Method for purifying coenzyme Q10 in fermentation thalli

Abstract

The invention provides a method for purifying coenzyme Q10 in fermentation thalli, which comprises the following steps: carrying out extraction treatment on the fermentation thalli containing coenzyme Q10 to obtain a raffinate; the extraction treatment comprises supercritical carbon dioxide extraction and / or subcritical fluid extraction; an extracting solution of the raffinate is obtained, and after the extracting solution is adsorbed through macroporous adsorption resin, a first eluent containing coenzyme Q10 is collected; the first eluent is sequentially subjected to vacuum concentration treatment, normal phase chromatography filler chromatography and crystallization, and the coenzyme Q10 with the purity not lower than 98% is obtained. The method is simple to operate and low in cost, the yield and the purity of the coenzyme Q10 are remarkably improved, the extraction and purification processes are simplified on the whole, the use types and the use quantity of organic solvents are reduced, and the operation complexity and the cost requirement are reduced.

Description

Technical Field

[0001] This invention belongs to the field of bioextraction technology, specifically relating to a method for purifying coenzyme Q10 from fermented bacterial cells. Background Technology

[0002] Coenzyme Q10 (CoQ10), also known as ubiquinone (UQ) or coenzyme Q (CoQ), is a benzoquinone lipid-soluble compound and one of the most important coenzymes in mitochondria. It is a coenzyme present in all eukaryotes that perform aerobic respiration. As an important component of the aerobic respiratory chain in eukaryotic cells, it acts as an activator in cellular metabolism and respiration, promoting metabolic processes in the body. It has multiple benefits, including enhancing non-specific immunity, scavenging free radicals, strengthening antioxidant capacity, stabilizing cell membrane structure, inhibiting apoptosis, delaying aging, and protecting the heart. It is widely used in medicine, dietary supplements, health products, and cosmetics.

[0003] There are three main methods for preparing coenzyme Q10: extraction from animal and plant tissues, chemical synthesis, and microbial fermentation. Among these, microbial coenzyme Q10 production is economical, not limited by raw materials, easy to scale up, and produces products with good activity, making it a promising area for development. However, the composition of microbial fermentation broth is complex, and the extraction and purification of coenzyme Q10 are challenging aspects. The purification process is as follows: after filtration, the fermentation broth is freeze-thawed and broken up; the cells are then extracted by soaking in a hydrophilic organic solvent; the extract is concentrated under reduced pressure, and then extracted again with a hydrophobic organic solvent to separate the organic layer containing coenzyme Q10; the extract is then subjected to silica gel column chromatography, washed with hexane, and eluted with a mixed solvent; the eluent is concentrated, ethanol is added, and the mixture is crystallized and filtered to obtain the coenzyme Q10 product. However, this process suffers from low coenzyme Q10 extraction efficiency due to limitations in cell disruption efficiency; furthermore, it involves a wide variety of organic solvents and is time-consuming. Summary of the Invention

[0004] To address the technical problems of low extraction efficiency, lengthy operation, and complex organic solvent usage in commonly used techniques for coenzyme Q10 extraction, this invention provides a method for purifying coenzyme Q10 from fermentation cells, comprising the following steps:

[0005] Fermented cells containing coenzyme Q10 are extracted to obtain a residue; the extraction process includes supercritical carbon dioxide extraction and / or subcritical fluid extraction.

[0006] Obtain the extract of the residue, and after adsorbing the extract through a macroporous adsorption resin, collect the first eluent containing coenzyme Q10.

[0007] The first eluent was subjected to vacuum concentration, normal phase chromatography, and crystallization to obtain coenzyme Q10 with a purity of not less than 98%.

[0008] Furthermore, in the subcritical fluid extraction process, the ratio of fermentation cells to extractant is 1 kg: 1.0 to 1.6 L, the extraction temperature is 35°C to 55°C, the number of extractions is 2 to 5, and the extraction time for each extraction is 30 to 60 minutes.

[0009] Furthermore, during the supercritical carbon dioxide extraction process, the extraction temperature is 40–50°C, the extraction time is 2–3 h, and the carbon dioxide flow rate is 20–30 L / h.

[0010] Furthermore, the extraction of the residue includes: stirring and extracting the residue with a first solvent, and collecting the extract; the first solvent includes one or more of methanol, ethanol, ethyl acetate, butyl acetate, n-hexane, acetone, n-butanol, and dichloromethane; wherein, when the first solvent is a combination of two solvents, the volume ratio of the two solvents is 10:2-5.

[0011] Furthermore, the step of collecting the first eluent containing coenzyme Q10 after adsorbing the extract through macroporous adsorption resin includes: after adsorbing the extract through the macroporous adsorption resin, prewashing with a second solvent, and then eluting with a third solvent to obtain the eluent containing coenzyme Q10.

[0012] The second solvent is one or more of methanol, ethanol, ethyl acetate, acetone, and isopropanol, and the third solvent is one or more of methanol, ethanol, dichloromethane, ethyl acetate, n-hexane, n-heptane, petroleum ether, and n-pentane.

[0013] Furthermore, the flow rate of the macroporous adsorption resin when eluted with the third solvent is 0.5-3 times column volume / hour.

[0014] Furthermore, the normal phase chromatographic packing material chromatography includes silica gel chromatography.

[0015] Furthermore, during the normal phase chromatography process, the product obtained by the vacuum concentration treatment is chromatographically separated by the normal phase chromatography packing to obtain a second eluent containing coenzyme Q10.

[0016] The volume ratio of the fourth solvent to the fifth solvent in the eluent used in the normal phase chromatography packing process is 1-10:0-2;

[0017] The fourth solvent includes one or more of methanol, ethanol, and isopropanol, and the fifth solvent includes one or more of acetone, ethyl acetate, n-hexane, n-pentane, n-heptane, petroleum ether, dichloromethane, trichloromethane, and toluene.

[0018] Furthermore, during the normal phase chromatography process, the elution flow rate of the eluent is 0.5-2 column volumes per hour.

[0019] Furthermore, the crystallization includes: dissolving the concentrated product of the second eluent in a sixth solvent at a temperature of 40-80°C, and crystallizing at a temperature of -10 to 0°C for 12-48 hours to obtain coenzyme Q10;

[0020] The sixth solvent includes methanol and / or ethanol.

[0021] Compared with the prior art, the present invention has at least the following advantages:

[0022] This invention provides a method for purifying coenzyme Q10 from fermented cells. While ensuring the yield and purity of coenzyme Q10, it simplifies the extraction and purification process, reduces the types and quantities of organic solvents used, and lowers the complexity and cost of operation.

[0023] Specifically, this invention employs subcritical or supercritical carbon dioxide extraction technology to efficiently extract coenzyme Q10 from bacterial cells. This eliminates the need for complex cell disruption steps, resulting in rapid and efficient extraction with high yields. The low-temperature operation is gentle, energy-saving, and environmentally friendly. Furthermore, it simplifies subsequent processes such as separation and concentration, significantly reducing energy consumption and production costs, and effectively improving the extraction efficiency of coenzyme Q10. Compared to traditional methods, subcritical or supercritical carbon dioxide extraction offers higher extraction rates and faster extraction speeds, while avoiding the degradation of coenzyme Q10 activity caused by high temperatures.

[0024] This invention uses macroporous adsorption resin, which has good adsorption performance for coenzyme Q10, simple desorption operation, good stability and high reusability, thus making industrial production operation simple, alleviating environmental pressure, and the solvents used can be easily recycled. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0026] Figure 1This is a schematic flowchart of a method for purifying coenzyme Q10 from fermented cells in one embodiment of the present invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention, as well as the prior art known to those skilled in the art and the description of the invention, may be implemented using any prior art methods, devices, and materials similar to or equivalent to the methods, devices, and materials in the embodiments of the present invention.

[0030] like Figure 1 As shown, this invention provides a method for purifying coenzyme Q10 from fermented bacterial cells, comprising the following steps:

[0031] S1. Fermented cells containing coenzyme Q10 are extracted to obtain a residue; the extraction process includes supercritical carbon dioxide extraction and / or subcritical fluid extraction.

[0032] In this invention, the mass percentage concentration of coenzyme Q10 in the fermentation cells containing coenzyme Q10 is 2.8% to 3.5%.

[0033] In this invention, the fermentation cells containing coenzyme Q10 include Rhodopseudomonas aeruginosa, which can be purchased from CGMCC with accession number CGMCCN0.1.2569.

[0034] In this invention, during the subcritical fluid extraction process, the ratio of fermentation cells to extractant is 1 kg: 1.0 to 1.6 L, the extraction temperature is 35°C to 55°C, the number of extractions is 2 to 5, and the duration of each extraction is 30 to 60 minutes.

[0035] In some embodiments of the present invention, the types of extractants used in the subcritical extraction process include n-butane, propane, and n-hexane.

[0036] In this invention, during the supercritical carbon dioxide extraction process, the extraction temperature is 40-50°C, the extraction time is 2-3 hours, and the carbon dioxide flow rate is 20-30 L / h.

[0037] In some embodiments of the present invention, the supercritical carbon dioxide extraction can be performed 2 to 3 times.

[0038] S2. Obtain the extract of the residue, and after adsorbing the extract through a macroporous adsorption resin, collect the first eluent containing coenzyme Q10.

[0039] In this invention, obtaining the extract of the residue includes: stirring and extracting the residue with a first solvent, and collecting the extract; the first solvent includes one or more of methanol, ethanol, ethyl acetate, butyl acetate, n-hexane, acetone, n-butanol, and dichloromethane; wherein, when the first solvent is a combination of two solvents, the volume ratio of the two solvents is 10:2-5.

[0040] In some embodiments of the present invention, the volume ratio of the first solvent to the raffinate can be 8 to 12:1.

[0041] In this invention, the macroporous adsorption resin can be a nonpolar macroporous adsorption resin.

[0042] In this invention, the step of collecting the first eluent containing coenzyme Q10 after adsorbing the extract through a macroporous adsorption resin comprises: adsorbing the extract through the macroporous adsorption resin, pre-washing with a second solvent, and then eluting with a third solvent to obtain the eluent containing coenzyme Q10; wherein the second solvent is one or more of methanol, ethanol, ethyl acetate, acetone, and isopropanol, and the third solvent is one or more of methanol, ethanol, dichloromethane, ethyl acetate, n-hexane, n-heptane, petroleum ether, and n-pentane.

[0043] Pre-washing is typically used to remove non-target components or impurities adsorbed on the macroporous adsorption resin, employing a relatively mild first solvent or combination of first solvents. Then, a third solvent or combination of third solvents with good solubility for coenzyme Q10 is used to elute the target component (coenzyme Q10) from the macroporous adsorption resin.

[0044] In some more specific embodiments of the present invention, the third solvent may be a solvent combination; for example, the third solvent may be ethyl acetate mixed with dichloromethane, or formic acid mixed with ethyl acetate.

[0045] In some embodiments of the present invention, the flow rate of the macroporous adsorption resin when eluted with the third solvent is 0.5-3 times column volume / hour.

[0046] S3. The first eluent is subjected to vacuum concentration, normal phase chromatography, and crystallization to obtain coenzyme Q10 with a purity of not less than 98%.

[0047] In this invention, the vacuum degree during the vacuum concentration process can be 60-100 Pa, the temperature can be 45-55℃, and the concentration ratio can be 2-10 times.

[0048] In this invention, normal-phase chromatography using silica gel chromatography is employed. When silica gel chromatography is used as the packing material for normal-phase chromatography, combining macroporous adsorption resin with the silica gel chromatography method effectively reduces the purification pressure on silica gel. Compared to silica gel chromatography alone, this invention offers higher silica gel utilization, requires less silica gel, and allows for a higher number of silica gel reuses.

[0049] In this invention, during normal phase chromatography, the product of the vacuum concentration treatment (i.e., the concentrate, hereinafter the same) is chromatographically separated by the normal phase chromatography packing to obtain a second eluent containing coenzyme Q10.

[0050] In some embodiments of the present invention, the eluent used in the normal phase chromatography packing process includes a fourth solvent and a fifth solvent, wherein the volume ratio of the fourth solvent to the fifth solvent is 1-10:0-2.

[0051] In some specific embodiments of the present invention, the concentrate can be dissolved in a fourth solvent first, and then subjected to normal phase chromatography.

[0052] In some more specific embodiments of the present invention, the volume ratio of the fourth solvent to the fifth solvent can be 2-5:1.

[0053] In some embodiments of the present invention, the fourth solvent includes one or more of methanol, ethanol, and isopropanol, and the fifth solvent includes one or more of acetone, ethyl acetate, n-hexane, n-pentane, n-heptane, petroleum ether, dichloromethane, trichloromethane, and toluene.

[0054] In some more specific embodiments of the present invention, the combination of the fourth solvent and the fifth solvent can be: the fourth solvent is one or more of ethanol, isopropanol, and methanol, and the fifth solvent can be one or more of n-hexane and petroleum ether; for example, the combination of the fourth solvent and the fifth solvent can be ethanol and n-hexane, or isopropanol and n-hexane, or methanol and petroleum ether, or methanol and n-hexane.

[0055] The commonly used purification process includes the following steps: after filtration of the fermentation broth, it is freeze-thawed and crushed; the cells are then extracted by soaking in a hydrophilic organic solvent; the extract is concentrated under reduced pressure, and then extracted again with a hydrophobic organic solvent to obtain an organic layer containing coenzyme Q10; the extract is then subjected to silica gel chromatography, washed with hexane, eluted with a mixed solvent, the eluent is concentrated, ethanol is added, crystallized, and filtered to obtain the coenzyme Q10 product. In silica gel chromatography, hexane and another hydrophobic organic solvent are often miscible as eluents, which makes separation difficult and poses significant challenges to subsequent solvent recovery.

[0056] In contrast, this invention, by limiting the types of the fourth and fifth solvents, selecting solvents with significant polarity differences, and precisely controlling their volume ratio, can form an elution system with a clear polarity gradient. This elution system can achieve selective elution based on the polarity differences of different substances, thereby effectively overcoming the separation difficulties present in commonly used techniques.

[0057] In some embodiments of the present invention, the elution flow rate of the eluent during the normal phase chromatography process is 0.5-2 column volumes / hour.

[0058] In this invention, crystallization includes: dissolving the concentrated product of the second eluent in a sixth solvent at a temperature of 40–80°C, and crystallizing at -10–0°C for 12–48 hours to obtain coenzyme Q10. In some embodiments of this invention, crystallization includes: concentrating the second eluent under reduced pressure, dissolving it in a fourth solvent at 40–80°C, crystallizing at -10–0°C for 12–48 hours, then filtering, drying the filter cake, and obtaining coenzyme Q10.

[0059] In some embodiments of the present invention, the vacuum degree of the reduced pressure concentration process during crystallization can be 60-100 Pa, the temperature can be 45-55℃, and the concentration ratio can be 2-10 times.

[0060] In some embodiments of the present invention, the sixth solvent includes methanol and / or ethanol.

[0061] Compared with the prior art, the present invention has at least the following advantages:

[0062] This invention provides a method for purifying coenzyme Q10 from fermented cells. While ensuring the yield and purity of coenzyme Q10, it simplifies the extraction and purification process, reduces the types and quantities of organic solvents used, and lowers the complexity and cost of operation.

[0063] Specifically, this invention employs subcritical or supercritical carbon dioxide extraction technology to efficiently extract coenzyme Q10 from bacterial cells. This eliminates the need for complex cell disruption steps, resulting in rapid and efficient extraction with high yields. The low-temperature operation is gentle, energy-saving, and environmentally friendly. Furthermore, it simplifies subsequent processes such as separation and concentration, significantly reducing energy consumption and production costs, and effectively improving the extraction efficiency of coenzyme Q10. Compared to traditional methods, subcritical or supercritical carbon dioxide extraction offers higher extraction rates and faster extraction speeds, while avoiding the degradation of coenzyme Q10 activity caused by high temperatures.

[0064] This invention uses macroporous adsorption resin, which has good adsorption performance for coenzyme Q10, simple desorption operation, good stability and high reusability, thus making industrial production operation simple, alleviating environmental pressure, and the solvents used can be easily recycled.

[0065] In this invention, the purity of the final coenzyme Q10 product is not less than 98%, and the coenzyme Q10 yield in the purification method of fermented cells provided by this invention is not less than 94%.

[0066] To facilitate a further understanding of the present invention by those skilled in the art, the following examples are provided:

[0067] Example 1

[0068] Take 3 kg of fermentation cell raw material containing 3.3% coenzyme Q10, place it in a supercritical carbon dioxide extraction vessel, and run the supercritical carbon dioxide extraction device to extract the fermentation cell for 2 hours to separate the fermentation cell and the extract residue; extract the fermentation cell again and combine the two extract residues.

[0069] The supercritical fluid extract residue was extracted with 10 times the amount of acetone and stirred. The extract was then directly adsorbed onto a non-polar macroporous adsorption resin, followed by pre-washing with acetone and finally elution with n-heptane at a flow rate of 1 BV / h. The macroporous adsorption resin eluent containing coenzyme Q10 was collected as the first eluent.

[0070] After the first eluent was concentrated under reduced pressure, it was dissolved in ethanol and then chromatographically filtered through normal phase chromatography packing material. The eluent was then eluted with ethanol:n-hexane (volume ratio 4:1) at a flow rate of 1 BV / h, and the second eluent containing coenzyme Q10 was collected.

[0071] After the second eluent was concentrated, it was dissolved in ethanol at 60°C, then placed at -5°C for 36 hours to crystallize. After low-temperature filtration and drying, 95.5g of coenzyme Q10 was finally obtained, with a content of 99.1% and a yield of 95.6%.

[0072] Example 2

[0073] Take 3 kg of fermentation cell raw material containing 3.3% coenzyme Q10, put it in a filter cloth bag, place it in a subcritical extraction vessel, introduce 3 L of subcritical fluid n-butane, extract 3 times at 45℃ for 45 min each time, and collect the raffinate.

[0074] The residue from subcritical extraction was extracted with 10 times the amount of ethyl acetate by stirring. The extract was then directly adsorbed onto a non-polar macroporous adsorption resin, prewashed with ethyl acetate, and finally eluted with ethyl acetate:dichloromethane (volume ratio 2:3) at a flow rate of 1 BV / h. The macroporous adsorption resin eluent containing coenzyme Q10 was collected, which is the first eluent.

[0075] After the first eluent was concentrated under reduced pressure, it was dissolved in methanol and then precipitated by normal phase chromatography. The eluent was then eluted with methanol:petroleum ether (volume ratio 2:1) at a flow rate of 2 BV / h, and the second eluent containing coenzyme Q10 was collected.

[0076] After the second eluent was concentrated, it was dissolved in ethanol at 60°C, then placed at -5°C for 36 hours to crystallize. After low-temperature filtration and drying, 94.9g of coenzyme Q10 was finally obtained, with a content of 98.8% and a yield of 94.7%.

[0077] Example 3

[0078] Take 3 kg of fermentation cell raw material containing 3.3% coenzyme Q10, place it in a supercritical carbon dioxide extraction vessel, run the supercritical carbon dioxide extraction device to extract the fermentation cell for 2 hours, separate the cell and the extract residue, extract the cell again, and combine the two extract residues.

[0079] The residue from supercritical fluid extraction was extracted with 10 times its volume of ethanol under stirring. The extract was then directly adsorbed onto a non-polar macroporous adsorption resin, followed by pre-washing with ethanol and finally elution with n-hexane at a flow rate of 1.5 BV / h. The macroporous adsorption resin eluent containing coenzyme Q10 was collected as the first eluent.

[0080] After the first eluent was concentrated under reduced pressure, it was dissolved in isopropanol and then chromatographically filtered through normal phase chromatography packing material. The eluent was then eluted with isopropanol:n-hexane (volume ratio 4:1) at a flow rate of 1 BV / h, and the second eluent containing coenzyme Q10 was collected.

[0081] After the second eluent was concentrated, it was dissolved in methanol at 60°C, then placed at -5°C for 36 hours to crystallize. After low-temperature filtration and drying, 95.2g of coenzyme Q10 was finally obtained, with a content of 98.5% and a yield of 94.7%.

[0082] Example 4

[0083] Take 3 kg of fermentation cell raw material containing 3.3% coenzyme Q10, put it in a filter cloth bag, place it in a subcritical extraction vessel, introduce 3 L of subcritical fluid n-butane, extract 3 times at 45℃ for 45 min each time, and collect the raffinate.

[0084] The residue from subcritical extraction was extracted with 10 times its volume of methanol by stirring. The extract was then directly adsorbed onto a nonpolar macroporous adsorption resin, followed by pre-washing with methanol, and finally eluted with methanol:ethyl acetate (volume ratio 1:6) at a flow rate of 0.5 BV / h. The macroporous adsorption resin eluent containing coenzyme Q10 was collected, i.e., the first eluent.

[0085] After the first eluent was concentrated under reduced pressure, it was dissolved in ethanol and then subjected to reversed-phase chromatography. The eluent was then eluted with ethanol:n-hexane (volume ratio 4:1) at a flow rate of 1 BV / h, and the second eluent containing coenzyme Q10 was collected.

[0086] The second eluent was concentrated and dissolved in methanol at 60°C, then allowed to crystallize at -5°C for 36 hours. The crystals were then filtered under low temperature and dried. Finally, 94.9 g of coenzyme Q10 was obtained, with a purity of 98.8% and a yield of 94.7%.

[0087] Comparative Example 1

[0088] (1) Disruption: 5L of fermentation broth was obtained by fermenting Rhodococcus-like bacteria (purchased from CGMCC, preservation number CGMCCN0.1.2569). After filtration, the filter cake was collected to obtain 782g of wet bacterial cells. 500g of the collected wet bacterial cells were taken and resuspended in 1.5mol / L hydrochloric acid solution with a volume equivalent to 5 times the volume of the wet bacterial cells. The bacterial cell wall was broken by ultrasonic disruption. The ultrasonic disruption conditions were 500W power, 0.6 frequency, and ultrasonic disruption was performed twice for 10min each time to obtain a bacterial suspension.

[0089] (2) Extraction: Add NaOH solution to adjust the pH of the bacterial suspension obtained in step (1) to 7.0, and extract twice with a mixed solution of ethyl acetate: petroleum ether 7:93. The volume of organic solvent added each time is 2:1 of bacterial suspension volume. Stir and extract for 2 hours each time. The extraction temperature is 40℃. Let stand and cool to separate the layers. Combine the organic phases to obtain the crude extract of coenzyme Q10.

[0090] Tests showed that the crude extract contained 67.2% coenzyme Q10, with an extraction rate of 92.0%.

[0091] After dehydration, the crude extract was ready for silica gel column chromatography. 50g of dried silica gel was packed into a column and equilibrated. 1000ml of the crude extract was injected into the equilibrated silica gel column at a flow rate of 1 BV / h. After loading, the column was washed with 1 column volume of petroleum ether, followed by elution with petroleum ether containing 7% ethyl acetate at a flow rate of 1 BV / h. 635ml of eluent was collected, and the coenzyme Q10 content was found to be 92.8%, with an extraction rate of 94.4%. The eluent was concentrated under reduced pressure to a coenzyme Q10:organic solvent ratio of 1:10. After cooling the concentrate to 30°C, 1wt% of the coenzyme Q10 content in the concentrate was added as seed crystals. The mixture was stirred at 50 rpm and kept at this temperature for 30 min. The temperature was then lowered to 20°C at a rate of 6°C / h and maintained for half an hour before filtration. The resulting crystals were washed and dried to obtain the purified coenzyme Q10 product. The obtained coenzyme Q10 product has a purity of 98.4% and a total yield of 82.7% from bacterial cells to product.

[0092] According to Comparative Example 1, the purity of the coenzyme Q10 product obtained through crushing, extraction, chromatography, and crystallization was 98.4%, and the total yield was only 82.7%. Furthermore, the ultrasonic crushing of the collected bacterial cells in Comparative Example 1 not only limited the number of cells crushed per batch but also damaged the structure of coenzyme Q10, resulting in a low yield and further restricting large-scale industrial production.

[0093] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for purifying coenzyme Q10 from fermented bacterial cells, characterized in that, Including the following steps: Fermented cells containing coenzyme Q10 are extracted to obtain a residue; the extraction process includes supercritical carbon dioxide extraction and / or subcritical fluid extraction. Obtain the extract of the residue, and after adsorbing the extract through a macroporous adsorption resin, collect the first eluent containing coenzyme Q10. The first eluent was subjected to vacuum concentration, normal phase chromatography, and crystallization to obtain coenzyme Q10 with a purity of not less than 98%.

2. The purification method according to claim 1, characterized in that, During the subcritical fluid extraction process, the ratio of fermentation cells to extractant is 1 kg: 1.0 to 1.6 L, the extraction temperature is 35℃ to 55℃, the number of extractions is 2 to 5, and the extraction time for each extraction is 30 min to 60 min.

3. The purification method according to claim 1, characterized in that, During the supercritical carbon dioxide extraction process, the extraction temperature is 40-50℃, the extraction time is 2-3h, and the carbon dioxide flow rate is 20-30L / h.

4. The purification method according to claim 1, characterized in that, The extraction solution for obtaining the residue comprises: stirring and extracting the residue with a first solvent, and collecting the extract; the first solvent comprises one or more of methanol, ethanol, ethyl acetate, butyl acetate, n-hexane, acetone, n-butanol, and dichloromethane; wherein, when the first solvent is a combination of two solvents, the volume ratio of the two solvents is 10:2-5.

5. The purification method according to any one of claims 1 to 4, characterized in that, The step of collecting the first eluent containing coenzyme Q10 after adsorbing the extract through a macroporous adsorption resin includes: adsorbing the extract through the macroporous adsorption resin, prewashing with a second solvent, and then eluting with a third solvent to obtain the eluent containing coenzyme Q10. The second solvent is one or more of methanol, ethanol, ethyl acetate, acetone, and isopropanol, and the third solvent is one or more of methanol, ethanol, dichloromethane, ethyl acetate, n-hexane, n-heptane, petroleum ether, and n-pentane.

6. The purification method according to claim 5, characterized in that, The flow rate of the macroporous adsorption resin when eluted with the third solvent is 0.5-3 times column volume / hour.

7. The purification method according to any one of claims 1, characterized in that, The normal phase chromatographic packing material chromatography includes silica gel chromatography.

8. The purification method according to any one of claims 1 to 7, characterized in that, During the normal phase chromatography process, the product of the vacuum concentration treatment is chromatographically purified by the normal phase chromatography packing to obtain a second eluent containing coenzyme Q10. The volume ratio of the fourth solvent to the fifth solvent in the eluent used in the normal phase chromatography packing process is 1-10:0-2; The fourth solvent includes one or more of methanol, ethanol, and isopropanol, and the fifth solvent includes one or more of acetone, ethyl acetate, n-hexane, n-pentane, n-heptane, petroleum ether, dichloromethane, trichloromethane, and toluene.

9. The purification method according to claim 8, characterized in that, During the normal phase chromatography process, the elution flow rate of the eluent is 0.5 to 2 column volumes per hour.

10. The purification method according to claim 7, characterized in that, The crystallization process includes: dissolving the concentrated product of the second eluent in a sixth solvent at a temperature of 40–80°C, and crystallizing at a temperature of -10–0°C for 12–48 hours to obtain coenzyme Q10; The sixth solvent includes methanol and / or ethanol.

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