CN108017530B - Method for continuously separating coenzyme Q10 from mushroom dregs - Google Patents

Abstract

The invention discloses a method for continuously separating coenzyme Q10 from bacterial residue, comprising: (1) dissolving a crude extract of coenzyme Q10 in a non-polar organic solvent to prepare a feed solution; (2) dissolving the feed solution and The eluent is continuously passed into the simulated moving bed chromatography system, and the raffinate is continuously collected from the raffinate port of the simulated moving bed chromatography system; (3) the raffinate obtained in step (2) is concentrated under reduced pressure and redissolved, and then passed through After crystallization, filtration and drying, a fine coenzyme Q10 with a purity greater than 98% is obtained. The method has the characteristics of high yield, high recovery rate, low solvent consumption and continuous production, and is suitable for industrialized large-scale popularization and application.

Description

A kind of method for continuously separating coenzyme Q10 from bacterial residue

technical field

The invention belongs to the technical field of chemical separation, in particular to a method for continuously separating coenzyme Q10 from bacterial residue.

Background technique

Coenzyme Q10, also known as ubiquinone, is a fat-soluble quinone compound that mainly exists in the heart, liver and kidney cells of animals. Its chemical name is 2-(3,7,11,15,19,23, 27,31,35,39-Decamethyl-2,6,10,14,18,22,26,30,34,38-tetradecenyl)-5,6-dimethoxy-3- Methyl-p-benzoquinone, the molecular formula is C ₅₉ H ₉₀ O ₄ , and the molecular weight is 863.34. Coenzyme Q10 has important physiological functions such as scavenging free radicals, improving intracellular respiration and enhancing immunity. Its market demand is constantly expanding, and it is widely used in the fields of medicine, cosmetics and food additives.

The production methods of coenzyme Q10 mainly include chemical synthesis method, animal and plant cell culture method and microbial fermentation method. Among them, microbial fermentation method has the advantages of high process stability, easy large-scale production, simple operation, high biological activity and easy absorption of the product. , is the current research focus of coenzyme Q10 production. The fermentation broth obtained by the microbial fermentation method is centrifuged, filtered, freeze-dried and pulverized to obtain bacterial residue, and the crude extract of coenzyme Q10 is obtained by extraction, and further purification is carried out to obtain a high-purity coenzyme Q10 product. Existing extraction methods include solvent extraction, saponification, supercritical fluid extraction, and then combined with silica gel column chromatography, recrystallization and other techniques to further purify the crude product of coenzyme Q10. However, coenzyme Q10 crude extracts mainly contain coenzyme Q homologues with different numbers of isopentenene units on the side chain, which are difficult to separate.

CN103819326A discloses a method for purifying coenzyme Q10 by successively using ultrasonic breaking, organic solvent extraction, silica gel column chromatography and crystallization. CN101429108A discloses a method for purifying coenzyme Q10 by sequential extraction with absolute ethanol, extraction with water and n-hexane, silica gel column chromatography and crystallization. CN102391092A discloses a method of extracting bacterial residues with supercritical carbon dioxide, followed by silica gel column chromatography and crystallization to obtain coenzyme Q10 with a purity greater than 99.5%. CN101987815A discloses a method combining adsorption resin and silica gel column chromatography to prepare coenzyme Q10 with a purity greater than 98%. These methods all require the use of silica gel column chromatography. Although high-purity coenzyme Q10 can be obtained, the above methods are all intermittent operation processes, and there are problems such as a large amount of organic solvent, a small amount of preparation, and a low utilization rate of silica gel, so the process is not economical. .

Simulated moving bed chromatography is currently the most promising preparative chromatography technology. The four inlets and outlets of eluent inlet, feed liquid inlet, extraction liquid outlet, and raffinate outlet divide all chromatographic columns into four zones with different flow rates. respectively undertake different functions. It uses the timing switching of four inlet and outlet materials to simulate the countercurrent movement of the eluent and the stationary phase, so as to realize the continuity of the inlet and outlet materials. The mixed solution containing the strong adsorption component and the eluent is continuously collected at the outlet of the extract, and the mixed solution containing the weak adsorption component and the eluent is continuously collected at the outlet of the raffinate. On the one hand, this operation allows continuous sample injection and thus high productivity; on the other hand, due to the recycling of the eluent, the solvent consumption is low, which can reduce the cost of large-scale preparation. By designing suitable flow rates for each zone, high-purity target components can be obtained.

SUMMARY OF THE INVENTION

In view of the shortcomings of the above methods, the present invention provides a method for continuously separating coenzyme Q10 from bacterial residues, which has simple process, large product preparation, high purity, low solvent consumption and low production cost.

In the present invention, coenzyme Q10 is separated from bacterial residue by a combined method of simulated moving bed chromatography and crystallization. The method uses the filler rich in polar groups on the surface as the stationary phase, adjusts the type and ratio of the eluent, and designs the appropriate flow rate and flow rate in each zone. Switching time, the use of simulated moving bed chromatography can achieve continuous separation of coenzyme Q10 and impurities, and then the separated coenzyme Q10 is purified by crystallization.

A method for continuously separating coenzyme Q10 from bacterial residue, comprising the following steps:

(1) dissolving the crude extract of coenzyme Q10 in a non-polar organic solvent to prepare a feed solution;

(2) the feed liquid and the eluent are continuously passed into the simulated moving bed chromatography system, and the raffinate is continuously collected from the raffinate port of the simulated moving bed chromatography system;

The simulated moving bed chromatographic system is composed of 4-32 chromatographic columns equipped with a stationary phase, and has four zones in total, and each zone is formed by connecting 1-8 chromatographic columns in series. The zones can be connected in series or disconnected, and the isocratic operation mode or the gradient operation mode can be adopted. The operating parameters such as the flow rate, switching time, switching times and column temperature of each zone are preset, and the feed liquid and eluent are continuously pumped. After the system reaches a steady state, the raffinate rich in coenzyme Q10 is continuously collected from the raffinate port.

Before the simulated moving bed chromatographic system runs, the stationary phase particles are packed into the chromatographic column by wet packing method, and the packing solvent is n-hexane or petroleum ether. Symmetry experiments were carried out to ensure that the performance indicators of each chromatographic column were consistent. According to the "triangle theory", the separable zone is preliminarily determined, and the flow rate and switching time of each zone are adjusted until the coenzyme Q10 and impurities are completely separated.

(3) After the raffinate obtained in step (2) is concentrated to remove the solvent, an organic solvent is added at 20 to 60 ° C until the solid is just dissolved, then the temperature is lowered to -5 to 5 ° C, and the crystallization is cooled for 12 to 36 hours and filtered. , the filter cake is washed with water and then dried in vacuum at 20-40 DEG C to obtain a fine coenzyme Q10 with a purity of more than 98%.

In the described step (1), the crude extract of coenzyme Q10 is extracted from the bacterial residue obtained by microbial fermentation, and the specific reference can be made to the method described in the patent applications with publication numbers of CN101314782A, CN101619330A or CN105886562A to cultivate the bacterial species, and filter the fermentation broth. , after drying and pulverizing, the bacterial residue is obtained; the extraction method for extracting the crude extract of Coenzyme Q10 from the bacterial residue is percolation extraction method, organic solvent extraction method, alcohol-alkali saponification method or supercritical fluid extraction method, which can be specifically disclosed as follows No. CN106146278A, CN101381747A, CN102391092A or CN104694613A.

The non-polar organic solvent is one or a mixture of any two in n-hexane, cyclohexane, n-heptane, n-octane and petroleum ether. These hydrophobic organic solvents have high solubility for CoQ10 crude extract.

The total concentration of the feed solution is 5-500 g/L, more preferably 50-300 g/L. If the feed concentration is too low, the production capacity will be reduced, and the process economy will be reduced; if the feed concentration is too high, the complete separation zone will be significantly reduced, the difficulty of designing operating conditions will increase, and the separation difficulty will increase.

The stationary phase of the simulated moving bed chromatography system is polar macroporous adsorption resin, ion exchange resin, silica gel or alumina. These stationary phases are rich in polar groups such as hydroxyl groups, which can form hydrogen bonds with carbon groups in coenzyme Q-like homologues, and identify small structural differences between homologues according to the size of the hydrogen bonding force.

The stationary phase is spherical particles with uniform particle size, uniform pore size and high mechanical strength.

The particle size of the stationary phase is controlled to be 5-200 μm, more preferably 10-100 μm. If the particle size is too large, the column efficiency will be reduced, which is not conducive to the separation of coenzyme Q10 and impurities; if the particle size is too small, the column pressure will be too high, which is not conducive to operation.

The pore size of the stationary phase is controlled to be 5-100 nm, more preferably 10-50 nm. If the pore size of the filler is too small, the coenzyme Q10 molecules will not easily enter the pore, reducing the separation effect; if the pore size of the filler is too large, the diffusion in the pores will be slow and the mass transfer resistance will be large.

Described eluent is n-hexane, cyclohexane, n-heptane, n-octane, petroleum ether, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide and carbon atoms One or a mixture of any two of the monohydric alcohols numbered from 1 to 4. A further preferred eluent is a mixture of n-hexane and ethyl acetate.

Preferably, the volume percentage of ethyl acetate in the eluent is 1-20%. If the proportion of ethyl acetate is too high, the polarity of the mobile phase will be strong, and the separation of coenzyme Q10 and impurities will be poor; if the proportion of ethyl acetate is too low, the polarity of the mobile phase will be weak, and the retention time of coenzyme Q10 in the system will be too long. , the system is difficult to achieve stability.

The size of the chromatographic column of the simulated moving bed chromatography system is 5-500 mm in diameter and 50-1000 mm in length, more preferably 10-100 mm in diameter and 100-500 mm in length. If the size of the chromatographic column is too small, the production capacity will be low; if the size of the chromatographic column is too large, the packing of the filler will be difficult, the wall effect will be obvious, and the separation ability of the chromatographic column will decrease.

The operating parameters of the simulated moving bed chromatography system are controlled as follows: the flow rate of the eluent is 1-1000mL/min, the flow rate of the feed liquid is 1-100mL/min, the flow rate of the extract is 1-100mL/min, and the flow rate of the raffinate is 1-100mL. /min, the switching time is 1～50min. The switching time is further preferably 3-10 min. If the switching time is too short, the switching valve is easily damaged; if the switching time is too long, the system is difficult to reach a steady state.

The separation temperature of the simulated moving bed chromatography system is 0-60°C, more preferably 20-50°C. If the temperature is too low, the solubility of coenzyme Q10 in the solvent will be significantly reduced, and the concentration of the feed solution will be limited; if the temperature is too high, coenzyme Q10 will be easily oxidized and deteriorated during the separation process.

The organic solvent is one or a mixture of any two of monohydric alcohol with 1-4 carbon atoms, acetonitrile, acetone, ethyl acetate, n-hexane and n-heptane.

The volume-to-mass ratio of the added amount of the organic solvent and the solid obtained after the raffinate is concentrated is 20-80 L/kg.

Compared with the prior art, the present invention has the following advantages:

1. Adopt continuous chromatography technology to improve the utilization rate of stationary phase and reduce the consumption of stationary phase.

2. Realize the continuous production of coenzyme Q10, the production process is fully automated, the labor intensity is low, and the production cost is low.

3. The purity of coenzyme Q10 obtained by the purification method of the present invention reaches more than 98%, the yield is high, and the solvent consumption is low, which is suitable for large-scale popularization and application.

Description of drawings

FIG. 1 is a liquid chromatogram of the coenzyme Q10 product in Example 1 of the present invention.

Detailed ways

In order to further understand the present invention, a method for continuously separating coenzyme Q10 from bacterial residues provided by the present invention will be specifically described below with reference to the examples, but the present invention is not limited to these examples, and those skilled in the art are in the core guiding ideology of the present invention. The non-essential improvements and adjustments made below still belong to the protection scope of the present invention.

In the following examples, the simulated moving bed device adopts the German CESP C9116 (Knoll, Germany), which is equipped with a multi-port rotary valve and can be connected to a maximum of 16 chromatographic columns. 4 sets of S-100 liquid phase pumps, in which the flow rate of the feed pump is 0-10mL/min, and the flow rate of the eluent pump, extraction liquid pump and raffinate pump is 0-50mL/min. The eluent is injected between zone 4 and zone 1, the feed liquid is injected between zone 2 and zone 3, coenzyme Q10 is collected at the raffinate outlet between zone 3 and zone 4, and impurity 10 is injected between zones 1 and 2 The extraction liquid outlet between the zones is collected. Every other switching time (note: this switching time is adjustable), the column switches one position in the opposite direction of the eluent flow.

The determination of coenzyme Q10 content in the following examples of the present invention is carried out according to the method described in the Chinese Pharmacopoeia, and the analytical conditions of liquid chromatography are: Waters Atlantis T3 analytical column (250mm×4.6mm, 5μm), methanol-anhydrous Ethanol (1:1) was used as the mobile phase, the flow rate was 1 mL/min, the injection volume was 20 μL, and the detector was an ultraviolet detector with a detection wavelength of 275 nm.

The calculation method for the purity of coenzyme Q10 is as follows: take a small sample of the collected coenzyme Q10 product and dissolve it in absolute ethanol and dilute it to make a solution containing about 0.2 mg of coenzyme Q10 per 1 mL. The external standard method is used to calculate its purity by peak area.

The calculation method of purity of the present invention and recovery rate is as follows:

Purity = mass of coenzyme Q10 in product ÷ total mass of product × 100%

Recovery rate = mass of coenzyme Q10 in product ÷ mass of coenzyme Q10 in raw material × 100%.

Example 1

The crude extract of coenzyme Q10 was dissolved in n-hexane to prepare a feed solution with a solid concentration of 50 g/L, wherein the content of coenzyme Q10 was about 62.3%.

The simulated moving bed is equipped with 8 chromatographic columns, the size of which is 1cm×25cm; the stationary phase is silica gel with a particle size of 45μm and a pore size of 10nm; the eluent is a mixture of n-hexane and ethyl acetate, wherein the volume percentage of ethyl acetate is 10 %; the operating temperature is 30°C; the operating parameters are optimized as follows: the flow rate of the eluent is 16mL/min, the flow rate of the feed liquid is 2mL/min, the flow rate of the extract liquid is 9.5mL/min, the flow rate of the raffinate is 8.5mL/min, and the switching time is 5min . After 32 consecutive switches, the system reached equilibrium, and the coenzyme Q10-rich solution was collected from the raffinate outlet. Analysis showed that the content of coenzyme Q10 in the raffinate was 95.6%.

Concentrate the raffinate into a solid, add ethanol at 50 °C until it is just completely dissolved, the solid-to-liquid ratio is 1:50, gradually reduce the temperature to 5 °C, cool and crystallize for 24 hours, filter, and put it in a vacuum drying box at 30 °C for drying for 24 hours Get Coenzyme Q10 boutique. The purity of the coenzyme Q10 product obtained by liquid chromatography was 99.2%, and the recovery rate of the whole process was 95.5%.

Example 2

The crude extract of coenzyme Q10 was dissolved in n-hexane to prepare a feed solution with a solid concentration of 100 g/L, wherein the content of coenzyme Q10 was about 68.7%.

The simulated moving bed is equipped with 8 chromatographic columns, the size of which is 1cm×25cm; the stationary phase is silica gel with a particle size of 20μm and a pore size of 22nm; the eluent is a mixture of n-hexane and ethanol, in which the volume percentage of ethanol is 5%; the operating temperature 30°C; the operating parameters were optimized and determined to be: eluent flow rate 15.4mL/min, feed liquid flow rate 1.5mL/min, extract liquid flow rate 8.9mL/min, raffinate flow rate 8.0mL/min, switching time 4min. After 32 consecutive switches, the system reached equilibrium, and the coenzyme Q10-rich solution was collected from the raffinate outlet. Analysis showed that the content of coenzyme Q10 in the raffinate was 96.5%.

The raffinate was concentrated into a solid, and ethyl acetate was added at 30°C until it was just completely dissolved. The solid-to-liquid ratio was 1:20. The temperature was gradually reduced to 0°C. After cooling and crystallizing for 24 hours, it was filtered and placed in a vacuum drying oven at 30°C. Dry for 24h to obtain Coenzyme Q10 fines. The purity of the coenzyme Q10 product obtained by liquid chromatography was 99.2%, and the recovery rate of the whole process was 95.9%.

Example 3

The crude extract of coenzyme Q10 was dissolved in n-hexane to prepare a feed solution with a solid concentration of 80 g/L, wherein the content of coenzyme Q10 was about 70.3%.

The simulated moving bed is equipped with 16 chromatographic columns, with a size of 1cm×15cm; the stationary phase is 200-300 mesh neutral alumina; the eluent is a mixture of n-hexane and ethyl acetate, wherein the volume percentage of ethyl acetate is 10% ; The operating temperature is 40°C; the operating parameters are optimized as follows: the flow rate of the eluent is 3mL/min, the flow rate of the feed liquid is 1.5mL/min, the flow rate of the extract liquid is 2.2mL/min, the flow rate of the raffinate is 2.3mL/min, and the switching time is 10min. . After 48 consecutive switches, the system reached equilibrium and a solution rich in CoQ10 was collected from the raffinate outlet. Analysis showed that the content of coenzyme Q10 in the raffinate was 95.2%.

Concentrate the raffinate into a solid, add ethanol at 50 °C until it is just completely dissolved, the solid-liquid ratio is 1:50, gradually reduce the temperature to 5 °C, cool and crystallize for 12 hours, filter, and put it in a vacuum drying box at 35 °C for drying for 12 hours Get Coenzyme Q10 boutique. The purity of the coenzyme Q10 product obtained by liquid chromatography was 98.2%, and the recovery rate of the whole process was 94.5%.

Example 4

The crude extract of coenzyme Q10 was dissolved in cyclohexane to prepare a feed solution with a solid concentration of 150 g/L, wherein the content of coenzyme Q10 was about 72.7%.

The simulated moving bed is equipped with 16 chromatographic columns, the size is 1cm×15cm; the stationary phase is neutral alumina of 200-300 mesh; the eluent is a mixture of cyclohexane and methanol, wherein the volume percentage of methanol is 5%; the operating temperature 20°C; the operating parameters are optimized and determined as: eluent flow rate 7mL/min, feed liquid flow rate 2mL/min, extract liquid flow rate 4.8mL/min, raffinate flow rate 4.2mL/min, switching time 5min. After 48 consecutive switches, the system reached equilibrium and a solution rich in CoQ10 was collected from the raffinate outlet. Analysis showed that the content of coenzyme Q10 in the raffinate was 95.0%.

Concentrate the raffinate into a solid, add acetone at 30 °C until it is just completely dissolved, the solid-liquid ratio is 1:20, gradually reduce the temperature to 5 °C, cool and crystallize for 24 hours, filter, and put it in a vacuum drying box at 35 °C for drying for 12 hours Get Coenzyme Q10 boutique. The purity of the coenzyme Q10 product obtained by liquid chromatography was 98.0%, and the recovery rate of the whole process was 93.8%.

Claims (5)

1. a method for continuously separating coenzyme Q10 from bacterial residue, comprising the following steps: (1) Dissolving the crude extract of coenzyme Q10 in a non-polar organic solvent to prepare a feed solution; the total concentration of the feed solution is 5~500g/L; (2) The feed liquid and the eluent are continuously passed into the simulated moving bed chromatography system, and the raffinate is continuously collected from the raffinate port of the simulated moving bed chromatography system; the operating parameters of the simulated moving bed chromatography system are controlled as follows : The separation temperature is 0~60℃, the flow rate of the eluent is 1~1000mL/min, the flow rate of the feed liquid is 1~100mL/min, the flow rate of the extract liquid is 1~100mL/min, the flow rate of the raffinate is 1~100mL/min, and the switching time 1~50min; The described simulated moving bed chromatographic system is composed of 4-32 chromatographic columns equipped with stationary phases, a total of four zones, and each zone is formed by connecting 1-8 chromatographic columns in series; The size of the chromatographic column of the simulated moving bed chromatography system is 10-100 mm in diameter and 100-500 mm in length; The stationary phase of the simulated moving bed chromatography system is silica gel or alumina, and the particle size of the stationary phase is controlled at 10-100 μm, and the pore size is controlled at 10-50 nm; (3) After concentrating the raffinate obtained in step (2) to remove the solvent, at 20~60°C, add organic solvent to dissolve, cooling and crystallizing at -5~5°C for 12~36h, filtering, and washing the filter cake with water for 20 Vacuum-drying at ~40°C to obtain Coenzyme Q10 with a purity of more than 98%; The volume-to-mass ratio of the solid obtained after the described organic solvent addition amount and the raffinate are concentrated is 20 to 80 L/kg. 2. the method for continuously separating coenzyme Q10 from bacterial residue according to claim 1, is characterized in that, described non-polar organic solvent is n-hexane, cyclohexane, n-heptane, n-octane and sherwood oil One or a mixture of any two. 3. the method for continuously separating coenzyme Q10 from bacterial residue according to claim 1, is characterized in that, described eluent is n-hexane, cyclohexane, n-heptane, n-octane, sherwood oil, acetonitrile , ethyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide and monohydric alcohol with 1 to 4 carbon atoms or a mixture of any two. 4. the method for continuously separating coenzyme Q10 from bacterial residue according to claim 3, is characterized in that, described eluent is the mixture of normal hexane and ethyl acetate, and wherein the volume percent of ethyl acetate is 1～ 20%. 5. The method for continuously separating coenzyme Q10 from bacterial residues according to claim 1, wherein in step (3), the organic solvent is a monohydric alcohol with 1 to 4 carbon atoms, acetonitrile, acetone , ethyl acetate, n-hexane and n-heptane or a mixture of any two.

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