CN111202788A - Extraction process for optimizing green tea polyphenol by using response surface method complex enzyme - Google Patents
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Abstract
The invention relates to an extraction process of green tea polyphenol, and particularly discloses an extraction process for optimizing green tea polyphenol by using a response surface method complex enzyme, which comprises the following steps: preparing materials; measuring the content of tea polyphenol; single factor experiment; optimizing a tea polyphenol extraction process by a response surface analysis method; the method adopts a complex enzyme assisted organic solvent extraction method to extract green tea polyphenol, takes the principles of environmental protection, low carbon and low cost into consideration, takes low-price nontoxic ethanol as an extractant, optimizes the process through single factor tests, Box-Behnken test design and response surface analysis, and obtains the optimal extraction process conditions of cellulase, pectinase dosage, solvent concentration, material-liquid ratio, extraction temperature, extraction time and pH value, wherein the extraction rate can reach 15.61% under the conditions.
Description
Technical Field
The invention relates to an extraction process of green tea polyphenol, in particular to an extraction process for optimizing green tea polyphenol by using a response surface method complex enzyme.
Background
Tea is one of three major drinks in the world, contains various biological active ingredients, and the natural physiological health-care efficacy of the tea is favored by more and more consumers, wherein tea polyphenol is one of important active ingredients, and the tea polyphenol is a general name of compounds such as catechins, flavonoids, phenolic acids and anthocyanidins in the tea, and has the functions of removing free radicals in vivo, resisting aging, cancers, radiation, bacteria and bacteria, and the like. The tea polyphenol has wide application prospect as a natural antioxidant. There are several methods for extracting tea polyphenol, and each has advantages and disadvantages. The invention takes old leaves of tea trees in Tibet Linzhi Yigong tea field as raw materials, optimizes the process for extracting tea polyphenol by using compound enzyme by using a response surface method, explores the optimal extraction conditions and realizes the highest efficiency extraction rate. The method is expected to provide theoretical support for fully utilizing old tea leaves, increasing the added value of the tea industry.
The Response Surface analysis method (RSM) is a statistical method that uses a reasonable experimental design method and obtains certain data through experiments, uses a multiple quadratic regression equation to fit the functional relationship between factors and Response values, and seeks optimal process parameters through the analysis of the regression equation to solve the multivariate problem.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the process for optimizing the extraction of the green tea polyphenol by using the response surface method complex enzyme, and the process has a simple and reasonable structure and is convenient to popularize.
A process for optimizing extraction of green tea polyphenol by using a response surface method complex enzyme comprises the following steps:
(1) material preparation
a. Selecting clean tea leaves, deactivating enzymes at high temperature, airing, crushing, sieving with a 40-mesh sieve to obtain tea powder, and refrigerating for later use;
(2) determination of tea polyphenol content
a. Accurately weighing 0.110g of gallic acid standard, placing the gallic acid standard in a 100mL volumetric flask, dissolving the gallic acid standard in deionized water, and obtaining a gallic acid standard stock solution after constant volume;
b. accurately sucking gallic acid solution with concentration of 0.0, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL in 100mL volumetric flask, respectively, diluting to constant volume with water, shaking to make the concentration of gallic acid solution be 0, 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL;
c. respectively taking 1mL of the gallic acid solution with different concentrations, transferring the gallic acid solution into a test tube, respectively adding 5mL of forinophenol reagent, shaking up, and reacting for 3-8 min;
d. respectively adding 4.0mL of sodium carbonate solution with the mass fraction of 75g/L into the solution in the step c, shaking up, and standing for 60min at room temperature;
e. d, measuring the absorbance of the mixed solution in the step d at the position of 765nm of the wavelength, and performing linear regression by using the gallic acid content (Y) and the absorbance (X) to obtain a standard curve equation: y is 0.0129X +0.0844, R2＝0.9996；
f. The calculation formula of the tea polyphenol yield is as follows:
(3) single factor experiment
a. Accurately weighing 1.000g of tea powder, and mixing with 70% ethanol solution as solvent;
b. carrying out water bath leaching single-factor test by using different cellulase, pectinase, solvent concentration, liquid-material ratio, extraction temperature, extraction time and pH value;
c. setting 5 different treatment levels for each single factor, making 3 treatments in parallel, and averaging to obtain the tea polyphenol content;
(4) response surface analysis method optimized tea polyphenol extraction process
a. On the basis of a single-factor experiment, selecting four single factors with larger influence as response variables, and carrying out Box-Behnken Design (BBD) experiment Design by using default expert8.0.6 software;
b. each factor is provided with 3 treatment levels, and each treatment is carried out in 3 parallels, so that the extraction condition is further optimized.
Preferably, the tea leaves in the step (1) are old tea leaves in Yigong tea farm in Bomiprefecture of Tibet Linzhi.
Preferably, the calculation formula of the yield of tea polyphenol in the step (2) is as follows: c. CTP: tea polyphenol content (%); a: the absorbance of the sample test solution; a. the0: absorbance of reagent blank liquid; SLOPEStd: the slope of the gallic acid standard curve; m: sample mass (g); v: sample extract volume (mL); d: dilution factor (typically 1mL to 100mL, then 100); ω: sample dry matter content (%).
Preferably, the cellulase used in step (3) is: 2.4, 6, 8 and 10mg/g, the dosage of the pectinase is as follows: 2.4, 6, 8 and 10mg/g, and the solvent concentration is as follows: 20. 40, 60, 80 and 100 percent, the material-liquid ratio is 1:10, 1:20, 1:30, 1:40 and 1:50g/mL, the extraction temperature is as follows: 30. 45, 60, 75 and 90 ℃, and the extraction time is as follows: 30. 70, 110, 150 and 190min, wherein the pH value is as follows: 3. 4, 5, 6 and 7.
Preferably, the four single factors selected in the step (4) are solvent concentration, feed-liquid ratio, extraction temperature and extraction time.
Compared with the prior art, the invention has the beneficial effects that:
the method adopts a complex enzyme assisted organic solvent extraction method to extract green tea polyphenol, gives consideration to the principles of green environmental protection, low carbon and low cost, takes low-price nontoxic ethanol as an extractant, optimizes the process through single factor test, Box-Behnken test design and response surface analysis, and obtains the optimal extraction process conditions of 6mg/g of cellulase dosage, 8mg/g of pectinase dosage, 68% of solvent concentration and 1 of feed-liquid ratio: 42, the extraction temperature is 56 ℃, the extraction time is 129min, the pH value is 5.0, the extraction rate can reach 15.61% under the condition, and compared with the extraction rate of the traditional tea polyphenol, the method adopts the complex enzyme for assistance, not only improves the extraction rate, but also shortens the extraction time, is easy for industrial operation, and has certain practical value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the effect of cellulase dosage on extraction yield;
FIG. 2 is a graph showing the effect of pectinase dosage on extraction yield;
FIG. 3 is a graph showing the effect of solvent concentration on tea polyphenol extraction yield;
FIG. 4 shows the effect of feed liquid ratio on tea polyphenol extraction rate;
FIG. 5 is a graph showing the effect of extraction temperature on the extraction rate of tea polyphenols;
FIG. 6 is a graph showing the effect of extraction time on the extraction rate of tea polyphenols;
FIG. 7 is a graph showing the effect of extraction time on the extraction rate of tea polyphenols;
FIG. 8 is a response graph and contour lines of the effect of solvent concentration and feed liquid ratio on the extraction rate of tea polyphenols;
FIG. 9 is a response graph and contour lines of the effect of solvent concentration and extraction temperature on the extraction rate of tea polyphenols;
FIG. 10 is a response graph and contour lines of the effect of solvent concentration and extraction time on the extraction rate of tea polyphenols;
FIG. 11 is a response graph and contour lines of the effect of feed-liquid ratio and extraction temperature on the extraction rate of tea polyphenols;
FIG. 12 is a response graph and contour lines of the effect of feed-liquid ratio and extraction time on the extraction rate of tea polyphenols;
FIG. 13 is a response graph and contour lines of the effect of extraction temperature and extraction time on the extraction rate of tea polyphenols.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention easier to clearly understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
(1) Material preparation
a. Selecting clean tea leaves, using old tea leaves of tea trees in Yigong tea farm in Bomicounty of the Tibetan Linzhi area, deactivating enzymes at high temperature, drying, crushing, sieving with a 40-mesh sieve to obtain tea powder, and refrigerating for later use;
(2) determination of tea polyphenol content
b. Accurately weighing 0.110g of gallic acid standard, placing the gallic acid standard in a 100mL volumetric flask, dissolving the gallic acid standard in deionized water, and obtaining a gallic acid standard stock solution after constant volume;
c. accurately sucking gallic acid solution with concentration of 0.0, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL in 100mL volumetric flask, respectively, diluting to constant volume with water, shaking to make the concentration of gallic acid solution be 0, 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL;
d. respectively taking 1mL of the gallic acid solution with different concentrations, transferring the gallic acid solution into a test tube, respectively adding 5mL of forinophenol reagent, shaking up, and reacting for 3-8 min;
e. respectively adding 4.0mL of sodium carbonate solution with the mass fraction of 75g/L into the solution in the step c, shaking up, and standing for 60min at room temperature;
f. d, measuring the absorbance of the mixed solution in the step d at the position of 765nm of the wavelength, and performing linear regression by using the gallic acid content (Y) and the absorbance (X) to obtain a standard curve equation: y is 0.0129X +0.0844, R2＝0.9996；
g. The calculation formula of the tea polyphenol yield is as follows:
in the formula:
cTP: tea polyphenol content,%;
a: the absorbance of the sample test solution;
A0: absorbance of reagent blank liquid;
SLOPEStd: the slope of the gallic acid standard curve;
m: sample mass in grams (g);
v: sample extract volume in milliliters (mL);
d: dilution factor (typically 1mL to 100mL, then 100);
ω: sample dry matter content (mass fraction),%;
(3) single factor experiment
a. Accurately weighing 1.000g of tea powder, and mixing with 70% ethanol solution as solvent;
b. using compound enzyme to assist organic solvent extraction method, setting 5 different treatment levels for each single factor; the dosage of the cellulase is as follows: 2.4, 6, 8 and 10mg/g, the dosage of the pectinase is as follows: 2.4, 6, 8 and 10mg/g, and the solvent concentration is as follows: 20. 40, 60, 80 and 100 percent, the material-liquid ratio is 1:10, 1:20, 1:30, 1:40 and 1:50g/mL, the extraction temperature is as follows: 30. 45, 60, 75 and 90 ℃, and the extraction time is as follows: 30. 70, 110, 150 and 190min, wherein the pH value is as follows: 3. 4, 5, 6 and 7.
c. Setting 5 different treatment levels for each single factor, making 3 treatments in parallel, and averaging to obtain the tea polyphenol content;
the specific experimental results are as follows:
i. effect of cellulase dosage on extraction yield
Most of tea polyphenol exists in cells, and cellulase and pectinase are used in the extraction process of tea polyphenol, and can effectively destroy plant cell walls, thereby increasing the dissolution amount of tea polyphenol and improving the extraction speed. Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, taking 70% ethanol solution as a solvent, adjusting the pH value of an extraction system to 5.0, and mixing the components according to a material-liquid ratio of 1:30 (g/ml), placing into a Vickers bottle, adding cellulase 2, 4, 6, 8 and 10mg respectively, extracting in 40 deg.C constant temperature water bath for 50min, filtering, and determining tea polyphenol content in the extractive solution according to step (2). And comparing the influence of different cellulase dosage on the tea polyphenol extraction rate to obtain the optimal cellulase dosage.
As shown in FIG. 1, the optimum amount of cellulase is 6mg/g, the extraction rate of tea polyphenols increases rapidly with the increase of the amount of cellulase, when the amount of cellulase reaches 6mg/g, the extraction rate is the highest, and when the amount of cellulase is increased, the extraction rate decreases slowly and gradually becomes stable.
Effect of pectinase dosage on extraction yield
Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, taking 70% ethanol solution as a solvent, adjusting the pH value of an extraction system to 5.0, and mixing the components according to a material-liquid ratio of 1:30 (g/ml), putting into a vitamin bottle, respectively adding 6mg of cellulase, then adding 2, 4, 6, 8 and 10mg of pectinase, extracting for 50min in a constant-temperature water bath at 40 ℃, filtering, and determining the content of tea polyphenol in the extracting solution according to the step (2). And comparing the influence of different pectinase dosages on the extraction rate of the tea polyphenol to obtain the optimal pectinase dosage.
As shown in FIG. 2, the best dosage of pectinase is 8mg/g, and within a certain range, the tea polyphenol extraction rate increases with the increase of pectinase dosage, and when the extraction rate reaches 8mg/g, the extraction rate begins to decrease slowly with the increase of enzyme dosage. It is possible that the enzyme dosage is saturated, so that the enzyme activity is inhibited, and the enzyme activity is combined with tea polyphenol to form precipitate, thereby reducing the yield of the tea polyphenol.
influence of solvent concentration on extraction yield of tea polyphenols
Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, respectively adding 6mg of cellulase and 8mg of pectinase, respectively placing the mixture into a Vickers, respectively taking 20%, 40%, 60%, 80% and 100% ethanol solution as solvents, adjusting the pH value of an extraction system to be 5.0, and mixing the materials according to a material-liquid ratio of 1:30 (g/ml), extracting for 50min in a constant-temperature water bath at 40 ℃, filtering, taking supernatant, and determining the content of the tea polyphenol in the extracting solution according to the step (2). Comparing the influence of solvents with different concentrations on the extraction rate of tea polyphenol.
As shown in FIG. 3, it can be seen from FIG. 3 that the extraction rate is increased with the increase of the concentration, and the extraction rate is maximized at a concentration of 60%, and then the extraction rate is decreased with the increase of the concentration, and the extraction rate of the absolute ethyl alcohol is minimized. The extraction rate is related to the polarity of the solvent, and the extraction rate of the organic solvent aqueous solution is better.
iv influence of feed liquid ratio on extraction rate of tea polyphenols
Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, taking a 60% ethanol solution as a solvent, adjusting the pH value of an extraction system to 5.0, respectively adding 6mg of cellulase and 8mg of pectinase, respectively placing the materials in a Vickers according to the material-liquid ratio of 1:10, 1:20, 1:30, 1:40 and 1:50g/mL, respectively extracting for 50min in a constant-temperature water bath at 40 ℃, filtering, and determining the content of tea polyphenol in an extracting solution according to the operation of the step (2). Comparing the influence of different feed liquid ratios on the extraction rate of tea polyphenols.
As a result, as shown in fig. 4, the liquid-to-liquid ratio increased and the extraction rate also increased, and when the liquid-to-liquid ratio reached 40:1, the extraction rate began to increase slowly, and 40:1 was determined as the optimum liquid-to-liquid ratio in consideration of the extraction cost and the separation and purification process.
v. influence of feed liquid ratio on extraction rate of tea polyphenols
Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, taking a 60% ethanol solution as a solvent, adjusting the pH value of an extraction system to 5.0, respectively adding 6mg of cellulase and 8mg of pectinase, putting the mixture into a Vietnam according to the material-liquid ratio of 1:40, respectively extracting for 50min in constant-temperature water bath at 30, 45, 60, 75 and 89 ℃, filtering, and determining the content of tea polyphenol in the extract according to the operation of the step (2). Comparing the influence of different temperatures on the extraction rate of tea polyphenol.
As shown in FIG. 5, it can be seen from FIG. 5 that the extraction rate is increased by increasing the temperature, and when the extraction temperature is 60 ℃, the extraction rate is maximized, and then the extraction rate is decreased by increasing the temperature, and it is likely that the enzyme activity is inactivated by the high temperature, which also partially degrades the tea polyphenols. vi. influence of extraction time on extraction rate of tea polyphenols
Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, taking a 60% ethanol solution as a solvent, adjusting the pH value of an extraction system to 5.0, respectively adding 6mg of cellulase and 8mg of pectinase, putting the mixture into a Vietnamese bottle according to the material-liquid ratio of 1:40, respectively extracting in a constant-temperature water bath at 60 ℃ for 30 min, 70 min, 110min, 150 min and 190min, filtering, and determining the content of tea polyphenol in an extracting solution according to the operation of the step (2). Comparing the influence of different extraction time on the extraction rate of tea polyphenol.
As shown in FIG. 6, it is understood from FIG. 6 that the extraction rate is increased with the increase of time, the extraction rate is the highest when the extraction time is 110min, and the extraction rate is decreased instead when the extraction time is prolonged, which may be caused by the partial degradation due to the temperature tolerance of tea polyphenols.
Influence of pH value on extraction rate of tea polyphenols
Accurately weighing 1.000g of tea powder, respectively weighing 5 parts, taking 60% ethanol solution as a solvent, respectively adding 6mg of cellulase and 8mg of pectinase, putting the mixture into a Vickers according to the material-liquid ratio of 1:40, adjusting the pH value of an extraction system to be 3.0, 4.0, 5.0, 6.0 and 7.0, respectively extracting in a constant-temperature water bath at 60 ℃ for 110min, filtering, and determining the content of tea polyphenol in an extracting solution according to the operation of the step (2). Comparing the influence of pH value on tea polyphenol extraction rate.
The results are shown in FIG. 7. from FIG. 7, it can be seen that the optimum pH for extraction of tea polyphenols is 5, indicating that tea polyphenols are stable in slightly acidic environment.
(4) Response surface analysis method optimized tea polyphenol extraction process
a. On the basis of a single-factor experiment, selecting four single factors with larger influence as response variables, and carrying out Box-Behnken Design (BBD) experiment Design by using default expert8.0.6 software;
b. each factor is provided with 3 treatment levels, and each treatment is carried out in 3 parallels, so that the extraction condition is further optimized.
TABLE 1 Box-Behnken Experimental design factors and horizon
On the basis of a single-factor experiment, 4 factors including solvent concentration, feed-liquid ratio, extraction temperature and extraction time are selected, and Box-Behnken Design (BBD) optimization experiment Design is carried out by using default expert8.0.6 software.
Table 2 BBD experimental design and results
Performing multiple regression fitting on the experimental data in the table 2 to obtain a coding multiple quadratic regression equation which takes the tea polyphenol yield as a dependent variable and takes 4 single factors of solvent concentration, material-liquid ratio, extraction time and extraction temperature as independent variables:
Y＝15.54-0.88A+10.84B-1.04C-0.24D-0.60AB+0.89AC+0.77AD-0.11BC+0.21BD+0.51CD-2.91A2-2.44B2-1.72C2-2.79D2
TABLE 3 analysis of variance of multiple regression equation model
Wherein, differences were very significant (P < 0.01); significant differences (P < 0.05); the remaining differences were not significant (P > 0.05).
The analysis of the model variance is the basis for judging whether the model is reliable, and the data analysis in table 3 shows that the P value in the model is far lower than 0.01, which indicates that the model has extremely strong significance, the correlation coefficient R2 is 0.9738, the coefficient of variation CV is 4.260%, and the signal-to-noise ratio is 11.56, which indicates that the quadratic regression equation has good fitting degree and can be used for the optimization and prediction of the tea polyphenol extraction process conditions. Meanwhile, the magnitude of the F value directly reflects the closeness degree of the relationship between each factor and the experimental result, namely: extraction temperature (C) ═ 46.11> solvent concentration (a) > 32.85> feed-to-liquid ratio ═ 30.05(B) > extraction time (D) > 2.45.
(5) Response surface analysis and extraction process optimization
As shown in FIGS. 8-13, which are response graphs and contour graphs of the extraction rate of tea polyphenol and four factors, the influence of the interaction of four single-factor variables on the yield Y of tea polyphenol is visually reflected, the steeper the curved surface of the three-dimensional response graph shows that the single-factor interaction is more obvious, and the more the contour line tends to be elliptical, the more obvious the single-factor interaction is shown [9]. Solving the fitting equation to obtain the optimal extraction conditions of the four factors as follows: the solvent concentration is 68.48%, the material-liquid ratio is 1:42 · 18, the extraction temperature is 55.82 ℃, the extraction time is 129.26min, and the highest extraction rate of tea polyphenol is 15.67% under the condition. For convenience of operation, the predicted optimal process conditions are modified as follows: the solvent concentration is 68%, the ratio of material to liquid is 1:42, the extraction temperature is 56 deg.C, and the extraction time is 129 min. After 3 times of repeated verification experiments, the average value of the tea polyphenol extraction rate is 15.61%, and the difference between the average value and the predicted value is 15.67%, which indicates that the model fitting degree is good.
The method adopts a complex enzyme assisted organic solvent extraction method to extract green tea polyphenol, gives consideration to the principles of environmental protection, low carbon and low cost, takes low-price nontoxic ethanol as an extractant, optimizes the process through single factor test, Box-Behnken test design and response surface analysis, and obtains the optimal extraction process conditions that the dosages of cellulase and pectinase are respectively 6mg/g and 8mg/g, the solvent concentration is 68%, and the feed-liquid ratio is 1:42, the extraction temperature is 56 ℃, the extraction time is 129min, the pH value is 5.0, the extraction rate can reach 15.61 percent under the condition, and compared with the extraction rate of the traditional tea polyphenol, the method adopts the complex enzyme for assistance, not only improves the extraction rate, but also shortens the extraction time, is easy for industrial operation, and has certain practical value.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. A process for optimizing extraction of green tea polyphenol by using a response surface method complex enzyme is characterized by comprising the following steps:
(1) material preparation
a. Selecting clean tea leaves, deactivating enzymes at high temperature, airing, crushing, sieving with a 40-mesh sieve to obtain tea powder, and refrigerating for later use;
(2) determination of tea polyphenol content
a. Accurately weighing 0.110g of gallic acid standard, placing the gallic acid standard in a 100mL volumetric flask, dissolving the gallic acid standard in deionized water, and obtaining a gallic acid standard stock solution after constant volume;
b. accurately sucking gallic acid solution with concentration of 0.0, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL in 100mL volumetric flask, respectively, diluting to constant volume with water, shaking to make the concentration of gallic acid solution be 0, 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ g/mL, 50 μ g/mL;
c. respectively taking 1mL of the gallic acid solution with different concentrations, transferring the gallic acid solution into a test tube, respectively adding 5mL of forinophenol reagent, shaking up, and reacting for 3-8 min;
d. respectively adding 4.0mL of sodium carbonate solution with the mass fraction of 75g/L into the solution in the step c, shaking up, and standing for 60min at room temperature;
e. d, measuring the absorbance of the mixed solution in the step d at the position of 765nm of the wavelength, and performing linear regression by using the gallic acid content (Y) and the absorbance (X) to obtain a standard curve equation: y is 0.0129X +0.0844, R2＝0.9996；
f. The calculation formula of the tea polyphenol yield is as follows:
(3) single factor experiment
a. Accurately weighing 1.000g of tea powder, and mixing with 70% ethanol solution as solvent;
b. carrying out water bath leaching single-factor test by using different cellulase, pectinase, solvent concentration, liquid-material ratio, extraction temperature, extraction time and pH value;
c. setting 5 different treatment levels for each single factor, making 3 treatments in parallel, and averaging to obtain the tea polyphenol content;
(4) response surface analysis method optimized tea polyphenol extraction process
a. On the basis of a single-factor experiment, selecting four single factors with larger influence as response variables, and carrying out Box-Behnken Design (BBD) experiment Design by using desingexpert8.0.6 software;
b. 3 treatment levels are set for each factor, each treatment is carried out in 3 parallel, and the extraction conditions are further optimized to obtain the optimal process conditions for extracting the green tea polyphenol.
2. The process for extracting green tea polyphenol by using the response surface method complex enzyme optimization, according to claim 1, is characterized in that: the tea leaves in the step (1) are old tea leaves in Yigong tea field in Bomicounty of Tibet Linzhi area.
3. The process for extracting green tea polyphenol by using the response surface method complex enzyme optimization, according to claim 1, is characterized in that: the calculation formula of the tea polyphenol yield in the step (2) is as follows: c. CTP: tea polyphenol content (%); a: the absorbance of the sample test solution; a. the0: absorbance of reagent blank liquid; SLOPEStd: the slope of the gallic acid standard curve; m: sample mass (g); v: sample extract volume (mL); d: dilution factor (typically 1mL to 100mL, then 100); ω: sample dry matter content (%).
4. The process for extracting green tea polyphenol by using the response surface method complex enzyme optimization, according to claim 1, is characterized in that: the dosage of the cellulase in the step (3) is as follows: 2.4, 6, 8 and 10mg/g, the dosage of the pectinase is as follows: 2.4, 6, 8 and 10mg/g, and the solvent concentration is as follows: 20. 40, 60, 80 and 100 percent, the material-liquid ratio is 1:10, 1:20, 1:30, 1:40 and 1:50g/mL, the extraction temperature is as follows: 30. 45, 60, 75 and 90 ℃, and the extraction time is as follows: 30. 70, 110, 150 and 190min, wherein the pH value is as follows: 3. 4, 5, 6 and 7.
5. The process for extracting green tea polyphenol by using the response surface method complex enzyme optimization, according to claim 1, is characterized in that: the four single factors selected in the step (4) are solvent concentration, feed-liquid ratio, extraction temperature and extraction time.
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| CN103655928A (en) * | 2013-11-26 | 2014-03-26 | 北京联合大学生物化学工程学院 | Complex enzyme method for extracting tea polyphenol from tea leaves |
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| CN105343335A (en) * | 2015-11-17 | 2016-02-24 | 南昌大学 | Method for optimizing extraction technology of aquilaria sinensis leaf tea polyphenol by utilizing response surface process |
| CN111704593A (en) * | 2020-06-16 | 2020-09-25 | 浙江中医药大学 | Technological method for optimizing extraction of tanshinone IIA and salvianolic acid B in salvia miltiorrhiza based on biological enzyme method of response surface |
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