Extraction method and application of frosted mulberry leaf extract

Abstract

The invention relates to the technical field of extraction of frosted mulberry leaves, in particular to an extraction method and application of a frosted mulberry leaf extract. The extraction method sequentially comprises the steps of pretreatment of frosted mulberry leaves, preparation of a compound enzyme aqueous solution, preparation of a hyaluronic acid aqueous solution, enzymolysis treatment and extraction of the frosted mulberry leaf extract, and the frosted mulberry leaf extract with the mass content of 1-deoxynojirimycin as high as 5.2-7.0% is obtained through specific selection of raw materials in cooperation with a specific extraction process. Meanwhile, the limitation that in the prior art, the mulberry leaf extract needs to be stored under the severe condition of 2-8 DEG C is broken through, and after the frosted mulberry leaf extract extracted through a specific extraction process is stored for 300 days under the condition of 18 DEG C in a shading mode, the retention rate of the mass content of 1-deoxynojirimycin contained in the frosted mulberry leaf extract can be larger than or equal to 89%; and the preservation cost is further reduced for popularization of subsequent hypoglycemic products.

Description

Technical Field

[0001] The invention relates to the technical field of frost-removed mulberry leaf extraction, and in particular to an extraction method and application of a frost-removed mulberry leaf extract. Background Art

[0002] 1-Deoxynojirimycin (DNJ) is a polyhydroxy alkaloid with significant physiological activities such as lowering blood sugar, blood pressure and blood lipids. In the journal "Agricultural Products Processing", the author Ren Xiangdong et al. mentioned in the article "Research Progress on Extraction and Purification Process of 1-Deoxynojirimycin in Mulberry Leaves and Its Hypoglycemic Effect": DNJ has an inhibitory effect on α-glucosidase (IC 50 =0.350 mg / mL) is the hypoglycemic drug glucagon (IC 50 =0.982 mg / mL), which is 2.7 times of that in the development of blood sugar-lowering foods.

[0003] The extraction methods of 1-deoxynojirimycin (DNJ) mainly include water extraction, organic solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, etc., but the relevant extraction and purification parameters are different, and a systematic extraction and purification process system has not yet been formed to guide industrial production. Since the content of DNJ in dry mulberry leaves is only about 0.1%, it is difficult to obtain 1-deoxynojirimycin (DNJ) with higher purity even after multiple purifications.

[0004] For example, CN114177218B discloses a mulberry leaf extract rich in 1-deoxynojirimycin and its preparation method. The preparation method comprises the following steps: S1, pretreatment: washing, drying and crushing the mulberry leaves to obtain mulberry leaf powder; S2, ultrasonic treatment: adding the mulberry leaf powder to water, ultrasonic treatment, centrifugation, taking the supernatant, obtaining supernatant 1 and residue; S3, enzymatic hydrolysis: adding equal mass water to the residue, first enzymolysis with composite enzyme 1, then enzymolysis with composite enzyme 2, centrifugation, taking the supernatant, obtaining supernatant 2; S4, filtration, concentration, drying: combining supernatant 1 in S2 and supernatant 2 in S3, performing membrane filtration, membrane concentration and spray drying. The highest content of DNJ obtained in the mulberry leaf extract by this technical solution is only 1.21% in Example 1.

[0005] For another example, CN119174493A discloses an extraction method and application of sugar-controlling components in mulberry leaves. The extraction method comprises the following steps: mixing mulberry leaf powder and acidic ethanol solution, ultrasonically extracting to obtain a first extract and a first extract residue; enzymatically hydrolyzing the first extract residue, collecting a second extract; combining the first extract and the second extract, concentrating and decolorizing to obtain a decolorized solution; microfiltration, nanofiltration and membrane concentration of the decolorized solution and then drying; the enzymatic hydrolysis treatment includes the first enzymatic hydrolysis treatment and the second enzymatic hydrolysis treatment; a composite decolorizer is added during the decolorization process; the preparation method of the composite decolorizer comprises the following steps: mixing activated carbon and attapulgite, acid washing and calcining to obtain a first mixture; mixing the first mixture, chitosan and acetic acid for reaction. It can be seen from the examples that the highest content of DNJ in the extract is 1.48% in Example 1.

[0006] After frost, the mulberry leaves are induced to express related genes due to environmental factors such as low temperature, which increases the synthesis and accumulation of DNJ. The content of DNJ can reach about 0.8%-1.5%, which is basically the same as the DNJ content obtained after extraction from mulberry leaves, or even higher. However, since the mulberry leaves are frosted in the later stage of growth, in order to adapt to the external environment in a low temperature environment, the mulberry leaves will adjust the composition of their cell walls, increase the cellulose content relatively, and enhance the toughness of the cell walls, thus playing a better protective role.

[0007] Since the cell walls of mulberry leaves after frost may be denser, when extracting intracellular components (such as 1-deoxynojirimycin (DNJ), etc.), the solvent or extraction method needs to more effectively destroy the cell wall structure in order to dissolve the components smoothly. For example, when using solvent extraction, a stronger solvent or auxiliary means (such as ultrasound, microwave, etc.) may be needed to penetrate the cell wall of mulberry leaves after frost; if enzymatic extraction is used, the effect of the enzyme on the cell wall of mulberry leaves after frost will also be different. Due to the differences in cell wall composition and structure, the enzymatic hydrolysis of the cell wall of mulberry leaves after frost may require adjustment or increase of the type and amount of enzymes. However, there is currently no practical extraction method that can maximize the extraction of effective ingredients in mulberry leaves after frost. Most of them are still traditional methods for extracting mulberry leaf components. Under the condition that the annual output of mulberry leaves after frost is lower than that of mulberry leaves, a large amount of mulberry leaf resources after frost are wasted, which increases the extraction cost of enterprises.

[0008] At the same time, 1-deoxynojirimycin (DNJ) needs to be stored at a low temperature of 2-8°C and must be kept away from light and sealed. Therefore, the existing mulberry leaf extract is also stored at 2-8°C, which also increases the storage cost.

[0009] Based on the above-mentioned prior art, since the cell wall structure of mulberry leaves after frost is more compact, the use of traditional mulberry leaf extraction methods has technical problems that need to be solved urgently, such as increased amount of extraction raw materials, increased types, or high cost of extract preservation. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides a method for extracting a frost-treated mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-covered mulberry leaves: selecting fresh, disease-free and insect-free frost-covered mulberry leaves, removing petioles and impurities, washing, drying or air-drying, and then crushing into frost-covered mulberry leaf powder with a particle size of 40-90 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1-0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of (3-5): (2-3): (1-4); Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water to obtain a hyaluronic acid aqueous solution with a hyaluronic acid content of 0.2-0.4% by mass; Step 4, enzymatic hydrolysis treatment: adding the frost mulberry leaf powder and the complex enzyme aqueous solution into a reaction container, and simultaneously dropping the hyaluronic acid aqueous solution to perform an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-treated mulberry leaf extract: heating the enzymatic hydrolysis reaction solution to inactivate the enzyme, filtering to remove the residue, concentrating and drying the filtrate to obtain a powdered frost-treated mulberry leaf extract containing 1-deoxynojirimycin (DNJ); The mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 5.2-7.0%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is greater than or equal to 89%.

[0011] Furthermore, the test method for the mass content of 1-deoxynojirimycin is to use a gas phase method for detection.

[0012] Furthermore, the cleaning in step 1 is cleaning with clean water.

[0013] Furthermore, the drying or air-drying temperature in step 1 is 30-60° C., and the water content of the frosted mulberry leaves is ≤10wt%.

[0014] Furthermore, the cellulase in step 2 includes one or more of endoglucanase, exoglucanase and β-glucosidase.

[0015] Furthermore, the hemicellulase in step 2 includes one or more of xylanase, arabinofuranosidase, galactomannanase and glucomannanase.

[0016] Furthermore, the pectinase in step 2 includes one or more of pectin methylesterase, polygalacturonase, and pectin lyase.

[0017] Furthermore, the dissolution temperatures in step 2 and step 3 are both at room temperature.

[0018] Furthermore, the molecular weight of the hyaluronic acid in step 3 is 3000-5000 Daltons.

[0019] Furthermore, in step 4, the solid-to-liquid ratio of the frost mulberry leaf powder to the complex enzyme aqueous solution is 1:(10-30), the solid mass unit is g, and the liquid volume unit is mL.

[0020] Furthermore, in step 4, the solid-to-liquid ratio of the frosted mulberry leaf powder to the hyaluronic acid aqueous solution is 1:(2-7), the solid mass unit is g, and the liquid volume unit is mL.

[0021] Furthermore, in step 4, the pH value of the enzymatic hydrolysis reaction is 4.0-6.0, the temperature is 40-60° C., and the time is 1-3 h.

[0022] Furthermore, the time of the dripping in step 4 is the same as the time of the enzymatic hydrolysis reaction.

[0023] Furthermore, during the enzymatic hydrolysis in step 4, stirring is continued at a speed of 150-300 rad / min.

[0024] Furthermore, the temperature of the enzyme inactivation treatment in step 5 is 80-90° C. and the time is 10-20 min.

[0025] Furthermore, the filtering method in step 5 includes but is not limited to reduced pressure filtration.

[0026] Furthermore, the pressure of the reduced pressure filtration is -0.07MPa to -0.09MPa.

[0027] Furthermore, the concentration in step 5 is to concentrate the filtrate to 1 / 4 of the original filtrate volume.

[0028] Furthermore, the drying in step 5 is spray drying, wherein the air inlet temperature is 150-190°C and the air outlet temperature is 60-80°C.

[0029] The present invention also provides a frost-frozen mulberry leaf extract, which is obtained by extracting through the above-mentioned method for extracting the frost-frozen mulberry leaf extract.

[0030] The present invention also provides a blood sugar lowering product, which is the above-mentioned frost mulberry leaf extract, or includes the above-mentioned frost mulberry leaf extract, or is prepared from the above-mentioned frost mulberry leaf extract.

[0031] The beneficial effects of the present invention are: 1. The present invention provides a method for extracting a frost-frozen mulberry leaf extract, which comprises pretreatment of the frost-frozen mulberry leaf, preparation of a composite enzyme aqueous solution, preparation of a hyaluronic acid aqueous solution, enzymatic hydrolysis, and extraction of the frost-frozen mulberry leaf extract. By selecting the raw materials specifically and coordinating with a specific extraction process, 1-deoxynojirimycin in the frost-frozen mulberry leaf with denser cell walls is successfully enriched and extracted. Compared with the traditional mulberry leaf extract extraction method in the prior art, under the condition that the annual output of the frost-frozen mulberry leaf itself is lower than that of the mulberry leaf, the resource utilization of the frost-frozen mulberry leaf is effectively maximized, and a frost-frozen mulberry leaf extract with a 1-deoxynojirimycin (DNJ) mass content of up to 5.2-7.0% is obtained; at the same time, the limitation of the prior art that the extract needs to be stored under harsh conditions of 2-8°C is broken. After the frost-frozen mulberry leaf extract extracted by the present invention is stored in a dark environment at 18°C ​​for 300 days, the mass content retention rate of the 1-deoxynojirimycin rich therein can be greater than or equal to 89%, which further reduces the storage cost for its subsequent popularization as a hypoglycemic product; 2. The present invention makes a specific selection of the particle size of the frost mulberry leaf powder, and specifically selects the particle size of the frost mulberry leaf powder to be between 40-90 meshes. If the particle size is too small, although the cell wall will be broken to a certain extent, it will also cause the effective substance to be exposed in advance, because 1-deoxynojirimycin is a polyhydroxy alkaloid, and some chemical bonds and functional groups (such as hydroxyl groups) in its molecular structure are sensitive to light. Under light conditions, especially ultraviolet (UV) and part of the visible light band, light energy may be absorbed by DNJ molecules. This process can enable the molecule to obtain sufficient energy, causing the chemical bonds inside it to break, rearrange or react with substances in the surrounding environment. Therefore, if the particle size of the frost mulberry leaf powder is too small, 1-deoxynojirimycin will fail prematurely to a certain extent, resulting in extraction failure; and if the particle size is too large, the dense cell wall of the frost mulberry leaf cannot be effectively broken in the pre-treatment part, so that the rich 1-deoxynojirimycin therein cannot be extracted to the maximum extent; 3. In the extraction method of the present invention, a composite enzyme aqueous solution is specifically selected to enzymolyze the frost mulberry leaf powder, that is, cellulase, hemicellulase, and pectinase in a specific mass ratio of (3-5): (2-3): (1-4) are selected, rather than a single enzyme hydrolyzate for enzymolysis. This is because the frost mulberry leaves are frosted in the later growth period. In a low temperature environment, in order to adapt to the external environment, the mulberry leaves will adjust the material composition of the cell wall, so that the content of the substances in the cell wall is relatively increased or the proportion is changed. These substances are cross-linked to form a solid structure to enhance the toughness of the cell wall and play a better protective role. This protective structure makes it more difficult for the effective substances in the cell to be released. Therefore, while selecting the frost mulberry leaf powder of a specific particle size, the type and ratio of the composite enzyme are also specifically limited, which can effectively disintegrate this dense structure and fully extract the rich 1-deoxynojirimycin therein; 4. The present invention also specifically adds a hyaluronic acid aqueous solution that has not been used in the existing extraction method, and adds it to the system in the process of enzymatic hydrolysis by dripping, because the chemical formula of 1-deoxynojirimycin is C6H 13 NO4 contains multiple hydroxyl groups (-OH) in its molecule. These hydroxyl groups make DNJ have a certain hydrophilicity. Hyaluronic acid is an acidic mucopolysaccharide, and its molecules contain a large number of carboxyl groups (-COOH) and hydroxyl groups (-OH). These functional groups make it highly hydrophilic and water-retaining, and can bind to other molecules through interactions such as hydrogen bonds. Therefore, in the process of enzymatic hydrolysis, the carboxyl groups and hydroxyl groups on hyaluronic acid can effectively and gradually capture the gradually released 1-deoxynojirimycin by dripping, and form stable hydrogen bonds or chemical bonds with the hydroxyl groups on 1-deoxynojirimycin. This microscopic interaction can improve the stability of 1-deoxynojirimycin in the system on the one hand, and effectively improve the distribution of 1-deoxynojirimycin in the system on the other hand, and reduce the entanglement and interference of other substances in the frost mulberry leaves on 1-deoxynojirimycin. Therefore, the frost mulberry leaf extract obtained by the present invention through specific raw materials and processes can be stored in a dark environment at 18°C ​​for 300 days, and the retention rate of the mass content of 1-deoxynojirimycin rich in it can be greater than or equal to 89%; At the same time, the present invention selects small molecular weight hyaluronic acid, which not only ensures the advantage of long-term storage of 1-deoxynojirimycin in the frost mulberry leaf extract, but also can maintain the safety performance of the product after being prepared into the corresponding blood sugar-lowering product and consumed by the human body. It is very friendly to people with high blood sugar and can be absorbed or excreted through the matrix's own metabolic process, further laying the foundation for the popularization of the blood sugar-lowering product. DETAILED DESCRIPTION

[0032] Examples 1-3 are extractions performed using frost-post-mulberry leaves produced in 2019; Examples 6-9 are extractions using frost-post-mulberry leaves produced in 2021; Examples 10-12 are extractions performed using frost-post-mulberry leaves produced in 2023; The test method for the mass content of 1-deoxynojirimycin is to use the gas phase method for detection, specifically according to the method disclosed in the journal "Northern Sericulture", authored by Li Hong et al., and titled "Gas Phase Determination of -Deoxynojirimycin (DNJ) in Mulberry Leaves".

[0033] At the same time, the present invention also determines the natural mass content of 1-deoxynojirimycin in frost-post-mulberry leaves harvested in different years, as shown in Table 1.

[0034] Example 1 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: select fresh, disease-free and insect-free frost-post-mulberry leaves, remove petioles and impurities, wash with clean water, dry at 30° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then grind into frost-post-mulberry leaf powder with a particle size of 40-50 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 3:3:2; In this embodiment, the cellulase is endoglucanase, the hemicellulase is arabinofuranosidase, and the pectinase is pectin lyase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 100 mL of complex enzyme aqueous solution are added into a reaction container, and 30 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 150 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 40°C, the time is 1.5 h, and the dropping time is also controlled at 1.5 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 80° C., the time is 20 min, and the pressure is controlled to be between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 150° C., and the outlet air temperature is 80° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frosted mulberry leaf extract is 6.1%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 94%.

[0035] Example 2 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, drying at 60° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 50-60 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.3% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 5:2:4; In this embodiment, the cellulase is endoglucanase, the hemicellulase is galactomannanase, and the pectinase is polygalacturonase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.2%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 200 mL of complex enzyme aqueous solution are added into a reaction container, and 70 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 200 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 50°C, the time is 1 h, and the dropping time is also controlled at 1 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 85° C., the time is 15 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 170° C., and the outlet air temperature is 70° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 5.3%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 93%.

[0036] Example 3 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, naturally drying until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 70-80 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 4:2:3; In this embodiment, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 300 mL of complex enzyme aqueous solution are added into a reaction container, and 50 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 300 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 60°C, the time is 2 h, and the dropping time is also controlled at 2 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 90° C., the time is 10 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 190° C., and the outlet air temperature is 60° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 6.8%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 96%.

[0037] Example 4 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, drying at 30° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 60-70 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 3:3:2; In this embodiment, the cellulase is exoglucanase, the hemicellulase is xylanase, and the pectinase is pectin methylesterase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 3000-4000 Daltons; Step 4, enzymatic hydrolysis treatment: 10 g of frost mulberry leaf powder and 100 mL of complex enzyme aqueous solution are added into a reaction container, and 30 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 150 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 40°C, the time is 3 h, and the dropping time is also controlled at 1.5 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 80° C., the time is 20 min, and the pressure is controlled to be between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 150° C., and the outlet air temperature is 80° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 6.5%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 91%.

[0038] Example 5 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, drying at 60° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 80-90 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.3% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 5:2:1; In this embodiment, the cellulase is exoglucanase, the hemicellulase is galactomannanase, and the pectinase is pectin lyase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.2%; wherein the molecular weight of the hyaluronic acid is controlled between 3000-4000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 200 mL of complex enzyme aqueous solution are added into a reaction container, and 20 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 200 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 50°C, the time is 1 h, and the dropping time is also controlled at 1 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 85° C., the time is 15 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 170° C., and the outlet air temperature is 70° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 7.0%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 94%.

[0039] Example 6 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, naturally drying until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 70-90 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 4:2:3; In this embodiment, the cellulase is exoglucanase, the hemicellulase is glucomannanase, and the pectinase is polygalacturonase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 3000-4000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 300 mL of complex enzyme aqueous solution are added into a reaction container, and 50 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 300 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 60°C, the time is 2 h, and the dropping time is also controlled at 2 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 90° C., the time is 10 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 190° C., and the outlet air temperature is 60° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 5.6%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 95%.

[0040] Example 7 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: select fresh, disease-free and insect-free frost-post-mulberry leaves, remove petioles and impurities, wash with clean water, dry at 30° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then grind into frost-post-mulberry leaf powder with a particle size of 40-50 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 3:3:2; In this embodiment, the cellulase is β-glucosidase, the hemicellulase is xylanase, and the pectinase is pectin methylesterase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 3000-4000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 100 mL of complex enzyme aqueous solution are added into a reaction container, and 30 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 150 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 40°C, the time is 1.5 h, and the dropping time is also controlled at 1.5 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 80° C., the time is 20 min, and the pressure is controlled to be between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 150° C., and the outlet air temperature is 80° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 5.2%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 90%.

[0041] Example 8 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, drying at 60° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 50-60 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.3% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 5:2:4; In this embodiment, the cellulase is β-glucosidase, the hemicellulase is galactomannanase, and the pectinase is pectin lyase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.2%; wherein the molecular weight of the hyaluronic acid is controlled between 3000-4000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 200 mL of complex enzyme aqueous solution are added into a reaction container, and 70 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 200 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 50°C, the time is 1 h, and the dropping time is also controlled at 1 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 85° C., the time is 15 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 170° C., and the outlet air temperature is 70° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 5.3%.

[0042] The test method for the mass content of 1-deoxynojirimycin is to use a gas phase method for detection, specifically according to the method disclosed in the journal "Northern Sericulture", authored by Li Hong et al., and titled "Gas Phase Determination of -Deoxynojirimycin (DNJ) in Mulberry Leaves"; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 96%.

[0043] Example 9 This embodiment provides a method for extracting frost-removed mulberry leaf extract, comprising the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, naturally drying until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 70-90 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 4:2:3; In this embodiment, the cellulase is β-glucosidase, the hemicellulase is arabinofuranosidase, and the pectinase is polygalacturonase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 3000-4000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 300 mL of complex enzyme aqueous solution are added into a reaction container, and 50 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 300 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 60°C, the time is 2 h, and the dropping time is also controlled at 2 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 90° C., the time is 10 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 190° C., and the outlet air temperature is 60° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this embodiment, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 6.8%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 89%.

[0044] Comparative Example 1 This comparative example provides a method for extracting frost-frozen mulberry leaf extract relative to Example 1. The difference from Example 1 is that the particle size of the frost-frozen mulberry leaf powder in this comparative example is between 20-30 meshes, and specifically comprises the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, drying at 30° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 20-30 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 3:3:2; In this embodiment, the cellulase is endoglucanase, the hemicellulase is arabinofuranosidase, and the pectinase is pectin lyase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 100 mL of complex enzyme aqueous solution are added into a reaction container, and 30 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 150 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 40°C, the time is 1.5 h, and the dropping time is also controlled at 1.5 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 80° C., the time is 20 min, and the pressure is controlled to be between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 150° C., and the outlet air temperature is 80° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this comparative example, the mass content of 1-deoxynojirimycin in the frost-frosted mulberry leaf extract is 3.9%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 68%.

[0045] Comparative Example 2 This comparative example provides a method for extracting a frost-frozen mulberry leaf extract relative to Example 1. The difference from Example 1 is that the particle size of the frost-frozen mulberry leaf powder in this comparative example is between 100-150 meshes, and specifically comprises the following steps: Step 1, pretreatment of frost-post-mulberry leaves: select fresh, disease-free and insect-free frost-post-mulberry leaves, remove petioles and impurities, wash with clean water, dry at 30° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then grind into frost-post-mulberry leaf powder with a particle size of 100-150 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 3:3:2; In this embodiment, the cellulase is endoglucanase, the hemicellulase is arabinofuranosidase, and the pectinase is pectin lyase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 100 mL of complex enzyme aqueous solution are added into a reaction container, and 30 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 150 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 40°C, the time is 1.5 h, and the dropping time is also controlled at 1.5 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 80° C., the time is 20 min, and the pressure is controlled to be between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 150° C., and the outlet air temperature is 80° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this comparative example, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 2.7%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 79%.

[0046] Comparative Example 3 This comparative example provides a method for extracting frost-frozen mulberry leaf extract relative to Example 2. The difference from Example 2 is that a single enzyme aqueous solution is used in this comparative example, and specifically includes the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, drying at 60° C. until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 50-60 mesh; Step 2, preparing a single enzyme aqueous solution: dissolving cellulase in water at room temperature to obtain a single enzyme aqueous solution with a cellulase content of 0.3% by mass; In this comparative example, the cellulase is endoglucanase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.2%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frosted mulberry leaf powder and 200 mL of a single enzyme aqueous solution are added into a reaction container, and 70 mL of a hyaluronic acid aqueous solution is added dropwise to perform an enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 200 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 50° C., the time is 1 h, and the dropping time is also controlled at 1 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 85° C., the time is 15 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 170° C., and the outlet air temperature is 70° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this comparative example, the mass content of 1-deoxynojirimycin in the frost-frosted mulberry leaf extract is 3.1%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 74%.

[0047] Comparative Example 4 This comparative example provides a method for extracting frost-removed mulberry leaf extract relative to Example 3. The difference from Example 3 is that no hyaluronic acid aqueous solution is used in this comparative example. Specifically, the method comprises the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, naturally drying until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 70-80 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 4:2:3; In this comparative example, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase; Step 3, enzymolysis treatment: 10 g of frosted mulberry leaf powder and 300 mL of a composite enzyme aqueous solution are added into a reaction container to perform an enzymolysis reaction, and the enzymolysis reaction is continuously stirred during the reaction, wherein the stirring speed is 300 rad / min, the pH value of the enzymolysis reaction is controlled at 4.0-6.0, the temperature is 60° C., and the time is 2 h to obtain an enzymolysis reaction solution; Step 4, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 90° C., the time is 10 min, and the pressure is controlled to be between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 190° C., and the outlet air temperature is 60° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this comparative example, the mass content of 1-deoxynojirimycin in the frost-frosted mulberry leaf extract is 3.6%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 37%.

[0048] Comparative Example 5 This comparative example provides a method for extracting frost-frosted mulberry leaf extract relative to Example 3. The difference from Example 3 is that in this comparative example, the hyaluronic acid aqueous solution is added to the reaction system at one time, and specifically comprises the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, naturally drying until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 70-80 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 4:2:3; In this comparative example, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 Daltons; Step 4, enzymatic hydrolysis treatment: 10 g of frost mulberry leaf powder, 300 mL of complex enzyme aqueous solution, and 50 mL of hyaluronic acid aqueous solution are added into a reaction container for enzymatic hydrolysis reaction, and stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 300 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 60° C., and the time is 2 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 90° C., the time is 10 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 190° C., and the outlet air temperature is 60° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this comparative example, the mass content of 1-deoxynojirimycin in the frost-frosted mulberry leaf extract was 2.9%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 52%.

[0049] Comparative Example 6 This comparative example provides a method for extracting frost mulberry leaf extract relative to Example 3. The difference from Example 3 is that the molecular weight of hyaluronic acid in this comparative example is between 20,000 and 50,000 Daltons, and specifically comprises the following steps: Step 1, pretreatment of frost-post-mulberry leaves: selecting fresh, disease-free and insect-free frost-post-mulberry leaves, removing petioles and impurities, washing with clean water, naturally drying until the moisture content of the frost-post-mulberry leaves is ≤10wt%, and then crushing into frost-post-mulberry leaf powder with a particle size of 70-80 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of 4:2:3; In this comparative example, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase; Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a hyaluronic acid mass content of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 20,000 and 50,000 Daltons; Step 4, enzymatic hydrolysis: 10 g of frost mulberry leaf powder and 300 mL of complex enzyme aqueous solution are added into a reaction container, and 50 mL of hyaluronic acid aqueous solution is added dropwise to perform enzymatic hydrolysis reaction. Stirring is continued during the enzymatic hydrolysis reaction, wherein the stirring speed is 300 rad / min, the pH value of the enzymatic hydrolysis reaction is controlled at 4.0-6.0, the temperature is 60°C, the time is 2 h, and the dropping time is also controlled at 2 h to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction liquid to inactivate the enzyme, wherein the temperature of the inactivation treatment is 90° C., the time is 10 min, and the pressure is controlled between -0.07 MPa and -0.09 MPa to perform reduced pressure filtration to remove the residue, and the filtrate is concentrated to 1 / 4 of the original filtrate volume, and then spray-dried, wherein the inlet air temperature is 190° C., and the outlet air temperature is 60° C., to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin (DNJ); In this comparative example, the mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 2.2%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 45%.

[0050] Comparative Example 7 In this comparative example, an equal amount of mulberry leaves in Example 1 disclosed in the prior art CN114177218B are replaced with frost-post-mulberry leaves in the present invention (produced in 2019, the same raw materials as the frost-post-mulberry leaves in Examples 1-3 of the present invention), and the experimental steps are exactly the same as those in Example 1 disclosed in the technical scheme to obtain a powdery finished product prepared according to CN114177218B.

[0051] The finished product in this comparative example was tested, and the mass content of 1-deoxynojirimycin in the finished product was 1.7%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract was 51%.

[0052] The mass content of 1-deoxynojirimycin in Example 1-9 and Comparative Example 1-7 and the mass content retention rate after being stored in a dark place at 18° C. for 300 days were compared. The comparison results are shown in Table 1: Table 1 Comparison of the mass content of 1-deoxynojirimycin in Examples 1-9 and Comparative Examples 1-7

[0053] Through the above comparison, the present invention obtains a frost-post-mulberry leaf extract with a 1-deoxynojirimycin (DNJ) mass content of up to 5.2-7.0% under the conditions of using specific raw materials and specific extraction processes. Compared with the traditional mulberry leaf extract extraction method in the prior art, under the condition that the annual output of frost-post-mulberry leaves is lower than that of mulberry leaves, the resource utilization of frost-post-mulberry leaves is effectively maximized, and at the same time, it breaks the limitation of the prior art that mulberry leaf extracts need to be stored under harsh conditions of 2-8°C. After the frost-post-mulberry leaf extract extracted by the present invention through a specific extraction process is stored in a dark place at 18°C ​​for 300 days, the retention rate of the mass content of 1-deoxynojirimycin rich therein can be greater than or equal to 89%, which further reduces the storage cost for its subsequent popularization as a blood sugar-lowering product.

[0054] It should be understood that the present invention is not limited to what has been described above and that various modifications and changes may be made without departing from its scope. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for extracting frost-frosted mulberry leaf extract, characterized in that: The steps include: Step 1, pretreatment of frost-covered mulberry leaves: selecting fresh, disease-free and insect-free frost-covered mulberry leaves, removing petioles and impurities, washing, drying or air-drying, and then crushing into frost-covered mulberry leaf powder with a particle size of 40-90 mesh; Step 2, preparing a composite enzyme aqueous solution: dissolving the composite enzyme in water to obtain a composite enzyme aqueous solution with a composite enzyme content of 0.1-0.5% by mass; The complex enzyme is composed of cellulase, hemicellulase and pectinase in a mass ratio of (3-5): (2-3): (1-4); Step 3, preparing a hyaluronic acid aqueous solution: dissolving hyaluronic acid in water to obtain a hyaluronic acid aqueous solution with a hyaluronic acid content of 0.2-0.4% by mass; Step 4, enzymatic hydrolysis treatment: adding the frost mulberry leaf powder and the complex enzyme aqueous solution into a reaction container, and simultaneously dropping the hyaluronic acid aqueous solution to perform an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysis reaction solution; Step 5, extracting the frost-frozen mulberry leaf extract: heating the enzymatic hydrolysis reaction solution to inactivate the enzyme, filtering to remove the residue, concentrating and drying the filtrate to obtain a powdered frost-frozen mulberry leaf extract containing 1-deoxynojirimycin; The mass content of 1-deoxynojirimycin in the frost-frozen mulberry leaf extract is 5.2-7.0%; After being stored in a dark environment at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is greater than or equal to 89%.

2. The method for extracting the frost-frosted mulberry leaf extract according to claim 1, characterized in that: The drying or air-drying temperature in step 1 is 30-60° C., and the moisture content of the frost-free mulberry leaves is ≤10wt%.

3. The method for extracting the frost-frosted mulberry leaf extract according to claim 1, characterized in that: The cellulase in step 2 includes one or more of endoglucanase, exoglucanase, and β-glucosidase; The hemicellulase includes one or more of xylanase, arabinofuranosidase, galactomannanase and glucomannanase; The pectinase includes one or more of pectin methylesterase, polygalacturonase and pectin lyase.

4. The method for extracting the frost-frosted mulberry leaf extract according to claim 1, characterized in that: The molecular weight of the hyaluronic acid described in step 3 is 3000-5000 Daltons.

5. The method for extracting the frost-frosted mulberry leaf extract according to claim 4, characterized in that: In step 4, the solid-to-liquid ratio of the frost mulberry leaf powder to the complex enzyme aqueous solution is 1:(10-30), the solid mass unit is g, and the liquid volume unit is mL; The solid-to-liquid ratio of the frosted mulberry leaf powder and the hyaluronic acid aqueous solution in step 4 is 1:(2-7), the solid mass unit is g, and the liquid volume unit is mL.

6. The method for extracting the frost-frosted mulberry leaf extract according to claim 1, characterized in that: The pH value of the enzymatic hydrolysis reaction in step 4 is 4.0-6.0, the temperature is 40-60° C., and the time is 1-3 h.

7. The method for extracting the frost-frosted mulberry leaf extract according to claim 6, characterized in that: The temperature of the enzyme inactivation treatment in step 5 is 80-90° C. and the time is 10-20 min.

8. The method for extracting the frost-frosted mulberry leaf extract according to claim 7, characterized in that: The drying in step 5 is spray drying, wherein the air inlet temperature is 150-190°C and the air outlet temperature is 60-80°C.

9. A frost-frosted mulberry leaf extract, characterized in that: The frost-frozen mulberry leaf extract is obtained by extracting the frost-frozen mulberry leaf extract according to any one of claims 1 to 8.

10. A blood sugar lowering product, characterized in that: The blood sugar lowering product is the frosted mulberry leaf extract according to claim 9, or includes the frosted mulberry leaf extract according to claim 9, or is prepared from the frosted mulberry leaf extract according to claim 9.

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