreases, possibly because excessively high ethanol concentrations cause protein denaturation, forming a dense cell wall structure that is not conducive to polyphenol dissolution. There is no significant difference in the yield of matsutake polyphenols at 40% and 50% ethanol concentrations. From the perspective of conserving reagents, an ethanol concentration of 40% is more suitable for extraction.

[0048] Line graphs showing the relationship between different temperatures and matsutake polyphenol yield are shown below. Figure 3 As shown. By Figure 3 It is known that different extraction temperatures lead to varying polyphenol yields. When the extraction temperature is between 30-50℃, the polyphenol yield increases with increasing temperature, reaching its maximum at 50℃. Further increasing the temperature beyond this point results in a decrease in polyphenol yield. This is because as temperature increases, molecular motion intensifies, leading to increased dissolution of polyphenols. However, temperatures exceeding 50℃ cause the decomposition and oxidation of matsutake polyphenols, and also result in ethanol evaporation, altering the material-to-liquid ratio and thus decreasing the polyphenol yield. Therefore, 50℃ is considered the most suitable temperature.

[0049] Line graphs showing the relationship between different extraction times and matsutake polyphenol yield are shown below. Figure 4 As shown. By Figure 4 It was found that the yield of matsutake polyphenols reached its maximum when the extraction time was 60 min. When the extraction time was less than 60 min, the yield of matsutake polyphenols was positively correlated with the extraction time; when the extraction time was greater than 60 min, the yield was negatively correlated with the extraction time. Generally, different extraction times lead to differences in the contact time between the sample and the solvent. When the extraction time is short, the contact time between the matsutake and the solvent is short, resulting in incomplete extraction of matsutake polyphenols. As the extraction time increases, the extraction rate of matsutake polyphenols gradually increases, reaching the maximum yield. When the extraction time exceeds 60 min, matsutake polyphenols undergo oxidation and decomposition reactions, leading to a decrease in polyphenol yield. Therefore, an extraction time of 60 min was chosen.

[0050] Line graphs showing the relationship between different solvent dosages and matsutake polyphenol yield are shown below. Figure 5 As shown. By Figure 5It can be seen that the yield of matsutake polyphenols increases slowly and then decreases with increasing solvent dosage, reaching its maximum at a solid-liquid ratio of 1:40 (g / mL). This may be because insufficient solvent cannot adequately wet the matsutake mushrooms, resulting in incomplete polyphenol dissolution and a lower yield. As the solid-liquid ratio increases, polyphenols in the matsutake mushrooms are gradually extracted, and the polyphenol yield gradually increases. Within a solid-liquid ratio range of 1:40-1:50 (g / mL), the polyphenol yield decreases, possibly because the polyphenols have already been sufficiently dissolved, and further increases in the solid-liquid ratio produce a dilution effect, leading to a decrease in polyphenol yield. Therefore, a solid-liquid ratio of 1:40 (g / mL) is considered appropriate.

[0051] Example 2: Response Surface Optimization Experiment for Matsutake Polyphenol Extraction Based on the results of single-factor experiments, a Box-Behnken design was used to conduct response surface methodology optimization experiments to obtain the optimal conditions for extracting matsutake polyphenols. The optimal process was then used to extract more matsutake polyphenols for the determination of their antioxidant activity. A four-factor, three-level response surface methodology experiment was conducted using ultrasonic time (A), ultrasonic temperature (B), material-to-liquid ratio (C), and ethanol concentration (D). The factor levels and coding design are shown in Table 3.

[0052] Table 3 Response Surface Experimental Factors and Levels Based on the results of the single-factor experiments, the effects of the interaction between four factors (A (ultrasonic time), B (ultrasonic temperature), C (solid-to-material ratio), and D (ethanol concentration) on the polyphenol yield were further discussed. Therefore, a four-factor, three-level response surface methodology experiment was conducted using Design-Expert 13 software. The experimental results are shown in Table 4.

[0053] Table 4 Results of Response Surface Experimental Design The response surface experimental results in Table 4 were analyzed using Design-Expert 13 software, and a quadratic multinomial regression equation was obtained: Y=4.11+0.022A-0.212B+0.144C+0.043D-0.107AB+0.009AC+0.013AD-0.085BC-0.094BD+0.030CD-0.223A 2 -0.490B 2 -0.332C 2 -0.218D 2 .

[0054] The analysis of variance for the regression model is shown in Table 5.

[0055] Table 5. Analysis of Variance for Regression Models Note: "*" indicates a significant difference. P <0.05); "**" indicates a highly significant difference ( P <0.01).

[0056] As shown in Table 5, in the regression model P Values ​​< 0.0001 indicate highly significant model differences; the lack-of-fit term... P Value > 0.05, lack of fit test not significant; coefficient of determination R 2 The correction factor R is 0.9464. Adj 2 The F-value of 0.8929 indicates a high degree of fit between the model and the experimental results on polyphenol yield, demonstrating good experimental stability and the ability to effectively predict and analyze matsutake polyphenol yield. Generally, a larger F-value indicates a more significant impact of the factor on polyphenol yield. The comparison of F-values ​​shows that the order of factors affecting polyphenol yield is: ultrasonic temperature (B) > material-to-liquid ratio (C) > ethanol concentration (D) > ultrasonic time (A). Among these, B, C, and A... 2 B 2 C 2 D 2 The yield of matsutake polyphenols differed significantly from that of matsutake mushrooms. P <0.01), and other factors showed no significant differences ( P >0.05).

[0057] The interaction results between pairwise factors affecting polyphenol yield were obtained through Design-Expert 13 software analysis. See details below. Figure 6 .

[0058] In a response surface plot, the flatter the surface, the smaller the effect of the factor on the polyphenol yield; conversely, the steeper the surface, the greater the effect. Figure 6 The surfaces of a, d, and e are relatively steep, indicating that the interaction between ultrasonic temperature and ultrasonic time, ultrasonic temperature and liquid-to-material ratio, and ultrasonic temperature and ethanol concentration have a significant impact on the yield of matsutake polyphenols. Figure 6 The relatively gentle curves in b and c indicate that the interaction between ultrasonic time, material-liquid ratio, and ethanol concentration has a relatively small impact on polyphenol yield.

[0059] Based on the obtained model, the optimal process conditions were predicted as follows: ultrasonic time 62.479 min, ultrasonic temperature 47.307℃, solid-liquid ratio 1:42.611 (g / mL), and ethanol concentration 41.779%. Under these conditions, the theoretical yield of matsutake polyphenols could reach 4.163 mg / g. Considering the feasibility of actual operation, the conditions were adjusted to an extraction temperature of 50℃, ultrasonic time 60 min, solid-liquid ratio 1:40 (g / mL), and ethanol concentration 40%. The experiment was repeated three times under these conditions, and the yield of matsutake polyphenols was calculated. The average value was 4.108 mg / g, with a relative error of 1.32%, which is close to the model prediction result, indicating that the model's optimization of the matsutake polyphenol extraction process is reasonable.

[0060] Example 3: Antioxidant Activity Experiment of Matsutake Polyphenols This embodiment tests the antioxidant activity of the extracted matsutake polyphenols, specifically including DPPH free radical scavenging experiments, hydroxyl free radical scavenging experiments, and ABTS+ free radical scavenging experiments.

[0061] (1) Experiment on the DPPH free radical scavenging effect of matsutake polyphenols: Take 2 mL of sample solutions of different concentrations and 2 mL of 0.25 mmol / L DPPH stock solution respectively and add them to the same test tube. Shake well and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm as Ai. The control is the extract of different concentrations and 2 mL of anhydrous ethanol solution, and the absorbance is measured as Aj. Mix the same volume of DPPH solution and water, and the absorbance is measured as A0. Use 2 mL of VC solution of the same concentration as a positive control. Calculate the clearance rate according to formula (2): Phenolic compounds, as important antioxidants in fruits and vegetables, are often used to evaluate the antioxidant properties of natural plants or compounds. DPPH free radicals, stable nitrogen-centered free radicals, exhibit a deep purple color in ethanol solution with a strong absorption peak at 517 nm. The presence of antioxidants in the solution can eliminate DPPH free radicals, causing the solution color to change from purple to yellow, while simultaneously reducing absorbance.

[0062] Depend on Figure 7 It can be seen that within the measured concentration range (2-10 mg / mL), the DPPH free radical scavenging ability of matsutake polyphenol solution increases with increasing concentration. C The DPPH free radical scavenging rate of the solution remained at around 80%, V C The solution exhibits good scavenging ability. With increasing concentration, the DPPH free radical scavenging ability of the matsutake polyphenol extract solution gradually approaches V. CThe scavenging rate of matsutake polyphenol extract solution reached its maximum at 10 mg / mL, which was 79.40 ± 0.66%. 50 The value was 3.92 mg / mL.

[0063] (2) Experiment on the scavenging of hydroxyl radicals by matsutake polyphenols: This experiment uses a slightly modified version of the method described in the reference to determine the ability of a sample to scavenge ·OH using the salicylic acid colorimetric method. The specific steps are as follows: Take 2 mL of sample solutions of different concentrations and add them to stoppered test tubes. Add 2 mL of 6 mmol / L ferrous sulfate solution and 2 mL of 6 mmol / L hydrogen peroxide solution sequentially. Shake well and let stand for 10 min. Then add 2 mL of 6 mmol / L salicylic acid solution and react in the dark for 30 min. Measure the absorbance at 510 nm. i Replace the sample with distilled water as a blank and measure its absorbance as A0; replace the salicylic acid solution with distilled water and measure its absorbance as A. j Test V using the same method. C The absorbance of the solution was used as a positive control. The clearance rate was calculated according to formula (3): The salicylic acid colorimetric method uses H2O2 and Fe... 2+ The Fenton reaction initiated by this reaction generates ·OH and exhibits a corresponding color, with a characteristic absorption wavelength at 510 nm. When a hydroxyl radical scavenger is introduced into the system, it competes with salicylic acid for the binding of ·OH, thereby causing a decrease in absorbance.

[0064] like Figure 8 As shown, V C Both matsutake polyphenols and pine mushroom polyphenols have the ability to scavenge hydroxyl radicals, among which V C The scavenging ability of V was significantly higher than that of matsutake polyphenols. Within the measured concentration range (6-36 mg / mL), V C The scavenging rate of ·OH free radicals remained above 99%. The scavenging rate of hydroxyl radicals by the matsutake polyphenol extract solution increased with increasing concentration; at a concentration of 36 mg / mL, the scavenging rate reached 97.33 ± 0.61%, IC50... 50 The value was 4.89 mg / mL.

[0065] (3) Experiment on the scavenging effect of matsutake polyphenols on ABTS+ free radicals: Take 0.1 mL of samples of different concentrations into test tubes, add 3.9 mL of ABTS+ solution (7.4 mmol / L ABTS+ stock solution, diluted with 1 mL of 1 mL of anhydrous ethanol after standing at low temperature for 12-16 h), shake well, and react at room temperature in the dark for 10 min. Measure the absorbance at 734 nm and record it as A. i The absorbance was measured using 3.9 mL of anhydrous ethanol instead of 3.9 mL of ABTS+ solution, and denoted as A. j A blank control was prepared by replacing 0.1 mL of sample solution with 0.1 mL of distilled water, denoted as A0. V C The solution served as a positive control. Each group was measured in triplicate, and the average value was used to calculate the clearance rate using formula (4): Depend on Figure 9 It can be seen that V C The extract exhibits strong scavenging ability against ABTS+ free radicals. The scavenging ability of matsutake polyphenol extract against ABTS+ free radicals increases with increasing concentration. When the concentration of matsutake polyphenols reaches 14 mg / mL, the scavenging rate of matsutake polyphenol extract against ABTS+ free radicals reaches 98.75±1.23%, IC50. 50 The value is 3.78 mg / mL. At the same concentration, V C The solution scavenged ABTS+ free radicals at a rate of 99.47%, and the scavenging effects of the two solutions were comparable.

[0066] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for extracting polyphenols from matsutake mushrooms, characterized in that, Matsutake mushroom raw material was extracted at least once using an ethanol solution under heating and ultrasonic conditions to obtain matsutake mushroom polyphenol extract; wherein the extraction temperature was 45℃-55℃, the ratio of matsutake mushroom raw material to ethanol was 1:38-42, and the ultrasonic conditions were used for 55-65 minutes.

2. The method for extracting matsutake polyphenols according to claim 1, characterized in that, The ethanol solution has a mass percentage of 35%-45%.

3. The method for extracting matsutake polyphenols according to claim 2, characterized in that, In the ultrasonic conditions, the ultrasonic frequency is 35-45Hz.

4. The method for extracting matsutake polyphenols according to claim 3, characterized in that, The extraction temperature is 50℃, the ultrasonic time is 60 min, the material-to-liquid ratio is 1:40, the mass percentage of the ethanol solution is 40%, and the ultrasonic frequency is 40 Hz.

5. A method for extracting matsutake polyphenols according to any one of claims 1-4, characterized in that, The matsutake mushroom raw material is freeze-dried matsutake mushroom powder.

6. A method for extracting matsutake polyphenols according to any one of claims 1-4, characterized in that, Each extraction includes the following steps: S1. Mix the matsutake mushroom raw material or the filter residue obtained from the previous extraction with the ethanol solution to obtain a mixture; S2. Extract the mixture under heating and ultrasonic conditions; S3. After cooling the extracted mixture, centrifuge and filter it to obtain filter residue and supernatant; S4. Concentrate the supernatant to obtain the matsutake polyphenol extract.

7. The method for extracting matsutake polyphenols according to claim 6, characterized in that, In step S3, the centrifugation speed is 3500-4500 r / min, and the time is 5-15 min.

8. The method for extracting matsutake polyphenols according to claim 6, characterized in that, In step S3, the temperature of the cooled mixture is 20℃-25℃.

9. The method for extracting matsutake polyphenols according to claim 6, characterized in that, In step S3, the filtration method is vacuum filtration, and in step S4, the concentration method is rotary evaporation.

10. A matsutake mushroom polyphenol preparation, characterized in that, The matsutake polyphenols in the preparation are extracted using the method described in any one of claims 1-9.

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