CN112107604B - Method for extracting sweet tea antioxidant polyphenol - Google Patents

Method for extracting sweet tea antioxidant polyphenol Download PDF

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CN112107604B
CN112107604B CN202010477289.1A CN202010477289A CN112107604B CN 112107604 B CN112107604 B CN 112107604B CN 202010477289 A CN202010477289 A CN 202010477289A CN 112107604 B CN112107604 B CN 112107604B
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sweet tea
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tea
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甘人友
刘毅
夏宇
刘宏艳
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Chengdu Agricultural Science And Technology Center
Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
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Abstract

The invention discloses a method for extracting sweet tea antioxidant polyphenol, which comprises the following steps: 1) drying sweet tea, pulverizing, and sieving to obtain tea powder; 2) and (3) adding ethanol water into the tea powder obtained in the step 1), performing microwave extraction, centrifuging, and collecting supernatant to obtain the tea powder. The sweet tea obtained by the preparation method has high content of antioxidant polyphenol and strong antioxidant activity, and tests prove that the antioxidant polyphenol of the sweet tea is 13.35 percent, 35.30 percent and 50.05 percent higher than TEAC, FRAP and TPC values of tea polyphenol extracted without microwave assistance (soaking method).

Description

Method for extracting sweet tea antioxidant polyphenol
Technical Field
The invention relates to the technical field of natural product extraction, and particularly relates to a method for extracting sweet tea antioxidant polyphenol.
Background
Lithocarpus litseifolius (Hance) Chun, also known as Rubus suavissimus, stevia rebaudiana, hydrangea strigosa, Lithocarpus polystachyus, etc., belongs to the genus Lithocarpus of the family Fagaceae. The method is mainly distributed in Guangxi, Yunnan, Sichuan and Hunan provinces in China. The lithocarpus litseifolius sweet tea mainly comprises polyphenol (8.9%), flavone (3.2%), rubusoside (1.5%) and the like, is a good health-care tea, and has high development value of antioxidant polyphenol. According to reports, lithocarpus litseifolius has good effects of resisting oxidation, inhibiting bacteria, reducing blood sugar, reducing blood fat, resisting allergy, resisting virus, improving blood circulation, improving memory and the like, and is safe and nontoxic. In recent years, the application range of Lithocarpus litseifolius is gradually expanded, and the Lithocarpus litseifolius has been widely noticed.
So far, few research reports about the extraction method of sweet tea antioxidant polyphenol exist. Among them, chinese patent publication No. CN101190276 describes a method for extracting an extract from dry sweet tea by an alcohol extraction and water precipitation method, which has the disadvantages of long production cycle, large ethanol consumption, complex process, high cost, low product purity, etc.; chinese patent publication No. CN108752231A describes a method for extracting polyphenol from sweet tea by adsorption method, which has the following disadvantages: the cost is high, and the purity is low; chinese patent publication No. CN106820144A describes a method for extracting polyphenol from fresh sweet tea leaves by ultrasonic-microwave synergistic extraction, which has the following disadvantages: the extraction times are many, the temperature is high, and the loss of polyphenol is easy to cause. In summary, a few existing related extraction methods have the following disadvantages: long time, large consumption of organic solvent, high cost, low yield, low safety and low purity. Therefore, the extraction process of the sweet tea antioxidant polyphenol still needs to be further researched and improved.
The microwave-assisted extraction method provided by the invention has the following advantages that: high selectivity, low cost, high efficiency, short extraction time, simple operation, energy conservation, environmental protection and strong industrial application value.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting sweet tea antioxidant polyphenol, which comprises the following steps:
1) drying sweet tea, pulverizing, and sieving to obtain tea powder;
2) and (3) adding ethanol water into the tea powder obtained in the step 1), performing microwave extraction, centrifuging, and collecting supernatant to obtain the tea powder.
Further, the drying temperature in the step 1) is 40-60 ℃; the grinding mode is grinding, the grinding speed is 14500rpm, and the grinding time is 1-4 min; the sieving is 100 mesh sieving.
Further, the mass volume ratio of the tea powder and the ethanol water solution in the step 2) is 1 g: 30-35 ml.
Furthermore, the mass volume ratio of the tea powder to the ethanol water solution is 1 g: 35ml or 1 g: 30 ml.
Furthermore, the concentration of the ethanol water solution is 58-60%, preferably 60%.
Further, the microwave extraction power in step 2) is 400-800W, the temperature is 30-70 ℃, and the time is 20-25 min.
Furthermore, the microwave extraction power is 600W, the temperature is 50 ℃, and the time is 20min or 25 min.
Further, the centrifugation speed of the step 2) is 4500Xg, and the time is 6-10 min.
The invention also provides sweet tea antioxidant polyphenol which is an extract prepared by the method.
Further, the extract contains sweet tea total phenols more than 130mg GAE/g, antioxidant activity TEAC value more than 610 μmol Trolox/g, and FRAP value more than 380 μmol Fe2+/g。
Compared with the prior art, the extraction method of sweet tea antioxidant polyphenol has the following remarkable effects:
(1) the liquid-material ratio and the ethanol concentration are reduced, the use of organic solvents is reduced, and a series of problems of high cost, high risk, high harm, high pollution and the like caused by the large use of the organic solvents in the traditional chemical extraction of the sweet tea antioxidant polyphenol are fundamentally solved.
(2) The extraction temperature is reduced, the energy consumption is reduced, and a series of problems of high energy consumption and cost and the like in the common extraction technology of the sweet tea antioxidant polyphenol are fundamentally solved.
(3) The extraction time is shortened, the extraction efficiency is improved, and the problem of long production cycle of the common extraction technology of the sweet tea antioxidant polyphenol is fundamentally solved.
(4) Compared with the conventional extraction method, the method has simple operation and time saving.
(5) The sweet tea obtained by the preparation method has high content of antioxidant polyphenol and strong antioxidant activity, and tests prove that the antioxidant polyphenol of the sweet tea is 13.35 percent, 35.30 percent and 50.05 percent higher than TEAC, FRAP and TPC values of tea polyphenol extracted without microwave assistance (soaking method).
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 comparison of microwave extraction with microwave-free extraction of TEAC, FRAP antioxidant activity and polyphenol content
FIG. 2 gallic acid standard curve.
FIG. 3 is a graph showing the effect of ethanol concentration on the total phenol content and antioxidant activity of sweet tea.
FIG. 4 is a graph showing the effect of liquid-material ratio on the total phenol content and antioxidant activity of sweet tea.
FIG. 5 is a graph showing the effect of extraction temperature on the total phenol content and antioxidant activity of sweet tea.
FIG. 6 is a graph showing the effect of microwave power on the total phenol content and antioxidant activity of sweet tea.
FIG. 7 is a graph showing the effect of extraction time on the total phenol content and antioxidant activity of sweet tea.
Detailed Description
Example 1 extraction of antioxidant Polyphenol of sweet tea of the present invention (bench test)
Drying sweet tea at 50 deg.C, grinding at 14500rpm for 2min, sieving with 100 mesh sieve to obtain tea powder, sealing in a dry container, and storing at 4 deg.C; putting 1g of sample powder into a centrifuge tube, mixing the sample powder with 35mL of ethanol water solution (60%), performing microwave extraction at 50 ℃ for 25min, cooling the mixture with running water, centrifuging the mixture at 4500Xg for 8min, and collecting the supernatant to obtain an extracting solution, wherein the microwave power is 400-. Diluting the extractive solution by 5 times, measuring antioxidant activity of the obtained diluted sample by TEAC method and FRAP method, and measuring sweet tea total phenol content by modified Folin phenol method, wherein the TEAC, FRAP and TPC values are 613.113 + -9.321 μmol Trolox/g, 381.290 + -4.419 μmol Fe respectively2+/g，135.940±0.516mg GAE/g。
Comparative example 1
Drying sweet tea at 50 deg.C, grinding at 14500rpm for 2min, sieving with 100 mesh sieve to obtain tea powder, and mixingSealing in a dry container, and storing at 4 deg.C; extracting tea polyphenol with microwave-free assistance (soaking method, soaking for 25min) according to the extraction conditions of 35:1v/w of extract, 25min of time and 60% of ethanol concentration: the TEAC, FRAP and TPC values at this time were 540.885 + -7.496. mu. mol Trolox/g, 281.815 + -9.208. mu. mol Fe2+G, 90.594. + -. 0.671mg GAE/g. The TEAC, FRAP and TPC values of the sweet tea antioxidant polyphenol extracted without microwave assistance (soaking method) are respectively 13.35%, 35.30% and 50.05% lower than those of the sweet tea antioxidant polyphenol extracted with microwave assistance, as shown in figure 1.
Comparative example 2
Drying sweet tea at 50 deg.C, grinding at 14500rpm for 2min, sieving with 100 mesh sieve to obtain tea powder, sealing in a dry container, and storing at 4 deg.C; the sweet tea antioxidant polyphenol is extracted by an ultrasonic-microwave extraction method, and the extraction process parameters are as follows: 60-80W of ultrasonic waves, 400-900W of microwaves, 35:1v/W of extracting solution, 25min of extracting time and 60% of ethanol concentration. The TEAC, FRAP and TPC values at this time were 580.329 + -8.056. mu. mol Trolox/g, 326.460 + -7.268. mu. mol Fe2+G, 107.940. + -0.741 mg GAE/g. The ultrasound-microwave extraction method has the TEAC, FRAP and TPC values respectively lower than those of microwave-assisted extraction of sweet tea antioxidant polyphenol by 5.35%, 14.38% and 21.07%.
Example 2 extraction of antioxidant Polyphenol of sweet tea of the present invention (pilot test)
Taking a proper amount of sweet tea, drying at 50 ℃, grinding for 2min at the rotating speed of 14500rpm, sieving with a 100-mesh sieve to obtain tea powder, sealing in a dry container, and storing at 4 ℃ for later use; mixing 200g sample powder with 8L ethanol water solution (60%), performing microwave extraction at 50 deg.C for 25min, cooling the mixture with flowing water, centrifuging for 8min, and collecting supernatant to obtain extractive solution, wherein the microwave power is 400-900W. Diluting the extractive solution by 5 times, measuring antioxidant activity of the obtained diluted sample by TEAC method and FRAP method, and measuring sweet tea total phenol content by modified Folin phenol method, wherein the TEAC, FRAP and TPC values are 152.2746 + -7.144 mmol Trolox/g, 113.4064 + -5.014 mmol Fe2+/g，51.8421±0.906g GAE/g。
The advantageous effects of the present invention are described below by way of test examples.
Test example 1 extraction and component Activity analysis of sweet tea antioxidant Polyphenol
1. Extraction and analysis method
(1) Drying sweet tea (Yaan, Sichuan) at 50 deg.C, grinding at 14500rpm for 2min, sieving with 100 mesh sieve to obtain tea powder, sealing in dry container, and storing at 4 deg.C.
(2) Putting 1g of the tea powder obtained in the step 1 into a centrifuge tube, and mixing the tea powder with ethanol aqueous solutions with different concentrations and volumes. After extraction, the mixture was cooled with running water, centrifuged at 4500Xg for 8min, and the supernatant was collected for subsequent experiments.
(3) Diluting 25mL of forinophenol reagent (2mol/L) to 250mL by using double distilled water, uniformly mixing to obtain 10 times of diluted forinophenol reagent (0.2mol/L), namely forinophenol working solution, and storing at room temperature in a dark place for later use;
(4) preparation of gallic acid standard series solution: weighing 0.1g of gallic acid, dissolving the gallic acid in 100mL of double distilled water to obtain a gallic acid standard stock solution with the concentration of 1mg/mL, transferring different volumes of the standard stock solution into centrifuge tubes respectively, complementing all volumes to scale with the double distilled water to obtain gallic acid standard series solutions with different concentrations, and storing at room temperature for later use.
(5) Drawing a gallic acid standard curve: respectively adding 2.0mL of the Folin phenol working solution obtained in the step (3) and 0.5mL of the gallic acid series standard solution obtained in the step (4), uniformly mixing, reacting at room temperature for 4min, and then respectively adding 2mL of NaCO3Mixing the solution (75g/L), reacting at room temperature in dark for 2h, and measuring absorbance value at 765nm wavelength; meanwhile, a blank experiment was performed as described above using 0.5mL of double distilled water instead of the gallic acid standard solution. Finally, a gallic acid concentration standard curve (as shown in FIG. 2) is prepared to obtain a linear regression equation, which shows that the gallic acid standard substance presents a good linear relationship in the range of 0.025-0.200 mg/mL.
(6) And (3) determining the content of the antioxidant total phenols in the sweet tea extract: respectively adding 2.5mL of Fulinfen working solution and 0.5mL of diluted sample, mixing, reacting at room temperature for 4min, and respectively adding 2mL of NaCO3The solution (75g/L) is mixed evenly and is reacted for 2 hours in the dark at room temperatureMeasuring the absorbance value at 765nm wavelength; meanwhile, replacing the gallic acid standard solution with 0.5mL double distilled water, and carrying out a blank experiment according to the method; calculating the total phenol content of the sweet tea extract according to a gallic acid concentration standard curve, wherein the formula is as follows:
total phenol content (mg GAE/g dry tea powder) 10-3N C V/M, wherein GAE is gallic acid equivalent, N is the dilution multiple of the sweet tea extract, C is the concentration (mg/mL) of GAE corresponding to the absorbance of the sample solution, V is the volume (mL) of the sweet tea extract, M is the dry weight (g) of the sweet tea powder, and the total phenol content is expressed as mg GAE/g DW;
(7) preparing a Trolox stock solution: taking 0.125g of Trolox, dissolving with a small amount of methanol, and diluting to 50mL with double distilled water to prepare 50mL of Trolox solution with the concentration of 10000 mu mol/L for later use.
(8) Drawing a standard curve in a TEAC method: trolox solution with final concentration of 0-1000 mu mmol/L is used as reference standard, 100 mu L of Trolox solution with different concentrations is fully mixed with 3.8mL of diluted ABTS reaction solution, and the mixture is reacted for 6min at room temperature. Then, the absorbance at 734nm was measured, and a standard curve was drawn to determine the regression equation.
(9) FRAP working solution: the method comprises the following steps of (1) in a ratio of 10: 1: 1 in a volume ratio of 300mmol/L of sodium acetate buffer, 10 mmol/L of TPTZ solution and 20mmol/L of ferric chloride solution.
(10) Drawing of standard curve in FRAP method FeSO in concentration range of 0-2000 mu mol/L4Adding 100 μ L of the solution into 3mL of FRAP reaction solution, reacting for 4min, measuring absorbance of the mixture at 593nm, and taking the absorbance as ordinate, and Fe2+Drawing a standard curve with the concentration as a horizontal coordinate, and solving a regression equation;
(11) diluting the extractive solution by 5 times, and determining folium hydrangeae strigosae total phenol content by Folin phenol method, which is expressed as mg GAE/g DW; the antioxidant activity of sweet tea against oxidation polyphenol is determined by TEAC method and FRAP method, wherein TEAC value is expressed as mu mol Trolox/g DW, FRAP value is expressed as mu mol Fe2+/DW。
2. Sweet tea antioxidant polyphenol extraction process optimization
(1) 5 factors influencing the total phenol content of the sweet tea are screened out according to the earlier research result, wherein the factors comprise microwave temperature, ethanol concentration, liquid-material ratio of ethanol solvent to tea powder, microwave auxiliary power and time, and a single-factor test is carried out. The total phenol content and antioxidant activity of the sweet tea are used as indexes, and the treatment is repeated three times each time.
The single factor test result shows that: ethanol concentration: 60% (as shown in fig. 3), liquid-to-material ratio: 30:1 (as shown in fig. 4), extraction temperature: 50 ℃ (as shown in fig. 5), extraction power: 600W (as shown in FIG. 6), extraction time: when 20min (as shown in FIG. 7), the folium hydrangeae strigosae has high content of total phenols and strong antioxidant activity.
(2) According to the single-factor test result, the extraction temperature is kept at 50 ℃, the extraction power is 600W, and three factors (X) which have obvious influence on the total phenol content of the sweet tea are selected to extract the liquid material ratio1) Extraction time (X)2) And ethanol concentration (X)3) Taking the total phenol content of the sweet tea and the antioxidant activity of TEAC and FRAP as evaluation indexes (Y) as independent variables, designing an analysis test with a three-factor five-level by adopting a central combination design method, wherein the design factors and the levels of the test are shown in table 1, and the design scheme and the result of the test are shown in table 2.
TABLE 1 test design factors and levels
Figure BDA0002516249490000061
TABLE 2 Experimental design and results
Figure BDA0002516249490000062
Figure BDA0002516249490000071
From the test results of table 2, it can be seen that: when the ethanol concentration is 58.15%, the liquid-material ratio is 35.40v/w, and the extraction time is 24.86min, the sweet tea total phenol content of the extract is highest, and the anti-oxidation activity FRAP value is largest; when the concentration of ethanol is 60%, the liquid-material ratio is 30v/w, and the extraction time is 20min, the TEAC value of antioxidant activity is maximum.
3. Extraction Process verification experiment
According to the investigation result of the extraction process and the production practice, the optimal extraction process is determined to be 60% of ethanol concentration, 35v/w of liquid-material ratio and 25min of extraction time, 3 batches of sweet tea antioxidant polyphenol are respectively prepared according to the extraction process to carry out verification experiments, and the measured values of TEAC, FRAP and TPC are verified to be 613.113 +/-9.321 mu mol Trolox/g and 381.290 +/-4.419 mu mol Fe2+135.940 +/-0.516 mg GAE/g, 58.15% concentration of ethanol, 35.40v/w of liquid-material ratio and 24.86min of extraction time, and test values of TEAC, FRAP and TPC of 614.516 mu mol Trolox/g and 383.225 mu mol Fe2+And/g, 133.618mg GAE/g are close. The verification result fully proves that the sweet tea antioxidant polyphenol extracting solution has high content of antioxidant polyphenol and strong antioxidant activity.
In conclusion, the extraction method of the sweet tea antioxidant polyphenol reduces the extraction temperature, the liquid-material ratio and the ethanol concentration, reduces the energy consumption and the usage amount of the organic solvent, obtains the extract with high content of sweet tea total phenols and strong antioxidant activity, improves the quality of the extract under the condition of greatly improving the extraction efficiency, and has practical popularization and application values.

Claims (7)

1. A method for extracting Lithocarpus litseifolius antioxidant polyphenol is characterized in that: it comprises the following steps:
1) oven drying Lithocarpus litseifolius at 40-60 deg.C, grinding at 14500rpm for 1-4min, and sieving with 100 mesh sieve to obtain tea powder;
2) taking the tea powder obtained in the step 1), adding 58-60% ethanol water solution for microwave extraction, centrifuging, and collecting supernatant to obtain the tea powder;
the mass volume ratio of the tea powder to a 58-60% ethanol water solution is 1 g: 30-35 ml; the microwave extraction power is 400-800W, the temperature is 30-70 ℃, and the time is 20-25 min.
2. The extraction method according to claim 1, characterized in that: the mass volume ratio of the tea powder to a 58-60% ethanol water solution is 1 g: 35ml or 1 g: 30 ml.
3. The extraction method according to claim 1 or 2, characterized in that: the concentration of the ethanol water solution is 60%.
4. The extraction method according to claim 1, characterized in that: the microwave extraction power is 600W, the temperature is 50 ℃, and the time is 20min or 25 min.
5. The extraction method according to claim 1, characterized in that: the centrifugation speed of step 2) is 4500Xg, and the time is 6-10 min.
6. An Lithocarpus litseifolius antioxidant polyphenol is characterized in that: the extract is prepared by the method of any one of claims 1 to 5.
7. The lithocarpus litseifolius antioxidant polyphenol as claimed in claim 6, wherein; the extract contains sweet tea total phenols more than 130mg GAE/g, antioxidant activity TEAC value more than 610 μmol Trolox/g, and FRAP value more than 380 μmol Fe2+/g。
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