Method for preparing mulberry twig crude extract by utilizing ultrasonic-assisted enzyme method

Abstract

The invention belongs to the field of preparation of ramulus mori crude extracts, and particularly relates to a method for preparing a ramulus mori crude extract by utilizing an ultrasonic-assisted enzyme method, which comprises the following steps: S1, weighing 0.5-1g of mulberry leaf powder in 100mL of distilled water; s2, adding pectinase, neutral protease, trypsin and papain; s3, taking the enzyme addition amount, the material-liquid ratio, the extraction temperature and the ultrasonic power as single-factor indexes, taking the total flavone extraction rate as an evaluation standard, establishing a regression equation through Box-Behnken design test, and collecting test data; s4, variance analysis and response surface analysis are conducted on the data, the enzyme adding amount, the material-liquid ratio, the extraction temperature and the ultrasonic power serve as single-factor indexes, the total flavone extraction rate serves as an evaluation standard, a regression equation is established through a Box-Behnken design test, and test data are collected; and performing variance analysis and response surface analysis on the data to prepare the optimal mulberry branch and leaf crude extract.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of crude extracts from mulberry branches, and particularly to a method for preparing crude extracts from mulberry branches by using ultrasonic-assisted enzymatic method. Background Art

[0002] The efficacy and functions of the crude extracts from mulberry branches mainly include anti-inflammatory, hypoglycemic, hypolipidemic, anti-atherosclerotic, antiviral, diuretic, heat-clearing, dampness-draining, detumescence, pain-relieving, promoting blood circulation, dispelling wind-dampness, benefiting joints, relaxing tendons and activating collaterals, enhancing immunity, and promoting metabolism, etc.

[0003] In the prior art, the effect of the prepared crude extracts from mulberry branches cannot reach the best. Therefore, we propose a method for preparing crude extracts from mulberry branches by using ultrasonic-assisted enzymatic method to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to solve the disadvantage that the effect of the prepared crude extracts from mulberry branches in the prior art cannot reach the best, and to propose a method for preparing crude extracts from mulberry branches by using ultrasonic-assisted enzymatic method.

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

[0006] A method for preparing crude extracts from mulberry branches by using ultrasonic-assisted enzymatic method, comprising the following steps:

[0007] S1. Weigh 0.5 - 1 g of mulberry leaf powder into 100 mL of distilled water;

[0008] [[ID=Z8]]S2. Add pectinase, neutral protease, trypsin, and papain;

[0009] S3. Take the enzyme addition amount, solid-liquid ratio, extraction temperature, and ultrasonic power as single-factor indexes, take the total flavonoid extraction rate as the evaluation standard, and establish a regression equation through Box-Behnken design experiments, and collect experimental data;

[0010] S4. Conduct variance analysis and response surface analysis on the data to prepare the best crude extracts from mulberry leaves.

[0011] Preferably, in step S3, an experimental design software is used to create a Box-Behnken design. In the software, input the number of factors, names, ranges, and the number of midpoint tests. Run the design wizard, and the software will generate a series of experimental combinations, which constitute the experimental plan of the Box-Behnken design. Conduct experiments according to the plan and record the response values. Input the experimental data into the design software, and the software will help analyze the data, construct a response surface model. Through analysis of variance, determine which factors are significant and whether there are significant interactions between factors. Use the graphical function of the software to visually view the response surface model and understand the relationship between factors and responses. By analyzing the response surface model, find the optimal combination of experimental parameters to achieve the purpose of process optimization. After finding the optimal parameter combination, conduct experimental verification to ensure accuracy.

[0012] Preferably, in step S2, pectinase, neutral protease, trypsin, and papain are added. The mixture is placed in an ultrasonic device for ultrasonic treatment. After the ultrasonic treatment, insoluble impurities are removed by filtration to obtain a filtrate containing the target extract. Then, the filtrate is concentrated to increase the concentration and purity of the target extract.

[0013] Preferably, in step S4, the basic steps of analysis of variance mainly include the following points: propose the null hypothesis and the alternative hypothesis, collect data, and ensure the integrity and accuracy of the data. Calculate the mean of each group and the total mean, and preliminarily judge the differences between different groups. Calculate the within-group variance and the between-group variance to reflect the degree of variation between the observed values within the group and the differences between different groups. Determine the total degrees of freedom, between-group degrees of freedom, and within-group degrees of freedom. Divide the sum of squares corresponding to each source of variation by the corresponding degrees of freedom to obtain the mean square of this effect. Divide the mean square of each effect to be tested by the mean square of the error to construct an F statistic. Select an appropriate significance level to judge whether the result is significant. According to the F statistic and degrees of freedom, look up the F distribution table or use statistical software to determine the critical F value. Compare the calculated F value with the critical F value, and make a conclusion based on the comparison of the P value and the significance level. Judge whether to reject the null hypothesis, conduct an in-depth discussion of the results, and interpret and analyze them in combination with professional knowledge.

[0014] Preferably, in step S4, the response surface analysis is as follows: use the Box-Behnken design to construct an experimental matrix to ensure that the experiment covers all possible variable combinations. Conduct experiments according to the experimental matrix and collect the corresponding response variable data, ensuring the accuracy and reliability of the data. Use the experimental data to fit a quadratic polynomial model and establish a response surface equation. Analyze the change trend of the response variable through the response surface graph and the contour map. Use an optimization algorithm to find the experimental conditions that make the response variable reach the best value. Verify the accuracy and reliability of the model through additional experimental data and find the optimal parameter combination.

[0015] Preferably, in S1, the mulberry leaves and twigs are naturally dried and then crushed by a pulverizer, passed through a 200-mesh sieve and stored in a refrigerator at 4°C to obtain mulberry leaf powder. Weigh 0.5 g of mulberry leaf powder and place it in 100 mL of distilled water.

[0016] Preferably, in S2, pectinase specifically decomposes pectin to convert it into soluble substances, thereby reducing the viscosity of the mulberry leaf powder. Through the decomposition of pectinase, the cell wall structure in the mulberry leaf powder is destroyed, and the effective components in the cells, such as proteins, polysaccharides, flavonoids, etc., can be more easily dissolved in the extraction solvent, thereby improving the extraction efficiency and the quality of the extract.

[0017] Preferably, in S2, when neutral protease reacts with the mulberry leaf powder, the enzyme acts on the protein molecules in the mulberry leaf powder and decomposes them into smaller polypeptide or amino acid fragments. This process is an enzymatic hydrolysis reaction, which can be carried out under relatively mild conditions and has high efficiency and specificity.

[0018] Preferably, in S1, weigh 0.5 g of mulberry leaf powder and place it in 100 mL of distilled water, stir and mix evenly, and the stirring time is 5 - 10 min.

[0019] Preferably, the preparation method of the pectinase is as follows: Prepare fresh fruits or vegetables as raw materials, wash and cut them into pieces, grind and homogenize them. Grind the cut raw materials quickly in a pre-cooled mortar and add a 10% (mass fraction) NaCl solution to make a homogenate. Filter the homogenate, use a funnel and filter paper or 5 layers of gauze to filter the homogenate to remove the solid impurities therein, and collect the filtrate. Next, perform centrifugation. Centrifuge the filtrate at a centrifugation rate of 4000 r / min for 15 minutes to further separate and purify the pectinase. After centrifugation, the pectinase mainly exists in the supernatant of the centrifuge tube. Finally, adjust the pH of the supernatant and store it. Pour the supernatant into a clean small beaker, adjust the pH to 3 with 0.1 mol / L glacial acetic acid, and then store it at 4°C for later use.

[0020] In the present invention, the beneficial effects of the method for preparing crude mulberry twig extract by ultrasonic-assisted enzymatic method are as follows:

[0021] In the present invention, the enzyme addition amount, solid-liquid ratio, extraction temperature, and ultrasonic power are used as single-factor indicators, the total flavonoid extraction rate is used as the evaluation standard, and a regression equation is established through Box-Behnken design experiments to collect experimental data; variance analysis and response surface analysis are performed on the data to prepare the best crude mulberry leaf extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a flow chart of the method for preparing crude mulberry twig extract by ultrasonic-assisted enzymatic method proposed by the present invention. Detailed Implementation Modes

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0024] Embodiment 1

[0025] Referring to Figure 1 , a method for preparing crude extracts of mulberry branches by ultrasonic-assisted enzymatic method, comprising the following steps:

[0026] S1. Weigh 0.5 g of mulberry leaf powder into 100 mL of distilled water;

[0027] S2. Add pectinase, neutral protease, trypsin, and papain;

[0028] S3. Using the enzyme addition amount, solid-liquid ratio, extraction temperature, and ultrasonic power as single-factor indicators, and the total flavonoid extraction rate as the evaluation standard, establish a regression equation through Box-Behnken design experiments, and collect experimental data;

[0029] S4. Conduct variance analysis and response surface analysis on the data to prepare the best crude extract of mulberry leaves.

[0030] In this embodiment, in S3, use experimental design software to create a Box-Behnken design. In the software, input the number of factors, names, ranges, and the number of midpoint experiments, run the design wizard, and the software will generate a series of experimental combinations, which constitute the experimental plan of the Box-Behnken design. Execute the experiments according to the plan and record the response values. Input the experimental data into the design software, and the software will help analyze the data, construct a response surface model, determine which factors are significant through variance analysis, and whether there are significant interactions between factors. Use the graphical function of the software to visually view the response surface model, understand the relationship between factors and responses, find the optimal combination of experimental parameters through analyzing the response surface model to achieve the purpose of process optimization. After finding the optimal parameter combination, conduct experimental verification to ensure accuracy.

[0031] In this embodiment, in S2, add pectinase, neutral protease, trypsin, and papain, place the mixture in an ultrasonic device for ultrasonic treatment. After the ultrasonic treatment, filter to remove insoluble impurities to obtain a filtrate containing the target extract. Then, concentrate the filtrate to increase the concentration and purity of the target extract.

[0032] In this embodiment, in S4, the basic steps of variance analysis mainly include the following points: proposing the null hypothesis and the alternative hypothesis, collecting data, and ensuring the integrity and accuracy of the data, calculating the means of each group and the total mean, preliminarily judging the differences between different groups, calculating the within-group variance and the between-group variance to reflect the degree of variation between the observed values within the group and the differences between different groups, determining the total degrees of freedom, the between-group degrees of freedom, and the within-group degrees of freedom, dividing the sum of squares corresponding to each source of variation by the corresponding degrees of freedom to obtain the mean square of this effect, dividing the mean square of each effect to be tested by the mean square of the error to construct the F statistic, selecting an appropriate significance level to judge whether the result is significant, looking up the F distribution table according to the F statistic and the degrees of freedom or using statistical software to determine the critical F value, comparing the calculated F value with the critical F value, making a conclusion based on the comparison between the P value and the significance level, judging whether to reject the null hypothesis, deeply discussing the results, and making interpretations and analyses in combination with professional knowledge.

[0033] In this embodiment, in S4, the response surface analysis is as follows: using the Box-Behnken design to construct an experimental matrix to ensure that the experiment covers all possible variable combinations, conducting the experiment according to the experimental matrix, and collecting the corresponding response variable data to ensure the accuracy and reliability of the data, fitting a quadratic polynomial model using the experimental data to establish a response surface equation, analyzing the change trend of the response variable through the response surface plot and the contour plot, using an optimization algorithm to find the experimental conditions that make the response variable reach the optimal value, verifying the accuracy and reliability of the model through additional experimental data, and finding the optimal parameter combination.

[0034] In this embodiment, in S$1$, the mulberry leaves and twigs are naturally dried and then crushed with a pulverizer, passed through a 200-mesh sieve, and stored in a 4°C refrigerator to obtain mulberry leaf powder. Weigh 0.5 g of mulberry leaf powder into 100 mL of distilled water.

[0035] In this embodiment, in S2, pectinase specifically decomposes pectin and converts it into soluble substances, thereby reducing the viscosity of the mulberry leaf powder. Through the decomposition of pectinase, the cell wall structure in the mulberry leaf powder is destroyed, and the effective components in the cells, such as proteins, polysaccharides, flavonoids, etc., can be more easily dissolved in the extraction solvent, thereby improving the extraction efficiency and the quality of the extract.

[0036] In this embodiment, in S2, when neutral protease reacts with the mulberry leaf powder, the enzyme acts on the protein molecules in the mulberry leaf powder and decomposes them into smaller polypeptide or amino acid fragments. This process is an enzymatic hydrolysis reaction, which can be carried out under relatively mild conditions and has high efficiency and specificity.

[0037] In this embodiment, in S1, weigh 0.� g of mulberry leaf powder into 100 mL of distilled water, stir and mix evenly, and the stirring time is 5 min.

[0038] In this embodiment, the preparation method of pectinase is as follows: Prepare fresh fruits or vegetables as raw materials, wash and cut them into pieces, grind and homogenize them. Grind the cut raw materials quickly in a pre-cooled mortar, and add a NaCl solution with a mass fraction of 10%, to make a homogenate. Filter the homogenate, using a funnel and filter paper or 5 layers of gauze to filter the homogenate to remove the solid impurities therein, and collect the filtrate. Next, perform centrifugation. Centrifuge the filtrate at a centrifugation rate of 4000 r / min for 15 minutes to further separate and purify pectinase. After centrifugation, pectinase mainly exists in the supernatant of the centrifuge tube. Finally, adjust the pH of the supernatant and store it. Pour the supernatant into a clean small beaker, adjust the pH to 3 with 0.1 mol / L glacial acetic acid, and then store it at 4°C for standby.

[0039] Example Two

[0040] A method for preparing crude extracts of mulberry branches by ultrasonic-assisted enzymatic method, comprising the following steps:

[0041] S1. Weigh 0.7 g of mulberry leaf powder into 100 mL of distilled water;

[0042] S2. Add pectinase, neutral protease, trypsin, and papain;

[0043] S3. Take the enzyme addition amount, solid-liquid ratio, extraction temperature, and ultrasonic power as single-factor indicators, take the total flavonoid extraction rate as the evaluation standard, and establish a regression equation through Box-Behnken design experiments, and collect experimental data;

[0044] S4. Perform variance analysis and response surface analysis on the data to prepare the best crude extract of mulberry branches and leaves.

[0045] In this embodiment, in S3, use experimental design software to create a Box-Behnken design. In the software, input the number of factors, names, ranges, and the number of midpoint experiments, run the design wizard, and the software will generate a series of experimental combinations, which constitute the experimental plan of the Box-Behnken design. Perform experiments according to the plan and record the response values. Input the experimental data into the design software, and the software will help analyze the data, construct a response surface model. Through variance analysis, determine which factors are significant and whether there are significant interactions between factors. Use the graphical function of the software to visually view the response surface model and understand the relationship between factors and responses. Through analyzing the response surface model, find the optimal combination of experimental parameters to achieve the purpose of process optimization. After finding the optimal parameter combination, conduct experimental verification to ensure accuracy.

[0046] In this embodiment, in S2, pectinase, neutral protease, trypsin, and papain are added. The mixture is placed in an ultrasonic device for ultrasonic treatment. After the ultrasonic treatment ends, insoluble impurities are removed by filtration to obtain a filtrate containing the target extract. Then, the filtrate is concentrated to increase the concentration and purity of the target extract.

[0047] In this embodiment, in S4, the basic steps of variance analysis mainly include the following points: proposing a null hypothesis and an alternative hypothesis, collecting data, and ensuring the integrity and accuracy of the data, calculating the mean of each group and the total mean, preliminarily judging the differences between different groups, calculating the within-group variance and the between-group variance to reflect the degree of variation between the observed values within the group and the differences between different groups, determining the total degrees of freedom, between-group degrees of freedom, and within-group degrees of freedom, dividing the sum of squares corresponding to each source of variation by the corresponding degrees of freedom to obtain the mean square of this effect, dividing the mean square of each effect to be tested by the mean square of the error to construct an F statistic, selecting an appropriate significance level to judge whether the result is significant, looking up the F distribution table according to the F statistic and degrees of freedom or using statistical software to determine the critical F value, comparing the calculated F value with the critical F value, making a conclusion based on the comparison of the P value and the significance level, judging whether to reject the null hypothesis, deeply discussing the results, and interpreting and analyzing them in combination with professional knowledge.

[0048] In this embodiment, in S4, the response surface analysis is as follows: using the Box-Behnken design to construct an experimental matrix to ensure that the experiment covers all possible variable combinations, conducting the experiment according to the experimental matrix, and collecting the corresponding response variable data to ensure the accuracy and reliability of the data, fitting a quadratic polynomial model using the experimental data to establish a response surface equation, analyzing the change trend of the response variable through the response surface plot and contour plot, using an optimization algorithm to find the experimental conditions that make the response variable reach the optimal value, verifying the accuracy and reliability of the model through additional experimental data, and finding the optimal parameter combination.

[0049] In this embodiment, in S1, the mulberry leaves and twigs are naturally dried and then pulverized by a pulverizer, passed through a 200-mesh sieve, and stored in a 4°C refrigerator to obtain mulberry leaf powder.

[0050] In this embodiment, in S2, pectinase specifically decomposes pectin and converts it into soluble substances, thereby reducing the viscosity of the mulberry leaf powder. Through the decomposition of pectinase, the cell wall structure in the mulberry leaf powder is destroyed, and the effective components in the cells, such as proteins, polysaccharides, flavonoids, etc., can be more easily dissolved in the extraction solvent, thereby improving the extraction efficiency and the quality of the extract.

[0051] In this embodiment, in S2, when neutral protease reacts with mulberry leaf powder, the enzyme acts on the protein molecules in the mulberry leaf powder and decomposes them into smaller polypeptide or amino acid fragments. This process is an enzymatic hydrolysis reaction, which can be carried out under relatively mild conditions and has high efficiency and specificity.

[0052] In this embodiment, in S1, 0.7 g of mulberry leaf powder is weighed into 100 mL of distilled water, stirred and mixed evenly, and the stirring time is 7 min.

[0053] In this embodiment, the preparation method of pectinase is as follows: Prepare fresh fruits or vegetables as raw materials, wash and cut them into pieces, grind and homogenize them. The cut raw materials are quickly ground in a pre-cooled mortar, and a 10% (mass fraction) NaCl solution is added to make a homogenate. The homogenate is filtered, and the homogenate is filtered using a funnel and filter paper or 5 layers of gauze to remove the solid impurities therein, and the filtrate is collected. Next, centrifugation is carried out. The filtrate is centrifuged at a centrifugation rate of 4000 r / min for 15 minutes to further separate and purify pectinase. After centrifugation, pectinase mainly exists in the supernatant of the centrifuge tube. Finally, the pH of the supernatant is adjusted and stored. The supernatant is poured into a clean small beaker, and the pH is adjusted to 3 with 0.1 mol / L glacial acetic acid, and then stored at 4 °C for standby.

[0054] Example Three

[0055] A method for preparing crude extracts of mulberry branches by ultrasonic-assisted enzymatic method, comprising the following steps:

[0056] S1. Weigh 1 g of mulberry leaf powder into 100 mL of distilled water;

[0057] S2. Add pectinase, neutral protease, trypsin, and papain;

[0058] S3. Take the enzyme addition amount, solid-liquid ratio, extraction temperature, and ultrasonic power as single-factor indicators, take the total flavonoid extraction rate as the evaluation standard, and establish a regression equation through Box-Behnken design experiments, and collect experimental data;

[0059] S4. Perform variance analysis and response surface analysis on the data to prepare the best crude extract of mulberry leaves and branches.

[0060] In this embodiment, in S3, an experimental design software is used to create a Box-Behnken design. In the software, input the number of factors, names, ranges, and the number of center point tests. Run the design wizard, and the software will generate a series of experimental combinations, which constitute the experimental plan of the Box-Behnken design. Execute the experiment according to the plan and record the response values. Input the experimental data into the design software, and the software will help analyze the data, construct a response surface model. Through analysis of variance, determine which factors are significant and whether there are significant interactions between factors. Use the graphical function of the software to visually view the response surface model and understand the relationship between factors and responses. By analyzing the response surface model, find the optimal combination of experimental parameters to achieve the purpose of process optimization. After finding the optimal parameter combination, conduct experimental verification to ensure accuracy.

[0061] In this embodiment, in S2, pectinase, neutral protease, trypsin, and papain are added. The mixture is placed in an ultrasonic device for ultrasonic treatment. After the ultrasonic treatment, insoluble impurities are removed by filtration to obtain a filtrate containing the target extract. Then, the filtrate is concentrated to increase the concentration and purity of the target extract.

[0062] In this embodiment, in S4, the basic steps of analysis of variance mainly include the following points: propose a null hypothesis and an alternative hypothesis, collect data, and ensure the integrity and accuracy of the data. Calculate the mean of each group and the total mean, and preliminarily judge the differences between different groups. Calculate the within-group variance and between-group variance to reflect the degree of variation between the observed values within the group and the differences between different groups. Determine the total degrees of freedom, between-group degrees of freedom, and within-group degrees of freedom. Divide the sum of squares corresponding to each source of variation by the corresponding degrees of freedom to obtain the mean square of this effect. Divide the mean square of each effect to be tested by the mean square of the error to construct an F statistic. Select an appropriate significance level to judge whether the result is significant. Look up the F distribution table according to the F statistic and degrees of freedom or use statistical software to determine the critical F value. Compare the calculated F value with the critical F value. Make a conclusion based on the comparison of the P value and the significance level, judge whether to reject the null hypothesis, conduct an in-depth discussion of the results, and interpret and analyze them in combination with professional knowledge.

[0063] In this embodiment, in S4, the response surface analysis is as follows: use a Box-Behnken design to construct an experimental matrix to ensure that the experiment covers all possible variable combinations. Conduct the experiment according to the experimental matrix and collect the corresponding response variable data to ensure the accuracy and reliability of the data. Use the experimental data to fit a quadratic polynomial model and establish a response surface equation. Analyze the changing trend of the response variable through the response surface graph and contour graph. Use an optimization algorithm to find the experimental conditions that make the response variable reach the best value. Verify the accuracy and reliability of the model through additional experimental data to find the optimal parameter combination.

[0064] In this embodiment, in S1, the mulberry leaves and twigs are naturally dried and then crushed by a pulverizer, passed through a 200-mesh sieve, and stored in a 4°C refrigerator to obtain mulberry leaf powder.

[0065] In this embodiment, in S2, pectinase specifically decomposes pectin to convert it into soluble substances, thereby reducing the viscosity of the mulberry leaf powder. Through the decomposition of pectinase, the cell wall structure in the mulberry leaf powder is destroyed, and the effective components in the cells, such as proteins, polysaccharides, flavonoids, etc., can be more easily dissolved in the extraction solvent, thus improving the extraction efficiency and the quality of the extract.

[0066] In this embodiment, in S2, when neutral protease reacts with the mulberry leaf powder, the enzyme acts on the protein molecules in the mulberry leaf powder and decomposes them into smaller polypeptide or amino acid fragments. This process is an enzymatic hydrolysis reaction, which can be carried out under relatively mild conditions and has high efficiency and specificity.

[0067] In this embodiment, the preparation method of pectinase is as follows: Prepare fresh fruits or vegetables as raw materials, wash and cut them into pieces, grind and homogenize them. Grind the cut raw materials quickly in a pre-cooled mortar and add a 10% (mass fraction) NaCl solution to make a homogenate. Filter the homogenate, use a funnel and filter paper or 5 layers of gauze to filter the homogenate to remove the solid impurities therein, and collect the filtrate. Next, perform centrifugation. Centrifuge the filtrate at a centrifugation rate of 4000 r / min for 15 minutes to further separate and purify pectinase. After centrifugation, pectinase mainly exists in the supernatant of the centrifuge tube. Finally, adjust the pH of the supernatant and store it. Pour the supernatant into a clean small beaker, adjust the pH to 3 with 0.1 mol / L glacial acetic acid, and then store it at 4°C for later use. [[ID=,12]]

[0068] As mentioned above, the above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. A method for preparing crude extracts of mulberry branches by ultrasonic-assisted enzymatic method, characterized in that, It includes the following steps: S1. Weigh 0.5 - 1 g of mulberry leaf powder into 100 mL of distilled water; S2. Add pectinase, neutral protease, trypsin, and papain; S3. Take the enzyme dosage, solid - liquid ratio, extraction temperature, and ultrasonic power as single - factor indexes, take the total flavonoid extraction rate as the evaluation standard, establish a regression equation through Box - Behnken design experiments, and collect experimental data; S4. Conduct variance analysis and response surface analysis on the data to prepare the best crude extract of mulberry leaves and branches.

2. The method for preparing crude mulberry twig extract by ultrasonic-assisted enzymatic method according to claim 1, characterized in that In S3, use experimental design software to create a Box - Behnken design. In the software, input the number of factors, names, ranges, and the number of mid - point experiments. Run the design wizard, and the software will generate a series of experimental combinations. These combinations constitute the experimental plan of the Box - Behnken design. Perform experiments according to the plan and record the response values. Input the experimental data into the design software, and the software will help analyze the data, construct a response surface model. Through variance analysis, determine which factors are significant and whether there are significant interactions between factors. Use the graphical function of the software to visually view the response surface model, understand the relationship between factors and responses. Through analyzing the response surface model, find the optimal combination of experimental parameters to achieve the purpose of process optimization. After finding the optimal parameter combination, conduct experimental verification to ensure accuracy.

3. A method for preparing a crude extract of mulberry branches by using ultrasonic-assisted enzymatic method according to claim 2, characterized in that, In S2, add pectinase, neutral protease, trypsin, and papain, place the mixture in an ultrasonic device for ultrasonic treatment. After the ultrasonic treatment, filter to remove insoluble impurities to obtain a filtrate containing the target extract. Then, concentrate the filtrate to increase the concentration and purity of the target extract.

4. A method for preparing a crude extract of mulberry branches by ultrasonic-assisted enzymatic method according to claim 3, characterized in that, In S4, the basic steps of variance analysis mainly include the following points: propose the null hypothesis and the alternative hypothesis, collect data, and ensure the integrity and accuracy of the data. Calculate the mean of each group and the total mean, and preliminarily judge the differences between different groups. Calculate the within - group variance and between - group variance to reflect the degree of variation between the observed values within the group and the differences between different groups. Determine the total degrees of freedom, between - group degrees of freedom, and within - group degrees of freedom. Divide the sum of squares corresponding to each source of variation by the corresponding degrees of freedom to obtain the mean square of this effect. Divide the mean square of each effect to be tested by the mean square of the error to construct an F - statistic. Select an appropriate significance level to judge whether the result is significant. According to the F - statistic and degrees of freedom, look up the F - distribution table or use statistical software to determine the critical F - value. Compare the calculated F - value with the critical F - value, make a conclusion based on the comparison of the P - value and the significance level, judge whether to reject the null hypothesis, conduct in - depth discussion on the results, and interpret and analyze them in combination with professional knowledge.

5. A method for preparing a crude extract of mulberry branches by using ultrasonic-assisted enzymatic method according to claim 4, characterized in that, In S4, the response surface analysis is specifically as follows: Use the Box-Behnken design to construct an experimental matrix to ensure that the experiment covers all possible variable combinations. Conduct the experiment according to the experimental matrix and collect the corresponding response variable data, ensuring the accuracy and reliability of the data. Use the experimental data to fit a quadratic polynomial model, establish a response surface equation, analyze the change trend of the response variable through the response surface plot and contour plot, use an optimization algorithm to find the experimental conditions that make the response variable reach the optimal value, and verify the accuracy and reliability of the model through additional experimental data to find the optimal parameter combination.

6. A method for preparing a crude extract of mulberry branches by using ultrasonic-assisted enzymatic method according to claim 5, characterized in that, In S1, the mulberry leaves and twigs are naturally dried and then crushed by a pulverizer, passed through a 2oo-mesh sieve, and stored in a 4°C refrigerator to obtain mulberry leaf powder. Weigh 0.5 g of mulberry leaf powder into 100 mL of distilled water.

7. A method for preparing a crude extract of mulberry branches by ultrasonic-assisted enzymatic method according to claim 6, characterized in that, In S2, pectinase specifically decomposes pectin and converts it into soluble substances, thereby reducing the viscosity of the mulberry leaf powder. Through the decomposition of pectinase, the cell wall structure in the mulberry leaf powder is destroyed, and the effective components in the cells, such as proteins, polysaccharides, flavonoids, etc., can be more easily dissolved in the extraction solvent, thereby improving the extraction efficiency and the quality of the extract.

8. A method for preparing a crude extract of mulberry twigs by using ultrasonic-assisted enzymatic method according to claim 7, characterized in that, In S2, when neutral protease reacts with the mulberry leaf powder, the enzyme acts on the protein molecules in the mulberry leaf powder and decomposes them into smaller polypeptide or amino acid fragments. This process is an enzymatic hydrolysis reaction, which can be carried out under relatively mild conditions and has high efficiency and specificity.

9. A method for preparing a crude extract of mulberry twigs by using ultrasonic-assisted enzymatic method according to claim 8, characterized in that, In S1, weigh 0.5 g of mulberry leaf powder into 100 mL of distilled water, stir and mix evenly, and the stirring time is 5 - 10 min.

10. A method for preparing a crude extract of mulberry twigs by ultrasonic-assisted enzymatic method according to claim 9, characterized in that, The preparation method of the pectinase is as follows: Prepare fresh fruits or vegetables as raw materials, wash and cut them into pieces, grind and homogenize them. Grind the cut raw materials quickly in a pre-cooled mortar and add a 10% (mass fraction) NaCl solution to make a homogenate. Filter the homogenate, use a funnel and filter paper or 5 layers of gauze to filter the homogenate to remove the solid impurities therein, and collect the filtrate. Next, perform centrifugation. Centrifuge the filtrate at a centrifugation rate of 4000 r / min for 15 minutes to further separate and purify the pectinase. After centrifugation, the pectinase mainly exists in the supernatant of the centrifuge tube. Finally, adjust the pH of the supernatant and store it. Pour the supernatant into a clean small beaker, adjust the pH to 3 with 0.1 mol / L glacial acetic acid, and then store it at 4°C for later use.

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