A method for preparing xanthan oligosaccharide by ion liquid assisted enzymatic hydrolysis

Abstract

The application discloses a method for preparing xanthan oligosaccharide by ion liquid assisted enzymatic hydrolysis, and belongs to the technical field of biology. The method comprises the following steps: (1) preparing a xanthan gum solution with a final concentration of 0.1 mg / mL-10 mg / mL; (2) adding commercial cellulase into the xanthan gum solution obtained in the step (1) and stirring until the xanthan gum is dissolved; (3) adding ion liquid into the xanthan gum solution obtained in the step (2) and stirring until the ion liquid is fully dissolved; (4) performing enzymatic hydrolysis reaction, and after the reaction is completed, the solution is filtered through a membrane and is freeze-dried to obtain xanthan oligosaccharide. The method solves the dependence of traditional biological methods on microbial fermentation technology and enzyme purification technology, and high-activity xanthan oligosaccharide is prepared, which lays a foundation for industrialized production and application of xanthan oligosaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing xanthan oligosaccharide by ion liquid assisted enzymatic hydrolysis, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Xanthan oligosaccharide prepared by xanthan degradation is a new type of oligosaccharide, which has attracted extensive attention of researchers due to its excellent antioxidant performance and various physiological functions such as treatment of osteoarthritis and inhibition of cell apoptosis.

[0003] At present, the methods for preparing xanthan oligosaccharide include physical method, chemical method and biological method. The physical method has high requirements for equipment, high energy consumption and large molecular weight of prepared oligosaccharide with low activity. The chemical method can effectively degrade xanthan, but its strong dependence on chemical reagents limits the application of the product, and the irregular cutting action of the chemical reagents causes the mixing of oligosaccharide in the xanthan degradation product. The biological method is a relatively mild degradation method, and the degradation product is controllable, but due to the lack of effective xanthan degrading enzyme, the activity of the obtained oligosaccharide is poor. At the same time, the biological method involves microbial fermentation and enzyme purification, which requires additional fermentation equipment and support of purification technology. Therefore, the current technology cannot meet our requirements for industrial application of xanthan oligosaccharide.

[0004] Cellulase is composed of the following three enzymes, which are (1) endo-cellulase: cutting the internal beta-1, 4 glycosidic bond of cellulose to produce new chain ends and oligosaccharides with different polymerization degrees; (2) cellobiohydrolase: cutting from the reducing end (or non-reducing end) of short-chain cellulose to produce cellobiose; (3) beta-glucosidase: hydrolyzing cellobiose into glucose. As an enzyme preparation that can effectively hydrolyze cellulose to produce oligosaccharides and is relatively easy to obtain and low in price, cellulase has been widely used in the fields of textiles, detergents, food, pharmaceuticals, biofuels, etc.

[0005] Ionic liquid refers to a kind of salt that is in liquid state at room temperature and is composed of organic cations with large molecular weight and inorganic / organic anions with small molecular weight. Due to the large difference in molecular weight between the two ions in ionic liquid, the crystal structure arrangement is unstable, resulting in weak van der Waals force between ions, low lattice energy and small hydrogen bond force. Ionic liquid has the advantages of weak volatility, strong antioxidant property, high polarity and easy separation, and has become a green solvent that can replace traditional organic solvents to dissolve cellulose. SUMMARY

[0006] To solve the above technical problems, the present application aims to provide a method for preparing xanthan oligosaccharide by ion liquid assisted enzymatic hydrolysis.

[0007] Because the xanthan gum main chain composed of glucose connected by β-1,4 glycosidic bond is the same as the structure of cellulose, and the ionic liquid can effectively destroy the dense structure of polysaccharide. Based on the above two points, the xanthan oligosaccharide is efficiently prepared by using ionic liquid assisted commercial cellulase method, and then the reaction conditions are optimized, and the activity of the product is characterized, which proves that the ionic liquid can efficiently degrade xanthan gum to prepare xanthan oligosaccharide. The application of commercial cellulase in the application breaks the dependence of traditional industrial biological method on microbial fermentation technology, protein purification technology and biological equipment. At the same time, the application of ionic liquid makes the structure of xanthan gum open, thereby further improving the activity of xanthan gum enzymolysis product. The application provides an effective theoretical basis for industrial production and application of xanthan oligosaccharide.

[0008] A preparation method of xanthan oligosaccharide, comprising the following steps:

[0009] (1) preparing a xanthan gum solution with a final concentration of 0.1 mg / mL-10 mg / mL;

[0010] (2) adding cellulase to the xanthan gum solution obtained in step (1) and stirring to dissolve, and the final concentration of cellulase in the obtained solution is 0.1 mg / mL-15 mg / mL;

[0011] (3) adding ionic liquid to the solution obtained in step (2), and the final concentration of ionic liquid in the obtained solution is 0.1% (v / v)-5% (v / v);

[0012] (4) performing enzymolysis reaction on the solution obtained in step (3), after the reaction is completed, heating the solution in boiling water, membrane filtering and freeze-drying, to obtain xanthan oligosaccharide.

[0013] Further, in the above technical solution, the xanthan gum solution in step (1) is an aqueous solution of xanthan gum.

[0014] Further, in the above technical solution, the ionic liquid in step (3) is an imidazole ionic liquid.

[0015] Further, in the above technical solution, the imidazole ionic liquid includes 1-butyl-3-methylimidazole chloride, 1-ethyl-3-methylimidazole acetate and 1-ethyl-3-methylimidazole diethyl phosphate.

[0016] Further, in the above technical solution, the enzymolysis reaction in step (4) is a reaction at 10-80℃ for 0.05-48h.

[0017] Further, in the above technical solution, the boiling water heating time in step (4) is 1-60min.

[0018] Further, the above technical solution, the freeze-drying time is 12h-72h.

[0019] Inventive benefits

[0020] The conventional method of preparing xanthan oligosaccharide by using xanthan degrading enzyme (or bacteria) has problems of low enzyme activity, poor product activity, dependence on microbial fermentation technology and protein purification technology. The present application can get oligosaccharide with biological activity by using commercial cellulase, and can prepare xanthan oligosaccharide with high activity (hydroxyl radical scavenging activity IC 50 = 0.87-1.88mg / mL) by introducing ionic liquid into the reaction system, which lays a foundation for the industrial production and application of xanthan oligosaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Determination of specific activity of enzyme-degraded xanthan gum for Comparative Example 1 and Examples 1-9.

[0022] Figure 2 Determination of hydroxyl radical scavenging activity of xanthan oligosaccharide prepared in Comparative Example 1 and Examples 1-9.

[0023] Figure 3 Ion chromatography characterization of xanthan oligosaccharide prepared in Comparative Example 1 and Examples 1-3.

[0024] Figure 4 Ion chromatography characterization of xanthan oligosaccharide prepared in Comparative Example 1 and Examples 4-6.

[0025] Figure 5 Ion chromatography characterization of xanthan oligosaccharide prepared in Comparative Example 1 and Examples 7-9. DETAILED DESCRIPTION

[0026] The following non-limiting examples can enable those skilled in the art to more fully understand the present application, but in no way limit the present application.

[0027] The xanthan gum used in the examples of the present application is produced by spectrum, the commercial cellulase is purchased from Sigma-Aldrich, the ionic liquid [Bmim]Cl (1-butyl-3-methylimidazole chloride) is purchased from Shanghai Dibo Biological Technology Co., Ltd., the ionic liquid [Emim]OAc (1-ethyl-3-methylimidazole acetate) is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., and the ionic liquid [Emim]DEP (1-ethyl-3-methylimidazole diethyl phosphate) is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0028] Comparative Example 1

[0029] Take 20 mg of xanthan gum and dissolve in water solution, the final concentration of xanthan gum is 0.2 mg / mL. Take 150 mg of commercial cellulase, dissolve in a small amount of water and then add into the xanthan gum solution, the final concentration of cellulase is 1.5 mg / mL. Then put the reaction solution into 37℃ for 20 min, after the reaction, heat the reaction solution in boiling water for 20 min. The reaction solution is filtered through 0.22 μm filter membrane and then the reducing sugar content is determined to measure the enzyme activity. Then freeze-dry the reaction solution for 48 h to obtain xanthan oligosaccharide. Finally, the xanthan oligosaccharide is determined for hydroxyl radical scavenging rate and ion chromatography characterization.

[0030] BCA solution configuration: A solution: 0.2 g 2,2'-biquinoline-4,4'-dicarboxylic acid disodium, 5.4 g Na2CO3, 2.4 g NaHCO3, distilled water to 100 mL, 4℃ light storage; B solution: 0.12 g CuSO4·5H2O, 0.12 g L-serine, distilled water to 100 mL, 4℃ light storage; BCA reaction solution: equal volume of "A solution" and "B solution" are mixed, and it is prepared immediately before use.

[0031] Reducing sugar content determination method: glucose is dried in an oven at 110℃ until constant weight. Then 0-100 μM of glucose standard solution is prepared (see Table 1), and then 200 μL of glucose solution is mixed with 200 μL of BCA solution, shaken well, and 3 groups of parallel samples are prepared. After 75℃ reaction for 30 min, it is cooled in ice water, and then 200 μL of reaction solution is taken from each tube and placed in a 96-well plate, and the absorbance is measured at OD 562 nm. The standard curve is obtained according to the glucose concentration and absorbance value. Then 200 μL of reaction solution is mixed with 200 μL of BSA solution, and the subsequent steps are the same as above to obtain the absorbance value, which is finally substituted into the standard curve to measure the reducing sugar content.

[0032] Table 1 Preparation of glucose standard solution

[0033]

[0034] Specific enzyme activity is defined as 1 U / g = 1 μmol of glucose equivalent released per gram of protein per minute

[0035] The specific enzyme activity of commercial cellulase hydrolyzing xanthan gum is measured to be 477.68 U / g (see Table 2). Figure 1 )

[0036] Hydroxyl radical scavenging rate determination method: the solution is prepared according to the following formula: 0.5 mL of 0.2 M pH 7.5 phosphate buffer, 0.1 mL of 0.52 mg / mL safranine and 0.5 mL of 2 M EDTA Na2-Fe 2+, 0.1 mL 1% H2O2 solution was added respectively, and the absorbance was measured at 520 nm after mixing and incubating at 40°C for 30 min. The blank group used pure water instead of xanthan oligosaccharide solution, and the control group used pure water instead of xanthan oligosaccharide solution and EDTANa2-Fe 2+ solution, and the result was expressed as clearance rate.

[0037] Clearance rate = (OD sample - OD blank) / (OD control - OD blank) x 100%.

[0038] The result was expressed as the half-clearance concentration (IC 50 ), that is, the oligosaccharide content required when the clearance rate reached 50%.

[0039] The IC 50 of hydroxyl radicals was measured to be 3.80 mg / mL (see Figure 2 )

[0040] Ion chromatography characterization method of xanthan oligosaccharide: IC5000 ion chromatography (IC) was used to qualitatively analyze the degree of polymerization of xanthan oligosaccharide with a silver ion detector. 0.2 mg / mL xanthan oligosaccharide sample was filtered through a 0.22 μm filter to remove impurities. ICS-5000 system (Dionex, Thermo Fisher, USA) was used, with Carbo Pac PA100 (Guard, 50 mm x 4 mm, Dionex) as the anion exchange guard column and Carbo Pac PA100 (Analytical, 250 mm x 4 mm, Dionex) as the anion exchange analysis column. AgCl detection was selected, the column temperature was set to 30°C, the volume flow rate was 1 mL / min, and the xanthan oligosaccharide sample was eluted by gradient elution with 200 mmol / L NaOH and 1 mol / L NaAc, and the signal was collected at 20 Hz wave frequency. The xanthan oligosaccharide components in the product were determined. Inulin was used as a standard for analysis.

[0041] The content of xanthan oligosaccharide with a degree of polymerization of 3-7 was low (see Figure 3 )

[0042] Conclusion: The activity of commercial cellulase in degrading xanthan gum is low, and the activity of the obtained xanthan oligosaccharide is also weaker than that of the prior art (IC 50 is about 2.23 mg / mL), and the content of xanthan oligosaccharide with a degree of polymerization of 3-7 is also low.

[0043] Example 1

[0044] Take 20 mg xanthan gum into water solution, the final concentration of xanthan gum is 0.2 mg / mL. Take 150 mg commercial cellulase, which is dissolved in a small amount of water, and then add into the xanthan gum solution, the final concentration of cellulase is 1.5 mg / mL. Take 0.1 mL [Bmim]Cl into the solution and dissolve completely, the final concentration of [Bmim]Cl is 0.1% (v / v). Then put the reaction solution into 37 ℃ for 20 min, after the reaction, heat the reaction solution in boiling water for 20 min to remove protein. The obtained reaction solution is filtered through 0.22 μm filter membrane, and then the reducing sugar content is determined to determine the enzyme activity. Then freeze-dry the reaction solution for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide is determined for hydroxyl radical scavenging rate and ion chromatography characterization. The determination method is the same as that of Comparative Example 1.

[0045] Example 2

[0046] Take 20 mg xanthan gum into water solution, the final concentration of xanthan gum is 0.2 mg / mL. Take 150 mg commercial cellulase, which is dissolved in a small amount of water, and then add into the xanthan gum solution, the final concentration of cellulase is 1.5 mg / mL. Take 1 mL [Bmim]Cl into the solution and dissolve completely, the final concentration of [Bmim]Cl is 1% (v / v). Then put the reaction solution into 37 ℃ for 20 min, after the reaction, heat the reaction solution in boiling water for 20 min. The obtained reaction solution is filtered through 0.22 μm filter membrane, and then the reducing sugar content is determined to determine the enzyme activity. Then freeze-dry the reaction solution for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide is determined for hydroxyl radical scavenging rate and ion chromatography characterization. The determination method is the same as that of Comparative Example 1.

[0047] Example 3

[0048] Take 20 mg xanthan gum into water solution, the final concentration of xanthan gum is 0.2 mg / mL. Take 150 mg commercial cellulase, which is dissolved in a small amount of water, and then add into the xanthan gum solution, the final concentration of cellulase is 1.5 mg / mL. Take 5 mL [Bmim]Cl into the solution and dissolve completely, the final concentration of [Bmim]Cl is 5% (v / v). Then put the reaction solution into 37 ℃ for 20 min, after the reaction, heat the reaction solution in boiling water for 20 min. The obtained reaction solution is filtered through 0.22 μm filter membrane, and then the reducing sugar content is determined to determine the enzyme activity. Then freeze-dry the reaction solution for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide is determined for hydroxyl radical scavenging rate and ion chromatography characterization. The determination method is the same as that of Comparative Example 1.

[0049] The specific enzyme activity of the hydrolysis of xanthan gum by the commercial cellulase assisted by different concentrations of [Bmim]CI was measured to be 524.16 U / g, 642.14 U / g, and 583.70 U / g, respectively (see Table 1) Figure 1 )

[0050] The content of xanthan oligosaccharide with a degree of polymerization of 3-7 was obviously increased (see Table 2) Figure 3 )

[0051] In addition, the product of Example 2 with the highest specific enzyme activity was selected to determine the hydroxyl radical scavenging activity, and the product of Example 2 had the highest hydroxyl radical scavenging activity, with an IC 50 = 1.39 mg / mL (see Table 3) Figure 2 )

[0052] Conclusion: It can be seen that [Bmim]CI can assist the commercial cellulase in hydrolyzing xanthan gum to prepare xanthan oligosaccharide, and the optimal enzyme activity and radical scavenging activity are 1.10 and 2.73 times that of Comparative Example 1. At the same time, the content of xanthan oligosaccharide is also increased.

[0053] Example 4

[0054] 20 mg of xanthan gum was weighed and dissolved in an aqueous solution, and the final concentration of xanthan gum was 0.2 mg / mL. 150 mg of commercial cellulase was weighed, dissolved in a small amount of water, and then added to the xanthan gum solution, and the final concentration of cellulase was 1.5 mg / mL. 0.1 mL of [Emim]OAc was weighed and added to the solution and dissolved, and the final concentration of [Emim]OAc was 0.1% (v / v). Then the reaction solution was placed in a 37°C environment for 20 min, and after the reaction was completed, the reaction solution was heated in boiling water for 20 min to remove protein. The obtained reaction solution was filtered through a 0.22 μm filter membrane, and then the reducing sugar content was determined to determine the enzyme activity. Then the reaction solution was freeze-dried for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide was determined for hydroxyl radical scavenging rate and ion chromatography characterization. The determination method was the same as that of Comparative Example 1.

[0055] Example 5

[0056] Example 4 20 mg of xanthan gum was dissolved in water solution, the final concentration of xanthan gum was 0.2 mg / mL. 150 mg of commercial cellulase was dissolved in a small amount of water and then added into the xanthan gum solution, the final concentration of cellulase was 1.5 mg / mL. 5 mL of [Emim]OAc was added into the solution and dissolved completely, the final concentration of [Emim]OAc was 5% (v / v). Then the reaction solution was placed in 37 °C for 20 min, after the reaction, the reaction solution was heated in boiling water for 20 min to remove protein. The obtained reaction solution was filtered through 0.22 μm filter membrane and then the reducing sugar content was determined to determine the enzyme activity. Then the reaction solution was freeze-dried for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide was determined for hydroxyl radical scavenging activity and ion chromatography characterization. The determination method was the same as that of Comparative Example 1.

[0057] Example 6

[0058] 20 mg of xanthan gum was dissolved in water solution, the final concentration of xanthan gum was 0.2 mg / mL. 150 mg of commercial cellulase was dissolved in a small amount of water and then added into the xanthan gum solution, the final concentration of cellulase was 1.5 mg / mL. 5 mL of [Emim]OAc was added into the solution and dissolved completely, the final concentration of [Emim]OAc was 5% (v / v). Then the reaction solution was placed in 37 °C for 20 min, after the reaction, the reaction solution was heated in boiling water for 20 min to remove protein. The obtained reaction solution was filtered through 0.22 μm filter membrane and then the reducing sugar content was determined to determine the enzyme activity. Then the reaction solution was freeze-dried for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide was determined for hydroxyl radical scavenging activity and ion chromatography characterization. The determination method was the same as that of Comparative Example 1.

[0059] The specific enzyme activity of the xanthan oligosaccharide obtained in Example 4 was the highest, which was 823.42 U / g (see Table 1). Figure 1 )

[0060] The content of xanthan oligosaccharide with a degree of polymerization of 3-7 was significantly improved (see Table 2). Figure 4 )

[0061] In addition, the hydroxyl radical scavenging activity of the product was determined by selecting the highest specific enzyme activity of Example 4, and the hydroxyl radical scavenging activity of the product of Example 4 was the highest, IC 50 = 1.09 mg / mL (see Table 3). Figure 2

[0062] ​Conclusion: It can be seen that [Emim]OAc can assist commercial cellulase to hydrolyze xanthan gum to prepare xanthan oligosaccharide, and the optimal enzyme activity and free radical scavenging activity are 1.37 and 3.49 times of those of Comparative Example 1. At the same time, the content of xanthan oligosaccharide is also obviously improved.

[0063] Example 7

[0064] 20 mg of xanthan gum was weighed into an aqueous solution, and the final concentration of xanthan gum was 0.2 mg / mL. 150 mg of commercial cellulase was weighed, and a small amount of water was used to fully dissolve it and then added to the xanthan gum solution, and the final concentration of cellulase was 1.5 mg / mL. Then 0.1 mL of [Emim]DEP was weighed and added to the solution and fully dissolved, and the final concentration of [Emim]DEP was 0.1% (v / v). Subsequently, the reaction solution was placed in a 37°C environment for 20 min, and after the reaction was completed, the reaction solution was heated in boiling water for 20 min. The obtained reaction solution was filtered through a 0.22 μm filter membrane, and the reducing sugar content was determined to determine the enzyme activity. Then the reaction solution was freeze-dried for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide was determined for hydroxyl radical scavenging rate and ion chromatography characterization. The determination method was the same as that of Comparative Example 1.

[0065] Example 8

[0066] 20 mg of xanthan gum was weighed into an aqueous solution, and the final concentration of xanthan gum was 0.2 mg / mL. 150 mg of commercial cellulase was weighed, and a small amount of water was used to fully dissolve it and then added to the xanthan gum solution, and the final concentration of cellulase was 1.5 mg / mL. Then 1 mL of [Emim]DEP was weighed and added to the solution and fully dissolved, and the final concentration of [Emim]DEP was 1% (v / v). Subsequently, the reaction solution was placed in a 37°C environment for 20 min, and after the reaction was completed, the reaction solution was heated in boiling water for 20 min. The obtained reaction solution was filtered through a 0.22 μm filter membrane, and the reducing sugar content was determined to determine the enzyme activity. Then the reaction solution was freeze-dried for 48 h to obtain xanthan oligosaccharide. Finally, the obtained xanthan oligosaccharide was determined for hydroxyl radical scavenging rate and ion chromatography characterization. The determination method was the same as that of Comparative Example 1.

[0067] Example 9

[0068] Take 20 mg xanthan gum and dissolve in water, the final concentration of xanthan gum is 0.2 mg / mL. Take 150 mg commercial cellulase, dissolve in a small amount of water, then add into the xanthan gum solution, the final concentration of cellulase is 1.5 mg / mL. Take 5 mL [Emim]DEP and add into the solution, then dissolve completely, the final concentration of [Emim]DEP is 5% (v / v). Then put the solution into 37 ℃ and react for 20 min, after the reaction, heat the solution in boiling water for 20 min. Take the solution through 0.22 μm filter membrane, then determine the reducing sugar content to determine the enzyme activity. Then freeze dry the solution for 48 h to get xanthan oligosaccharide. Finally, determine the hydroxyl radical scavenging rate of the xanthan oligosaccharide and determine the ion chromatography. The determination method is the same as Comparative Example 1.

[0069] The specific enzyme activity of the xanthan oligosaccharide prepared by the commercial cellulase assisted by [Emim]DEP in Example 7-9 is 669.55 U / g, 885.27 U / g, 405.40 U / g respectively (see Table 1). Figure 1 )

[0070] The content of xanthan oligosaccharide with a degree of polymerization of 3-7 is significantly improved (see Table 2). Figure 5

[0071] In addition, the highest specific enzyme activity of Example 8 is selected to determine the hydroxyl radical scavenging activity of the product, and the hydroxyl radical scavenging activity of the product of Example 8 is the highest, IC 50 = 0.87 mg / mL (see Table 3). Figure 2

[0072] Conclusion: It can be seen that [Emim]DEP can assist commercial cellulase to hydrolyze xanthan gum to prepare xanthan oligosaccharide, and the optimal enzyme activity and radical scavenging activity are 1.47 and 4.37 times of Comparative Example 1. At the same time, the content of xanthan oligosaccharide is also significantly improved.​​

Claims

1. A method for preparing xanthan gum oligosaccharides, characterized in that, Includes the following steps: (1) Prepare xanthan gum solutions with a final concentration of 0.1 mg / mL to 10 mg / mL; (2) Add cellulase to the xanthan gum solution obtained in step (1) and stir until dissolved. The final concentration of cellulase in the resulting solution is 0.1 mg / mL-15 mg / mL. (3) Add the ionic liquid to the solution obtained in step (2), and the final concentration of the ionic liquid in the solution is 0.1% (v / v) - 5% (v / v); (4) The solution obtained in step (3) is subjected to enzymatic hydrolysis. After the reaction is completed, the solution is heated in boiling water, filtered through a membrane, and freeze-dried to obtain xanthan gum oligosaccharide. The ionic liquid mentioned in step (3) is an imidazole ionic liquid.

2. The preparation method according to claim 1, characterized in that, The xanthan gum solution mentioned in step (1) is an aqueous solution of xanthan gum.

3. The preparation method according to claim 1, characterized in that, The imidazole ionic liquids include 1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium acetate, and 1-ethyl-3-methylimidazolium diethyl phosphate.

4. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis reaction described in step (4) is carried out at 10℃-80℃ for 0.05-48h.

5. The preparation method according to claim 1, characterized in that, The boiling water heating time in step (4) is 1-60 min.

6. The preparation method according to claim 1, characterized in that, The freeze-drying time is 12h-72h.

Images