CN112094828B - Method for extracting SOD superoxide dismutase and theanine from camellia - Google Patents
Method for extracting SOD superoxide dismutase and theanine from camellia Download PDFInfo
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- DATAGRPVKZEWHA-YFKPBYRVSA-N N(5)-ethyl-L-glutamine Chemical compound CCNC(=O)CC[C@H]([NH3+])C([O-])=O DATAGRPVKZEWHA-YFKPBYRVSA-N 0.000 title claims abstract description 138
- 229940026510 theanine Drugs 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 29
- 235000018597 common camellia Nutrition 0.000 title claims abstract description 18
- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 17
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 17
- 240000001548 Camellia japonica Species 0.000 title abstract description 15
- 238000000605 extraction Methods 0.000 claims abstract description 11
- 239000003463 adsorbent Substances 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 62
- 239000003480 eluent Substances 0.000 claims description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 238000000108 ultra-filtration Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 16
- 238000002386 leaching Methods 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 239000012065 filter cake Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 229910021536 Zeolite Inorganic materials 0.000 claims description 8
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 8
- 239000008151 electrolyte solution Substances 0.000 claims description 8
- 239000002808 molecular sieve Substances 0.000 claims description 8
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000010457 zeolite Substances 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 241000628997 Flos Species 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000000643 oven drying Methods 0.000 claims description 5
- 238000010298 pulverizing process Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 3
- 229920002521 macromolecule Polymers 0.000 claims description 3
- 238000001728 nano-filtration Methods 0.000 claims description 3
- 150000003384 small molecules Chemical class 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 241000209507 Camellia Species 0.000 claims 3
- 230000008569 process Effects 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 239000003960 organic solvent Substances 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
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- 229910021645 metal ion Inorganic materials 0.000 abstract description 2
- 238000000053 physical method Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000001953 recrystallisation Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract 1
- 241001122767 Theaceae Species 0.000 description 12
- 150000008442 polyphenolic compounds Chemical class 0.000 description 8
- 235000013824 polyphenols Nutrition 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 2
- CITFYDYEWQIEPX-UHFFFAOYSA-N Flavanol Natural products O1C2=CC(OCC=C(C)C)=CC(O)=C2C(=O)C(O)C1C1=CC=C(O)C=C1 CITFYDYEWQIEPX-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 235000005487 catechin Nutrition 0.000 description 2
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 2
- 229950001002 cianidanol Drugs 0.000 description 2
- 150000002206 flavan-3-ols Chemical class 0.000 description 2
- 235000011987 flavanols Nutrition 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- ZAHXYMFVNNUHCP-UHFFFAOYSA-N Naphazoline nitrate Chemical compound O[N+]([O-])=O.C=1C=CC2=CC=CC=C2C=1CC1=NCCN1 ZAHXYMFVNNUHCP-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 1
- 150000002216 flavonol derivatives Chemical class 0.000 description 1
- 235000011957 flavonols Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000009048 phenolic acids Nutrition 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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Abstract
The invention relates to a method for extracting SOD superoxide dismutase and theanine from camellia, which can simultaneously extract the theanine and the SOD through the camellia, has safe, simple and convenient operation flow, low energy consumption, small use amount of organic solvent, no use of toxic organic solvent or toxic metal ions, repeated use of the adsorbent, preparation of high-purity theanine product through recrystallization, and the SOD extraction method is an all-physical method, can be completed by only common simple equipment, and has simple process and no danger; the temperature used in the extraction process is low, the condition is mild, and the finished SOD can be stored at normal temperature; no environmental pollution and no endogenous chemical pollution; the method for extracting the SOD can reduce development cost, is a method for extracting the plant SOD on a large scale, has simple equipment and process, easily obtained raw materials and low cost, ensures the quality of finished products and obtains the SOD with higher purity.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to a method for extracting SOD superoxide dismutase and theanine from camellia.
Background
Superoxide dismutase (SOD for short) is extremely important for human health, and medical and biochemical research results show that the superoxide dismutase has the functions of preventing aging, cancer and cardiovascular and cerebrovascular diseases. Thus, SOD formulations are listed as a state-of-the-art technology with priority, item 31. Since the artificial synthesis of SOD is not possible, it is only possible to extract SOD from animals and plants. The oxide dismutase SOD can resist and block the damage to cells caused by oxygen free radicals, repair damaged cells in time and restore the damage to cells caused by the free radicals. Due to modern living pressure, environmental pollution, various radiation and excessive movement can cause oxygen free radicals to be formed in a large quantity; therefore, SOD is increasingly important in biological antioxidant mechanisms, and many patents have been issued for methods for extracting SOD from animal blood. The common characteristics are long process flow, poor quality, single product and resource waste.
Tea polyphenols and theanine are two organic substances common in tea. Wherein the tea polyphenols are the general names of polyphenols in tea, including flavanols, anthocyanin, flavonoids, flavonols, phenolic acids, etc. Mainly comprises flavanols (catechin), and catechin accounts for 60-80%. Tea polyphenols, also called tea-making or tea tannin, are one of the main components forming the color, aroma and taste of tea leaves, and also one of the main components of tea leaves with health care function. Research shows that the active substances such as tea polyphenol have the functions of detoxification and radiation resistance, can effectively prevent radioactive substances from invading bone marrow, and can enable the saw 90 and the drill 60 to be rapidly discharged out of the body, and are known as 'radiation clera' by the health and medical community. The content of tea polyphenols in tea leaves is generally 20-35%, and in view of the value of tea polyphenols and their high content in tea leaves, tea polyphenols are generally obtained by separating and extracting from tea leaves.
However, SOD and theanine can be extracted from camellia simultaneously, and the method has high environmental protection and utilization value.
Disclosure of Invention
The invention aims to provide a method for extracting SOD superoxide dismutase and theanine from camellia.
In order to solve the technical problems, the invention adopts the following technical scheme: a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one: cleaning flos Camelliae Japonicae, oven drying, pulverizing, performing ultrasonic wall breaking, leaching, and collecting leaching solution;
step two: centrifuging the leaching solution, and collecting clear liquid for later use;
step three: ultrafiltering the clear solution to obtain a solution A with macromolecular substances, a solution B with small molecular substances and an SOD filter cake;
step four: adjusting the solution B at 5-75 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of 1-5 to flow through an inorganic zeolite molecular sieve adsorbent through a galvanostat to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using ammonia water solution or NaCl or KC1 electrolyte solution as eluent, and collecting the eluent, namely theanine solution;
step five: removing the eluent from the eluent; concentrating, adding 3-5 times of anhydrous ethanol to dry and crystallize, cooling to 0deg.C, and vacuum filtering to obtain theanine crude product; dissolving theanine crude product in small amount of hot water, adding 3-5 times of anhydrous ethanol, re-drying for crystallization, cooling to 0deg.C, vacuum filtering, and re-crystallizing for 1-3 times; vacuum freeze drying to obtain refined theanine product;
step seven: dissolving the SOD filter cake to obtain a solution, and performing supercritical C0 on the solution 2 Extracting to obtain SOD concentrated solution, and lyophilizing to obtain SOD product.
Further, the solvent is water, ethanol or a mixture thereof.
Further, the ultrasonic wall breaking adopts an intermittent mode, wherein the ultrasonic is carried out for 5-10s, the ultrasonic is carried out for 3-5s, and the ultrasonic is carried out for 30-60 times.
Further, the ultrafiltration process adopts primary ultrafiltration and secondary ultrafiltration.
Further, the secondary ultrafiltration selects a nanofiltration membrane with a interception molecular weight of more than 2000, the filtrate is solution B, and the filter cake is SOD.
Furthermore, the primary ultrafiltration adopts an ultrafiltration membrane with the molecular weight of more than 5 ten thousand, and the filtrate only contains SOD, small molecules and ions.
Further, supercritical C0 2 The extraction pressure in the extraction is 13kPa, and the extraction temperature is 35-45 ℃.
Further, in the fourth step, the eluent is 0.5mol L-3mol L ammonia water solution or 0.2-0.5mol L NaCKCl electrolyte solution.
Compared with the prior art, the invention has the advantages and positive effects that:
according to the method for extracting theanine and SOD, theanine and SOD can be extracted simultaneously through camellia, the operation flow is safe, simple and convenient, the energy consumption is low, the use amount of an organic solvent is small, a toxic organic solvent or toxic metal ions are not used, the adsorbent can be used repeatedly, a theanine product with higher purity can be prepared through recrystallization, and the SOD extraction method is an all-physical method for extracting SOD, and the method is completed only by common simple equipment, and has simple process and no danger; the temperature used in the extraction process is low, the condition is mild, and the finished SOD can be stored at normal temperature; no environmental pollution and no endogenous chemical pollution; the method for extracting the SOD can reduce development cost, is a method for extracting the plant SOD on a large scale, has simple equipment and process, easily obtained raw materials and low cost, ensures the quality of finished products and obtains the SOD with higher purity.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1:
a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one: cleaning flos Camelliae Japonicae, oven drying, pulverizing, performing ultrasonic wall breaking for 5s intermittently for 3s and 30 times, leaching with water as solvent, and collecting leaching solution;
step two: centrifuging the leaching solution, and collecting clear liquid for later use;
step three: ultrafiltering the clear liquid, wherein the ultrafiltration process adopts primary ultrafiltration and secondary ultrafiltration, the primary ultrafiltration adopts ultrafiltration membrane with molecular weight of more than 5 ten thousand, and the filtrate only contains SOD, small molecules and ions; the second-stage ultrafiltration selects a nanofiltration membrane with a intercepted molecular weight of more than 2000, the filtrate is solution B, the filter cake is SOD, and a solution A with macromolecular substances, a solution B with micromolecular substances and an SOD filter cake are obtained;
step four: adjusting the solution B at the temperature of 5 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of 1 to flow through an inorganic zeolite molecular sieve adsorbent through a constant flow device to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using 0.5mol L ammonia water solution as eluent, and collecting the eluent, namely theanine solution;
step five: removing the eluent from the eluent; concentrating, adding 3 times of absolute ethyl alcohol to dry and crystallize, cooling to 0 ℃, and suction filtering to obtain theanine crude product; dissolving theanine crude product in small amount of hot water, adding 3 times of anhydrous ethanol, drying, crystallizing, cooling to 0deg.C, vacuum filtering, and recrystallizing for 1 time; vacuum freeze drying to obtain refined theanine product;
step seven: dissolving SOD filter cake to obtain solution, and performing supercritical C0 treatment 2 Extracting under 13kPa at 35 deg.c to obtain SOD concentrated solution, and freeze drying to obtain SOD product.
Example 2:
a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one: cleaning flos Camelliae Japonicae, oven drying, pulverizing, performing ultrasonic wall breaking for 10s intermittently for 5s and 60 times, leaching with ethanol as solvent, and collecting leaching solution;
step two-step three as in example 1;
step four: adjusting the solution B at 75 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of 5 to flow through an inorganic zeolite molecular sieve adsorbent through a constant flow device to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using 3mol L ammonia water solution as eluent, and collecting the eluent, namely theanine solution;
step five: removing the eluent from the eluent; concentrating, adding 5 times of absolute ethyl alcohol to dry and crystallize, cooling to 0 ℃, and suction filtering to obtain theanine crude product; dissolving theanine crude product in small amount of hot water, adding 5 times of anhydrous ethanol, drying, crystallizing, cooling to 0deg.C, vacuum filtering, and recrystallizing for 3 times; vacuum freeze drying to obtain refined theanine product;
step seven: dissolving SOD filter cake to obtain solution, and processing the solution to obtain supercritical solutionBoundary C0 2 Extracting under 13kPa at 45 deg.c to obtain concentrated SOD liquid, and freeze drying to obtain SOD product.
Example 3:
a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one: cleaning flos Camelliae Japonicae, oven drying, pulverizing, performing ultrasonic wall breaking for 6s intermittently and 4s intermittently, performing ultrasonic treatment for 45 times, leaching with water, ethanol or their mixture, and collecting leaching solution;
step two-step three as in example 1;
step four: adjusting the solution B at 50 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of the solution to be 3, and enabling the aqueous solution to flow through an inorganic zeolite molecular sieve adsorbent through a constant flow device to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using 0.2mol L NaCl electrolyte solution as eluent, and collecting the eluent, namely theanine solution;
step five: removing the eluent from the eluent; concentrating, adding absolute ethyl alcohol with the volume of 4 times of the volume of the concentrated solution for drying and crystallizing, cooling to 0 ℃, and carrying out suction filtration to obtain a theanine crude product; dissolving theanine crude product in small amount of hot water, adding 4 times of anhydrous ethanol, drying, crystallizing, cooling to 0deg.C, vacuum filtering, and recrystallizing for 2 times; vacuum freeze drying to obtain refined theanine product;
step seven: dissolving SOD filter cake to obtain solution, and performing supercritical C0 treatment 2 Extracting under 13kPa at 40 deg.c to obtain SOD concentrated solution, and freeze drying to obtain SOD product.
Example 4:
a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one-step three is as in example 3;
step four: adjusting the solution B at 50 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of the solution to be 3, and enabling the aqueous solution to flow through an inorganic zeolite molecular sieve adsorbent through a constant flow device to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using 0.5mol L NaCl electrolyte solution as eluent, and collecting the eluent, namely theanine solution;
step five-step seven are the same as in example 3, and the obtained SOD concentrated solution is freeze-dried to obtain an SOD finished product.
Example 5:
a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one-step three is as in example 3;
step four: adjusting the solution B at 50 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of the solution to be 3, and enabling the aqueous solution to flow through an inorganic zeolite molecular sieve adsorbent through a constant flow device to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using 0.5mol L KCl electrolyte solution as eluent, and collecting the eluent, namely theanine solution;
step five-step seven are the same as in example 3, and the obtained SOD concentrated solution is freeze-dried to obtain an SOD finished product.
Example 6:
a method for extracting SOD superoxide dismutase and theanine from camellia comprises the following steps:
step one-step three is as in example 3;
step four: adjusting the solution B at 50 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of the solution to be 3, and enabling the aqueous solution to flow through an inorganic zeolite molecular sieve adsorbent through a constant flow device to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using 0.2mol L KCl electrolyte solution as eluent, and collecting the eluent, namely theanine solution;
step five-step seven are the same as in example 3, and the obtained SOD concentrated solution is freeze-dried to obtain an SOD finished product.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by this patent.
Claims (3)
1. A method for extracting SOD superoxide dismutase and theanine from camellia is characterized in that: the method comprises the following steps:
step one: cleaning flos Camelliae Japonicae, oven drying, pulverizing, performing ultrasonic wall breaking, leaching, and collecting leaching solution;
step two: centrifuging the leaching solution, and collecting clear liquid for later use;
step three: ultrafiltering the clear solution to obtain a solution A with macromolecular substances, a solution B with small molecular substances and an SOD filter cake;
step four: adjusting the solution B at 5-75 ℃ by hydrochloric acid, acetic acid and NaOH to enable the aqueous solution containing theanine with the pH value of 1-5 to flow through an inorganic zeolite molecular sieve adsorbent through a galvanostat to adsorb the theanine; washing with water until the effluent is colorless, eluting the adsorbed theanine by using ammonia water solution or NaCl or KC1 electrolyte solution as eluent, and collecting the eluent, namely theanine solution; wherein the concentration of the ammonia water solution is 0.5mol/L-3mol/L; the concentration of the NaCl or KC1 electrolyte solution is 0.2-0.5mol/L;
step five: removing the eluent from the eluent; concentrating, adding 3-5 times of anhydrous ethanol to dry and crystallize, cooling to 0deg.C, and vacuum filtering to obtain theanine crude product; dissolving theanine crude product in small amount of hot water, adding 3-5 times of anhydrous ethanol, re-drying for crystallization, cooling to 0deg.C, vacuum filtering, and re-crystallizing for 1-3 times; vacuum freeze drying to obtain refined theanine product;
step six: dissolving the SOD filter cake to obtain a solution, and performing supercritical CO on the solution 2 Extracting to obtain SOD concentrated solution, and lyophilizing the obtained SOD concentrated solution to obtain SOD product;
the ultrasonic wall breaking adopts an intermittent mode, wherein the ultrasonic is carried out for 5-10s, the ultrasonic is carried out for 3-5s, and the ultrasonic is carried out for 30-60 times;
the ultrafiltration process adopts primary ultrafiltration and secondary ultrafiltration, wherein the secondary ultrafiltration selects nanofiltration membranes with interception molecular weight of more than 2000, filtrate is solution B, filter cake is SOD, the primary ultrafiltration adopts ultrafiltration membranes with molecular weight of more than 5 ten thousand, and the filtrate only contains SOD, small molecules and ions.
2. A method for extracting SOD superoxide dismutase and theanine from camellia according to claim 1, wherein: the leaching solvent is water, ethanol or a mixture thereof.
3. A method for extracting SOD superoxide dismutase and theanine from camellia according to claim 1, wherein: supercritical CO 2 The extraction pressure in the extraction is 13kPa, and the extraction temperature is 35-45 ℃.
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