Method for extracting perigord truffle polysaccharide under assistance of compound enzyme and application of perigord truffle polysaccharide

Abstract

The invention relates to the technical field of natural plant extraction, in particular to a method for extracting perigord truffle polysaccharide with the assistance of a compound enzyme, neutral protease and chitinase are compounded, enzymolysis is carried out according to the characteristics of perigord truffle cell walls, action targets are clear, the cell wall degradation efficiency is high, extraction conditions are optimized, and the perigord truffle polysaccharide is obtained. The damage of high temperature to the polysaccharide structure is avoided, the natural conformation of the polysaccharide is better maintained, the yield of the polysaccharide is remarkably improved, and the biological activity of the polysaccharide is better maintained; the perigord truffle polysaccharide prepared by the invention has excellent in-vitro antioxidant activity, and lays a technical foundation for developing perigord truffle polysaccharide functional food raw materials with high added value.

Description

Technical Field

[0001] This invention relates to the field of natural plant extraction technology, specifically to a method and application of compound enzyme-assisted extraction of black truffle polysaccharides. Background Technology

[0002] Black truffle ( Tuber indicum Black truffle is a precious edible and medicinal fungus, rich in polysaccharides, proteins, amino acids, and other bioactive components. Modern research shows that black truffle polysaccharides are one of its key active ingredients, possessing various physiological functions such as antioxidation, immune regulation, antitumor activity, and blood sugar and lipid reduction, and have broad application prospects in the fields of functional foods and medicine.

[0003] The extraction method of polysaccharides directly affects their extraction rate and bioactivity. Currently, the main extraction methods for fungal polysaccharides include hot water extraction, ultrasound-assisted extraction, microwave-assisted extraction, and enzyme-assisted extraction. Hot water extraction is simple to operate, but it suffers from low extraction efficiency, long extraction time, and the risk of polysaccharide degradation and decreased activity due to high temperatures. While ultrasound and microwave-assisted methods can improve efficiency, they are costly and may adversely affect polysaccharide activity due to localized overheating. In contrast, enzyme-assisted extraction, through specific enzymatic hydrolysis of cell wall components, promotes the release of intracellular polysaccharides, offering advantages such as mild conditions, high efficiency, and better preservation of the polysaccharide's natural conformation and bioactivity.

[0004] Black truffles possess a unique cell wall structure, primarily composed of chitin and β-glucan as its backbone, interspersed with numerous structural proteins and extremely low in cellulose. This structural characteristic determines the varying extraction efficiencies of different hydrolytic enzymes. Current research on enzyme-assisted extraction of black truffle polysaccharides lacks systematic screening of different hydrolytic enzymes and in-depth comparison of compounding schemes. In particular, it fails to fully utilize the characteristics of its cell wall structure to select the optimal enzyme types and their compounding methods, resulting in extraction efficiency and product activity requiring further improvement. Therefore, optimizing the extraction process to efficiently extract black truffle polysaccharides while maximizing the preservation of their biological activity is of significant research importance. Summary of the Invention

[0005] To overcome the shortcomings of the above-mentioned technical defects, the present invention provides a method and application for extracting black truffle polysaccharides with the assistance of a compound enzyme, which can extract black truffle polysaccharides efficiently and gently, and can maintain their antioxidant activity to the maximum extent.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

[0007] On one hand, the present invention provides a method for extracting polysaccharides from black truffles using a compound enzyme-assisted extraction process, comprising the following steps: S1. Mix black truffle powder with water, then add a compound enzyme for enzymatic hydrolysis to obtain a black truffle polysaccharide solution, wherein the compound enzyme is composed of neutral protease and chitinase; S2. After inactivating the enzymes in the black truffle polysaccharide solution at high temperature, centrifuge and collect the supernatant; S3. Add ethanol to the supernatant to precipitate, centrifuge to collect the precipitate, and dry to obtain crude extract of black truffle polysaccharide.

[0008] Further, in S1, the mass-to-volume ratio of the black truffle to water is 1g:(25-55)mL, or 1g:(35-55)mL, or 1g:(45-55)mL, or 1g:(50-55)mL, or 1g:(52-55)mL, or 1g:53mL.

[0009] Furthermore, the amount of the compound enzyme added is 1-2.5%, or 1.5%-2.3%, or 1.5%-2%, or 1.7%-1.9%, or 1.8% of the weight of the black truffle.

[0010] Further, the mass ratio of the neutral protease to the chitinase is (1-3):1, or (1-3):2, or (1-3):3, or 1:(1-3), or 2:(1-3), or 3:(1-3), or 1:1.

[0011] Further, in S1, the enzymatic hydrolysis time is 50-80 min, or 50-70 min, or 50-65 min, or 50-60 min, or 55-60 min, or 55-57 min, or 56 min.

[0012] Further, in S1, the enzymatic hydrolysis temperature is 45-60℃, or 48-60℃, or 50-60℃, or 50-58℃, or 54-58℃, or 54-56℃, or 55℃.

[0013] Furthermore, in S1, the pH of the enzymatic hydrolysis system is 3-7, or 4-7, or 4-6, or 5.

[0014] Further, in step S2, the enzyme inactivation conditions are: heating in a water bath at 90-100℃ for 5-10 minutes.

[0015] Further, S3 specifically involves adding 3-6 times the volume of anhydrous ethanol to the supernatant and allowing it to stand at 1-5°C for 10-20 hours; removing the reaction solution, centrifuging, discarding the supernatant, collecting the precipitate, washing the precipitate with anhydrous ethanol, and freeze-drying it to obtain crude extract of black truffle polysaccharide.

[0016] Secondly, the present invention provides a black truffle polysaccharide, which is prepared by the above-described method.

[0017] Thirdly, this invention provides the application of black truffle polysaccharide in the preparation of immunomodulatory drugs or antioxidant drugs.

[0018] Furthermore, the drug uses black truffle polysaccharide as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0019] Further, the amount of the active ingredient is 0.001-90 wt% relative to the total weight of the drug; or 0.02-50 wt%; or 0.1-20%; or 0.005-5%.

[0020] Those skilled in the art will fully understand that the pharmaceutically acceptable carrier is generally recognized for this purpose and as an inactive ingredient in the pharmaceutical preparation.

[0021] The auxiliary agents include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0022] The diluent can be one or more of mannitol, sucrose, lactose, sorbitol, xylitol, polyethylene glycol, propylene glycol, vegetable oil, and mineral oil; the disintegrant can be one or more of croscarmellose sodium, colloidal silica, and citric acid; the binder can be one or more of starch paste, ethanol, water, and povidone alcohol solution; the preservative can be one or more of ethylparaben, propylparaben, sorbic acid, potassium sorbate, calcium propionate, sodium dehydroacetate, sodium diacetate, and sodium lactate; and the antioxidant can be ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, butylated hydroxytoluene, glycine, inositol, and other antioxidants. The flavoring agent may be one or more of the following: citric acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite; the flavoring agent may be one or more of the following: aspartame, sucrose, xylitol, steviol glycosides, cyclamate, sorbitol, cocoa, pure vanilla, vanillin, ethyl vanillin, chocolate, malt, and mint; the suspending agent may be one or more of the following: xanthan gum, polyvinylpyrrolidone, sodium alginate, aluminum stearate, and hydrogenated vegetable oil; the emulsifier may be one or more of the following: alkyl sulfate, soap, dodecylbenzene sulfonate, lactate, sulfosuccinate, monoglyceride sulfonate, phosphate ester, siloxane, and taurine.

[0023] In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0024] Pharmaceutical compositions may be prepared according to methods known in the art. For this purpose, if desired, the active ingredient may be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form a suitable administration or dosage form for human use.

[0025] In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0026] Compared with existing technologies, the method for extracting black truffle polysaccharides using a compound enzyme in this invention combines neutral protease and chitinase to enzymatically hydrolyze the cell walls of black truffles, resulting in a clear target and high cell wall degradation efficiency. Furthermore, the extraction conditions are optimized to avoid damage to the polysaccharide structure caused by high temperatures, thus better preserving the natural conformation of the polysaccharides. This significantly improves the yield of polysaccharides and better maintains their biological activity, laying a technical foundation for developing high-value-added functional food ingredients from black truffle polysaccharides. Attached Figure Description

[0027] Figure 1 The effects of different single enzyme treatments on the yield of polysaccharides from black truffles were investigated. 1A compares the polysaccharide yields of single enzyme treatments, while 1B compares the polysaccharide yields of single enzyme and multi-enzyme treatments. Figure 2 The effect of different ratios of complex enzymes on the yield of black truffle polysaccharides; Figure 3 The effect of different material-to-liquid ratios on the yield of black truffle polysaccharides; Figure 4 The effect of different enzyme addition amounts on the yield of black truffle polysaccharides; Figure 5 The effect of different enzymatic hydrolysis times on the yield of black truffle polysaccharides; Figure 6 The effect of different enzymatic hydrolysis temperatures on the yield of black truffle polysaccharides; Figure 7 The effect of pH value on the yield of black truffle polysaccharides in different systems; Figure 8 This invention compares the in vitro antioxidant activity of black truffle polysaccharides extracted by hot water method; wherein, 8A is a comparison of hydroxyl radical scavenging ability; 8B is a comparison of DPPH radical scavenging ability; and 8C is a comparison of ABTS radical scavenging ability. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0029] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0030] This invention aims to provide a method for extracting black truffle polysaccharides using a compound enzyme-assisted extraction process. By systematically screening different hydrolytic enzymes and precisely optimizing the enzymatic hydrolysis conditions, the optimal conditions are selected to prepare black truffle polysaccharides with the best in vitro antioxidant activity.

[0031] Based on the present invention, black truffle polysaccharide is prepared by the following steps: S1. Mix black truffle powder with water, then add a complex enzyme and perform enzymatic hydrolysis to obtain an enzymatic hydrolysate, wherein the complex enzyme is composed of neutral protease and chitinase. In some embodiments of this implementation, the mass-volume ratio of black truffles to water is 1:(35-55). In actual operation, it can be selected as 1:35, 1:40, 1:45, 1:50, 1:55. Other specific values ​​within this range can be selected, and will not be described in detail here. In some embodiments of this implementation, the amount of the compound enzyme added is 0.5%-2.5% of the mass of the black truffle. In actual operation, it can be selected as 0.5%, 1.0%, 1.5%, 2.0%, 2.5%. Other specific values ​​within this range can also be selected, and will not be described in detail here. In some embodiments of this implementation, the mass ratio of the neutral protease to the chitinase is (1-3):(1-3). In actual operation, it can be selected as 3:1, 2:1, 1:1, 1:2, 1:3. Other specific values ​​within this range can be selected, and will not be described in detail here. In some embodiments of this implementation, the enzymatic hydrolysis time is 50-90 min. In actual operation, it can be selected as 50 min, 60 min, 70 min, 80 min, or 90 min. Other specific values ​​within this range can also be selected, and will not be described in detail here. In some embodiments of this implementation, the enzymatic hydrolysis temperature is 40-60 ℃. In actual operation, it can be selected as 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃. Other specific values ​​within this range can also be selected, and will not be described in detail here. In some embodiments of this implementation, the enzymatic hydrolysis pH value is 3-7. In actual operation, it can be selected as 3, 4, 5, 6, or 7. Other specific values ​​within this range can also be selected, and will not be described in detail here. S2. After inactivating the enzymes in the black truffle polysaccharide solution, centrifuge and collect the supernatant; In some embodiments of this implementation, the enzyme hydrolysate is heated in a water bath at 90-100°C for 5-10 min to inactivate the enzyme, and then centrifuged at 4000 r / min for 20 min to collect the supernatant; in some preferred embodiments, the enzyme inactivation treatment conditions are heating in a water bath at 90-95°C for 5-8 min. S3. Add ethanol to the supernatant to precipitate, centrifuge to collect the precipitate, dry it to obtain crude extract of black truffle polysaccharide; In some embodiments of this implementation, 3-6 times the volume of anhydrous ethanol is added to the supernatant, and the mixture is allowed to stand at 1-5°C for 10-20 hours. The reaction solution is then removed, centrifuged, the supernatant is discarded, and the precipitate is collected. The precipitate is washed with anhydrous ethanol and then freeze-dried under vacuum to obtain crude extract of black truffle polysaccharide. In some preferred embodiments, the ethanol precipitation conditions are as follows: 4 times the volume of anhydrous ethanol is added to the supernatant, and the mixture is allowed to stand at 4°C for at least 12 hours.

[0032] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0033] Materials and reagents: Black truffles ( Tuber indicumThe dried mushrooms were purchased from the Kunming Wild Mushroom Market in Yunnan Province; neutral protease (50,000 U / g) and acidic protease (50,000 U / g) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; cellulase (10,000 U / g) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; chitinase ≥1000 U / g. The following reagents were obtained from preliminary screening in the laboratory: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), salicylic acid, potassium ferricyanide, ferric chloride, and ascorbic acid (Vc) were purchased from Sigma-Aldrich, USA; glucose standard (purity ≥99.0%) was purchased from the National Institutes for Food and Drug Control, China; total antioxidant capacity (T-AOC) test kit (ABTS method, catalog number A015-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute; hydroxyl radical scavenging capacity test kit (catalog number HH3402) and DPPH radical scavenging capacity test kit (catalog number HH3415) were purchased from Beijing Box Biotechnology Co., Ltd.; anhydrous ethanol was of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; and ultrapure water was used for the experiment.

[0034] Example 1: Screening of hydrolytic enzymes Weigh 1.0 g of black truffle powder (80 mesh) and mix with 30 mL of water. Then add hydrolytic enzyme and perform enzymatic hydrolysis at pH 5.0, 50 ℃, and 70 min to obtain the enzymatic hydrolysate. Heat the enzymatic hydrolysate in a 90 ℃ water bath for 5-8 min to inactivate the enzyme. Then centrifuge at 4000 r / min for 20 min and collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant and let it stand at 4 ℃ for 14 h. Take out the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain the crude extract of black truffle polysaccharide.

[0035] Example 1.1 The method of Example 1 was used, wherein the hydrolytic enzyme was a neutral protease, and the amount of enzyme added was 1% of the mass of the black truffle.

[0036] Example 1.2 The method of Example 1 was used, wherein the hydrolytic enzyme was chitinase, and the amount of enzyme added was 1% of the mass of the black truffle.

[0037] Example 1.3 The method of Example 1 was used, wherein the hydrolytic enzyme was an acidic protease, and the amount of enzyme added was 1% of the mass of the black truffle.

[0038] Example 1.4 The method of Example 1 was used, wherein the hydrolytic enzyme was cellulase, and the amount of enzyme added was 1% of the mass of the black truffle.

[0039] Example 1.5 The method of Example 1 was adopted, wherein the hydrolytic enzyme was a neutral protease and a chitinase in a mass ratio of 1:1, and the amount of enzyme added was 1% of the mass of the black truffle.

[0040] The content of the crude polysaccharide extracts obtained in Examples 1.1-1.5 above was determined. Figure 1 As shown in Figure A, compared with the control group without enzymes, the neutral protease treatment group showed the best extraction effect, with a polysaccharide extraction rate of 13.55%. The chitinase treatment group also showed good extraction effect, with a polysaccharide extraction rate of 10.60%, while the extraction rates of the acidic protease and cellulase treatment groups were lower than those of the control group without enzymes (9.45%). Figure 1 As shown in B, with a total enzyme addition of 1%, the polysaccharide extraction rate reached 16.40% when neutral protease and chitinase were combined at a mass ratio of 1:1, which was significantly higher than any single hydrolase treatment group. This indicates that the combination of the two may have a synergistic effect, and therefore it was selected as the enzyme system for subsequent process optimization.

[0041] Example 2: Investigating the effects of different complex enzymes Weigh 1.0g of black truffle powder (80 mesh) and mix with 30mL of water. Then add a compound enzyme at 1% of the black truffle mass. Perform enzymatic hydrolysis at pH 5.0, 50℃, and 70 min to obtain the hydrolysate. Heat the hydrolysate in a 90℃ water bath for 5-8 min to inactivate the enzyme. Then centrifuge at 4000 r / min for 20 min and collect the supernatant. Add 4 volumes of anhydrous ethanol to the supernatant and let it stand at 4℃ for 14 h. Remove the reaction solution, centrifuge, discard the supernatant, and collect the precipitate. Soak the precipitate in anhydrous ethanol. Wash and freeze-dry under vacuum to obtain crude extract of black truffle polysaccharide.

[0042] Example 2.1 The method of Example 2 was adopted, wherein the complex enzyme was a chitinase: neutral protease in a mass ratio of 1:3.

[0043] Example 2.2 The method of Example 2 was adopted, wherein the complex enzyme was a chitinase: neutral protease in a mass ratio of 1:2.

[0044] Example 2.3 The method of Example 2 was adopted, wherein the complex enzyme was a chitinase: neutral protease in a mass ratio of 1:1.

[0045] Example 2.4 The method of Example 2 was adopted, wherein the complex enzyme was a chitinase: neutral protease in a mass ratio of 2:1.

[0046] Example 2.5 The method of Example 2 was adopted, wherein the complex enzyme was a chitinase: neutral protease in a mass ratio of 3:1.

[0047] The content of the crude polysaccharide extracts obtained in Examples 2.1-2.5 above was determined. Figure 2 As can be seen, the polysaccharide extraction rate significantly increased when the enzyme ratio was between 1:3 and 1:1, reaching its maximum at an enzyme ratio of 1:1. At an enzyme ratio of 3:1, the polysaccharide extraction rate decreased slightly. This indicates that the ratio of chitinase to neutral protease exhibited the best synergistic effect within the range of 1:1 to 2:1, resulting in the most stable and higher extraction rate. This suggests that the synergistic effect between the two is balanced, with no primary or secondary inhibition.

[0048] Example 3: Investigating the effect of different feed-liquid ratios Weigh 1.0g of black truffle powder (80 mesh) and mix it with water. Then add a compound enzyme (chitinase: neutral protease in a mass ratio of 1:1), with the enzyme addition amount being 1% of the mass of the black truffle. Perform enzymatic hydrolysis at pH 5.0, a hydrolysis temperature of 50 ℃, and a hydrolysis time of 70 min to obtain the enzymatic hydrolysate. Place the hydrolysate in a 95 ℃ water bath and heat for 15 min to inactivate the enzyme. Then centrifuge at 4000 r / min for 20 min and collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant, let it stand at 4 ℃ for 14 h, take out the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain the crude extract of black truffle polysaccharides.

[0049] Example 3.1 The method of Example 3 was used, wherein the mass-to-volume ratio of black truffle to water was 1g:15mL.

[0050] Example 3.2 The method of Example 3 was used, wherein the mass-to-volume ratio of black truffle to water was 1g:25mL.

[0051] Example 3.3 The method of Example 3 was used, wherein the mass-to-volume ratio of black truffle to water was 1g:35mL.

[0052] Example 3.4 The method of Example 3 was used, wherein the mass-to-volume ratio of black truffle to water was 1g:45mL.

[0053] Example 3.5 The method of Example 3 was used, wherein the mass-to-volume ratio of black truffle to water was 1g:55mL.

[0054] The content of the crude polysaccharide extracts obtained in Examples 3.1-3.5 above was determined. Figure 3As can be seen, when the material-to-liquid ratio increases from 1:15 to 1:45, the polysaccharide extraction rate shows a continuous upward trend and reaches its maximum value; however, when the material-to-liquid ratio is further increased to 1:55, the yield decreases instead. This indicates that at a material-to-liquid ratio of 1:45, the compound enzyme can produce a more comprehensive degradation effect on the cell wall of black truffles, thereby improving the extraction efficiency of the target substance. However, an excessively high material-to-liquid ratio may lead to excessive degradation of polysaccharide components, which in turn reduces the extraction rate.

[0055] Example 4: Investigating the effect of different enzyme dosages Weigh 1.0g of black truffle powder (80 mesh) and mix with 45mL of water. Then add a compound enzyme (chitinase: neutral protease in a mass ratio of 1:1) and perform enzymatic hydrolysis at pH 5.0, 50℃, and 70 min to obtain the hydrolysate. Heat the hydrolysate in a 90℃ water bath for 5-8 min to inactivate the enzyme. Then centrifuge at 4000 r / min for 20 min and collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant and let it stand at 4℃ for 14 h. Take out the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain the crude extract of black truffle polysaccharide.

[0056] Example 4.1 The method of Example 4 was used, wherein the amount of enzyme added was 0.5% of the mass of the black truffle.

[0057] Example 4.2 The method of Example 4 was used, wherein the amount of enzyme added was 1.0% of the mass of the black truffle.

[0058] Example 4.3 The method of Example 4 was used, wherein the amount of enzyme added was 1.5% of the mass of the black truffle.

[0059] Example 4.4 The method of Example 4 was used, wherein the amount of enzyme added was 2% of the mass of the black truffle.

[0060] Example 4.5 The method of Example 4 was used, wherein the amount of enzyme added was 2.5% of the mass of the black truffle.

[0061] The content of the crude polysaccharide extracts obtained in Examples 4.1-4.5 above was determined. Figure 4 As can be seen, the enzyme addition amount is positively correlated with the extraction rate in the range of 0.5%–1.5%, and reaches a critical value at 1.5%. The enzyme addition amount is negatively correlated with the extraction rate in the range of 1.5%–2.5%, indicating that excessive enzyme addition may lead to excessive degradation of polysaccharide components, which in turn reduces the extraction rate.

[0062] Example 5: Investigating the effect of different enzymatic hydrolysis times Weigh 1.0g of black truffle powder (80 mesh) and mix with 45mL of water. Then add a compound enzyme (chitinase: neutral protease in a mass ratio of 1:1), with the enzyme addition amount being 1.5% of the black truffle mass. Perform enzymatic hydrolysis at pH 5.0 and a hydrolysis temperature of 50℃ to obtain an enzymatic hydrolysate. Place the hydrolysate in a 90℃ water bath and heat for 5-8 min to inactivate the enzyme. Then centrifuge at 4000 r / min for 20 min and collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant, let stand at 4℃ for 14 h, remove the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain crude black truffle polysaccharide extract.

[0063] Example 5.1 The method of Example 5 was used, wherein the enzymatic hydrolysis time was 50 min.

[0064] Example 5.2 The method of Example 5 was used, wherein the enzymatic hydrolysis time was 60 min.

[0065] Example 5.3 The method of Example 5 was used, wherein the enzymatic hydrolysis time was 70 min.

[0066] Example 5.4 The method of Example 5 was used, wherein the enzymatic hydrolysis time was 80 min.

[0067] Example 5.5 The method of Example 5 was used, wherein the enzymatic hydrolysis time was 90 min.

[0068] The content of the crude polysaccharide extracts obtained in Examples 5.1-5.5 above was determined. Figure 5 As can be seen, the polysaccharide extraction rate increased significantly when the enzymatic hydrolysis time was extended from 50 min to 70 min, reaching its maximum at 70 min. The polysaccharide extraction rate dropped when the enzymatic hydrolysis time was 90 min. This indicates that an enzymatic hydrolysis time of 50-70 min is conducive to polysaccharide dissolution, while an excessively long enzymatic hydrolysis time may lead to the degradation of the dissolved polysaccharides, resulting in a decrease in the polysaccharide extraction rate.

[0069] Example 6 investigates the effect of different enzymatic hydrolysis temperatures Weigh 1.0g of black truffle powder (80 mesh) and mix with 45mL of water. Then add a compound enzyme (chitinase: neutral protease in a mass ratio of 1:1), with the enzyme addition amount being 1.5% of the mass of black truffle. Enzymatic hydrolysis is carried out at pH 5.0 for 70min to obtain the enzymatic hydrolysate. The enzymatic hydrolysate is heated in a 90℃ water bath for 5-8min to inactivate the enzyme. Then, it is centrifuged at 4000 r / min for 20min, and the supernatant is collected. Four volumes of anhydrous ethanol are added to the supernatant, and the mixture is allowed to stand at 4℃ for 14h. The reaction solution is then removed, centrifuged, and the supernatant is discarded. The precipitate is collected, washed with anhydrous ethanol, and then freeze-dried under vacuum to obtain the crude extract of black truffle polysaccharides.

[0070] Example 6.1 The method of Example 6 was used, wherein the enzymatic hydrolysis temperature was 40 °C.

[0071] Example 6.2 The method of Example 6 was used, wherein the enzymatic hydrolysis temperature was 45°C.

[0072] Example 6.3 The method of Example 6 was used, wherein the enzymatic hydrolysis temperature was 50 °C.

[0073] Example 6.4 The method of Example 6 was used, wherein the enzymatic hydrolysis temperature was 55 °C.

[0074] Example 6.5 The method of Example 6 was used, wherein the enzymatic hydrolysis temperature was 60 °C.

[0075] The content of the crude polysaccharide extracts obtained in Examples 6.1-6.5 above was determined. Figure 6 As can be seen, the polysaccharide extraction rate increased significantly when the enzymatic hydrolysis time was extended from 40 ℃ to 55 ℃, reaching its maximum value at 55 ℃. Further increasing the enzymatic hydrolysis temperature caused the polysaccharide extraction rate to drop, indicating that the enzymatic hydrolysis temperature within the range of 40-55 ℃ is conducive to polysaccharide dissolution, while excessively high enzymatic hydrolysis temperatures may lead to enzyme denaturation and inactivation, resulting in a decrease in the polysaccharide extraction rate.

[0076] Example 7: Investigating the effect of different pH values Weigh 1.0g of black truffle powder (80 mesh) and mix with 45mL of water. Then add a compound enzyme (chitinase: neutral protease in a mass ratio of 1:1), with the enzyme addition amount being 1.5% of the mass of black truffle. Adjust the pH of the system, and carry out enzymatic hydrolysis at 55℃ for 70min to obtain the enzymatic hydrolysate. Heat the enzymatic hydrolysate in a 95℃ water bath for 15min to inactivate the enzyme, and then centrifuge at 4000 r / min for 20min. Collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant, let it stand at 4℃ for 14h, take out the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain the crude extract of black truffle polysaccharide.

[0077] Example 7.1 The method of Example 7 was used, wherein the pH of the enzymatic hydrolysis system was 3.

[0078] Example 7.2 The method of Example 7 was used, wherein the pH of the enzymatic hydrolysis system was 4.

[0079] Example 7.3 The method of Example 7 was used, wherein the pH of the enzymatic hydrolysis system was 5.

[0080] Example 7.4 The method of Example 7 was used, wherein the pH of the enzymatic hydrolysis system was 6.

[0081] Example 7.5 The method of Example 7 was used, wherein the pH of the enzymatic hydrolysis system was 7.

[0082] Example 7.6 The method of Example 7 was used, wherein the pH of the enzymatic hydrolysis system was 8.

[0083] The content of the crude polysaccharide extracts obtained in Examples 7.1-7.6 above was determined. Figure 7 As can be seen, the system pH significantly affects enzyme activity. When the system pH is between 3 and 5, the polysaccharide extraction rate is positively correlated with the pH value, reaching its maximum value at a certain pH. However, as the enzymatic hydrolysis system gradually changes from acidic to alkaline conditions, the polysaccharide extraction rate will decrease.

[0084] Example 8 Weigh 1.0 g of black truffle powder (80 mesh) and mix with 53 mL of water. Then add a compound enzyme (chitinase: neutral protease in a 1:1 mass ratio), with the enzyme addition amount being 1.8% of the black truffle mass. Perform enzymatic hydrolysis at pH 5.0, a hydrolysis temperature of 55 ℃, and a hydrolysis time of 56 min to obtain the enzymatic hydrolysate. Place the hydrolysate in a 90 ℃ water bath and heat for 5-8 min to inactivate the enzyme. Then centrifuge at 4000 r / min for 20 min and collect the supernatant. Add 4 times the volume of anhydrous ethanol to the supernatant, let stand at 4 ℃ for 14 h, remove the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain the crude extract of black truffle polysaccharides.

[0085] Comparative Example 1 The preparation conditions were the same as in Example 8, except that 1.8% neutral protease was used in the enzymatic hydrolysis process, and chitinase was not added.

[0086] Comparative Example 2 The preparation conditions were the same as in Example 8, except that 1.8% chitinase was used in the enzymatic hydrolysis process, and no neutral protease was added.

[0087] Comparative Example 3 Accurately weigh 1.0 g of black truffle powder (80 mesh), add distilled water at a liquid-to-solid ratio of 53:1 mL / g, and extract in a 90 ℃ water bath for 2 h; then centrifuge at 4000 r / min for 20 min and collect the supernatant; add 4 times the volume of anhydrous ethanol to the supernatant, let stand at 4 ℃ for 14 h, take out the reaction solution, centrifuge, discard the supernatant, collect the precipitate, wash the precipitate with anhydrous ethanol, and freeze-dry under vacuum to obtain crude extract of black truffle polysaccharide.

[0088] Test case (1) The content of the crude polysaccharide extracts obtained in Example 8 and Comparative Examples 1-3 was determined, and the results are shown in the table. As shown in Figure 1.

[0089] Table 1

[0090] As can be seen, the polysaccharide extraction rate of Example 8 was (20.03±0.21)%, which was significantly higher than that of Comparative Examples 1-3, with an increase of 26-52%. This indicates that the present invention has a higher polysaccharide extraction efficiency than the traditional hot water extraction method. Furthermore, the composite enzyme system composed of neutral protease and chitinase can better enhance the cell wall structure characteristics and synergistically and efficiently assist in the hydrolysis of black truffles, significantly improving the polysaccharide yield.

[0091] (2) The in vitro antioxidant activity of the black truffle polysaccharide samples extracted in Example 8 and Comparative Example 3 was determined: Accurately weigh black truffle polysaccharide samples and prepare solutions of 0.5, 1.0, 2.0, 4.0, and 8.0 mg / mL with distilled water to determine their antioxidant activity. Ascorbic acid (Vc) was used as a positive control. The in vitro antioxidant activity of black truffle polysaccharides was determined using standard kit methods, specifically including hydroxyl radical scavenging ability (detection wavelength 536 nm), DPPH radical scavenging ability (detection wavelength 515 nm), and total antioxidant capacity (T-AOC, ABTS method, detection wavelength 414 nm). All measurements were performed using a full-wavelength microplate reader. Radical scavenging ability was expressed as scavenging rate (%), and total antioxidant capacity (ABTS method) was expressed as Trolox equivalent (μmol Trolox / mL). Each experiment had three replicates, and the procedures were strictly followed according to the instructions of each kit. The results are as follows: Figure 8 As shown.

[0092] All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Design-Expert 12 and SPSS 26.0 software were used for experimental design, analysis of variance (ANOVA), and significance testing (P < 0.05 indicates significant difference).

[0093] Figure 8 As can be seen, within the concentration range of 0.5~4.0 mg / mL, the scavenging ability of polysaccharides extracted by both methods against ABTS free radicals, hydroxyl free radicals and DPPH free radicals is concentration-dependent. However, the scavenging rate of the polysaccharide extracted in Example 8 of this invention is significantly higher than that of the extract obtained in Comparative Example 3 at all concentrations. Furthermore, the polysaccharide extracted in this invention has significantly better activity in scavenging ABTS free radicals, hydroxyl free radicals and DPPH free radicals than the traditional hot water extraction method, indicating that the compound enzyme-assisted extraction process can better maintain the antioxidant activity of black truffle polysaccharides.

[0094] As can be seen, the composite enzyme system of neutral protease and chitinase used in this invention for the enzymatic hydrolysis of black truffles demonstrates that neutral protease can efficiently hydrolyze structural proteins in the cell wall without destroying polysaccharide activity, while chitinase can specifically degrade the chitin skeleton. Both act on different components of the cell wall and have a synergistic effect in degrading the cell wall. This not only efficiently assists in the hydrolysis of black truffles and significantly increases the polysaccharide yield, but also results in the extracted polysaccharides exhibiting superior in vitro antioxidant activity. This indicates that the extraction process of this invention achieves efficient extraction while better preserving the biological activity of the polysaccharides, laying a technical foundation for developing high-value-added functional food ingredients from black truffle polysaccharides.

[0095] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. A method for extracting polysaccharides from black truffles using a compound enzyme-assisted extraction process, characterized in that, Includes the following steps: S1. Mix black truffle powder with water, then add a compound enzyme to perform enzyme-assisted extraction of polysaccharides. After enzymatic hydrolysis, a black truffle polysaccharide solution is obtained. The compound enzyme consists of neutral protease and chitinase. S2. After inactivating the enzymes in the black truffle polysaccharide solution, centrifuge and collect the supernatant; S3. Add ethanol to the supernatant to precipitate, centrifuge to collect the precipitate, and dry to obtain crude extract of black truffle polysaccharide.

2. The method for extracting black truffle polysaccharides with the aid of a compound enzyme according to claim 1, characterized in that, In S1, the mass-to-volume ratio of the black truffle to water is 1 g: (15-55) mL; and / or, The amount of the compound enzyme added is 0.5%-2.5% of the mass of the black truffle; and / or, The mass ratio of the neutral protease to the chitinase is (1-3):(1-3); and / or, The enzymatic hydrolysis time is 50-90 min; and / or, The enzymatic hydrolysis temperature is 40-60℃; and / or, The enzymatic hydrolysis is performed at pH 3-7.

3. The method for extracting black truffle polysaccharides with the aid of a compound enzyme according to claim 2, characterized in that, In S1, the mass-to-volume ratio of the black truffle to water is 1g:(25-55)mL, or 1g:(35-55)mL, or 1g:(45-55)mL, or 1g:(50-55)mL, or 1g:(52-55)mL, or 1g:53mL.

4. The method for extracting black truffle polysaccharides with the aid of a compound enzyme according to claim 1, characterized in that, In step S1, the amount of the compound enzyme added is 1-2.5%, 1.5%-2.3%, 1.5%-2%, 1.7%-1.9%, or 1.8% of the mass of the black truffle; and, The mass ratio of the neutral protease to the chitinase is (1-3):1, or (1-3):2, or (1-3):3, or 1:(1-3), or 2:(1-3), or 3:(1-3), or 1:

1.

5. The method for extracting black truffle polysaccharides with the aid of a compound enzyme according to claim 1, characterized in that, In S1, the enzymatic hydrolysis time is 50-80 min, or 50-70 min, or 50-65 min, or 50-60 min, or 55-60 min, or 55-57 min, or 56 min.

6. The method for extracting black truffle polysaccharides with the aid of a compound enzyme according to claim 1, characterized in that, In S1, the enzymatic hydrolysis temperature is 45-60℃, or 48-60℃, or 50-60℃, or 50-58℃, or 54-58℃, or 54-56℃, or 55℃.

7. The method for extracting black truffle polysaccharides using a compound enzyme according to any one of claims 1-6, characterized in that, In step S2, the enzyme inactivation conditions are: heating in a water bath at 90-100℃ for 5-10 minutes.

8. The method for extracting black truffle polysaccharides with the aid of a compound enzyme according to claim 7, characterized in that, S3 specifically involves adding 3-6 times the volume of anhydrous ethanol to the supernatant and allowing it to stand at 1-5°C for 10-20 hours; removing the reaction solution, centrifuging, discarding the supernatant, collecting the precipitate, washing the precipitate with anhydrous ethanol, and freeze-drying it to obtain crude extract of black truffle polysaccharide.

9. A black truffle polysaccharide, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. The application of the black truffle polysaccharide according to claim 9 in the preparation of immunomodulatory drugs or antioxidant drugs.

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