CN118146184A - Method for extracting anthocyanin from fresh leaves of Zijuan tea trees - Google Patents
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Abstract
The invention discloses a method for extracting anthocyanin from fresh leaves of Zijuan tea trees, and belongs to the technical field of anthocyanin extraction. The method comprises the following steps: freeze-drying fresh tea leaves, crushing, adding freeze-dried tea powder into a mixed enzyme solution for ultrasonic enzymolysis, adding filtered filtrate into anthocyanin extract for ultrasonic extraction, carrying out suction filtration, extracting filter residues again according to the same method, carrying out suction filtration, and combining the two obtained filtrates to obtain anthocyanin extract; the mixed enzyme solution comprises cellulase, pectase and laccase; the anthocyanin extracting solution is prepared by mixing absolute ethyl alcohol, water and citric acid according to the volume ratio of 2:1:2. Compared with the existing anthocyanin extraction method, the method provided by the invention is more suitable for extracting anthocyanin from tea trees, has the advantages of simple process, high product purity and high yield, and provides theoretical support for industrial production of extracting anthocyanin by taking Zijuan tea as a raw material.
Description
Technical Field
The invention relates to the technical field of anthocyanin extraction, in particular to a method for extracting anthocyanin from fresh leaves of Zijuan tea trees.
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. The Zijuan tea is one of the highest anthocyanin-containing varieties of the existing tea, and has about 100 times higher content than the Japanese "Cuanku" variety. The buds, leaves and stems of the tender tips of the Zijuan tea trees are purple, so that the Zijuan tea trees can be used as a raw material for tea processing and a preferable raw material for extracting plant anthocyanin.
At present, more than 700 known compounds separated and identified in tea leaves are available, and the tea leaves are rich in polyphenols, including catechins, flavonoids, flavonols, anthocyanins, phenolic acids, depsipelas and the like. Anthocyanin is a polar compound which can be dissolved in water and organic polar solvent, so that the extraction method of anthocyanin based on the anthocyanin mainly comprises a water extraction method and a solvent extraction method. The water extraction method has high safety, low cost and simple process flow, and is convenient for realizing the industrialization of mass production. But has low extraction rate, low raw material utilization rate, high impurity content and low economic benefit. The extraction efficiency of the solvent extraction method is higher than that of the water extraction method, but on the one hand, when the anthocyanin is applied to the food industry, the organic solvent is not completely removed, so that the potential safety hazard exists; on the other hand, during the extraction process, the organic solvent dissolves other components in the tea leaves together, resulting in a high impurity content of the extracted product. Therefore, providing a method suitable for extracting anthocyanin from tea leaves is a current problem to be solved.
Disclosure of Invention
The invention aims to provide a method for extracting anthocyanin from fresh tea leaves of Zijuan tea trees, which solves the problems of the prior art and improves the yield and purity of the anthocyanin extracted from the tea leaves.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for extracting anthocyanin from fresh leaves of Zijuan tea trees, which comprises the following steps:
freeze-drying fresh tea leaves, crushing, adding freeze-dried tea powder into a mixed enzyme solution for ultrasonic enzymolysis, adding filtered filtrate into anthocyanin extract for ultrasonic extraction, carrying out suction filtration, extracting filter residues again according to the same method, carrying out suction filtration, and combining the two obtained filtrates to obtain anthocyanin extract;
The mixed enzyme solution comprises cellulase, pectase and laccase;
The anthocyanin extracting solution is prepared by mixing absolute ethyl alcohol, water and citric acid according to the volume ratio of 2:1:2.
Further, the mass ratio of the cellulase to the pectase to the laccase in the mixed enzyme solution is (2-3) (1-1.5).
Further, the mass volume ratio of the freeze-dried tea powder to the mixed enzyme solution is 6g:40-50mL; the concentration of the mixed enzyme solution is 30-40mg/mL.
Further, in the ultrasonic extraction, the volume ratio of the filtered filtrate to the anthocyanin extract is 1: (1.5-2).
Further, the power of ultrasonic extraction is 250W, and the time is 30-40min.
Further, during ultrasonic enzymolysis, the ultrasonic frequency is 50-60kHz, the ultrasonic time is 40-50min, and the temperature is 36-37 ℃.
Further, the anthocyanin extract has a pH of 3.
Further, the anthocyanin extract is obtained and then further comprises the steps of concentration, purification and vacuum drying.
The invention discloses the following technical effects:
Before extraction by a solvent method, firstly, the polyphenol substances with larger influence on anthocyanin in tea are oxidized by laccase, so that the extraction rate caused by anthocyanin oxidation is not high; meanwhile, the tea is extracted under the acidic condition containing citric acid, so that metal ions in the tea are chelated with the citric acid to generate a compound, and the purity of the product is improved.
Compared with the existing anthocyanin extraction method, the method provided by the invention is more suitable for extracting anthocyanin from tea trees, has the advantages of simple process, high product purity and high yield, and provides theoretical support for industrial production of extracting anthocyanin by taking Zijuan tea as a raw material.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The fresh tea leaves of the Zijuan tea tree used in the embodiment of the invention are picked from a test tea garden of the institute of tea leaves of the academy of agricultural sciences of Yunnan province, and one bud is picked from four and five leaves.
Example 1
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
S1: vacuum freeze drying fresh tea tree leaves of Zijuan tea tree, and pulverizing to obtain freeze-dried powder;
S2: uniformly mixing cellulase, pectase and laccase according to a mass ratio of 2:1:1 to prepare a mixed enzyme solution of 30 mg/mL; 6g of tea freeze-dried powder is weighed, added into 50mL of mixed enzyme solution, stirred uniformly and transferred into an ultrasonic reactor, and subjected to ultrasonic crushing for 40min at 37 ℃ with ultrasonic frequency of 55kHz. Filtering with cation exchange resin after the reaction is completed, and collecting filtrate;
S3: mixing absolute ethyl alcohol, water and citric acid uniformly according to absolute ethyl alcohol, water and citric acid=2:1:2 (V: V) to prepare anthocyanin extract;
Mixing the filtrate obtained in the step S2 with the anthocyanin extract according to the volume ratio of 1:2, fully and uniformly mixing, regulating the pH value to be 3 by using HCl, performing ultrasonic treatment for 30min at 250W, performing suction filtration, extracting filter residues again according to the same method, performing suction filtration, and combining the two filtrates to obtain the anthocyanin extract;
s4: concentrating anthocyanin extract in rotary evaporator to obtain extract;
s5: dissolving the extract in deionized water, centrifuging, collecting supernatant, adding into DM130 resin adsorption column, and adsorbing at adsorption rate of 2.5L/h; washing the adsorption column with distilled water after adsorption, eluting with 50% ethanol at an elution rate of 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: vacuum freeze drying fresh tea tree leaves of Zijuan tea tree, and pulverizing to obtain freeze-dried powder;
s2: uniformly mixing cellulase, pectase and laccase according to a mass ratio of 2.5:1:1.5 to prepare a mixed enzyme solution with a concentration of 40 mg/mL; 6g of tea freeze-dried powder is weighed, added into 40mL of mixed enzyme solution, stirred uniformly and transferred into an ultrasonic reactor, and subjected to ultrasonic crushing for 45min at 36 ℃ with the ultrasonic frequency of 50kHz. Filtering with cation exchange resin after the reaction is completed, and collecting filtrate;
S3: mixing absolute ethyl alcohol, water and citric acid uniformly according to absolute ethyl alcohol, water and citric acid=2:1:2 (V: V) to prepare anthocyanin extract;
Mixing the filtrate obtained in the step S2 with the anthocyanin extract according to the volume ratio of 1:1.5, fully and uniformly mixing, regulating the pH value to be 3 by using HCl, performing ultrasonic treatment for 35min at 250W, performing suction filtration, extracting filter residues again according to the same method, performing suction filtration, and combining the two obtained filtrates to obtain the anthocyanin extract;
s4: concentrating anthocyanin extract in rotary evaporator to obtain extract;
s5: dissolving the extract in deionized water, centrifuging, collecting supernatant, adding into DM130 resin adsorption column, and adsorbing at adsorption rate of 2.5L/h; washing the adsorption column with distilled water after adsorption, eluting with 50% ethanol at an elution rate of 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: vacuum freeze drying fresh tea tree leaves of Zijuan tea tree, and pulverizing to obtain freeze-dried powder;
S2: uniformly mixing cellulase, pectase and laccase according to a mass ratio of 3:1.5:1.5 to prepare a mixed enzyme solution of 35 mg/mL; 6g of tea freeze-dried powder is weighed, added into 50mL of mixed enzyme solution, stirred uniformly and transferred into an ultrasonic reactor, and subjected to ultrasonic crushing for 50min at 37 ℃ with ultrasonic frequency of 60kHz. Filtering with cation exchange resin after the reaction is completed, and collecting filtrate;
S3: mixing absolute ethyl alcohol, water and citric acid uniformly according to absolute ethyl alcohol, water and citric acid=2:1:2 (V: V) to prepare anthocyanin extract;
Mixing the filtrate obtained in the step S2 with the anthocyanin extract according to the volume ratio of 1:2, fully and uniformly mixing, regulating the pH value to be 3 by using HCl, performing ultrasonic treatment for 40min at 250W, performing suction filtration, extracting filter residues again according to the same method, performing suction filtration, and combining the two filtrates to obtain the anthocyanin extract;
s4: concentrating anthocyanin extract in rotary evaporator to obtain extract;
s5: dissolving the extract in deionized water, centrifuging, collecting supernatant, adding into DM130 resin adsorption column, and adsorbing at adsorption rate of 2.5L/h; washing the adsorption column with distilled water after adsorption, eluting with 50% ethanol at an elution rate of 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Comparative example 1
A method for extracting anthocyanin from fresh tea leaves comprises the following steps:
S1: vacuum freeze drying fresh tea tree leaves of Zijuan tea tree, and pulverizing to obtain freeze-dried powder;
S2: uniformly mixing cellulase, pectase and tannase according to a mass ratio of 2:1:1 to prepare a mixed enzyme solution of 30 mg/mL; 6g of tea freeze-dried powder is weighed, added into 50mL of mixed enzyme solution, stirred uniformly and transferred into an ultrasonic reactor, and subjected to ultrasonic crushing for 40min at 37 ℃ with ultrasonic frequency of 55kHz. Filtering with cation exchange resin after the reaction is completed, and collecting filtrate;
S3: mixing absolute ethyl alcohol, water and citric acid uniformly according to absolute ethyl alcohol, water and citric acid=2:1:2 (V: V) to prepare anthocyanin extract;
Mixing the filtrate obtained in the step S2 with the anthocyanin extract according to the volume ratio of 1:2, fully and uniformly mixing, regulating the pH value to be 3 by using HCl, performing ultrasonic treatment for 30min at 250W, performing suction filtration, extracting filter residues again according to the same method, performing suction filtration, and combining the two filtrates to obtain the anthocyanin extract;
s4: concentrating anthocyanin extract in rotary evaporator to obtain extract;
s5: dissolving the extract in deionized water, centrifuging, collecting supernatant, adding into DM130 resin adsorption column, and adsorbing at adsorption rate of 2.5L/h; washing the adsorption column with distilled water after adsorption, eluting with 50% ethanol at an elution rate of 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: vacuum freeze drying fresh tea tree leaves of Zijuan tea tree, and pulverizing to obtain freeze-dried powder;
s2: uniformly mixing cellulase, pectase and laccase according to a mass ratio of 2.5:1:1.5 to prepare a mixed enzyme solution with a concentration of 40 mg/mL; 6g of tea freeze-dried powder is weighed, added into 40mL of mixed enzyme solution, stirred uniformly and then subjected to enzymolysis for 3h in a constant-temperature water bath kettle at 35 ℃. Filtering with cation exchange resin after the reaction is completed, and collecting filtrate;
S3: mixing absolute ethyl alcohol, water and citric acid uniformly according to absolute ethyl alcohol, water and citric acid=2:1:2 (V: V) to prepare anthocyanin extract;
Mixing the filtrate obtained in the step S2 with the anthocyanin extract according to the volume ratio of 1:1.5, fully and uniformly mixing, regulating the pH value to be 3 by using HCl, performing ultrasonic treatment for 35min at 250W, performing suction filtration, extracting filter residues again according to the same method, performing suction filtration, and combining the two obtained filtrates to obtain the anthocyanin extract;
s4: concentrating anthocyanin extract in rotary evaporator to obtain extract;
s5: dissolving the extract in deionized water, centrifuging, collecting supernatant, adding into DM130 resin adsorption column, and adsorbing at adsorption rate of 2.5L/h; washing the adsorption column with distilled water after adsorption, eluting with 50% ethanol at an elution rate of 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: vacuum freeze drying fresh tea tree leaves of Zijuan tea tree, and pulverizing to obtain freeze-dried powder;
S2: uniformly mixing cellulase, pectase and laccase according to a mass ratio of 3:1.5:1.5 to prepare a mixed enzyme solution of 35 mg/mL; 6g of tea freeze-dried powder is weighed, added into 50mL of mixed enzyme solution, stirred uniformly and transferred into an ultrasonic reactor, and subjected to ultrasonic crushing for 50min at 37 ℃ with ultrasonic frequency of 60kHz. Filtering with cation exchange resin after the reaction is completed, and collecting filtrate;
s3: mixing absolute ethyl alcohol and water uniformly according to absolute ethyl alcohol water=2:1 (V: V) to prepare anthocyanin extract;
Mixing the filtrate obtained in the step S2 with the anthocyanin extract according to the volume ratio of 1:2, fully and uniformly mixing, regulating the pH value to be 3 by using HCl, performing ultrasonic treatment for 40min at 250W, performing suction filtration, extracting filter residues again according to the same method, performing suction filtration, and combining the two filtrates to obtain the anthocyanin extract;
s4: concentrating anthocyanin extract in rotary evaporator to obtain extract;
s5: dissolving the extract in deionized water, centrifuging, collecting supernatant, adding into DM130 resin adsorption column, and adsorbing at adsorption rate of 2.5L/h; washing the adsorption column with distilled water after adsorption, eluting with 50% ethanol at an elution rate of 4.5L/h; concentrating the ethanol eluent under reduced pressure, and vacuum drying to obtain anthocyanin.
Analysis of results
The quality (mg), purity (%) and yield (%) of anthocyanin in each of examples and comparative examples were counted, and the properties such as 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 Table 1, the anthocyanin in the tea is extracted by the method of the embodiments 1-3, the yield and the product purity are both high, and the product is clear and free of turbidity. In the comparative example 1, laccase is replaced by tannase, although the tannase can decompose tannin, tannic acid and other components contained in tea, as the tea also contains a large amount of polyphenols, the polyphenols have a certain oxidation effect on anthocyanin, the laccase can oxidize the polyphenols to eliminate the adverse effect on the anthocyanin, and the tannase does not act on the polyphenols, so that the anthocyanin yield and purity are reduced; in the comparative example 2, water bath constant temperature enzymolysis is used for replacing ultrasonic crushing enzymolysis, on one hand, the cell walls cannot be thoroughly crushed, so that the effective components in the tea can be rapidly transferred and diffused, and on the other hand, the water bath condition temperature is higher, the extraction of anthocyanin is not facilitated, and the yield is reduced; comparative example 3, in which citric acid was not added, resulted in poor removal of metal ions from tea leaves, thereby chelating anthocyanin with metal ions, resulting in difficulty in separation of anthocyanin, and low purity of the final product.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A method for extracting anthocyanin from fresh leaves of Zijuan tea trees is characterized by comprising the following steps:
freeze-drying fresh tea leaves, crushing, adding freeze-dried tea powder into a mixed enzyme solution for ultrasonic enzymolysis, adding filtered filtrate into anthocyanin extract for ultrasonic extraction, carrying out suction filtration, extracting filter residues again according to the same method, carrying out suction filtration, and combining the two obtained filtrates to obtain anthocyanin extract;
The mixed enzyme solution comprises cellulase, pectase and laccase;
The anthocyanin extracting solution is prepared by mixing absolute ethyl alcohol, water and citric acid according to the volume ratio of 2:1:2.
2. The method according to claim 1, wherein the mass ratio of cellulase, pectase and laccase in the mixed enzyme solution is (2-3): 1-1.5.
3. The method according to claim 1, wherein the mass to volume ratio of the lyophilized tea powder to the mixed enzyme solution is 6g:40-50mL; the concentration of the mixed enzyme solution is 30-40mg/mL.
4. The method according to claim 1, wherein the volume ratio of the filtered filtrate to the anthocyanin extract is 1 during ultrasonic extraction: (1.5-2).
5. The method of claim 4, wherein the power of the ultrasonic extraction is 250W for 30-40min.
6. The method according to claim 1, wherein the ultrasonic frequency is 50-60kHz, the ultrasonic time is 40-50min, and the temperature is 36-37 ℃ during ultrasonic enzymolysis.
7. The method of claim 1, wherein the anthocyanin extract has a pH of 3.
8. The method of claim 1, further comprising the steps of concentrating, purifying and vacuum drying after obtaining the anthocyanin extract.
Publications (1)
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