A method for extracting coenzyme Q10

Abstract

The present invention belongs to the technical field of biopharmaceuticals, and discloses a method for extracting coenzyme Q10. The coenzyme Q10 fermentation broth is slowly added into a mixed solution of petroleum ether and ethyl acetate, stirred, allowed to stand, and layered to obtain an aqueous phase and an organic phase. The organic phase is concentrated, and then repeatedly extracted with ethanol. The extraction solutions are combined to obtain a mixed solution containing coenzyme Q10. Petroleum ether is slowly added to the mixed solution, stirred, and allowed to stand to separate out the organic phase and the aqueous phase. The organic phase is concentrated to obtain an extract. After the extract is dissolved, activated carbon is added, stirred, and filtered to obtain a decolorized solution. The decolorized solution is cooled and filtered to obtain the finished product of coenzyme Q10. The extraction method of the present invention has a simple process and convenient operation, can effectively reduce the operating cost, greatly shorten the production cycle, and lay a foundation for realizing large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a method for extracting coenzyme Q10. Background Art

[0002] Coenzyme Q10 is a fat-soluble antioxidant, which has functions such as improving human immunity, enhancing antioxidant capacity, delaying aging, and enhancing human vitality. It is widely used in cardiovascular system diseases in medicine and is widely used as a nutritional health product and food additive at home and abroad. The preparation methods of coenzyme Q10 are mainly divided into three types: extraction from animal and plant tissues, chemical synthesis method, and microbial fermentation method; among them, the extraction method from animal and plant tissues is cumbersome and costly due to the conditions; the chemical synthesis method requires separation of cis-trans isomers, and the separation components are less used. The microbial fermentation method is widely used due to its low production cost and no isomers.

[0003] The patent with the publication number CN 115677468 A discloses a purification method of coenzyme Q10. Its process uses processes such as plate and frame filtration and drying of bacterial residues. It is difficult to match the production equipment, has high requirements for continuous operation equipment, and has a long production process cycle. New environmental protection problems are likely to occur during steps such as drying of bacterial residues.

[0004] The Chinese patent with the authorization number CN107337593B relates to a method for preparing pure coenzyme Q10. Its process steps are as follows: First, the coenzyme Q10 fermentation broth is microfiltered through a ceramic membrane and spray-dried to obtain a crude product, then the crude product is extracted with acetone, the obtained extract is phase-separated and concentrated under reduced pressure to obtain a coenzyme Q10 extraction concentrate, then extracted with petroleum ether, subjected to silica gel column chromatography, and the eluate is concentrated by vacuum distillation. Finally, coenzyme Q10 crystal seeds are added for crystallization, and vacuum drying is carried out to obtain pure coenzyme Q10. The present invention uses a ceramic membrane microfiltration and spray drying method to crush the mycelium, and then refluxes and extracts with acetone and separates with a ceramic membrane filter to break the cell wall of the mycelium and separate it, enriching coenzyme Q10. The crude coenzyme Q10 extract mainly contains coenzyme Q homologues with different numbers of isoprene units on the side chain, which are very similar in properties to coenzyme Q10. It is difficult to separate them by silica gel column chromatography technology, with a large amount of solvent used, a long time-consuming, and low efficiency. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to provide a method for extracting coenzyme Q10, which overcomes the defects in the prior art, has a simple process, is easy to operate, has a low operating cost, can effectively reduce energy consumption and shorten the production cycle, and lays a foundation for large-scale industrial production.

[0006] To solve the above technical problems, the technical solution of the present invention is:

[0007] A method for extracting coenzyme Q10, comprising the following steps:

[0008] a. Extraction: Slowly add a mixed solution of petroleum ether and ethyl acetate to the coenzyme Q10 fermentation broth at a volume ratio of 5:1 to 3 while continuously stirring. Stir at a speed of 200 - 400 rpm for 1 - 3 h, then let it stand for 20 - 40 min. After stratification, separate the organic phase and the aqueous phase;

[0009] b. Concentrate the organic phase obtained in step a under the conditions of a temperature of 40 - 70 °C and a vacuum degree of -0.10 Mpa to -0.09 Mpa until no condensed water flows out to obtain a concentrated solution. The concentrated solution is repeatedly extracted with ethanol 3 - 5 times. Each time, add ethanol while stirring at a speed of 300 - 600 rpm. After adding, stir at 40 - 60 °C for 1 - 3 h. After stirring, filter to obtain an ethanol extract solution, then cool it to 8 - 12 °C to obtain a mixed solution containing coenzyme Q10, and mix all the mixed solutions obtained each time together; The total amount of ethanol added is 1 - 3 times the volume of the concentrated solution;

[0010] c. Slowly add petroleum ether to the mixed solution containing coenzyme Q10 obtained in step b. The volume ratio of petroleum ether to the mixed solution is 1 - 3:1. During the addition process, continuously stir. Stir at a speed of 300 - 800 rpm until it becomes clear, then let it stand for 1 - 3 h, and then separate the organic phase and the aqueous phase, controlling the titer of the aqueous phase at 0.5 g / L;

[0011] d. Concentrate the organic phase obtained in step c under the conditions of a temperature of 40 - 60 °C and a vacuum degree of -0.10 Mpa to -0.09 Mpa until no condensed water flows out to obtain an extract;

[0012] e. Dissolve the extract obtained in step d at 40 - 70 °C. After dissolution and clarification, add 0.1 - 0.5% activated carbon, stir at 40 - 70 °C and a speed of 400 - 800 rpm for 30 min, then filter by suction to obtain a decolorized solution. Cool the decolorized solution to 20 - 25 °C at a rate of 3 - 5 °C / h, and then filter by suction to obtain the finished product of coenzyme Q10.

[0013] Preferably, in step a, the volume ratio of the coenzyme Q10 fermentation broth to the mixed solution of petroleum ether and ethyl acetate is 5:2, and the volume ratio of petroleum ether to ethyl acetate in the mixed solution of petroleum ether and ethyl acetate is 10:3 to 5; Stir at a speed of 300 rpm for 2 h, then let it stand for 30 min.

[0014] Further, the volume ratio of petroleum ether to ethyl acetate in the mixed solution of petroleum ether and ethyl acetate is 10:4.

[0015] Preferably, in step b, the organic phase is concentrated under the conditions of a temperature of 55°C and a vacuum degree of -0.095 Mpa; it is concentrated until no condensed water flows out to obtain a concentrated solution. The concentrated solution is repeatedly extracted with ethanol 4 times. Each time, ethanol is added while stirring at a speed of 400 rpm. After the addition is completed, stirring is carried out at 50°C for 2 hours. After stirring, filtration is performed to filter out the ethanol extract, and then the temperature is lowered to 10°C. The total amount of ethanol added is 2 times the volume of the concentrated solution.

[0016] Preferably, in step c, the volume ratio of petroleum ether to the mixed solution is 2:1. Stirring is continuously carried out during the addition process, and stirring is carried out at a speed of 500 rpm until it becomes clear, and then it is allowed to stand for 2 hours.

[0017] Preferably, in step d, the organic phase is concentrated under the conditions of a temperature of 50°C and a vacuum degree of -0.095 Mpa.

[0018] Preferably, in step e, the extract is dissolved at 55°C. After dissolution and clarification, 0.3% activated carbon is added, and stirring is carried out at 55°C and a speed of 600 rpm for 30 minutes, followed by suction filtration to obtain a decolorized solution. The decolorized solution is cooled to 22.5°C at a rate of 4°C / h, and then suction filtration is carried out.

[0019] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0020] 1. The process adopted by the present invention cancels steps such as plate and frame filtration, bacterial residue drying, and bacterial residue extraction in the traditional process, not only simplifies the equipment, but also shortens the production cycle, and at the same time avoids causing new pollution to the environment during the bacterial residue extraction process.

[0021] 2. The solvents used in the present invention are ethyl acetate, petroleum ether, and ethanol, with few types, low usage amounts, and low harm to the environment.

[0022] 3. The present invention uses ethanol crystallization to purify the crude coenzyme Q10. While ensuring the crystallization yield, it improves the quality of coenzyme Q10, and the obtained product has a good crystal form and a content ≥ 99.5%.

[0023] In summary, the extraction method of the present invention has a simple process, is easy to operate, can effectively reduce the operating cost, greatly shortens the production cycle, and the equipment used is all conventional equipment with low energy consumption, laying a foundation for realizing large-scale industrial production. Specific Embodiments

[0024] The technical solution of the present invention will be further described below in conjunction with embodiments:

[0025] Example 1:

[0026] a. Extraction: Take 1000 L of coenzyme Q10 fermentation broth, slowly add 200 L of a mixed solution of petroleum ether and ethyl acetate (where the volume ratio of petroleum ether to ethyl acetate is 10:3) at a volume ratio of 5:1, continuously stir while adding, stir at a speed of 200 rpm for 3 h, then let it stand for 40 min, and separate the organic phase and the aqueous phase after stratification;

[0027] b. Concentrate the organic phase obtained in step a under the conditions of a temperature of 40 °C and a vacuum degree of -0.10 Mpa until no condensed water flows out to obtain a concentrated solution. Add ethanol while stirring at 300 rpm, extract in 3 portions, stir at 40 °C for 3 h, filter out the ethanol extract, mix the three ethanol extracts, and cool down to 8 °C to obtain a mixed solution containing coenzyme Q10;

[0028] c. Slowly add petroleum ether to the coenzyme Q10 mixed solution, with the volume ratio of petroleum ether to the mixed solution being 1:1. Continuously stir during the addition, stir at a speed of 300 rpm until it becomes clear, then let it stand for 3 h, and then separate the organic phase and the aqueous phase, controlling the titer of the aqueous phase at 0.5 g / L;

[0029] d. Concentrate the organic phase obtained in step c under the conditions of a temperature of 40 °C and a vacuum degree of -0.10 Mpa until no condensed water flows out to obtain an extract;

[0030] e. Dissolve the extract obtained in step d at 40 °C. After dissolution and clarification, add 0.1% activated carbon, stir at 40 °C and a speed of 400 rpm for 30 min, filter by suction to obtain a decolorized solution. Cool the decolorized solution to 25 °C at a rate of 3 °C / h and filter by suction to obtain 2160 g of coenzyme Q10 finished product, where the content of coenzyme Q10 is 99.6%.

[0031] Example 2:

[0032] a. Extraction: Take 1000 L of coenzyme Q10 fermentation broth, slowly add 600 L of a mixed solution of petroleum ether and ethyl acetate (where the volume ratio of petroleum ether to ethyl acetate is 10:5) at a volume ratio of 5:3, continuously stir while adding, stir at a speed of 400 rpm for 1 h, then let it stand for 20 min, and separate the organic phase and the aqueous phase after stratification;

[0033] b. Concentrate the organic phase obtained in step a under the conditions of a temperature of 70 °C and a vacuum degree of -0.09 Mpa until no condensed water flows out to obtain a concentrated solution. Add ethanol while stirring at 600 rpm, extract in 5 portions, stir at 60 °C for 1 h, filter out the ethanol extract, mix the five ethanol extracts and cool down to 12 °C to obtain a mixed solution containing coenzyme Q10;

[0034] c. Slowly add petroleum ether to the obtained mixed solution containing coenzyme Q10. The volume ratio of petroleum ether to the mixed solution is 3:1. Stir continuously during the addition process and stir at a speed of 800 rpm until it becomes clear. Then let it stand for 1 h, and then separate the organic phase and the aqueous phase, controlling the titer of the aqueous phase at 0.5 g / L;

[0035] d. Concentrate the organic phase obtained in step c under the conditions of a temperature of 60 °C and a vacuum degree of -0.09 Mpa until no condensed water flows out to obtain an extract;

[0036] e. Dissolve the extract obtained in step d at 70 °C. After dissolution and clarification, add 0.5% activated carbon and stir at 70 °C and a speed of 800 rpm for 30 min, then filter by suction to obtain a decolorized solution. Cool the decolorized solution to 25 °C at a rate of 5 °C / h and filter by suction to obtain 2220 g of coenzyme Q10 finished product, where the content of coenzyme Q10 is 99.5%.

[0037] Example 3:

[0038] a. Extraction: Take 1000 L of coenzyme Q10 fermentation broth and slowly add 400 L of a mixed solution of petroleum ether and ethyl acetate (where the volume ratio of petroleum ether to ethyl acetate is 10:4) at a volume ratio of 5:2. Stir continuously at a speed of 300 rpm for 2 h, then let it stand for 30 min, and separate the organic phase and the aqueous phase after stratification;

[0039] b. Concentrate the organic phase in step a under the conditions of a temperature of 55 °C and a vacuum degree of -0.095 Mpa until no condensed water flows out to obtain a concentrated solution. Add ethanol while stirring at 400 rpm and extract it in 4 portions. Stir at 50 °C for 2 h, then filter out the ethanol extract solution, and mix the four ethanol extract solutions and cool to 10 °C to obtain a mixed solution containing coenzyme Q10;

[0040] c. Slowly add petroleum ether to the obtained mixed solution containing coenzyme Q10. The volume ratio of petroleum ether to the mixed solution is 2:1. Stir continuously during the addition process and stir at a speed of 500 rpm until it becomes clear. Then let it stand for 2 h, and then separate the organic phase and the aqueous phase, controlling the titer of the aqueous phase at 0.5 g / L;

[0041] d. Concentrate the organic phase obtained in step c under the conditions of a temperature of 50 °C and a vacuum degree of -0.095 Mpa until no condensed water flows out to obtain an extract;

[0042] e. Dissolve the extract obtained in step d at 55°C. After dissolution and clarification, add 0.3% activated carbon, stir for 30 min at 55°C and 600 rpm, then perform suction filtration to obtain a decolorized solution. Cool the decolorized solution to 22.5°C at a rate of 4°C / h and perform suction filtration to obtain 2430 g of Coenzyme Q10 finished product, with the Coenzyme Q10 content being 99.8%.

[0043] Example 4:

[0044] Except that the volume ratio of the Coenzyme Q10 fermentation broth to the mixed solution of ethyl acetate and petroleum ether in step a extraction is 5:2.5. Other process conditions are the same as those in Example 3, and 2280 g of Coenzyme Q10 finished product is obtained, with the Coenzyme Q10 content being 99.5%.

[0045] Example 5

[0046] Except that the volume ratio of the Coenzyme Q10 fermentation broth to the mixed solution of ethyl acetate and petroleum ether in step a extraction is 5:1.5. Other process conditions are the same as those in Example 3, and 2265 g of Coenzyme Q10 finished product is obtained, with the Coenzyme Q10 content being 99.6%.

[0047] Example 6

[0048] Except that the volume ratio of the mixed solution to petroleum ether in step c is 1:3, other process conditions are the same as those in Example 3, and 2190 g of Coenzyme Q10 finished product is obtained, with the Coenzyme Q10 content being 99.5%.

[0049] Comparative Example 1: An existing extraction method of Coenzyme Q10 - Method 1

[0050] Weigh 10 kg of wet mycelium with a water content of about 60%, add 10 L of prepared sodium sulfite aqueous solution, perform pulping under vigorous stirring, add 50 L of n - hexane and 1 kg of dodecyltrimethylammonium chloride. After adding, continue pulping and extracting for 6 hours at 40°C. Add 1 L of saturated sodium chloride solution, stir well, transfer to a liquid - separation device and let it stand, remove the aqueous phase. Wash the organic phase with saturated sodium chloride aqueous solution (1 L×3), dry the organic phase with anhydrous sodium sulfate, filter, and rotary - evaporate the solvent to obtain an extract. Separate the extract by silica gel column chromatography with a silica gel of 200 - 300 mesh and a mass 10 times that of the extract. The eluent is a mixed solvent of 1% acetone and n - hexane by volume ratio. Combine the eluent containing Coenzyme Q10, rotary - evaporate the solvent, add anhydrous ethanol at 60°C to just dissolve it again, control the crystallization temperature to drop 2°C per hour, slowly cool to 0°C and maintain for 3 hours. When a large amount of crystals appear, filter, wash, and dry to obtain 102.1 g of finished product with a purity of 99.0%.

[0051] Comparative Example 2: An existing extraction method of Coenzyme Q10 - Method 2

[0052] Weigh 10 kg of wet mycelium with a water content of about 50%, add 10 L of the prepared sodium sulfite aqueous solution, and carry out pulping under vigorous stirring. Then add 50 L of n-hexane and 1 kg of dodecyltrimethylammonium chloride. After adding, continue pulping and extracting for 7 hours at a temperature of 30 °C. Add 1 L of saturated sodium chloride solution, stir well, transfer to a liquid separation device and let it stand. Remove the aqueous phase. The organic phase is washed with saturated sodium chloride aqueous solution (1 L × 3), dried with anhydrous sodium sulfate, filtered, and the solvent is rotary evaporated to obtain an extract. The extract is separated by silica gel column chromatography with a silica gel of 200 - 300 mesh, and the mass of the silica gel is 10 times that of the extract. The eluent is a mixed solvent of acetone and n-hexane with a volume ratio of 0.8%. Combine the eluent containing coenzyme Q10, rotary evaporate the solvent, add anhydrous ethanol at 60 °C to just dissolve it again, control the crystallization temperature to drop by 2 °C per hour, slowly cool to 0 °C and maintain for 3 hours. A large amount of crystals appear, filter, wash, and dry to obtain 105.1 g of the finished product with a purity of 98.9%.

[0053] Comparative Example 3: The third existing extraction method of coenzyme Q10

[0054] Weigh 10 kg of wet mycelium with a water content of about 70%, add 10 L of the prepared sodium sulfite aqueous solution, and carry out pulping under vigorous stirring. Then add 50 L of n-hexane and 1 kg of dodecyltrimethylammonium chloride. After adding, continue pulping and extracting for 5 hours at a temperature of 50 °C. Add 1 L of saturated sodium chloride solution, stir well, transfer to a liquid separation device and let it stand. Remove the aqueous phase. The organic phase is washed with saturated sodium chloride aqueous solution (1 L × 3), dried with anhydrous sodium sulfate, filtered, and the solvent is rotary evaporated to obtain an extract. The extract is separated by silica gel column chromatography with a silica gel of 200 - 300 mesh, and the mass of the silica gel is 10 times that of the extract. The eluent is a mixed solvent of acetone and n-hexane with a volume ratio of 1.5%. Combine the eluent containing coenzyme Q10, rotary evaporate the solvent, add anhydrous ethanol at 60 °C to just dissolve it again, control the crystallization temperature to drop by 2 °C per hour, slowly cool to 0 °C and maintain for 3 hours. A large amount of crystals appear, filter, wash, and dry to obtain 103 g of the finished product with a purity of 99.1%.

[0055] Results and analysis:

[0056] Analyze the products of Examples 1 - 6 and Comparative Examples 1 - 3, and the data are shown in Table 1:

[0057] Table 1 Detection data of the products of Examples 1 - 6 and Comparative Examples 1 - 3

[0058] Yield (%) Coenzyme Q10 Content (%) Example 1 72.0％ 99.6％ Example 2 74.0％ 99.5％ Example 3 81.0％ 99.8％ Example 4 76.0％ 99.5％ Example 5 75.5％ 99.6％ Example 6 73.0％ 99.5％ Comparative Example 1 50.6％ 99.0％ Comparative Example 2 52.0％ 98.9％ Comparative Example 3 51.5％ 99.1％

[0059] As can be seen from the above results, the yield of Example 3 of the present invention is the highest, and the content of coenzyme Q10 is also relatively high. Therefore, its reaction conditions are relatively better. Using the reaction conditions of Example 3 and changing the volume ratio of the fermentation broth to the mixed solution of ethyl acetate and petroleum ether in step a, it is found that the yield of coenzyme Q10 decreases significantly, indicating that the volume ratio of the fermentation broth to the mixed solution of ethyl acetate and petroleum ether is a relatively important parameter. Changing the volume ratio of the mixed solution to petroleum ether in step c, it is found that the yield of coenzyme Q10 also decreases significantly, indicating that the volume ratio of the mixed solution to petroleum ether is also a relatively important parameter.

[0060] By comparing with the extraction of coenzyme Q10 in the prior art, it can be seen that the yield of coenzyme Q10 in the prior art is much lower than that of the present invention, and the content of coenzyme Q10 is also significantly lower than that of the present invention.

[0061] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for extracting coenzyme Q10, characterized in that, It includes the following steps: a. Extraction: Slowly add a mixed solution of petroleum ether and ethyl acetate to the coenzyme Q10 fermentation broth at a volume ratio of 5:1 to 3. Among them, the volume ratio of petroleum ether to ethyl acetate in the mixed solution of petroleum ether and ethyl acetate is 10:3 to 5; at the same time, continuously stir at a speed of 200 to 400 rpm for 1 to 3 hours, then let it stand for 20 to 40 minutes, and separate the organic phase and the aqueous phase after stratification; b. Concentrate the organic phase in step a under the conditions of a temperature of 40 to 70 °C and a vacuum degree of -0.10 Mpa to -0.09 Mpa until no condensed water flows out to obtain a concentrated solution. The concentrated solution is repeatedly extracted with ethanol 3 to 5 times. Each time, ethanol is added while stirring at a speed of 300 to 600 rpm. After the addition is completed, stir at 40 to 60 °C for 1 to 3 hours. After stirring, filter to filter out the ethanol extract, then cool down to 8 to 12 °C to obtain a mixed solution containing coenzyme Q10, and mix the mixed solutions obtained each time together; the total addition amount of ethanol is 1 to 3 times the volume of the concentrated solution; c. Slowly add petroleum ether to the mixed solution containing coenzyme Q10 obtained in step b. The volume ratio of petroleum ether to the mixed solution is 1 to 3:

1. During the addition process, continuously stir at a speed of 300 to 800 rpm until it becomes clear, then let it stand for 1 to 3 hours, and then separate the organic phase and the aqueous phase, and control the titer of the aqueous phase at 0.5 g / L; d. Concentrate the organic phase obtained in step c under the conditions of a temperature of 40 to 60 °C and a vacuum degree of -0.10 Mpa to -0.09 Mpa until no condensed water flows out to obtain an extract; e. Dissolve the extract obtained in step d at 40 to 70 °C. After dissolution and clarification, add 0.1 to 0.5% activated carbon, stir at 40 to 70 °C and a speed of 400 to 800 rpm for 30 minutes, filter by suction to obtain a decolorized solution, cool the decolorized solution to 20 to 25 °C at a rate of 3 to 5 °C / h, and filter by suction to obtain the finished product of coenzyme Q10.

2. The extraction method of coenzyme Q10 according to claim 1, characterized in that: In step a, the volume ratio of the coenzyme Q10 fermentation broth to the mixed solution of petroleum ether and ethyl acetate is 5:2; stir at a speed of 300 rpm for 2 hours, and then let it stand for 30 minutes.

3. The extraction method of coenzyme Q10 according to claim 1, characterized in that: In the mixed solution of petroleum ether and ethyl acetate in step a, the volume ratio of petroleum ether to ethyl acetate is 10:

4.

4. The extraction method of coenzyme Q10 according to claim 1, characterized in that: In step b, the organic phase is concentrated under the conditions of a temperature of 55 °C and a vacuum degree of -0.095 Mpa; concentrate until no condensed water flows out to obtain a concentrated solution. The concentrated solution is repeatedly extracted with ethanol 4 times. Each time, ethanol is added while stirring at a speed of 400 rpm. After the addition is completed, stir at 50 °C for 2 hours. After stirring, filter to filter out the ethanol extract, and then cool down to 10 °C; the total addition amount of ethanol is 2 times the volume of the concentrated solution.

5. The extraction method of coenzyme Q10 according to claim 1, characterized in that: In step c, the volume ratio of petroleum ether to the mixed solution is 2:

1. During the addition process, continuously stir at a speed of 500 rpm until it becomes clear, and then let it stand for 2 hours.

6. The extraction method of coenzyme Q10 according to claim 1, wherein: In step d, the organic phase is concentrated under the conditions of a temperature of 50 °C and a vacuum degree of -0.095 Mpa.

7. The extraction method of coenzyme Q10 according to claim 1, characterized in that: In step e, the extract is dissolved at 55°C. After dissolution and clarification, 0.3% activated carbon is added, and the mixture is stirred at 55°C and 600 rpm for 30 min, followed by suction filtration to obtain a decolorized solution. The decolorized solution is cooled to 22.5°C at a rate of 4°C / h and then subjected to suction filtration.