Extraction method of mulberry leaf polysaccharide

Abstract

The invention discloses a mulberry leaf polysaccharide extraction method which comprises the following steps: S1, crushing and sieving mulberry leaves dried at low temperature, and adding distilled water for soaking to obtain a mulberry leaf soaking solution; s2, mixing a compound enzyme preparation with water, and heating in a water bath to activate the enzyme; s3, adding a buffer solution into the mulberry leaf soaking solution to adjust the pH value, then adding an activated compound enzyme preparation, and heating in a water bath in ultrasonic equipment to obtain an extracting solution; and S4, filtering and concentrating the extracting solution, adding the concentrated solution into an ethanol solution, centrifuging, taking the precipitate, washing, and drying to obtain the mulberry leaf polysaccharide. The method can significantly improve the extraction rate of mulberry leaf polysaccharide, and is simple in process and easy to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product component extraction, and in particular relates to a method for extracting mulberry leaf polysaccharide. Background Art

[0002] Mulberry leaf polysaccharide is a macromolecular compound composed of a variety of monosaccharides in mulberry leaves. It has immunomodulatory function and can activate immune cells such as macrophages and T lymphocytes. After being activated, macrophages can enhance their ability to phagocytize pathogens, and the activation of T lymphocytes helps to initiate cellular immune responses, thereby enhancing the body's immunity and helping the human body resist the invasion of external pathogens. In addition, mulberry leaf polysaccharide also has a hypoglycemic effect. It can regulate the insulin signaling pathway, increase insulin sensitivity, promote the uptake and utilization of glucose by peripheral tissues, and thus lower blood sugar levels. In addition, mulberry leaf polysaccharide also has a certain contribution to anti-oxidation, which can remove free radicals in the body and reduce the damage to cells caused by oxidative stress.

[0003] At present, the methods for extracting mulberry leaf polysaccharides from mulberry leaves include water extraction, acid / alkali extraction, enzyme extraction, ultrasonic extraction, microwave extraction, ultra-high pressure extraction, etc. These methods each have their own advantages and disadvantages. For example, the water extraction method utilizes the increased solubility of polysaccharides in hot water to dissolve the polysaccharides from the mulberry leaf tissue by increasing the temperature. It is a relatively traditional, simple and mild extraction method, but its disadvantages are that the extraction time is long, the extraction rate is relatively low, and the impurity content is high; the acid-base extraction method effectively destroys the cell wall and accelerates the dissolution of polysaccharides, but the acid-base conditions will cause the polysaccharide hydrolysis, resulting in a decrease in the extraction rate; the enzyme extraction method has mild extraction conditions and can selectively decompose cell wall components, with a high extraction rate and purity, but the reaction conditions need to be strictly controlled; the ultrasonic extraction method, microwave extraction method and ultra-high pressure extraction method have fast extraction speed and high extraction rate, but the cost is high and the parameters need to be strictly controlled, otherwise the polysaccharide structure will be destroyed. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting mulberry leaf polysaccharides to solve the technical problem of low extraction rate of mulberry leaf polysaccharides in the above-mentioned prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0007] S1: crushing the low-temperature dried mulberry leaves, sieving them, and soaking them in distilled water to obtain a mulberry leaf soaking liquid;

[0008] S2: Mix the complex enzyme preparation with water and heat it in a water bath to activate the enzyme;

[0009] S3: adding a buffer solution to the mulberry leaf soaking solution to adjust the pH, then adding the activated complex enzyme preparation, and heating it in a water bath in an ultrasonic device to obtain an extract;

[0010] S4: Filter and concentrate the extract, add the concentrate into an ethanol solution and centrifuge, wash and dry the precipitate to obtain mulberry leaf polysaccharide.

[0011] Furthermore, in step S1, the temperature of low-temperature drying is 40-50° C.; during soaking, distilled water is added at a material-liquid ratio of 1:15-20 for soaking for 24 hours.

[0012] Furthermore, in step S2, the complex enzyme preparation includes cellulase, pectinase and β-glucosidase.

[0013] Furthermore, the cellulase, pectinase and beta-glucosidase are prepared in a mass ratio of 3-5:2-3:1-2.

[0014] Furthermore, the complex enzyme preparation is mixed at a mass ratio of complex enzyme preparation: water = 1:5-15.

[0015] Furthermore, the water bath heating condition is 30-50°C and the time is 15-30 min.

[0016] Furthermore, the buffer solution is an acetic acid-sodium acetate buffer solution, and the pH is adjusted to 4.5-5.5.

[0017] Furthermore, the ultrasonic power of the ultrasonic equipment is 50-80W, the water bath temperature is 30-50°C, and the time is 1-3h.

[0018] Furthermore, the filtered extract is concentrated to 1 / 3-1 / 2 of the original volume at 40-50° C. and a pressure of -0.08 to -0.09 MPa.

[0019] Furthermore, in step S4, the amount of ethanol used is 3-5 times the volume of the concentrated solution, the centrifugal speed is 4000-5000 r / min, and the time is 10-15 min.

[0020] The extraction process principle of the present invention is:

[0021] S1: Low-temperature drying can remove moisture from mulberry leaves, while preventing high temperature from damaging the bioactive components such as polysaccharides in mulberry leaves, such as glycosidic bond breakage and sugar chain degradation, thereby affecting their bioactivity and extraction rate; crushing and screening can increase the surface area of ​​mulberry leaves and the degree of cell fragmentation, which is conducive to the better penetration of solvents into the mulberry leaf tissues during the subsequent soaking process, so that polysaccharides and other components can be more fully dissolved, and at the same time ensure that the dispersed mulberry leaves have a uniform particle size, which is conducive to a more uniform reaction during the subsequent enzyme treatment and extraction process; distilled water is a good solvent, and the soaking process can make the mulberry leaf tissue absorb water and swell. After absorbing water, the structure of the cells becomes loose, which can make it easier for polysaccharides and other components originally present in the cells to diffuse out; on the other hand, it also provides a good substrate state for the subsequent enzymatic hydrolysis process, so that the enzyme can better contact the cell wall and the polysaccharides in the cells.

[0022] S2: Activate cellulase, pectinase, β-glucosidase, etc. in the complex enzyme preparation at a certain temperature to change the structure of the active center of the enzyme, thereby converting the enzyme from an inactive or low-activity state to a high-activity state, so that it can effectively decompose the corresponding components of the mulberry leaf cell wall in the subsequent extraction process and promote the release of polysaccharides.

[0023] S3: The buffer solution can resist the influence of a small amount of external acid or alkali, maintain the pH value of the reaction system relatively stable, thereby ensuring that the various enzymes in the complex enzyme work in a relatively stable pH environment, maintain the activity of the enzyme, and effectively decompose the cell wall components, thereby improving the extraction efficiency of mulberry leaf polysaccharides. At the same time, a stable pH environment also contributes to the stability of the polysaccharide components during the extraction process, avoiding structural changes or degradation due to pH changes. The mulberry leaf cell wall is decomposed by enzymatic hydrolysis, so that the polysaccharides wrapped by the cell wall can be fully released into the extract. Ultrasonic treatment produces cavitation, that is, tiny bubbles in the liquid form, grow and collapse under the action of ultrasound. The instantaneous high pressure and high temperature generated during the collapse can destroy the cell wall of the mulberry leaf cells, making it easier to release the polysaccharides in the cells. At the same time, ultrasound can also produce mechanical vibration and microflow effects, promote the penetration and diffusion of the extract in the mulberry leaf tissue, and accelerate the dissolution of polysaccharides. Water bath heating can maintain the appropriate temperature required for the enzymatic reaction on the one hand, and accelerate the thermal motion of molecules on the other hand, promoting the dissolution and diffusion of polysaccharides in the extract. Ultrasonication and water bath heating work synergistically to improve the extraction efficiency of polysaccharides. Ultrasonic assistance can enhance the destruction of cell walls, while water bath heating provides suitable temperature conditions for enzymatic hydrolysis and promotes the dissolution of polysaccharides. The combination of the two can achieve a higher polysaccharide extraction rate in a shorter time.

[0024] S4: Using the pore size of filter media such as filter paper or filter membrane, the solid impurities in the extract, such as undecomposed mulberry leaf residues, enzyme proteins, etc., are separated from the liquid part, and the insoluble impurities in the extract are removed to obtain a relatively clear solution containing polysaccharides, which provides a pure liquid basis for subsequent concentration and precipitation operations. The solvent in the extract is partially volatilized by reduced pressure concentration, thereby increasing the concentration of polysaccharides. Since adding ethanol for precipitation when the polysaccharide concentration is low not only consumes a large amount of ethanol, but also the precipitation effect may not be ideal, the volume of the extract is reduced by concentration, and the concentration of polysaccharides is increased, which can facilitate the subsequent alcohol precipitation operation. The polysaccharide is separated from the solution by precipitation and centrifugation to obtain a crude polysaccharide, and finally the impurities adsorbed on the surface of the precipitate and the volatile impurities are removed by washing and drying to obtain a mulberry leaf polysaccharide finished product.

[0025] The selection principle of the process parameters of the present invention is:

[0026] Low temperature drying temperature: In the temperature range of 40-50℃, the water content of mulberry leaves can be removed while minimizing the damage to the chemical structure of mulberry leaf polysaccharides. If the temperature is too high, it may cause partial degradation of polysaccharides, resulting in a reduction in the amount of polysaccharides extracted and their biological activity may be reduced. If the temperature is too low, the drying process will be too slow and inefficient. Moreover, if the mulberry leaves are kept in a low temperature drying environment for a long time, microorganisms may grow in the mulberry leaves, affecting the quality of the mulberry leaves and thus reducing the polysaccharide extraction rate.

[0027] Soaking liquid-to-solid ratio: A soaking liquid-to-solid ratio of 1:15-20 can provide sufficient solvent for dissolving the polysaccharides in mulberry leaves. A higher liquid-to-solid ratio can ensure that the polysaccharides are fully dissolved, while avoiding the burden of subsequent concentration operations due to excessively high liquid-to-solid ratios.

[0028] Mass ratio of enzyme preparation components: According to the composition characteristics of the plant components of mulberry leaves, cellulase, pectinase and β-glucosidase are selected in a ratio of 3-5:2-3:1-2 to prepare a composite enzyme preparation, which can ensure the hydrolysis of cellulose, pectin and glycosidic bonds in mulberry leaves while ensuring the synergistic effect between the three enzymes.

[0029] Composite enzyme preparation activation material-liquid ratio, activation temperature: At a material-liquid ratio of 1:5-15, the composite enzyme preparation can be evenly dispersed in water and the enzyme can be activated at an appropriate concentration. Considering the characteristics of the three enzymes, the activation temperature of 30-50 is selected to help the active center of the enzyme to fold and expose correctly, which can ensure the activation effect of the three enzymes and prevent the enzyme from being inactivated due to excessively high temperature or insufficient activation effect due to excessively low temperature, resulting in insufficient enzyme activity, thereby affecting the efficiency of enzymolysis.

[0030] pH: Considering the characteristics of the three enzymes, the pH is adjusted to 4.5-5.5, so that the three enzymes can maintain relatively high activity, thereby effectively decomposing the cell wall components and improving the extraction efficiency of mulberry leaf polysaccharides. Too high or too low pH values ​​will affect the enzyme structure. Too high or too low pH may change the charge state of the active center of the enzyme, affecting the binding ability of the enzyme to the substrate, resulting in reduced enzyme activity or even inactivation, affecting the extraction rate.

[0031] Ultrasonic power: Choosing an ultrasonic power of 50-80W can produce a certain degree of cavitation, mechanical vibration and microflow effect, which is used to destroy the cell wall of mulberry leaf cells and make it easier to release polysaccharides in the cells. Under this ultrasonic power, the various effects produced are relatively mild, which can not only achieve the purpose of promoting the release of polysaccharides, but also reduce the damage to the polysaccharide structure and enzyme activity.

[0032] Ultrasonic water bath temperature: Choose a water bath temperature of 30-50℃, which can take into account the optimal reaction temperature of the enzyme and the heat that may be generated during the ultrasonic process. Within this temperature range, the enzyme can maintain good catalytic activity, while avoiding enzyme inactivation or destruction of the polysaccharide structure due to excessive temperature. The appropriate temperature can also accelerate the thermal motion of molecules and promote the dissolution and diffusion of polysaccharides in the extract.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention can significantly improve the extraction rate of mulberry leaf polysaccharide by using a composite enzyme preparation in combination with ultrasound-assisted extraction of mulberry leaf polysaccharide. In Examples 1-5 of the present application, the extraction rates of mulberry leaf polysaccharide are 15.8%, 15.2%, 14.7%, 14.8%, and 15.4%, respectively, among which the extraction rate of Example 1 reaches 15.8%. The extraction method is simple, easy to operate, and has a high extraction rate.

[0035] 2. The present application adopts cellulase, pectinase and β-glucosidase to support the composite enzyme preparation. Through the interaction of the three enzymes, they can synergistically enhance the efficiency, thereby greatly improving the extraction rate of mulberry leaf polysaccharides. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, preferred embodiments are given below to further describe the present invention in detail. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0037] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0038] S1: crushing the mulberry leaves dried at 40-50°C, passing through an 80-mesh sieve, adding distilled water at a material-liquid ratio of 1:15-20 and soaking for 24 hours to obtain a mulberry leaf soaking liquid;

[0039] S2: Cellulase, pectinase, and β-glucosidase are prepared into a composite enzyme preparation in a mass ratio of 3-5:2-3:1-2, mixed with water in a mass ratio of 1:5-15, and heated in a water bath at 30-50°C for 15-30 minutes to activate the enzymes;

[0040] S3: Add acetic acid-sodium acetate to the mulberry leaf soaking liquid to slowly adjust the pH of the solution to 4.5-5.5, then add the activated complex enzyme preparation, and heat in a water bath at 30-50°C for 1-3h in an ultrasonic device with an ultrasonic power of 50-80W to obtain an extract;

[0041] S4: Filter the extract, concentrate the extract to 1 / 3-1 / 2 of the original volume at 40-50°C and a pressure of -0.08 to -0.09 MPa, add the concentrate into a 95% ethanol solution which is 3-5 times the volume of the concentrate, centrifuge at a speed of 4000-5000 r / min for 10-15 min, wash the precipitate with anhydrous ethanol and acetone for 3 times respectively, and dry to obtain mulberry leaf polysaccharide.

[0042] The above is described below by means of more specific examples.

[0043] Example 1

[0044] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0045] S1: crushing the mulberry leaves after low-temperature drying at 40°C, passing through an 80-mesh sieve, adding distilled water at a solid-liquid ratio of 1:17 and soaking for 24 hours to obtain a mulberry leaf soaking liquid;

[0046] S2: a composite enzyme preparation of cellulase, pectinase and β-glucosidase in a mass ratio of 5:3:1 was mixed with water in a mass ratio of 1:10, and heated in a water bath at 30°C for 18 min to activate the enzymes;

[0047] S3: Add acetic acid-sodium acetate to the mulberry leaf soaking liquid to slowly adjust the solution to pH 5, then add the activated complex enzyme preparation, and heat it in a water bath at 35°C for 1.5h in an ultrasonic device with an ultrasonic power of 50W to obtain an extract;

[0048] S4: Filter the extract, concentrate the extract to 1 / 2 of the original volume at 40°C and a pressure of -0.08MPa, add the concentrate into a 95% ethanol solution which is 4.2 times the volume of the concentrate, centrifuge at a speed of 4000r / min for 15min, wash the precipitate with anhydrous ethanol and acetone for 3 times respectively, and dry to obtain mulberry leaf polysaccharide.

[0049] Example 2

[0050] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0051] S1: crushing the mulberry leaves after low-temperature drying at 48°C, passing through an 80-mesh sieve, adding distilled water at a solid-liquid ratio of 1:20 and soaking for 24 hours to obtain a mulberry leaf soaking liquid;

[0052] S2: a composite enzyme preparation of cellulase, pectinase and β-glucosidase in a mass ratio of 4:2:1 was mixed with water in a mass ratio of 1:8, and heated in a water bath at 30°C for 46 min to activate the enzymes;

[0053] S3: Add acetic acid-sodium acetate to the mulberry leaf soaking liquid to slowly adjust the solution to pH 4.5, then add the activated complex enzyme preparation, and heat in a water bath at 50°C for 3 hours in an ultrasonic device with an ultrasonic power of 40W to obtain an extract;

[0054] S4: Filter the extract, concentrate the extract to 1 / 2 of the original volume at 42°C and a pressure of -0.09MPa, add the concentrate into a 95% ethanol solution that is 5 times the volume of the concentrate, centrifuge at a speed of 4000r / min for 12min, wash the precipitate with anhydrous ethanol and acetone for 3 times respectively, and dry to obtain mulberry leaf polysaccharide.

[0055] Example 3

[0056] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0057] S1: crushing the mulberry leaves dried at 43°C, passing through an 80-mesh sieve, adding distilled water at a solid-liquid ratio of 1:18 and soaking for 24 hours to obtain a mulberry leaf soaking liquid;

[0058] S2: a composite enzyme preparation of cellulase, pectinase, and β-glucosidase in a mass ratio of 3:3:2 was mixed with water in a mass ratio of 1:15, and heated in a water bath at 34°C for 26 min to activate the enzymes;

[0059] S3: Add acetic acid-sodium acetate to the mulberry leaf soaking liquid to slowly adjust the solution to pH 5.5, then add the activated complex enzyme preparation, and heat in a water bath at 30°C for 2h in an ultrasonic device with an ultrasonic power of 60W to obtain an extract;

[0060] S4: Filter the extract, concentrate the extract to 1 / 3 of the original volume at 50°C and a pressure of -0.09MPa, add the concentrate into a 95% ethanol solution that is 3 times the volume of the concentrate, centrifuge at a speed of 4000r / min for 10min, wash the precipitate with anhydrous ethanol and acetone for 3 times respectively, and dry to obtain mulberry leaf polysaccharide.

[0061] Example 4

[0062] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0063] S1: crushing the mulberry leaves after low-temperature drying at 50°C, passing through an 80-mesh sieve, adding distilled water at a solid-liquid ratio of 1:16 and soaking for 24 hours to obtain a mulberry leaf soaking liquid;

[0064] S2: a composite enzyme preparation of cellulase, pectinase and β-glucosidase in a mass ratio of 3:2:1 was mixed with water in a mass ratio of 1:13, and heated in a water bath at 50°C for 20 min to activate the enzymes;

[0065] S3: Add acetic acid-sodium acetate to the mulberry leaf soaking liquid to slowly adjust the solution to pH 4.5, then add the activated complex enzyme preparation, and heat in a water bath at 45°C for 1 hour in an ultrasonic device with an ultrasonic power of 80W to obtain an extract;

[0066] S4: Filter the extract, concentrate the extract to 1 / 2 of the original volume at 45°C and a pressure of -0.09MPa, add the concentrate into a 95% ethanol solution which is 3.5 times the volume of the concentrate, centrifuge at a speed of 5000r / min for 10min, wash the precipitate with anhydrous ethanol and acetone for 3 times respectively, and dry to obtain mulberry leaf polysaccharide.

[0067] Example 5

[0068] A method for extracting mulberry leaf polysaccharides comprises the following steps:

[0069] S1: crushing the mulberry leaves after low-temperature drying at 48°C, passing through an 80-mesh sieve, adding distilled water at a solid-liquid ratio of 1:15 and soaking for 24 hours to obtain a mulberry leaf soaking liquid;

[0070] S2: a composite enzyme preparation of cellulase, pectinase and β-glucosidase in a mass ratio of 5:3:2 was mixed with water in a mass ratio of 1:5, and heated in a water bath at 40°C for 30 min to activate the enzymes;

[0071] S3: Add acetic acid-sodium acetate to the mulberry leaf soaking solution to slowly adjust the solution to pH 5, then add the activated complex enzyme preparation, and heat in a 40°C water bath for 2.5 hours in an ultrasonic device with an ultrasonic power of 70W to obtain an extract;

[0072] S4: Filter the extract, concentrate the extract to 1 / 3 of the original volume at 48°C and a pressure of -0.08MPa, add the concentrate into a 95% ethanol solution which is 4.6 times the volume of the concentrate, centrifuge at a speed of 5000r / min for 15min, wash the precipitate with anhydrous ethanol and acetone for 3 times respectively, and dry to obtain mulberry leaf polysaccharide.

[0073] Comparative Example 1

[0074] It is basically the same as Example 1, except that no complex enzyme preparation is used in the extraction method.

[0075] Comparative Example 2

[0076] The method is basically the same as Example 1, except that pectinase and β-glucosidase are not added to the composite enzyme preparation, but only cellulase is used.

[0077] Comparative Example 3

[0078] The method is basically the same as Example 1, except that no cellulase and β-glucosidase are added to the composite enzyme preparation, but only pectinase is used.

[0079] Comparative Example 4

[0080] The method is basically the same as Example 1, except that no cellulase and pectinase are added to the composite enzyme preparation, but only β-glucosidase is used.

[0081] Comparative Example 5

[0082] The method is basically the same as Example 1, except that ultrasonic equipment is not used for extraction, but only complex enzyme preparation is used for extraction.

[0083] 1. Effect of compound enzyme preparation on the extraction rate of mulberry leaf polysaccharides

[0084] Take 1g of mulberry leaf polysaccharide obtained in Examples 1-5 and Comparative Examples 1-4, add distilled water to 100mL to obtain a sample solution; use the phenol-sulfuric acid method to measure the absorbance at a wavelength of 490nm, perform regression analysis, and obtain a regression equation. Take 1ml of the sample solution of Examples 1-5 and Comparative Examples 1-4, add 1mL of distilled water, 1mL of 5% phenol solution, and 5mL of concentrated sulfuric acid, react in a water bath for 15min, measure the absorbance at a wavelength of 490nm, substitute it into the above regression equation to determine the mass concentration of the solution, and calculate the extraction rate of mulberry leaf polysaccharide.

[0085] Table 1 Effect of compound enzyme preparation on the extraction rate of mulberry leaf polysaccharides

[0086] project Extraction rate (%) Example 1 15.8 Example 2 15.2 Example 3 14.7 Example 4 14.8 Example 5 15.4 Comparative Example 1 4.2 Comparative Example 2 8.4 Comparative Example 3 7.7 Comparative Example 4 6.3 Comparative Example 5 10.1

[0087] It can be seen from Table 1 that:

[0088] (1) In Examples 1-5 of the present application, the extraction rates of mulberry leaf polysaccharides are 15.8%, 15.2%, 14.7%, 14.8%, and 15.4%, respectively. Among them, the extraction rate of Example 1 reaches 15.8%, while the extraction rates of Comparative Examples 1-4, which do not use the composite enzyme preparation of the present application or do not fully use the components of the composite enzyme preparation of the present application, are 4.2%, 8.4%, 7.7%, and 6.3%, respectively, which are all inferior to Examples 1-5 of the present application. This shows that the present application can significantly improve the extraction rate of mulberry leaf polysaccharides by using the composite enzyme preparation composed of cellulase, pectinase, and β-glucosidase.

[0089] This is because: mulberry leaf cell walls contain a large amount of cellulose, and cellulase can specifically hydrolyze the β-1,4-glycosidic bonds in cellulose molecules, breaking them down into small molecules such as glucose, thereby destroying the cellulose structure in the cell wall, reducing the strength of the cell wall, and increasing the permeability of the cell wall, making it easier for the polysaccharide components in the mulberry leaf to diffuse into the extract, thereby increasing the extraction rate of polysaccharides. In addition, cellulase degrades the cellulose components in the plant cell wall, destroys the skeleton structure of the cell wall, and makes the structure of the plant cell loose, and the pectin and sugars originally wrapped in the cellulose are The substances are exposed, which increases the permeability of the cells, provides more action sites for pectinase and β-glucosidase, and improves the efficiency of pectinase and β-glucosidase. In the extraction process, as cellulase degrades cellulose, the macromolecular cellulose in the system is gradually decomposed into small molecular substances, which reduces the viscosity of the reaction system and enhances the fluidity of the extract, which is beneficial to the diffusion and transfer of pectinase and β-glucosidase in the system, so that pectinase and β-glucosidase can be more evenly distributed in the reaction system and fully contact with the substrate, thereby improving the rate and effect of the enzymatic reaction.

[0090] The cell walls of mulberry leaves contain a large amount of pectin, which forms a tight structure that wraps the effective ingredients such as polysaccharides inside the cells. Pectinase can specifically decompose the α-1,4-glycosidic bonds in the cell walls, making the structure of the cell walls loose, thereby destroying the integrity of the cell walls, allowing the polysaccharide components to be released, and creating a channel for the subsequent action of other enzymes and the release of polysaccharides.

[0091] Some polysaccharides in mulberry leaves exist in the form of glycosides, and the glycosidic bonds in them affect the solubility and biological activity of polysaccharides. β-glucosidase can specifically hydrolyze the β-glucosidase bonds in polysaccharide molecules, decomposing polysaccharides into smaller molecular fragments or monosaccharides, increasing the solubility of polysaccharides, making them easier to release from cells and dissolve in the extract, thereby improving the extraction rate of polysaccharides. In addition, since cellulase degrades cellulose, it will form intermediates such as cellobiose, and β-glucosidase can further decompose cellobiose, etc., reducing the accumulation of intermediates such as cellobiose in the reaction system, preventing the accumulation of intermediates such as cellobiose from inhibiting the activity of cellulase, and maintaining the activity of cellulase in the reaction system.

[0092] Therefore, the combined effects of cellulase, pectinase and β-glucosidase can synergistically enhance the extraction rate of mulberry leaf polysaccharides.

[0093] (2) The extraction rate of comparative example 5, which did not use ultrasonic equipment for ultrasonication, was 10.1%, which was also lower than that of examples 1-5 of the present application. This shows that the present application can further improve the extraction rate of cellulase by using enzyme combined with ultrasonic assisted extraction.

[0094] This is because: when ultrasonic waves propagate in a liquid medium, cavitation will occur, forming a large number of tiny bubbles in the liquid. These bubbles will expand and close rapidly. At the moment when the bubbles close, extremely high pressure and temperature will be generated locally, forming powerful shock waves and microjets, thereby destroying the cell wall structure of mulberry leaves, allowing the polysaccharides in the cells to be released more easily into the extract; and ultrasonic propagation will cause the vibration of the medium particles, causing the mulberry leaf tissue cells to be acted upon by mechanical force, loosening the connection between mulberry leaf cells, increasing the permeability of the cells, and promoting the exudation of polysaccharides. At the same time, mechanical vibration can also accelerate the penetration and diffusion of the extract in the mulberry leaf tissue, allowing the polysaccharides to dissolve into the extract more quickly. In addition, the cavitation and mechanical vibration of ultrasonic waves can make the enzyme and the substrate mix more fully, increase the contact opportunities between the enzyme and the substrate, and enable the substrate that was originally unevenly dispersed in the solution to better combine with the enzyme under the action of ultrasonic waves, thereby increasing the rate of the enzymatic reaction. Therefore, through the action of ultrasonic waves, the cell structure of mulberry leaves can be destroyed, and the reaction of the enzyme can be promoted, thereby synergistically improving the extraction rate of mulberry leaf polysaccharides.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting mulberry leaf polysaccharides, characterized in that: The following steps are involved: S1: crushing the low-temperature dried mulberry leaves, sieving them, and soaking them in distilled water to obtain a mulberry leaf soaking liquid; S2: Mix the complex enzyme preparation with water and heat it in a water bath to activate the enzyme; S3: adding a buffer solution to the mulberry leaf soaking solution to adjust the pH, then adding the activated complex enzyme preparation, and heating it in a water bath in an ultrasonic device to obtain an extract; S4: Filter and concentrate the extract, add the concentrate into an ethanol solution and centrifuge, wash and dry the precipitate to obtain mulberry leaf polysaccharide.

2. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In the step S1, the temperature of low-temperature drying is 40-50° C.; during soaking, distilled water is added at a material-liquid ratio of 1:15-20 and soaked for 24 hours.

3. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In the step S2, the composite enzyme preparation includes cellulase, pectinase and β-glucosidase.

4. The method for extracting mulberry leaf polysaccharides according to claim 3, characterized in that: The cellulase, pectinase and beta-glucosidase are prepared in a mass ratio of 3-5:2-3:1-2.

5. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In the step S2, the complex enzyme preparation is mixed at a mass ratio of complex enzyme preparation: water = 1:5-15.

6. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In step S2, the water bath heating condition is 30-50° C. and the time is 15-30 min.

7. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In step S3, the buffer solution is an acetic acid-sodium acetate buffer solution, and the pH is adjusted to 4.5-5.

5.

8. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In step S3, the ultrasonic power of the ultrasonic equipment is 50-80W, the water bath temperature is 30-50°C, and the time is 1-3h.

9. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In the step S4, the filtered extract is concentrated to 1 / 3-1 / 2 of the original volume at 40-50° C. and a pressure of -0.08 to -0.09 MPa.

10. The method for extracting mulberry leaf polysaccharides according to claim 1, characterized in that: In step S4, the amount of ethanol used is 3-5 times the volume of the concentrated solution, the centrifugal speed is 4000-5000 r / min, and the time is 10-15 min.