A method for the separation and purification of coenzyme Q10

Abstract

The present invention belongs to the field of separation and extraction of bioactive substances, and specifically discloses a method for separating and purifying coenzyme Q10, comprising the following steps: 1) obtaining bacterial cells; 2) pre-treating the bacterial cells; 3) obtaining solid bacterial residues; 4) extraction; 5) crystallization; 6) dissolution and chromatography; 7) vacuum concentration, water washing and drying. The present invention uses β-phenylethyl alcohol to replace the methanol solvent in the traditional method, reducing the use of toxic organic solvents. At the same time, isopentyl acetate and ethylene glycol monobutyl ether are used for extraction, improving the purity and yield of coenzyme Q10. Further, ultrasound and freeze-drying are used to further improve the purity and yield of coenzyme Q10. Meanwhile, the present invention has few steps, low preparation cost and high production efficiency, and can be produced on a large scale.

Description

Technical Field

[0001] The present invention belongs to the field of separation and extraction of bioactive substances, and specifically discloses a method for separating and purifying coenzyme Q10. Background Art

[0002] As the only coenzyme Q substance in the human body, coenzyme Q10 was first discovered by Professor Frederick Crane of the University of Wisconsin in the mitochondria of bovine heart cells in 1957. The concentration of coenzyme Q10 in the human heart is the highest, mainly derived from dietary supplements and self-synthesis. It can not only serve as an activator of cell metabolism and cell respiration, but also as an important natural antioxidant and non-specific immune enhancer. Reduced coenzyme Q10 can be oxidized by losing electrons to form oxidized coenzyme Q10, thus having the effect of scavenging free radicals and inhibiting the damage of free radicals to biological membranes. Coenzyme Q10 can also scavenge free radicals through synergistic action with vitamin E, inhibit lipid peroxidation under different oxidative stress conditions, and increase the activity of antioxidant enzymes. Coenzyme Q10 is rich in cardiomyocytes, can promote oxidative phosphorylation of ischemic myocardium, reduce mitochondrial oxygen consumption, improve the production efficiency of intracellular ATP, thereby improving the energy metabolism and function of cardiomyocytes under ischemic conditions, and contributing to the recovery of ischemic myocardium. Dietary supplementation of coenzyme Q10 is an effective way to increase the level of coenzyme Q10 in individuals. It has a relatively high content in foods such as animal viscera (heart, liver, kidney), beef, pork, saury, sardine, snakehead fish, peanuts, and soybeans. In the treatment of certain heart diseases such as viral myocarditis and chronic cardiac insufficiency, coenzyme Q10 can be supplemented for adjuvant treatment.

[0003] There have been reports on the extraction of coenzyme Q10 or reduced coenzyme Q10 in the prior art. For example, CN201610485022.0 discloses a process for simultaneously separating and preparing high-purity coenzyme Q10 and reduced coenzyme Q10. The mycelium residue is used as a raw material for percolation extraction to obtain a percolation extract containing coenzyme Q10 and reduced coenzyme Q10. Most of the coenzyme Q10 is separated by crystallization, and the crystallization mother liquor is reduced to obtain a reduced solution. The reduced solution is extracted to obtain an extract containing reduced coenzyme Q10, and the extract is crystallized to separate reduced coenzyme Q10. Finally, high-purity coenzyme Q10 with a purity of more than 98% and high-purity reduced coenzyme Q10 with a purity of more than 98% are obtained, and the total yield is more than 94%. CN202010991744.X discloses a method for extracting coenzyme Q10 from a coenzyme Q10 fermentation broth, and its process steps include pretreatment, leaching, crystallization, dissolution, chromatography, vacuum concentration, water washing, and drying of the coenzyme Q10 fermentation broth. By using the method of the present invention, the effective content of the coenzyme Q10 finished product can reach 99%, and the total extraction yield can reach 95%.

[0004] However, in existing extraction methods, it is necessary to add harmful organic solvents such as methanol for pretreatment of the bacterial cells, resulting in methanol residues. Or methods such as percolation extraction are used. Although the yield is relatively high, the extraction speed is slow and the production efficiency is low. It can only stay in the laboratory stage and is difficult to be used in actual production. Therefore, there is an urgent need to develop a method for separating and extracting coenzyme Q10 without polluting organic solvents such as methanol and with high speed. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a method for separating and purifying coenzyme Q10.

[0006] The technical solution of the present invention is as follows:

[0007] A method for separating and purifying coenzyme Q10, comprising the following steps:

[0008] 1) Obtaining bacterial cells:

[0009] Agrobacterium tumefaciens producing coenzyme Q10 is subjected to high-density fermentation, and then the wet bacterial cells are obtained by filtration and separation;

[0010] 2) Pretreatment of bacterial cells:

[0011] An excessive amount of β-phenylethyl alcohol is added to the wet bacterial cells to submerge the bacterial cells, and after stirring and standing, filtration is carried out to obtain wet bacterial residues;

[0012] 3) Obtaining solid bacterial residues:

[0013] An aqueous solution of sodium sulfite is added to the wet bacterial residues in step 2), and the mass ratio of the wet bacterial residues to the aqueous solution of sodium sulfite is 1:10-20; after stirring and standing for 1-2 h again, filtration is carried out, and the obtained wet bacterial residues are freeze-dried to obtain solid bacterial residues;

[0014] 4) Extraction:

[0015] Isoamyl acetate is added to the above solid bacterial residues, and the mass ratio of the solid bacterial residues to isoamyl acetate is 1:10-15; under ultrasonic conditions, stirring and standing are carried out for 1-2 h, and then filtration is carried out with a 0.45 μm filter cloth;

[0016] 5) Crystallization:

[0017] 1-2 volumes of water are added to the filtrate obtained in process 4), and the temperature is rapidly lowered to 0-5 °C within 30 min, and after stirring and standing, filtration is carried out with a 0.45 μm filter cloth to obtain solid coenzyme Q10;

[0018] 6) Dissolution and chromatography:

[0019] The above-obtained solid coenzyme Q10 is dissolved with butyl acetate, and the mass fraction of the solid coenzyme Q10 is 20-25%;

[0020] Then it is added to a chromatographic device, using butyl acetate as the mobile phase, and chromatography is carried out at a flow rate of 3-5 L / min. The liquid showing yellow or orange-yellow is collected to obtain a coenzyme Q10 butyl acetate solution;

[0021] 7) Vacuum concentration, washing with water and drying:

[0022] The coenzyme Q10 butyl acetate solution can be obtained as a pure product of coenzyme Q10 after vacuum concentration, washing with water, filtration and drying.

[0023] Furthermore, in the above method for separating and purifying coenzyme Q10, the Agrobacterium tumefaciens in step 1) is the existing Agrobacterium tumefaciens with the preservation number of ATCC 23308.

[0024] Furthermore, in the above method for separating and purifying coenzyme Q10, the high-density fermentation in step 2) includes the following steps: Agrobacterium tumefaciens is subjected to high-density aerobic fermentation for 72 hours with an inoculation amount of 7.5%; the components of the fermentation medium include 15% glucose, 1% ammonium sulfate, 0.34% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 1.5% corn steep liquor, 0.5% diammonium hydrogen phosphate, pH 6-7.5, and the fermentation temperature is 28-31 °C.

[0025] Furthermore, in the above method for separating and purifying coenzyme Q10, the mass fraction of the sodium sulfite aqueous solution in step 3) is 15-20%, preferably 17%.

[0026] Furthermore, in the above method for separating and purifying coenzyme Q10, the parameters of freeze-drying in step 3) are:

[0027] a) Vacuum drying: The wet bacterial residue is put into a vacuum drying oven and dried at low temperature and vacuum until the water content is lower than 15% to obtain product A;

[0028] b) Pre-freezing: Product A is spread out with a thickness of less than 1 cm, put into a freezing device, and quickly cooled to below -30 °C at a cooling rate of 5-8 °C / min to obtain product B;

[0029] c) Freeze-drying: Add a freeze-drying protectant with a final content of 5-10 wt% to product B, mix evenly, put it into a freeze-dryer for freeze-drying, and carry out the following freeze-drying cycles of S1-S4 2-4 times to obtain product C:

[0030] S1: -35 to -45 °C, pressure 1000-2000 Pa, freeze-dry for 30-60 min;

[0031] S2: -45 to -55 °C, pressure 500-1000 Pa, freeze-dry for 60-90 min;

[0032] S3: -55 to -65 °C, pressure 200 - 500 Pa, freeze-dry for 30 - 50 min;

[0033] S4: -65 to -80 °C, pressure 100 - 200 Pa, freeze-dry for 50 - 100 min;

[0034] d) Product collection: Slowly warm the product C to room temperature and collect the product.

[0035] Further, in the above method for separating and purifying coenzyme Q10, the stirring under ultrasonic conditions in step 4) is specifically: ultrasonic for 3 s and stop for 5 s, in a cycle; the ultrasonic power is 10 W / mL; the stirring speed is 300 - 500 rpm.

[0036] Further, in the above method for separating and purifying coenzyme Q10, the chromatography equipment in step 6) is a macroporous resin chromatography column, with a height-to-diameter ratio of 10 - 12:1, and the packing is D315.

[0037] Further, in the above method for separating and purifying coenzyme Q10, the

[0038] In step (7), in the vacuum concentration, control the temperature at 50 - 60 °C, and the pressure at -0.1 to 0 Mpa.

[0039] Further, in the above method for separating and purifying coenzyme Q10, in step (7), the water washing means washing once with purified water, and the amount used is 3 - 6 times the weight of the coenzyme Q10 solid; the drying means drying with a vacuum dryer for 2 - 4 h, the drying temperature is 35 - 40 °C, and the vacuum pressure is -0.02 to -0.08 Mpa.

[0040] Further, the present invention also discloses a coenzyme Q10 obtained by the separation and purification method described in any one of claims 1 - 9.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention discloses a method for separating and purifying coenzyme Q10. The present invention uses β-phenylethyl alcohol to replace the methanol solvent in the traditional method, reducing the use of toxic organic solvents. At the same time, isopentyl acetate and ethylene glycol monobutyl ether are used for extraction, improving the purity and yield of coenzyme Q10; further, ultrasonic and freeze-drying are used to further improve the purity and yield of coenzyme Q10. At the same time, the steps of the present invention are few, the preparation cost is low, the production efficiency is high, and it can be produced on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the process flow diagram for the separation and purification of coenzyme Q10;

[0044] Figure 2For the comparison of the purity of coenzyme Q10;

[0045] Figure 3 For the comparison of the yield of coenzyme Q10. Specific implementation mode

[0046] As Figure 1 shown, a method for separating and purifying coenzyme Q10 includes the following steps:

[0047] 1) Obtaining of bacterial cells:

[0048] Perform high-density fermentation on Agrobacterium tumefaciens producing coenzyme Q10, and then filter and separate to obtain wet bacterial cells;

[0049] 2) Pretreatment of bacterial cells:

[0050] Add an excessive amount of β-phenylethyl alcohol to the wet bacterial cells to submerge the cells, stir, let stand, and then filter to obtain wet bacterial residues;

[0051] 3) Obtaining solid bacterial residues:

[0052] Add an aqueous sodium sulfite solution to the wet bacterial residues in step 2), and the mass ratio of the wet bacterial residues to the aqueous sodium sulfite solution is 1:10 - 20; stir again, let stand for 1 - 2 h, and then filter. The obtained wet bacterial residues are freeze-dried to obtain solid bacterial residues;

[0053] 4) Extraction:

[0054] Add isopentyl acetate to the above solid bacterial residues, and the mass ratio of the solid bacterial residues to the isopentyl acetate is 1:10 - 15; stir and let stand for 1 - 2 h under ultrasonic conditions, and then filter with a 0.45 μm filter cloth;

[0055] 5) Crystallization:

[0056] Add 1 - 2 volumes of water to the filtrate obtained in process 4), quickly cool to 0 - 5 °C within 30 min, stir, let stand, and then filter with a 0.45 μm filter cloth to obtain solid coenzyme Q10;

[0057] 6) Dissolution and chromatography:

[0058] Dissolve the above-obtained solid coenzyme Q10 with butyl acetate, and the mass fraction of the solid coenzyme Q10 is 20 - 25%;

[0059] Then add it to a chromatography device, use butyl acetate as the mobile phase, perform chromatography under the condition of a flow rate of 3 - 5 L / min, collect the liquid showing yellow or orange-yellow color, and obtain a butyl acetate solution of coenzyme Q10;

[0060] 7) Vacuum concentration, water washing and drying:

[0061] The pure product of coenzyme Q10 can be obtained by subjecting the butyl acetate solution of coenzyme Q10 to reduced pressure concentration, washing with water, filtration, and drying.

[0062] The Agrobacterium tumefaciens in step 1) is the existing Agrobacterium tumefaciens with the preservation number of ATCC 23308.

[0063] The high-density fermentation in step 2) includes the following steps: subjecting Agrobacterium tumefaciens to high-density aerobic fermentation for 72 hours with an inoculation amount of 7.5%; the fermentation medium components include 15% glucose, 1% ammonium sulfate, 0.34% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 1.5% corn steep liquor, 0.5% diammonium hydrogen phosphate, pH 6 - 7.5, and the fermentation temperature is 28 - 31°C.

[0064] The mass fraction of the aqueous sodium sulfite solution in step 3) is 15 - 20%.

[0065] The parameters of freeze-drying in step 3) are as follows:

[0066] a) Vacuum drying: Put the wet bacterial residue into a vacuum drying oven and conduct low-temperature vacuum drying until the water content is lower than 15% to obtain product A.

[0067] b) Pre-freezing: Spread product A with a thickness of less than 1 cm, put it into a freezing device, and rapidly cool it to below -30°C at a cooling rate of 5 - 8°C / min to obtain product B.

[0068] c) Freeze-drying: Add a freeze-drying protectant with a final content of 5 - 10 wt% to product B, mix evenly, put it into a freeze dryer for freeze-drying, and conduct the following freeze-drying cycles of S1 - S4 2 - 4 times to obtain product C:

[0069] S1: -35 to -45°C, pressure 1000 - 2000 Pa, freeze-dry for 30 - 60 min;

[0070] S2: -45 to -55°C, pressure 500 - 1000 Pa, freeze-dry for 60 - 90 min;

[0071] S3: -55 to -65°C, pressure 200 - 500 Pa, freeze-dry for 30 - 50 min;

[0072] S4: -65 to -80°C, pressure 100 - 200 Pa, freeze-dry for 50 - 100 min;

[0073] d) Receiving the material: Slowly heat product C to room temperature and receive the material.

[0074] The specific stirring under ultrasonic conditions in step 4) is to ultrasound for 3 s and stop for 5 s in a cycle; the ultrasonic power is 10 W / mL; the stirring speed is 300 - 500 rpm.

[0075] The chromatography equipment in step 6) is a macroporous resin chromatography column with a height-to-diameter ratio of 10-12:1, and the packing material is D315;

[0076] In step (7), during the reduced-pressure concentration, the temperature is controlled at 50-60°C and the pressure is -0.1-0 Mpa.

[0077] In step (7), the water washing means washing once with purified water, and the dosage is 3-6 times the weight of the coenzyme Q10 solid; the drying means drying with a vacuum dryer for 2-4 h at a drying temperature of 35-40°C and a vacuum pressure of -0.02--0.08 MPa.

[0078] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0079] The reagents or instruments used in the embodiments of the present invention that are not indicated by the manufacturer can all be obtained as conventional reagent products through commercial purchase.

[0080] Example 1

[0081] A method for separating and purifying coenzyme Q10, comprising the following steps:

[0082] 1) Obtaining of thallus:

[0083] The Agrobacterium tumefaciens producing coenzyme Q10 is subjected to high-density fermentation, and then the wet thallus is obtained by filtration and separation;

[0084] 2) Thallus pretreatment:

[0085] An excessive amount of β-phenylethyl alcohol is added to the wet thallus to submerge the thallus, stirred, allowed to stand, and then filtered to obtain wet thallus residue;

[0086] 3) Obtaining of solid thallus residue:

[0087] Sodium sulfite aqueous solution is added to the wet thallus residue in step 2), and the mass ratio of the wet thallus residue to the sodium sulfite aqueous solution is 1:10; after stirring and standing for 1-2 h again and then filtering, the obtained wet thallus residue is freeze-dried to obtain a solid thallus residue;

[0088] 4) Extraction:

[0089] Isoamyl acetate is added to the above solid thallus residue, and the mass ratio of the solid thallus residue to the isoamyl acetate is 1:10; stirred and allowed to stand for 1-2 h under ultrasonic conditions and then filtered through a 0.45 μm filter cloth;

[0090] 5) Crystallization:

[0091] Add water with a volume 1 - 2 times that of the filtrate obtained in step 4), rapidly cool it to 0 - 5°C within 30 min, stir, let it stand, and then filter with a 0.45 μm filter cloth to obtain solid coenzyme Q10;

[0092] 6) Dissolution and chromatography:

[0093] Dissolve the obtained solid coenzyme Q10 with butyl acetate, and the mass fraction of the solid coenzyme Q10 is 20%;

[0094] Then add it to a chromatography device, use butyl acetate as the mobile phase, perform chromatography under the condition of a flow rate of 3 - 5 L / min, collect the liquid showing yellow or orange - yellow color, and obtain a butyl acetate solution of coenzyme Q10;

[0095] 7) Vacuum concentration, water washing and drying:

[0096] The butyl acetate solution of coenzyme Q10 can obtain pure coenzyme Q10 after vacuum concentration, water washing, filtration and drying;

[0097] The Agrobacterium tumefaciens in step 1) is the existing Agrobacterium tumefaciens with the preservation number of ATCC 23308;

[0098] The high - density fermentation in step 2) includes the following steps: perform high - density aerobic fermentation on Agrobacterium tumefaciens for 72 hours with an inoculation amount of 7.5%; the components of the fermentation medium include 15% glucose, 1% ammonium sulfate, 0.34% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 1.5% corn steep liquor, 0.5% diammonium hydrogen phosphate, pH 6 - 7.5, and the fermentation temperature is 28 - 31°C;

[0099] The mass fraction of the aqueous sodium sulfite solution in step 3) is 15%;

[0100] The parameters of freeze - drying in step 3) are as follows:

[0101] a) Vacuum drying: Put the wet bacterial residue into a vacuum drying oven, perform low - temperature vacuum drying until the water content is lower than 15% to obtain product A;

[0102] b) Pre - freezing: Spread product A with a thickness of less than 1 cm, put it into a freezing device, and rapidly cool it to below - 30°C at a cooling rate of 5 - 8°C / min to obtain product B;

[0103] c) Freeze - drying: Add a freeze - drying protectant with a final content of 5 - 10 wt% to product B, mix evenly, put it into a freeze - dryer for freeze - drying, and perform the following S1 - S4 freeze - drying cycles 2 - 4 times to obtain product C:

[0104] S1: -35 to -45 °C, pressure 1000 - 2000 Pa, freeze-dry for 30 - 60 min;

[0105] S2: -45 to -55 °C, pressure 500 - 1000 Pa, freeze-dry for 60 - 90 min;

[0106] S3: -55 to -65 °C, pressure 200 - 500 Pa, freeze-dry for 30 - 50 min;

[0107] S4: -65 to -80 °C, pressure 100 - 200 Pa, freeze-dry for 50 - 100 min;

[0108] d) Receiving the material: Slowly heat the product C to room temperature and receive the material.

[0109] The specific stirring under ultrasonic conditions in step 4) is to perform ultrasonic for 3 s and stop for 5 s in a cycle; the ultrasonic power is 10 W / mL; the stirring speed is 300 - 500 rpm.

[0110] The chromatography equipment in step 6) is a macroporous resin chromatography column with a height-to-diameter ratio of 10 - 12:1, and the packing is D315;

[0111] In step (7), in the vacuum concentration, control the temperature at 50 - 60 °C and the pressure at -0.1 to 0 Mpa.

[0112] In step (7), the water washing means washing once with purified water, and the dosage is 3 - 6 times the weight of the coenzyme Q10 solid; the drying means drying with a vacuum dryer for 2 - 4 h, the drying temperature is 35 - 40 °C, and the vacuum pressure is -0.02 to -0.08 MPa.

[0113] Example 2

[0114] A method for separating and purifying coenzyme Q10, comprising the following steps:

[0115] 1) Obtaining the bacterial cells:

[0116] Perform high-density fermentation on Agrobacterium tumefaciens producing coenzyme Q10, and then filter and separate to obtain wet bacterial cells;

[0117] 2) Pretreatment of the bacterial cells:

[0118] Add an excessive amount of β-phenylethyl alcohol to the wet bacterial cells to submerge the cells, stir, let stand, and then filter to obtain wet bacterial residues;

[0119] 3) Obtaining solid bacterial residues:

[0120] Add an aqueous sodium sulfite solution to the wet bacterial residue in step 2), and the mass ratio of the wet bacterial residue to the aqueous sodium sulfite solution is 1:15; stir again, let stand for 1 - 2 h, and then filter. The obtained wet bacterial residue is freeze-dried to obtain a solid bacterial residue;

[0121] 4) Extraction:

[0122] Add isopentyl acetate to the above solid bacterial residue, and the mass ratio of the solid bacterial residue to the isopentyl acetate is 1:12; stir and let stand for 1 - 2 h under ultrasonic conditions, and then filter with a 0.45 μm filter cloth;

[0123] 5) Crystallization:

[0124] Add 1 - 2 volumes of water to the filtrate obtained in process 4), quickly cool to 0 - 5 °C within 30 min, stir, let stand, and then filter with a 0.45 μm filter cloth to obtain solid coenzyme Q10;

[0125] 6) Dissolution and chromatography:

[0126] Dissolve the above-obtained solid coenzyme Q10 with butyl acetate, and the mass fraction of the solid coenzyme Q10 is 22%;

[0127] Then add it to a chromatography device, use butyl acetate as the mobile phase, perform chromatography at a flow rate of 3 - 5 L / min, collect the liquid showing yellow or orange-yellow color, and obtain a butyl acetate solution of coenzyme Q10;

[0128] 7) Vacuum concentration, washing with water and drying:

[0129] The butyl acetate solution of coenzyme Q10 can be obtained as a pure product of coenzyme Q10 after vacuum concentration, washing with water, filtering and drying;

[0130] The Agrobacterium tumefaciens in step 1) is the existing Agrobacterium tumefaciens with the preservation number of ATCC 23308;

[0131] The high-density fermentation in step 2) includes the following steps: perform high-density aerobic fermentation of Agrobacterium tumefaciens for 72 hours with an inoculation amount of 7.5%; the components of the fermentation medium include 15% glucose, 1% ammonium sulfate, 0.34% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 1.5% corn steep liquor, 0.5% diammonium hydrogen phosphate, pH 6 - 7.5, and the fermentation temperature is 28 - 31 °C;

[0132] The mass fraction of the aqueous sodium sulfite solution in step 3) is 17%;

[0133] The parameters of freeze-drying in step 3) are:

[0134] a) Vacuum drying: Put the wet bacterial residue into a vacuum drying oven, and perform low-temperature vacuum drying until the water content is lower than 15% to obtain product A;

[0135] b) Pre-freezing: Spread product A flat with a thickness of less than 1 cm, place it in a freezing device, and rapidly cool it to below -30°C at a cooling rate of 5 - 8°C / min to obtain product B;

[0136] c) Freeze-drying: Add a freeze-drying protectant with a final content of 5 - 10 wt% to product B, mix evenly, then place it in a freeze-dryer for freeze-drying, and perform the following freeze-drying cycles of S1 - S4 for 2 - 4 times to obtain product C:

[0137] S1: -35 to -45°C, pressure 1000 - 2000 Pa, freeze-dry for 30 - 60 min;

[0138] S2: -45 to -55°C, pressure 500 - 1000 Pa, freeze-dry for 60 - 90 min;

[0139] S3: -55 to -65°C, pressure 200 - 500 Pa, freeze-dry for 30 - 50 min;

[0140] S4: -65 to -80°C, pressure 100 - 200 Pa, freeze-dry for 50 - 100 min;

[0141] d) Receiving the material: Slowly warm product C to room temperature and receive the material.

[0142] The specific stirring under ultrasonic conditions in step 4) is to perform ultrasonic for 3 s and stop for 5 s in a cycle; the ultrasonic power is 10 W / mL; the stirring speed is 300 - 500 rpm.

[0143] The chromatography equipment in step 6) is a macroporous resin chromatography column with a height-to-diameter ratio of 10 - 12:1, and the packing is D315;

[0144] In step (7), in the reduced-pressure concentration, control the temperature at 50 - 60°C and the pressure at -0.1 to 0 Mpa.

[0145] In step (7), the water washing means washing once with purified water, and the dosage is 3 - 6 times the weight of coenzyme Q10 solid; the drying means drying with a vacuum dryer for 2 - 4 h, the drying temperature is 35 - 40°C, and the vacuum pressure is -0.02 to -0.08 MPa.

[0146] Example 3

[0147] A method for separating and purifying coenzyme Q10, comprising the following steps:

[0148] 1) Obtaining the bacterial cells:

[0149] Perform high-density fermentation on Agrobacterium tumefaciens producing coenzyme Q10, and then filter and separate to obtain wet bacterial cells;

[0150] 2) Pretreatment of bacterial cells:

[0151] Excessive β-phenylethyl alcohol is added to the wet bacterial cells to submerge the cells, followed by stirring, standing, and then filtration to obtain wet bacterial residues.

[0152] 3) Obtaining solid bacterial residues:

[0153] An aqueous sodium sulfite solution is added to the wet bacterial residues obtained in step 2), and the mass ratio of the wet bacterial residues to the aqueous sodium sulfite solution is 1:20. After stirring again and standing for 1 - 2 h, filtration is carried out. The obtained wet bacterial residues are freeze-dried to obtain solid bacterial residues.

[0154] 4) Extraction:

[0155] Isoamyl acetate is added to the above solid bacterial residues, and the mass ratio of the solid bacterial residues to the isoamyl acetate is 1:15. Under ultrasonic conditions, after stirring and standing for 1 - 2 h, filtration is carried out through a 0.45 μm filter cloth.

[0156] 5) Crystallization:

[0157] Water with a volume 1 - 2 times that of the filtrate obtained in process 4) is added, and the temperature is rapidly decreased to 0 - 5 °C within 30 min. After stirring and standing, filtration is carried out through a 0.45 μm filter cloth to obtain solid coenzyme Q10.

[0158] 6) Dissolution and chromatography:

[0159] The obtained solid coenzyme Q10 is dissolved with butyl acetate, and the mass fraction of the solid coenzyme Q10 is 25%.

[0160] Then it is added to a chromatography device, using butyl acetate as the mobile phase, and chromatography is carried out at a flow rate of 3 - 5 L / min. The liquid showing yellow or orange-yellow is collected to obtain a butyl acetate solution of coenzyme Q10.

[0161] 7) Vacuum concentration, water washing, and drying:

[0162] The butyl acetate solution of coenzyme Q10 is subjected to vacuum concentration, water washing, filtration, and drying to obtain pure coenzyme Q10.

[0163] The Agrobacterium tumefaciens in step 1) is the existing Agrobacterium tumefaciens with the preservation number of ATCC 23308.

[0164] The high-density fermentation in step 2) includes the following steps: Agrobacterium tumefaciens is subjected to high-density aerobic fermentation for 72 h with an inoculation amount of 7.5%. The components of the fermentation medium include 15% glucose, 1% ammonium sulfate, 0.34% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 1.5% corn steep liquor, 0.5% diammonium hydrogen phosphate, pH 6 - 7.5, and the fermentation temperature is 28 - 31 °C.

[0165] In step 3), the mass fraction of the sodium sulfite aqueous solution is 20%.

[0166] The parameters of the freeze-drying in step 3) are as follows:

[0167] a) Vacuum drying: Put the wet bacterial residue into a vacuum drying oven and conduct low-temperature vacuum drying until the water content is lower than 15% to obtain product A;

[0168] b) Pre-freezing: Spread product A flat with a thickness of less than 1 cm, put it into a freezing device, and rapidly cool it to below -30°C at a cooling rate of 5 - 8°C / min to obtain product B;

[0169] c) Freeze-drying: Add a freeze-drying protectant with a final content of 5 - 10 wt% to product B, mix evenly, then put it into a freeze-dryer for freeze-drying, and conduct the following freeze-drying cycles of S1 - S4 for 2 - 4 times to obtain product C:

[0170] S1: -35 to -45°C, pressure 1000 - 2000 Pa, freeze-dry for 30 - 60 min;

[0171] S2: -45 to -55°C, pressure 500 - 1000 Pa, freeze-dry for 60 - 90 min;

[0172] S3: -55 to -65°C, pressure 200 - 500 Pa, freeze-dry for 30 - 50 min;

[0173] S4: -65 to -80°C, pressure 100 - 200 Pa, freeze-dry for 50 - 100 min;

[0174] d) Receiving the material: Slowly heat product C to room temperature and receive the material.

[0175] The specific stirring under ultrasonic conditions in step 4) is to perform ultrasonic for 3 s and stop for 5 s in a cycle; the ultrasonic power is 10 W / mL; the stirring speed is 300 - 500 rpm.

[0176] The chromatography equipment in step 6) is a macroporous resin chromatography column with a height-to-diameter ratio of 10 - 12:1, and the packing is D315;

[0177] In step (7), in the reduced-pressure concentration, control the temperature at 50 - 60°C and the pressure at -0.1 to 0 Mpa.

[0178] In step (7), the water washing refers to washing once with purified water, and the amount used is 3 - 6 times the weight of the coenzyme Q10 solid; the drying refers to drying with a vacuum dryer for 2 - 4 h, the drying temperature is 35 - 40°C, and the vacuum pressure is -0.02 to -0.08 MPa.

[0179] Comparative Example 1

[0180] Step 2) In the pretreatment, methanol was used instead of β-phenethyl alcohol, and the rest was the same as in Example 2.

[0181] Comparative Example 2

[0182] Step 3) Vacuum drying was used to obtain freeze-drying, and the rest was the same as in Example 2.

[0183] Comparative Example 3

[0184] In Step 4), ethylene glycol monobutyl ether was used to replace isopentyl acetate, and the rest was the same as in Example 2.

[0185] Comparative Example 4

[0186] In Step 4), ultrasonic was not used, and the rest was the same as in Example 2.

[0187] Test Example 1

[0188] The coenzyme Q10 prepared in Examples 1-3 and Comparative Examples 1-4 was compared in terms of purity and yield. The results are shown in Table 1 and Figure 2 and Figure 3 as shown.

[0189] The purity was determined by the following method: An HPLC analysis method was established using a WATERS high-performance liquid chromatograph in the United States, and the detector was an ultraviolet detector; the chromatographic column was: WATERS Atlantis T3 column (250 mm × 4.6 mm, 5 μm); the injection volume was 10 μL; the mobile phase was methanol-ethanol (1:1, v / v); the flow rate was 1 mL / min; the column temperature was 30 °C.

[0190] Table 1 Purity and Yield of Coenzyme Q10

[0191] HPLC Purity (%) Yield (%) Example 1 99.3 98.2 Example 2 99.6 99.1 Example 3 99.4 99.0 Comparative Example 1 97.4 96.3 Comparative Example 2 95.9 96.1 Comparative Example 3 94.7 95.5 Comparative Example 4 93.3 94.2

[0192] From the data of Examples 1-3 and Comparative Examples 1-4, it can be seen that in the present invention, β-phenethyl alcohol was used to replace the methanol solvent, reducing the use of toxic organic solvents. At the same time, isopentyl acetate and ethylene glycol monobutyl ether were used for extraction, improving the purity and yield of coenzyme Q10; further, ultrasonic and freeze-drying were used, further improving the purity and yield of coenzyme Q10. At the same time, the steps of the present invention are few, the preparation cost is low, the production efficiency is high, and it can be produced on a large scale.

[0193] There are several preferred embodiments of the invention, which are described in more specific and detailed manner, but should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for separating and purifying coenzyme Q10, characterized in that, It includes the following steps: 1) Obtaining the bacterial cells: Perform high-density fermentation on Agrobacterium tumefaciens producing coenzyme Q10, and then filter and separate to obtain wet bacterial cells; 2) Pretreatment of the bacterial cells: Add an excessive amount of β-phenylethyl alcohol to the wet bacterial cells to submerge the cells, stir, let stand, and then filter to obtain wet bacterial residues; 3) Obtaining solid bacterial residues: Add an aqueous sodium sulfite solution to the wet bacterial residues in step 2), and the mass ratio of the wet bacterial residues to the aqueous sodium sulfite solution is 1:10 - 20; stir again, let stand for 1 - 2 h and then filter, and the obtained wet bacterial residues are freeze-dried to obtain solid bacterial residues; 4) Extraction: Add isopentyl acetate to the above solid bacterial residues, and the mass ratio of the solid bacterial residues to the isopentyl acetate is 1:10 - 15; stir and let stand for 1 - 2 h under ultrasonic conditions and then filter with a 0.45 μm filter cloth; 5) Crystallization: Add 1 - 2 volumes of water to the filtrate obtained in step 4), quickly cool to 0 - 5 °C within 30 min, stir, let stand, and then filter with a 0.45 μm filter cloth to obtain solid coenzyme Q10; 6) Dissolution and chromatography: Dissolve the obtained solid coenzyme Q10 with butyl acetate, and the mass fraction of the solid coenzyme Q10 is 20 - 25%; Then add it to a chromatography device, use butyl acetate as the mobile phase, perform chromatography under the condition of a flow rate of 3 - 5 L / min, collect the liquid showing yellow or orange-yellow, and obtain a butyl acetate solution of coenzyme Q10; 7) Vacuum concentration, water washing and drying: The butyl acetate solution of coenzyme Q10 is subjected to vacuum concentration, water washing, filtration and drying to obtain pure coenzyme Q10.

2. The method for separating and purifying coenzyme Q10 according to claim 1, characterized in that, The Agrobacterium tumefaciens in step 1) is the existing Agrobacterium tumefaciens with the preservation number of ATCC 23308.

3. The method for separating and purifying coenzyme Q10 according to claim 2, wherein, The high-density fermentation in step 2) includes the following steps: perform high-density aerobic fermentation on Agrobacterium tumefaciens with an inoculation amount of 7.5% for 72 hours; the components of the fermentation medium include 15% glucose, 1% ammonium sulfate, 0.34% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 1.5% corn steep liquor, 0.5% diammonium hydrogen phosphate, pH 6 - 7.5, and the fermentation temperature is 28 - 31 °C.

4. A method for separating and purifying coenzyme Q10 according to claim 1, characterized in that, The mass fraction of the aqueous sodium sulfite solution in step 3) is 15 - 20%.

5. The separation and purification method of coenzyme Q10 according to claim 1, characterized in that, The parameters of the freeze-drying in step 3) are: a) Vacuum drying: Put the wet bacterial residues into a vacuum drying oven, and perform low-temperature vacuum drying until the water content is lower than 15% to obtain product A; b) Pre-freezing: Spread product A with a thickness of less than 1 cm, put it into a freezing device, and quickly cool to below -30 °C at a cooling rate of 5 - 8 °C / min to obtain product B; c) Freeze-drying: Add a freeze-drying protectant with a final content of 5 - 10 wt% to product B, mix evenly, put it into a freeze-dryer for freeze-drying, and perform the following freeze-drying cycles of S1 - S4 for 2 - 4 times to obtain product C: S1: -35 to -45 °C, pressure 1000 - 2000 Pa, freeze-dry for 30 - 60 min; S2: -45 to -55 °C, pressure 500 - 1000 Pa, freeze-dry for 60 - 90 min; S3: -55 to -65 °C, pressure 200 - 500 Pa, freeze-dry for 30 - 50 min; S4: -65 to -80 °C, pressure 100 - 200 Pa, freeze-dry for 50 - 100 min; d) Collection of materials: Slowly warm the product C to room temperature and collect the materials.

6. The method for separating and purifying coenzyme Q10 according to claim 1, wherein The specific stirring under ultrasonic conditions in step 4) is as follows: Ultrasonic for 3 s and stop for 5 s, in a cycle; ultrasonic power is 10 W / mL; stirring speed is 300 - 500 rpm.

7. A method for separating and purifying coenzyme Q10 according to claim 1, characterized in that, The chromatography equipment in step 6) is a macroporous resin chromatography column, with a height-to-diameter ratio of 10 - 12:1, and the packing material is D315.

8. A method for separating and purifying coenzyme Q10 according to claim 1, characterized in that, In step (7), in the vacuum concentration, control the temperature at 50 - 60 °C, and the pressure at -0.1 - 0 Mpa.

9. A method for separating and purifying coenzyme Q10 according to claim 1, characterized in that, In step (7), the water washing means washing once with purified water, and the dosage is 3 - 6 times the weight of coenzyme Q10 solid; the drying means drying with a vacuum dryer for 2 - 4 h, the drying temperature is 35 - 40 °C, and the vacuum pressure is -0.02 - 0.08 MPa.

Images