CN108315367B - Method for producing creatine phosphate by two-step enzymolysis method - Google Patents
Method for producing creatine phosphate by two-step enzymolysis method Download PDFInfo
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Abstract
The invention belongs to the technical field of sports supplements, and particularly discloses a method for producing creatine phosphate by a two-step enzymolysis method. Taking natural phospholipid as a raw material, adding water, a creatine compound and biological enzyme, and carrying out primary enzymolysis reaction at 40-80 ℃ for 4-72 h; and after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out secondary enzymolysis reaction at 40-80 ℃ for 4-8 h, and purifying to obtain the phosphocreatine. The preparation method does not use any organic solvent such as normal hexane, ethanol, ethyl acetate, acetone and the like, the used raw materials are natural products, and the product has higher physiological activity.
Description
Technical Field
The invention belongs to the technical field of sports supplements, and particularly relates to a method for producing creatine phosphate by a two-step enzymolysis method.
Background
Creatine phosphate is a high-energy phosphate compound phospholipid in muscle or other excitable tissues (such as brain and nerves) and acts to replenish the energy reserve of Adenosine Triphosphate (ATP).
Phosphocreatine is typically synthesized using phosphoenolpyruvate and creatine. The following figures are shown in detail:
however, the raw material phosphoenolpyruvic acid used in the synthesis method is a chemical synthesis product, and an organic solvent is required during synthesis, so that the large-scale production and application of the creatine phosphate as a sports supplement are greatly limited.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for producing creatine phosphate by using natural phospholipid as a raw material through a two-step enzymolysis method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for producing creatine phosphate by a two-step enzymolysis method comprises the following steps: taking natural phospholipid as a raw material, adding water, a creatine compound and biological enzyme, and carrying out primary enzymolysis reaction at 40-80 ℃ for 4-72 h; after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out secondary enzymolysis reaction at 40-80 ℃ for 4-8 h, and purifying to obtain phosphocreatine; wherein the mass ratio of the natural phospholipid to the water to the creatine compound to the biological enzyme to the phospholipase C is 1: 0.1-20: 0.1-4: 0.001-0.1: 0.01-0.1; the enzyme activity of the biological enzyme is more than or equal to 1000U/g, and the enzyme activity of the phospholipase C is more than or equal to 10000U/g.
The natural phospholipid includes but is not limited to one or more of Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Phosphatidylserine (PS), Phosphatidic Acid (PA), Phosphatidylglycerol (PG) and Lysophospholipid (LP).
The natural phospholipid is derived from the following sources (1), (2) or (3):
(1) plants including but not limited to soybean, rapeseed, sunflower;
(2) animals including but not limited to shrimps, fish, birds, mammals, or extracts thereof;
(3) a mixture of two or more of (1) and (2).
The creatine compound comprises but is not limited to one or a mixture of more of anhydrous creatine, hydrate of creatine and salt of creatine.
The biological enzyme includes, but is not limited to, one or a mixture of two of phospholipase D and creatine kinase.
The purification method is a centrifugal filtration method, an organic solvent extraction method or a chromatographic column separation method.
The production method of the invention has the following route schematic diagram:
wherein, R1 and R2 in the natural phospholipid both represent H or fatty acid chain, and X represents small molecule compounds, such as: x = H, the natural phospholipid is phosphatidic acid; x = choline, the natural phospholipid being Phosphatidylcholine (PC); x = ethanolamine, and the natural phospholipid is Phosphatidylethanolamine (PE).
Has the advantages that: the preparation method does not use any organic solvent such as normal hexane, ethanol, ethyl acetate, acetone and the like, the used raw materials are natural products, and the product has higher physiological activity.
Drawings
FIG. 1: the invention relates to a process flow chart of a production method.
Detailed Description
In order to make the invention clearer and clearer, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The process flow diagram is shown in figure 1, and the specific steps are as follows:
adding water, creatine and biological enzyme into a mixture of rapeseed phosphatide (powder) and egg yolk lecithin (powder) (the content of phosphatide in the mixture is 95.6 wt percent through detection), and carrying out enzymolysis reaction for 16 hours at 45 ℃; after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out enzyme reaction for 6 h at 55 ℃, carrying out low-temperature (5 ℃) centrifugal separation, washing and filtering an obtained solid phase for 3 times by using distilled water with the weight being 10 times that of the solid phase to obtain a target product, and detecting by using a high performance liquid chromatography, wherein the content of creatine phosphate in the target product is 90.8%;
wherein the mass ratio of the mixture of the rapeseed phosphatide and the yolk phosphatide (calculated by the content of phosphatide in the mixture) to the water, the creatine, the biological enzyme and the phospholipase C is 1: 10: 1: 0.01; the biological enzyme is a mixture of phospholipase D (enzyme activity 1000U/g) and creatine kinase (enzyme activity 1000U/g) in a mass ratio of 1: 1; the enzyme activity of the phospholipase C is 10000U/g.
Example 2
The process flow diagram is shown in figure 1, and the specific steps are as follows:
adding water, creatine and biological enzyme into soybean concentrated phospholipid (extracted from soybean oil, wherein the content of phospholipid is 60 wt% by detection), and performing enzymolysis reaction at 55 deg.C for 72 h; after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out enzyme reaction for 4h at 60 ℃, carrying out low-temperature (5 ℃) centrifugal separation, washing and filtering an obtained solid phase for 3 times by using distilled water with the weight being 10 times that of the solid phase to obtain a target product, and detecting by using a high performance liquid chromatography, wherein the content of creatine phosphate in the target product is 91.2%;
wherein the mass ratio of the soybean concentrated phospholipid (based on the content of phospholipid) to the water, the creatine, the biological enzyme and the phospholipase C is 1: 0.1: 0.01; the biological enzyme is phospholipase D (enzyme activity is 1000U/g); the enzyme activity of the phospholipase C is 10000U/g.
Example 3
The process flow diagram is shown in figure 1, and the specific steps are as follows:
adding water, creatine and biological enzyme into medulla bovis Seu Bubali extract (phospholipid content is 15.6 wt% detected), and performing enzymolysis at 40 deg.C for 72 hr; after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out enzyme reaction for 4h at 55 ℃, carrying out low-temperature (5 ℃) centrifugal separation, washing and filtering an obtained solid phase for 3 times by using distilled water with the weight being 10 times that of the solid phase to obtain a target product, and detecting by using a high performance liquid chromatography, wherein the content of creatine phosphate in the target product is 90.2%;
wherein the mass ratio of the bovine brain extract (based on the content of phospholipid) to water, creatine, biological enzyme and phospholipase C is 1: 18: 2: 0.01; the biological enzyme is phospholipase D (enzyme activity is 1000U/g); the enzyme activity of the phospholipase C is 10000U/g.
Claims (4)
1. A method for producing creatine phosphate by a two-step enzymolysis method is characterized in that: taking natural phospholipid as a raw material, adding water, a creatine compound and biological enzyme, and carrying out primary enzymolysis reaction at 40-55 ℃ for 16-72 h; after the primary enzymolysis reaction is finished, adding phospholipase C, carrying out secondary enzymolysis reaction at 55-60 ℃ for 4-6 h, and purifying to obtain phosphocreatine; wherein the mass ratio of the natural phospholipid to the water to the creatine compound to the biological enzyme to the phospholipase C is 1: 0.1-20: 0.1-4: 0.001-0.01: 0.01-0.01; the enzyme activity of the biological enzyme is more than or equal to 1000U/g, and the enzyme activity of the phospholipase C is more than or equal to 10000U/g; the creatine compound is one or a mixture of more of anhydrous creatine, hydrate of creatine and salt of creatine; the biological enzyme is one or a mixture of two of phospholipase D and creatine kinase.
2. The production method according to claim 1, wherein: the natural phospholipid is one or a mixture of more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphatidylglycerol and lysophospholipid.
3. The production method according to claim 1 or 2, characterized in that: the natural phospholipid is derived from the following sources (1), (2) or (3):
(1) plants including but not limited to soybean, rapeseed, sunflower;
(2) animals including but not limited to shrimps, fish, birds, mammals, or extracts thereof;
(3) a mixture of two or more of (1) and (2).
4. The production method according to claim 1, wherein: the purification method is a centrifugal filtration method, an organic solvent extraction method or a chromatographic column separation method.
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| CN1478899A (en) * | 2002-08-27 | 2004-03-03 | 中国科学院生物物理研究所 | Production method of creatine phosphate |
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| CN102653778A (en) * | 2012-05-08 | 2012-09-05 | 广东省微生物研究所 | Enzymolysis preparation method of phosphatidylinositol |
| CN102808006A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for producing phosphocreatine by microbial enzyme method |
| CN103014083A (en) * | 2012-12-31 | 2013-04-03 | 湖南宝利士生物技术有限公司 | Method of preparing creatine phosphate sodium |
| CN103266126A (en) * | 2013-06-06 | 2013-08-28 | 上海兆维科技发展有限公司 | Method for producing creatine phosphate by using enzyme method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9937239B2 (en) * | 2014-05-21 | 2018-04-10 | The Johns Hopkins University | Preservation and reconstitution of cell-free protein expression systems |
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| CN1478899A (en) * | 2002-08-27 | 2004-03-03 | 中国科学院生物物理研究所 | Production method of creatine phosphate |
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| CN102653778A (en) * | 2012-05-08 | 2012-09-05 | 广东省微生物研究所 | Enzymolysis preparation method of phosphatidylinositol |
| CN102808006A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for producing phosphocreatine by microbial enzyme method |
| CN103014083A (en) * | 2012-12-31 | 2013-04-03 | 湖南宝利士生物技术有限公司 | Method of preparing creatine phosphate sodium |
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