阅读说明：本技术 芜菁总黄酮的提取工艺 (Process for extracting turnip total flavone ) 是由 张丽静 王凤忠 范蓓 张文会 陈锋 李淑英 夏建伟 卢聪 张娜娜 于 2020-06-01 设计创作，主要内容包括：本发明公开了一种芜菁总黄酮的提取工艺,包括：步骤一、确定每个影响因素的最优值；步骤二、筛选出对芜菁总黄酮提取率影响最显著的三个影响因素,定义为显著因素；步骤三、以显著因素的最优值为中心,并以显著因素作为响应面实验的自变量,以芜菁总黄酮提取率为响应值,对各自变量进行多项式拟合回归得出回归方程,通过对回归方程进行响应面分析,得出显著因素的最佳工艺参数；步骤四、分别以显著因素的最佳工艺参数、除显著因素以外的影响因素的最优值作为提取参数,进行芜菁总黄酮提取。发明通过单因素实验与响应面方法结合的方式优化了芜菁总黄酮提取工艺的工艺参数,与现有技术相比提高了芜菁总黄酮的提取率。(The invention discloses an extraction process of turnip total flavonoids, which comprises the following steps: step one, determining an optimal value of each influence factor; screening three influencing factors which have the most obvious influence on the extraction rate of the turnip total flavonoids and defining the three influencing factors as the remarkable factors; taking the optimal value of the significant factor as a center, taking the significant factor as an independent variable of a response surface experiment, taking the extraction rate of the turnip total flavonoids as a response value, performing polynomial fitting regression on respective variables to obtain a regression equation, and performing response surface analysis on the regression equation to obtain the optimal process parameter of the significant factor; and step four, respectively taking the optimal process parameters of the significant factors and the optimal values of the influencing factors except the significant factors as extraction parameters to extract the turnip total flavonoids. The invention optimizes the technological parameters of the turnip total flavone extraction process by combining the single-factor experiment and the response surface method, and improves the extraction rate of the turnip total flavone compared with the prior art.)

1. The extraction process of the turnip total flavone is characterized by comprising the following steps:

determining influence factors of a turnip total flavone extraction process, performing a single factor experiment aiming at each influence factor, and determining an optimal value of each influence factor by taking the turnip total flavone extraction rate as an index, wherein the number of the influence factors is not less than 3;

step two, determining the maximum range of the allowed value of each influence factor, taking the two end points of the maximum range of each influence factor as a low-level value and a high-level value respectively, taking the extraction rate of the total flavonoids of the turnips as a response value, screening out three influence factors which have the most obvious influence on the extraction rate of the total flavonoids of the turnips, and defining the three influence factors as significant factors;

taking the optimal value of the significant factor as a center, taking the significant factor as an independent variable of a response surface experiment, taking the extraction rate of the turnip total flavonoids as a response value, performing polynomial fitting regression on respective variables to obtain a regression equation, and performing response surface analysis on the regression equation to obtain the optimal process parameter of the significant factor;

and step four, respectively taking the optimal process parameters of the significant factors and the optimal values of the influencing factors except the significant factors as extraction parameters to extract the turnip total flavonoids.

2. The extraction process of turnip total flavonoids as claimed in claim 1, wherein the single factor experiment for each influencing factor is specifically as follows:

taking the influence factor as an independent variable, taking the other influence factors as determined values, extracting the total flavonoids of the turnips, and taking the extraction rate of the total flavonoids of the turnips as an index to determine the optimal value of the influence factor.

3. The extraction process of turnip total flavonoids in claim 2, wherein the regression equation in step three is as follows: Y-7.08531-0.017500A-0.089375B-0.052063C +0.010000 AB-3.75000E-003 AC +1.75000E-004 BC + 0.21500A²+6.12500E-004*B²+1.34375E-004*C²；

In the formula, Y is the extraction rate of turnip total flavonoids, A is the mixture ratio of complex enzyme, B is the ratio of material to liquid, and C is the ultrasonic power.

4. The extraction process of turnip total flavonoids in claim 3, wherein the influencing factors of the extraction process of turnip total flavonoids in the first step comprise: the compound enzyme comprises compound enzyme proportion, compound enzyme dosage, ethanol concentration, material-liquid ratio, enzymolysis time, enzymolysis temperature, ultrasonic power and ultrasonic time, wherein the compound enzyme is prepared by compounding cellulase and pectinase, and the material-liquid ratio refers to the ratio of the mass of the turnip powder to the volume of ethanol solution;

the process for extracting the turnip total flavone in the step four comprises the following specific steps:

s1, adding 75% ethanol solution and 2% complex enzyme into the turnip powder, and performing enzymolysis at 50 deg.C for 55min to obtain turnip enzymatic hydrolysate;

s2, performing high-temperature enzyme deactivation and cooling on the turnip enzymatic hydrolysate, and then performing ultrasonic treatment, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, so as to obtain the ultrasonic enzymatic hydrolysate;

s3, centrifuging the enzymatic hydrolysate after ultrasonic treatment to obtain supernatant, namely the turnip total flavone extracting solution;

wherein the feed-liquid ratio is 1:38g/mL, and the complex enzyme ratio is 1.9:1 g/g.

5. The process for extracting turnip total flavonoids as claimed in claim 4, wherein the time for deactivating enzymes in the turnip enzymatic hydrolysate at high temperature in step S2 is 5min, the speed of centrifugation of the enzymatic hydrolysate in step S3 is 3000r/min, and the centrifugation time is 10 min.

6. The extraction process of turnip total flavonoids as claimed in claim 4 or 5, wherein the specific steps of the ultrasonic treatment in step S2 are as follows: adding the cooled turnip enzymatic hydrolysate into an ultrasonic extractor, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, and extracting to obtain an ultrasonic primary extracting solution and an ultrasonic primary filter residue; adding 50% of ethanol with the volume of 38% of the mass of the ultrasonic primary filter residue into the ultrasonic primary filter residue, and then adding the mixture into an ultrasonic extractor, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, and extracting to obtain an ultrasonic secondary extracting solution and an ultrasonic secondary filter residue; adding 20% ethanol with the volume 38 times of the mass of the ultrasonic secondary filter residue into the ultrasonic secondary filter residue, adding the mixture into an ultrasonic extractor, extracting to obtain an ultrasonic tertiary extracting solution and ultrasonic tertiary filter residue with the ultrasonic power of 204W and the ultrasonic time of 60min, and mixing the ultrasonic primary extracting solution, the ultrasonic secondary extracting solution and the ultrasonic tertiary extracting solution to obtain the ultrasonic enzymatic hydrolysate.

Technical Field

The invention relates to the technical field of plant extraction. More particularly, the invention relates to an extraction process of turnip total flavonoids.

Background

Turnip is taken as vegetable for long-term eating at high altitude in Tibet, has rich nutrition and is deeply popular with the masses. According to the traditional Chinese medicine, the turnip is sweet, pungent and bitter in taste, warm in nature and non-toxic; entering stomach, liver and kidney meridians; has effects of promoting appetite and digestion, descending qi and relieving epigastric distention, relieving cough and eliminating phlegm, promoting diuresis and removing toxic substance, and warming spleen and stomach. Modern researches show that the turnip contains a large amount of biological active ingredients such as flavone, saponin, glucoside, alkaloid, volatile oil, phenols, tannin and the like, has the effects of resisting oxidation, reducing blood fat, reducing blood sugar, resisting fatigue, resisting oxygen deficiency and the like, and provides theoretical basis for developing turnip health-care food.

At present, the extraction process of the total flavonoids in turnips at home and abroad is optimized by adopting an orthogonal test design, and the orthogonal test design has the characteristics that only a single point can be analyzed, continuous variables cannot be analyzed, so that the precision of an experiment is insufficient, the interaction among all influencing factors cannot be well examined, and the optimal extraction process of the total flavonoids in the turnips cannot be well screened.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Still another object of the present invention is to provide a process for extracting turnip total flavonoids, which can screen out the optimum parameters of the process for extracting turnip total flavonoids and has a high extraction rate compared to the prior art.

To achieve these objects and other advantages in accordance with the present invention, there is provided a turnip total flavone extraction process comprising the steps of:

determining influence factors of a turnip total flavone extraction process, performing a single factor experiment aiming at each influence factor, and determining an optimal value of each influence factor by taking the turnip total flavone extraction rate as an index, wherein the number of the influence factors is not less than 3;

step two, determining the maximum range of the allowed value of each influence factor, taking the two end points of the maximum range of each influence factor as a low-level value and a high-level value respectively, taking the extraction rate of the total flavonoids of the turnips as a response value, screening out three influence factors which have the most obvious influence on the extraction rate of the total flavonoids of the turnips, and defining the three influence factors as significant factors;

taking the optimal value of the significant factor as a center, taking the significant factor as an independent variable of a response surface experiment, taking the extraction rate of the turnip total flavonoids as a response value, performing polynomial fitting regression on respective variables to obtain a regression equation, and performing response surface analysis on the regression equation to obtain the optimal process parameter of the significant factor;

and step four, respectively taking the optimal process parameters of the significant factors and the optimal values of the influencing factors except the significant factors as extraction parameters to extract the turnip total flavonoids.

Preferably, the single factor experiment performed for each influencing factor is specifically:

taking the influence factor as an independent variable, taking the other influence factors as determined values, extracting the total flavonoids of the turnips, and taking the extraction rate of the total flavonoids of the turnips as an index to determine the optimal value of the influence factor.

Preferably, the regression equation in step three is: Y-7.08531-0.017500A-0.089375B-0.052063C +0.010000 AB-3.75000E-003 AC +1.75000E-004 BC + 0.21500A²+6.12500E-004*B²+1.34375E-004*C²；

In the formula, Y is the extraction rate of turnip total flavonoids, A is the mixture ratio of complex enzyme, B is the ratio of material to liquid, and C is the ultrasonic power.

Preferably, the influencing factors of the turnip total flavone extraction process in the step one comprise: the compound enzyme comprises compound enzyme proportion, compound enzyme dosage, ethanol concentration, material-liquid ratio, enzymolysis time, enzymolysis temperature, ultrasonic power and ultrasonic time, wherein the compound enzyme is prepared by compounding cellulase and pectinase, and the material-liquid ratio refers to the ratio of the mass of the turnip powder to the volume of ethanol solution;

the process for extracting the turnip total flavone in the step four comprises the following specific steps:

s1, adding 75% ethanol solution and 2% complex enzyme into the turnip powder, and performing enzymolysis at 50 deg.C for 55min to obtain turnip enzymatic hydrolysate;

s2, performing high-temperature enzyme deactivation and cooling on the turnip enzymatic hydrolysate, and then performing ultrasonic treatment, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, so as to obtain the ultrasonic enzymatic hydrolysate;

s3, centrifuging the enzymatic hydrolysate after ultrasonic treatment to obtain supernatant, namely the turnip total flavone extracting solution;

wherein the feed-liquid ratio is 1:38g/mL, and the complex enzyme ratio is 1.9:1 g/g.

Preferably, the time for performing high-temperature enzyme deactivation on the turnip enzymatic hydrolysate in the step S2 is 5min, the rotation speed for centrifuging the enzymatic hydrolysate in the step S3 is 3000r/min, and the centrifuging time is 10 min.

Preferably, the specific steps of the ultrasonic treatment in step S2 are: adding the cooled turnip enzymatic hydrolysate into an ultrasonic extractor, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, and extracting to obtain an ultrasonic primary extracting solution and an ultrasonic primary filter residue; adding 50% of ethanol with the volume of 38% of the mass of the ultrasonic primary filter residue into the ultrasonic primary filter residue, and then adding the mixture into an ultrasonic extractor, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, and extracting to obtain an ultrasonic secondary extracting solution and an ultrasonic secondary filter residue; adding 20% ethanol with the volume 38 times of the mass of the ultrasonic secondary filter residue into the ultrasonic secondary filter residue, adding the mixture into an ultrasonic extractor, extracting to obtain an ultrasonic tertiary extracting solution and ultrasonic tertiary filter residue with the ultrasonic power of 204W and the ultrasonic time of 60min, and mixing the ultrasonic primary extracting solution, the ultrasonic secondary extracting solution and the ultrasonic tertiary extracting solution to obtain the ultrasonic enzymatic hydrolysate.

The invention at least comprises the following beneficial effects:

firstly, the invention optimizes the technological parameters of the turnip total flavone extraction process by combining the single-factor experiment and the response surface method, and improves the extraction rate of the turnip total flavone compared with the prior art.

Secondly, the cooled turnip enzymatic hydrolysate is extracted for three times by changing the ultrasonic power, the material-liquid ratio and the ethanol concentration, and on the premise of improving the extraction rate of the turnip total flavonoids, the extraction rate of the turnip polysaccharides is improved simultaneously to obtain the compound of the turnip total flavonoids and the polysaccharides.

Thirdly, in the process of extracting the turnip total flavone, the obtained turnip enzymatic hydrolysate is firstly subjected to high-temperature enzyme deactivation treatment and then ultrasonic treatment, so that the step of directly performing ultrasonic treatment on the obtained turnip enzymatic hydrolysate is replaced, and the aim of preventing the compound enzyme from excessively performing enzymolysis on the turnip powder is fulfilled, so that the extraction rate of the turnip total flavone is reduced.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows the influence of the complex enzyme ratio on the extraction rate of total flavonoids in turnip according to one of the technical schemes of the present invention;

FIG. 2 is a diagram illustrating the effect of the amount of the complex enzyme on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention;

FIG. 3 is a graph showing the effect of the feed liquid ratio on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention;

FIG. 4 is a diagram illustrating the effect of ethanol concentration on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention;

FIG. 5 is a diagram illustrating the influence of the enzymolysis temperature on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention;

FIG. 6 is a diagram illustrating the influence of the enzymolysis time on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention;

FIG. 7 is a diagram illustrating the effect of ultrasonic power on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention;

FIG. 8 is a diagram illustrating the effect of the ultrasonic treatment time on the extraction rate of total flavonoids in turnip according to one embodiment of the present invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

1. The materials include turnip sample from Cijue county of Changdu region of Tibet autonomous region, rutin control sample from Sigma company in America, antioxidant ability detection kit (ABTS) from Biyuntian biotechnology limited, and cellulase (5 × 10)⁴U/g), pectinase (40U/mg) was purchased from Beijing Solaibao Tech Co., Ltd; the total antioxidant capacity detection kit (ABTS) is purchased from Biyuntian biotechnology limited company; the reagents such as sodium nitrite, aluminum nitrate and the like are made in China and analytically and are purchased from chemical reagent companies of national drug group.

2. Turnip powder preparation method

Cleaning turnip tuber, drying at 50 deg.C for 24 hr, pulverizing, sieving with 60 mesh sieve, and packaging in a sealed bag.

3. Method for extracting turnip total flavone

3.1 adding ethanol solution and complex enzyme into the turnip powder, and then carrying out enzymolysis for a period of time at a certain temperature to obtain turnip enzymolysis liquid;

3.2, carrying out high-temperature enzyme deactivation and cooling on the turnip enzymatic hydrolysate, and then carrying out ultrasonic treatment to obtain an ultrasonic enzymatic hydrolysate;

3.3 centrifuging the enzymatic hydrolysate after ultrasonic treatment to obtain supernatant, namely the turnip total flavone extracting solution.

4. Detection of total flavone extraction rate

The reference uses NaNO₂-AlCl₃Drawing a rutin standard curve according to the experimental result of the colorimetric method, wherein the rutin standard curve is setThe detection wavelength was determined to be 510 nm. Drawing a standard curve according to the result of the light absorption value, and obtaining a linear equation of y-0.00823 x-0.004, R²0.9992. Taking 5mL of turnip total flavone extract, adding a certain amount of color reagent for color development, detecting a light absorption value in a colorimeter with the wavelength of 510nm, calculating the content of the total flavone through a standard curve, and then calculating the extraction rate of the total flavone according to the following formula. The average was calculated in triplicate.

The extraction rate of total flavone (C × V × N × 10)^-3/W)×100％

Wherein C is the concentration (mg/mL) of total flavone solution in the turnip determination sample, and V is the volume of the extracting solution; n is the dilution factor, W is the weight (g) of the weighed turnip powder.

5. Single factor experiment

5.1 determining the influence factors of the turnip total flavone extraction process as follows: the compound enzyme comprises compound enzyme proportion, compound enzyme dosage, ethanol concentration, material-liquid ratio, enzymolysis time, enzymolysis temperature, ultrasonic power and ultrasonic time, wherein the compound enzyme is prepared by compounding cellulase and pectinase, the material-liquid ratio refers to the mass ratio of the turnip powder to the volume of an ethanol solution, and the compound enzyme dosage refers to the percentage of the compound enzyme in the weight of the turnip powder.

5.2 influence of Complex enzyme ratio on the extraction yield of Total Flavonoids from Brassica rapa

Extracting the total flavonoids according to the method mentioned in the 3 turnip total flavonoids extraction method, wherein the influence factor values except the compound enzyme ratio are respectively set as 1: 35g/mL, the dosage of the complex enzyme accounts for 2% of the weight of the turnip powder, the concentration of ethanol is 75%, the enzymolysis temperature is 50 ℃, the enzymolysis time is 55min, the ultrasonic power is 200W, the ultrasonic time is 60min, and different complex enzyme ratios are set as follows: 1: 1g/g, 1.5: 1g/g, 2: 1g/g, 2.5: 1g/g, 3: 1g/g, after extraction, the extraction rate of the turnip total flavonoids is determined by the method mentioned in the detection of the extraction rate of the 4 total flavonoids when the complex enzymes are mixed, and a change curve is drawn, and the result is shown in figure 1.

As can be seen from FIG. 1, the extraction rate of total flavonoids in turnip shows a tendency of increasing and then slowly decreasing with the increase of the addition ratio of cellulase. When the cellulase is less, the cell wall is not completely damaged, the dissolution rate of the total flavone is increased along with the increase of the proportion of the cellulase, and the extraction rate is gradually increased to the maximum value of 0.95 percent. When the proportion of the cellulase is continuously increased, the extraction rate of the total flavonoids is in a slow descending trend. Probably because the cellulase is excessive, part of the fiber is attached to the surface of the turnip granules, the leaching of the total flavone is influenced, and the extraction rate of the total flavone is slightly reduced, so the optimal value of the ratio of the cellulase to the pectinase, namely the optimal value of the complex enzyme ratio is 2: 1g/g, wherein the compound enzyme ratio is 2: 1g/g was used further in subsequent experiments.

5.3 Effect of Complex enzyme dosage on the extraction yield of Total Flavonoids from Brassica rapa

Extracting total flavonoids according to the method mentioned in the method for extracting total flavonoids from turnip in 3, setting different compound enzyme dosages: 1%, 1.5%, 2%, 2.5%, 3%, and the complex enzyme ratio is 2: 1g/g, the numerical settings of ethanol concentration, feed-liquid ratio, enzymolysis time, enzymolysis temperature, ultrasonic power and ultrasonic time are the same as the numerical settings mentioned in the influence of 5.2 complex enzyme ratio on the extraction rate of the total flavonoids in the turnip, the extraction rate of the total flavonoids in the turnip is determined by the method mentioned in the detection of the extraction rate of the total flavonoids in 4 after extraction, and a change curve is drawn, and the result is shown in figure 2.

As can be seen from FIG. 2, the total flavone extraction rate of turnip shows a trend of increasing first and then stabilizing with the increase of the compound enzyme dosage, and when the compound enzyme dosage reaches 2%, the total flavone extraction rate reaches the maximum value of 1.14%. When the dosage of the compound enzyme is continuously increased, the extraction rate of the total flavone is in a stable trend. The probable reason is that with the increase of the using amount of the complex enzyme, the cell wall can be effectively destroyed, the dissolution of the total flavone is promoted, and the extraction rate is gradually increased; when the dosage of the complex enzyme exceeds 2 percent, the concentration of the substrate can not lead the enzyme to reach saturation with the increase of the dosage, so the extraction rate of the total flavone is not increased any more. According to the experimental result, 2% is selected as the optimal value of the compound enzyme dosage, wherein 2% of the compound enzyme dosage is continuously used in the subsequent experiment.

5.4 Effect of feed liquid ratio on extraction ratio of total flavonoids from Brassica rapa

Extracting the total flavonoids according to the method mentioned in the extraction method of the turnip total flavonoids in 3, and setting different material-liquid ratios: 1: 25g/mL, 1: 30g/mL, 1: 35g/mL, 1: 40g/mL, 1: 45g/mL, complex enzyme ratio 2: 1g/g of complex enzyme, 2% of the amount of the complex enzyme, the numerical settings of ethanol concentration, enzymolysis time, enzymolysis temperature, ultrasonic power and ultrasonic time and the numerical settings mentioned in the influence of 5.2 complex enzyme proportion on the extraction rate of the total flavonoids in the turnip, measuring the extraction rate of the total flavonoids in the turnip at different material-liquid ratios by the method mentioned in the detection of the extraction rate of the total flavonoids in 4 after extraction, and drawing a change curve, wherein the result is shown in figure 3.

As can be seen from FIG. 3, the extraction rate of total flavonoids in turnip increases and then gradually decreases as the ratio of the ingredients to the liquids increases. With the increase of the feed-liquid ratio, the solvent is gradually increased, the dissolution of flavone is increased, the enzymolysis is increased, and when the feed-liquid ratio is 1: 35g/mL, the extraction rate of total flavone reaches the maximum value of 1.24%, so the optimal value of the selected feed-liquid ratio is 1: 35g/mL, wherein the ratio of material to liquid is 1: 35g/mL was used continuously in subsequent experiments.

5.5 influence of ethanol concentration on the extraction yield of Total Flavonoids from Brassica rapa

Extracting the total flavonoids according to the method mentioned in the method for extracting the total flavonoids from the turnip in 3, and setting different ethanol concentrations: 55%, 65%, 75%, 85% and 95%, and the compound enzyme proportion is 2: 1g/g, the using amount of the complex enzyme is 2%, and the material-liquid ratio is 1: 35g/mL, the numerical settings of enzymolysis time, enzymolysis temperature, ultrasonic power and ultrasonic time and the numerical settings mentioned in the influence of 5.2 complex enzyme ratio on the extraction rate of the turnip total flavonoids, the extraction rate of the turnip total flavonoids at different ethanol concentrations is determined by the method mentioned in the detection of the extraction rate of the total flavonoids in step 4 after extraction, and a change curve is drawn, and the result is shown in figure 4.

As can be seen from fig. 4, with the increase of the ethanol concentration, the concentration difference between the inside and the outside of the cells is increased, which is beneficial to leaching of the total flavonoids, the extraction rate is gradually increased, the highest ethanol concentration is reached, the ethanol concentration is continuously increased, the elution amount of the alcohol-soluble pigments and the lipophilic impurities is increased, the components compete with the flavonoids to be combined with ethanol-water molecules, and the extraction rate of the total flavonoids is reduced, so that the optimal value of the ethanol concentration is selected to be 75%, wherein the ethanol concentration is 75% and is continuously used in subsequent experiments.

5.6 Effect of enzymolysis temperature on the extraction ratio of turnip Total Flavonoids

Extracting the total flavonoids according to the method mentioned in the method for extracting the total flavonoids from the turnip in the step 3, and setting different enzymolysis temperatures: 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, and the complex enzyme ratio is 2: 1g/g, the using amount of the complex enzyme is 2%, and the material-liquid ratio is 1: 35g/mL, the ethanol concentration is 75%, the numerical settings of the enzymolysis time, the ultrasonic power and the ultrasonic time and the numerical settings mentioned in the influence of the 5.2 complex enzyme ratio on the extraction rate of the turnip total flavonoids are set, the extraction rates of the turnip total flavonoids at different enzymolysis temperatures are determined by the method mentioned in the detection of the extraction rate of the total flavonoids in step 4 after extraction, and a change curve is drawn, and the result is shown in figure 5.

As can be seen from FIG. 5, the extraction rate of total turnip flavonoids gradually decreases after increasing with the increase of the enzymolysis temperature. Under the condition of low temperature, the activity of the enzyme is not completely excited, the activity of the enzyme is gradually increased along with the increase of the temperature, the reaction rate is increased, the extraction rate of the total flavone is 0.97 percent at most when the enzymolysis temperature is 50 ℃, and the enzymolysis efficiency is reduced when the temperature exceeds 50 ℃, so that the 50 ℃ is selected as the optimal value of the enzymolysis temperature, wherein the enzymolysis temperature is 50 ℃ and is continuously used in subsequent experiments.

5.7 Effect of enzymolysis time on the extraction ratio of turnip Total Flavonoids

Extracting total flavonoids according to the method mentioned in the method for extracting total flavonoids from turnip in 3, and setting different enzymolysis times: 35min, 45min, 55min, 65min and 75min, and the compound enzyme ratio is 2: 1g/g, the using amount of the complex enzyme is 2%, and the material-liquid ratio is 1: 35g/mL, the ethanol concentration is 75%, the enzymolysis temperature is 50 ℃, the numerical settings of the ultrasonic power and the ultrasonic time are the same as the numerical settings mentioned in the influence of the 5.2 complex enzyme ratio on the extraction rate of the total flavonoids of the turnips, the extraction rates of the total flavonoids of the turnips at different enzymolysis times are measured by the method mentioned in the detection of the extraction rate of the total flavonoids in step 4 after extraction, and a change curve is drawn, and the result is shown in figure 6.

As can be seen from FIG. 6, as the time of enzymolysis is prolonged, the enzyme is fully activated, the enzymolysis effect is gradually increased, the extraction rate of turnip total flavonoids is also gradually increased, and then the stability is maintained. The extraction rate of total flavone is 1% when the enzymolysis time is 60 min. After the time exceeds 55min, the enzymolysis efficiency is basically stable, the extraction rate of the total flavonoids is kept unchanged, so the optimal value of the enzymolysis time is set to 55min, wherein the enzymolysis time is 55min, and the total flavonoids can be continuously used in subsequent experiments.

5.8 Effect of ultrasonic Power on the extraction yield of Total Flavonoids from Brassica rapa

Extracting the total flavonoids according to the method mentioned in the method for extracting the total flavonoids from the turnip in 3, and setting different ultrasonic powers: 160W, 180W, 200W, 220W and 240W, and the compound enzyme proportion is 2: 1g/g, the using amount of the complex enzyme is 2.0 percent, and the material-liquid ratio is 1: 35g/mL, 75% of ethanol, 50% of enzymolysis temperature, 55min of enzymolysis time and 60min of ultrasonic time, measuring the extraction rate of the turnip total flavonoids at different ultrasonic powers by the method mentioned in the detection of the extraction rate of the total flavonoids in the step 4 after extraction, and drawing a change curve, wherein the result is shown in figure 7.

As can be seen from FIG. 7, the extraction rate of turnip total flavonoids increases with the increase of the ultrasonic power, and when the ultrasonic power exceeds 200W, the flow of the extracting agent is accelerated, the stay time of the ultrasonic wave is reduced, and the extraction rate of the total flavonoids is gradually reduced, so that the optimal value of the selected ultrasonic power is 200W, wherein the ultrasonic power is 200W and is continuously used in the subsequent experiments.

5.9 Effect of ultrasound time on the extraction yield of Total Flavonoids from Brassica rapa

Extracting the total flavonoids according to the method mentioned in the method for extracting the total flavonoids from the turnip in 3, and setting different ultrasonic time: 40min, 50min, 60min, 70min and 80min, and the compound enzyme ratio is 2: 1g/g, the using amount of the complex enzyme is 2.0 percent, and the material-liquid ratio is 1: 35g/mL, the ethanol concentration is 75%, the enzymolysis temperature is 50 ℃, the ultrasonic power is 200W, the extraction rate of the turnip total flavonoids at different enzymolysis time is measured by the method mentioned in the detection of the extraction rate of the total flavonoids in step 4 after extraction, and a change curve is drawn, and the result is shown in figure 8.

As can be seen from FIG. 8, the extraction rate of total flavonoids in turnip showed a tendency of increasing and then keeping stable as the ultrasound time is prolonged. When the ultrasonic treatment time reaches 60min, the extraction rate of the total flavonoids reaches the maximum, and is 1.05%. After 60min, the extraction rate of the total flavonoids is basically kept unchanged. The extraction cost is increased along with the extension of the ultrasonic time, and the total flavone in the turnip can be fully extracted within 60min, so the optimal value of the ultrasonic time is set to be 60 min.

6. Plackett-Burman experiment

6.1 level gauge design

Determining the maximum range of the allowed value of each influence factor, taking the optimal value of each influence factor as the center in the maximum range of the allowed value, and taking a low-level value and a high-level value respectively around the optimal value of each influence factor, wherein the maximum range of the allowed value is an interval in which the change of the extraction rate of the total flavonoids in the turnips is relatively stable, the high-level value is about 1.25-1.5 times of the low-level value and is generally not more than 2 times, the low-level value is coded as-1, the high-level value is coded as 1, and the design combination is shown in table 1.

TABLE 1Plackett-Burman experiment influencing factors and levels

6.2Plackett-Burman Experimental design, results and significance test

The experiment has 8 influencing factors, and in order to accurately and quickly find out the most obvious influencing factor on the turnip total flavone extraction process, 4 virtual variables are required to be added for estimating experiment errors, so that at least 12 groups of experiments are designed in the experiment, and each group of experiments has 8 influencing factors and 4 virtual variables. And determining the significance of each influence factor by analyzing each influence factor by taking a low-level value and a high-level value and comparing the difference between the two levels of values of each influence factor. And (3) carrying out experiment design on the data in the table 1 by using minitab software to obtain 12 groups of experiments, wherein the design results are shown in the table 2, the virtual variables are not shown in the table 2, and then carrying out multiple regression analysis on the data in the table 2 by using the minitab software to obtain the significance of each influence factor, and the results are shown in the table 3.

TABLE 2Plackett-Burman experiment influence factor levels and results

Experiment number	A	B	C	D	E	F	G	H	Extraction ratio (%)
										1	1	-1	1	1	-1	1	-1	-1	0.94
2	1	1	-1	1	-1	-1	-1	1	0.76
										3	1	-1	1	-1	-1	-1	1	1	1.05
4	-1	-1	-1	-1	-1	-1	-1	-1	1.02
										5	-1	1	1	1	-1	1	1	-1	0.89
6	-1	1	1	-1	1	-1	-1	-1	0.73
										7	1	-1	-1	-1	1	1	1	-1	0.65
8	-1	-1	1	1	1	-1	1	1	0.97
										9	1	1	1	-1	1	1	-1	1	0.82
10	-1	-1	-1	1	1	1	-1	1	1.17
										11	-1	1	-1	-1	-1	1	1	1	1.09
12	1	1	-1	1	1	-1	1	-1	0.88

TABLE 3 significance test of the influencing factors

Model (model)	Regression coefficient	Standard deviation of	Value of T	P value	Significance of
						A	0.0975	0.018	5.43	0.012	*
B	-0.0258	0.018	-1.44	0.246	--
						C	-0.0575	0.018	3.20	0.049	*
D	0.0192	0.018	1.07	0.364	--
						E	-0.0308	0.018	-1.72	0.185	--
F	0.0375	0.018	2.09	0.128	--
						G	-0.0692	0.018	-3.85	0.031	*
H	0.0192	0.018	1.07	0.364	--

Note: indicates significant effect (p < 0.05).

As can be seen from Table 3, the influence factors A (complex enzyme ratio), C (feed-liquid ratio) and G (ultrasonic power) among the eight influence factors have the most significant influence on the extraction rate of the total flavonoids, and therefore, the three influence factors are selected for carrying out a response surface optimization experiment.

7. Box-Behnken experimental design and response surface result analysis

7.1Box-Behnken Experimental design

By taking the optimal values of the significant factors screened by 6 and Plackett-Burman experiments as the center, 1 horizontal value is taken around the optimal value of each significant factor as the level of a response surface, and the levels are respectively coded as-1, 0 and 1, wherein the value of 1 is about 1.25-1.5 times of the value of-1 and generally not more than 2, and a Box-Behnken response surface optimization experiment with three factors and three levels is designed, and the experimental design is shown in Table 4.

TABLE 4Box-Behnken response surface experiment influence factors and horizon table

7.2 response surface Experimental design combination and turnip Total flavonoid extraction Rate

The data in Table 4 were processed using software and the Box-Behnken experimental design combinations and turnip total flavone extraction rates are shown in Table 5.

TABLE 5Box-Behnken response surface Experimental design combinations and turnip Total Flavonoids extraction rates

7.3 establishing regression equation and significance test

And (3) performing polynomial fitting regression by using design expert 8.0 software and data in table 5 to obtain a regression equation:

Y＝7.08531-0.017500*A-0.089375*B-0.052063*C+0.010000*AB-3.75000E-003*AC+1.75000E-004*BC+0.21500*A²+6.12500E-004*B²+1.34375E-004*C²table 6 shows the results of analysis of variance of the regression equation. According to regression analysis, the mismatching value is less than 0.05, which shows that no mismatching factor exists and the fitting degree is good. Significant coefficient R of regression equation²0.9583, simulation adjustment factor R² _Adj0.9046, the regression equation is shown to be reliable.

TABLE 6 analysis of variance of regression equation

Sources of variance	Degree of freedom	Sum of squares	Mean square	F value	P value	Significance of
							Model (model)	9	5.288	0.573	17.86	<0.0001	**
A	1	0.237	0.237	17.564	0.0073	**
							B	1	0.311	0.311	14.251	0.0045	**
C	1	0.458	0.458	15.779	0.0381	--
							AB	1	0.104	0.104	3.124	0.0206	*
AC	1	0.562	0.562	1.765	0.2268	--
							BC	1	0.491	0.491	1.533	0.2561	--
A2	1	0.039	0.039	12.241	0.0001	**
							B2	1	0.058	0.058	18.04	0.0038	**
C2	1	0.0312	0.0312	9.82	0.7639	--
							Missimilitude term	3	0.022	0.0074	2.83	0.1245	--
Pure error	4	0.2051	0.0--912	--	--	--
							Sum of	16	5.42	--	--	--	--
--	--	R2＝0.9683	R2 Adj＝0.9446	--	--	--

Note: indicates significant effect (p <0.05) and indicates very significant effect (p < 0.01).

As can be seen from Table 6, the three influencing factors of the turnip total flavone extraction rate from large to small are as follows: the compound enzyme preparation ratio (A), the liquid-material ratio (B) and the ultrasonic power (D), wherein the compound enzyme preparation ratio (A), the liquid-material ratio (B) and the influence on the extraction rate are extremely obvious (P is less than 0.001), and the interaction of the ultrasonic power (D), the compound enzyme preparation ratio (A) and the liquid-material ratio (B) also has obvious influence on the extraction rate (P is less than 0.05). The optimal extraction process of the turnip total flavonoids predicted by the regression model comprises the following steps: the compound enzyme is proportioned as follows: 1.935:1 g/g; the feed-liquid ratio is 1:37.54g/mL, the ultrasonic power is 204W, the extraction rate is 1.489%, and the optimal value of a single-factor experiment is selected under other conditions. In order to facilitate the actual operation, the extraction conditions are set as complex enzyme ratio: 1.9:1 g/g; the material-liquid ratio is 1:38g/mL, the ultrasonic power is 204W, three average experiments are carried out, the average extraction rate is 1.458%, and the difference with the predicted value is 2.08%, which shows that the model has high reliability and can be used for extracting the turnip total flavonoids.

8. Detection of polysaccharide extraction

Reference the experimental results for the sulfuric acid-phenol colorimetric method use a dextran standard curve in which the set detection wavelength is 485 nm. Drawing a standard curve according to the result of the light absorption value, and obtaining a linear equation of y ═ 0.5634x-0.035, R²0.9992. Taking 5mL of turnip total flavone extract, adding a certain amount of color reagent for color development, detecting a light absorption value in a colorimeter with the wavelength of 485nm, calculating the content of polysaccharide through a standard curve, and then calculating the polysaccharide extraction rate according to the following formula. The average was calculated in triplicate.

Polysaccharide extraction (%) ═ C_{Polysaccharides}×V_{Polysaccharides}×N_{Polysaccharides}×10^-3/W_{Polysaccharides})×100％

Wherein, C_{Polysaccharides}Determination of polysaccharide concentration (mg/mL), V, in samples for turnip_{Polysaccharides}Is the volume of the extracting solution; n is a radical of_{Polysaccharides}To dilution factor, W_{Polysaccharides}Is the weighed mass (g) of the turnip powder.

< example 1>

The optimum values of the compound enzyme ratio, the compound enzyme dosage, the ethanol concentration, the material-liquid ratio, the enzymolysis time, the enzymolysis temperature, the ultrasonic power and the ultrasonic time screened by a 5-factor experiment are taken as the extraction process parameters of the turnip total flavone, and the total flavone is extracted by the method mentioned in the 3-factor turnip total flavone extraction method.

< example 2>

The optimal values of the compound enzyme dosage, the ethanol concentration, the enzymolysis time, the enzymolysis temperature and the ultrasonic time screened by a 5-factor experiment and the optimal process parameters of the compound enzyme proportion, the material-liquid ratio and the ultrasonic power determined in the 7Box-Behnken experiment design and the response surface result analysis are taken as the total flavone extraction process parameters of the turnip, and the total flavone extraction is carried out by the method mentioned in the 3-way total flavone extraction method.

< example 3>

The experimental procedure is the same as example 2, except that: adding the cooled turnip enzymatic hydrolysate into an ultrasonic extractor, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, and extracting to obtain an ultrasonic primary extracting solution and an ultrasonic primary filter residue; adding 50% of ethanol with the volume of 38% of the mass of the ultrasonic primary filter residue into the ultrasonic primary filter residue, and then adding the mixture into an ultrasonic extractor, wherein the ultrasonic power is 204W, and the ultrasonic time is 60min, and extracting to obtain an ultrasonic secondary extracting solution and an ultrasonic secondary filter residue; adding 20% ethanol with the volume 38 times of the mass of the ultrasonic secondary filter residue into the ultrasonic secondary filter residue, adding the mixture into an ultrasonic extractor, extracting to obtain an ultrasonic tertiary extracting solution and ultrasonic tertiary filter residue with the ultrasonic power of 204W and the ultrasonic time of 60min, mixing the ultrasonic primary extracting solution, the ultrasonic secondary extracting solution and the ultrasonic tertiary extracting solution to obtain an ultrasonic enzymolysis solution, and centrifuging the ultrasonic enzymolysis solution to obtain a supernatant, namely the turnip total flavone and polysaccharide compound extracting solution.

< analysis of extraction efficiency of Total Flavonoids from turnip >

The extraction rates of the total flavonoids and the polysaccharides of the turnips obtained in the extraction solutions of the total flavonoids in the turnips obtained in the embodiments 1, 2 and 3 are respectively detected, the extraction rate of the total flavonoids of the turnips is detected by the method mentioned in the detection of the extraction rate of the total flavonoids of 4, and the extraction rate of the polysaccharides is detected by the method mentioned in the detection of the extraction rate of the polysaccharides of 8. As a result, the extraction rate of total flavonoids from turnip in example 1 was 1.05%, the extraction rate of polysaccharides was 0.87%, the extraction rate of total flavonoids from turnip in example 2 was 1.46%, the extraction rate of polysaccharides was 0.92%, the extraction rate of total flavonoids from turnip in example 3 was 1.82%, and the extraction rate of polysaccharides was 7.34%.

Through comparison between the embodiment 1 and the embodiment 2, the invention discovers that the process parameters of the turnip total flavone extraction process are optimized through a mode of combining a single-factor experiment and a response surface method, and the extraction rate of the turnip total flavone is improved compared with the prior art. Example 2 and example 3 show that the cooled turnip enzymatic hydrolysate is extracted for three times by changing the concentration of ethanol, the extraction rate of turnip polysaccharide is improved on the premise of improving the extraction rate of the total flavone of turnip, and the compound of the total flavone of turnip and polysaccharide is obtained.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.