CN114213276A - 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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Abstract
The invention discloses a method for extracting and purifying theanine from an enzyme catalytic reaction, which comprises the following steps: (1) carrying out enzyme catalysis on a reaction liquid for generating theanine, and obtaining a clarified liquid after adjusting the pH value, vacuum filtration and decolorization; (2) filtering the clear solution to obtain filtrate, evaporating the filtrate to dryness to obtain evaporated material, extracting the evaporated material with ethanol water solution under stirring, heat filtering, and evaporating the filtrate to dryness to obtain low-purity theanine; (3) purifying the low-purity theanine by using sephadex chromatography, and evaporating eluent to dryness to obtain a crude product of theanine; (4) and recrystallizing the crude theanine, concentrating and drying to obtain the refined theanine. The method has the advantages of less solvent types, simple process, convenient operation, suitability for industrialization, and higher safety of the obtained 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) belongs to amide compounds, and has a molecular formula of C7H14N2O3. A large number of research results show that the L-theanine has a plurality of important physiological functions, so the L-theanine is widely applied to the food, health care products and pharmaceutical industries. L-theanine can be extracted and separated from tea leaves, but only accounts for 1% -2% of the dry weight of the tea leaves; in addition, the methods for extracting and separating high-purity L-theanine from tea leaves reported at present usually involve time-consuming, high-cost and complicated procedures due to the lack of simple, specific and efficient separation methods, which are not suitable for industrial production. On the contrary, the chemical synthesis of L-theanine has the advantages of simple process, low cost, high yield, etc., but the synthesized theanine is racemic mixture of L-and D-forms, and the separation technique is complicated.
With the research on the application of biotechnology to theanine synthesis, in addition to the application of genetic engineering and fermentation engineering techniques to theanine synthesis, more studies have been made on enzyme engineering, particularly enzyme immobilization techniques, which improve the efficiency of theanine synthesis. Through the combination of four major projects of biotechnology, there is an opportunity to produce theanine by adopting a more efficient and high-quality method. The L-theanine catalytically 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 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 theanine synthesized by the enzyme method has the advantages, the reaction system for synthesizing theanine by the enzyme method also contains a large amount of inorganic impurities and catalytic enzyme (one or more mixed enzymes) besides reaction raw materials, so that the subsequent purification of 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 enzyme, uses a small amount of solvents, is environment-friendly, has simple process, low cost and high theanine recovery rate, and is beneficial to industrial mass production.
The above object of the present invention can be achieved by the following technical solutions: a method for extracting and purifying theanine from enzyme catalysis reaction comprises the following steps:
(1) carrying out enzyme catalysis on a reaction liquid for generating theanine, and obtaining a clarified liquid after adjusting the pH value, vacuum filtration and decolorization;
(2) filtering the clear solution to obtain filtrate, evaporating the filtrate to dryness to obtain evaporated material, extracting the evaporated material with ethanol water solution under stirring, heat filtering, and evaporating the filtrate to dryness to obtain low-purity theanine;
(3) purifying the low-purity theanine by using sephadex chromatography, and evaporating eluent to dryness to obtain a crude product of theanine;
(4) and recrystallizing the crude theanine, concentrating and drying to obtain the refined theanine.
In the method for extracting and purifying theanine from enzyme catalytic reaction:
preferably, the reaction solution for enzymatically producing theanine in step (1) is derived from free enzyme solution for catalytically synthesizing theanine or immobilized enzyme for catalytically synthesizing theanine (enzyme is dropped into the reaction solution), and in any way, a large amount of inorganic salt and other auxiliary reagents (such as ATP) are required to be added besides ethylamine (or ethylamine hydrochloride) and glutamic acid (or sodium glutamate).
For example, the reaction solution for enzymatically producing theanine may be derived from "research on the enzymatic synthesis of L-theanine using γ -glutamyl methylamine synthetase, master thesis at tianjin science university: a reaction solution for synthesizing L-theanine by enzyme catalysis disclosed in Junjia, 6 months 2019.
Preferably, the concentration of theanine in the reaction solution for enzymatically producing theanine in step (1) is 27 to 30 g/L.
Preferably, acetic acid or phosphoric acid is adopted to adjust the pH value in the step (1), and the pH value is adjusted to 6-6.5.
In the present invention, hydrochloric acid or phosphoric acid is used to adjust the pH value for several reasons:
(a) theanine is stable under acidic conditions, especially when heated;
(b) the ethylamine hydrochloride is easier to remove by using an alcohol solvent under an acidic condition;
(c) if other acids are used for adjustment, other acid radical ions are artificially introduced (salt species are increased), so that the system is more complicated, and the separation difficulty is increased.
Preferably, the auxiliary agent adopted in the vacuum filtration in the step (1) is diatomite, perlite or activated carbon.
During vacuum filtration, the adoption of the auxiliary agent can reduce the filtration difficulty and shorten the filtration time.
Preferably, the decolorizing agent adopted in the decolorizing in the step (1) is activated carbon, the decolorizing temperature is 70-80 ℃, and the decolorizing time is 4-6 hours.
The active carbon is adopted for decoloring, so that the pigment in the reaction liquid for generating theanine through enzyme catalysis can be removed, the color is inevitably deepened in the reaction process no matter the enzyme liquid or the immobilized enzyme is used for catalyzing, the active carbon can be used for aiming at the pigment brought by the enzyme liquid or the immobilized enzyme, the pigment is all organic matters and can be obviously removed under the adsorption action of the active carbon, and if other decoloring methods are used, the effect is poor, and chemical methods such as hydrogen peroxide and the like cannot be used. The decolorization temperature range is preferably 70-80 ℃, the reflux amount is large when the temperature is too high, the physical adsorption capacity of the activated carbon is reduced when the temperature is too high, the added activated carbon is increased, the decolorization time is prolonged when the temperature is too low, and part of theanine is lost when being adsorbed by the activated carbon.
Preferably, in the step (2), when the ethanol aqueous solution is extracted, the mass percentage of the ethanol in the ethanol aqueous solution is 90-95%, the temperature during extraction is 70-80 ℃, and the mass-volume ratio of the steam-dried substance to the ethanol aqueous solution is 1 g: 10-15 mL, the extraction time is 6-8 h, and the hot filtration temperature is 50-55 ℃.
Most catalytic enzymes can be removed by adopting 90-95% by mass of ethanol aqueous solution for extraction, the catalytic enzymes belong to proteins, most catalytic enzymes can be separated out in the presence of 90-95% by mass of ethanol aqueous solution, and a small amount of catalytic enzymes (such as soluble proteins) which cannot be separated out can be completely removed in the subsequent glucose gel chromatography purification.
Preferably, the mass percentage content of the low-purity theanine in the step (2) is 55-65%.
Preferably, in the step (3), the low-purity theanine is dissolved in deionized water and then purified by sephadex chromatography, wherein the dosage relationship between the low-purity theanine and the deionized water is 1 g: 10-15 mL.
Preferably, the type of the column filler used for 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 filler is 1G: 8-10 g of deionized water as eluent, and the elution speed is 1V/2-3 h (V is the wet volume of the filler).
Sephadex chromatography is used primarily for desalting (such as 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 in ethanol water at 70-75 ℃ and then recrystallized, wherein the mass percentage of ethanol in the ethanol water is 80-88%, and the mass volume relationship between the crude theanine and the ethanol water is 1 g: 30-40 mL, and the crystallization temperature during recrystallization is 20-30 ℃ and the time is 10-15 h.
In the ethanol aqueous solution, the mass percentage of ethanol is 80-88%, and the material-liquid ratio is 1 g: 30-40 mL, the crystallization temperature is 20-30 ℃, the time is 10-15 h', the recrystallization is carried out according to the residual impurities to be removed, such as sodium chloride, magnesium chloride and sodium glutamate, and the content of chloride ions and other amino acids is not over standard due to precipitation only under the condition.
Preferably, the refined theanine in the step (4) has a mass percentage of more than 98%.
Compared with the prior art, the invention has the following advantages:
(1) the method adopts 90-95% of ethanol water solution for extraction, so that most of catalytic enzyme can be removed, and the catalytic enzyme can be completely removed by combining with sephadex chromatography purification;
(2) the method adopts sephadex chromatography purification to remove inorganic impurities in the enzyme catalysis reaction liquid;
(3) the method adopts acetic acid or phosphoric acid to adjust the pH value, can stabilize theanine under acidic conditions, particularly during heating, can remove ethylamine hydrochloride more easily, and can not introduce other acid radical ions artificially;
(4) the method for extracting and purifying theanine from the enzyme catalysis reaction provided by the invention avoids a series of problems of too complex process, low yield, various solvents, high cost and the like in the existing reported purification method, the purity of the product obtained by the method can reach more than 98%, and the yield can reach more than 80%;
(5) the method has the advantages of less solvent types, high solvent application degree, repeated use of materials and easy regeneration, and is a method suitable for industrial production.
Detailed Description
The present invention is further specifically described below by way of examples, but the embodiments of the present invention are not limited thereto. The starting materials used in the examples are all commercially available products unless otherwise specified.
Example 1
Taking 500mL of enzyme catalysis reaction liquid (the concentration of theanine is 30g/L), adjusting the pH of the reaction liquid to 6.2 by using acetic acid, then carrying out vacuum filtration, using diatomite as a filter aid during the filtration, filtering to obtain a filtrate, then adding activated carbon into the filtrate for decolorization at the temperature of 75 ℃ for 5 hours, filtering after the decolorization is finished, and evaporating the filtrate to dryness to obtain 90.3g of a distillate.
Adding 903mL of 95% (mass percentage, the same below) ethanol aqueous solution into the evaporated material, then stirring and extracting at 75 ℃ for 7 hours, and after the extraction is finished, carrying out hot filtration when the system is cooled to 50 ℃. The filtrate was evaporated to dryness to obtain theanine with low purity, about 22.1g in total, and the theanine content (mass percentage, the same below) was 60.7% by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/12mL, then loading the sample into a chromatographic column filled with 221G of sephadex G-10, after loading the sample, using deionized water as an eluent for elution with the elution speed of 1V/2.0h (V is the wet volume of the filler), collecting the eluent according to 20mL of each part, monitoring the elution progress by TLC (ninhydrin color development), combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.6G of crude theanine, and detecting the theanine content (mass percentage content, the same below) to be 82.6% by HPLC.
Dissolving the crude product in 480mL of 85% (mass percent of ethanol, the same below) ethanol water solution at 75 ℃, recrystallizing at 20 ℃ for 10h, filtering to obtain 12.4g of refined theanine product with the HPLC detection content of 98.7%.
Example 2
Taking 500mL of enzyme catalysis reaction liquid (the concentration of theanine is 30g/L), adjusting the pH of the reaction liquid to 6 by using phosphoric acid, then carrying out vacuum filtration, using activated carbon as a filter aid during the filtration to obtain a filtrate after filtration, then adding the activated carbon into the filtrate to decolor at 75 ℃ for 4h, filtering after the decoloration is finished, and evaporating the filtrate to dryness to obtain 90.8g of a evaporated material.
And adding 908mL of 90% ethanol aqueous solution into the evaporated substances, then stirring and extracting, wherein the extraction temperature is 70 ℃, the extraction time is 6 hours, after the extraction is finished, when the system is cooled to 50 ℃, hot filtering is carried out, the filtrate is evaporated to dryness to obtain low-purity theanine, the total amount of the low-purity theanine is 23.4g, and the content of the theanine is 59.8% through HPLC detection.
Dissolving the product with deionized water to make the concentration of theanine be 1G/10mL, then loading the sample into a chromatographic column filled with 187.2G of sephadex G-10, after loading the sample, using deionized water as an eluent for elution, wherein the elution speed is 1V/2h (V is the wet volume of the filler), collecting the eluent according to 20mL of each fraction, monitoring the elution progress by TLC (ninhydrin color development), combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.6G of crude theanine, and detecting the theanine content to be 85.8% by HPLC.
Dissolving the crude product with 468mL of 80% ethanol aqueous solution at 70 ℃, recrystallizing at 20 ℃, standing for 10h, filtering to obtain refined theanine 12.9g, with HPLC detection content of 98.3%.
Example 3
Taking 500mL of enzyme catalysis reaction liquid (the concentration of theanine is 30g/L), adjusting the pH of the reaction liquid to 6.5 by using acetic acid, then carrying out vacuum filtration, using perlite as a filter aid during the filtration to obtain a filtrate, and then adding activated carbon into the filtrate to decolor at the temperature of 80 ℃ for 6 hours;
filtering after decoloring, evaporating the filtrate to dryness to obtain 90.2g of evaporated material, adding 1353mL of 95% ethanol aqueous solution into the evaporated material, then stirring and extracting at the extraction temperature of 80 ℃ for 8h, and performing hot filtration when the system is cooled to 55 ℃ after extraction. The filtrate was evaporated to dryness to obtain low-purity theanine, amounting to about 22.7g, with a theanine content of 62.1% by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/15mL, then loading the product into a chromatographic column filled with 227G of sephadex G-15, after loading, using deionized water as an eluent for elution at the elution speed of 1V/3h (V is the wet volume of the filler), collecting the eluent according to 20mL of each fraction, monitoring the elution progress by TLC (ninhydrin color development), combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.9G of crude theanine, and detecting the theanine content by HPLC to be 85.6%.
Dissolving the crude product with 628mL of 88% ethanol water solution at 70 deg.C, recrystallizing at 30 deg.C for 15 hr, filtering to obtain refined theanine 12.4g with HPLC content of 98.5%.
Example 4
500mL of enzyme catalysis reaction liquid (the concentration of theanine is 30g/L) is taken, phosphoric acid is used for adjusting the pH of the reaction liquid to be 6.3, then vacuum filtration is carried out, perlite is used as a filter aid during the filtration, and the filtrate is obtained after the filtration. Then adding activated carbon into the filtrate for decolorization at 75 ℃ for 5 hours;
filtering after decoloring, evaporating the filtrate to dryness to obtain 90.4g of evaporated material, adding 1176mL of 98% ethanol aqueous solution into the evaporated material, then stirring and extracting at the extraction temperature of 75 ℃ for 7h, and performing hot filtration after the system is cooled to 53 ℃ after extraction. The filtrate was evaporated to dryness to obtain low-purity theanine, amounting to 21.9g, with a theanine content of 61.9% by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/13mL, then loading the product into a chromatographic column filled with 197G of sephadex G-15, after loading, using deionized water as an eluent for elution at the elution speed of 1V/2.5h (V is the wet volume of the filler), collecting the eluent according to 20mL of each fraction, monitoring the elution progress by TLC (ninhydrin color development), combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.6G of crude theanine, and detecting the theanine content to be 85.7% by HPLC.
Dissolving the crude product with 546mL of 86% ethanol aqueous solution at 70 ℃, recrystallizing at 25 ℃ for 13h, and filtering to obtain refined theanine product 12.2g with HPLC detection content of 98.6%.
Example 5
500mL of enzyme catalysis reaction liquid (the concentration of theanine is 30g/L) is taken, acetic acid is used for adjusting the pH of the reaction liquid to be 6.4, then vacuum filtration is carried out, diatomite is used as a filter aid during the filtration, and the filtrate is obtained after the filtration. Then adding activated carbon into the filtrate for decolorization at 75 ℃ for 5 hours;
filtering after decoloring, evaporating the filtrate to dryness to obtain 90.7g of evaporated material, adding 907mL of 92% ethanol aqueous solution into the evaporated material, then stirring and extracting at 75 ℃ for 7h, and performing hot filtration after the system is cooled to 50 ℃ after extraction is finished. The filtrate was evaporated to dryness to obtain low-purity theanine, amounting to 21.8g, with a theanine content of 61.1% by HPLC.
Dissolving the product with deionized water to make the concentration of theanine be 1G/10ml, then loading the product into a chromatographic column filled with 218G of sephadex G-15, after loading, using deionized water as eluent to carry out elution at the elution speed of 1V/2.5h (V is the wet volume of the filler), collecting the eluent according to 20ml of each fraction, monitoring the elution progress by TLC (ninhydrin color development), combining the eluents containing theanine, evaporating the combined eluents to dryness to obtain 15.1G of crude theanine, and detecting the theanine content to be 86.1% by HPLC.
Dissolving the crude product with 480mL of 85% ethanol aqueous solution at 75 ℃, recrystallizing at 20 ℃, standing for 10h, and filtering to obtain 12.5g of refined theanine product with the content of 98.4% detected by HPLC.
The present invention is illustrated by the following examples, which are not intended to limit the scope of the invention. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.
Claims (10)
1. A method for extracting and purifying theanine from enzyme catalytic reaction is characterized by comprising the following steps:
(1) carrying out enzyme catalysis on a reaction liquid for generating theanine, and obtaining a clarified liquid after adjusting the pH value, vacuum filtration and decolorization;
(2) filtering the clear solution to obtain filtrate, evaporating the filtrate to dryness to obtain evaporated material, extracting the evaporated material with ethanol water solution under stirring, heat filtering, and evaporating the filtrate to dryness to obtain low-purity theanine;
(3) purifying the low-purity theanine by using sephadex chromatography, and evaporating eluent to dryness to obtain a crude product of theanine;
(4) and recrystallizing the crude theanine, concentrating and drying to obtain the refined theanine.
2. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: the concentration of the theanine in the reaction liquid for generating the theanine through the enzyme catalysis in the step (1) is 27-30 g/L.
3. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: adjusting the pH value by adopting acetic acid or phosphoric acid in the step (1), and adjusting the pH value to 6-6.5; the auxiliary agent adopted in the vacuum filtration in the step (1) is diatomite, perlite or active carbon; and (2) the decoloring agent adopted in the decoloring in the step (1) is activated carbon, the decoloring temperature is 70-80 ℃, and the decoloring time is 4-6 hours.
4. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: when the ethanol aqueous solution is extracted in the step (2), the mass percentage of ethanol in the ethanol aqueous solution is 90-95%, the extraction temperature is 70-80 ℃, and the mass volume ratio of the steam-dried substance to the ethanol aqueous solution is 1 g: 10-15 mL, the extraction time is 6-8 h, and the hot filtration temperature is 50-55 ℃.
5. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: the mass percentage content of the low-purity theanine in the step (2) is 55-65%.
6. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: in the step (3), the low-purity theanine is dissolved by deionized water, and then purified by adopting sephadex chromatography, wherein the dosage relationship of the low-purity theanine and the deionized water is 1 g: 10-15 mL.
7. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: the type of the column filler used for the sephadex chromatography in the step (3) is sephadex G-10 or sephadex G-15, and the mass part ratio of the material (low-purity theanine) to the filler is 1: 8-10, the eluent is deionized water, and the elution speed is 1V/(2-3) h (V is the wet volume of the filler).
8. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: in the step (3), the mass percentage of the crude theanine is 80-88%.
9. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: in the step (4), the crude theanine is dissolved by 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 relationship between the crude theanine and the ethanol water solution is 1 g: 30-40 mL, and the crystallization temperature during recrystallization is 20-30 ℃ and the time is 10-15 h.
10. The method for extracting and purifying theanine from enzyme-catalyzed reaction as claimed in claim 1, wherein: the quality theanine in the step (4) has a mass percentage of more than 98%.
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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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