CN112341504A - Method for optimizing conditions for extracting tannin from grape seeds by organic solvent extraction method - Google Patents
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Abstract
The invention provides a condition optimization method for extracting tannin from grape seeds by an organic solvent extraction method, and belongs to the technical field of tannin extraction. The condition optimization method for extracting tannin from grape seeds by the organic solvent extraction method disclosed by the invention considers the influence of the material-liquid ratio, the volume fraction of ethanol, the extraction time and the extraction temperature on the extraction rate of tannin in the grape seeds, and obtains the optimal extraction conditions of tannin in the grape seeds by the result analysis of a single-factor experiment and a response surface optimization experiment, wherein the optimal extraction conditions of tannin in the grape seeds are as follows: the volume fraction of the ethanol is 51.70%, the extraction time is 3.08h, the extraction temperature is 61.87, and the extraction rate of the tannin is as high as 6.36%.
Description
The present application is a divisional application of the Chinese patent application with application number CN201510959651.8, entitled "method for extracting tannin from grape seeds" filed by the Chinese patent office of 2015, 12, month 21, and the entire content of the application is incorporated by reference in the present application.
Technical Field
The invention relates to the technical field of tannin extraction, in particular to a condition optimization method for extracting tannin from grape seeds by an organic solvent extraction method.
Background
Grapes (grapes) are deciduous vine plants of the genus Vitis (vitasis) of the family Vitaceae. The fruits can be made into products such as grape juice, raisins, wine and the like, and with the increasing production of grape products, wastes such as grape skin, grape seeds, grape waste residues and the like generated in the processing process are urgently needed to be comprehensively utilized.
Relevant researches show that the grape wastes contain rich grape seed oil, protein, tannin, resveratrol and other substances, the protein content is high, the amino acid types are very complete, the mineral substances are rich, the requirements of human bodies are met, and the substances are widely applied to the industries of medicines, health products, cosmetics and the like, so that the comprehensive development and utilization of the grape seed wastes are realized, and the significance of changing waste into valuables is profound. If the treatment is proper, the pollution to the environment can be reduced, and huge economic benefits can be created, thereby achieving the win-win effect.
Tannin is a water-soluble polyphenol substance widely existing in a plurality of plants, is a very strong in-vivo active functional factor, and a plurality of researches show that the tannin has stronger oxidation resistance and better pharmacological activity due to the own polyphenol hydroxyl structure, and is widely applied to the fields of food, health care products, leather production, printing and dyeing industry, medicines, cosmetics and the like. However, because the structure of tannin is complex and the artificial synthesis is difficult, the tannin is mainly extracted from natural plants such as gallnut at home and abroad nowadays, and because natural resources are limited, the price of tannin in the market at home and abroad is very expensive. In addition, the yield and the planting area of grapes in China are in the first place in the world, grapes can generate a large amount of byproducts such as grape seeds and grape skins in the processes of juicing and brewing, tannin content in the grape seeds is extremely rich, and some special tannins also have the effect of preventing cardiovascular diseases. At present, the tannin extraction from grape seeds has great development potential in China, but the research on the tannin extraction in China is less. Therefore, the method has very important practical significance in exploring the optimized process for extracting the tannin from the grape seeds.
According to the method, the extraction process of the tannin in the grape seeds is optimized and researched by applying a response surface analysis method, a multiple quadratic regression equation is used as a tool for function estimation, and a contour line model and a curved surface model are established by combining mathematics and statistics, so that the extraction rate of the tannin in the grape seeds is the highest, and a certain scientific basis is provided for comprehensive development and utilization of the grape seeds.
Disclosure of Invention
The invention aims to provide a condition optimization method for extracting tannin from grape seeds by an organic solvent extraction method, which considers the influence of a material-liquid ratio, an ethanol volume fraction, extraction time and extraction temperature on the extraction rate of tannin in the grape seeds, and obtains the optimal extraction conditions of tannin in the grape seeds by result analysis of a single-factor experiment and a response surface optimization experiment: the volume fraction of the ethanol is 51.70%, the extraction time is 3.08h, the extraction temperature is 61.87 ℃, and the extraction rate of the tannin is as high as 6.36% under the condition.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a condition optimization method for extracting tannin from grape seeds by an organic solvent extraction method, which comprises the following steps:
1) the influence of the material-liquid ratio, the volume fraction of ethanol, the extraction time and the extraction temperature on the extraction rate of tannin in grape seeds is researched:
effect of volume fraction of extractant ethanol on tannin extraction yield:
taking 21 triangular flasks, dividing into 7 groups, wherein each group comprises 3 parallel triangular flasks, adding 1g of pretreated raw materials, adding 30mL of ethanol, 40% of ethanol, 50% of ethanol, 60% of ethanol, 70% of ethanol, 80% of ethanol and 90% of ethanol into each group of triangular flasks, sealing with a preservative film, and leaching in a constant-temperature water bath kettle at 60 ℃ for 3 hours; taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min; sucking 1mL of supernatant from each sample solution, placing the supernatant in a 50mL volumetric flask containing 30mL of distilled water, adding 2.5mLF-D reagent, adding 5mL of saturated sodium carbonate solution, and adding water to dilute to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
influence of different feed liquid ratios on tannin extraction rate:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel triangular bottles, adding 1g of pretreated raw materials, adding 20mL, 30mL, 40mL, 50mL, 60mL, 70mL and 80mL of 50% ethanol into each group of triangular bottles, sealing by using a preservative film, and leaching for 3 hours in a constant-temperature water bath kettle at 60 ℃; taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min; 2mL of supernatant liquid is sucked from each sample liquid, the supernatant liquid is placed in a 50mL volumetric flask containing 30mL of distilled water, 2.5mLF-D reagent is added, 5mL of saturated sodium carbonate solution is added, and water is added for dilution to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
effect of different extraction temperatures on tannin extraction yield:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials, adding 30mL of 50% ethanol into each group of triangular bottles, sealing with preservative film, and leaching in a constant temperature water bath kettle at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 3 h. Taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min. Sucking 1mL of supernatant from a sample solution with the extraction temperature of 60 ℃, sucking 2mL of supernatant from each of the other groups, placing the supernatant into a 50mL volumetric flask containing 30mL of distilled water, adding 2.5mLF-D reagent, adding 5mL of saturated sodium carbonate solution, and adding water to dilute to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
effect of different extraction times on tannin extraction yield:
taking 15 triangular bottles, dividing the 15 triangular bottles into 5 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials into the bottles, respectively adding 30mL of 50% ethanol into each group of triangular bottles, sealing the bottles by using preservative films, and then placing the bottles in a constant-temperature water bath kettle at 60 ℃ for leaching for 1 hour, 2 hours, 3 hours, 4 hours and 5 hours respectively; taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min; 2mL of supernatant liquid is sucked from each sample liquid, the supernatant liquid is placed in a 50mL volumetric flask containing 30mL of distilled water, 2.5mL of F-D reagent is added, 5mL of saturated sodium carbonate solution is added, and the mixture is diluted to 50mL by adding water; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
2) through the result analysis of the single-factor experiment and the response surface optimization experiment, the optimal extraction conditions of the tannin in the grape seeds are obtained:
according to the experiment design principle of a Box-Behnken center group, the experiment result of the influence of single factors on the tannin extraction rate is synthesized, 3 optimal levels of 3 factors including the material-liquid ratio, the extraction time and the extraction temperature are selected, an experiment design is carried out by adopting a response surface analysis method of 3 factors and 3 levels on the basis of a single factor experiment, a mathematical model is obtained, and the interaction among the factors and the relation between the factors and the response value are analyzed;
3) on the basis of a single-factor experiment, Design-expert.8.05b software is applied, a Box-Behnken Design is adopted to establish a mathematical model, three factors of ethanol volume fraction X1, extraction time X2 and extraction temperature X3 are selected as investigation objects, the average value of tannin extraction rate Y is taken as a response value, and a response surface with three factors and three levels is adopted to carry out experiment Design;
4) after regression fitting is carried out on the experimental result obtained in the step 3), a multiple quadratic regression equation of the light absorption value to the volume fraction, the extraction times and the extraction time of the ethanol is obtained:
y-6.1 +0.27X1+0.17X2+0.32X3-0.083X1X2+0.071X1X3+0.086X2X3-0.83X12-1.05X22-0.91X 32; in the formula: the extraction rate of Y-tannin; x1-ethanol volume fraction; x2-leaching time; x3-leaching temperature;
5) solving and seeking the maximum value point according to a regression equation obtained by the operation of a response surface method:
Y＝6.1+0.27X1+0.17X2+0.32X3-0.083X1X2+0.071X1X3+0.086X2X3-0.83X12-1.05X22-0.91X32；
the maximum value of Y is obtained when X1 is 0.185, X2 is 0.088, and X3 is 0.203, i.e. the optimum process conditions are: the volume fraction of the ethanol is 51.70%, the extraction time is 3.08h, the extraction temperature is 61.87 ℃, and under the condition, the extraction rate of the tannin is as high as 6.36%.
The invention has the following positive technical effects:
the application provides a condition optimization method for extracting tannin from grape seeds by an organic solvent extraction method, which inspects the influence of a material-liquid ratio, an ethanol volume fraction, extraction time and extraction temperature on the extraction rate of tannin in the grape seeds, and obtains the best extraction condition of tannin in the grape seeds by result analysis of a single-factor experiment and a response surface optimization experiment: the volume fraction of the ethanol is 51.70%, the extraction time is 3.08h, the extraction temperature is 61.87, and the extraction rate of the tannin is as high as 6.36%.
Drawings
FIG. 1 is the effect of volume fraction of extractant ethanol on tannin extraction yield;
FIG. 2 is the effect of different feed liquid ratios on tannin extraction yield;
FIG. 3 is a graph of the effect of different extraction temperatures on tannin extraction yield;
FIG. 4 is a graph of the effect of different extraction times on tannin extraction yield;
FIG. 5 is a plot of the effect of ethanol volume fraction and extraction time interaction on tannin extraction yield (contour plot);
FIG. 6 is a graph of the effect of ethanol volume fraction and extraction time interaction on tannin extraction yield (response surface 3D plot);
FIG. 7 is a plot of the effect of ethanol volume fraction and extraction temperature interaction on tannin extraction yield (contour plot);
FIG. 8 is a graph of the effect of the interaction of ethanol volume fraction and extraction temperature on tannin extraction yield (3D plot);
FIG. 9 is a plot of the effect of extraction time and extraction temperature on tannin extraction yield (contour plot);
FIG. 10 is a graph of the effect of extraction time and extraction temperature on tannin extraction yield (3D plot).
Detailed Description
The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The application provides a condition optimization method for extracting tannin in grape seeds by an organic solvent extraction method,
1) the influence of the material-liquid ratio, the volume fraction of ethanol, the extraction time and the extraction temperature on the extraction rate of tannin in grape seeds is researched:
effect of volume fraction of extractant ethanol on tannin extraction yield:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials, adding 30mL of 30%, 40%, 50%, 60%, 70%, 80% and 90% ethanol into each group of triangular bottles, sealing with preservative film, and leaching in a constant-temperature water bath kettle at 60 ℃ for 3 h. Taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min. 1mL of the supernatant was aspirated from each sample solution, and the resulting supernatant was placed in a 50mL volumetric flask containing 30mL of distilled water, and 2.5mLF-D reagent was added, followed by 5mL of saturated sodium carbonate solution and water to dilute the solution to 50 mL. Shaking to mix well, and measuring the absorbance at 650nm after 30 min.
Influence of different feed liquid ratios on tannin extraction rate:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials, adding 20mL, 30mL, 40mL, 50mL, 60mL, 70mL and 80mL of 50% ethanol into each group of triangular bottles, sealing with preservative film, and leaching in a constant-temperature water bath kettle at 60 ℃ for 3 h. Taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min. 2mL of the supernatant was aspirated from each sample solution, and the resulting supernatant was placed in a 50mL volumetric flask containing 30mL of distilled water, and 2.5mLF-D reagent was added, followed by 5mL of saturated sodium carbonate solution and water to dilute the solution to 50 mL. Shaking to mix well, and measuring the absorbance at 650nm after 30 min.
Effect of different extraction temperatures on tannin extraction yield:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials, adding 30mL of 50% ethanol into each group of triangular bottles, sealing with preservative film, and leaching in a constant temperature water bath kettle at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 3 h. Taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min. 1mL of supernatant was taken from the sample solution at 60 ℃ and 2mL of supernatant was taken from each of the other groups, and the supernatant was put into a 50mL volumetric flask containing 30mL of distilled water, and 2.5mLF-D reagent and 5mL of saturated sodium carbonate solution were added, and diluted to 50mL with water. Shaking to mix well, and measuring the absorbance at 650nm after 30 min.
Effect of different extraction times on tannin extraction yield:
taking 15 triangular bottles, dividing into 5 groups, wherein 3 triangular bottles in each group are parallel, adding 1g of pretreated raw materials, respectively adding 30mL of 50% ethanol into each triangular bottle, sealing with a preservative film, and leaching in a constant-temperature water bath kettle at 60 ℃ for 1h, 2h, 3h, 4h and 5 h. Taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min. 2mL of the supernatant was aspirated from each sample solution, and the resulting supernatant was placed in a 50mL volumetric flask containing 30mL of distilled water, and 2.5mL of F-D reagent was added, followed by 5mL of saturated sodium carbonate solution and water to dilute the solution to 50 mL. Shaking to mix well, and measuring the absorbance at 650nm after 30 min.
2) Through the result analysis of the single-factor experiment and the response surface optimization experiment, the optimal extraction conditions of the tannin in the grape seeds are obtained:
according to the experiment design principle of Box-Behnken center group sum, the experiment result of influence of single factors on the tannin extraction rate is synthesized, 3 optimal levels of 3 factors including the material-liquid ratio, the extraction time and the extraction temperature are selected, an experiment design is carried out by adopting a response surface analysis method of 3 factors and 3 levels on the basis of single factor experiment, a mathematical model is obtained, and the interaction among the factors and the relation between the factors and the response value are analyzed. The experimental factors and levels are shown in table 1.
TABLE 1 tannin extraction Process response surface Experimental factors and levels
As shown in figure 1, the tannin of grape seeds is extracted by using ethanol aqueous solution as an extracting agent and setting different proportions of ethanol and water, and the magnitude of light absorption values is compared. Searching the tannin concentration corresponding to the measured light absorption value according to the drawn standard curve, and calculating the tannin content and the extraction rate; as can be seen from FIG. 1, the volume fraction of ethanol is about 50% and the dissolution capacity of tannin is strongest, and the extraction rate is reduced when the proportion is increased or decreased, which is related to the complex structure of tannin and the physicochemical properties of ethanol. Relevant researches show that the extraction amount of tannin by adding a little water is higher than that of a pure ethanol solution, so that the dissolution of bound tannin can be promoted, and the degradation and deterioration of tannin in the long-time heat-preservation extraction process can be reduced.
As shown in fig. 2, 1g of raw material is taken and added with ethanol aqueous solution with different volumes, different material liquid ratios are set for extracting tannin of grape seeds, and the tannin extraction rates of different material liquid ratios are calculated. Calculating the tannin concentration corresponding to the measured light absorption value according to the drawn standard curve, and calculating the tannin content and the extraction rate; as is apparent from FIG. 2, the extraction rate of tannin increases with the increase of the volume of the extractant, reaches a maximum value when the ratio of the feed to the liquid is about 1:60, and then the extraction rate of tannin does not change greatly with the increase of the volume of the extractant, and basically tends to be flat. Due to the fact that the ratio of the material to the liquid is too large, resource waste is caused, and the subsequent work of suction filtration, centrifugation, drying and the like is more difficult. Therefore, 1:60 is selected as the best feed-to-liquid ratio for tannin extraction.
As shown in fig. 3, 7 gradient levels were set according to the suitable temperature range for tannin extraction to compare the tannin extraction rates in the grape seeds, and the tannin extraction rates at different extraction temperatures were calculated. The tannin concentration corresponding to the measured light absorption value is calculated according to the drawn standard curve, and the tannin content and the extraction rate are calculated, as is apparent from fig. 3, the extraction rate of tannin in grape seeds is increased with the increase of the extraction temperature, the extraction rate of tannin is the highest at 60 ℃, and the extraction rate of tannin is gradually reduced when the temperature is continuously increased. This is because at lower temperatures, the increase in temperature promotes the dissolution of the tannin in the aqueous ethanol solution, so that the content of extracted tannin increases gradually. However, tannin is prone to chemical reactions such as degradation, condensation, oxidation and the like at too high temperature, and the tannin extraction rate is reduced.
As shown in fig. 4, 5 levels are set according to the suitable time range for tannin extraction in the relevant data to compare the tannin extraction rates in the grape seeds, the light absorption values of the sample solutions at different extraction times are compared, the measured tannin concentration corresponding to the light absorption values is searched according to the drawn standard curve, and the tannin content and the extraction rate are calculated, as is apparent from fig. 4, the tannin extraction rate increases with the increase of the extraction time, the tannin extraction rate reaches the maximum value about 3 hours of extraction, and the tannin extraction rate is reduced with the increase of the extraction time. The data show that prolonged isothermal leaching results in a slight decrease in tannin extraction yield, probably due to tannin complex denaturation.
3) On the basis of a single-factor experiment, Design-expert.8.05b software is applied, a Box-Behnken Design is adopted to establish a mathematical model, three factors of ethanol volume fraction X1, extraction time X2 and extraction temperature X3 are selected as investigation objects, the average value of tannin extraction rate Y is taken as a response value, a response surface of three factors at three levels is adopted to carry out experiment Design, and the experiment result obtained by the Box-Behnken Design scheme is shown in Table 2.
TABLE 2 response surface optimization experiment design and response values
4) And (3) after regression fitting is carried out on the experimental results, obtaining a multiple quadratic regression equation of the light absorption value to the volume fraction, the extraction times and the extraction time of the ethanol:
Y＝6.1+0.27X1+0.17X2+0.32X3-0.083X1X2+0.071X1X3+0.086X2X3-0.83X12-1.05X22-0.91X32
in the formula: the extraction rate of Y-tannin;
x1-ethanol volume fraction;
x2-leaching time;
x3-leaching temperature.
To test the reliability of the regression equation, analysis of variance was performed, and the results are shown in table 3.
TABLE 3 ANOVA TABLE
Note: "+" is significant (p <0.05) and "+" is highly significant (p < 0.01).
It can be seen from table 3 that the mismatch term P is 0.5647>0.05, indicating that the mismatch is not significant; p <0.01 for the regression model, indicating that the model is highly significant; the correction decision coefficient (correlation coefficient) R2Adj of the model is 0.9597, indicating that the model reflects a change in 95.97% response value. The three points all show that the fitting degree of the model is good, so that the regression model can be used for analyzing experimental data and accurately predicting the optimal extraction condition.
The magnitude of the F value of the primary term reflects the influence degree of each factor on the response value, and the larger the F value is, the larger the influence degree of the factor on the response value is. As can be seen from Table 3, the influence of each factor on the tannin extraction yield is in the following order: leaching temperature > ethanol volume fraction > leaching time.
As shown in fig. 5-10, the contour map and 3D map of the response surface can visually reflect the influence of each factor on the extraction rate of tannin, and the interaction among the three factors of the volume fraction of ethanol, the extraction time and the extraction temperature, so that the optimal process parameters can be found out more accurately. Therefore, on the basis of the regression model analysis of variance results, a response surface and a contour map of the influence of the extraction time, the extraction temperature and the volume fraction of ethanol on the extraction rate of tannin in grape seeds are prepared by using software Design-expert8.05b, and the results are shown in fig. 7 to 9.
The contour diagrams and 3D surface diagrams in fig. 5-10 visually reflect the influence of the interaction between the factors on the tannin extraction rate, i.e., the interaction between the other two factors when one of the factors is fixed affects the tannin extraction rate, and the optimal process parameters can be found.
The shape of the contour line can reflect the existence of interaction and the strength of the interaction, the ellipse represents the interaction of the two factors, the circle is opposite to the interaction, and the density degree of the contour line is related to the significance of the interaction. In addition, the degree of influence of the two on the response value can also be determined by the inclination of the 3D map surface map, and the higher the inclination is, namely the steeper the inclination is, the more remarkable the interaction between the two is. The 3 contour diagrams of this application are the ellipse, and the density degree of contour lines is similar, and 3D curved surface graph gradient is close unanimous, shows that the interaction of each factor has an influence to tannin extraction rate, but each group's interaction experiment is little to the influence of tannin extraction rate.
5) Optimizing and verifying the optimal process conditions:
(1) optimization of optimal parameters
And solving and seeking the maximum value point of the regression equation obtained by the operation of the response surface method.
Y＝6.1+0.27X1+0.17X2+0.32X3-0.083X1X2+0.071X1X3+0.086X2X3-0.83X12-1.05X22-0.91X32；
The Y value is the largest when X1 is 0.185, X2 is 0.088 and X3 is 0.203, i.e. the optimum process conditions are 51.70% by volume of ethanol, 3.08h extraction time, 61.87 ℃ extraction temperature, and tannin extraction rate is as high as 6.36%.
(2) Verification of optimal conditions
And (5) carrying out verification test by using the obtained optimal process conditions. The average value of three parallel tests is used as an experimental result, the obtained tannin extraction rate is 6.35 percent and is 0.01 percent different from a theoretical value, the relative error is 0.16 percent, and the actual measured value basically accords with the theoretical predicted value, so that the extraction condition parameters obtained by adopting a response surface method are reliable and have practical value.
In summary, the conditions of the extraction process of tannin in grape seeds are optimized and researched by an organic solvent extraction method, the influence of the material-liquid ratio, the volume fraction of ethanol, the extraction time and the extraction temperature on the extraction rate of tannin in grape seeds is investigated, and the optimal extraction conditions of tannin in grape seeds are obtained by analyzing the results of a single-factor experiment and a response surface optimization experiment: the volume fraction of the ethanol is 51.70%, the extraction time is 3.08h, the extraction temperature is 61.87, and the extraction rate of the tannin is as high as 6.36%.
The above description is only for the purpose of illustrating the present invention, and it should be understood that the present invention is not limited to the above embodiments, and various modifications conforming to the spirit of the present invention are within the scope of the present invention.
Claims (1)
1. A condition optimization method for extracting tannin from grape seeds by an organic solvent extraction method is characterized by comprising the following steps:
1) the influence of the material-liquid ratio, the volume fraction of ethanol, the extraction time and the extraction temperature on the extraction rate of tannin in grape seeds is researched:
effect of volume fraction of extractant ethanol on tannin extraction yield:
taking 21 triangular flasks, dividing into 7 groups, wherein each group comprises 3 parallel triangular flasks, adding 1g of pretreated raw materials, adding 30mL of ethanol, 40% of ethanol, 50% of ethanol, 60% of ethanol, 70% of ethanol, 80% of ethanol and 90% of ethanol into each group of triangular flasks, sealing with a preservative film, and leaching in a constant-temperature water bath kettle at 60 ℃ for 3 hours; taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min; sucking 1mL of supernatant from each sample solution, placing the supernatant in a 50mL volumetric flask containing 30mL of distilled water, adding 2.5mLF-D reagent, adding 5mL of saturated sodium carbonate solution, and adding water to dilute to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
influence of different feed liquid ratios on tannin extraction rate:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel triangular bottles, adding 1g of pretreated raw materials, adding 20mL, 30mL, 40mL, 50mL, 60mL, 70mL and 80mL of 50% ethanol into each group of triangular bottles, sealing by using a preservative film, and leaching for 3 hours in a constant-temperature water bath kettle at 60 ℃; taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min; 2mL of supernatant liquid is sucked from each sample liquid, the supernatant liquid is placed in a 50mL volumetric flask containing 30mL of distilled water, 2.5mLF-D reagent is added, 5mL of saturated sodium carbonate solution is added, and water is added for dilution to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
effect of different extraction temperatures on tannin extraction yield:
dividing 21 triangular bottles into 7 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials, adding 30mL of 50% ethanol into each group of triangular bottles, sealing with preservative film, and leaching in a constant temperature water bath kettle at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 3 h. Taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min. Sucking 1mL of supernatant from a sample solution with the extraction temperature of 60 ℃, sucking 2mL of supernatant from each other, placing the supernatant into a 50mL volumetric flask containing 30mL of distilled water, adding 2.5mL of F-D reagent, adding 5mL of saturated sodium carbonate solution, and adding water to dilute to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
effect of different extraction times on tannin extraction yield:
taking 15 triangular bottles, dividing the 15 triangular bottles into 5 groups, wherein each group comprises 3 parallel bottles, adding 1g of pretreated raw materials into the bottles, respectively adding 30mL of 50% ethanol into each group of triangular bottles, sealing the bottles by using preservative films, and then placing the bottles in a constant-temperature water bath kettle at 60 ℃ for leaching for 1 hour, 2 hours, 3 hours, 4 hours and 5 hours respectively; taking out, vacuum filtering with Buchner funnel, transferring filtrate into centrifuge tube, and centrifuging at 3000r/min for 30 min; 2mL of supernatant liquid is sucked from each sample liquid, the supernatant liquid is placed in a 50mL volumetric flask containing 30mL of distilled water, 2.5mLF-D reagent is added, 5mL of saturated sodium carbonate solution is added, and water is added for dilution to 50 mL; shaking to mix thoroughly, and measuring light absorption value at 650nm after 30 min;
2) through the result analysis of the single-factor experiment and the response surface optimization experiment, the optimal extraction conditions of the tannin in the grape seeds are obtained:
according to the experiment design principle of a Box-Behnken center group, the experiment result of the influence of single factors on the tannin extraction rate is synthesized, 3 optimal levels of 3 factors including the material-liquid ratio, the extraction time and the extraction temperature are selected, an experiment design is carried out by adopting a response surface analysis method of 3 factors and 3 levels on the basis of a single factor experiment, a mathematical model is obtained, and the interaction among the factors and the relation between the factors and the response value are analyzed;
3) on the basis of a single-factor experiment, Design-expert.8.05b software is applied, a Box-Behnken Design is adopted to establish a mathematical model, three factors of ethanol volume fraction X1, extraction time X2 and extraction temperature X3 are selected as investigation objects, the average value of tannin extraction rate Y is taken as a response value, and a response surface with three factors and three levels is adopted to carry out experiment Design;
4) after regression fitting is carried out on the experimental result obtained in the step 3), a multiple quadratic regression equation of the light absorption value to the volume fraction, the extraction times and the extraction time of the ethanol is obtained:
y-6.1 +0.27X1+0.17X2+0.32X3-0.083X1X2+0.071X1X3+0.086X2X3-0.83X12-1.05X22-0.91X 32; in the formula: the extraction rate of Y-tannin; x1-ethanol volume fraction; x2-leaching time; x3-leaching temperature;
5) solving and seeking the maximum value point according to a regression equation obtained by the operation of a response surface method:
Y＝6.1+0.27X1+0.17X2+0.32X3-0.083X1X2+0.071X1X3+0.086X2X3-0.83X12-1.05X22-0.91X32；
the maximum value of Y is obtained when X1 is 0.185, X2 is 0.088, and X3 is 0.203, i.e. the optimum process conditions are: the volume fraction of the ethanol is 51.70%, the extraction time is 3.08h, the extraction temperature is 61.87 ℃, and under the condition, the extraction rate of the tannin is as high as 6.36%.
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