CN111440252B - Method for extracting tea polysaccharide from tea leaves - Google Patents
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Abstract
The invention discloses a method for extracting tea polysaccharide from tea leaves, which comprises the following steps: the method comprises the steps of firstly, tea residue pretreatment, secondly, microwave wall breaking, thirdly, variable-frequency ultrasonic composite enzymolysis, fourthly, centrifugal concentration, fifthly, alcohol precipitation, sixthly, washing and drying. The invention combines the advantages of various extraction methods, effectively combines the technical characteristics of microwave, ultrasonic wave and enzyme treatment, effectively improves the wall breaking efficiency, does not damage the polysaccharide structure, and obviously improves the yield and the antioxidant activity of the polysaccharide.
Description
Technical Field
The invention belongs to the field of tea deep processing, and particularly relates to a method for extracting tea polysaccharide from tea leaves.
Background
A large amount of tea residues are generated in a tea deep processing factory of large tea drinks, tea polyphenols, catechins and the like, only water-soluble components in the tea leaves are utilized in the normal production process, certain effective substances such as tea polyphenols, amino acids, crude proteins, crude fibers and the like are still remained in the tea residues, and the tea residues have high utilization value; the tea polysaccharide is a compound polysaccharide, and consists of saccharides, pectin and protein, and specifically comprises the following components: 30.92% of total sugar, 17.87% of protein, 8.89% of water, 16.48% of ash, 12.92% of pectin and 12.92% of others. The tea polysaccharide has the physiological functions of reducing blood sugar, reducing blood pressure, resisting anoxia, increasing coronary blood flow, enhancing non-specific immunity of human body, resisting cancer, resisting oxidation and the like, and has obvious economic benefit when being extracted from tea leaves.
The method for extracting the tea polysaccharide mainly comprises a hot water extraction method, an ultrafiltration extraction method, an enzyme extraction method, an ultrasonic extraction method, a microwave extraction method and the like, wherein the hot water extraction method is the most common, the operation is simple, but the extraction efficiency is low, the high-temperature extraction is easy to influence the biological activity of the tea polysaccharide, the enzyme extraction method is mild in condition, the release of polysaccharide substances is accelerated by adding saccharifying enzyme or protein enzyme, the ultrasonic extraction method utilizes the cavitation effect of ultrasound, the extraction efficiency and the yield of the tea polysaccharide can be improved, and the microwave extraction method is short in extraction time and high in efficiency through microwave energy, but is only suitable for heat-stable extracts. Despite various extraction methods, the extraction method in the prior report still has the problems of low extraction rate, low purity and the like when the tea residue is used as a raw material for extraction, and a comprehensive extraction method with high yield is rarely reported.
Disclosure of Invention
Based on the background, the invention combines the advantages of various extraction methods, effectively combines the technical characteristics of microwave, ultrasonic wave and enzyme treatment, develops a comprehensive extraction method, effectively improves the wall breaking efficiency, does not damage the polysaccharide structure, and obviously improves the yield and the antioxidant activity of the polysaccharide.
The invention provides a method for extracting tea polysaccharide from tea leaves, which comprises the following steps:
(1) tea residue pretreatment: crushing the tea leaves, sieving the crushed tea leaves by a sieve of 100-200 meshes, degreasing the tea leaves by absolute ethyl alcohol, centrifuging the degreased tea leaves, and then drying the degreased tea leaves in vacuum to obtain fine tea leaf powder;
(2) microwave wall breaking: adding water into the tea residue powder, controlling water temperature, setting microwave power, and performing microwave wall breaking;
(3) carrying out variable frequency ultrasonic composite enzymolysis: adjusting the temperature of the solution treated in the step (2), adjusting the pH value of the solution, adding cellulase and pectinase, setting ultrasonic frequency, and performing low-frequency ultrasonic enzymolysis; regulating the temperature and the ultrasonic frequency of the solution again, and carrying out high-frequency ultrasonic enzymolysis;
(4) and (3) centrifugal concentration: after the enzymatic hydrolysate treated in the step (3) is subjected to high-temperature enzyme deactivation, centrifugal separation is carried out, vacuum filtration is carried out to remove residues, and filtrate is subjected to rotary evaporation concentration to obtain concentrated solution;
(5) alcohol precipitation: adding ethanol into the concentrated solution obtained in the step (4), adjusting the alcohol content, standing, performing centrifugal separation, and removing supernatant to obtain precipitate;
(6) washing and drying: and (4) washing the precipitate obtained in the step (5) by sequentially adopting absolute ethyl alcohol, acetone and absolute ethyl ether, and then carrying out vacuum freeze drying to obtain the tea polysaccharide.
Preferably, the step (1) of degreasing with absolute ethyl alcohol is to wash the tea residue powder with absolute ethyl alcohol circularly at 75 ℃ for 30-50 min.
Preferably, the ratio of the materials to the liquids added in the step (2) is 1: 35-45, the water temperature is 70-80 ℃, the microwave power for microwave wall breaking is 500-600W, and the time is 60-80 min.
The high-energy microwave pre-treats the tea residue feed liquid, so that after tea residue cells absorb the high-energy microwave energy, the intracellular pressure is increased, cell walls are broken, and a large amount of active ingredients overflow.
Preferably, the total mass of the cellulase and the pectinase added in the step (3) is 0.1-0.2% of the mass of the solution, and the mass ratio of the cellulase to the pectinase is 1:1 to 2.
Preferably, the temperature of the solution subjected to low-frequency enzymolysis in the step (3) is 45-55 ℃, the pH value of the solution is 6.5-6.8, the ultrasonic frequency is 35-45 KHz, and the time is 40-50 min.
The low-frequency ultrasonic wave helps the enzyme substances to obtain effective acceleration and kinetic energy, and the enzyme substances are quickly and uniformly dispersed in water, and meanwhile, the effective components in the feed liquid are quickly dissolved in the water after the ultrasonic kinetic energy is obtained.
Preferably, the temperature of the solution subjected to high-frequency enzymolysis in the step (3) is 65-75 ℃, the ultrasonic frequency is 60-70 KHz, and the time is 30-40 min.
After enzymolysis, cell walls become thinner and are partially broken, high-frequency ultrasonic waves generate strong shock waves, the cell walls of plants are more effectively destroyed, effective components in the raw materials are bombed, and the extraction efficiency and the extraction speed are improved.
Preferably, in the step (4), the temperature of enzyme deactivation is 110-120 ℃ for 10-15 min, the centrifugal separation is performed at a rotating speed of 3000-3500 r/min for 10-12 min, and the temperature of rotary evaporation is 90-95 ℃ for 15-30 min.
Preferably, the alcohol content of the alcohol precipitation in the step (5) is 80-85%, the temperature is 35-40 ℃, the standing time is 4-5 h, and the centrifugal separation is performed at a rotating speed of 3000-3500 r/min for 10-15 min.
Preferably, in the step (6), the washing is carried out for 2 times by using each reagent, each time lasts for 8-10 min, and the vacuum freeze drying is carried out for 120-150 min at 55 ℃ and under the vacuum degree of 50-80 MPa.
Compared with the prior art, the invention has the beneficial effects that:
(1) the tea residue liquid is pretreated by high-energy microwaves, so that after tea residue cells absorb the high-energy microwave energy, the intracellular pressure is increased, cell walls are broken, and a large amount of active ingredients overflow.
(2) By utilizing the cavitation effect and the mechanical effect of the ultrasonic wave, the low-frequency ultrasonic wave in the first stage is beneficial to the enzyme substances to obtain effective acceleration and kinetic energy, and the enzyme substances are quickly and uniformly dispersed in water, and meanwhile, the effective components in the feed liquid are quickly dissolved in the water after the ultrasonic kinetic energy is obtained;
(3) the reactive enzyme can act on the cell wall of the tea residue cell, after enzymolysis is carried out for a period of time, the cell wall becomes thinner and is partially broken, after the solution temperature is properly increased, the high-frequency ultrasonic waves at the second stage are used for generating strong shock waves, the cell wall of the plant is more effectively damaged, the effective components in the raw materials are bombed, and the extraction efficiency and the extraction speed are improved.
(4) The invention effectively combines the technical characteristics of microwave, ultrasonic wave and enzyme treatment, effectively improves the wall breaking efficiency, does not damage the polysaccharide structure, obviously improves the yield and the antioxidant activity of the polysaccharide, and the prepared polysaccharide
The polysaccharide elution amount of the tea polysaccharide finished product reaches 11.3 percent, the tea polysaccharide content in the finished product reaches 73.4 percent, and the antioxidant capacity reaches 86.6 percent.
Drawings
FIG. 1 is a process flow diagram for extracting tea polysaccharide from tea leaves.
Detailed Description
Based on the above background, the present invention provides a method for extracting tea polysaccharide from tea leaves, comprising the steps of:
(1) tea residue pretreatment: crushing the tea leaves, sieving the crushed tea leaves by a sieve of 100-200 meshes, degreasing the tea leaves by absolute ethyl alcohol, centrifuging the degreased tea leaves, and then drying the degreased tea leaves in vacuum to obtain fine tea leaf powder;
(2) microwave wall breaking: adding water into the tea residue powder, controlling water temperature, setting microwave power, and performing microwave wall breaking;
(3) carrying out variable frequency ultrasonic composite enzymolysis: adjusting the temperature of the solution treated in the step (2), adjusting the pH value of the solution, adding cellulase and pectinase, setting ultrasonic frequency, and performing low-frequency ultrasonic enzymolysis; regulating the temperature and the ultrasonic frequency of the solution again, and carrying out high-frequency ultrasonic enzymolysis;
(4) and (3) centrifugal concentration: after the enzymatic hydrolysate treated in the step (3) is subjected to high-temperature enzyme deactivation, centrifugal separation is carried out, vacuum filtration is carried out to remove residues, and filtrate is subjected to rotary evaporation concentration to obtain concentrated solution;
(5) alcohol precipitation: adding ethanol into the concentrated solution obtained in the step (4), adjusting the alcohol content, standing, performing centrifugal separation, and removing supernatant to obtain precipitate;
(6) washing and drying: and (4) washing the precipitate obtained in the step (5) by sequentially adopting absolute ethyl alcohol, acetone and absolute ethyl ether, and then carrying out vacuum freeze drying to obtain the tea polysaccharide.
Preferably, the step (1) of degreasing with absolute ethyl alcohol is to wash the tea residue powder with absolute ethyl alcohol circularly at 75 ℃ for 30-50 min.
Preferably, the ratio of the materials to the liquids added in the step (2) is 1: 35-45, the water temperature is 70-80 ℃, the microwave power for microwave wall breaking is 500-600W, and the time is 60-80 min.
Preferably, the total mass of the cellulase and the pectinase added in the step (3) is 0.1-0.2% of the mass of the solution, and the mass ratio of the cellulase to the pectinase is 1:1 to 2.
Preferably, the temperature of the solution subjected to low-frequency enzymolysis in the step (3) is 45-55 ℃, the pH value of the solution is 6.5-6.8, the ultrasonic frequency is 35-45 KHz, and the time is 40-50 min.
Preferably, the temperature of the solution subjected to high-frequency enzymolysis in the step (3) is 65-75 ℃, the ultrasonic frequency is 60-70 KHz, and the time is 30-40 min.
Preferably, in the step (4), the temperature of enzyme deactivation is 110-120 ℃ for 10-15 min, the centrifugal separation is performed at a rotating speed of 3000-3500 r/min for 10-12 min, and the temperature of rotary evaporation is 90-95 ℃ for 15-30 min.
Preferably, the alcohol content of the alcohol precipitation in the step (5) is 80-85%, the temperature is 35-40 ℃, the standing time is 4-5 h, and the centrifugal separation is performed at a rotating speed of 3000-3500 r/min for 10-15 min.
Preferably, in the step (6), the washing is carried out for 2 times by using each reagent, each time lasts for 8-10 min, and the vacuum freeze drying is carried out for 120-150 min at 55 ℃ and under the vacuum degree of 50-80 MPa.
The technical solution of the present invention will be further described in detail with reference to specific embodiments. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1
Pulverizing tea residue, sieving with 100 mesh sieve, defatting with anhydrous ethanol, circularly washing tea residue powder at 75 deg.C for 30min, centrifuging at 2000r/min for 10min, removing supernatant, and drying at 40 deg.C under 50MPa for 30min to obtain tea residue fine powder; adding water into tea residue powder at a material-to-liquid ratio of 1:35, controlling water temperature at 70 deg.C, setting microwave power at 500W, and microwave wall breaking for 80 min; adding cellulase and pectinase with the mass percent of 0.1% of the solution according to the mass ratio of 1:1, adjusting the temperature of the solution to 45 ℃, the pH value to 6.5, setting the ultrasonic frequency to 35KHz, carrying out low-frequency enzymolysis for 50min, adjusting the temperature of the solution to 65 ℃, setting the ultrasonic frequency to 60KHz, and carrying out high-frequency enzymolysis for 40 min; inactivating enzyme at 110 deg.C for 15min, centrifuging at 3000r/min for 10min, vacuum filtering, removing residue, placing filtrate in rotary evaporator, and rotary evaporating and concentrating at 90 deg.C for 30min to obtain concentrated solution; adding ethanol into the concentrated solution, adjusting the ethanol content to 80%, adjusting the temperature of the solution to 35 deg.C, standing for 5h, centrifuging in a centrifuge at 3000r/min for 15min, and removing the supernatant to obtain precipitate; washing the precipitate with anhydrous ethanol, acetone, and anhydrous ether sequentially for 8min for 2 times each time; vacuum freeze drying at 55 deg.C and 50MPa for 150min to obtain tea polysaccharide product.
Example 2
Pulverizing tea residue, sieving with 150 mesh sieve, degreasing with anhydrous ethanol, circularly washing tea residue powder at 75 deg.C for 40min, centrifuging at 2000r/min for 10min, removing supernatant, and drying at 40 deg.C under 50MPa for 30min to obtain tea residue fine powder; adding water into tea residue powder according to the material-liquid ratio of 1:40, controlling the water temperature at 75 deg.C, setting microwave power of 550W, and microwave wall breaking for 70 min; adding cellulase and pectinase with the mass percent of 0.15% of the solution according to the mass ratio of 1:1.5, adjusting the temperature of the solution to 50 ℃, the pH value to 6.6, setting the ultrasonic frequency to 40KHz, carrying out low-frequency enzymolysis for 45min, adjusting the temperature of the solution to 70 ℃, setting the ultrasonic frequency to 65KHz, and carrying out high-frequency enzymolysis for 35 min; inactivating enzyme at 115 deg.C for 12min, centrifuging at 3300r/min for 11min, vacuum filtering, removing residue, placing the filtrate in rotary evaporator, and rotary evaporating at 93 deg.C for 20min to obtain concentrated solution; adding ethanol into the concentrated solution, adjusting the ethanol content to 83%, adjusting the temperature of the solution to 38 deg.C, standing for 4.5h, centrifuging in a centrifuge at 3300r/min for 12min, and removing the supernatant to obtain precipitate; washing the precipitate with anhydrous ethanol, acetone, and anhydrous ether sequentially for 2 times (9 min each time); vacuum freeze drying at 55 deg.C and 65MPa for 135min to obtain tea polysaccharide product.
Example 3
Pulverizing tea residue, sieving with 200 mesh sieve, defatting with anhydrous ethanol, circularly washing tea residue powder at 75 deg.C for 50min, centrifuging at 2000r/min for 10min, removing supernatant, and drying at 40 deg.C under 50MPa for 30min to obtain tea residue fine powder; adding water into tea residue powder according to the material-liquid ratio of 1:45, controlling the water temperature at 80 deg.C, setting microwave power of 600W, and microwave wall breaking for 60 min; adding cellulase and pectinase with the mass percent of 0.2% of the solution according to the mass ratio of 1:2, adjusting the temperature of the solution to 55 ℃, the pH value to 6.8, setting the ultrasonic frequency to 45KHz, performing low-frequency enzymolysis for 40min, adjusting the temperature of the solution to 75 ℃, setting the ultrasonic frequency to 70KHz, and performing high-frequency enzymolysis for 30 min; inactivating enzyme at 120 deg.C for 10min, centrifuging at 3500r/min for 10min, vacuum filtering, removing residue, placing filtrate in rotary evaporator, and rotary evaporating and concentrating at 95 deg.C for 15min to obtain concentrated solution; adding ethanol into the concentrated solution, adjusting the ethanol content to 85%, adjusting the solution temperature to 40 deg.C, standing for 5h, centrifuging in a centrifuge at 3500r/min for 10min, and removing the supernatant to obtain precipitate; washing the precipitate with anhydrous ethanol, acetone, and anhydrous ether sequentially for 10min 2 times each time; vacuum freeze drying at 55 deg.C and 80MPa for 120min to obtain tea polysaccharide product.
The polysaccharide elution amount in the tea leaves and the tea polysaccharide content in the finished product are measured by adopting an anthrone-sulfuric acid method, the tea polysaccharide oxidation resistance is measured by adopting a DPPH method, and the table 1 shows the relevant indexes of the tea polysaccharide finished products prepared in the examples 1-3.
| Amount of dissolution/%) | Content of polysaccharide/%) | Oxidation resistance% | |
| Example 1 | 10.4 | 70.1 | 84.9 |
| Example 2 | 11.3 | 73.4 | 86.6 |
| Example 3 | 9.7 | 68.6 | 83.8 |
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for extracting tea polysaccharide from tea leaves is characterized by comprising the following steps:
(1) tea residue pretreatment: crushing the tea leaves, sieving the crushed tea leaves by a sieve of 100-200 meshes, degreasing the tea leaves by absolute ethyl alcohol, centrifuging the degreased tea leaves, and then drying the degreased tea leaves in vacuum to obtain fine tea leaf powder;
(2) microwave wall breaking: adding water into the tea residue powder, controlling the water temperature, setting microwave power, and performing microwave wall breaking, wherein the ratio of the water to the material to the liquid is 1: 35-45, the water temperature is 70-80 ℃, the microwave power for microwave wall breaking is 500-600W, and the time is 60-80 min;
(3) carrying out variable frequency ultrasonic composite enzymolysis: adjusting the temperature of the solution treated in the step (2), adjusting the pH value of the solution, adding cellulase and pectinase, setting ultrasonic frequency, and performing low-frequency ultrasonic enzymolysis, wherein the total mass of the added cellulase and pectinase is 0.1-0.2% of the mass of the solution, and the mass ratio of the cellulase to the pectinase is 1: 1-2, wherein the temperature of the solution subjected to low-frequency ultrasonic enzymolysis is 45-55 ℃, the pH value of the solution is 6.5-6.8, the ultrasonic frequency is 35-45 KHz, and the time is 40-50 min; adjusting the temperature and the ultrasonic frequency of the solution again, and carrying out high-frequency ultrasonic enzymolysis, wherein the temperature of the solution for high-frequency ultrasonic enzymolysis is 65-75 ℃, the ultrasonic frequency is 60-70 KHz, and the time is 30-40 min;
(4) and (3) centrifugal concentration: after the enzymatic hydrolysate treated in the step (3) is subjected to high-temperature enzyme deactivation, centrifugal separation is carried out, vacuum filtration is carried out to remove residues, and filtrate is subjected to rotary evaporation concentration to obtain concentrated solution;
(5) alcohol precipitation: adding ethanol into the concentrated solution obtained in the step (4), adjusting the alcohol content, standing, performing centrifugal separation, and removing supernatant to obtain precipitate;
(6) washing and drying: and (4) washing the precipitate obtained in the step (5) by sequentially adopting absolute ethyl alcohol, acetone and absolute ethyl ether, and then carrying out vacuum freeze drying to obtain the tea polysaccharide.
2. The method for extracting tea polysaccharide from tea leaves as claimed in claim 1, wherein the step (1) of degreasing with absolute ethanol is to wash tea leaf powder with absolute ethanol at 75 ℃ for 30-50 min in a circulating manner.
3. The method for extracting tea polysaccharide from tea leaves as claimed in claim 1, wherein the temperature of the enzyme deactivation in the step (4) is 110-120 ℃ for 10-15 min, the centrifugal separation is performed at a rotating speed of 3000-3500 r/min for 10-12 min, and the temperature of the rotary evaporation is 90-95 ℃ for 15-30 min.
4. The method for extracting tea polysaccharide from tea leaves as claimed in claim 1, wherein the alcohol content of the alcohol precipitation in the step (5) is 80-85%, the temperature is 35-40 ℃, the standing time is 4-5 h, and the centrifugal separation is 3000-3500 r/min, and the rotating speed is 10-15 min.
5. The method for extracting tea polysaccharide from tea leaves as claimed in claim 1, wherein the washing in the step (6) is 2 times of washing for each reagent, each time for 8-10 min, and the vacuum freeze drying is performed at 55 ℃ and under a vacuum degree of 50-80 MPa for 120-150 min.
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