CN111303109B - Method for extracting anthocyanin from fresh tea leaves - Google Patents
Method for extracting anthocyanin from fresh tea leaves Download PDFInfo
- Publication number
- CN111303109B CN111303109B CN202010166637.3A CN202010166637A CN111303109B CN 111303109 B CN111303109 B CN 111303109B CN 202010166637 A CN202010166637 A CN 202010166637A CN 111303109 B CN111303109 B CN 111303109B
- Authority
- CN
- China
- Prior art keywords
- leaching
- anthocyanin
- tea leaves
- fresh tea
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
Abstract
The invention discloses a method for extracting anthocyanin from fresh tea leaves, which comprises the following steps: (1) making fresh tea leaves into slurry; (2) Performing enzymolysis on the slurry, and collecting filtrate after enzymolysis; (3) Leaching the filtrate obtained in the step (2) at least twice to obtain leaching liquor; (4) Distilling the leaching solution at 70-75 ℃ and 0.05-0.1 MPa to obtain extractum; (5) Adsorbing and eluting the extract with DM130 resin adsorption column, concentrating the eluate, and drying to obtain anthocyanin. The method provided by the invention has the advantages that the anthocyanin yield is more than 2%, the product purity is more than 40%, and the problems of low extraction efficiency and low purity in the process of extracting tea anthocyanin by adopting the prior art can be effectively solved.
Description
Technical Field
The invention belongs to the technical field of natural medicine extraction, and particularly relates to a method for extracting anthocyanin from fresh tea leaves.
Background
Anthocyanin, also known as anthocyanidin, is a water-soluble natural pigment widely existing in plants in nature, belongs to flavonoid compounds, and can enable plants to present colorful colors along with the change of pH value of cell sap. Anthocyanin is a secondary metabolic substance produced by plants and helps the plants to resist adverse conditions, such as freeze injury, salt injury, low-phosphorus stress and the like. Many researches show that the anthocyanin is also a safe, nontoxic and beneficial antioxidant and antibacterial agent, and has physiological functions of reducing blood pressure, reducing blood fat, preventing colon cancer, resisting inflammation and the like. Therefore, the method has great application value in the aspects of food, chemical industry, medicine and the like, and how to extract anthocyanin from plants becomes a key problem for development and application of the anthocyanin.
At present, anthocyanin-rich plants such as blueberries, grapes and purple potatoes are common, but tea trees containing anthocyanin are rare. Generally, the young shoots of the tea trees are green or yellow-green, but the tea trees are easy to generate purple bud phenomenon at high temperature and high drought in summer, mainly because of the color development of anthocyanin in the young shoots under adverse conditions. It is reported that the young shoots of tea tree varieties with different genetic backgrounds are purple or deep purple all the year round, such as the silk, the purple sweet, and the like, the leaf color is stable, and the anthocyanin content can account for 3 percent of the dry matter content and is far higher than that of the common tea tree varieties. The study shows that anthocyanin serving as a water-soluble pigment can be directly taken into a body through brewing, so that the anthocyanin becomes a great hot spot in the tea industry.
In the prior art, a solvent extraction method is mostly adopted when anthocyanin is extracted, but simple solvent extraction is not suitable for extracting tea with complex components, and the solvent extraction is low in extraction efficiency and is easily influenced by other components in the tea in the operation process, so that the purity of the anthocyanin is low. Therefore, it is necessary to develop a simple, efficient extraction method capable of improving the purity of anthocyanin.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for extracting anthocyanin from fresh tea leaves, which aims to solve the problems of low extraction efficiency and low purity when the prior art is adopted to extract the anthocyanin from the tea leaves.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: preparing fresh tea into slurry;
s2: performing enzymolysis on the slurry, and collecting filtrate after enzymolysis;
s3: leaching the filtrate obtained in the step S2 at least twice to obtain leaching liquor;
s4: distilling the leaching solution at 70-75 ℃ and 0.05-0.1 MPa to obtain extractum;
s5: adsorbing and eluting the extract with DM130 resin adsorption column, concentrating the eluate, and drying to obtain anthocyanin.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the preparation method of the slurry comprises the following steps: grinding fresh tea leaves with liquid nitrogen, and mixing the ground materials with citric acid solution according to a feed-liquid ratio of 1:5-10 g/mL to obtain slurry; the concentration of the citric acid solution is 0.1-0.5 g/mL.
Further, the concentration of the citric acid solution is 0.4g/mL; the fresh tea grinding material and the citric acid solution are mixed according to the feed liquid ratio of 1:8 g/mL.
According to the invention, citric acid solution and fresh tea grinding materials are mixed to prepare slurry, and citric acid can chelate components such as metal ions in tea so as to avoid influencing the separation and purification of the subsequent anthocyanin; in addition, the citric acid solution can be used as a carrier solution to dissolve the mixed enzyme, so that the enzyme can exert better enzymolysis effect, and the extraction efficiency of anthocyanin is greatly improved.
Further, the method for enzymolysis of the slurry and collection of the filtrate in S2 comprises the following steps: mixing the slurry and the mixed enzyme according to the mass ratio of 1000:1-5, carrying out enzymolysis for 45-60 min at 37-40 ℃, then filtering by using cation exchange resin coated with montmorillonite, and collecting filtrate; the mixed enzyme comprises cellulase, pectase and tannase with the mass ratio of 1:0.4-0.8:0.2-0.5.
Further, the mass ratio of cellulase, pectase and tannase in the mixed enzyme is 1:0.5:0.3.
Further, the enzymolysis is carried out under the ultrasonic condition, and the ultrasonic frequency is 40-50 kHz.
In the invention, the fresh tea slurry is treated by ultrasonic enzymolysis, so that the problem that anthocyanin is difficult to extract is fundamentally solved, and the extraction efficiency of anthocyanin is greatly improved. The principle is as follows: the ultrasonic energy can generate and transmit strong energy, can penetrate through tissue cells of tea leaves to cause plant cell rupture of cavitation operators, is beneficial to transfer and diffusion of active ingredients in plants, and has the advantages of shortening extraction time, improving yield and the like; however, the ultrasonic frequency cannot be too high or too low, the excessive high can cause other components in the tea to flow out together, the anthocyanin is polluted, the anthocyanin is easy to oxidize, the anthocyanin cannot smoothly flow out of the cell tissues due to the too low frequency, and the extraction efficiency is reduced, so that the ultrasonic frequency is limited to be within the range of 40-50 kHz. The enzymolysis is mainly due to the anthocyanin contained in the cell wall, and the cell wall of the specific tissue of the tea can be degraded by utilizing enzyme; the enzyme used in the invention is a mixed enzyme, wherein, cellulose in the cell wall of the fresh tea is mainly degraded by cellulase, which is helpful for cell rupture and anthocyanin outflow; pectic enzyme decomposes pectin and other components in tea, which is beneficial to the extraction of anthocyanin; the tannase can decompose tannin, tannic acid and other components contained in the tea, so that the separation and purification of anthocyanin are easier; the three components act together to accelerate the anthocyanin in the tea to diffuse into the enzymolysis liquid and prevent the anthocyanin from oxidizing, and meanwhile, the impurity in the anthocyanin is decomposed, which is beneficial to the separation and purification of the anthocyanin, and the purity of the final product is ensured.
According to the invention, the cation exchange resin coated with montmorillonite is used for filtering the enzymolysis liquid, so that not only can large-particle impurities be removed, but also components such as theanine in the enzymolysis liquid can be filtered out, the separation and purification of the subsequent anthocyanin are facilitated, and the purity of the final product is further improved.
Further, 3 leaches are carried out in S3, the leaching comprising the steps of:
SS1: mixing the filtrate with the leaching agent according to the volume ratio of 1:1.5, leaching for 1-3 hours at the temperature of 0-4 ℃, centrifuging, and collecting supernatant;
SS2: mixing the supernatant obtained by SS1 with a leaching agent according to the volume ratio of 1:1.5, leaching for 1-3 hours at 0-4 ℃, centrifuging, and collecting the supernatant;
SS3: mixing the supernatant obtained by SS2 with a leaching agent according to the volume ratio of 1:1.5, leaching for 10-15 hours at the temperature of 0-4 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
the leaching agent is 1% HCl-MeOH solution.
Further, the leaching temperature in SS1 is 0 ℃, and the leaching time is 2 hours; the leaching temperature in SS2 is 2 ℃, and the leaching time is 2 hours; the leaching temperature in SS3 is 4 ℃ and the leaching time is 12h.
In the present invention, 1% HCl-MeOH solution was used as the leaching agent because: although anthocyanin is a pigment which is easily dissolved in water, since anthocyanin forms a stable molecular complex with proteins, polysaccharides, etc. usually by hydrogen bonding in plants, it is difficult to directly extract with water even after cell wall is broken. The formation of hydrogen bonds is due to the formation of covalent bonds between H and the more electronegative group, resulting in a strong shift of the common electron pair to the vicinity of the more electronegative group, such that H is almost uncovered and exhibits some ability to attract electrons in another molecule. The invention adopts HCl-MeOH solution as leaching agent, and the solution contains more H + In the presence of H + Under the condition of existence, hydrogen bonds are easy to damage, moreover, anthocyanin is better in stability in an acidic environment, is not easy to be oxidized and decomposed under a high-temperature condition, and is easier to dissolve in methanol, so that the solution is used as a leaching agent, the leaching efficiency of the anthocyanin can be improved, and the stability of the anthocyanin can be improved.
When the method is used for leaching, the temperature is in a lower range, so that the loss of anthocyanin can be reduced to the greatest extent, and the yield of anthocyanin is ensured. And the temperature is gradually increased during leaching, so that the anthocyanin is completely leached, and the yield of the anthocyanin is further improved.
Further, the method for adsorbing and eluting the extractum in the step S5 comprises the following steps:
(1) Soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column;
(2) Dissolving the extract in deionized water, centrifugally separating, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h;
(3) After the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h;
(4) Concentrating the ethanol eluent under reduced pressure, and drying to obtain anthocyanin.
According to the invention, the DM130 resin adsorption column is utilized to carry out adsorption elution treatment on the extract, so that hygroscopic components in the extract can be removed, the stability of the product is improved, harmful components such as heavy metals can be removed, the refining degree of the finally obtained anthocyanin is high, and the quality of the product is ensured.
The beneficial effects of the invention are as follows: the anthocyanin in the fresh tea is extracted through pulping, enzymolysis, repeated leaching, adsorption elution and other treatments, so that the phenolic substances in the tea can be prevented from oxidizing anthocyanin, the interference of other phenolic substances is overcome, and the high-purity anthocyanin is obtained; the method has the advantages of high extraction efficiency, low anthocyanin loss rate and good reproducibility, and is beneficial to large-scale industrialized application.
Drawings
FIG. 1 is a standard chromatogram of anthocyanin;
FIG. 2 is a chromatogram of the anthocyanin product of the tea plant line "ziyan";
FIG. 3 is a chromatogram of the anthocyanin product of the tea tree line "rhododendron".
Detailed Description
The following describes the present invention in detail with reference to examples.
Example 1
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: accurately weighing 5g of fresh leaves of the purple sweet tea tree, adding the fresh leaves into a pre-cooled mortar, and then adding liquid nitrogen for rapid grinding; transferring the ground tea leaves into a beaker, adding 40mL of citric acid solution with the concentration of 0.4g/mL, and uniformly mixing to obtain slurry;
s2: weighing 1g of cellulase, 0.5g of pectase and 0.3g of tannase, and uniformly mixing the cellulase, the pectase and the tannase to obtain mixed enzyme; 180mg of mixed enzyme is weighed and added into the slurry, the slurry is transferred into an ultrasonic reactor after being stirred uniformly, ultrasonic enzymolysis is carried out for 60min at 37 ℃, and the ultrasonic frequency is controlled to be about 45 kHz; then filtering with cation exchange resin coated with montmorillonite, and collecting filtrate;
s3: mixing the filtrate with 1% HCl-MeOH solution at a volume ratio of 1:1.5, leaching at 0deg.C for 2h, centrifuging, and collecting supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching at 2 ℃ for 2h, centrifuging, and collecting the supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching for 12h at 4 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
s4: distilling the leaching solution at 70deg.C and 0.1MPa to obtain extract;
s5: soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column; then dissolving the extract in deionized water, centrifugally separating, taking supernatant, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h; after the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Example 2
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: accurately weighing 5g of fresh tea leaves of rhododendron sinensis, adding the fresh tea leaves into a pre-cooled mortar, and then adding liquid nitrogen for rapid grinding; transferring the ground tea leaves into a beaker, adding 50mL of citric acid solution with the concentration of 0.1g/mL, and uniformly mixing to obtain slurry;
s2: weighing 1g of cellulase, 0.4g of pectase and 0.5g of tannase, and uniformly mixing the cellulase, the pectase and the tannase to obtain mixed enzyme; 275mg of mixed enzyme is weighed and added into the slurry, the slurry is transferred into an ultrasonic reactor after being stirred uniformly, ultrasonic enzymolysis is carried out for 45min at 40 ℃, and the ultrasonic frequency is controlled to be about 50 kHz; then filtering with cation exchange resin coated with montmorillonite, and collecting filtrate;
s3: mixing the filtrate with 1% HCl-MeOH solution at a volume ratio of 1:1.5, leaching at 2deg.C for 2h, centrifuging, and collecting supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching at 2 ℃ for 2h, centrifuging, and collecting the supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching for 10 hours at 2 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
s4: distilling the leaching solution at 75deg.C under 0.05MPa to obtain extract;
s5: soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column; then dissolving the extract in deionized water, centrifugally separating, taking supernatant, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h; after the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Example 3
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: accurately weighing 5g of fresh tea leaves of Ziyan, adding the fresh tea leaves into a pre-cooled mortar, and then adding liquid nitrogen for rapid grinding; transferring the ground tea leaves into a beaker, adding 25mL of citric acid solution with the concentration of 0.5g/mL, and uniformly mixing to obtain slurry;
s2: weighing 1g of cellulase, 0.4g of pectase and 0.5g of tannase, and uniformly mixing the cellulase, the pectase and the tannase to obtain mixed enzyme; weighing 60mg of mixed enzyme, adding the mixed enzyme into the slurry, uniformly stirring, transferring the slurry into an ultrasonic reactor, and carrying out enzymolysis for 45min at 37 ℃; then filtering with cation exchange resin coated with montmorillonite, and collecting filtrate;
s3: mixing the filtrate with 1% HCl-MeOH solution at a volume ratio of 1:1.5, leaching at 0deg.C for 3h, centrifuging, and collecting supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching at 0 ℃ for 3 hours, centrifuging, and collecting the supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching for 15h at 0 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
s4: distilling the leaching solution at 75deg.C and 0.08MPa to obtain extract;
s5: soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column; then dissolving the extract in deionized water, centrifugally separating, taking supernatant, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h; after the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
The anthocyanidins prepared in example 1 and example 2 were subjected to HPLC detection, respectively.
The specific process is as follows:
HPLC detection hardware conditions: agilent1260 (Agilent, usa); column Titank-C18 (250 mm. Times.4.6 mm,5 μm Fimbrance, USA).
Elution conditions: mobile phase a, water/acetonitrile/formic acid, 87/3/10 (v/v/v); mobile phase B, acetonitrile; 0min,15% b;30min,30% B.
Detection conditions: loading, 10 microliters; column temperature, left and right 35 ℃; the detection wavelength is 520nm.
Standard sample: delphinidin pigment, cyanidin, pelargonidin, malvidin, and paeoniflorin.
The detection results of the anthocyanin extracted from the "purple sweet" tea sample in the example 1 are shown in fig. 1 and fig. 2, wherein fig. 1 is a standard chromatogram of anthocyanin, and fig. 2 is a chromatogram of "purple sweet" tea anthocyanin; the detection results of the anthocyanin extracted from the rhododendron leaf sample in the embodiment 2 are shown in fig. 1 and 3, wherein fig. 1 is a standard chromatogram of the anthocyanin, and fig. 3 is a chromatogram of the rhododendron leaf anthocyanin.
Comparative example 1
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: accurately weighing 5g of fresh tea leaves of Ziyan, adding the fresh tea leaves into a pre-cooled mortar, and then adding liquid nitrogen for rapid grinding; transferring the grinded tea leaves into a beaker, adding 40mL of deionized water, and uniformly mixing to obtain slurry;
s2: weighing 1g of cellulase, 0.5g of pectase and 0.3g of tannase, and uniformly mixing the cellulase, the pectase and the tannase to obtain mixed enzyme; 180mg of mixed enzyme is weighed and added into the slurry, the slurry is transferred into an ultrasonic reactor after being stirred uniformly, ultrasonic enzymolysis is carried out for 60min at 37 ℃, and the ultrasonic frequency is controlled to be about 45 kHz; then filtering with cation exchange resin coated with montmorillonite, and collecting filtrate;
s3: mixing the filtrate with 1% HCl-MeOH solution at a volume ratio of 1:1.5, leaching at 0deg.C for 2h, centrifuging, and collecting supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching at 2 ℃ for 2h, centrifuging, and collecting the supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching for 12h at 4 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
s4: distilling the leaching solution at 70deg.C and 0.1MPa to obtain extract;
s5: soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column; then dissolving the extract in deionized water, centrifugally separating, taking supernatant, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h; after the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Comparative example 2
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: accurately weighing 5g of fresh tea leaves of Ziyan, adding the fresh tea leaves into a pre-cooled mortar, and then adding liquid nitrogen for rapid grinding; transferring the ground tea leaves into a beaker, adding 40mL of citric acid solution with the concentration of 0.4g/mL, and uniformly mixing to obtain slurry;
s2: mixing the slurry with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching for 2 hours at 0 ℃, centrifuging, and collecting supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching at 2 ℃ for 2h, centrifuging, and collecting the supernatant; mixing the obtained supernatant with 1% HCl-MeOH solution according to the volume ratio of 1:1.5, leaching for 12h at 4 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
s3: distilling the leaching solution at 70deg.C and 0.1MPa to obtain extract;
s4: soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column; then dissolving the extract in deionized water, centrifugally separating, taking supernatant, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h; after the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Comparative example 3
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
s1: accurately weighing 5g of fresh tea leaves of Ziyan, adding the fresh tea leaves into a pre-cooled mortar, and then adding liquid nitrogen for rapid grinding; transferring the ground tea leaves into a beaker, adding 40mL of citric acid solution with the concentration of 0.4g/mL, and uniformly mixing to obtain slurry;
s2: weighing 1g of cellulase, 0.5g of pectase and 0.3g of tannase, and uniformly mixing the cellulase, the pectase and the tannase to obtain mixed enzyme; 180mg of mixed enzyme is weighed and added into the slurry, the slurry is transferred into an ultrasonic reactor after being stirred uniformly, ultrasonic enzymolysis is carried out for 60min at 37 ℃, and the ultrasonic frequency is controlled to be about 45 kHz; then filtering with cation exchange resin coated with montmorillonite, and collecting filtrate;
s3: mixing the filtrate with absolute ethyl alcohol according to the volume ratio of 1:1.5, leaching for 2 hours at 0 ℃, centrifuging, and collecting supernatant; mixing the obtained supernatant with absolute ethyl alcohol according to the volume ratio of 1:1.5, leaching for 2 hours at the temperature of 2 ℃, centrifuging, and collecting the supernatant; mixing the obtained supernatant with absolute ethyl alcohol according to the volume ratio of 1:1.5, leaching for 12 hours at the temperature of 4 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
s4: distilling the leaching solution at 70deg.C and 0.1MPa to obtain extract;
s5: soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column; then dissolving the extract in deionized water, centrifugally separating, taking supernatant, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h; after the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Analysis of results
The anthocyanin yield (mg), purity (%) and yield were each counted in each of examples and comparative examples, and the properties such as the appearance color of the obtained products were observed, and the results are shown in Table 1.
Table 1 statistics of the products obtained for each experimental group
As can be seen from the table, the anthocyanin in the tea is extracted by adopting the method (examples 1-3) in the invention, the yield and the product purity are both higher, and the anthocyanin can be stably stored. Compared with the embodiment 1, the comparison document 1 has the advantages that citric acid is not added when the slurry is prepared, so that the subsequent enzyme cannot be well dissolved, the enzymolysis effect is limited, the anthocyanin yield is reduced, the metal ions in the tea cannot be well removed, the anthocyanin is difficult to separate, and the purity of the final product is not high. Comparative example 2 compared with example 1, anthocyanin in tea leaves could not smoothly flow out from cell tissues without enzymolysis treatment, resulting in a much lower yield than example 1. In comparative example 3, absolute ethanol was used as a leaching agent, and hydrogen bonds formed by anthocyanin could not be broken, resulting in lower final anthocyanin yield.
While specific embodiments of the invention have been described in detail in connection with the examples, it should not be construed as limiting the scope of protection of the patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.
Claims (7)
1. A method for extracting anthocyanin from fresh tea leaves, which is characterized by comprising the following steps:
s1: grinding fresh tea leaves with liquid nitrogen, and mixing the ground materials with citric acid solution according to a feed-liquid ratio of 1:5-10 g/mL to obtain slurry; the concentration of the citric acid solution is 0.1-0.5 g/mL;
s2: mixing the slurry and the mixed enzyme according to the mass ratio of 1000:1-5, carrying out enzymolysis for 45-60 min at 37-40 ℃, then filtering by using cation exchange resin coated with montmorillonite, and collecting filtrate; the mixed enzyme comprises cellulase, pectase and tannase with the mass ratio of 1:0.4-0.8:0.2-0.5;
s3: leaching the filtrate obtained in the step S2 at least twice to obtain leaching liquor;
s4: distilling the leaching solution at 70-75 ℃ and 0.05-0.1 MPa to obtain extractum;
s5: adsorbing and eluting the extract with DM130 resin adsorption column, concentrating the eluate, and drying to obtain anthocyanin.
2. The method for extracting anthocyanin from fresh tea leaves according to claim 1, wherein: the concentration of the citric acid solution is 0.4g/mL; the fresh tea grinding material and the citric acid solution are mixed according to the feed liquid ratio of 1:8 g/mL.
3. The method for extracting anthocyanin from fresh tea leaves according to claim 1, wherein: the mass ratio of the cellulase to the pectase to the tannase in the mixed enzyme is 1:0.5:0.3.
4. The method for extracting anthocyanin from fresh tea leaves according to claim 1, wherein: the enzymolysis is carried out under the ultrasonic condition, and the ultrasonic frequency is 40-50 kHz.
5. The method of extracting anthocyanin from fresh tea leaves of claim 1 wherein the 3 times of leaching in S3 comprises the steps of:
SS1: mixing the filtrate with the leaching agent according to the volume ratio of 1:1.5, leaching for 1-3 hours at the temperature of 0-4 ℃, centrifuging, and collecting supernatant;
SS2: mixing the supernatant obtained by SS1 with a leaching agent according to the volume ratio of 1:1.5, leaching for 1-3 hours at 0-4 ℃, centrifuging, and collecting the supernatant;
SS3: mixing the supernatant obtained by SS2 with a leaching agent according to the volume ratio of 1:1.5, leaching for 10-15 hours at the temperature of 0-4 ℃, centrifuging, and collecting the supernatant to obtain leaching liquor;
the leaching agent is 1% HCl-MeOH solution.
6. The method for extracting anthocyanin from fresh tea leaves of claim 5, wherein the method comprises the steps of: the leaching temperature in SS1 is 0 ℃, and the leaching time is 2 hours; the leaching temperature in SS2 is 2 ℃, and the leaching time is 2 hours; the leaching temperature in SS3 is 4 ℃ and the leaching time is 12h.
7. The method for extracting anthocyanin from fresh tea leaves according to claim 1, wherein the specific process of S5 is as follows:
(1) Soaking and swelling DM130 resin with ethanol, washing with distilled water until no ethanol exists, and loading into an adsorption column to obtain DM130 resin adsorption column;
(2) Dissolving the extract in deionized water, centrifugally separating, adding the supernatant into a DM130 resin adsorption column, and adsorbing at an adsorption rate of 2.5L/h;
(3) After the adsorption is finished, washing the adsorption column by distilled water, and then eluting by 50% ethanol, wherein the eluting rate is 4.5L/h;
(4) Concentrating the ethanol eluent under reduced pressure, and drying to obtain anthocyanin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010166637.3A CN111303109B (en) | 2020-03-11 | 2020-03-11 | Method for extracting anthocyanin from fresh tea leaves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010166637.3A CN111303109B (en) | 2020-03-11 | 2020-03-11 | Method for extracting anthocyanin from fresh tea leaves |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111303109A CN111303109A (en) | 2020-06-19 |
| CN111303109B true CN111303109B (en) | 2023-04-28 |
Family
ID=71145607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010166637.3A Active CN111303109B (en) | 2020-03-11 | 2020-03-11 | Method for extracting anthocyanin from fresh tea leaves |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111303109B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102020868A (en) * | 2010-05-21 | 2011-04-20 | 湖北紫鑫生物科技有限公司 | Method for extracting purple sweet potato anthocyanin |
| CN104356106A (en) * | 2014-10-21 | 2015-02-18 | 山东省科学院生物研究所 | Extraction and purification method of purple potato anthocyanin |
| CN104764846A (en) * | 2015-04-14 | 2015-07-08 | 四川农业大学 | Method for extracting, purifying and identifying anthocyanin from fresh tea leaves |
| CN105777697A (en) * | 2016-05-11 | 2016-07-20 | 黔南民族师范学院 | Method for extracting high-purity anthocyanin from purple sweet potatoes |
| CN109022513A (en) * | 2018-09-14 | 2018-12-18 | 中国热带农业科学院海口实验站 | The method of ultrasonic wave auxiliary biological complex enzyme enzymolysis and extraction hill gooseberry's anthocyanidin |
-
2020
- 2020-03-11 CN CN202010166637.3A patent/CN111303109B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102020868A (en) * | 2010-05-21 | 2011-04-20 | 湖北紫鑫生物科技有限公司 | Method for extracting purple sweet potato anthocyanin |
| CN104356106A (en) * | 2014-10-21 | 2015-02-18 | 山东省科学院生物研究所 | Extraction and purification method of purple potato anthocyanin |
| CN104764846A (en) * | 2015-04-14 | 2015-07-08 | 四川农业大学 | Method for extracting, purifying and identifying anthocyanin from fresh tea leaves |
| CN105777697A (en) * | 2016-05-11 | 2016-07-20 | 黔南民族师范学院 | Method for extracting high-purity anthocyanin from purple sweet potatoes |
| CN109022513A (en) * | 2018-09-14 | 2018-12-18 | 中国热带农业科学院海口实验站 | The method of ultrasonic wave auxiliary biological complex enzyme enzymolysis and extraction hill gooseberry's anthocyanidin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111303109A (en) | 2020-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102127125B (en) | The modularization preparation of series of purification Weibull multiplexed combination purification technique and products thereof | |
| CN101284022B (en) | Complexation extraction method of plant polyphenols substances | |
| CN101973984B (en) | Method for extracting general flavones from bamboo leaves | |
| CN106008442A (en) | Method for extracting anthocyanin from nitraria tangutorum peel residues | |
| CN108948102B (en) | Method for extracting tea polyphenol | |
| CN107722132B (en) | Method for co-producing algal polysaccharide and algal protein from algae | |
| CN108272831A (en) | The extracting method of Korean Needle polyphenol compound and its application | |
| CN111303109B (en) | Method for extracting anthocyanin from fresh tea leaves | |
| CN103571221A (en) | Extracting and purifying method of red-brown pigment of chestnut shells | |
| CN112250565A (en) | Method for simultaneously purifying and extracting phenolic compounds and flavone compounds from phellinus igniarius | |
| CN116987056A (en) | Method for extracting dihydroquercetin from larch | |
| CN108434043B (en) | Pomegranate extract for skin whitening and preparation method thereof | |
| CN116925163A (en) | Method for obtaining rose glycoside by low-frequency ultrasound | |
| CN112442136A (en) | Method for extracting functional components from tremella | |
| CN108976307B (en) | Extraction method of pitaya peel polysaccharide | |
| CN114917265A (en) | Method for extracting ginsenoside | |
| CN116139055A (en) | Preparation method and application of vitamin C-enriched roxburgh rose extract | |
| CN114031498A (en) | Method for extracting high-purity honeysuckle chlorogenic acid by membrane separation method | |
| WO2021243856A1 (en) | Method for extracting myrica rubra leaf proanthocyanidins | |
| CN107556348A (en) | Acryloyl acid esters compound and preparation method thereof | |
| CN108517000B (en) | Method for separating and preparing petunidin-3-O-arabinoside | |
| CN111437236A (en) | Dendrobium extract, preparation method and application thereof | |
| CN111544494A (en) | HPEF extraction method of tea seed phenolic acid | |
| CN113796536B (en) | Method for enriching phenolic compounds in peanut skin, peanut skin extract prepared by method and application of peanut skin extract | |
| CN115944575B (en) | Method for extracting effective components from purslane and purslane extracting solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |