CN114213276B - Method for extracting and purifying theanine from enzyme catalytic reaction - Google Patents
Method for extracting and purifying theanine from enzyme catalytic reaction Download PDFInfo
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- CN114213276B CN114213276B CN202111368881.9A CN202111368881A CN114213276B CN 114213276 B CN114213276 B CN 114213276B CN 202111368881 A CN202111368881 A CN 202111368881A CN 114213276 B CN114213276 B CN 114213276B
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- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
Abstract
The invention discloses a method for extracting and purifying theanine from an enzyme catalytic reaction, which comprises the following steps: (1) The reaction liquid of the theanine generated by the enzyme catalysis is subjected to pH value adjustment, vacuum suction filtration and decoloration to obtain clarified liquid; (2) Filtering the clarified liquid to obtain filtrate, evaporating the filtrate to obtain an evaporated matter, stirring and extracting the evaporated matter with ethanol water solution, then performing hot filtration, and evaporating the filtrate to obtain low-purity theanine; (3) Purifying the low-purity theanine by adopting sephadex chromatography, evaporating eluent to dryness to obtain crude theanine; (4) The crude theanine is concentrated and dried after recrystallization treatment to obtain the refined theanine. The method has the advantages of less solvent, simple process, convenient operation, suitability for industrialization, and high safety of theanine product.
Description
Technical Field
The invention belongs to the technical field of theanine extraction, and particularly relates to a method for extracting and purifying theanine from an enzyme catalytic reaction.
Background
L-theanine (L-theanine) is an amide compound with a molecular formula of C 7 H 14 N 2 O 3 . A large number of research results show that the L-theanine has a plurality of important physiological functions, so that the L-theanine is widely applied to the industries of foods, health care products and medicines. L-theanineThe acid can be extracted and separated from the tea leaves, but the acid accounts for 1-2% of the dry mass of the tea leaves; in addition, the currently reported methods for extracting and separating high-purity L-theanine from tea leaves generally involve time-consuming, high-cost and complex operation processes due to the lack of simple, specific and efficient separation methods, which are not beneficial to industrial production. In contrast, chemical synthesis of L-theanine has the advantages of simple process, low cost, high yield, etc., but the synthesis of L-and D-form of theanine is a racemic mixture and the separation technique is complicated.
Along with the intensive research on the use of biotechnology in theanine synthesis, in addition to the use of genetic engineering and fermentation engineering techniques in theanine synthesis, enzyme engineering, particularly enzyme immobilization techniques, have been studied more in this respect, and these techniques have improved the efficiency of theanine synthesis. By combining the four projects of biotechnology, the method has the opportunity to adopt a more efficient and high-quality method for producing theanine. The L-theanine synthesized by the biological enzyme method is more in line with the green development concept, no organic solvent is involved in the production process, the pollution production link is less, the production process is greatly simplified, the product has almost no solvent residue, and the product is more in line with the pursuit of the public on healthy products. Although the enzymatic synthesis of theanine has the advantages, the enzymatic synthesis reaction system contains a large amount of inorganic impurities and catalytic enzymes (one or more mixed enzymes) besides the reaction raw materials, so that the subsequent purification of the theanine is very difficult.
Disclosure of Invention
The invention aims to provide a method for extracting and purifying theanine from an enzyme catalytic reaction, which can remove a large amount of inorganic impurities and catalytic enzymes, has the advantages of few types of used solvents, environmental protection, simple process, low cost and high recovery rate of the theanine, and is favorable for industrialized mass production.
The above object of the present invention can be achieved by the following technical solutions: a method for extracting purified theanine from an enzyme-catalyzed reaction comprising the steps of:
(1) The reaction liquid of the theanine generated by the enzyme catalysis is subjected to pH value adjustment, vacuum suction filtration and decoloration to obtain clarified liquid;
(2) Filtering the clarified liquid to obtain filtrate, evaporating the filtrate to obtain an evaporated matter, stirring and extracting the evaporated matter with ethanol water solution, then performing hot filtration, and evaporating the filtrate to obtain low-purity theanine;
(3) Purifying the low-purity theanine by adopting sephadex chromatography, evaporating eluent to dryness to obtain crude theanine;
(4) The crude theanine is concentrated and dried after recrystallization treatment to obtain the refined theanine.
In the above method for extracting and purifying theanine from the enzyme-catalyzed reaction:
preferably, the reaction solution for enzyme-catalyzed production of theanine in the step (1) is derived from free enzyme solution-catalyzed synthesis of theanine or immobilized enzyme-catalyzed synthesis of theanine (enzyme may fall off into the reaction solution), and in either way, a large amount of inorganic salt and other auxiliary reagent (such as ATP) are required to be added in addition to ethylamine (or ethylamine hydrochloride) and glutamic acid (or sodium glutamate).
For example, the reaction liquid for enzymatically producing theanine can be derived from "research on synthesizing L-theanine by using gamma-glutamylmethylamine synthetase", university of Tianjin science and technology, ind., shu: zhu Xinya, 2019, 6 ", the reaction solution for the enzymatic synthesis of L-theanine.
Preferably, the concentration of theanine in the reaction liquid for producing theanine catalyzed by the enzyme in the step (1) is 27 to 30g/L.
Preferably, in the step (1), acetic acid or phosphoric acid is adopted to adjust the pH value, and the pH value is adjusted to 6-6.5.
In the invention, hydrochloric acid or phosphoric acid is necessary to adjust the pH value, and the following reasons are:
(a) Theanine is stable under acidic conditions, especially when heated;
(b) The ethylamine hydrochloride is easier to remove under acidic conditions by using an alcohol solvent;
(c) If other acid is used for regulation, other acid radical ions (the salt types are increased) are artificially introduced, so that the system is more complex, and the separation difficulty is increased.
Preferably, the auxiliary agent adopted in the step (1) in the vacuum filtration is diatomite, perlite or active carbon.
When in vacuum filtration, the auxiliary agent is adopted to reduce the difficulty of the filtration and shorten the filtration time.
Preferably, the decoloring agent adopted in the step (1) is activated carbon, the decoloring temperature is 70-80 ℃, and the decoloring time is 4-6 h.
The active carbon is used for decoloring, the pigment in the reaction liquid for catalyzing and generating theanine can be removed, the color can be deepened in the reaction process by using the enzyme liquid or immobilized enzyme, the active carbon is used for the pigment brought by the enzyme liquid or immobilized enzyme, the pigment is organic matter, the pigment is obviously removed under the adsorption effect of the active carbon, and if other decoloring methods are poor in effect, chemical methods such as hydrogen peroxide and the like cannot be used. The decolorizing temperature is preferably 70-80 deg.c, and the decolorizing temperature is too high, and this results in great reflux amount, low active carbon adsorbing capacity, increased active carbon amount, long decolorizing time and partial theanine adsorbing loss.
Preferably, in the step (2), the mass percentage of ethanol in the ethanol aqueous solution is 90-95%, the temperature in the extraction is 70-80 ℃, and the mass volume ratio of the evaporated matter to the ethanol aqueous solution is 1g: 10-15 mL, the extraction time is 6-8 h, and the hot filtration temperature is 50-55 ℃.
The method adopts an ethanol water solution with the mass percent of 90-95% to extract and remove most of catalytic enzyme, the catalytic enzyme belongs to protein, most of the catalytic enzyme can be separated out in the presence of the ethanol water solution with the mass percent of 90-95%, and a small amount of catalytic enzyme (such as soluble protein) which cannot be separated out can be completely removed in the subsequent glucose gel chromatography purification.
Preferably, the mass percentage of the low-purity theanine in the step (2) is 55-65%.
Preferably, in the step (3), the low-purity theanine is dissolved by deionized water and then purified by sephadex chromatography, wherein the dosage relation of the low-purity theanine and the deionized water is 1g: 10-15 mL.
Preferably, the column packing used in the sephadex chromatography in the step (3) is sephadex G-10 or sephadex G-15, and the mass ratio of the material (low-purity theanine) to the packing is 1G: 8-10 g, the eluent is deionized water, and the elution speed is 1V/2-3 h (V is the wet volume of the filler).
The use of sephadex chromatography is mainly desalting (e.g. inorganic salts) and soluble proteins.
Preferably, the mass percentage of the crude theanine in the step (3) is 80-88%.
Preferably, in the step (4), the crude theanine is dissolved by adopting an ethanol aqueous solution at 70-75 ℃ and then recrystallized, wherein the mass percentage of ethanol in the ethanol aqueous solution is 80-88%, and the mass volume relation between the crude theanine and the ethanol aqueous solution is 1g: 30-40 mL, the crystallization temperature is 20-30 ℃ and the time is 10-15 h during recrystallization.
The mass percentage of ethanol in the ethanol water solution is 80-88%, and the feed liquid ratio is 1g: 30-40 mL, the crystallization temperature is 20-30 ℃ and the time is 10-15 h' the recrystallization is carried out according to the residual impurities which need to be removed, such as sodium chloride, magnesium chloride and sodium glutamate, and chlorine ions and other amino acid contents can not be separated out to exceed the standard only under the conditions.
Preferably, the quality theanine in the step (4) is 98% or more by mass.
Compared with the prior art, the invention has the following advantages:
(1) The method adopts ethanol aqueous solution with the mass percentage of 90-95% to extract and remove most of catalytic enzyme, and then combines sephadex chromatography purification to completely remove the catalytic enzyme;
(2) The method adopts sephadex chromatography purification to remove inorganic impurities in enzyme catalytic reaction liquid;
(3) The method adopts acetic acid or phosphoric acid to adjust the pH value, so that theanine is stable under acidic conditions, particularly when being heated, ethylamine hydrochloride can be removed more easily, and other acid radical ions can not be artificially introduced;
(4) The method for extracting and purifying theanine from the enzyme catalytic reaction provided by the invention avoids a series of problems of excessively complex process, low yield, multiple solvent types, high cost and the like in the prior reported purification method, and the purity of the product obtained by the method can reach more than 98 percent and the yield can reach more than 80 percent;
(5) The method of the invention has the advantages of less types of used solvents, high solvent application degree, repeated use of materials and easy regeneration, and is a method which is more suitable for industrial production.
Detailed Description
The present invention will be described in further detail by way of examples, but embodiments of the present invention are not limited thereto. The raw materials used in the examples, unless otherwise specified, were all commercially available products.
Example 1
Taking 500mL of enzyme catalytic reaction liquid (the concentration of theanine is 30 g/L), regulating the pH of the reaction liquid to 6.2 by acetic acid, then carrying out vacuum filtration, using diatomite as a filter aid during the vacuum filtration, filtering to obtain filtrate, adding active carbon into the filtrate for decoloring at the temperature of 75 ℃ for 5h, filtering after decoloring, and evaporating the filtrate to dryness to obtain 90.3g of evaporated matter.
903mL of 95% (mass percent, the same applies below) ethanol water solution is added into the evaporated matter, then stirring extraction is carried out, the extraction temperature is 75 ℃, the extraction time is 7h, and after the extraction is finished, the system is cooled to 50 ℃ and then filtered by heat. The filtrate was evaporated to dryness to give low purity theanine, about 22.1g in total, and the theanine content (mass% content, the same applies hereinafter) was 60.7% by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/12mL, then loading the solution into a chromatographic column filled with 221G of sephadex G-10, eluting with deionized water as eluent at the eluting speed of 1V/2.0h (V is the wet volume of the filler), collecting the eluent according to 20mL each part, monitoring the eluting progress (ninhydrin color development) by TLC, combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.6G of crude theanine, and detecting the theanine content (mass percent content, the same applies below) to be 82.6% by HPLC.
The crude product is dissolved by 480mL of 85% (mass percent of ethanol, the same applies below) ethanol water solution at 75 ℃ and then recrystallized, the crystallization temperature is 20 ℃, the standing time is 10 hours, and the theanine refined product is 12.4g and the HPLC detection content is 98.7 percent.
Example 2
Taking 500mL of enzyme catalytic reaction liquid (the concentration of theanine is 30 g/L), regulating the pH value of the reaction liquid to 6 by phosphoric acid, then carrying out vacuum suction filtration, using active carbon as a filter aid during suction filtration, filtering to obtain filtrate, adding active carbon into the filtrate for decolorization at the temperature of 75 ℃ for 4 hours, filtering after the decolorization is finished, and evaporating the filtrate to dryness to obtain 90.8g of evaporated matter.
908mL of 90% ethanol water solution is added into the evaporated matter, then stirring extraction is carried out, the extraction temperature is 70 ℃, the extraction time is 6 hours, after the extraction is finished, the system is cooled to 50 ℃, hot filtration is carried out, the filtrate is evaporated to dryness, and low-purity theanine is obtained, the total amount of the theanine is about 23.4g, and the theanine content is 59.8% detected by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/10mL, then loading the solution into a chromatographic column filled with 187.2G of sephadex G-10, eluting with deionized water as eluent at the eluting speed of 1V/2h (V is the wet volume of the filler), collecting the eluent according to 20mL each time, monitoring (ninhydrin color development) eluting progress by TLC, combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.6G of crude theanine, and detecting the theanine content by HPLC to be 85.8%.
The crude product is dissolved by 468mL of 80% ethanol water solution at 70 ℃ and then recrystallized, the crystallization temperature is 20 ℃, the standing time is 10 hours, and 12.9g of theanine refined product is obtained by filtering, and the content detected by HPLC is 98.3%.
Example 3
Taking 500mL of enzyme catalytic reaction liquid (the concentration of theanine is 30 g/L), regulating the pH of the reaction liquid to 6.5 by acetic acid, then carrying out vacuum filtration, using perlite as a filter aid during the vacuum filtration, filtering to obtain filtrate, adding active carbon into the filtrate for decolorization, wherein the temperature is 80 ℃ and the time is 6 hours;
filtering after decoloring, evaporating filtrate to obtain 90.2g of evaporated matter, adding 1353mL of 95% ethanol water solution into the evaporated matter, stirring and extracting at 80 ℃ for 8h, and thermally filtering when the system is cooled to 55 ℃ after extracting. The filtrate was evaporated to dryness to give low purity theanine, about 22.7g total, with 62.1% theanine content as measured by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/15mL, then loading the solution into a chromatographic column filled with 227G of sephadex G-15, eluting with deionized water as eluent at the eluting speed of 1V/3h (V is the wet volume of the filler), collecting the eluent according to 20mL each time, monitoring the eluting progress (ninhydrin color development) by TLC, combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.9G of crude theanine, and detecting the theanine content to be 85.6% by HPLC.
The crude product is dissolved by 628mL of 88% ethanol water solution at 70 ℃ and then recrystallized, the crystallization temperature is 30 ℃, the standing time is 15 hours, and 12.4g of theanine refined product is obtained after filtration, and the HPLC detection content is 98.5%.
Example 4
500mL of enzyme catalytic reaction solution (the concentration of theanine is 30 g/L) is taken, the pH value of the reaction solution is regulated to 6.3 by phosphoric acid, then vacuum filtration is carried out, perlite is used as a filter aid during the suction filtration, and the filtrate is obtained after the filtration. Adding active carbon into the filtrate for decolorization at 75 ℃ for 5 hours;
filtering after decoloring, evaporating the filtrate to obtain 90.4g of evaporated matter, adding 1176mL of 98% ethanol water solution into the evaporated matter, stirring and extracting at 75 ℃ for 7h, and thermally filtering after the extraction is finished and the system is cooled to 53 ℃. The filtrate was evaporated to dryness to give low purity theanine, about 21.9g total, with a theanine content of 61.9% as measured by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/13mL, then loading the theanine into a chromatographic column filled with 197G of sephadex G-15, eluting with deionized water as eluent at the eluting speed of 1V/2.5h (V is the wet volume of the filler), collecting the eluent according to 20mL each time, monitoring (ninhydrin color development) eluting progress by TLC, combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.6G of crude theanine, and detecting the theanine content by HPLC to be 85.7%.
The crude product is dissolved by 546mL of 86% ethanol water solution at 70 ℃ and then recrystallized, the crystallization temperature is 25 ℃, the standing time is 13h, and 12.2g of theanine refined product is obtained by filtering, and the HPLC detection content is 98.6%.
Example 5
500mL of enzyme catalytic reaction solution (the concentration of theanine is 30 g/L) is taken, the pH value of the reaction solution is regulated to 6.4 by acetic acid, then vacuum filtration is carried out, diatomite is used as a filter aid during the suction filtration, and a filtrate is obtained after the filtration. Adding active carbon into the filtrate for decolorization at 75 ℃ for 5 hours;
filtering after decoloring, evaporating filtrate to obtain 90.7g of evaporated matter, adding 907mL of 92% ethanol water solution into the evaporated matter, stirring and extracting at 75 ℃ for 7h, and thermally filtering when the system is cooled to 50 ℃ after the extraction is completed. The filtrate was evaporated to dryness to give low purity theanine, about 21.8g total, with a theanine content of 61.1% as measured by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/10ml, then loading the solution into a chromatographic column filled with 218G of sephadex G-15, eluting with deionized water as eluent at the eluting speed of 1V/2.5h (V is the wet volume of the filler), collecting the eluent according to 20ml of each part, monitoring (ninhydrin color development) eluting progress by TLC, combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.1G of crude theanine, and detecting the theanine content by HPLC to be 86.1%.
The crude product is dissolved by 480mL of 85% ethanol water solution at 75 ℃ and then recrystallized, the crystallization temperature is 20 ℃, the standing time is 10 hours, and 12.5g of theanine refined product is obtained by filtration, and the HPLC detection content is 98.4%.
The invention has been described with reference to a few specific embodiments, it being necessary to note that the above specific embodiments are provided for the purpose of further illustration and are not intended to limit the scope of the invention. Some insubstantial modifications and adaptations of the invention by others are within the scope of the invention.
Claims (8)
1. A method for extracting purified theanine from an enzyme-catalyzed reaction, comprising the steps of:
(1) The reaction liquid of the theanine generated by the enzyme catalysis is subjected to pH value adjustment, vacuum suction filtration and decoloration to obtain clarified liquid;
(2) Filtering the clarified liquid to obtain filtrate, evaporating the filtrate to obtain an evaporated matter, stirring and extracting the evaporated matter with ethanol water solution, then performing hot filtration, and evaporating the filtrate to obtain low-purity theanine;
(3) Purifying the low-purity theanine by adopting sephadex chromatography, evaporating eluent to dryness to obtain crude theanine;
(4) Recrystallizing crude theanine, concentrating, and drying to obtain refined theanine;
in the step (1), acetic acid or phosphoric acid is adopted to adjust the pH value, and the pH value is adjusted to 6-6.5; the auxiliary agent adopted in the step (1) in the vacuum filtration is diatomite, perlite or active carbon;
in the step (2), when the ethanol aqueous solution is extracted, the mass percentage of ethanol in the ethanol aqueous solution is 90-95%, the temperature during extraction is 70-80 ℃, and the mass volume ratio of the evaporated matter to the ethanol aqueous solution is 1g: 10-15 mL, wherein the extraction time is 6-8 h;
the column packing used in the sephadex chromatography in the step (3) is sephadex G-10 or sephadex G-15, and the mass ratio of the low-purity theanine to the packing is 1: 8-10, wherein the eluent is deionized water, the elution speed is 1V/2-3 h, and V is the wet volume of the filler;
in the step (4), the crude theanine is dissolved by adopting an ethanol water solution at 70-75 ℃ and then recrystallized, wherein the mass percentage of ethanol in the ethanol water solution is 80-88%, and the mass volume relation between the crude theanine and the ethanol water solution is 1g: 30-40 mL, the crystallization temperature is 20-30 ℃ during recrystallization, and the time is 10-15 h.
2. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: the concentration of theanine in the reaction liquid for producing theanine by the enzyme catalysis in the step (1) is 27-30 g/L.
3. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: and (3) the decoloring agent adopted in the step (1) is activated carbon, the decoloring temperature is 70-80 ℃, and the decoloring time is 4-6 h.
4. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: the hot filtration temperature in the step (2) is 50-55 ℃.
5. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: the mass percentage of the low-purity theanine in the step (2) is 55-65%.
6. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: in the step (3), the low-purity theanine is dissolved by deionized water and then purified by sephadex chromatography, wherein the dosage relation of the low-purity theanine and the deionized water is 1g: 10-15 mL.
7. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: in the step (3), the mass percentage of the crude theanine is 80-88%.
8. The method for extracting purified theanine from an enzyme-catalyzed reaction according to claim 1, wherein: the quality percentage of the refined theanine in the step (4) is more than 98 percent.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557841A (en) * | 2004-01-14 | 2004-12-29 | 中国农业科学院茶叶研究所 | Process for synthetic extraction of polysaccharides, tea-polyphenol, theanine, caffeine from tea |
| CN101805269A (en) * | 2010-04-08 | 2010-08-18 | 晋江市恒源科技开发有限公司 | Method for separating and extracting natural theanine |
| CN102719367A (en) * | 2012-07-10 | 2012-10-10 | 湖南农业大学 | Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557841A (en) * | 2004-01-14 | 2004-12-29 | 中国农业科学院茶叶研究所 | Process for synthetic extraction of polysaccharides, tea-polyphenol, theanine, caffeine from tea |
| CN101805269A (en) * | 2010-04-08 | 2010-08-18 | 晋江市恒源科技开发有限公司 | Method for separating and extracting natural theanine |
| CN102719367A (en) * | 2012-07-10 | 2012-10-10 | 湖南农业大学 | Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase |
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