Extraction method and application of mulberry leaf extract after frost

Abstract

The present invention relates to the technical field of extraction of mulberry leaves after frost, and in particular to an extraction method and application of an extract of mulberry leaves after frost. The extraction method sequentially includes pretreatment of mulberry leaves after frost, preparation of a composite enzyme aqueous solution, preparation of a hyaluronic acid aqueous solution, enzymatic hydrolysis treatment, and extraction of an extract of mulberry leaves after frost. Through specific selection of raw materials and cooperation with a specific extraction process, an extract of mulberry leaves after frost with a 1-deoxynojirimycin mass content as high as 5.2-7.0% is obtained; at the same time, it breaks through the limitation that the existing mulberry leaf extract needs to be stored under harsh conditions of 2-8°C. After the extract of mulberry leaves after frost extracted by a specific extraction process is stored in the dark at 18°C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin it is rich in can be greater than or equal to 89%, further reducing the storage cost for its subsequent popularization as a hypoglycemic product.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of mulberry leaves after frost, and particularly relates to an extraction method and application of an extract of mulberry leaves after frost. Background Art

[0002] 1-Deoxynojirimycin (DNJ) is a polyhydroxy alkaloid with significant physiological activities such as reducing blood sugar, blood pressure, and blood lipid. The journal "Processing of Agricultural Products", authors Ren Xiangdong et al. in the article named "Research Progress on the Extraction and Purification Process of 1-Deoxynojirimycin in Mulberry Leaves and Its Hypoglycemic Effect" mentioned that the inhibitory effect of DNJ on α-glucosidase (IC 50 =0.350 mg / mL) is 2.7 times that of the hypoglycemic drug acarbose (IC 50 =0.982 mg / mL), and it has broad application prospects in the development of hypoglycemic foods.

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

[0004] For example, CN114177218B discloses a mulberry leaf extract rich in 1-deoxynojirimycin and a preparation method thereof. The preparation method includes the following steps: S1. Pretreatment: cleaning, drying, and pulverizing mulberry leaves to obtain mulberry leaf powder; S2. Ultrasonic treatment: adding the mulberry leaf powder into water, performing ultrasonic treatment, centrifuging, and taking the supernatant to obtain supernatant 1 and residue; S3. Enzymatic hydrolysis treatment: adding the same mass of water to the residue, first hydrolyzing with complex enzyme 1, then hydrolyzing with complex enzyme 2, centrifuging, and taking the supernatant to obtain supernatant 2; S4. Filtration, concentration, and drying: combining supernatant 1 in S2 and supernatant 2 in S3, and 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 a method for extracting and applying sugar-control components in mulberry leaves. The extraction method includes the following steps: mixing mulberry leaf powder and acidic ethanol solution and performing ultrasonic extraction to obtain a first extract and a first extraction residue; enzymatically treating the first extraction residue and collecting a second extract; combining the first extract and the second extract, concentrating and decolorizing to obtain a decolorized solution; performing microfiltration, nanofiltration and membrane concentration on the decolorized solution and then drying; the enzymatic treatment includes a first enzymatic treatment and a second enzymatic treatment; a composite decolorizing agent is added during the decolorization process; the preparation method of the composite decolorizing agent includes the following steps: mixing activated carbon and attapulgite, performing pickling and calcination to obtain a first mixture; reacting the first mixture, chitosan and acetic acid. As can be seen from its examples, the highest content of DNJ in the extract is 1.48% in Example 1.

[0006] After the mulberry leaves are frosted, due to the induction of the expression of related genes by environmental factors such as low temperature, the synthesis and accumulation of DNJ increase, and its content can reach about 0.8%-1.5%, which is basically the same as or even higher than the DNJ content obtained after extracting mulberry leaves. However, because the mulberry leaves are frosted in the later growth stage and are in a low-temperature environment, in order to adapt to the external environment, the mulberry leaves will adjust the composition of the cell wall, increasing the cellulose content relatively to enhance the toughness of the cell wall and play a better protective role.

[0007] Since the cell wall of frosted mulberry leaves 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 to enable the components to dissolve smoothly. For example, when using a solvent for extraction, a stronger solvent or auxiliary means (such as ultrasound, microwave, etc.) may be required to penetrate the cell wall of frosted mulberry leaves; if an enzymatic extraction method is used, the effect of the enzyme on the cell wall of frosted mulberry leaves will also be different. Due to the differences in the composition and structure of the cell wall, the enzymatic hydrolysis of the cell wall of frosted mulberry leaves may require adjusting or increasing the types and dosages of enzymes. However, there is currently no practical extraction method that can maximize the extraction of the effective components in frosted mulberry leaves. Most are still traditional extraction methods for mulberry leaf components. Under the condition that the annual output of frosted mulberry leaves is itself lower than that of mulberry leaves, a large amount of frosted mulberry leaf resources are wasted, increasing the extraction cost of enterprises.

[0008] At the same time, 1-deoxynojirimycin (DNJ) needs to be stored in a low-temperature environment of 2-8°C, and it is necessary to ensure light-proof and sealed storage. Therefore, the existing technology mulberry leaf extract is also stored under the condition of 2-8°C, which also increases the storage cost.

[0009] Based on the above situation of the prior art, in the prior art, due to the more compact structure of the cell wall of mulberry leaves after frost, there are technical problems that need to be solved urgently, such as an increase in the amount of extraction raw materials used, an increase in types, or a high cost of preserving the extract when using traditional mulberry leaf extraction methods. Summary of the Invention

[0010] To solve the above technical problems, the present invention provides a method for extracting an extract of mulberry leaves after frost, which includes the following steps:

[0011] Step 1, pretreatment of mulberry leaves after frost: Select fresh and pest-free mulberry leaves after frost, remove the petioles and impurities, wash them, and then dry them by drying or natural air drying, and then crush them into mulberry leaf powder after frost with a particle size of 40-90 mesh;

[0012] Step 2, prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water to obtain a composite enzyme aqueous solution with a mass content of the composite enzyme of 0.1-0.5%;

[0013] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase according to a mass ratio of (3-5):(2-3):(1-4);

[0014] Step 3, prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water to obtain a hyaluronic acid aqueous solution with a mass content of hyaluronic acid of 0.2-0.4%;

[0015] Step 4, enzymatic hydrolysis treatment: Add the mulberry leaf powder after frost and the composite enzyme aqueous solution into a reaction vessel, and at the same time dropwise add the hyaluronic acid aqueous solution to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysis reaction solution;

[0016] Step 5, extract the extract of mulberry leaves after frost: Heat the enzymatic hydrolysis reaction solution to carry out enzyme inactivation treatment, filter to remove the residue, and after concentrating and drying the filtrate, obtain a powdery extract of mulberry leaves after frost containing 1-deoxynojirimycin (DNJ);

[0017] The mass content of 1-deoxynojirimycin in the extract of mulberry leaves after frost is 5.2-7.0%;

[0018] Stored in the dark at 18°C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the extract of mulberry leaves after frost is greater than or equal to 89%.

[0019] Further, the test method for the mass content of 1-deoxynojirimycin is to detect it by using gas phase method.

[0020] Further, the washing in Step 1 is washing with clean water.

[0021] Further, the temperature for drying or natural air drying in Step 1 is 30-60°C, and at this time the water content of the mulberry leaves after frost ≤ 10wt%.

[0022] Further, the cellulase described in step 2 includes one or more of endoglucanase, exoglucanase, and β-glucosidase.

[0023] Further, the hemicellulase described in step 2 includes one or more of xylanase, arabinofuranosidase, galactomannanase, and glucomannanase.

[0024] Further, the pectinase described in step 2 includes one or more of pectin methylesterase, polygalacturonase, and pectin lyase.

[0025] Further, the temperatures for dissolution in both step 2 and step 3 are normal temperatures.

[0026] Further, the molecular weight of the hyaluronic acid described in step 3 is 3,000 - 5,000 Daltons.

[0027] Further, the solid-liquid ratio of the defrosted mulberry leaf powder to the complex enzyme aqueous solution in step 4 is 1:(10 - 30), where the unit of solid mass is g and the unit of liquid volume is mL.

[0028] Further, the solid-liquid ratio of the defrosted mulberry leaf powder to the hyaluronic acid aqueous solution in step 4 is 1:(2 - 7), where the unit of solid mass is g and the unit of liquid volume is mL.

[0029] Further, 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.

[0030] Further, the dropping time in step 4 is the same as the time of the enzymatic hydrolysis reaction.

[0031] Further, continuous stirring is performed during the enzymatic hydrolysis in step 4, and the stirring speed is 150 - 300 rad / min.

[0032] Further, the temperature of the enzyme inactivation treatment in step 5 is 80 - 90 °C, and the time is 10 - 20 min.

[0033] Further, the filtration method in step 5 includes, but is not limited to, vacuum filtration.

[0034] Further, the pressure of the vacuum filtration is -0.07 MPa to -0.09 MPa.

[0035] Further, the concentration in step 5 is to concentrate the filtrate to 1 / 4 of the original filtrate volume.

[0036] Further, the drying in step 5 is spray drying, where the inlet air temperature is 150 - 190 °C and the outlet air temperature is 60 - 80 °C.

[0037] The present invention also provides an extract of mulberry leaves after frost, which is obtained by the extraction method of the above-mentioned extract of mulberry leaves after frost.

[0038] The present invention also provides a blood sugar-lowering product, which is the above-mentioned extract of mulberry leaves after frost, or includes the above-mentioned extract of mulberry leaves after frost, or is prepared from the above-mentioned extract of mulberry leaves after frost.

[0039] The beneficial effects of the present invention are as follows:

[0040] 1. The present invention provides an extraction method for an extract of mulberry leaves after frost, which successively includes pretreatment of mulberry leaves after frost, preparation of a composite enzyme aqueous solution, preparation of a hyaluronic acid aqueous solution, enzymatic hydrolysis treatment, and extraction of the extract of mulberry leaves after frost. Through specific selection of raw materials and in combination with a specific extraction process, 1-deoxynojirimycin in mulberry leaves after frost with denser cell walls is successfully enriched and extracted. Compared with the traditional extraction method of mulberry leaf extract in the prior art, under the condition that the annual output of mulberry leaves after frost is lower than that of mulberry leaves itself, the resource utilization of mulberry leaves after frost is effectively maximized, and an extract of mulberry leaves after frost with a 1-deoxynojirimycin (DNJ) mass content as high as 5.2 - 7.0% is obtained; at the same time, it breaks through the limitation that the existing technology extract needs to be stored under harsh conditions of 2 - 8°C. The extract of mulberry leaves after frost extracted by the specific extraction process of the present invention can maintain a retention rate of the mass content of the rich 1-deoxynojirimycin greater than or equal to 89% after being stored in the dark at 18°C for 300 days, further reducing the storage cost for its subsequent popularization as a blood sugar-lowering product;

[0041] 2. The present invention makes a specific selection of the particle size of the mulberry leaf powder after frost. The particle size of the mulberry leaf powder after frost is specifically selected to be between 40 - 90 mesh. If the particle size is too small, although it will break the cell wall to a certain extent, it will also cause the premature exposure of effective substances. Since 1-deoxynojirimycin is a polyhydroxy alkaloid, some chemical bonds and functional groups (such as hydroxyl groups) in its molecular structure are sensitive to light. Under light conditions, especially the irradiation of ultraviolet (UV) and some visible light bands, light energy may be absorbed by the DNJ molecule. This process can enable the molecule to obtain sufficient energy, resulting in the breaking, rearrangement of its internal chemical bonds or reaction with substances in the surrounding environment. Therefore, too small a particle size of the mulberry leaf powder after frost will cause 1-deoxynojirimycin to become ineffective prematurely to a certain extent, resulting in extraction failure; while if the particle size is too large, the dense cell wall of the mulberry leaf after frost cannot be effectively broken in the pretreatment part, thus resulting in the inability to maximize the extraction of the rich 1-deoxynojirimycin therein;

[0042] 3. In the extraction method of the present invention, a composite enzyme aqueous solution is specifically selected to enzymatically hydrolyze the post - frost mulberry leaf powder. That is, cellulase, hemicellulase, and pectinase with a specific mass ratio of (3 - 5):(2 - 3):(1 - 4) are selected, rather than a single - enzyme hydrolysis solution for enzymatic hydrolysis. This is because after the post - frost mulberry leaves are frosted in the late growth stage, 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, causing the relative increase or change in the proportion of substances in the cell wall. These substances are cross - linked with each other to form a strong structure to enhance the toughness of the cell wall and play a better protective role for the cell wall. And this protective structure makes it more difficult for the effective substances inside the cell to be released. Therefore, while selecting post - frost mulberry leaf powder with a specific particle size, the types and proportions of the composite enzyme are also specifically limited, which can effectively break down this dense structure and fully extract the 1 - deoxynojirimycin rich in it;

[0043] 4. In the extraction method of the present invention, a hyaluronic acid aqueous solution that has not been used in the existing extraction method is also specifically added, and it is added to the system in a drop - by - drop manner during the enzymatic hydrolysis process. This is because the chemical formula of 1 - deoxynojirimycin is C 6 H 13 NO 4 , and the molecule contains multiple hydroxyl groups (-OH). These hydroxyl groups make DNJ have a certain hydrophilicity. Hyaluronic acid is an acidic mucopolysaccharide, and its molecule contains a large number of carboxyl groups (-COOH) and hydroxyl groups (-OH). These functional groups make it have strong hydrophilicity and water - retention capacity, and can bind to other molecules through interactions such as hydrogen bonds. Therefore, during the enzymatic hydrolysis process, by using the drop - by - drop method, the carboxyl and hydroxyl groups on hyaluronic acid can effectively and gradually capture the gradually released 1 - deoxynojirimycin, and form stable hydrogen bonds or chemical bonds with the hydroxyl groups on 1 - deoxynojirimycin. This microscopic interaction, on the one hand, will improve the stability of 1 - deoxynojirimycin in the system, on the other hand, can effectively improve the distribution of 1 - deoxynojirimycin in the system, and also reduce the entanglement and interference of other substances in the post - frost mulberry leaves on 1 - deoxynojirimycin. Therefore, the post - frost mulberry leaf extract obtained by the present invention through specific raw materials and processes can have the mass content retention rate of the rich 1 - deoxynojirimycin greater than or equal to 89% after being stored in the dark at 18°C for 300 days;

[0044] At the same time, the present invention selects small - molecular - weight hyaluronic acid. Besides ensuring the advantage of long - term storage of 1 - deoxynojirimycin in the post - frost mulberry leaf extract, even when prepared into corresponding hypoglycemic products and consumed by humans, it can still maintain the safety performance of the products, which is very friendly to people with high blood sugar and can be absorbed or excreted through the body's own metabolic processes, further laying a foundation for the popularization of the hypoglycemic products. Detailed implementation mode

[0045] Examples 1-3 were extracted using mulberry leaves after frost harvested in 2019;

[0046] Examples 6-9 were extracted using mulberry leaves after frost harvested in 2021;

[0047] Examples 10-12 were extracted using mulberry leaves after frost harvested in 2023;

[0048] The test method for the mass content of 1-deoxynojirimycin is to detect it by gas chromatography method. Specifically, it is determined according to the method disclosed in the journal "Northern Sericulture", authors Li Hong et al., titled "Determination of 1-Deoxynojirimycin (DNJ) in Mulberry Leaves by Gas Chromatography Method".

[0049] At the same time, the present invention also determines the natural mass content of 1-deoxynojirimycin in mulberry leaves after frost harvested in different years. See Table 1 for details.

[0050] Example 1

[0051] This example provides a method for extracting an extract of mulberry leaves after frost, including the following steps:

[0052] Step 1, pretreatment of mulberry leaves after frost: Select fresh and pest-free mulberry leaves after frost, remove the petioles and impurities, wash with clean water, dry at 30 °C until the water content of the mulberry leaves after frost ≤ 10 wt%, and then crush them into mulberry leaf powder with a particle size of 40-50 mesh;

[0053] Step 2, prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.1% of the composite enzyme;

[0054] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 3:3:2;

[0055] In this example, the cellulase is endoglucanase, the hemicellulase is arabinofuranosidase, and the pectinase is pectin lyase;

[0056] Step 3, prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 4000 and 5000 daltons;

[0057] Step 4, enzymatic hydrolysis treatment: Add 10 g of post - frost mulberry leaf powder and 100 mL of complex enzyme aqueous solution into the reaction vessel, and simultaneously dropwise add 30 mL of hyaluronic acid aqueous solution to carry out enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, where 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;

[0058] Step 5, extract the post - frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 80 °C, the time is 20 min, control the pressure between - 0.07 MPa and - 0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, where the inlet air temperature is 150 °C and the outlet air temperature is 80 °C to obtain a powdery post - frost mulberry leaf extract containing 1 - deoxynojirimycin (DNJ);

[0059] In this example, the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 6.1%;

[0060] Store in the dark at 18 °C for 300 days, and the retention rate of the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 94%.

[0061] Example 2

[0062] This example provides a method for extracting post - frost mulberry leaf extract, including the following steps:

[0063] Step 1, pre - treatment of post - frost mulberry leaves: Select fresh, pest - and - disease - free post - frost mulberry leaves, remove the petioles and impurities, wash with clean water, dry at 60 °C until the water content of the post - frost mulberry leaves ≤ 10 wt%, and then crush them into post - frost mulberry leaf powder with a particle size of 50 - 60 mesh;

[0064] Step 2, prepare a complex enzyme aqueous solution: Dissolve the complex enzyme in water at room temperature to obtain a complex enzyme aqueous solution with a mass content of 0.3% of the complex enzyme;

[0065] Wherein the complex enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 5:2:4;

[0066] In this example, the cellulase is endoglucanase, the hemicellulase is galactomannanase, and the pectinase is polygalacturonase;

[0067] Step 3, prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.2% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 4000 - 5000 daltons;

[0068] Step 4, enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder and 200 mL of complex enzyme aqueous solution into the reaction vessel, and simultaneously dropwise add 70 mL of hyaluronic acid aqueous solution to carry out enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0069] Step 5, extract the post-frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment, the temperature of the enzyme inactivation treatment is 85 °C, the time is 15 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove the residue, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 170 °C and the outlet air temperature is 70 °C to obtain a powdery post-frost mulberry leaf extract containing 1-deoxynojirimycin (DNJ);

[0070] In this embodiment, the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 5.3%;

[0071] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 93%.

[0072] Example 3

[0073] This embodiment provides a method for extracting post-frost mulberry leaf extract, including the following steps:

[0074] Step 1, pre-treatment of post-frost mulberry leaves: Select fresh, pest-free post-frost mulberry leaves, remove the petioles and impurities, wash with clean water, and naturally dry until the water content of the post-frost mulberry leaves ≤ 10 wt%, and then crush them into post-frost mulberry leaf powder with a particle size of 70 - 80 mesh;

[0075] Step 2, prepare a complex enzyme aqueous solution: Dissolve the complex enzyme in water at room temperature to obtain a complex enzyme aqueous solution with a mass content of 0.5% of the complex enzyme;

[0076] Wherein the complex enzyme is composed of cellulase, hemicellulase, and pectinase according to a mass ratio of 4:2:3;

[0077] In this embodiment, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase;

[0078] Step 3. Prepare the hyaluronic acid aqueous solution: Dissolve 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 and 5000 Daltons;

[0079] Step 4. Enzymatic hydrolysis treatment: Add 10 g of post - frost mulberry leaf powder and 300 mL of the complex enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 50 mL of the hyaluronic acid aqueous solution to carry out enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0080] Step 5. Extract the post - frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme - inactivating treatment, the temperature of the enzyme - inactivating treatment is 90 °C, the time is 10 min, control the pressure between - 0.07 MPa and - 0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 190 °C and the outlet air temperature is 60 °C to obtain a powdery post - frost mulberry leaf extract containing 1 - deoxynojirimycin (DNJ);

[0081] In this example, the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 6.8%;

[0082] Store it in the dark at 18 °C for 300 days, and the retention rate of the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 96%.

[0083] Example 4

[0084] This example provides a method for extracting post - frost mulberry leaf extract, including the following steps:

[0085] Step 1. Pretreat the post - frost mulberry leaves: Select fresh and pest - free post - frost mulberry leaves, remove the petioles and impurities, wash them with water, dry them at 30 °C until the water content of the post - frost mulberry leaves ≤ 10 wt%, and then crush them into post - frost mulberry leaf powder with a particle size of 60 - 70 mesh;

[0086] Step 2. Prepare the complex enzyme aqueous solution: Dissolve the complex enzyme in water at room temperature to obtain a complex enzyme aqueous solution with a complex enzyme mass content of 0.1%;

[0087] Wherein the complex enzyme is composed of cellulase, hemicellulase, and pectinase according to the mass ratio of 3:3:2;

[0088] In this example, the cellulase is exoglucanase, the hemicellulase is xylanase, and the pectinase is pectin methylesterase;

[0089] Step 3: Prepare the hyaluronic acid aqueous solution: Dissolve 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 and 4000 Daltons;

[0090] Step 4: Enzymatic hydrolysis treatment: Add 10 g of mulberry leaves powder after frosting and 100 mL of composite enzyme aqueous solution into the reaction vessel, and simultaneously dropwise add 30 mL of hyaluronic acid aqueous solution for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0091] Step 5: Extract the mulberry leaves extract after frosting: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 80 °C, the time is 20 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then perform spray drying, wherein the inlet air temperature is 150 °C and the outlet air temperature is 80 °C to obtain a powdery mulberry leaves extract after frosting containing 1 - deoxynojirimycin (DNJ);

[0092] In this example, the mass content of 1 - deoxynojirimycin in the mulberry leaves extract after frosting is 6.5%;

[0093] Store in the dark at 18 °C for 300 days, and the retention rate of the mass content of 1 - deoxynojirimycin in the mulberry leaves extract after frosting is 91%.

[0094] Example 5

[0095] This example provides a method for extracting mulberry leaves extract after frosting, including the following steps:

[0096] Step 1: Pretreat the mulberry leaves after frosting: Select fresh and pest - free mulberry leaves after frosting, remove the petioles and impurities, wash with clean water, dry at 60 °C until the water content of the mulberry leaves after frosting ≤ 10 wt%, and then crush them into mulberry leaves powder with a particle size of 80 - 90 mesh;

[0097] Step 2: Prepare the composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme mass content of 0.3%;

[0098] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 5:2:1;

[0099] In this example, the cellulase is exoglucanase, the hemicellulase is galactomannanase, and the pectinase is pectin lyase;

[0100] Step 3: Prepare the hyaluronic acid aqueous solution: Dissolve 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 and 4000 Daltons.

[0101] Step 4: Enzymatic hydrolysis treatment: Add 10 g of post - frost mulberry leaf powder and 200 mL of composite enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 20 mL of hyaluronic acid aqueous solution for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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.

[0102] Step 5: Extract the post - frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 85 °C, the time is 15 min, control the pressure between - 0.07 MPa and - 0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then perform spray drying. Among them, the inlet air temperature is 170 °C, the outlet air temperature is 70 °C, to obtain a powdery post - frost mulberry leaf extract containing 1 - deoxynojirimycin (DNJ).

[0103] In this example, the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 7.0%.

[0104] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 94%.

[0105] Example 6

[0106] This example provides a method for extracting post - frost mulberry leaf extract, including the following steps:

[0107] Step 1: Pretreatment of post - frost mulberry leaves: Select fresh and pest - free post - frost mulberry leaves, remove the petioles and impurities, wash with clean water, and naturally dry until the water content of the post - frost mulberry leaves ≤ 10 wt%, and then crush them into post - frost mulberry leaf powder with a particle size of 70 - 90 mesh.

[0108] Step 2: Prepare the composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a composite enzyme mass content of 0.5%.

[0109] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 4:2:3.

[0110] In this embodiment, the cellulase is exoglucanase, the hemicellulase is glucomannanase, and the pectinase is polygalacturonase;

[0111] Step 3: Prepare the hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 3000 and 4000 daltons;

[0112] Step 4: Enzymatic hydrolysis treatment: Add 10 g of powdered mulberry leaves after frosting and 300 mL of the composite enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 50 mL of the hyaluronic acid aqueous solution to carry out the enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuous stirring is carried out, 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 the enzymatic hydrolysis reaction solution;

[0113] Step 5: Extract the mulberry leaves extract after frosting: Heat the enzymatic hydrolysis reaction solution to carry out enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 90 °C, the time is 10 min, control the pressure between -0.07 MPa and -0.09 MPa to carry out vacuum filtration to remove the residue, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 190 °C and the outlet air temperature is 60 °C to obtain a powdery mulberry leaves extract after frosting containing 1-deoxynojirimycin (DNJ);

[0114] In this embodiment, the mass content of 1-deoxynojirimycin in the mulberry leaves extract after frosting is 5.6%;

[0115] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the mulberry leaves extract after frosting is 95%.

[0116] Example 7

[0117] This embodiment provides a method for extracting the mulberry leaves extract after frosting, including the following steps:

[0118] Step 1: Pretreatment of mulberry leaves after frosting: Select fresh and pest-free mulberry leaves after frosting, remove the petioles and impurities, wash with clean water, dry at 30 °C until the water content of the mulberry leaves after frosting ≤ 10 wt%, and then crush them into powdered mulberry leaves after frosting with a particle size of 40 - 50 mesh;

[0119] Step 2: Prepare the composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.1% of the composite enzyme;

[0120] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase according to the mass ratio of 3:3:2;

[0121] In this embodiment, the cellulase is β-glucosidase, the hemicellulase is xylanase, and the pectinase is pectin methylesterase;

[0122] Step 3: Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of hyaluronic acid of 0.4%; wherein the molecular weight of the hyaluronic acid is controlled between 3000 and 4000 Daltons;

[0123] Step 4: Enzymatic hydrolysis treatment: Add 10 g of mulberry leaves powder after frosting and 100 mL of the complex enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 30 mL of the hyaluronic acid aqueous solution to carry out an enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0124] Step 5: Extract the mulberry leaves extract after frosting: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment, the temperature of the enzyme inactivation treatment is 80 °C, the time is 20 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 150 °C and the outlet air temperature is 80 °C to obtain a powdery mulberry leaves extract after frosting containing 1-deoxynojirimycin (DNJ);

[0125] In this embodiment, the mass content of 1-deoxynojirimycin in the mulberry leaves extract after frosting is 5.2%;

[0126] Store in the dark at 18 °C for 300 days, and the retention rate of the mass content of 1-deoxynojirimycin in the mulberry leaves extract after frosting is 90%.

[0127] Example 8

[0128] This embodiment provides a method for extracting a mulberry leaves extract after frosting, including the following steps:

[0129] Step 1: Pretreatment of mulberry leaves after frosting: Select fresh and pest-free mulberry leaves after frosting, remove the petioles and impurities, wash with clean water, dry at 60 °C until the water content of the mulberry leaves after frosting ≤ 10 wt%, and then crush them into mulberry leaves powder with a particle size of 50 - 60 mesh;

[0130] Step 2: Prepare a complex enzyme aqueous solution: Dissolve the complex enzyme in water at room temperature to obtain a complex enzyme aqueous solution with a mass content of complex enzyme of 0.3%;

[0131] Wherein the complex enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 5:2:4;

[0132] In this embodiment, the cellulase is β-glucosidase, the hemicellulase is galactomannanase, and the pectinase is pectin lyase;

[0133] Step 3: Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.2% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 3000 and 4000 daltons;

[0134] Step 4: Enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder and 200 mL of complex enzyme aqueous solution into a reaction vessel, and simultaneously add dropwise 70 mL of hyaluronic acid aqueous solution for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0135] Step 5: Extract the post-frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 85 °C, the time is 15 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then perform spray drying, wherein the inlet air temperature is 170 °C and the outlet air temperature is 70 °C to obtain a powdery post-frost mulberry leaf extract containing 1-deoxynojirimycin (DNJ);

[0136] In this embodiment, the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 5.3%.

[0137] The test method for the mass content of 1-deoxynojirimycin is to detect it by gas phase method, specifically according to the method disclosed in the journal "Northern Sericulture", authors Li Hong et al., titled "Determination of -deoxynojirimycin (DNJ) in Mulberry Leaves by Gas Phase Method";

[0138] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 96%.

[0139] Example 9

[0140] This embodiment provides a method for extracting a post-frost mulberry leaf extract, including the following steps:

[0141] Step 1. Pretreatment of mulberry leaves after frost: Select fresh mulberry leaves after frost without pests and diseases, remove petioles and impurities, wash with clean water, and naturally dry until the water content of the mulberry leaves after frost ≤ 10 wt%, and then crush them into mulberry leaf powder after frost with a particle size of 70 - 90 mesh;

[0142] Step 2. Prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.5% of the composite enzyme;

[0143] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 4:2:3;

[0144] In this embodiment, the cellulase is β - glucosidase, the hemicellulase is arabinofuranosidase, and the pectinase is polygalacturonase;

[0145] Step 3. Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 3000 - 4000 daltons;

[0146] Step 4. Enzymatic hydrolysis treatment: Add 10 g of mulberry leaf powder after frost and 300 mL of the composite enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 50 mL of the hyaluronic acid aqueous solution to carry out an enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0147] Step 5. Extract the mulberry leaf extract after frost: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment, the temperature of the enzyme inactivation treatment is 90 °C, the time is 10 min, control the pressure between - 0.07 MPa and - 0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 190 °C and the outlet air temperature is 60 °C to obtain a powdery mulberry leaf extract after frost containing 1 - deoxynojirimycin (DNJ);

[0148] In this embodiment, the mass content of 1 - deoxynojirimycin in the mulberry leaf extract after frost is 6.8%;

[0149] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1 - deoxynojirimycin in the mulberry leaf extract after frost is 89%.

[0150] Comparative Example 1

[0151] This comparative example provides an extraction method of post - frost mulberry leaf extract relative to Example 1. The difference from Example 1 is that the particle size of the post - frost mulberry leaf powder in this comparative example is between 20 - 30 mesh, and the specific steps are as follows:

[0152] Step 1. Pretreatment of post - frost mulberry leaves: Select fresh and pest - free post - frost mulberry leaves, remove petioles and impurities, wash with clean water, dry at 30 °C until the water content of the post - frost mulberry leaves ≤ 10 wt%, and then crush them into post - frost mulberry leaf powder with a particle size of 20 - 30 mesh;

[0153] Step 2. Prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.1% of the composite enzyme;

[0154] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 3:3:2;

[0155] In this example, the cellulase is endoglucanase, the hemicellulase is arabinofuranosidase, and the pectinase is pectin lyase;

[0156] Step 3. Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 4000 - 5000 daltons;

[0157] Step 4. Enzymatic hydrolysis treatment: Add 10 g of post - frost mulberry leaf powder and 100 mL of the composite enzyme aqueous solution into a reaction vessel, and simultaneously add 30 mL of the hyaluronic acid aqueous solution dropwise for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0158] Step 5. Extract the post - frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 80 °C, the time is 20 min, control the pressure between - 0.07 MPa and - 0.09 MPa for vacuum filtration to remove residues, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then perform spray drying, wherein the inlet air temperature is 150 °C and the outlet air temperature is 80 °C to obtain a powdery post - frost mulberry leaf extract containing 1 - deoxynojirimycin (DNJ);

[0159] In this comparative example, the mass content of 1 - deoxynojirimycin in the post - frost mulberry leaf extract is 3.9%;

[0160] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 68%.

[0161] Comparative Example 2

[0162] This comparative example provides an extraction method of post-frost mulberry leaf extract relative to Example 1. The difference from Example 1 is that the particle size of the post-frost mulberry leaf powder in this comparative example is between 100 and 150 mesh, and the specific steps are as follows:

[0163] Step 1, Pretreatment of post-frost mulberry leaves: Select fresh post-frost mulberry leaves without diseases and pests, remove the petioles and impurities, wash them with clean water, dry them at 30 °C until the water content of the post-frost mulberry leaves ≤ 10 wt%, and then crush them into post-frost mulberry leaf powder with a particle size of 100 - 150 mesh;

[0164] Step 2, Prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.1% of the composite enzyme;

[0165] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 3:3:2;

[0166] In this example, the cellulase is endoglucanase, the hemicellulase is arabinofuranosidase, and the pectinase is pectin lyase;

[0167] Step 3, Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 4000 and 5000 daltons;

[0168] Step 4, Enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder and 100 mL of the composite enzyme aqueous solution into the reaction vessel, and simultaneously drip 30 mL of the hyaluronic acid aqueous solution to carry out an enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuous stirring is carried out, 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 dripping time is also controlled at 1.5 h to obtain an enzymatic hydrolysis reaction solution;

[0169] Step 5, Extract the post-frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 80 °C, the time is 20 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove the residue, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 150 °C and the outlet air temperature is 80 °C to obtain a powdery post-frost mulberry leaf extract containing 1-deoxynojirimycin (DNJ);

[0170] In this comparative example, the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 2.7%;

[0171] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 79%.

[0172] Comparative Example 3

[0173] This comparative example provides an extraction method of post-frost 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, which specifically includes the following steps:

[0174] Step 1. Pretreatment of post-frost mulberry leaves: Select fresh post-frost mulberry leaves without pests and diseases, remove the petioles and impurities, wash with clean water, dry at 60 °C until the water content of the post-frost mulberry leaves ≤ 10 wt%, and then crush them into post-frost mulberry leaf powder with a particle size of 50-60 mesh;

[0175] Step 2. Prepare a single enzyme aqueous solution: Dissolve cellulase in water at room temperature to obtain a single enzyme aqueous solution with a mass content of cellulase of 0.3%;

[0176] In this comparative example, the cellulase is endoglucanase;

[0177] Step 3. Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of hyaluronic acid of 0.2%; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 daltons;

[0178] Step 4. Enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder and 200 mL of the single enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 70 mL of the hyaluronic acid aqueous solution for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuous stirring is carried out, 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;

[0179] Step 5. Extract the post-frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 85 °C, the time is 15 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove the residue, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 170 °C and the outlet air temperature is 70 °C to obtain a powdery post-frost mulberry leaf extract containing 1-deoxynojirimycin (DNJ);

[0180] In this comparative example, the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 3.1%;

[0181] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 74%.

[0182] Comparative Example 4

[0183] This comparative example provides an extraction method of post-frost mulberry leaf extract relative to Example 3. The difference from Example 3 is that in this comparative example, an aqueous hyaluronic acid solution is not used, and it specifically includes the following steps:

[0184] Step 1. Pretreatment of post-frost mulberry leaves: Select fresh post-frost mulberry leaves without pests and diseases, remove the petioles and impurities, wash with clean water, and air-dry naturally until the water content of the post-frost mulberry leaves ≤ 10 wt%, and then crush them into post-frost mulberry leaf powder with a particle size of 70 - 80 mesh;

[0185] Step 2. Prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.5% of the composite enzyme;

[0186] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 4:2:3;

[0187] In this comparative example, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase;

[0188] Step 3. Enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder and 300 mL of the composite enzyme aqueous solution into a reaction vessel for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuous stirring is carried out. Among them, 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;

[0189] Step 4. Extract the post-frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 90 °C, the time is 10 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove the residue, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying, wherein the inlet air temperature is 190 °C and the outlet air temperature is 60 °C to obtain a powdery post-frost mulberry leaf extract containing 1-deoxynojirimycin (DNJ);

[0190] In this comparative example, the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 3.6%;

[0191] Stored in the dark 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%.

[0192] Comparative Example 5

[0193] This comparative example provides an extraction method of post-frost 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 the specific steps are as follows:

[0194] Step 1. Pretreatment of post-frost mulberry leaves: Select fresh post-frost mulberry leaves without diseases and pests, remove the petioles and impurities, wash with clean water, and naturally dry until the water content of the post-frost mulberry leaves ≤ 10 wt%, and then crush them into post-frost mulberry leaf powder with a particle size of 70-80 mesh;

[0195] Step 2. Prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.5% of the composite enzyme;

[0196] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 4:2:3;

[0197] In this comparative example, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase;

[0198] Step 3. Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 4000-5000 daltons;

[0199] Step 4. Enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder, 300 mL of composite enzyme aqueous solution, and 50 mL of hyaluronic acid aqueous solution to the reaction vessel for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuous stirring is carried out. Among them, 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;

[0200] Step 5. Extract the post-frost mulberry leaf extract: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 90 °C, the time is 10 min, control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove the residue, concentrate the filtrate to 1 / 4 of the original filtrate volume, and then carry out spray drying. Among them, the inlet air temperature is 190 °C, and the outlet air temperature is 60 °C to obtain a powdery post-frost mulberry leaf extract containing 1-deoxynojirimycin (DNJ);

[0201] In this comparative example, the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 2.9%;

[0202] Stored in the dark at 18 °C for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the post-frost mulberry leaf extract is 52%.

[0203] Comparative Example 6

[0204] This comparative example provides an extraction method of post-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 it specifically includes the following steps:

[0205] Step 1. Pretreatment of post-frost mulberry leaves: Select fresh post-frost mulberry leaves without diseases and pests, remove the petioles and impurities, wash them with clean water, and naturally dry them until the water content of the post-frost mulberry leaves ≤ 10 wt%, and then crush them into post-frost mulberry leaf powder with a particle size of 70 - 80 mesh;

[0206] Step 2. Prepare a composite enzyme aqueous solution: Dissolve the composite enzyme in water at room temperature to obtain a composite enzyme aqueous solution with a mass content of 0.5% of the composite enzyme;

[0207] Wherein the composite enzyme is composed of cellulase, hemicellulase, and pectinase in a mass ratio of 4:2:3;

[0208] In this comparative example, the cellulase is endoglucanase, the hemicellulase is glucomannanase, and the pectinase is pectin methylesterase;

[0209] Step 3. Prepare a hyaluronic acid aqueous solution: Dissolve hyaluronic acid in water at room temperature to obtain a hyaluronic acid aqueous solution with a mass content of 0.4% of hyaluronic acid; wherein the molecular weight of the hyaluronic acid is controlled between 20,000 and 50,000 Daltons;

[0210] Step 4. Enzymatic hydrolysis treatment: Add 10 g of post-frost mulberry leaf powder and 300 mL of the composite enzyme aqueous solution into a reaction vessel, and simultaneously dropwise add 50 mL of the hyaluronic acid aqueous solution for enzymatic hydrolysis reaction. During the enzymatic hydrolysis reaction, continuously stir, 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;

[0211] Step 5: Extract the extract of mulberry leaves after frosting: Heat the enzymatic hydrolysis reaction solution for enzyme inactivation treatment. The temperature of the enzyme inactivation treatment is 90°C, and the time is 10 min. Control the pressure between -0.07 MPa and -0.09 MPa for vacuum filtration to remove residues. Concentrate the filtrate to 1 / 4 of the original filtrate volume, and then perform spray drying. The inlet air temperature is 190°C, and the outlet air temperature is 60°C to obtain a powdery extract of mulberry leaves after frosting containing 1-deoxynojirimycin (DNJ);

[0212] In this comparative example, the mass content of 1-deoxynojirimycin in the extract of mulberry leaves after frosting is 2.2%;

[0213] Store in the dark at 18°C for 300 days, and the retention rate of the mass content of 1-deoxynojirimycin in the extract of mulberry leaves after frosting is 45%.

[0214] Comparative Example 7

[0215] In this comparative example, the mulberry leaves in Example 1 disclosed in the prior art CN114177218B were replaced with the mulberry leaves after frosting in the present invention (produced in 2019, the same as the raw materials of the mulberry leaves after frosting in Examples 1-3 of the present invention). The experimental steps were exactly the same as those in Example 1 disclosed in this technical solution to obtain a powdery finished product prepared according to CN114177218B.

[0216] Detect the finished product in this comparative example. The mass content of 1-deoxynojirimycin in the finished product is 1.7%;

[0217] Store in the dark at 18°C for 300 days, and the retention rate of the mass content of 1-deoxynojirimycin in the extract of mulberry leaves after frosting is 51%.

[0218] Compare the mass content of 1-deoxynojirimycin and the retention rate of the mass content after storing in the dark at 18°C for 300 days in Examples 1-9 and Comparative Examples 1-7. The comparison results are shown in Table 1:

[0219] Table 1 Comparison of the mass content of 1-deoxynojirimycin in Examples 1-9 and Comparative Examples 1-7

[0220]

[0221] Through the above comparison, under the conditions of using specific raw materials and a specific extraction process, the present invention obtains a post-frost mulberry leaf extract with a 1-deoxynojirimycin (DNJ) mass content as high as 5.2 - 7.0%. Compared with the extraction methods of traditional mulberry leaf extracts in the prior art, under the condition that the annual output of post-frost mulberry leaves is itself lower than that of mulberry leaves, the present invention effectively maximizes the utilization of post-frost mulberry leaf resources. At the same time, it breaks through the limitation that mulberry leaf extracts in the prior art need to be stored under harsh conditions of 2 - 8°C. After being stored in the dark for 300 days at 18°C, the retention rate of the mass content of 1-deoxynojirimycin rich in the post-frost mulberry leaf extract obtained by the specific extraction process of the present invention can be greater than or equal to 89%, further reducing the storage cost for its subsequent popularization as a hypoglycemic product.

[0222] It should be understood that the present invention is not limited to what has been described above and can be variously modified and changed without departing from its scope. The scope of the present invention is only limited 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 place at 18° C. for 300 days, the retention rate of the mass content of 1-deoxynojirimycin in the frost-frosted mulberry leaf extract is greater than or equal to 89%; The molecular weight of the hyaluronic acid described in step 3 is 3000-5000 Daltons.

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: 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.

5. 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.

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

7. The method for extracting the frost-frosted mulberry leaf extract according to claim 6, 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.

8. 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 7.

9. A blood sugar lowering product, characterized in that: The blood sugar lowering product is the frosted mulberry leaf extract according to claim 8, or includes the frosted mulberry leaf extract according to claim 8, or is prepared from the frosted mulberry leaf extract according to claim 8.

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