CN107337593B - Preparation method of coenzyme Q10 pure product - Google Patents
Preparation method of coenzyme Q10 pure product Download PDFInfo
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- CN107337593B CN107337593B CN201710606521.5A CN201710606521A CN107337593B CN 107337593 B CN107337593 B CN 107337593B CN 201710606521 A CN201710606521 A CN 201710606521A CN 107337593 B CN107337593 B CN 107337593B
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- C07C46/00—Preparation of quinones
- C07C46/10—Separation; Purification; Stabilisation; Use of additives
Abstract
The invention relates to a method for preparing a coenzyme Q10 pure product, which comprises the following process steps: firstly, carrying out microfiltration and spray drying on coenzyme Q10 fermentation liquor by a ceramic membrane to obtain a crude product, then leaching the crude product by acetone, carrying out phase separation and reduced pressure concentration on the obtained leaching liquor to obtain a coenzyme Q10 extraction concentrated solution, then carrying out petroleum ether extraction, silica gel column chromatography and reduced pressure distillation to concentrate the eluent, finally adding coenzyme Q10 seed crystals for crystallization, and carrying out reduced pressure drying to obtain a coenzyme Q10 pure product. The method adopts ceramic membrane microfiltration and spray drying methods to crush hyphae, and then carries out acetone reflux extraction and ceramic membrane filter separation to break the cell wall of the hyphae and separate the hyphae and enrich coenzyme Q10, and the method can improve the extraction yield by more than 7 percent and is beneficial to improving the economic benefit; the method is simple and suitable for industrial production; the method reduces the discharge amount of waste water and is beneficial to environmental protection treatment.
Description
Technical Field
The invention belongs to the technical field of biological fermentation and extraction, and particularly relates to a preparation method of a pure coenzyme Q10 product.
Background
Coenzyme Q10(CoenzymeQ10), also known as ubiquinone, of formula C59H90O4，The vitamin has molecular weight of 863.4, has quinoid structure, and is a retinoid. Coenzyme Q10 has effects in scavenging free radicals, improving intracellular respiration, and enhancing immunity. Can be clinically used for the adjuvant treatment of cancer; it can be used for treating various heart diseases such as coronary heart disease, heart failure, arrhythmia, acute and chronic viral hepatitis, diabetes, odontoseisis, myasthenia, aplastic anemia, gastroduodenal ulcer, and Parkinson's disease. With the continuous and intensive research on the application of the coenzyme Q10, the coenzyme Q10 is widely applied to the fields of medicines, food additives, health care and the like. At present, the usage amount of the world coenzyme Q10 is increased rapidly year by year, and the market demand is extremely large.
The coenzyme Q10 is produced mainly by chemical synthesis, plant cell culture and microbial fermentation. The microbial fermentation method has the advantages of high process stability, easy control, high product purity, high yield and easy large-scale production. Therefore, the production of coenzyme QIO by microbial fermentation is the mainstream of the development of the production process.
The coenzyme Q10 is yellow or light yellow crystal at room temperature, has a melting point of 48-52 ℃, is unstable to light, is easily decomposed into reddish substances, is insensitive to humidity and temperature, and is relatively stable. Coenzyme Q10 is insoluble in methanol and water, slightly soluble in ethanol, soluble in ether and acetone, and soluble in n-hexane, chloroform, benzene, carbon tetrachloride, etc.
Coenzyme Q10 is an ester-soluble vitamin, and coenzyme QIO produced by microbial fermentation mainly exists in bacterial cells, and is secreted out of the cells in a small amount. In the prior art, for the extraction of coenzyme Q10, the following reports are published:
1. an alcohol-alkali saponification extracting method includes firstly, centrifugally separating a fermentation liquor of coenzyme Q10 to obtain mycelia, adding the mycelia into a system containing pyrogallic acid, ethanol, sodium hydroxide and n-hexane, refluxing and extracting, saponifying and hydrolyzing free fatty acid and fatty acid glyceride in the mycelia to transfer into a water phase, transferring coenzyme Q10 with strong ester solubility into the n-hexane with low polarity, extracting for many times by petroleum ether, transferring the coenzyme Q10 into a petroleum ether-n-hexane organic solvent system, washing the organic solvent extracting solution to be neutral, carrying out vacuum concentration and rotary evaporation to remove the organic solvent, dissolving the organic solvent by absolute ethyl alcohol, filtering, adding water for precipitation and filtering to obtain a coenzyme Q10 pure product. In the process, due to long-time saponification reflux, the transposition of methoxyl in coenzyme Q10 and ethoxyl in ethanol is easily caused, and a mono-or di-ethoxyl derivative is generated, so that the extraction yield and quality of coenzyme Q10 are influenced.
2. The alkalization saponification extraction method comprises the steps of firstly carrying out centrifugal separation on a coenzyme Q10 fermentation liquor to obtain mycelia, refluxing the mycelia with acidic water and alkaline water in sequence, extracting the mycelia with an organic solvent, washing the organic solvent extract to be neutral, carrying out vacuum concentration and rotary evaporation to remove the organic solvent, dissolving the organic solvent with absolute ethyl alcohol, filtering, adding water for precipitation, and filtering to obtain a coenzyme Q10 pure product. In the process, the mono-or di-ethoxy derivatives are not generated, but the emulsification in the extraction process is serious, so that the extraction yield and quality are influenced.
3. An organic solvent stirring and crushing extraction method includes firstly, centrifugally separating a coenzyme Q10 fermentation liquor to obtain mycelium, adding acetone, strongly stirring, centrifuging, taking supernatant, removing the acetone by vacuum concentration and rotary evaporation, adding petroleum ether or n-hexane for multiple extraction to obtain a coenzyme Q10 extracting solution, concentrating the extracting solution to thick silk liquid by a vacuum rotary evaporator, adding absolute ethyl alcohol, freezing, cooling, standing for precipitation, filtering, recovering filtrate, adding water for precipitation, and filtering to obtain a pure coenzyme Q10 product. In the process, the acetone stirring and crushing time is short, the release yield of the coenzyme Q10 is low, the acetone stirring and crushing time is long, and the coenzyme Q10 is easy to deform, so that the product quality is influenced.
4. A grinding-crushing extraction method includes such steps as centrifugal separation of fermented coenzyme Q10 liquid to obtain mycelium, adding acetone, adding quartz sand and antioxidizing agent, quick grinding, centrifugal separation, collecting supernatant, vacuum concentrating, rotary evaporating to remove acetone, adding petroleum ether or n-hexane, extracting to obtain the liquid extract of coenzyme Q10, vacuum concentrating, adding absolute alcohol, freezing, cooling, laying aside, filtering, recovering filtrate, adding water, deposition and filtering to obtain pure coenzyme Q10. In the process, special grinding equipment is needed, grinding force and time have great influence on the release rate of the coenzyme Q10, and in order to prevent oxidation loss of the coenzyme Q10 caused by over grinding, an antioxidant is also needed to be added, so that the production cost and the extraction difficulty are increased.
5. An ultrasonic crushing and extracting method includes firstly, centrifugally separating a fermentation liquor of coenzyme Q10 to obtain mycelia, adding acetone to prepare a bacterial suspension, freezing and cooling, crushing by ultrasonic waves according to certain power, frequency and interval time, centrifuging, taking supernate, removing acetone by vacuum concentration and rotary evaporation, adding petroleum ether or n-hexane for multiple extraction to obtain a coenzyme Q10 extracting solution, concentrating the extracting solution to thick silk liquid by a vacuum rotary evaporator, adding absolute ethyl alcohol, freezing and cooling, standing for precipitation, filtering, recovering filtrate, adding water for precipitation and filtering to obtain a pure coenzyme Q10 product. In the process, a large amount of heat is released in the ultrasonic treatment process, refrigeration cooling equipment is needed, the energy attenuation of ultrasonic waves is fast along with the increase of the thickness of the treatment layer, the treatment capacity is limited, the value of the ultrasonic equipment is high, the production cost of large-scale production and application is high, and the process is not suitable for industrial production.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a coenzyme Q10 pure product, which effectively improves the yield of the coenzyme Q10 and the product quality, has simple process and less solvent loss and can reduce environmental pollution.
The technical scheme adopted for realizing the purpose is as follows:
a method for preparing a coenzyme Q10 pure product is characterized by comprising the following process steps: firstly, carrying out microfiltration and spray drying on coenzyme Q10 fermentation liquor by a ceramic membrane to obtain a crude product, then leaching the crude product by acetone, carrying out phase separation and reduced pressure concentration on the obtained leaching liquor to obtain a coenzyme Q10 extraction concentrated solution, then carrying out petroleum ether extraction, silica gel column chromatography and reduced pressure distillation to concentrate the eluent, finally adding coenzyme Q10 seed crystals for crystallization, and carrying out reduced pressure drying to obtain a coenzyme Q10 pure product.
The ceramic membrane microfiltration refers to filtering by adopting a ceramic membrane with the filter diameter of 0.1-0.45 um, controlling the feeding temperature to be 25-30 ℃, controlling the dialysis ratio to be 1: 1.5-2.0 and controlling the concentration multiple to be 1.8-2.6 times.
The spray drying conditions are that the air inlet temperature is controlled to be 140-160 ℃, and the air outlet temperature is controlled to be 60-70 ℃.
The acetone extraction refers to adding acetone and zeolite into the obtained crude product, slowly heating to 49-52 ℃, refluxing for 2.0-2.5 hr, and filtering with a ceramic filter element filter while the crude product is hot, wherein the addition of the acetone is 3-5 times of the volume of the crude product, and the addition of the zeolite is 0.5-0.8 per mill of the crude product by mass volume.
The leaching solution phase separation means that the leaching solution is placed into a phase separation tank, a saturated NaCL solution with the mass volume of 15-20% is added, the temperature is 28-32 ℃, the stirring is carried out for 30-40 min, the standing is carried out for 20-30 min, supernatant liquid is collected, and reduced pressure distillation is carried out to obtain a coenzyme Q10 extraction concentrated solution.
And the petroleum ether extraction is to add petroleum ether into the acetone extraction concentrated solution obtained by decompression concentration for dissolving, wherein the addition amount is 3-5 times of the volume of the coenzyme Q10 concentrated solution, add equal volume of water, stir for 10-20 min, stand, divide water, and collect a petroleum ether layer.
The silica gel column chromatography and reduced pressure distillation concentration of the eluent refers to that petroleum ether extract containing coenzyme Q10 is subjected to purification and separation in a silica gel column, the eluent is petroleum ether, the feeding speed is 2-5 BV/h, the elution speed is 1.5-3.5 BV/h, the feeding temperature is 25-30 ℃, eluent containing 10 sections of coenzyme Q is collected, and reduced pressure distillation is carried out to obtain a purified coenzyme Q10 concentrated solution.
The crystallization is to add 2-3 times volume of absolute ethyl alcohol into coenzyme Q10 eluent obtained by reduced pressure distillation, heat the mixture to 48-52 ℃, stir the mixture for dissolution, and filter the mixture while the mixture is hot; slowly cooling the filtrate to 28-30 ℃ under stirring, adding coenzyme Q10 seed crystals with the mass volume of 0.02-0.05 per mill, adding deionized water until crystals are separated out, continuously adding deionized water, controlling the temperature to 20-23 ℃, keeping the temperature for 9.0-10.5 hours, filtering, and drying wet crystals under reduced pressure to obtain a pure coenzyme Q10 product, wherein the total water addition amount is 3-5 times of the volume of the coenzyme Q10 concentrated solution.
The technical advantages of the invention are embodied in that:
the invention provides a method for extracting coenzyme Q10 by smashing hypha by a ceramic membrane microfiltration and spray drying method and acetone reflux leaching, wherein the total yield is averagely more than 75%, and is improved by more than 7% compared with the existing yield.
2, the method utilizes the characteristic that the melting point of the coenzyme Q10 is 48-52 ℃, and the coenzyme Q10 crude product is extracted completely by acetone reflux and separation through a ceramic membrane filter, and the impurity separation method is simple and convenient.
3, the invention has simple operation process and strong operability.
4, the invention has less waste water amount and reduces the environmental protection treatment intensity.
Detailed description of the invention
The invention is illustrated below by way of examples, which are to be understood as being illustrative and not limiting. The scope and core content of the invention are to be determined by the claims.
Coenzyme Q10 fermentation broth sources in the following examples: the method is obtained according to the following process flow:
freezing pipe
Bevel spore
First-class seed liquid
Second-stage seed liquid
Fermentation liquor
Coenzyme Q10 is produced by adopting a three-stage fermentation mode, the production strain is rhodopseudomonas palustris, carbon sources in a culture medium mainly comprise starch and glucose, nitrogen sources comprise bean cake powder, corn steep liquor and yeast extract, and the titer is generally about 2600-3000 ug/ml after fermentation culture for 96 hours.
500L of coenzyme Q10 fermentation broth (titer 2900 u/ml) was prepared for use.
Example 1
100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor is taken, and filtered by a ceramic membrane microfiltration membrane (aperture 0.1 um), the feeding temperature is about 25 ℃, the dialysis ratio is 1:1.5, the concentration multiple is 1.8 times, and 139L (titer 2050 u/ml) of membrane pulp is obtained, and the yield is 98.18%.
And (3) spray-drying the membrane pulp, controlling the air inlet temperature to be 140-160 ℃ and the air outlet temperature to be 60-70 ℃, and obtaining 307g of a coenzyme Q10 crude product, wherein the water content is 9.2% and the yield is 97.90%.
Spray drying to obtain crude product, adding into a distiller, adding acetone (921ml) 3 times the weight of the crude product, adding 0.5 ‰ (w/v) zeolite, slowly heating to 49 deg.C, refluxing for 2.0hr, filtering with ceramic filter core filter (pore diameter of 0.5 μm), and repeatedly leaching the residue once again with unchanged leaching condition. Collecting the leaching solution, transferring into a phase separation tank, adding 15% (w/v) NaCl saturated solution, stirring at 28 deg.C for 30min, standing for 20min, collecting the supernatant, and distilling under reduced pressure to obtain 411g of coenzyme Q10 extract concentrate, water content of 34.30%, and yield of 96.83%.
Dissolving the acetone extract concentrate in petroleum ether, adding 3.0 times of coenzyme Q10 concentrate (1233ml), adding equal volume of water, stirring for 10min, standing, separating, collecting petroleum ether layer, purifying and separating with silica gel column at feeding speed of 2BV/h, eluting speed of 1.5BV/h and feeding temperature of 25 deg.C, collecting eluate containing coenzyme Q10, and distilling under reduced pressure to obtain 326g of purified coenzyme Q10 extract concentrate, water content of 20.78% and yield of 95.68%.
Adding 2 times volume of anhydrous ethanol (652ml) into the purified coenzyme Q10 concentrate obtained by distilling and concentrating the eluent under reduced pressure, heating to 48 ℃, stirring for dissolving, and filtering while the solution is hot; slowly cooling the filtrate to 28 ℃ under stirring, adding 0.02 per mill w/v coenzyme Q10 seed crystal, adding deionized water until crystal precipitation, continuously adding deionized water until the total added water amount is 3 times (978ml) of the weight of the purified coenzyme Q10 concentrate, controlling the temperature at 20 ℃, keeping the temperature for 9.0 hours, filtering, and drying the wet crystal under reduced pressure to obtain 217g of a coenzyme Q10 pure product, wherein the water content is 0.12%, the yield is 83.90% and the total yield is 74.74%.
Example 2
100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor is taken, and filtered by a ceramic membrane microfiltration membrane (aperture 0.1 um), the feeding temperature is about 27 ℃, the dialysis ratio is 1:1.7, the concentration multiple is 2.0 times, and membrane pulp 135L (titer 2130 u/ml) is obtained, and the yield is 99.16%.
And (3) spray-drying the membrane pulp, controlling the air inlet temperature to be 140-160 ℃ and the air outlet temperature to be 60-70 ℃, and obtaining 308g of a coenzyme Q10 crude product, wherein the water content is 7.6% and the yield is 98.97%.
Spray drying to obtain crude product, adding into a distiller, adding acetone (1078ml) with a volume of 3.5 times of the weight of the crude product, adding 0.6 ‰ (w/v) zeolite, slowly heating to 50 deg.C, refluxing for 2.2hr, filtering with ceramic filter core filter (aperture of 0.5um), and repeatedly leaching the residue once again with unchanged leaching condition. Collecting the leaching solution, transferring into a phase separation tank, adding 16% (w/v) NaCl saturated solution, stirring at 29 ℃, standing for 25min, collecting supernatant, and distilling under reduced pressure to obtain 408g of coenzyme Q10 extract concentrate, wherein the water content is 32.12%, and the yield is 97.41%.
Dissolving the acetone extraction concentrate in petroleum ether, adding 3.5 times (1430ml) of the weight of the coenzyme Q10 extraction concentrate, adding equal volume of water, stirring for 15min, standing, separating water, collecting a petroleum ether layer, purifying and separating in a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 3BV/h, the elution speed is 2BV/h, the feeding temperature is 27 ℃, collecting the eluent containing 10 sections of coenzyme Q, and distilling under reduced pressure to obtain 331g of purified coenzyme Q10 extraction concentrate, the water content is 19.41 percent, and the yield is 96.25 percent.
Adding anhydrous ethanol (828ml) with the volume of 2.5 times of the weight of the coenzyme Q10 concentrate obtained by distilling and concentrating the eluent under reduced pressure, heating to 49 ℃, stirring for dissolving, and filtering while the solution is hot; slowly cooling the filtrate to 29 ℃ under stirring, adding 0.03 thousandth w/v coenzyme Q10 seed crystal, adding deionized water until crystal precipitation, continuously adding deionized water until the total added water amount is 4 times (1324ml) of the weight of the purified coenzyme Q10 concentrate, controlling the temperature at 22 ℃, preserving the heat for 10.0 hours, filtering, and drying the wet crystal under reduced pressure to obtain 219g of a pure coenzyme Q10 product, wherein the water content is 0.07%, the yield is 84.80%, and the total yield is 75.46%.
Example 3
100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor is taken, and filtered by a ceramic membrane microfiltration membrane (aperture 0.22 um), the feeding temperature is about 29 ℃, the dialysis ratio is 1:1.8, and the concentration multiple is 2.2 times, so that 127L (titer 2230 u/ml) of membrane pulp is obtained, and the yield is 97.87%.
And (3) spray-drying the membrane pulp, controlling the air inlet temperature to be 140-160 ℃ and the air outlet temperature to be 60-70 ℃, and obtaining 301g of a coenzyme Q10 crude product, wherein the water content is 8.9% and the yield is 96.61%.
Spray drying to obtain crude product, adding into a distiller, adding acetone (1204ml) 4 times the weight of the crude product, adding 0.7 ‰ (w/v) zeolite, slowly heating to 51 deg.C, refluxing for 2.0hr, filtering with ceramic filter core filter (aperture of 0.5 μm), and repeatedly leaching the residue once again with unchanged leaching condition. Collecting the leaching liquor, transferring into a phase separation tank, adding 17% (w/v) NaCl saturated solution, stirring at 30 ℃ for 35min, standing for 20-30 min, collecting supernatant, and distilling under reduced pressure to obtain 412g of coenzyme Q10 extract concentrate, 35.55% of water and 96.91% of yield.
Dissolving the acetone extract concentrate in petroleum ether, adding the amount of 1648ml which is 4 times the weight of the coenzyme Q10 concentrate, adding equal volume of water, stirring for 15min, standing, separating water, collecting a petroleum ether layer, purifying and separating in a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 4BV/h, the elution speed is 2.5BV/h, the feeding temperature is 28 ℃, collecting the eluent containing the coenzyme Q10 sections, and distilling under reduced pressure to obtain 327g of purified coenzyme Q10 concentrate, the water content is 21.38 percent, and the yield is 96.75 percent.
Adding 3 times of anhydrous ethanol (981ml) into the purified coenzyme Q10 concentrate obtained by distilling and concentrating the eluent under reduced pressure, heating to 50 ℃, stirring for dissolving, and filtering while the solution is hot; slowly cooling the filtrate to 30 ℃ under stirring, adding 0.03 thousandth w/v coenzyme Q10 seed crystal, adding deionized water until crystal precipitation, continuously adding deionized water until the total added water volume is 5 times (1635 ml) of the volume of the purified coenzyme Q10 concentrate, controlling the temperature at 22 ℃, keeping the temperature for 10.5 hours, filtering, and drying the wet crystal under reduced pressure to obtain 212g of a coenzyme Q10 pure product, wherein the water content is 0.04%, the yield is 82.40%, and the total yield is 73.07%.
Example 4
100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor is taken, and filtered by a ceramic membrane microfiltration membrane (aperture 0.22 um), the feeding temperature is about 30 ℃, the dialysis ratio is 1:1.9, the concentration multiple is 2.4 times, and membrane pulp 121L (titer 2340 u/ml) is obtained, and the yield is 97.50%.
And (3) spray-drying the membrane pulp, controlling the air inlet temperature to be 140-160 ℃ and the air outlet temperature to be 60-70 ℃, and obtaining 302g of a coenzyme Q10 crude product, wherein the water content is 6.4% and the yield is 99.97%.
Spray drying to obtain crude product, adding into a distiller, adding acetone (1360ml) with volume 4.5 times of the weight of the crude product, adding 0.8 ‰ (w/v) zeolite, slowly heating to 51 deg.C, refluxing for 2.5hr, filtering with ceramic filter core filter (pore diameter of 0.5 μm), and repeatedly leaching the residue once again with unchanged leaching condition. Collecting the leaching solution, transferring into a phase separation tank, adding 18% (w/v) NaCl saturated solution, stirring at 30 ℃ for 35min, standing for 25min, collecting the supernatant, and distilling under reduced pressure to obtain coenzyme Q10 extract concentrate 402g, water content of 31.70% and yield of 97.20%.
Dissolving the acetone extract concentrate in petroleum ether, adding 4.5 times of coenzyme Q10 concentrate (1810ml), adding equal volume of water, stirring for 15min, standing, separating water, collecting petroleum ether layer, purifying and separating in silica gel column, eluting with petroleum ether at 5BV/h, 3BV/h and 29 deg.C, collecting eluate containing 10 segments of coenzyme Q, and distilling under reduced pressure to obtain 340g of purified coenzyme Q10 concentrate, 22.53% of water and 95.86% of yield.
Adding 2.5 times volume of anhydrous ethanol (850mL) into the purified coenzyme Q10 concentrate obtained by distilling and concentrating the eluent under reduced pressure, heating to 51 ℃, stirring for dissolving, and filtering while the solution is hot; slowly cooling the filtrate to 29 ℃ under stirring, adding 0.04% w/v coenzyme Q10 seed crystal, adding deionized water until crystal is separated out, continuously adding deionized water until the total water addition amount is 4.5 times (1530 ml) of the volume of the purified coenzyme Q10 concentrate, controlling the temperature at 22 ℃, keeping the temperature for 10.5 hours, filtering, and drying wet crystals under reduced pressure to obtain 216g of a coenzyme Q10 pure product, wherein the water content is 0.09%, the yield is 81.92%, and the total yield is 74.42%.
Example 5
100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor is taken, and filtered by a ceramic membrane microfiltration membrane (aperture 0.45 um), the feeding temperature is about 28 ℃, the dialysis ratio is 1:2.0, and the concentration multiple is 2.6 times, so that 115L (titer 2480 u/ml) of membrane pulp is obtained, and the yield is 98.67%.
And (3) spray-drying the membrane slurry, controlling the air inlet temperature to be 140-160 ℃ and the air outlet temperature to be 60-70 ℃, and obtaining 298g of a coenzyme Q10 crude product, wherein the water content is 5.5% and the yield is 98.41%.
Spray drying to obtain crude product, adding into a distiller, adding acetone (1490ml) 5 times the weight of the crude product, adding 0.7 ‰ (w/v) zeolite, slowly heating to 52 deg.C, refluxing for 2.5hr, filtering with ceramic filter core filter (aperture of 0.5 μm), and repeatedly leaching the residue once again with unchanged leaching condition. Collecting the leaching solution, transferring into a phase separation tank, adding 20% (w/v) NaCl saturated solution, stirring at 32 ℃, standing for 30min, collecting supernatant, and distilling under reduced pressure to obtain 407g of coenzyme Q10 extract concentrate, 32.31% of water and 97.80% of yield.
Dissolving the acetone extract concentrate in petroleum ether, adding 5 times (2035ml) of the weight of the coenzyme Q10 concentrate, adding equal volume of water, stirring for 20min, standing, separating water, collecting petroleum ether layer, purifying and separating in silica gel column, wherein the eluent is petroleum ether, the feeding speed is 5BV/h, the eluting speed is 3.5BV/h, the feeding temperature is 30 ℃, collecting the eluent containing 10 sections of coenzyme Q, and distilling under reduced pressure to obtain 338g of purified coenzyme Q10 concentrate, the water content is 22.05%, and the yield is 95.66%.
Adding 2.5 times volume of anhydrous ethanol (845ml) into the purified coenzyme Q10 concentrate obtained by distilling and concentrating the eluent under reduced pressure, heating to 52 ℃, stirring for dissolving, and filtering while the solution is hot; slowly cooling the filtrate to 30 ℃ under stirring, adding 0.05 thousandth w/v coenzyme Q10 seed crystal, adding deionized water until crystal precipitation, continuously adding deionized water until the total added water amount is 5 times (1690 ml) the weight of the purified coenzyme Q10 concentrate, controlling the temperature at 23 ℃, keeping the temperature for 10.5 hours, filtering, and drying the wet crystal under reduced pressure to obtain 225g of a coenzyme Q10 pure product, wherein the water content is 0.07%, the yield is 85.30%, and the total yield is 77.53%.
Comparative example 1 (alcohol-base saponification extraction method):
taking 100L (2900 u/ml in fermentation unit) of coenzyme Q10 fermentation liquor, performing solid-liquid separation in a centrifuge at the rotation speed of 1200 rpm, after the feed liquid is fed completely, continuously operating the centrifuge for 20 minutes, spin-drying the mushroom dregs to obtain 32kg of wet mushroom dregs, putting the wet mushroom dregs into a 200L reaction kettle, adding 2kg of pyrogallic acid, 50L of alkaline ethanol (PH 7.8-8.0) and 50L of n-hexane, controlling the temperature of the reaction kettle to be about 60 ℃, performing reflux extraction on the coenzyme Q10 in the mushroom dregs, after refluxing for 2 hours, transferring the liquid in the reaction kettle into a phase splitting tank with a stirrer, adding 20L of petroleum ether into the phase splitting tank, controlling the rotation speed of the phase splitting tank to be 40-60 rpm at room temperature, stirring for 20 minutes, standing, layering, separating an upper clear liquid, extracting the coenzyme Q10 remained in a lower layer liquid for three times, adding 10L of petroleum ether extract each time, and summarizing the petroleum ether extract, washing the extracting solution to be neutral, dividing water, collecting a petroleum ether layer, purifying and separating in a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 5BV/h, the eluting speed is 1.5BV/h, the feeding temperature is 30 ℃, collecting 10 sections of eluent containing coenzyme Q, and carrying out reduced pressure distillation to obtain 324g of purified coenzyme Q10 concentrate, the water content is 21.22%, and the yield is 88.02%.
Transferring the concentrate into a 200L dissolving tank, adding 90L of absolute ethyl alcohol, heating to 48-50 ℃, dissolving while stirring, filtering with a ceramic filter element filter (the aperture is 0.5um) while hot, putting the filtrate into a crystallizing tank, slowly cooling the filtrate to 30 ℃ while stirring, adding 0.05 thousandth w/v coenzyme Q10 seed crystal, adding deionized water until crystals are separated out, continuously adding deionized water until the total water addition amount reaches 180L, controlling the temperature to 23-25 ℃, preserving the heat for 10.5 hours, filtering, drying wet crystals under reduced pressure to obtain 198g of a coenzyme Q10 pure product, wherein the water content is 0.06%, the yield is 77.42%, and the total yield is 68.14%.
Comparative example 2 (alkalizing saponification extraction method):
taking 100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor, performing solid-liquid separation in a centrifuge at the rotation speed of 1200 revolutions per minute, after the feed liquid is fed completely, continuously operating the centrifuge for 20 minutes, drying the mushroom dregs to obtain 32kg of wet mushroom dregs, putting the wet mushroom dregs into a 500L reaction kettle, firstly adding 100L of oxalic acid water (PH 3.5-3.8) at the temperature of about 60 ℃, performing reflux extraction on the coenzyme Q10 in the mushroom dregs, refluxing for 3 hours, then adding 150L of liquid caustic soda (5 percent sodium hydroxide), continuously preserving heat and refluxing for 3 hours, after the reflux is finished, transferring the reaction kettle liquid into a phase splitting tank with a stirrer, adding 20L of petroleum ether into the phase splitting tank, controlling the rotation speed at room temperature for 60 revolutions per minute, stirring for 20 minutes, standing for 10 minutes, layering, separating the supernatant, then adding the residual coenzyme Q10 in the lower layer, extracting for three times, adding 10L of petroleum ether each time, the petroleum ether extract is collected.
Adding equal volume of water into the petroleum ether extracting solution, stirring for 15min, standing, separating water, collecting a petroleum ether layer, purifying and separating in a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 5BV/h, the eluting speed is 1.5BV/h, the feeding temperature is 30 ℃, collecting 10 sections of eluent containing coenzyme Q, and carrying out reduced pressure distillation to obtain 318g of purified coenzyme Q10 concentrate, the water content is 22.05 percent, and the yield is 85.48 percent.
Transferring the concentrate into a 200L dissolving tank, adding 90L of absolute ethyl alcohol, heating to 48-50 ℃, dissolving under stirring, filtering with a ceramic filter element filter (the aperture is 0.5um) while hot, entering a crystallizing tank, slowly cooling the filtrate to 30 ℃ under stirring, adding 0.5 per thousand w/v coenzyme Q10 seed crystal, adding deionized water until crystals are separated out, continuously adding deionized water until the total water addition amount is 200L, controlling the temperature at 23 ℃, keeping the temperature for 10.5hr, filtering, and drying wet crystals under reduced pressure to obtain 208g of a coenzyme Q10 pure product, 0.07% of water, 83.85% of yield and 71.67% of total yield.
Comparative example 3 (organic solvent agitation and crushing extraction method):
taking 100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor, putting the fermentation liquor into a centrifuge for solid-liquid separation, controlling the rotating speed at 1200 r/min, after the feed liquid is fed completely, continuously operating the centrifuge for 20min, carrying out spin-drying on the mushroom dregs to obtain 32kg of wet mushroom dregs, putting the wet mushroom dregs into a 2000L stirring tank, adding 700L of acetone, regulating the rotating speed to 1000-1200 r/min, stirring for 5hr, taking supernatant, removing the acetone by using a vacuum rotary evaporator, and concentrating the concentrate to 322g, wherein the water content is 19.68% and the yield is 89.18%.
Transferring the concentrate into a 200L dissolving tank, transferring the liquid in a reaction kettle into a phase splitting tank with a stirrer, adding 20L petroleum ether into the phase splitting tank, controlling the rotating speed at room temperature for 60 r/min, stirring for 20min, standing for 10min, layering, separating out supernatant, extracting the residual coenzyme Q10 in the lower layer liquid for three times, adding 10L petroleum ether into the lower layer liquid each time, collecting the petroleum ether extract, washing the extract to be neutral, dividing into water, collecting the petroleum ether layer, purifying and separating by a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 5BV/h, the eluting speed is 1.5BV/h, the feeding temperature is 30 ℃, collecting the eluent containing 10 segments of coenzyme Q, and carrying out reduced pressure distillation to obtain 275g of the purified coenzyme Q10 concentrate, the water content is 16.72 percent, and the yield is 88.55 percent.
Transferring the concentrate into a 200L dissolving tank, adding 90L anhydrous ethanol, heating to 48-50 deg.C, dissolving under stirring, filtering with a ceramic filter element filter (with pore diameter of 0.5um), and crystallizing in a crystallizing tank. Slowly cooling the filtrate to 30 ℃ under stirring, adding 0.05 thousandth w/v coenzyme Q10 seed crystal, adding deionized water until crystal precipitation, continuously adding deionized water until the total water addition amount reaches 180L, controlling the temperature at 23 ℃, keeping the temperature for 10.5 hours, filtering, and drying wet crystals under reduced pressure to obtain 190g of coenzyme Q10 pure product, wherein the water content is 0.04%, the yield is 82.93%, and the total yield is 65.49%.
Comparative example 4 (grind crush extraction):
taking 100L (fermentation unit 2900 u/ml) of coenzyme Q10 fermentation liquor, putting the fermentation liquor into a centrifuge for solid-liquid separation, controlling the rotating speed at 1200 rpm, after the feed liquid is fed completely, continuously operating the centrifuge for 20 minutes, carrying out spin-drying on the mushroom dregs to obtain 32kg of wet mushroom dregs, adding the wet mushroom dregs into a micro powder grinding device, respectively adding 60L of acetone, 2kg of quartz sand and 0.2kg of sodium thiosulfate, quickly grinding for 1.5-2.0 hr, after the grinding is finished, filtering the feed liquid, taking filtrate, and removing the acetone by vacuum concentration and rotary evaporation to obtain 326g of concentrate, 20.23% of water and 89.67% of yield.
Transferring the concentrate into a 200L dissolving tank, transferring the liquid in a reaction kettle into a phase splitting tank with a stirrer, adding 20L petroleum ether into the phase splitting tank, controlling the rotating speed at room temperature for 60 r/min, stirring for 20min, standing for 10min, layering, separating out supernatant, extracting the residual coenzyme Q10 in the lower layer liquid for three times, adding 10L petroleum ether into the lower layer liquid each time, collecting the petroleum ether extract, washing the extract to be neutral, collecting a petroleum ether layer after water separation, purifying and separating the petroleum ether layer by a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 5BV/h, the eluting speed is 1.5BV/h, the feeding temperature is 30 ℃, collecting the eluent containing 10 segments of coenzyme Q, and carrying out reduced pressure distillation to obtain 290g of the purified coenzyme Q10 concentrate, the water content is 19.28%, and the yield is 90.02%.
Transferring the concentrated solution into a 200L dissolving tank, adding 90L anhydrous ethanol, heating to 48-50 deg.C, dissolving under stirring, filtering with a ceramic filter element filter (with aperture of 0.5um) while hot, and feeding into a crystallizing tank; slowly cooling the filtrate to 30 ℃ under stirring, adding 0.5 thousandth w/v coenzyme Q10 seed crystal, adding deionized water until crystal precipitation, continuously adding deionized water until the total water addition amount reaches 180L, controlling the temperature at 23 ℃, keeping the temperature for 10.5 hours, filtering, and drying wet crystals under reduced pressure to obtain 185g of a coenzyme Q10 pure product, wherein the water content is 0.07%, the yield is 82.02%, and the total yield is 66.21%.
Claims (5)
1. A method for preparing a coenzyme Q10 pure product is characterized by comprising the following process steps: firstly, carrying out microfiltration and spray drying on coenzyme Q10 fermentation liquor by using a ceramic membrane to obtain a crude product, then leaching the crude product by using acetone, carrying out phase separation on the obtained leaching liquor by using a NaCL saturated solution with the mass volume of 15-20%, carrying out reduced pressure concentration to obtain a coenzyme Q10 extraction concentrated solution, then carrying out petroleum ether extraction, silica gel column chromatography, reduced pressure distillation and concentration on the eluent, finally adding coenzyme Q10 seed crystals for crystallization, and carrying out reduced pressure drying to obtain a coenzyme Q10 pure product;
the ceramic membrane microfiltration refers to filtering by adopting a ceramic membrane with the filter diameter of 0.1-0.45 mu m, controlling the feeding temperature to be 25-30 ℃, controlling the dialysis ratio to be 1: 1.5-2.0 and the concentration multiple to be 1.8-2.6 times;
the acetone extraction refers to adding acetone and zeolite into the obtained crude product, slowly heating to 49-52 ℃, refluxing for 2.0-2.5 hr, and filtering with a ceramic filter element filter while the crude product is hot;
the petroleum ether extraction, silica gel column chromatography and reduced pressure distillation of concentrated eluent refer to that the coenzyme Q10 extracted concentrated solution is added with petroleum ether for dissolution, the adding amount is 3-5 times of the volume of the coenzyme Q10 concentrated solution, equal volume of water is added, the mixture is stirred for 10-20 min, the mixture is kept stand, water is separated, a petroleum ether layer is collected, then the petroleum ether layer is fed into a silica gel column for purification and separation, the eluent is petroleum ether, the feeding speed is 2-5 BV/h, the eluting speed is 1.5-3.5 BV/h, the feeding temperature is 25-30 ℃, eluent containing coenzyme Q10 sections is collected, and reduced pressure distillation is carried out, so that the purified coenzyme Q10 concentrated solution is obtained.
2. The method for preparing the pure coenzyme Q10 product according to claim 1, wherein the spray drying conditions are that the inlet air temperature is controlled to be 140-160 ℃ and the outlet air temperature is controlled to be 60-70 ℃.
3. The method for preparing the coenzyme Q10 pure product according to claim 1, wherein the addition amount of acetone is 3-5 times of the volume of the crude product, and the addition amount of zeolite is 0.5-0.8 per mill of the crude product by mass volume.
4. The method for preparing the coenzyme Q10 pure product according to claim 1, wherein the phase separation means that the leaching solution is put into a phase separation tank, added with NaCL saturated liquid with the mass volume of 15-20% and stirred at the temperature of 28-32 ℃ for 30-40 min, kept stand for 20-30 min, and collected with supernatant.
5. The method for producing a purified coenzyme Q10 according to claim 1, wherein the crystallization is carried out by adding 2 to 3 times the volume of absolute ethanol to an eluate of coenzyme Q10 obtained by vacuum distillation, heating to 48 to 50 ℃, stirring for dissolution, and filtering while hot; slowly cooling the filtrate to 28-30 ℃ under stirring, adding coenzyme Q10 seed crystals with the mass volume of 0.02-0.05 per mill, adding deionized water until crystals are separated out, continuously adding deionized water, controlling the temperature to 20-23 ℃, keeping the temperature for 9.0-10.5 hours, filtering, and drying wet crystals under reduced pressure to obtain a pure coenzyme Q10 product, wherein the total water addition amount is 3-5 times of the volume of the coenzyme Q10 concentrated solution.
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