CN114621991A - Method for preparing neohesperidin dihydrochalcone by biological enzyme method - Google Patents
Method for preparing neohesperidin dihydrochalcone by biological enzyme method Download PDFInfo
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- CN114621991A CN114621991A CN202011478840.0A CN202011478840A CN114621991A CN 114621991 A CN114621991 A CN 114621991A CN 202011478840 A CN202011478840 A CN 202011478840A CN 114621991 A CN114621991 A CN 114621991A
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- neohesperidin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
Abstract
The invention discloses a method for preparing neohesperidin dihydrochalcone by a biological enzyme method, which comprises the following steps: (1) selecting neohesperidin, adding an alkaline solution, adjusting the reaction temperature to perform reaction, adding acid to adjust the pH value after the reaction is completed, and cooling to separate out neohesperidin chalcone; (2) dissolving neohesperidin chalcone in an alcohol solvent, adjusting the pH value by adopting a phosphate buffer solution, and then adding alkene reductase, coenzyme and a coenzyme regeneration system for reaction; (3) filtering after the reaction is finished, concentrating and crystallizing the filtrate, and performing post-treatment to obtain the neohesperidin dihydrochalcone. The method adopts alkene reductase to replace metal catalyst, avoids using chemical reagent, uses a coenzyme regeneration system to reduce the use of expensive coenzyme, and has the advantages of safety, environmental protection, low cost and high yield.
Description
Technical Field
The invention belongs to the technical field of neohesperidin dihydrochalcone, and particularly relates to a method for preparing neohesperidin dihydrochalcone by a biological enzyme method.
Background
The application with the publication number of CN103804439A discloses a synthesis method of neohesperidin dihydrochalcone, which prepares the neohesperidin dihydrochalcone by hydrogenation reaction of neohesperidin in an alkaline solution hydrogen environment.
Application with publication number CN104193786A discloses a synthesis method of neohesperidin dihydrochalcone, which takes neohesperidin as a starting material, methanol and ethanol as reaction media, and sodium borohydride and potassium borohydride as reducing agents to prepare the neohesperidin dihydrochalcone by reaction under heating conditions. Although the method is simple, the sodium borohydride and the potassium borohydride have large using amount, the reduction selectivity is poor, and side reaction is easy to occur
CN103334119A discloses a synthesis method of neohesperidin dihydrochalcone, which comprises the steps of dissolving neohesperidin in alkali to obtain neohesperidin chalcone, adding an electrolyte and the neohesperidin chalcone into an anode chamber and a cathode chamber by an electrolysis method, and electrolyzing to obtain the neohesperidin dihydrochalcone. Although the method does not need to use a catalyst and gaseous hydrogen, active hydrogen is provided by electrolyzed water to achieve the aim of hydrogenation. However, there are also disadvantages: the yield is low, and a large amount of electric energy is consumed.
In the existing preparation method, hydrogen is used in the production process of neohesperidin dihydrochalcone, a special hydrogenation reaction kettle is needed, the production operation risk is high, the yield of other production methods which do not use hydrogen is low, and most of the preparation methods adopt metal catalysts and are not environment-friendly.
Disclosure of Invention
The invention aims to provide a method for preparing neohesperidin dihydrochalcone by a biological enzyme method, which adopts alkene reductase to replace a metal catalyst, avoids using chemical reagents, uses a coenzyme regeneration system to reduce the use of expensive coenzyme, and has the advantages of safety, environmental protection, low cost and high yield.
The above object of the present invention can be achieved by the following technical solutions: a method for preparing neohesperidin dihydrochalcone by a biological enzyme method comprises the following steps:
(1) selecting neohesperidin, adding an alkaline solution, adjusting the reaction temperature to perform reaction, adding acid to adjust the pH value after the reaction is completed, and cooling to separate out neohesperidin chalcone;
(2) dissolving neohesperidin chalcone in an alcohol solvent, adjusting the pH value by adopting a phosphate buffer solution, and then adding alkene reductase, coenzyme and a coenzyme regeneration system for reaction;
(3) filtering after the reaction is finished, concentrating and crystallizing the filtrate, and then carrying out post-treatment to obtain the neohesperidin dihydrochalcone.
In the method for preparing the neohesperidin dihydrochalcone by the biological enzyme method:
preferably, in the step (1), the mass part ratio of the neohesperidin to the alkaline solution is 1: 3-10, wherein the mass percentage of the alkaline solution is 5-20%.
Preferably, the alkaline solution in step (1) is a sodium hydroxide solution or a potassium hydroxide solution.
Preferably, the reaction temperature in the step (1) is 20-50 ℃, and the reaction time is 4-20 hours.
Preferably, the reaction temperature in the step (1) is 30-40 ℃, and the reaction time is 6-8 hours.
Preferably, in the step (1), acid is added to adjust the pH value to 3-4.
Preferably, the acid includes, but is not limited to, hydrochloric acid.
Preferably, the alcohol solvent in the step (2) is methanol or ethanol, and the dosage relationship between the alcohol solvent and the neohesperidin chalcone is 5-10 mL: 1g of the total weight of the composition.
Preferably, the concentration of the phosphate buffer solution in the step (2) is 50-100 mmol/L, and the dosage relationship of the phosphate buffer solution and the neohesperidin chalcone is 10-20 mL: 1 g.
Preferably, the pH value is adjusted to be 5-9 in the step (2).
Preferably, the dosage of the alkene reductase in the step (2) is one ten thousandth to five thousandth of the total mass of the neohesperidin chalcone.
Preferably, the coenzyme in the step (2) is NAD + or NADP +, and the dosage of the coenzyme is five to one ten-thousandth of the total mass of the neohesperidin chalcone.
Preferably, the coenzyme regeneration system in the step (2) is glucose dehydrogenase and glucose, wherein the dosage of the glucose dehydrogenase is one thousandth to one hundredth of the total mass of the neohesperidin chalcone; the dosage of the glucose is ten percent to fifty percent of the total mass of the neohesperidin chalcone.
Preferably, the reaction temperature in the step (2) is 20-40 ℃, and the reaction time is 8-24 h.
Preferably, the post-treatment described in step (3) comprises filtration, washing and drying.
The invention opens ring reaction of neohesperidin in alkaline solution to generate neohesperidin chalcone, then adds phosphate buffer solution, alkene reductase, coenzyme and coenzyme regeneration system in neohesperidin dihydrochalcone alcoholic solution to react to generate neohesperidin dihydrochalcone, and then carries out subsequent treatments such as concentration, cooling, crystallization, filtration, washing with water, drying and the like to obtain the finished product.
Compared with the prior art, the invention has the following advantages:
(1) the method adopts alkene reductase to replace metal catalysts such as raney or palladium carbon and the like to catalyze the neohesperidin chalcone to convert the neohesperidin dihydrochalcone, avoids the use of chemical reagents, and is safe and environment-friendly;
(2) the coenzyme regeneration system is used, so that the use of expensive coenzyme NAD + or NADP + can be reduced;
(3) the invention adopts biological enzyme to prepare the neohesperidin dihydrochalcone, has good selectivity, and has high yield and content of the neohesperidin dihydrochalcone.
Detailed Description
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the present invention and to enable the above and other objects, features, and advantages of the present invention to be more readily understood, the following detailed description of the preferred embodiments is provided, wherein the materials are commercially available without specific recitation.
Example 1
The method for preparing neohesperidin dihydrochalcone by using the biological enzyme method provided by the embodiment comprises the following steps of:
(1) adding 100 g of neohesperidin into 500 ml of water, adding 50 g of sodium hydroxide, heating to 50 ℃, stirring for dissolving, reacting for 5 hours, adding dilute hydrochloric acid to adjust the pH value of the solution to 3.0, and cooling for crystallization to obtain 97 g of neohesperidin chalcone;
(2) the resulting neohesperidin chalcone was dissolved in 500 ml of methanol, 1000 ml of phosphate-potassium phosphate buffer (50mmol/L, pH 7.0 adjusted), 0.05 g of ene reductase, 0.25g of coenzyme NAD +, 0.5 g of glucose dehydrogenase, and 30 g of glucose were added, and the reaction was stirred at 30 ℃ for 24 hours.
Filtering after the reaction is finished, concentrating and crystallizing the filtrate, filtering and washing to obtain 95 g of neohesperidin dihydrochalcone with the mass percentage of 97%.
Example 2
The method for preparing neohesperidin dihydrochalcone by using the biological enzyme method provided by the embodiment comprises the following steps of:
(1) selecting neohesperidin, adding an alkaline solution, adjusting the reaction temperature to perform reaction, adding an acid to adjust the pH value after the reaction is completed, and cooling to separate out neohesperidin chalcone;
wherein the mass ratio of the neohesperidin to the alkaline solution is 1: 8.
the alkaline solution potassium hydroxide solution is prepared by adding potassium hydroxide into water, and the mass percentage of the alkaline solution is 20%.
The reaction temperature was 30 ℃ and the reaction time was 8 hours.
Hydrochloric acid is added to adjust the pH value to 3.5.
(2) Dissolving neohesperidin chalcone in an alcohol solvent, adjusting the pH value by adopting a phosphate buffer solution, and then adding alkene reductase, coenzyme and a coenzyme regeneration system for reaction;
the alcohol solvent is ethanol, and the dosage relationship of the alcohol solvent and the neohesperidin chalcone is 8 mL: 1g of the total weight of the composition.
The pH was adjusted to 6.
The dosage of the alkene reductase is one thousandth of the total mass of the neohesperidin chalcone.
The coenzyme is NADP +, and the dosage of the coenzyme is five thousandths of the total mass of the neohesperidin chalcone.
The coenzyme regeneration system comprises glucose dehydrogenase and glucose, and the dosage of the glucose dehydrogenase is 0.5 percent of the total mass of the neohesperidin chalcone; the dosage of the glucose is 15% of the total mass of the neohesperidin chalcone.
The reaction temperature is 40 ℃, and the reaction time is 10 h.
(3) Filtering after the reaction is finished, concentrating and crystallizing the filtrate, and then carrying out post-treatment including filtering, washing and drying to obtain the neohesperidin dihydrochalcone.
Example 3
(1) Selecting neohesperidin, adding an alkaline solution, adjusting the reaction temperature to perform reaction, adding acid to adjust the pH value after the reaction is completed, and cooling to separate out neohesperidin chalcone;
wherein the mass ratio of the neohesperidin to the alkaline solution is 1: 10.
the alkaline solution potassium hydroxide solution is prepared by adding potassium hydroxide into water, and the mass percentage of the alkaline solution is 10%.
The reaction temperature was 40 ℃ and the reaction time was 6 hours.
Adding hydrochloric acid to adjust pH to 4.
(2) Dissolving neohesperidin chalcone in an alcohol solvent, adjusting the pH value by adopting a phosphate buffer solution, and then adding alkene reductase, coenzyme and a coenzyme regeneration system for reaction;
the alcohol solvent is ethanol, and the dosage relationship of the alcohol solvent and the neohesperidin chalcone is 10 mL: 1g of the total weight of the composition.
The pH was adjusted to 8.
The dosage of the alkene reductase is eight ten-thousandth of the total mass of the neohesperidin chalcone.
The coenzyme is NAD + and the dosage of the coenzyme is eight ten-thousandth of the total mass of the neohesperidin chalcone.
The coenzyme regeneration system comprises glucose dehydrogenase and glucose, and the dosage of the glucose dehydrogenase is eight thousandth of the total mass of the neohesperidin chalcone; the dosage of the glucose is 40% of the total mass of the neohesperidin chalcone.
The reaction temperature is 30 ℃, and the reaction time is 15 h.
(3) Filtering after the reaction is finished, concentrating and crystallizing the filtrate, and then carrying out post-treatment including filtering, washing and drying to obtain the neohesperidin dihydrochalcone.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for preparing neohesperidin dihydrochalcone by a biological enzyme method is characterized by comprising the following steps:
(1) selecting neohesperidin, adding an alkaline solution, adjusting the reaction temperature to perform reaction, adding acid to adjust the pH value after the reaction is completed, and cooling to separate out neohesperidin chalcone;
(2) dissolving neohesperidin chalcone in an alcohol solvent, adjusting the pH value by adopting a phosphate buffer solution, and then adding alkene reductase, coenzyme and a coenzyme regeneration system for reaction;
(3) filtering after the reaction is finished, concentrating and crystallizing the filtrate, and then carrying out post-treatment to obtain the neohesperidin dihydrochalcone.
2. The method for preparing neohesperidin dihydrochalcone by using the bio-enzymatic method according to claim 1, wherein the method comprises the following steps: in the step (1), the mass part ratio of the neohesperidin to the alkaline solution is 1: 3-10 percent of alkaline solution, wherein the mass percentage of the alkaline solution is 5-20 percent.
3. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: in the step (1), the reaction temperature is 20-50 ℃, and the reaction time is 4-20 hours.
4. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: and (2) adding acid to adjust the pH value to 3-4 in the step (1).
5. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: the alcohol solvent in the step (2) is methanol or ethanol, and the dosage relationship of the alcohol solvent and the neohesperidin chalcone is 5-10 mL: 1g of the total weight of the composition.
6. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: the concentration of the phosphate buffer solution in the step (2) is 50-100 mmol/L, and the dosage relation of the phosphate buffer solution and the neohesperidin chalcone is 10-20 mL: 1g of the total weight of the composition.
7. The method for preparing neohesperidin dihydrochalcone by using the bio-enzymatic method according to claim 1, wherein the method comprises the following steps: and (3) adjusting the pH value to 5-9 in the step (2).
8. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: the dosage of the alkene reductase in the step (2) is one ten thousandth to five thousandth of the total mass of the neohesperidin chalcone; the coenzyme in the step (2) is NAD + or NADP +, and the dosage of the coenzyme is five to one ten-thousandth of the total mass of the neohesperidin chalcone.
9. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: the coenzyme regeneration system comprises glucose dehydrogenase and glucose, wherein the dosage of the glucose dehydrogenase is one thousandth to one hundredth of the total mass of the neohesperidin chalcone; the dosage of the glucose is ten percent to fifty percent of the total mass of the neohesperidin chalcone.
10. The method for preparing neohesperidin dihydrochalcone according to claim 1, comprising the steps of: in the step (2), the reaction temperature is 20-40 ℃, and the reaction time is 8-24 h.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4087558A (en) * | 1976-03-16 | 1978-05-02 | Linke Harald A B | Sweetening foods with neohesperidin chalcone |
| US20140045233A1 (en) * | 2012-07-31 | 2014-02-13 | Symrise Ag | Method for Biotechnological Production of Dihydrochalcones |
| CN106008619A (en) * | 2016-08-08 | 2016-10-12 | 四川生科力科技有限公司 | Preparation method of neohesperidin dihydrochalcone |
| CN110498821A (en) * | 2019-08-22 | 2019-11-26 | 湖南省农产品加工研究所 | A method of synthesis neohesperidin dihydrochalcone |
-
2020
- 2020-12-14 CN CN202011478840.0A patent/CN114621991A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4087558A (en) * | 1976-03-16 | 1978-05-02 | Linke Harald A B | Sweetening foods with neohesperidin chalcone |
| US20140045233A1 (en) * | 2012-07-31 | 2014-02-13 | Symrise Ag | Method for Biotechnological Production of Dihydrochalcones |
| CN106008619A (en) * | 2016-08-08 | 2016-10-12 | 四川生科力科技有限公司 | Preparation method of neohesperidin dihydrochalcone |
| CN110498821A (en) * | 2019-08-22 | 2019-11-26 | 湖南省农产品加工研究所 | A method of synthesis neohesperidin dihydrochalcone |
Non-Patent Citations (3)
| Title |
|---|
| BEATA ŻYSZKA ET AL.: "Highly effective, regiospecific reduction of chalcone by cyanobacteria leads to the formation of dihydrochalcone: two steps towards natural sweetness", 《MICROBIAL CELL FACTORIES》, pages 136 * |
| MECHTHILD GALL ET AL: "Enzymatic conversion of flavonoids using bacterial chalcone isomerase and enoate reductase", 《ANGEW CHEM INT ED ENGL》, pages 1439 - 1442 * |
| 李爱平: "新橙皮苷二氢查耳酮的制备及应用研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》, pages 024 - 489 * |
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