CN101712969A - Preparation method of L-carnitine - Google Patents
Preparation method of L-carnitine Download PDFInfo
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- CN101712969A CN101712969A CN200810121542A CN200810121542A CN101712969A CN 101712969 A CN101712969 A CN 101712969A CN 200810121542 A CN200810121542 A CN 200810121542A CN 200810121542 A CN200810121542 A CN 200810121542A CN 101712969 A CN101712969 A CN 101712969A
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Abstract
The invention discloses a preparation method of L-carnitine. Pseudomonas elodea obtained from a soil sample by separation is adopted to be screened with a microwave mutagenesis method to obtain high-yield enzyme bacterial strain; then the high-yield enzyme bacterial strain is carried out with increment cultivation; after micro-capsule fossilization, reactant, i.e. crotonbetaine is added into bacterial strain cells so as to obtain L-carnitine by enzymolysis reaction under the ultrasonic assisted condition. The percent conversion of the reactant reaches 80% after 24 hours, thus obviously improving the percent conversion of the reactant.
Description
Technical field
The invention relates to a kind of preparation method of L-carnitine, belong to bioengineering field.
Background technology
From the beginning of this century, scientist has absorbed out from animal meat since the L-carnitine (L-carnitine), and its health protection effect is subjected to people's attention always.The L-carnitine is that two scholar Gulewjisch and Krimberg find in the meat concentrated solution that in 1905 the chemical structure of nineteen twenty-seven L-carnitine is proved.Nineteen fifty-two Fracuke isolates L-carnitine and called after VITAMIN-B in the concentrated solution of liver.Frize in 1958 finds out the L-carnitine the fats oxidn effect, and carnitine can stimulate the speed of fats oxidn.Think that the L-carnitine is the essential nutritive substance of L body in " sesame state nutrition institute magazine " report in 1986 symposium of nutrition " must ".
L-carnitine (L-carnitine) is a kind of functional factor of biostearin, chemistry L-3-hydroxyl by name-4-TMA (TriMethylAmine) butyric acid, molecular formula C
7H
15NO
3, good stability, the high temperature of ability more than 200 ℃ can be placed in the solution of PH3-6 1 year.It is the compound relevant with energy metabolism in the animal body, and biochemical action has: 1. promote the lipid acid β-Yang Hua; 2. regulate acyl group ratio in the plastosome; 3. enlist the services of excessive or irrational acyl group group; 4. influence amino acid whose metabolism; 5. combine the discharge that promotes ammonia in the body with urea, prevent the toxicity that excess ammonia produces.In addition, L-carnitine also has effect to the absorption of liposoluble vitamin and Ca, P; Can participate in glycogen heteroplasia process indirectly; Prevent hyperammonemia; Participate in the ripening process of sperm etc.Along with development of modern science, L-carnitine is applied to food, medicines and health protection field gradually, and by Switzerland, France, the U.S. and the World Health Organization legal be the multipurpose nutritive agent.
Preparation about L-carnitine has a variety of methods, and people are inquiring into better technology always and producing L-carnitine.The L-carnitine of very early time is separated from meat medicinal extract, and typical method is nineteen fifty-twos such as Carter to have extracted the pure brilliant carnitine of 0.6g with the 450g beef extract.Second method is the biological synthesis process that starts from Methionin and methionine(Met).The third method is a chemical synthesis, and the known chemical synthesis method has tens approach at present, is raw material with epoxy chloropropane, chloroacetyl acetacetic ester, mannitol etc., and operational path is all longer.Developed into the method for mesotomy afterwards again, comprised that chemistry split, biology splits and enzyme process splits and the utilization again of dextrorotation carnitine.Along with the continuous development of science and technology, microbe fermentation method, change method and gene recombination technology also in the middle of the production that constantly applies to L-carnitine from the carnitine derivative of dehydrogenation carnitine.
Enzyme transforming process also is a kind of comparatively advanced method of L-carnitine of producing, and it is to utilize the enzyme of purifying or somatic cells that the precursor substance (comprising 7-butyrobetaine, crotonbetaine, D carnitine or 3-dehydrogenation carnitine at present) of L-metabolism of carnitine is converted into the L-carnitine.Enzyme transforming process has characteristics such as environmental friendliness, cost are low, Product Safety height, is very attractive method.
Summary of the invention
One of purpose of the present invention provides a kind of preparation method of L-carnitine.
The said preparation method of L-carnitine of the present invention, be with pick up from pedotheque in the false pseudomonas bacillus that obtains of separation, adopt the method for microwave irradiation that it is screened, obtain high bacterium producing multi enzyme preparation and the cultivation of rising in value, again with the immobilization of strain cell micro-capsule, add the reaction substrate crotonbetaine then, under the ultrasonic wave subsidiary conditions, react.The effectively hydrolyzing enzyme that false pseudomonas bacillus is produced is converted into L-carnitine with crotonbetaine.
Technical solution of the present invention is as follows:
(1) cultivation of bacterium producing multi enzyme preparation, screening
Behind the sample natural air drying that contains false pseudomonas bacillus that picks up from soil, pulverize with mortar, carry out lock out operation according to gradient dilution method and spread plate method.Coated flat board places incubator to cultivate, after treating that bacterium colony grows, observe with the uv analyzer of 254nm, the bacterium colony that picking grows fine is connected to fresh solid plate, through being transferred to the test tube slant behind the unit cell separation and purification several generations, it is standby to place 4 ℃ of refrigerators to preserve.
With the sterilized water wash-out of the bacterial strain on the test tube slant, the inoculum size by 10% is connected to 30 ℃ of joltings (160r/min) cultivation 24h in the seed culture fluid.Get the above-mentioned seed liquor that is diluted to 10mL and be divided into the 2mL small test tube of packing into, eliminate the heat effect of microwave with low temperature dissipation of heat method.Adopt microwave oven that bacterium liquid is carried out mutagenic treatment.Be applied in the plate culture medium getting 0.2mL after the bacterium liquid dilution after the microwave irradiation processing, be inverted for 30 ℃ and cultivate 1-3 days to single bacterium colony appearance.The single preferably bacterium colony of picking growing way carries out shake flask fermentation repeatedly and sieves again.
Sieve again by fermentation, we are transferred to seed culture medium with the high bacterial strain of yield of enzyme that obtains and shake in the bottle, and under 35 ℃, rotating speed 160r/rain cultivates 24h, promptly obtains the cell seed liquor.
(2) immobilization of high yield enzyme strain cell
Add people's seed nutrient solution in sterilized water, the seed liquor of getting after the dilution is mixed with NaCS (Ushercell), stirs, and is used for dripping a glue capsule.After this solution degassing, dripping people's quality concentration by the air-flow generator for microcapsules is in 6%PDMDAAC (Poly Dimethyl Diallyl Ammonium Chloride) solution.Mild stirring under the room temperature, reaction is cultivated contains the cell microcapsule.Behind the 30min, the remaining PDMDAAC in flush away surface promptly gets immobilized cell to be cultivated.
(3) preparation of L-carnitine
Immobilized cell is cultivated: will wrap the capsule behind the bacterium, join contain (pH7.0) in the immobilized cell substratum shake in the bottle rotating speed 150r/min shaking table cultivation 120h.
After immobilized cell is cultivated, add the reaction substrate crotonbetaine in substratum, use the ultrasonic wave auxiliary treatment, temperature of reaction is 37 ℃, measures through behind the 24h, and the transformation efficiency of substrate has reached 80%.
Screen high bacterium producing multi enzyme preparation with the microwave irradiation facture among the present invention, remedied deficiency, by the microwave irradiation method with the traditional method screening, removed the low bacterial strain of yield of enzyme, obtain the higher bacterial strain of yield of enzyme, cultivated through increment again, obtained to test required high bacterium producing multi enzyme preparation.Then with microencapsulation with the somatic cells embedding, purpose is to improve the utilization ratio of enzyme that somatic cells produces.Increased the ultrasonic wave ancillary technique again in the enzyme digestion reaction stage, ultrasonic wave has increased the permeability of cytolemma to reaction substrate under the prerequisite of not destroying cell, and substrate can be combined with enzyme fully, has significantly improved the rate of utilization of the transformation efficiency and the enzyme of substrate.
L-carnitine (L-Carnitine; have another name called levocarnitine, carnitine, DL-carnitine chloride); be a kind of special acid that extensively is present in body tissue, it gives off energy oxidation in the long-chain acyl base band human mitochondrion, Intramitochondrial short chain acyl can be transported to outside the film again.Regulate the ratio of the inside and outside acyl-CoA/CoA of plastosome etc., so it is lipometabolic necessary cofactor.Be found in 1905 from L-carnitine, people constantly explore the new purposes of expanding it since the century, and aspect such as L-carnitine, protective foods clinical in China has obtained using widely in recent years.At first, the application aspect chronic kidney hypofunction peritonaeum hemodialysis.L-carnitine, adds when giving uremia hemodialysis patients promoting erythrocyte element (EPO) conventional treatment and uses L-carnitine in maximum surely belonging in the application aspect the hemodialysis clinically as pharmaceutical applications now, and patient Hb and Hct are significantly raise.Simultaneously, a large amount of animal and human's body experiments all confirm, replenish L-carnitine the treatment cardiovascular disorder is benefited.It can improve the energy metabolism of cardiac muscle, can reduce the case fatality rate of patients with congestive heart failure again, improves natural history in heart failure.Replenish L-carnitine, can make the hypoxic-ischemic cardiac muscle get back to the Fatty Acid Oxidation mode from the anaerobic glycolysis mode, thereby myocardial cell's energy metabolism is recovered, reduced the accumulation of objectionable impurities in the myocardial cell that anaerobic glycolysis produces, play prevention and alleviated myocardial damage, raising patient heart tolerance, reduced effects such as angina pectoris attacks frequency.In addition, L-carnitine can promote patient's metabolism of fat, increases the particularly utilization of stomach fat tissue of fatty tissue, alleviates the centre type obesity.Reduce patients serum's triglyceride levels, help to prevent atherosclerosis.The left-handed carnitine that revolves can also reduce fat rapidly, releases energy, and regains one's strength, the existing a kind of indispensable raw material that has become in the diet products.
In a word, verified, L-carnitine is a kind of generally acknowledged safe, nontoxic nutrition-fortifying agent, and its physiological function aspect metabolism of fat can make it to be widely used in treatment, diet food, baby humanized milk powder, player functions beverage, person in middle and old age's nutritious supplementary of clinical disease and make an addition to feed pig, ox, sheep, chicken, fish in the feed.Along with the continuous progress of medical science state of the art, the raising of living standards of the people, the purposes that L-carnitine is new also will obtain bigger expansion.
Claims (6)
1. preparation method of L-carnitine is characterized in that may further comprise the steps:
(1) cultivation of bacterium producing multi enzyme preparation, screening
Behind the sample natural air drying that contains false pseudomonas bacillus that picks up from soil, pulverize with mortar, carry out lock out operation according to gradient dilution method and spread plate method.Coated flat board places incubator to cultivate, after treating that bacterium colony grows, observe with the uv analyzer of 254nm, the bacterium colony that picking grows fine is connected to fresh solid plate, through being transferred to the test tube slant behind the unit cell separation and purification several generations, it is standby to place 4 ℃ of refrigerators to preserve.
With the sterilized water wash-out of the bacterial strain on the test tube slant, the inoculum size by 10% is connected to 30 ℃ of joltings (160r/min) cultivation 24h in the seed culture fluid.Get the above-mentioned seed liquor that is diluted to 10mL and be divided into the 2mL small test tube of packing into, eliminate the heat effect of microwave with low temperature dissipation of heat method.Adopt microwave oven that bacterium liquid is carried out mutagenic treatment.Be applied in the plate culture medium getting 0.2mL after the bacterium liquid dilution after the microwave irradiation processing, be inverted for 30 ℃ and cultivate 1-3 days to single bacterium colony appearance.The single preferably bacterium colony of picking growing way carries out shake flask fermentation repeatedly and sieves again.
Sieve again by fermentation, we are transferred to seed culture medium with the high bacterial strain of yield of enzyme that obtains and shake in the bottle, and under 35 ℃, rotating speed 160r/rain cultivates 24h, promptly obtains the cell seed liquor.
(2) immobilization of high yield enzyme strain cell
Add people's seed nutrient solution in sterilized water, the seed liquor of getting after the dilution is mixed with NaCS (Ushercell), stirs, and is used for dripping a glue capsule.After this solution degassing, dripping people's quality concentration by the air-flow generator for microcapsules is in 6%PDMDAAC (Poly Dimethyl Diallyl Ammonium Chloride) solution.Mild stirring under the room temperature, reaction is cultivated contains the cell microcapsule.Behind the 30min, the remaining PDMDAAC in flush away surface promptly gets immobilized cell to be cultivated.
(3) preparation of L-carnitine
Immobilized cell is cultivated: will wrap the capsule behind the bacterium, join contain (pH7.0) in the immobilized cell substratum shake in the bottle rotating speed 150r/min shaking table cultivation 120h.
After immobilized cell is cultivated, add the reaction substrate crotonbetaine in substratum, use the ultrasonic wave auxiliary treatment, temperature of reaction is 37 ℃, measures through behind the 24h, and the transformation efficiency of substrate has reached 80%.
2. place incubator to cultivate according to the coated flat board described in the claim (1), it is characterized in that culture temperature is 26 ℃-28 ℃, incubation time is 36h-48h.
3. according to the employing microwave oven described in the claim (1) bacterium liquid is carried out mutagenic treatment, the frequency of utilization that it is characterized in that microwave is 2450Hz, and output rating is 800W, and the treatment time is 140s-160s.
4. according to several substratum described in the right 1, it is characterized in that the moiety of substratum is as follows: inclined-plane (flat board) substratum: agar 1.5%, peptone 0.5%, extractum carnis 3%, MnSO
4.H
2O0.05%, yeast powder 0.3%, NaCl 2.4%, and KCl 0.01%, CaCl
2.H
2O 0.15%, Fe
2(PO
4)
30.001%, MgCl
2.6H
2O 0.55%, MgSO
4.7H
2O 0.69%.PH7.3-7.4 is transferred in the distilled water preparation, sterilization.
Seed culture medium: corn steep liquor 3.0%; Sodium-chlor 0.3%; Glucose 1.5%.PH7.3-7.4 is transferred in the distilled water preparation, sterilization.
Fermention medium: corn steep liquor 2.5%, sodium-chlor 0.3%, glucose 2.0%, sulfate of ammoniac 0.1%, sal epsom 0.05%, cobalt chloride 0.01% is transferred pH7.3-7.4, sterilization.
The immobilized cell substratum: 2% fumaric acid, 1% yeast extract, 1% peptone, 0.3% crotonobetaine alkali salt, all the other are distilled water, transfer pH7.0, sterilization.
5. mix with NaCS (Ushercell) according to said seed liquor of getting after the dilution in the right (2), seed liquor after it is characterized in that diluting is meant that adding sterilized water is 1/5 of original content with former seed liquor dilution, and the added amount of NaCS (Ushercell) is 4% (m/v) of dilution back seed liquor.
6. according to the said ultrasonic wave auxiliary treatment of using of right (3), it is characterized in that said hyperacoustic frequency of utilization is 20kHz, power is 10W.
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|---|---|---|---|
| CN200810121542A CN101712969A (en) | 2008-10-07 | 2008-10-07 | Preparation method of L-carnitine |
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|---|---|---|---|
| CN200810121542A CN101712969A (en) | 2008-10-07 | 2008-10-07 | Preparation method of L-carnitine |
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| Publication Number | Publication Date |
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| CN101712969A true CN101712969A (en) | 2010-05-26 |
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|---|---|---|---|
| CN200810121542A Pending CN101712969A (en) | 2008-10-07 | 2008-10-07 | Preparation method of L-carnitine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100831A (en) * | 2018-10-25 | 2020-05-05 | 中国科学院微生物研究所 | Recombinant bacterium for producing L-carnitine and construction method and application thereof |
-
2008
- 2008-10-07 CN CN200810121542A patent/CN101712969A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100831A (en) * | 2018-10-25 | 2020-05-05 | 中国科学院微生物研究所 | Recombinant bacterium for producing L-carnitine and construction method and application thereof |
| CN111100831B (en) * | 2018-10-25 | 2022-04-05 | 中国科学院微生物研究所 | Recombinant bacterium for producing L-carnitine and construction method and application thereof |
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Open date: 20100526 |