CN115584357B - Fermentation extraction method of coenzyme Q10 - Google Patents
Fermentation extraction method of coenzyme Q10 Download PDFInfo
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- CN115584357B CN115584357B CN202211359776.3A CN202211359776A CN115584357B CN 115584357 B CN115584357 B CN 115584357B CN 202211359776 A CN202211359776 A CN 202211359776A CN 115584357 B CN115584357 B CN 115584357B
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- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C46/00—Preparation of quinones
- C07C46/10—Separation; Purification; Stabilisation; Use of additives
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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Abstract
The invention provides a fermentation extraction method of coenzyme Q10. The method has proved that the corn starch and the corn steep liquor are adopted to replace a carbon source and a nitrogen source in the traditional fermentation medium respectively, so that the yield of the coenzyme Q10 can be obviously improved, and meanwhile, when the isopropanol is adopted as an extraction solvent, the extraction effect of the coenzyme Q10 can be further improved. Compared with other carbon sources and nitrogen sources, the corn starch and the corn steep liquor have the advantages of low component cost, high yield and the like. The percolation extraction method has the advantages of simple process, convenient operation, easy scale-up production and the like.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and relates to an extraction and purification method for producing coenzyme Q10 by a fermentation method.
Background
Coenzyme Q10 (C0 enzyme Ql0, coQl 0), also known as Ubiquinone (Ubiquinone), is a class of fat-soluble quinone compounds, one of the most important coenzymes in the mitochondria of cells. As an important constituent of the aerobic respiratory chain of eukaryotic cells, the eukaryotic cell can act on cell metabolism and cell respiration as an activator, has multiple effects of promoting the metabolism process in a human body, improving the nonspecific immunity of the human body, scavenging free radicals, enhancing the antioxidant capacity, stabilizing the cell membrane structure, inhibiting apoptosis, delaying aging, protecting the heart and the like, and is widely applied to the fields of medical treatment, dietary supplement, health care products, cosmetics and the like.
Related studies have expected that the annual composite growth rate of market demand for coenzyme Q10 will reach over 20% during the next decade. The development and the utilization of coenzyme Q10 in China are relatively late, and the main consumer market is in Europe and America. With the understanding of the health care efficacy of coenzyme Q10 by the chinese public, the domestic demand has begun to proliferate.
At present, the domestic source for producing coenzyme Q10 comprises chemical synthesis, plant cell culture, a microbial fermentation method and the like besides natural sources. The content of coenzyme Q10 in the natural source is relatively low, and the extraction component is relatively high; the chemical synthesis method has complex reaction, more steps, more byproducts and higher production cost; in contrast, microbial fermentative production of coenzyme Q10 has been of interest in the industry, and major research and development emphasis has focused on the same: constructing and screening high-yield strains, optimizing culture conditions of the strains, developing a proper precursor substance for promoting biosynthesis of coenzyme Q10, optimizing fermentation production process, optimizing separation and purification process and the like. The coenzyme Q10 produced by microbial fermentation has the following advantages: 1. the fermentation product is a natural product, has good biological activity and is easy to be absorbed by human body; 2. has no restriction of raw materials and is suitable for industrial production. However, the existing processes for extracting and purifying coenzyme Q10 from fermentation liquor have certain defects, and the problems of high consumption of solvent and silica gel, high pollution of waste water and solid waste and the like exist in the application to industrial production. Therefore, the technology needs to be simplified and optimized on the basis of the prior art, and a method for extracting high-purity coenzyme Q10 from fermentation broth, which has the advantages of simple technology, low cost and high yield, is found.
Disclosure of Invention
In view of the above-described circumstances, the present invention provides a method for fermentation extraction of coenzyme Q10. Compared with other carbon sources and nitrogen sources, the corn starch and the corn steep liquor have the advantages of low component cost, high yield and the like. The percolation extraction method has the advantages of simple process, convenient operation, easy scale-up production and the like.
Specifically, the invention provides a fermentation extraction method of coenzyme Q10, which comprises the following steps:
(1) Activating the rhodobacter sphaeroides strain, inoculating the rhodobacter sphaeroides strain into a seed culture medium, and performing shake cultivation for 20-30 hours at the temperature of 28-35 ℃ at the rotating speed of 180-220 rpm to obtain a seed solution, wherein the seed culture medium comprises the following components: naCl 2g/L, K 2 HPO 4 1.3g/L，KH 2 PO 4 0.5g/L，MgSO 4 0.125g/L，FeSO 4 0.1g/L, 3g/L glucose, 8g/L yeast extract powder, and adjusting the pH of the culture medium to 7.0.
Preferably, the rotation speed is 200 revolutions per minute, the temperature is 30 ℃, and the incubation time is 24 hours.
(2) Transferring the seed liquid into a fermentation medium with an inoculum size (volume/volume) of 8-15%, and carrying out shaking table fermentation culture at a rotation speed of 280-320 rpm at 28-35 ℃ for 100-150 hours to obtain a fermentation liquid, and introducing sterile air into the fermentation liquid in the fermentation process, wherein the ventilation ratio of the sterile air is 25-35L/min; wherein the composition of the fermentation medium is: mgSO (MgSO) 4 6.3g/L，KH 2 PO 4 3g/L，NaCl 2g/L，CaCO 3 2g/L, 3g/L of sodium glutamate, 40g/L of corn starch and 4g/L of corn steep liquor, and regulating the pH of the culture medium to 7.0.
Preferably, the inoculation amount is 10%, the rotating speed is 300 rpm, the temperature is 30 ℃, the culture time is 120 hours, and the ventilation ratio is 30L/min.
(3) Centrifuging the fermentation liquor at a rotating speed of 7000-9000 rpm for 20-40 minutes, collecting bottom solids, and washing the bottom solids with deionized water to obtain thalli; preferably, the rotation speed is 8000 revolutions per minute, and the centrifugation time is 30 minutes.
(4) Extracting coenzyme Q10 from thallus by percolation extraction, continuously adding isopropanol from the top of column, and controlling flow rate to 2.0-3.0mL/min until the volume of the percolate is 500mL. Repeatedly collecting enough percolate as raw material liquid for subsequent operation;
(5) Mixing percolates, and concentrating under reduced pressure with rotary evaporator; purifying the concentrated mixture by silica gel column chromatography and identifying by thin layer chromatography, crystallizing after identifying, quantitatively analyzing by HPLC, and calculating the yield.
Further preferably, the coenzyme Q10 producing bacteria is a strain type selected from the disclosure of US11390893B2, which may be of the genus Agrobacterium, aspergillus, acetobacter, aminococcus, agrobacterium, acidophilia, bulleromyces, bullera, brevundimonas, cryptococcus, chionosphaerophaera, candida, pseudomonas Cerinosterus, exisophiala, exobasidium, fellomyces, filobasidiella, filobasidium, geotrichum, graphiolaceae, gluconobacter, kockovaella, kurtzmanomyces, lalaria, leucosporidium, legionella, methylobacterium, mycoplana, oosporidium, pseudomonas, paracoccus, petroleum, rhodotorula, rhodosporidium, rhodotorula, sporogenes, saitoella, shizosaccharomyces, sphingomonas, sporotrichum, sypodiomycopsis, strigmatosporidium, tapporillena, tremella, trichospororia, tilletiopsis, ustilago, udeniomyces, xanthophilomyces, xanthobacter, paecilomyces, acremonium, hyphomonas or Rhizobium.
Further, the coenzyme Q10-producing microorganism is rhodobacter sphaeroides; more preferably, the rhodobacter sphaeroides is a rhodobacter sphaeroides strain with a collection number of CGMCC No.5997, a rhodobacter sphaeroides strain with a collection number of CGMCC No.5998 and/or a rhodobacter sphaeroides strain with a collection number of CGMCC No. 5999.
The invention has the following advantages: the invention provides a fermentation extraction method of coenzyme Q10. The method has proved that the corn starch and the corn steep liquor are adopted to replace a carbon source and a nitrogen source in the traditional fermentation medium respectively, so that the yield of the coenzyme Q10 can be obviously improved, and meanwhile, when the isopropanol is adopted as an extraction solvent, the extraction effect of the coenzyme Q10 can be further improved. Compared with other carbon sources and nitrogen sources, the corn starch and the corn steep liquor have the advantages of low component cost, high yield and the like. The percolation extraction method has the advantages of simple process, convenient operation, easy scale-up production and the like.
Drawings
FIG. 1 is a carbon source selection;
FIG. 2 is a nitrogen source selection;
FIG. 3 is an extraction solvent selection.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
A fermentation extraction method of coenzyme Q10, comprising the following steps:
(1) Activating rhodobacter sphaeroides strain (with a preservation number of CGMCC No. 5997), inoculating the rhodobacter sphaeroides strain into a seed culture medium, and performing shake culture at a rotation speed of 200 rpm at 30 ℃ for 24 hours to obtain a seed solution, wherein the seed culture medium comprises the following components: naCl 2g/L, K 2 HPO 4 1.3g/L，KH 2 PO 4 0.5g/L，MgSO 4 0.125g/L，FeSO 4 0.1g/L, 3g/L glucose, 8g/L yeast extract powder, and adjusting the pH of the culture medium to 7.0.
(2) Transferring the seed liquid into a fermentation medium with an inoculum size (volume/volume) of 10%, and carrying out shaking table fermentation culture at a rotation speed of 300 rpm at 30 ℃ for 120 hours to obtain a fermentation liquid, and introducing sterile air into the fermentation liquid in the fermentation process, wherein the ventilation ratio of the sterile air is 30L/min; wherein the composition of the fermentation medium is: mgSO (MgSO) 4 6.3g/L，KH 2 PO 4 3g/L，NaCl 2g/L，CaCO 3 2g/L, 3g/L of sodium glutamate, 40g/L of glucose and 4g/L of yeast extract powder, and regulating the pH of the culture medium to 7.0.
(3) Centrifuging the fermentation liquor for 30 minutes at a rotation speed of 8000 revolutions per minute, collecting bottom solids, washing the bottom solids with deionized water, wherein the solid-to-liquid ratio of the bottom solids to the deionized water is 1:25 (g/mL) to obtain a cell;
(4) Extracting coenzyme Q10 from thallus by percolation extraction, continuously adding n-hexane from the top of column, and controlling flow rate to 2.0-3.0mL/min until the volume of the percolate is 500mL. Repeatedly collecting enough percolate as raw material liquid for subsequent operation;
(5) Mixing percolates, and concentrating under reduced pressure with rotary evaporator; purifying the concentrated mixture by silica gel column chromatography and identifying by thin layer chromatography, crystallizing after identifying, quantitatively analyzing by HPLC, and calculating the yield.
Example 2
A fermentation extraction method of coenzyme Q10, the method steps of which are as shown in example 1, except that the carbon source in the fermentation medium is replaced by 40g/L of glucose and 40g/L of sucrose.
Example 3
A fermentation extraction method of coenzyme Q10, the method steps of which are as shown in example 1, except that the carbon source in the fermentation medium is replaced by 40g/L of glucose to 40g/L of corn starch.
Example 4
A fermentation extraction method of coenzyme Q10, the method steps are as shown in example 1, the difference is that the carbon source in the fermentation culture medium is replaced by 40g/L glucose and 40g/L corn starch, and the nitrogen source is replaced by 4g/L yeast extract powder (NH) 4 ) 2 SO 4 4g/L。
Example 5
A fermentation extraction method of coenzyme Q10, the method steps are as shown in example 1, the difference is that carbon source carbon in a fermentation culture medium is replaced by 40g/L glucose and 40g/L corn starch, and nitrogen source is replaced by 4g/L yeast extract and 4g/L corn steep liquor.
Example 6
A fermentation extraction method of coenzyme Q10, the method steps are as shown in example 1, the difference is that the carbon source in the fermentation culture medium is replaced by 40g/L of glucose and 40g/L of corn starch, and the nitrogen source is 4g/L of corn steep liquor. The solvent in step 4 was replaced with n-butanol by n-hexane.
Example 7
A fermentation extraction method of coenzyme Q10, the method steps are as shown in example 1, the difference is that the carbon source in the fermentation culture medium is replaced by 40g/L of glucose and 40g/L of corn starch, and the nitrogen source is 4g/L of corn steep liquor. The solvent in step 4 was replaced by isopropanol with n-hexane.
As shown in the results of the test shown in FIG. 1-3, the yield of coenzyme Q10 after separation and purification of the rhodobacter sphaeroides strain in fermentation media of different carbon sources is different, the yield of coenzyme Q10 can reach 21mg/L when glucose is used as a carbon source, and the yield of coenzyme Q10 can reach 53mg/L when corn starch is used as the carbon source. Therefore, to further increase the production of coenzyme Q10, corn starch was selected as a carbon source in the fermentation medium in subsequent experiments. In addition to the carbon source, the nitrogen source is also one of the key factors influencing the growth of microorganisms, and in order to further optimize the nitrogen source suitable for fermentation of rhodobacter sphaeroides strains, the inventor immediately performs a nitrogen source selection optimization experiment, and as a result, the yield of coenzyme Q10 can obviously reach 65mg/L when corn steep liquor is used as the nitrogen source, and can be improved by 25% compared with yeast extract. In addition, the extraction process also can correspond to the yield of the coenzyme Q10, after optimizing the fermentation medium, the inventor subsequently performs an optimization experiment on the extraction solvent in the percolation extraction method, and the analysis shows that the extraction efficiency by adopting different solvents is obviously different, and the conventional percolation extraction method generally adopts n-hexane as the extraction solvent, but in the experiment, the inventor surprisingly finds that compared with n-hexane, the extraction effect of isopropanol is more excellent, the yield of the coenzyme Q10 can be up to 100mg/L, the yield of the coenzyme Q10 is obviously improved, and the method is further suitable for the subsequent industrialized amplification production.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, but the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. A fermentation extraction method of coenzyme Q10, characterized in that the method comprises the following steps:
(1) Activating the rhodobacter sphaeroides strain, inoculating the rhodobacter sphaeroides strain into a seed culture medium, and carrying out shake cultivation for 24 hours at the temperature of 30 ℃ at the rotating speed of 200 revolutions per minute to obtain seed liquid; the composition of the seed culture medium is as follows: naCl 2g/L, K 2 HPO 4 1.3 g/L，KH 2 PO 4 0.5g/L， MgSO 4 0.125 g/L，FeSO 4 0.1g/L, glucose 3g/L, yeast extract 8g/L, and regulating pH of a culture medium to 7.0, wherein the preservation number of the strain is CGMCC No. 5997;
(2) Transferring the seed liquid into a fermentation culture medium with an inoculation amount of 10%, and carrying out shaking table fermentation culture at a rotation speed of 300 rpm at 30 ℃ for 120 hours to obtain a fermentation liquid, and introducing sterile air into the fermentation liquid in the fermentation process, wherein the ventilation ratio of the sterile air is 30L/min; wherein, corn starch and corn steep liquor are respectively used as a carbon source and a nitrogen source in the fermentation culture medium; the composition of the fermentation medium is as follows: mgSO (MgSO) 4 6.3 g/L， KH 2 PO 4 3 g/L，NaCl 2 g/L，CaCO 3 2g/L, 3g/L of sodium glutamate, 40g/L of corn starch, 4g/L of corn steep liquor, and adjusting the pH of the culture medium to 7.0;
(3) Centrifuging the fermentation liquor at a rotating speed of 7000-9000 rpm for 20-40 minutes, collecting bottom solids, and washing the bottom solids with deionized water to obtain thalli;
(4) Extracting coenzyme Q10 from thallus by percolation extraction, continuously adding extraction solvent from the top of column, controlling flow rate to 2.0-3.0mL/min until the volume of the collected percolate is 500mL; repeatedly collecting enough percolate as raw material liquid for subsequent operation; wherein the extraction solvent is isopropanol;
(5) Mixing percolates, and concentrating under reduced pressure with rotary evaporator; purifying the concentrated mixture by silica gel column chromatography and identifying by thin layer chromatography, crystallizing after identifying, quantitatively analyzing by HPLC, and calculating the yield.
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018003974A1 (en) * | 2016-07-01 | 2018-01-04 | 株式会社カネカ | Method for producing coenzyme q10 |
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