CN112174796B - Method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor - Google Patents
Method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor Download PDFInfo
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Abstract
The invention relates to a method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor, which comprises the process steps of pretreatment, leaching, crystallization, dissolution, chromatography, reduced pressure concentration, water washing and drying of the coenzyme Q10 fermentation liquor. By adopting the method, the effective content of the coenzyme Q10 finished product can reach 99 percent, and the total extraction yield can reach 95 percent. Compared with the prior domestic conventional process, the method has the advantages of high extraction yield, high product quality, simple extraction process, low production cost and the like, and is favorable for enhancing the market competitiveness of the product at home and abroad.
Description
Technical Field
The invention belongs to the technical field of antioxidant extraction, and particularly relates to a method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor generated by rhodobacter sphaeroides fermentation.
Background
Coenzyme Q10 is a fat-soluble antioxidant, can activate human cells and nutrition of cellular energy, has the functions of improving human immunity, enhancing antioxidation, delaying senility, enhancing human vitality and the like, is widely used for cardiovascular system diseases in medicine, and is widely used for nutritional health products and food additives at home and abroad.
At present, in domestic related documents, a ceramic membrane filtration process and a chromatography process are generally adopted for extraction and purification of coenzyme Q10, and the two extraction methods have respective advantages and disadvantages.
For example, chinese patent "a method and apparatus for extracting coenzyme Q10" (CN 201911412590.8) adopts a ceramic membrane filtration process, which comprises the following steps: the method comprises the steps of filtering coenzyme Q10 fermentation liquor to obtain bacterial powder, leaching and evaporating to dryness to obtain a coenzyme Q10 crude product, filtering by a small-aperture ceramic membrane, alkalifying, saponifying and filtering by a ceramic microfiltration membrane, and distilling a concentrated solution of the ceramic microfiltration membrane under reduced pressure to remove a solvent to obtain the coenzyme Q10. The problems of the method are that:
1. the method does not carry out pretreatment on the fermentation liquor of the coenzyme Q10 before extraction and purification, so that the content of impurities in a crude product is higher and exceeds more than 40 percent, and the method is not beneficial to the continuous operation of a downstream extraction and purification process.
2. The whole extraction and purification process has more steps, wherein 2 membrane filtration processes and saponification reactions are performed, so that the extraction period is longer, and the production efficiency is reduced.
3. The problems of high impurity content of coenzyme Q10, particularly pigment in finished products, are not effectively solved by membrane filtration and saponification reaction, and the quality of the products is influenced.
For another example, chinese patent "a method for extracting and purifying coenzyme Q10" (CN 201810796126.2) adopts a chromatography method to extract and purify coenzyme Q10, and the process thereof is as follows: the adopted process comprises the steps of extraction, precipitation, decoloration and chromatographic refining, and the technology achieves the technical effects that the purity of the coenzyme Q10 product can reach more than 99.8 percent, the total yield can reach more than 98.5 percent, but the problems exist:
1. in the extraction process, the adsorption process and the chromatography process are combined respectively, so that the extraction period is longer and the production efficiency is lower.
2. In the adsorption process, the adsorbent is at least one of activated clay, diatomite and activated carbon, which affects the yield of the product and reduces the yield of coenzyme Q10 by 1-2%.
3. The adsorbent used in the chromatography process is silica gel. In the coenzyme Q10 adsorption process, the silica gel adsorbent is found to have the conditions of small specific surface area, low adsorption quantity, low adsorption efficiency, poor adsorption effect and the like. Under the condition of mass production, the consumption of the silica gel adsorbent is large, the adsorption period is long, and the content of impurities in the collected coenzyme Q10 is higher than 2 percent through detection.
4. The embodiment of the invention has smaller scale and fails to embody the technical level of industrial mass production. Pilot-scale tests prove that the scale of the bacterial residues is 50kg, and after three times of extraction and purification, the average yield of the final product is 90-92%, the purity is 93-95%, and the technical level cannot be achieved.
For another example, the Chinese patent "a method for preparing a pure coenzyme Q10" (CN 201710606521.5) adopts the following processes: firstly, microfiltration and spray drying of 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 concentration on the eluent, finally adding coenzyme Q10 seed crystal for crystallization, and carrying out reduced pressure drying to obtain a coenzyme Q10 pure product. The problems of the technology are as follows:
1. the technology does not describe the technical effect of the process, and the product extraction yield is low. In five examples, the total extraction yield is 73-7%, and the total yield is low.
3. The silica gel chromatography process is adopted, and the same problem exists in the extraction and purification method of coenzyme Q10 (CN 201810796126.2).
Other patents, for example "a process for extracting and separating coenzyme Q10 from mushroom dregs" (CN 201610481472.2) disclose a process for extracting and preparing high-purity coenzyme Q10 from mushroom dregs, in which the mushroom dregs are used as raw materials to perform percolation extraction to obtain a coenzyme Q10 percolation extract, the coenzyme Q10 extract is subjected to multi-stage extraction to remove impurities to obtain a raffinate, and the raffinate is subjected to crystallization treatment to finally obtain the high-purity coenzyme Q10 with the purity of more than 98%, and the yield is more than 95%. The percolation process is known as a dynamic leaching process. Firstly, the patent does not describe a coenzyme Q10 fermentation liquor pretreatment process, and secondly, fat-soluble impurities in the mushroom dregs can enter an organic solution through a percolation process; and finally, multi-stage extraction is adopted to remove impurities, so that most of impurities can be removed only, and the removal amount of the impurities is limited. Only by adopting the processes of percolation, multistage extraction and crystallization, the technical effect described in the patent can not be achieved at all through verification.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the method for extracting the coenzyme Q10 from the coenzyme Q10 fermentation liquor, which can effectively improve the extraction yield and content of the coenzyme Q10, reduce the production cost and has simple extraction process.
The technical scheme adopted for realizing the purpose is as follows:
a method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor is characterized by comprising the following process steps:
(1) Pretreatment of
Filtering coenzyme Q10 fermentation liquor by using a plate frame, adding methanol into the obtained wet mushroom dregs (1), stirring, standing, filtering, adding water and sodium sulfite into the obtained wet mushroom dregs (2), stirring again, standing, filtering, and performing flash evaporation and drying on the obtained wet mushroom dregs (3) to obtain solid mushroom dregs;
(2) Leaching
Adding ethylene glycol-butyl ether into the solid fungus dregs, stirring, standing and filtering;
(3) Crystallization of
Adding water into the filtrate obtained in the process (2), quickly cooling to 0-5 ℃ within 30min, stirring, standing and filtering to obtain solid coenzyme Q10;
(4) Dissolving and chromatography
Dissolving the solid coenzyme Q10 by using butyl acetate, adding the solution into chromatography equipment, carrying out chromatography by using butyl acetate as a mobile phase under the condition of a flow rate of 3-5L/min, and collecting a liquid showing yellow or orange yellow to obtain a coenzyme Q10 butyl acetate solution;
(6) Concentrating under reduced pressure, washing with water and drying
And carrying out reduced pressure concentration, washing, filtering and drying on the coenzyme Q10 butyl acetate solution to obtain the pure coenzyme Q10.
In the step (1), the amount of the methanol is V Methanol :M Wet mushroom dregs (1) 10kg of a compound (1) =2 to 6L); the water dosage is according to V Water (W) :M Wet mushroom dregs (1) 10kg of sodium sulfite at a ratio of =6 to 10L, and the amount of sodium sulfite is M Sodium sulfite :M Wet mushroom dregs (1) 10kg of water at a ratio of =1 to 2g; the standing time is controlled to be 20-60 min.
In the step (2), the dosage of the ethylene glycol-butyl ether is V Ethylene glycol monobutyl ether :M Solid mushroom dregs 1kg of (= 4-8l); the standing time is controlled to be 180-220 min.
In the step (3), the water consumption is V Ethylene glycol monobutyl ether :V Purified water 8 to 12L; the standing time is controlled to be 120-160 min.
In the step (4), the solubility of the solid coenzyme Q10 is controlled to be 20-30%.
In the step (4), the height-diameter ratio of the glass column in the chromatographic equipment is 5-6: 1, the filler is a porous silicon carbide microsphere composite material filled with polystyrene gel, and the dosage of the porous silicon carbide microsphere composite material is M Filler :M Total amount of coenzyme Q10 fermentation broth = 2-4kg Total amount of coenzyme Q10 fermentation broth (kg) = fermentation liquor unit (mg/L) × fermentation liquor volume (m) 3 ）/1000。
The preparation process of the porous silicon carbide microsphere composite material filled with polystyrene gel comprises the following steps:
1) Dissolving PEG2000 and gulonic acid in deionized water, adding 80% of nano silicon carbide powder in the total amount by 3-5 times under the condition of slow stirring by a magnetic stirrer, continuously adding a polyurethane solution with the mass concentration of 5% after uniform mixing, continuously stirring, slowly adding the rest 20% of nano silicon oxide powder, continuously stirring for 120-150 min to obtain gel-like nano silicon carbide slurry,
the dosage of the substances is as follows:
M PEG2000 :M gulonic acid :M Nano silicon carbide powder : V 5% polyurethane solution =3kg:1～2kg:5～6kg:14～15L；
2) Adding vacuum pump oil and Tween 20 into the gel-like nanometer silicon carbide slurry, stirring at the rotating speed of 700r/min for 40-60 min, adding saturated calcium sulfate solution, continuously stirring for 10-20 min, adding absolute ethyl alcohol, stirring for 80-100 min, standing for 60-80 min, pouring out upper pump oil after liquid level layering, collecting silicon carbide microspheres at the bottom of a beaker, thoroughly cleaning the microspheres with 40-50% ethanol and water, drying at 50-60 ℃, sieving the bonded microspheres with an 80-mesh standard sieve to obtain monodisperse silicon carbide ceramic microsphere green bodies,
the dosage of the substances is as follows: m Nano silicon carbide powder :M Vacuum pump oil :M Tween 20 ：L Saturated calcium sulfate solution ：L Anhydrous ethanol =1kg:3～4kg：0.5～0.6kg：4～5L：15～20L；
3) Sintering the silicon carbide ceramic microsphere green blank by adopting a high-temperature muffle furnace, which specifically comprises the following steps: firstly, raising the temperature to 200 ℃ at the speed of 10 ℃/min, and preserving the temperature for 30min; then raising the temperature to 300 ℃ at the speed of 10 ℃/min, preserving the heat for 30min, finally raising the temperature to 500 ℃ at the speed of 5 ℃/min, and preserving the heat for 20min; naturally cooling to room temperature to obtain porous silicon carbide microspheres;
4) Soaking the porous silicon carbide microspheres in 10-20% polystyrene-butyl acetate solution, keeping the temperature at 90-100 ℃ for 60-80 min under aseptic conditions, slowly cooling to room temperature, adding purified water for grinding after the polystyrene gel is solidified, and sieving to remove fragments to obtain the porous silicon carbide microsphere composite material filled with the polystyrene gel,
the dosage of the substances is as follows:
M porous silicon carbide microspheres : L 10-20% polystyrene-butyl acetate solution =1kg:3～5L。
In the step (5), the temperature is controlled to be 90-100 ℃ and the pressure is controlled to be-0.1-0 MPa in the vacuum concentration.
In the step (5), the washing is that purified water is used for washing for 1 time, and the dosage of the purified water is 5-10 times of the solid weight of the coenzyme Q10; the drying is that a double-cone rotary vacuum dryer is adopted for drying for 14-15 h, the drying temperature is 30-40 ℃, the vacuum pressure is-0.02 to-0.08 MPa, and the operation frequency is adjusted to be 8-15 HZ according to the humidity of the material.
The technical advantages of the invention are embodied in that:
1. the method comprises the steps of firstly, leaching coenzyme Q10 fermentation liquor by using methanol (the action is that an organic solvent is used for leaching the coenzyme Q10 in fungus dregs), pretreating sodium sulfite (the action is that the coenzyme Q10 is in contact with oxygen in the air, so that an oxidation phenomenon exists, the coenzyme Q10 is protected by adding the sodium sulfite and is not easy to be oxidized), and the like, and then, further extracting by using ethylene glycol-butyl ether, so that the extraction yield of the coenzyme Q10 reaches 98%.
2. The invention adopts a low-temperature crystallization mode, effectively removes water-soluble impurities and ensures that the crystallization yield is more than 98 percent.
3. The invention uses the novel synthesized porous silicon carbide microsphere composite material filled with polystyrene gel as the filler in the chromatography process, the filler can effectively separate coenzyme Q10 and impurities, the chromatography period is shorter, and the period is shortened by about 7 hours compared with a silica gel filler (the period is more than 18 hours). After the chromatography is finished, the yield can reach more than 97 percent, and the effective content reaches 99 percent.
In conclusion, the method can lead the effective content of the coenzyme Q10 finished product to reach 99 percent and the total extraction yield to reach 95 percent. Compared with the prior domestic conventional process, the method has the advantages of high extraction yield, excellent product quality, simple and convenient extraction process, low production cost and the like, and is favorable for enhancing the market competitiveness of the product at home and abroad.
Detailed Description
The invention is illustrated below by way of examples, which are to be understood as being illustrative and not limiting. The scope and spirit of the present invention are defined by the appended claims.
In the following examples, the coenzyme 10 fermentation broth is prepared by fermentation of rhodobacter sphaeroides, and the process is the technology disclosed at present.
In the following examples, the aspect ratio of the glass column of the chromatography device was 5: 1, and the filler was a porous silicon carbide microsphere composite filled with polystyrene gel in the interior, and the amount of the filler was M Filler :M Total amount of coenzyme Q10 fermentation broth 1kg, wherein M is =3kg Total amount (kg) of coenzyme Q10 fermentation broth = fermentation liquor unit (mg/L) × fermentation liquor volume (m) 3 )/1000. Before use, the filler is added into the glass column, the butyl acetate is added into the column, and the column is soaked for 140min. After the infiltration, the rinsing with butyl acetate was continued for 2 times. During the chromatography, the flow rate was controlled at 4L/min.
The preparation process of the porous silicon carbide microsphere composite material filled with polystyrene gel comprises the following steps:
1) Dissolving PEG2000 and gulonic acid in deionized water, adding 80% of nanometer silicon carbide powder in 4 times under the condition of slowly stirring by a magnetic stirrer, after uniformly mixing, continuously adding polyurethane solution with the mass concentration of 5%, continuously stirring, slowly adding the rest 20% of nanometer silicon oxide powder, continuously stirring for 130min to obtain nanometer silicon carbide slurry with uniformly dispersed particles and gelatinous slurry,
M PEG2000 :M gulonic acid :M Nano silicon carbide powder : V 5% polyurethane solution =3kg:1kg:6kg:14L；
2) Adding vacuum pump oil and Tween 20 into the gel-like nanometer silicon carbide slurry, stirring at the rotating speed of 700r/min for 60min, adding saturated calcium sulfate solution, continuously stirring for 15min, adding absolute ethyl alcohol, stirring for 100min, standing for 70min, pouring upper layer pump oil after liquid level layering, collecting silicon carbide microspheres at the bottom of a beaker, thoroughly cleaning the microspheres with 50% ethanol and water, drying at the temperature of 50-60 ℃, sieving the microspheres bonded together by a standard sieve of 80 meshes to obtain monodisperse silicon carbide ceramic microsphere green bodies,
the dosage of the substances is as follows: m Nano silicon carbide powder :M Vacuum pump oil :M Tween 20 ：L Saturated calcium sulfate solution ：L Anhydrous ethanol =1kg: 4kg：0.5kg：4.5L：17L；
3) Sintering the silicon carbide ceramic microsphere green blank by adopting a high-temperature muffle furnace, which specifically comprises the following steps: firstly, raising the temperature to 200 ℃ at the speed of 10 ℃/min, and preserving the temperature for 30min; then raising the temperature to 300 ℃ at the speed of 10 ℃/min, preserving the heat for 30min, finally raising the temperature to 500 ℃ at the speed of 5 ℃/min, and preserving the heat for 20min; naturally cooling to room temperature to obtain porous silicon carbide microspheres;
4) Soaking the porous silicon carbide microspheres in a 15% polystyrene-butyl acetate (butyl acetate is used as a solvent) solution, preserving the heat for 80min at 90-100 ℃ under aseptic conditions, and then slowly cooling the porous silicon carbide microspheres to room temperature, so that the porous silicon carbide microspheres are filled with the polystyrene solution; after the polystyrene gel is solidified, taking out the silicon carbide microspheres deposited at the bottom, adding purified water for grinding, removing polystyrene fragments and crushed silicon carbide fragments through standard sieve screening and a fluidized bed to obtain the porous silicon carbide microsphere composite material filled with the polystyrene gel, after the polystyrene gel is solidified,
the dosage of the substances is as follows:
M porous silicon carbide microspheres : L 10-20% polystyrene-butyl acetate solution =1kg:4L。
Example 1
Coenzyme Q10 fermentation liquor 100m 3 The fermentation unit is 2980mg/L, and the total amount is 298kg.
The coenzyme Q10 fermentation liquor is filtered by a plate frame to obtain wet mushroom dregs (1).
13920L of primary water and 2.32kg of sodium sulfite are added into the wet mushroom dregs (2), the wet mushroom dregs (3) are obtained by stirring, standing for 20min and filtering, and the solid mushroom dregs (4135 kg) are obtained by flash evaporation and drying, and the water content is 1.9%.
Adding 16540L ethylene glycol-butyl ether into the solid bacterial residue, stirring and standing for 180min, and filtering to obtain filtrate.
Adding 132320L of purified water into the obtained filtrate, rapidly cooling to 5 ℃ within 30min, stirring and standing for 120min, and filtering to obtain 10 294kg of solid coenzyme Q with the yield of 98%.
Adding 1460L of butyl acetate into solid coenzyme Q10 to dissolve, and controlling the concentration at 20%.
In the chromatographic equipment, the glass column diameter is 0.5 m, height is 2.5 m, and 596kg of filling agent is added. Adding coenzyme Q10 butyl acetate solution into the chromatographic column treated by butyl acetate, and taking butyl acetate as a mobile phase at the flow rate of 3L/min. The liquid showing yellow or orange-yellow color was collected to obtain a coenzyme Q10 butyl acetate solution.
The coenzyme Q10 butyl acetate solution is decompressed and concentrated to obtain 290kg of coenzyme Q10 solid, and the yield is 98.6%.
Adding purified water 1420L into the solid coenzyme Q10, washing with water for 60min, filtering, and drying with double cones to obtain pure coenzyme Q10 289.7kg.
Through measurement and calculation, the total yield is as follows: 97.2 percent and the effective content of the finished product is 98.4 percent.
Example 2
100m coenzyme Q10 fermentation liquor 3 The fermentation unit is 2763mg/L, and the total amount is 276kg.
The coenzyme Q10 fermentation broth is filtered by a plate-and-frame filter to obtain wet mushroom dregs (1), 22.8 tons of the wet mushroom dregs are added with 6840L of methanol, stirred and kept stand for 30min, and the wet mushroom dregs (2) are obtained by filtration.
Adding 15960L of primary water and 2.74kg of sodium sulfite into the wet mushroom dregs (2), stirring, standing for 30min, filtering to obtain wet mushroom dregs (3), and flash-drying to obtain solid mushroom dregs 4024kg with water content of 2.1%.
Adding ethylene glycol-butyl ether 20120L into the solid fungus residue, stirring and standing for 190min, and filtering to obtain filtrate.
Adding purified water 181080L into the obtained filtrate, rapidly cooling to 4 deg.C within 30min, stirring and standing for 130min, and filtering to obtain solid coenzyme Q10 kg with yield of 98.5%.
Butyl acetate 1232L is added into solid coenzyme Q10 for dissolution, and the concentration is controlled at 22%.
The diameter of the glass column is 0.5 m, the height is 2.6 m, the filling agent 678kg is added, the coenzyme Q10 butyl acetate solution is added into the chromatography column after butyl acetate treatment, butyl acetate is taken as a mobile phase, and the flow rate is 3.5L/min. The liquid showing yellow or orange-yellow color was collected to obtain a coenzyme Q10 butyl acetate solution.
The coenzyme Q10 butyl acetate solution is decompressed and concentrated to obtain 268.6kg of coenzyme Q10 solid, and the yield is 98.7%.
1600L of purified water is added into the solid coenzyme Q10, and the mixture is washed for 70min, filtered and then bipyramidally dried to obtain 268.5kg of pure coenzyme Q10.
Through measurement and calculation, the total yield is as follows: 97.3 percent and the effective content of the finished product is 98.5 percent.
Example 3
Coenzyme Q10 fermentation liquor 100m 3 The fermentation unit is 2860mg/L, and the total amount is 286kg.
The coenzyme Q10 fermentation liquor is filtered by a plate frame to obtain wet mushroom dregs (1).
Adding 18480L primary water and 3.47kg sodium sulfite into wet mushroom residue (2), stirring and standing for 40min, filtering to obtain wet mushroom residue (3), and flash drying to obtain solid mushroom residue 4052kg with water content of 2.2%.
Adding 24312L ethylene glycol-butyl ether into the obtained solid fungus residue, stirring, standing for 200min, and filtering to obtain filtrate.
Adding 243120L of purified water into the filtrate, rapidly cooling to 3 deg.C within 30min, stirring, standing for 140min, and filtering to obtain solid coenzyme Q10 kg with yield of 98.6%.
Adding butyl acetate 1128L into solid coenzyme Q10, and dissolving to control the concentration at 25%.
The diameter of the glass column is 0.5 m, the height is 2.8 m, 715kg of filling agent is added, coenzyme Q10 butyl acetate solution is added into the chromatography column after butyl acetate treatment, butyl acetate is taken as a mobile phase, and the flow rate is 4L/min. The liquid showing yellow or orange-yellow color was collected to obtain a coenzyme Q10 butyl acetate solution.
The coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 278.6kg of coenzyme Q10 solid with a yield of 98.8%.
Adding purified water 2230L into solid coenzyme Q10, washing with water for 80min, filtering, and drying with double cones to obtain pure coenzyme Q10.5 kg.
Through measurement and calculation, the total yield is as follows: 97.4 percent and the effective content of the finished product is 98.7 percent.
Example 4
Coenzyme Q10 fermentation liquor 100m 3 Fermentation unit 2900mg/L, total 290kg.
The coenzyme Q10 fermentation liquor is filtered by a plate frame to obtain wet mushroom residue (1).
Adding 20970L of primary water and 4.19kg of sodium sulfite into the wet mushroom residue (2), stirring and standing for 50min, filtering to obtain wet mushroom residue (3), and flash-drying to obtain 4101kg of solid mushroom residue with water content of 2.4%.
Adding 28707L of ethylene glycol-butyl ether into the solid fungus residues, stirring, standing for 210min, and filtering to obtain filtrate.
Adding 315777L of purified water into the filtrate, rapidly cooling to 2 deg.C within 30min, stirring and standing for 150min, and filtering to obtain 10kg of solid coenzyme Q with yield of 98.5%.
Adding 1020L butyl acetate into solid coenzyme Q10 for dissolution, and controlling the concentration at 28%.
The diameter of the glass column is 0.5 m, the height is 2.9 m, the filling agent is added with 1001kg, the chromatography column after butyl acetate treatment is added with coenzyme Q10 butyl acetate solution, butyl acetate is taken as a mobile phase, and the flow rate is 4.5L/min. The liquid showing yellow or orange-yellow color was collected to obtain a coenzyme Q10 butyl acetate solution.
The coenzyme Q10 butyl acetate solution thus obtained was concentrated under reduced pressure to give 282.3kg of coenzyme Q10 as a solid, and the yield was 98.7%.
2500L of purified water is added into the solid coenzyme Q10, the mixture is washed for 90min, and then is filtered and then is subjected to bipyramid drying to obtain 282.2kg of pure coenzyme Q10.
Through measurement and calculation, the total yield is as follows: 97.3 percent and the effective content of the finished product is 98.5 percent.
Example 5
Coenzyme Q10 fermentation liquor 100m 3 The fermentation unit is 2751mg/L, and the total amount is 275.1kg.
The coenzyme Q10 fermentation liquor is filtered by a plate frame to obtain wet mushroom dregs (1) 22.1 tons, added with methanol 16506L, stirred and kept stand for 60min, and filtered to obtain the wet mushroom dregs (2).
Adding 22100L of primary water into the wet mushroom residue (2), adding 4.42kg of sodium sulfite, stirring, standing for 60min, filtering to obtain wet mushroom residue (3), and flash-evaporating and drying to obtain 4085kg of solid mushroom residue with water content of 2.0%.
Adding 32680L ethylene glycol-butyl ether into the fungus residue, stirring, standing for 220min, and filtering to obtain filtrate.
Adding 392160L of purified water into the filtrate, rapidly cooling to 0 deg.C within 30min, stirring and standing for 160min, and filtering to obtain solid coenzyme Q10 kg with yield of 98.4%.
Butyl acetate 903L was added to the solid coenzyme Q10 to dissolve the coenzyme Q, and the concentration was controlled at 30%.
The diameter of the glass column is 0.5 m, the height is 3m, the filling agent is 1100kg is added, the coenzyme Q10 butyl acetate solution is added into the chromatography column after butyl acetate treatment, butyl acetate is taken as a mobile phase, and the flow rate is 5L/min. The liquid showing yellow or orange-yellow color was collected to obtain a coenzyme Q10 butyl acetate solution.
The coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to give 267.2kg of coenzyme Q10 solid with a yield of 98.6%.
2500L of purified water is added into the solid coenzyme Q10, the mixture is washed for 90min, and then is filtered and then is subjected to bipyramid drying to obtain 267.1kg of pure coenzyme Q10.
Through measurement and calculation, the total yield is as follows: 97.1 percent and the effective content of the finished product is 98.4 percent.
Comparative example 1
Coenzyme Q10 fermentation liquor 100m 3 The fermentation unit is 2834mg/L, and the total amount of the coenzyme Q10 is 283.4kg.
The fermentation liquor is extracted by ethyl acetate-methanol solvent and evaporated to dryness to obtain 278.9kg of coenzyme Q10 crude product, and the yield is 98.4%. Adding a nonpolar solvent n-hexane into the crude coenzyme Q10 product to dissolve the coenzyme Q10.
Filtering the obtained solution by using a small-aperture ceramic membrane to remove insoluble impurities and high-molecular-weight impurities; in the step, the average aperture of the small-aperture ceramic membrane is 1-100nm, preferably 5-10nm, and some suspended particles and macromolecular impurities are filtered.
Adding alkali liquor into the obtained filtrate to carry out saponification reaction; the alkali solution is sodium hydroxide with the concentration of 5wt%, the addition amount of the alkali solution is 0.4 times of that of the filtrate, and the temperature is controlled at 24 ℃.
Sequentially adding methanol, ethanol and water into the saponification reaction liquid, simultaneously filtering by using a ceramic microfiltration membrane, and distilling the concentrated solution of the ceramic microfiltration membrane under reduced pressure to remove the solvent to obtain coenzyme Q10; the total volume of the added methanol and ethanol is controlled to be 5 times of the volume of the saponification reaction liquid, and the water washing amount of the added water is controlled to be 1 time of the volume of the saponification reaction liquid. The microfiltration membrane is subjected to hydrophobic modification treatment, and the water drop contact angle of the surface of the microfiltration membrane ranges from 100 degrees to 160 degrees.
258kg of finished coenzyme Q10 product, the content is 90.4 percent by detection, and the yield is 91.0 percent.
Comparative example 2
1000kg of the fermented microbial cells and 71.2kg of the total amount of coenzyme Q10. Performing continuous countercurrent ultrasonic extraction (5% of ethyl acetate-95% of n-hexane as an extractant), feeding the extract into a dynamic circulation low-temperature evaporation concentrator for evaporation and concentration, wherein the concentration temperature is 35 ℃, the vacuum degree is-0.085 MPa, 68.2kg of coenzyme Q10 extract is obtained, the content of coenzyme Q10 in the extract is measured to be 65.72kg, the extraction yield is 92.3%, and the content of a coenzyme Q10 crude product is 87.5%.
Comparative example 3
Collecting coenzyme Q10 fermentation broth 1m 3 (fermentation unit 2900 mg/L), and filtering with ceramic membrane microfiltration membrane (aperture 0.1 um), wherein the feed temperature is about 25 ℃, the dialysis ratio is 1.5, and the concentration multiple is 1.8 times, thus obtaining 231L of membrane pulp.
The membrane slurry is subjected to spray drying, the air inlet temperature is controlled to be 140-160 ℃, the air outlet temperature is controlled to be 60-70 ℃, and 3.1kg of coenzyme Q10 crude product is obtained, the effective content is 87.9%, and the yield is 91.3%.
Adding acetone 3 times the weight and volume 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 (pore diameter of 0.5 μm), and repeatedly leaching the residue once again under the same 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 supernatant, and distilling under reduced pressure to obtain coenzyme Q10 extract concentrate 3.8kg, with effective content of 66.11% and yield of 92.2%.
Dissolving the acetone extraction concentrate in petroleum ether, adding the amount of the coenzyme Q10 concentrate to be 3.0 times of the volume of the coenzyme Q10 concentrate, adding equal volume of water, stirring for 10min, standing, dividing water, collecting a petroleum ether layer, purifying and separating the petroleum ether layer in a silica gel column, wherein the eluent is petroleum ether, the feeding speed is 2BV/h, the eluting speed is 1.5BV/h, the feeding temperature is 25 ℃, collecting the eluent containing 10 sections of the coenzyme Q, and distilling under reduced pressure to obtain 2.8kg of purified coenzyme Q10 extraction concentrate, wherein the effective content is 80.5 percent, and the yield is 88 percent.
Adding absolute ethanol with the volume of 2 times of the weight of the purified coenzyme Q10 concentrate obtained by distilling and concentrating the eluent under reduced pressure into the concentrate, heating the concentrate to 48 ℃, stirring the concentrate for dissolving, and filtering the concentrate while the concentrate is hot; slowly cooling the filtrate to 28 ℃ under stirring, adding 0.02 per thousand w/v coenzyme Q10 seed crystal, adding deionized water until crystals are separated out, continuously adding deionized water, controlling the total water addition amount to be 3 times (978 ml) of the weight of the purified coenzyme Q10 concentrate, keeping the temperature at 20 ℃, keeping the temperature for 9.0 hours, filtering, and drying wet crystals under reduced pressure to obtain 2.2kg of a coenzyme Q10 pure product, wherein the content of the coenzyme Q10 pure product is 98.0%, and the total yield of the coenzyme Q10 pure product is 75.8%.
Comparative example 4
Weighing 1000g (0.06 kg) of bacterial residues (wherein the mass percent of the coenzyme Q10 is 2.6%), filling the bacterial residues into a percolation column (phi 20.0 multiplied by 35 cm) by a wet method, after uniform filling, keeping the stacking volume of the bacterial residues about 1300mL, soaking the bacterial residues for 2 hours at the constant temperature of 10 ℃ to fully swell the bacterial residues, starting an outlet valve of the percolation column, simultaneously and continuously adding n-hexane from the top of the column, and controlling the flow rate to be 2.5-3.0 mL/min until the volume of the collected percolation liquid is 1250mL. The operation is repeated for 1 time according to the steps to obtain the percolate with the volume of 2480mL and the coenzyme Q10 content of 0.023kg/L, and the yield is 95.1 percent.
Concentrating the percolate to remove solvent, dissolving with n-hexane, and making into solution with solid concentration of 300 mg/mL. Three-stage cross-flow extraction is carried out, and N, N-dimethylformamide with the same amount is added into each stage to be used as an extracting agent. After the extraction is finished, the total extraction yield is 89.6 percent
The solution is cooled and crystallized for 24h, then filtered and washed with a suitable amount of cold ethanol. Fully pumping and drying the mixture for 6 hours at 30 ℃ to obtain 0.044kg of yellow coenzyme Q10 refined product with the purity of 98.0 percent, wherein the coenzyme Q10 in the fungus residue is taken as the reference, and the yield of the coenzyme Q10 in the whole process is 74.2 percent.
Comparative example 5
Coenzyme Q10 fermentation liquor 100m 3 The fermentation unit is 2843mg/L, and the total amount is 2.84kg.
Carrying out solid-liquid separation on fermentation liquor, drying, crushing the bacterial residues to obtain 4.2 tons of bacterial residues, mixing the bacterial residues with n-hexane according to the mass ratio of 1: 2, stirring and leaching after uniformly mixing, leaching for 1.5 hours at the temperature of 55 ℃, filtering through a microfiltration membrane, and collecting an extracting solution and retentate; the microfiltration membrane is an inorganic ceramic membrane, the interception molecular weight is 2000Da, and the filtering temperature is 40 ℃;
mixing the obtained extractive solution with alkaline alcohol solution (1L solution containing 80g sodium chloride, 20g sodium hydroxide, 80mL methanol, and the balance water) at volume ratio of 1: 2, oscillating for 5min, standing for layering, collecting organic phase, rotary evaporating at 55 deg.C and vacuum degree of 0.04Mpa for concentration, and re-dissolving the obtained concentrated solution with n-hexane;
performing silica gel column chromatography on the obtained liquid, and eluting by using a normal hexane solution containing 3% (V/V) isopropyl ether at the flow rate of 1 column volume per hour until no obvious yellow color exists; then decompressing and concentrating the coenzyme Q10 eluent under the conditions of 55 ℃ and 0.04Mpa of vacuum degree; dissolving the obtained concentrated solution in anhydrous ethanol, refrigerating at 0 deg.C overnight, vacuum-filtering to obtain coenzyme Q10 colloid; finally, vacuum drying is carried out at the temperature of 2 ℃ to obtain pure coenzyme Q102.4kg with the purity of 98.01 percent; the yield thereof was found to be 86%.
Claims (7)
1. A method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor is characterized by comprising the following process steps:
(1) Pretreatment of
Filtering coenzyme Q10 fermentation liquor by using a plate frame, adding methanol into the obtained wet mushroom residue (1), stirring, standing, filtering, adding water and sodium sulfite into the obtained wet mushroom residue (2), stirring again, standing, filtering, and performing flash evaporation drying on the obtained wet mushroom residue (3) to obtain solid mushroom residue;
(2) Leaching
Adding ethylene glycol-butyl ether into the solid fungus dregs, stirring, standing and filtering;
(3) Crystallization of
Adding water into the filtrate obtained in the process (2), quickly cooling to 0-5 ℃ within 30min, stirring, standing and filtering to obtain solid coenzyme Q10;
(4) Dissolving and chromatography
Dissolving the solid coenzyme Q10 by using butyl acetate, adding the solution into chromatography equipment, performing chromatography by using the butyl acetate as a mobile phase under the condition of a flow rate of 3-5L/min, and collecting a yellow or orange liquid to obtain a coenzyme Q10 butyl acetate solution; the height-diameter ratio of a glass column in the chromatographic equipment is 5-6: 1, and the filler is a porous silicon carbide microsphere composite material filled with polystyrene gel, and the dosage of the filler is M, namely the total amount of M-coenzyme Q10 fermentation liquor = 2-4 kg; the preparation process of the porous silicon carbide microsphere composite material filled with polystyrene gel comprises the following steps:
1) Dissolving PEG2000 and gulonic acid in deionized water, adding 80% of nano silicon carbide powder in the total amount by 3-5 times under the condition of slowly stirring by a magnetic stirrer, continuously adding a polyurethane solution with the mass concentration of 5% after uniformly mixing, continuously stirring, slowly adding the rest 20% of nano silicon oxide powder, continuously stirring for 120-150 min to obtain gel-like nano silicon carbide slurry,
the dosage of the substances is as follows:
MPEG2000, M gulonic acid, M nano silicon carbide powder, V5% polyurethane solution = 3kg;
2) Adding vacuum pump oil and Tween 20 into the gel-like nanometer silicon carbide slurry, stirring for 40-60 min at the rotating speed of 700r/min, adding saturated calcium sulfate solution, continuously stirring for 10-20 min, adding absolute ethyl alcohol, stirring for 80-100 min, standing for 60-80 min, after liquid level layering, dumping upper layer pump oil, collecting silicon carbide microspheres at the bottom of a beaker, thoroughly cleaning the microspheres with 40-50% ethanol and water, drying at 50-60 ℃, sieving the bonded microspheres with an 80-mesh standard sieve to obtain monodisperse silicon carbide ceramic microsphere green bodies,
the dosage of the substances is as follows: m nano silicon carbide powder, M vacuum pump oil, M Tween 20: l saturated calcium sulfate solution: l absolute ethanol =1kg: 0.5-0.6 kg: 4-5L: 15-20L;
3) Sintering the silicon carbide ceramic microsphere green blank by adopting a high-temperature muffle furnace, which specifically comprises the following steps: firstly, raising the temperature to 200 ℃ at the speed of 10 ℃/min, and preserving the temperature for 30min; then raising the temperature to 300 ℃ at the speed of 10 ℃/min, preserving the heat for 30min, finally raising the temperature to 500 ℃ at the speed of 5 ℃/min, and preserving the heat for 20min; naturally cooling to room temperature to obtain porous silicon carbide microspheres;
4) Soaking the porous silicon carbide microspheres in 10-20% polystyrene-butyl acetate solution, keeping the temperature at 90-100 ℃ for 60-80 min under aseptic conditions, slowly cooling to room temperature, adding purified water for grinding after the polystyrene gel is solidified, and sieving to remove fragments to obtain the porous silicon carbide microsphere composite material filled with the polystyrene gel,
the dosage of the substances is as follows:
m porous silicon carbide microspheres, wherein L10-20% of polystyrene-butyl acetate solution = 1kg;
(5) Concentrating under reduced pressure, washing with water and drying
And (3) carrying out reduced pressure concentration, washing, filtering and drying on the coenzyme Q10 butyl acetate solution to obtain a pure coenzyme Q10.
2. The method for extracting coenzyme Q10 from a coenzyme Q10 fermentation broth according to claim 1, wherein in the step (1), the amount of methanol is added in a ratio of V methanol to M wet residue (1) =2 to 6L; the water dosage is added according to the proportion of V water to M wet bacteria residue (1) = 6-10L; the standing time is controlled to be 20-60 min.
3. The method for extracting coenzyme Q10 from a coenzyme Q10 fermentation broth according to claim 1, wherein in the step (2), the amount of the ethylene glycol butyl ether is 1kg of the total amount of the ethylene glycol butyl ether, M solid fungal residue = 4-8L; the standing time is controlled to be 180-220 min.
4. The method for extracting coenzyme Q10 from a coenzyme Q10 fermentation broth according to claim 1, wherein in the step (3), the amount of water is 8 to 12L in terms of V ethylene glycol-butyl ether; the standing time is controlled to be 120-160 min.
5. The method for extracting coenzyme Q10 from a coenzyme Q10 fermentation broth according to claim 1, wherein in the step (4), the solubility of the solid coenzyme Q10 is controlled to be 20 to 30%.
6. The method of extracting coenzyme Q10 from a coenzyme Q10 fermentation broth according to claim 1, wherein in the step (5), the temperature is controlled to 90 to 100 ℃ and the pressure is controlled to-0.1 to 0MPa in the concentration under reduced pressure.
7. The method for extracting coenzyme Q10 from a coenzyme Q10 fermentation broth according to claim 1, wherein in the step (5), the washing is 1 time of washing with purified water in an amount of 5 to 10 times the weight of the coenzyme Q10 solid; the drying is that a double-cone rotary vacuum dryer is adopted for drying for 14-15 h, the drying temperature is 30-40 ℃, the vacuum pressure is-0.02 to-0.08 MPa, and the operating frequency is adjusted to be 8-15 HZ according to the humidity of the material.
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