WO2022057603A1 - Method for extracting coenzyme q10 from fermentation broth of coenzyme q10 - Google Patents

Method for extracting coenzyme q10 from fermentation broth of coenzyme q10 Download PDF

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WO2022057603A1
WO2022057603A1 PCT/CN2021/115304 CN2021115304W WO2022057603A1 WO 2022057603 A1 WO2022057603 A1 WO 2022057603A1 CN 2021115304 W CN2021115304 W CN 2021115304W WO 2022057603 A1 WO2022057603 A1 WO 2022057603A1
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coenzyme
silicon carbide
add
added
water
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PCT/CN2021/115304
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French (fr)
Chinese (zh)
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徐淑芬
任勇
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宁夏泰胜生物科技有限公司
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Publication of WO2022057603A1 publication Critical patent/WO2022057603A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C46/00—Preparation of quinones
    • C07C46/10—Separation; Purification; Stabilisation; Use of additives

Definitions

  • the invention relates to a method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquid produced by fermentation of Rhodobacter sphaeroides, and belongs to the technical field of antioxidant extraction.
  • Coenzyme Q10 is a fat-soluble antioxidant, which can activate the nutrition of human cells and cell energy. It has the functions of improving human immunity, enhancing anti-oxidation, delaying aging and enhancing human vitality. It is widely used in medicine for cardiovascular system diseases. It is widely used in nutritional health care products and food additives.
  • Chinese patent CN111153786A adopts the ceramic membrane filtration process, and the process is as follows: the bacterial powder obtained by filtering the coenzyme Q10 fermentation broth is respectively leached and evaporated to obtain the crude coenzyme Q10 product, and then filtered through a small pore ceramic membrane, Alkalization, saponification reaction and ceramic microfiltration membrane are carried out for filtration treatment, and after the concentrated solution of ceramic microfiltration membrane is distilled under reduced pressure to remove solvent, coenzyme Q10 is obtained.
  • the problem with this method is:
  • the fermentation broth of coenzyme Q10 is not pretreated before extraction and purification, resulting in a high impurity content in the crude product, exceeding 40%, which is not conducive to the continuation of the downstream extraction and purification process.
  • Membrane filtration and saponification did not effectively solve the problem of high impurity content of coenzyme Q10, especially the pigment problem in the finished product, which affected the quality of the product.
  • Chinese patent CN108863743A adopts the method of chromatography to extract and purify coenzyme Q10, and its technological process is as follows: the adopted technology is extraction, precipitation, decolorization and chromatographic refining steps, and the technical effect achieved by this technology is that the purity of product coenzyme Q10 can reach More than 99.8%, the total yield can reach more than 98.5%, but the existing problems are:
  • the adsorbents in the adsorption process are activated clay, diatomite and activated carbon (at least one), which affects the yield of the product and reduces the yield of coenzyme Q10 by 1-2%.
  • the adsorbent used in the chromatography process is silica gel.
  • the silica gel adsorbent had small specific surface area, low adsorption capacity, low adsorption efficiency, and poor adsorption effect.
  • the amount of silica gel adsorbent is large, and the adsorption cycle time is long.
  • the collected coenzyme Q10 has a high impurity content, exceeding 2%.
  • the scale shown in the embodiments of this invention is small, and fails to reflect the technical level achieved by industrialized large-scale production. According to this process, the pilot test has been carried out.
  • the scale of the fungus residue is 50kg. After three extractions and purifications, the average yield of the final product is 90-92%, and the purity is 93-95%, which fails to reach the level described in the patent. technical level.
  • the extraction yield of the product is low.
  • the total extraction yield is 73-7%, and the total yield is relatively low.
  • Chinese patent CN106146278A discloses a process for preparing high-purity coenzyme Q10 by extracting and preparing high-purity coenzyme Q10 from bacterial residue.
  • the raffinate is mixed to obtain the raffinate, and then the raffinate is crystallized to finally obtain coenzyme Q10.
  • the percolation process is a dynamic leaching process.
  • the patent does not describe the coenzyme Q10 fermentation broth pretreatment process.
  • the fat-soluble impurities in the bacterial residue can enter the organic solution through the percolation process.
  • multi-stage extraction is used to remove impurities, only Most impurities can be removed, and the amount of impurities removed is limited.
  • Patent Document 1 CN111153786A;
  • Patent document 2 CN108863743A;
  • Patent document 3 CN107337593A
  • Patent Document 4 CN106146278A.
  • the object of the present invention is to overcome the above-mentioned defects in the prior art, and to provide a method for extracting coenzyme Q10 from coenzyme Q10 fermentation broth with a simple extraction process that effectively improves the extraction yield and content of coenzyme Q10, reduces production costs, and has a simple extraction process.
  • a method for extracting coenzyme Q10 from coenzyme Q10 fermentation broth comprising the following steps:
  • the coenzyme Q10 fermentation broth is filtered with a plate and frame, methanol is added to the obtained wet mushroom residue 1, stirred and allowed to stand, and then filtered, water and sodium sulfite are added to the obtained wet mushroom residue 2, stirred again, and filtered after standing, and the obtained wet bacteria Slag (3) is obtained by flash drying to obtain solid bacterial slag;
  • the above solid coenzyme Q10 is dissolved with butyl acetate, and then added to a chromatography device, using butyl acetate as a mobile phase, and performing chromatography under the condition of a flow rate of 3 to 5 L/min, collecting a yellow or orange-yellow liquid to obtain Coenzyme Q10 butyl acetate solution;
  • the coenzyme Q10 can be obtained by concentrating the coenzyme Q10 butyl acetate solution under reduced pressure, washing with water, filtering and drying.
  • step (4) the solubility of the solid coenzyme Q10 is controlled at 20-30%.
  • the preparation process of the porous silicon carbide microsphere composite filled with polystyrene gel is as follows:
  • the dosage of each of the above substances is:
  • the dosage of each of the above substances is:
  • step (5) in the concentration under reduced pressure, the temperature is controlled to be 90-100° C. and the pressure to be -0.1-0 MPa.
  • the washing refers to washing with purified water once, and the dosage is 5 to 10 times the solid weight of coenzyme Q10;
  • the drying refers to using double Cone rotary vacuum dryer is used for drying for 14 ⁇ 15h, drying temperature is 30 ⁇ 40°C, vacuum pressure is -0.02 ⁇ -0.08MPa, and operating frequency is 8 ⁇ 15Hz.
  • methanol is used for the pretreatment of the coenzyme Q10 fermentation broth, and the coenzyme Q10 in the bacterial residue is extracted with an organic solvent, and sodium sulfite is added to protect the coenzyme Q10 from being oxidized, and then the ethylene glycol-butyl ether is used for further extraction, thereby
  • the extraction yield of coenzyme Q10 is greatly improved, preferably reaching 98%.
  • the invention adopts a low-temperature crystallization method, which effectively removes water-soluble impurities, so that the crystallization yield is greatly improved, for example, more than 98%.
  • the effective content of the finished product of Coenzyme Q10 can be greatly increased, for example, it can preferably reach 99%, and the total extraction yield can also be improved, for example, it can preferably reach 95%.
  • the method of the invention has the advantages of high extraction yield, excellent product quality, simple extraction process, low production cost and the like.
  • the height-diameter ratio of the glass column of the chromatography equipment is 5:1
  • the filler is a porous silicon carbide microsphere composite filled with polystyrene gel
  • the amount thereof is M filler :
  • porous silicon carbide microspheres Soak the above-mentioned porous silicon carbide microspheres in a 15% polystyrene-butyl acetate (butyl acetate is a solvent) solution, keep warm at 90-100° C. for 80 min under sterile conditions, and then slowly cool it to room temperature , in this way, the porous silicon carbide microspheres are filled with polystyrene solution; after the polystyrene gel is solidified, take out the silicon carbide microspheres deposited at the bottom, add purified water for grinding, add purified water for grinding, and pass the standard Screening and fluidized bed removal of polystyrene fragments and broken silicon carbide fragments, the porous silicon carbide microsphere composite filled with polystyrene gel can be obtained. After the polystyrene gel is solidified,
  • the dosage of each of the above substances is:
  • the present invention uses a porous silicon carbide microsphere composite filled with polystyrene gel as a filler in the chromatography process.
  • the filler can effectively separate coenzyme Q10 and impurities, and the chromatography cycle is short, which is similar to the silica filler. (The cycle is more than 18h), the cycle is shortened, for example, it can be shortened by 7h.
  • Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2980mg/L, and the total amount is 298kg.
  • the coenzyme Q10 fermentation broth was filtered by plate and frame to obtain 23.2 tons of wet bacterial residue 1, 4640 L of methanol was added, stirred and allowed to stand for 10 min, and filtered to obtain wet bacterial residue 2.
  • the diameter of the glass column is 0.5 meters and the height is 2.5 meters, and 596kg of filler is added.
  • Coenzyme Q10 butyl acetate solution was added to the chromatography column treated with butyl acetate, butyl acetate was used as the mobile phase, and the flow rate was 3 L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
  • the coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 290 kg of solid coenzyme Q10, and the yield was 98.6%.
  • the total yield is 97.2%, and the effective content of the finished product is 98.4%.
  • Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2763mg/L, and the total amount is 276kg.
  • the coenzyme Q10 fermentation broth was filtered through the plate and frame to obtain 22.8 tons of wet bacterial residue 1, 6840 L of methanol was added, stirred and allowed to stand for 30 minutes, and filtered to obtain wet bacterial residue 2.
  • the glass column has a diameter of 0.5 meters and a height of 2.6 meters. Add 678 kg of filler, and add coenzyme Q10 butyl acetate solution to the column treated with butyl acetate. Use butyl acetate as the mobile phase and the flow rate is 3.5 L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
  • the coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 268.6 kg of coenzyme Q10 solid, and the yield was 98.7%.
  • the total yield is 97.3%, and the effective content of the finished product is 98.5%.
  • Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2860mg/L, and the total amount is 286kg.
  • Coenzyme Q10 fermentation broth was filtered by plate and frame to obtain 23.1 tons of wet bacterial residue 1, 9240 L of methanol was added, stirred and allowed to stand for 40 min, and filtered to obtain wet bacterial residue 2.
  • the glass column has a diameter of 0.5 meters and a height of 2.8 meters. 715 kg of filler is added. Coenzyme Q10 butyl acetate solution is added to the chromatography column treated with butyl acetate. The mobile phase is butyl acetate, and the flow rate is 4 L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
  • the total yield is 97.4%, and the effective content of the finished product is 98.7%.
  • Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2900mg/L, and the total amount is 290kg.
  • the coenzyme Q10 fermentation broth was filtered through the plate and frame to obtain 23.3 tons of wet bacterial residue 1, 11650 L of methanol was added, stirred and allowed to stand for 50 minutes, and filtered to obtain wet bacterial residue 2.
  • the glass column has a diameter of 0.5 meters and a height of 2.9 meters. Add 1001kg of filler, and add coenzyme Q10 butyl acetate solution to the column treated with butyl acetate. Use butyl acetate as the mobile phase and the flow rate is 4.5L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
  • the total yield is 97.3%, and the effective content of the finished product is 98.5%.
  • Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2751mg/L, and the total amount is 275.1kg.
  • the coenzyme Q10 fermentation broth was filtered through the plate and frame to obtain 22.1 tons of wet bacterial residue 1, 16506 L of methanol was added, stirred and allowed to stand for 60 min, and filtered to obtain wet bacterial residue 2.
  • the glass column has a diameter of 0.5 meters and a height of 3 meters. Add 1100kg of filler, and add coenzyme Q10 butyl acetate solution to the chromatography column treated with butyl acetate. Use butyl acetate as the mobile phase and the flow rate is 5L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
  • the coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 267.2 kg of coenzyme Q10 solid, and the yield was 98.6%.
  • the total yield is 97.1%, and the effective content of the finished product is 98.4%.
  • the coenzyme Q10 fermentation broth is 100m 3 , the fermentation unit is 2834mg/L, and the total amount of coenzyme Q10 is 283.4kg.
  • the fermentation broth was extracted with ethyl acetate-methanol solvent and evaporated to dryness to obtain 278.9 kg of crude coenzyme Q10 product, and the yield was 98.4%.
  • the non-polar solvent n-hexane was added to the crude product of coenzyme Q10 to dissolve coenzyme Q10.
  • the obtained solution is filtered with a small pore size ceramic membrane to remove insoluble impurities and large molecular weight impurities; in this step, the average pore size of the small pore size ceramic membrane is 1-100 nm, preferably 5-10 nm, to filter out some suspended particles and macromolecular impurities .
  • lye to the obtained filtrate to carry out saponification reaction;
  • the lye is sodium hydroxide, the concentration is 5wt%, the amount of lye added is 0.4 times that of the filtrate, and the temperature is controlled at 24°C.
  • Methanol, ethanol and water were added to the saponification reaction solution in turn, and at the same time, a ceramic microfiltration membrane was used for filtration treatment.
  • a ceramic microfiltration membrane was used for filtration treatment.
  • the concentrated solution of the ceramic microfiltration membrane was distilled under reduced pressure to remove the solvent, coenzyme Q10 was obtained; the total volume of methanol and ethanol added was The amount is controlled at 5 times the volume of the saponification reaction solution, and the amount of washing with water is controlled at 1 time the volume of the saponification reaction solution.
  • the microfiltration membrane has been hydrophobically modified, and the water droplet contact angle on the surface of the microfiltration membrane is in the range of 100-160°.
  • the finished product of coenzyme Q10 is 258kg, and the detected content is 90.4%, and the yield is 91.0%.
  • the fermented cell is 1000kg, and the total amount of coenzyme Q10 is 71.2kg.
  • Continuous countercurrent ultrasonic extraction extract 5% ethyl acetate-95% n-hexane
  • the extract enters into a dynamic circulation low-temperature evaporative concentrator for evaporation and concentration, the concentration temperature is 35 ° C, and the vacuum degree is -0.085MPa to obtain coenzyme Q10 extract 68.2 kg
  • the measured coenzyme Q10 content in the extract was 65.72kg
  • the extraction yield was 92.3%
  • the crude coenzyme Q10 content was 87.5%.
  • the above membrane slurry is spray-dried, and the inlet air temperature is controlled at 140-160° C. and the outlet air temperature is 60-70° C. to obtain 3.1 kg of crude coenzyme Q10 with an effective content of 87.9% and a yield of 91.3%.
  • the above acetone leaching concentrate was added into petroleum ether to dissolve, and the added amount was 3.0 times the volume of the coenzyme Q10 concentrate, then an equal volume of water was added, stirred for 10 min, allowed to stand, separated from water, collected the petroleum ether layer, and put into a silica gel column for purification and separation,
  • the eluent is petroleum ether, the feed rate is 2BV/h, the elution rate is 1.5BV/h, and the feed temperature is 25°C.
  • the eluate containing coenzyme Q10 is collected and distilled under reduced pressure to obtain purified coenzyme Q10 extract concentrate 2.8kg, the effective content is 80.5%, and the yield is 88%.
  • the purified coenzyme Q10 concentrate obtained by concentrating the eluent by distillation under reduced pressure was added with 2 times the volume of absolute ethanol by weight, heated to 48°C, stirred to dissolve, and filtered while hot; under stirring, the filtrate was slowly cooled to 28°C , add 0.02 ⁇ w/v coenzyme Q10 seed crystals, add deionized water until crystals are precipitated, continue to add deionized water, the total amount of water added is 3 times the weight of purified coenzyme Q10 concentrate (978ml), the temperature is controlled at 20°C, and the temperature is kept for 9.0hr. , filtered, and the wet crystals were dried under reduced pressure to obtain 2.2 kg of pure coenzyme Q10 with a content of 98.0% and a total yield of 75.8%.
  • the permeate was concentrated to remove the solvent, and dissolved in n-hexane to prepare a solution with a solid concentration of 300 mg/mL. Carry out three-stage cross-flow extraction, and add equal amount of N,N-dimethylformamide to each stage as extractant. At the end of extraction, the total extraction yield is 89.6%
  • the solution was cooled and crystallized for 24 hours, filtered, and washed with an appropriate amount of cold ethanol. After being fully drained, vacuum dried at 30°C for 6 hours to obtain 0.044kg of yellow coenzyme Q10 fine product with a purity of 98.0%. Based on the coenzyme Q10 in the bacterial residue, the yield of coenzyme Q10 in the whole process was 74.2%.
  • Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2843mg/L, and the total amount is 2.84kg.
  • the fermentation broth was separated from solid and liquid, dried and pulverized the bacterial residue to obtain 4.2 tons of bacterial residue, which was mixed with n-hexane at a mass ratio of 1:2, and then stirred and leached at a temperature of 55°C for 1.5 hours.
  • the microfiltration membrane is filtered to collect the extract and the retentate;
  • the microfiltration membrane is an inorganic ceramic membrane, the molecular weight cut-off is 2000Da, and the filtration temperature is 40°C;
  • the obtained extract and alkali-alcohol solution (containing 80 g of sodium chloride, 20 g of sodium hydroxide, and 80 mL of methanol in 1 L of solution, and the remainder is water) were mixed in a volume ratio of 1:2, shaken for 5 min, left to stand for stratification, and the organic phase was taken. , concentrated by rotary evaporation at a temperature of 55°C and a vacuum of 0.04Mpa, and the obtained concentrated solution was redissolved in n-hexane;
  • the obtained liquid was subjected to silica gel column chromatography, eluted with a n-hexane solution containing 3% (V/V) isopropyl ether, and the elution flow rate was 1 column volume per hour, and the elution was carried out until there was no obvious yellow color; then the obtained
  • the coenzyme Q10 eluate was concentrated under reduced pressure at a temperature of 55 °C and a vacuum of 0.04 Mpa; the obtained concentrated solution was dissolved in absolute ethanol, refrigerated at 0 °C overnight, and vacuum filtered to obtain coenzyme Q10 colloid; finally at a temperature of 2 °C Under vacuum drying conditions, 2.4 kg of pure coenzyme Q10 was obtained, the purity was 98.01%, and the yield was 86%.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to a method for extracting coenzyme Q10 from a fermentation broth of coenzyme Q10, comprising subjecting the fermentation broth of coenzyme Q10 to pre-processing, leaching, crystallization, dissolution, chromatography, concentration under reduced pressure, water washing, and drying. The method of the present invention has the advantages of high extraction yield, excellent product quality, simple extraction process, low production costs, etc. and is beneficial for enhancing market competitiveness of products.

Description

一种从辅酶Q10发酵液中提取辅酶Q10的方法A kind of method for extracting coenzyme Q10 from coenzyme Q10 fermentation broth 技术领域technical field
本发明涉及一种从类球红细菌发酵产生的辅酶Q10发酵液中提取辅酶Q10的方法，属于抗氧化剂提取技术领域。The invention relates to a method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquid produced by fermentation of Rhodobacter sphaeroides, and belongs to the technical field of antioxidant extraction.
背景技术Background technique
辅酶Q10是一种脂溶性抗氧化剂，能激活人体细胞和细胞能量的营养，具有提高人体免疫力、增强抗氧化、延缓衰老和增强人体活力等功能，医学上广泛用于心血管***疾病，国内外广泛将其用于营养保健品及食品添加剂。Coenzyme Q10 is a fat-soluble antioxidant, which can activate the nutrition of human cells and cell energy. It has the functions of improving human immunity, enhancing anti-oxidation, delaying aging and enhancing human vitality. It is widely used in medicine for cardiovascular system diseases. It is widely used in nutritional health care products and food additives.
目前，国内相关文献中对于辅酶Q10的提取纯化，通常采用陶瓷膜过滤工艺和层析工艺，这两种提取方法各具优缺。At present, the extraction and purification of coenzyme Q10 in the relevant domestic literature usually adopts the ceramic membrane filtration process and the chromatography process. These two extraction methods have their own advantages and disadvantages.
例如，中国专利CN111153786A采用的是陶瓷膜过滤工艺，其工艺过程为：将辅酶Q10发酵液经过滤得到的菌粉，分别经浸提和蒸干得到辅酶Q10粗品，然后经过小孔径陶瓷膜过滤、碱化、皂化反应和陶瓷微滤膜进行过滤处理，陶瓷微滤膜的浓缩液减压蒸馏除溶剂之后，得到辅酶Q10。该方法存在的问题是：For example, Chinese patent CN111153786A adopts the ceramic membrane filtration process, and the process is as follows: the bacterial powder obtained by filtering the coenzyme Q10 fermentation broth is respectively leached and evaporated to obtain the crude coenzyme Q10 product, and then filtered through a small pore ceramic membrane, Alkalization, saponification reaction and ceramic microfiltration membrane are carried out for filtration treatment, and after the concentrated solution of ceramic microfiltration membrane is distilled under reduced pressure to remove solvent, coenzyme Q10 is obtained. The problem with this method is:
①、该方法在提取纯化前未对辅酶Q10的发酵液进行预处理，导致粗品中的杂质含量较高，超过了40％以上，不利于下游提取纯化工艺的继续进行。①. In this method, the fermentation broth of coenzyme Q10 is not pretreated before extraction and purification, resulting in a high impurity content in the crude product, exceeding 40%, which is not conducive to the continuation of the downstream extraction and purification process.
②、整个提取纯化工艺步骤较多，其中有2次膜过滤工序和皂化反应，导致提取周期较长，降低了生产效率。②. There are many steps in the whole extraction and purification process, including two membrane filtration processes and saponification reactions, which lead to a long extraction cycle and reduce production efficiency.
③、膜过滤和皂化反应并没有有效解决辅酶Q10杂质含量高的问题，特别是成品中的色素问题，影响了产品的品质。3. Membrane filtration and saponification did not effectively solve the problem of high impurity content of coenzyme Q10, especially the pigment problem in the finished product, which affected the quality of the product.
又如，中国专利CN108863743A采用层析的方法提取纯化辅酶Q10，其工艺过程为：采用的工艺是萃取、沉淀、脱色和层析精制步骤，该技术达到的技术效果是产品辅酶Q10的纯度可达到99.8％以上，总收率可达到98.5％以上，但是存在的问题有：Another example, Chinese patent CN108863743A adopts the method of chromatography to extract and purify coenzyme Q10, and its technological process is as follows: the adopted technology is extraction, precipitation, decolorization and chromatographic refining steps, and the technical effect achieved by this technology is that the purity of product coenzyme Q10 can reach More than 99.8%, the total yield can reach more than 98.5%, but the existing problems are:
①、提取过程中，分别采用了吸附工艺和层析工艺相结合，导致提取周期较长，生产效率较低。① In the extraction process, the combination of adsorption process and chromatography process was adopted respectively, resulting in a long extraction cycle and low production efficiency.
②、吸附工艺中吸附剂是活性白土、硅藻土和活性炭(至少一种)，影 响了产品的收率，使辅酶Q10收率下降了1～2％。②. The adsorbents in the adsorption process are activated clay, diatomite and activated carbon (at least one), which affects the yield of the product and reduces the yield of coenzyme Q10 by 1-2%.
③、层析工艺中使用的吸附剂是硅胶。辅酶Q10吸附过程中，发现硅胶吸附剂存在比表面积小、吸附量低、吸附效率低、吸附效果差等情况。在大生产的条件下，硅胶吸附剂用量很大，而且吸附周期时间较长，收集到的辅酶Q10经检测，杂质含量较高，超过了2％。③ The adsorbent used in the chromatography process is silica gel. During the adsorption process of coenzyme Q10, it was found that the silica gel adsorbent had small specific surface area, low adsorption capacity, low adsorption efficiency, and poor adsorption effect. Under the conditions of large-scale production, the amount of silica gel adsorbent is large, and the adsorption cycle time is long. The collected coenzyme Q10 has a high impurity content, exceeding 2%.
④、该发明中实施例展现的规模较小，未能体现出工业化大生产达到的技术水平。依据该工艺进行了中试验证，菌渣的规模为50kg，进过三次提取和纯化，最终产品的平均收率在90～92％，纯度在93～95％，未能达到该专利所中阐述的技术水平。④. The scale shown in the embodiments of this invention is small, and fails to reflect the technical level achieved by industrialized large-scale production. According to this process, the pilot test has been carried out. The scale of the fungus residue is 50kg. After three extractions and purifications, the average yield of the final product is 90-92%, and the purity is 93-95%, which fails to reach the level described in the patent. technical level.
再如，中国专利CN107337593A采用的工艺是：首先将辅酶Q10发酵液经陶瓷膜微滤和喷雾干燥得到粗品，然后将该粗品用丙酮浸提，所得浸提液分相、减压浓缩得到辅酶Q10提取浓缩液，之后用石油醚萃取、硅胶柱层析、减压蒸馏浓缩洗脱液，最后加入辅酶Q10晶种结晶，减压干燥即可得到辅酶Q10纯品。该技术存在的问题是：Another example, the technique adopted in Chinese patent CN107337593A is: first, the coenzyme Q10 fermentation broth is subjected to ceramic membrane microfiltration and spray drying to obtain a crude product, then the crude product is extracted with acetone, the obtained extract is phase-separated and concentrated under reduced pressure to obtain coenzyme Q10. The concentrate is extracted, followed by extraction with petroleum ether, silica gel column chromatography, and vacuum distillation to concentrate the eluate. Finally, coenzyme Q10 seed crystals are added to crystallize and dried under reduced pressure to obtain pure coenzyme Q10. The problems with this technology are:
①、该技术中没有阐述工艺的技术效果，而且产品提取收率低。五个实施例中，提取总收率在73～7％，总收率较低。①. The technical effect of the process is not described in this technology, and the extraction yield of the product is low. In the five embodiments, the total extraction yield is 73-7%, and the total yield is relatively low.
②、采用硅胶层析工艺，中国专利CN108863743A存在同样的问题。②, using silica gel chromatography process, Chinese patent CN108863743A has the same problem.
中国专利CN106146278A公开了一种从菌渣中提取制备高纯度辅酶Q10的工艺，该技术以菌渣为原料进行渗漉提取得辅酶Q10渗漉提取液，将辅酶Q10提取液经多级萃取进行除杂得萃余液，再将萃余液进行结晶处理，最终得到辅酶Q10。众所周知，渗漉工艺就是动态的浸提工艺，该专利没有阐述辅酶Q10发酵液预处理工艺，其次菌渣中的脂溶性杂质可以通过渗漉工艺进入有机溶液；最后采用多级萃取除杂，只能去除大部分杂质，杂质去除量有限。Chinese patent CN106146278A discloses a process for preparing high-purity coenzyme Q10 by extracting and preparing high-purity coenzyme Q10 from bacterial residue. The raffinate is mixed to obtain the raffinate, and then the raffinate is crystallized to finally obtain coenzyme Q10. As we all know, the percolation process is a dynamic leaching process. The patent does not describe the coenzyme Q10 fermentation broth pretreatment process. Secondly, the fat-soluble impurities in the bacterial residue can enter the organic solution through the percolation process. Finally, multi-stage extraction is used to remove impurities, only Most impurities can be removed, and the amount of impurities removed is limited.
引证文件列表List of citing documents
专利文献Patent Literature
专利文献1 CN111153786A；Patent Document 1 CN111153786A;
专利文献2 CN108863743A；Patent document 2 CN108863743A;
专利文献3 CN107337593A；Patent document 3 CN107337593A;
专利文献4 CN106146278A。Patent Document 4 CN106146278A.
发明内容SUMMARY OF THE INVENTION
技术问题technical problem
本发明的目的在于克服上述现有技术中的缺陷，提供一种有效提高辅酶Q10提取收率和含量，降低生产成本，且提取工艺简单的从辅酶Q10发酵液中提取辅酶Q10的方法。The object of the present invention is to overcome the above-mentioned defects in the prior art, and to provide a method for extracting coenzyme Q10 from coenzyme Q10 fermentation broth with a simple extraction process that effectively improves the extraction yield and content of coenzyme Q10, reduces production costs, and has a simple extraction process.
解决方案solution
为了解决上述技术问题，根据本发明的一实施例，提供了一种从辅酶Q10发酵液中提取辅酶Q10的方法，包括如下步骤：In order to solve the above-mentioned technical problems, according to an embodiment of the present invention, a method for extracting coenzyme Q10 from coenzyme Q10 fermentation broth is provided, comprising the following steps:
(1)预处理(1) Preprocessing
将辅酶Q10发酵液用板框过滤，在得到的湿菌渣①中加入甲醇，搅拌、静置后过滤，所得湿菌渣②中加入水和亚硫酸钠，再次搅拌、静置后过滤，所得湿菌渣③经闪蒸干燥得到固体菌渣；The coenzyme Q10 fermentation broth is filtered with a plate and frame, methanol is added to the obtained wet mushroom residue ①, stirred and allowed to stand, and then filtered, water and sodium sulfite are added to the obtained wet mushroom residue ②, stirred again, and filtered after standing, and the obtained wet bacteria Slag (3) is obtained by flash drying to obtain solid bacterial slag;
(2)浸提(2) leaching
在上述固体菌渣中加入乙二醇-丁醚，搅拌、静置后过滤；Add ethylene glycol-butyl ether to the above-mentioned solid bacterial residue, stir and filter after standing;
(3)结晶(3) Crystallization
在步骤(2)所得滤液中加入水，在30min内快速降温至0～5℃，搅拌、静置后过滤得到固体辅酶Q10；Water is added to the filtrate obtained in step (2), the temperature is rapidly lowered to 0 to 5° C. within 30 min, stirred and left to stand, and then filtered to obtain solid coenzyme Q10;
(4)溶解、层析(4) Dissolution and chromatography
用醋酸丁酯将上述固体辅酶Q10溶解，然后加入到层析设备中，以醋酸丁酯为流动相，在流速3～5L/min条件下进行层析，收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液；The above solid coenzyme Q10 is dissolved with butyl acetate, and then added to a chromatography device, using butyl acetate as a mobile phase, and performing chromatography under the condition of a flow rate of 3 to 5 L/min, collecting a yellow or orange-yellow liquid to obtain Coenzyme Q10 butyl acetate solution;
(5)减压浓缩、水洗和干燥(5) Concentration under reduced pressure, washing with water and drying
将辅酶Q10醋酸丁酯溶液经减压浓缩、水洗、过滤、干燥后即可得到辅酶Q10。The coenzyme Q10 can be obtained by concentrating the coenzyme Q10 butyl acetate solution under reduced pressure, washing with water, filtering and drying.
对于上述方法，在一种可能的实现方式中，步骤(1)中，所述甲醇用量按照V 甲醇:M 湿菌渣①＝2～6L:10kg比例添加；所述水用量按照V 水:M 湿菌渣①＝6～10L:10kg比例添加，亚硫酸钠用量按照M 亚硫酸钠:M 湿菌渣①＝1～2g:10kg比例添加；所述静置时间控制在20～60min。 For aforesaid method, in a possible implementation, in step (1), the methanol consumption is added according to V methanol : M wet mushroom residue① =2～6L:10kg ratio; the water consumption is added according to V water : M Wet mushroom residue① =6～10L:10kg ratio is added, and sodium sulfite consumption is added according to M sodium sulfite :M wet mushroom residue① =1～2g:10kg ratio; Described standing time is controlled at 20～60min.
对于上述方法，在一种可能的实现方式中，步骤(2)中，所述乙二醇-丁醚用量按照V 乙二醇-丁醚:M 固体菌渣＝4～8L:1kg；所述静置时间控制在180～ 220min。 For the above method, in a possible implementation manner, in step (2), the consumption of the ethylene glycol-butyl ether is according to V ethylene glycol-butyl ether : M solid bacterial residue =4～8L: 1kg; The standing time is controlled at 180-220min.
对于上述方法，在一种可能的实现方式中，步骤(3)中，所述水用量按照V 乙二醇-丁醚:V 纯化水＝1L:8～12L；所述静置时间控制在120～160min。 For the above method, in a possible implementation manner, in step (3), the water consumption is according to V ethylene glycol-butyl ether : V purified water =1L:8～12L; the standing time is controlled at 120 ~160min.
对于上述方法，在一种可能的实现方式中，步骤(4)中，所述固体辅酶Q10的溶解度控制在20～30％。For the above method, in a possible implementation manner, in step (4), the solubility of the solid coenzyme Q10 is controlled at 20-30%.
对于上述方法，在一种可能的实现方式中，步骤(4)中，所述层析设备中的玻璃柱高径比为5～6:1，填充剂为内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料，其用量为M 填充剂:M 辅酶Q10发酵液＝2～4kg:1kg。 For the above method, in a possible implementation manner, in step (4), the height-diameter ratio of the glass column in the chromatography equipment is 5-6:1, and the filler is a porous glass column filled with polystyrene gel inside. Silicon carbide microsphere composite material, its dosage is M filler : M coenzyme Q10 fermentation broth =2～4kg:1kg.
对于上述方法，在一种可能的实现方式中，所述内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料的制备工艺为：For the above method, in a possible implementation manner, the preparation process of the porous silicon carbide microsphere composite filled with polystyrene gel is as follows:
1)将PEG2000、古龙酸溶于去离子水中，在磁力搅拌器缓慢搅拌状态下分3～5次将总加入量80％的纳米碳化硅粉末加入，混合均匀后，继续加入质量浓度为5％的聚氨酯溶液，继续搅拌，再缓慢加入总加入量剩余20％纳米氧化硅粉末，持续搅拌120～150min，即可得到凝胶状纳米碳化硅料浆，1) Dissolve PEG2000 and gulonic acid in deionized water, add nano-silicon carbide powder with a total amount of 80% in 3 to 5 times under the slow stirring state of a magnetic stirrer, and after mixing evenly, continue to add a mass concentration of 5%. The polyurethane solution was continuously stirred, and then slowly added the remaining 20% nano-silicon oxide powder in the total amount, and continued stirring for 120-150 min to obtain a gel-like nano-silicon carbide slurry.
上述各物质用量为：The dosage of each of the above substances is:
M PEG2000:M 古龙酸:M 纳米碳化硅粉末:V 5％的聚氨酯溶液＝3kg:1～2kg:5～6kg:14～15L； M PEG2000 : M coronic acid : M nano silicon carbide powder : V 5% polyurethane solution =3kg: 1～2kg: 5～6kg: 14～15L;
2)在上述凝胶状纳米碳化硅料桨中加入真空泵油和吐温20，在700r/min的转速下搅拌40～60min，后加入饱和硫酸钙溶液继续搅拌10～20min，再加入无水乙醇，搅拌80～100min后静置60～80min，待液面分层后，倾去上层泵油，收集烧杯底部的碳化硅微球，用浓度为40～50％乙醇和水将微球彻底清洗干净，在50～60℃下烘干，用80目标准筛将粘结在一起微球筛去，即可得到单分散的碳化硅陶瓷微球生胚，2) Add vacuum pump oil and Tween 20 to the above-mentioned gelatinous nano-silicon carbide paddle, stir at a speed of 700r/min for 40-60min, then add saturated calcium sulfate solution and continue stirring for 10-20min, and then add absolute ethanol After stirring for 80-100min, let stand for 60-80min. After the liquid level is stratified, pour off the upper pump oil, collect the silicon carbide microspheres at the bottom of the beaker, and thoroughly clean the microspheres with 40-50% ethanol and water. , drying at 50-60 °C, and sieving the bonded microspheres with an 80-mesh standard sieve to obtain monodisperse silicon carbide ceramic microsphere green embryos.
上述各物质用量为：M 纳米碳化硅粉末:M 真空泵油:M 吐温20:V 饱和硫酸钙溶液:V 无水乙醇＝1kg:3～4kg：0.5～0.6kg：4～5L：15～20L； The dosage of each above-mentioned substance is: M nano silicon carbide powder : M vacuum pump oil : M Tween 20 : V saturated calcium sulfate solution : V absolute ethanol =1kg: 3～4kg: 0.5～0.6kg: 4～5L: 15～20L ;
3)采用高温马弗炉对碳化硅陶瓷微球生胚进行烧结，具体为：首先以10℃/min的速度将温度升至200℃，保温30min；接着以10℃/min的速度将温度升至300℃，保温30min，最后以5℃/min的速度将温度升至500℃，保温20min；自然冷却到室温即可得到多孔碳化硅微球；3) Use a high temperature muffle furnace to sinter the silicon carbide ceramic microsphere green embryos, specifically: first, the temperature is raised to 200°C at a speed of 10°C/min, and the temperature is kept for 30 minutes; then the temperature is raised at a speed of 10°C/min. to 300°C, hold for 30min, and finally raise the temperature to 500°C at a rate of 5°C/min, hold for 20min; naturally cool to room temperature to obtain porous silicon carbide microspheres;
4)将上述多孔碳化硅微球浸泡在10～20％聚苯乙烯-乙酸丁酯溶液中，在无菌条件，90～100℃下保温60～80min，之后使其缓慢冷却至室温，待聚 苯乙烯凝胶凝固后，加纯化水进行研磨，筛去碎片即可得到内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料，4) Soak the above-mentioned porous silicon carbide microspheres in a 10-20% polystyrene-butyl acetate solution, keep the temperature at 90-100° C. for 60-80 min under sterile conditions, and then slowly cool it to room temperature. After the styrene gel is solidified, purified water is added for grinding, and the fragments are sieved to obtain a porous silicon carbide microsphere composite filled with polystyrene gel.
上述各物质用量为：The dosage of each of the above substances is:
M 多孔碳化硅微球:V 10～20％聚苯乙烯-乙酸丁酯溶液＝1kg:3～5L。 M Porous silicon carbide microspheres : V 10～20% polystyrene-butyl acetate solution =1kg:3～5L.
对于上述方法，在一种可能的实现方式中，步骤(5)中，所述减压浓缩中，控制温度90～100℃，压力-0.1～0MPa。For the above method, in a possible implementation manner, in step (5), in the concentration under reduced pressure, the temperature is controlled to be 90-100° C. and the pressure to be -0.1-0 MPa.
对于上述方法，在一种可能的实现方式中，步骤(5)中，所述水洗是指用纯化水水洗1次，用量是辅酶Q10固体重量的5～10倍；所述干燥是指采用双锥回转真空干燥机干燥14～15h，干燥温度30～40℃，真空压力为-0.02～-0.08MPa，运行频率为8～15Hz。For the above method, in a possible implementation manner, in step (5), the washing refers to washing with purified water once, and the dosage is 5 to 10 times the solid weight of coenzyme Q10; the drying refers to using double Cone rotary vacuum dryer is used for drying for 14~15h, drying temperature is 30~40℃, vacuum pressure is -0.02~-0.08MPa, and operating frequency is 8~15Hz.
有益效果beneficial effect
本发明对于辅酶Q10发酵液采用甲醇进行预处理，采用有机溶剂浸提菌渣中的辅酶Q10，加入亚硫酸钠，保护辅酶Q10，不易被氧化，然后用乙二醇-丁醚进行进一步的提取，从而使得辅酶Q10的提取收率大幅提高，优选地达到了98％。In the present invention, methanol is used for the pretreatment of the coenzyme Q10 fermentation broth, and the coenzyme Q10 in the bacterial residue is extracted with an organic solvent, and sodium sulfite is added to protect the coenzyme Q10 from being oxidized, and then the ethylene glycol-butyl ether is used for further extraction, thereby The extraction yield of coenzyme Q10 is greatly improved, preferably reaching 98%.
本发明采用低温结晶方式，有效去除了水溶性杂质，使得结晶收率大幅提高，例如98％以上。The invention adopts a low-temperature crystallization method, which effectively removes water-soluble impurities, so that the crystallization yield is greatly improved, for example, more than 98%.
综上所述，采用本发明方法，可使辅酶Q10成品有效含量大幅提高，例如优选地可以达到99％，提取总收率也得以提高，例如优选地可以达到95％。本发明的方法具有提取收率高，产品质量优，提取工艺简捷，生产成本低等优势。To sum up, by using the method of the present invention, the effective content of the finished product of Coenzyme Q10 can be greatly increased, for example, it can preferably reach 99%, and the total extraction yield can also be improved, for example, it can preferably reach 95%. The method of the invention has the advantages of high extraction yield, excellent product quality, simple extraction process, low production cost and the like.
根据下面参考附图对示例性实施例的详细说明，本发明的其它特征及方面将变得清楚。Other features and aspects of the present invention will become apparent from the following detailed description of exemplary embodiments with reference to the accompanying drawings.
具体实施方式detailed description
以下将参考附图详细说明本发明的各种示例性实施例、特征和方面。附图中相同的附图标记表示功能相同或相似的元件。尽管在附图中示出了实施例的各种方面，但是除非特别指出，不必按比例绘制附图。Various exemplary embodiments, features and aspects of the present invention will be described in detail below with reference to the accompanying drawings. The same reference numbers in the figures denote elements that have the same or similar functions. While various aspects of the embodiments are shown in the drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实例中，对于本领域技术人员熟知的方法、手段、元件和电路未作详细描述，以便于凸显本发明的主旨。In addition, in order to better illustrate the present invention, numerous specific details are given in the following detailed description. It will be understood by those skilled in the art that the present invention may be practiced without certain specific details. In other instances, methods, means, components and circuits well known to those skilled in the art have not been described in detail so as not to obscure the subject matter of the present invention.
在本发明一个具体的实例中，层析设备的玻璃柱高径比为5:1，填充剂为内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料，其用量为M 填充剂:M 辅酶Q10 发酵液总量＝3kg:1kg，其中M 辅酶Q10发酵液总量(kg)＝发酵液单位(mg/L)×发酵液体积(m 3)/1000。在使用前，先往玻璃柱中加入填充剂，醋酸丁酯加入柱中，浸润140min。浸润结束继续用醋酸丁酯冲洗2遍。层析过程中，流速控制在4L/min。 In a specific example of the present invention, the height-diameter ratio of the glass column of the chromatography equipment is 5:1, the filler is a porous silicon carbide microsphere composite filled with polystyrene gel, and the amount thereof is M filler : M Total amount of coenzyme Q10 fermentation broth =3kg:1kg, wherein M total amount of coenzyme Q10 fermentation broth (kg)=unit of fermentation broth (mg/L)×volume of fermentation broth (m 3 )/1000. Before use, add filler to the glass column, add butyl acetate to the column, soak for 140min. Continue to rinse 2 times with butyl acetate after the infiltration is over. During the chromatography, the flow rate was controlled at 4 L/min.
上述内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料的制备工艺为：The preparation process of the above-mentioned porous silicon carbide microsphere composite filled with polystyrene gel is as follows:
1)将PEG2000、古龙酸溶于去离子水中，在磁力搅拌器缓慢搅拌状态下分4次将总量80％的纳米碳化硅粉末加入，混合均匀后，继续加入质量浓度为5％的聚氨酯溶液，继续搅拌，再缓慢加入剩余20％纳米氧化硅粉末，持续搅拌130min，即可得到颗粒分散均匀、料浆呈凝胶状的纳米碳化硅料桨，1) Dissolve PEG2000 and coronic acid in deionized water, add 80% of the nano-silicon carbide powder in four batches under the state of slow stirring with a magnetic stirrer, and after mixing evenly, continue to add a 5% mass concentration of polyurethane solution , continue to stir, then slowly add the remaining 20% of the nano-silicon oxide powder, and continue to stir for 130 min to obtain a nano-silicon carbide paddle with uniformly dispersed particles and a gel-like slurry.
M PEG2000:M 古龙酸:M 纳米碳化硅粉末:V 5％的聚氨酯溶液＝3kg:1kg:6kg:14L； M PEG2000 : M gulonic acid : M nano silicon carbide powder : V 5% polyurethane solution =3kg:1kg:6kg:14L;
2)在上述凝胶状纳米碳化硅料桨中加入真空泵油和吐温20，在700r/min的转速下搅拌60min，后加入饱和硫酸钙溶液继续搅拌15min，再加入无水乙醇，搅拌100min后静置70min，待液面分层后，倾去上层泵油，收集烧杯底部的碳化硅微球，用浓度为50％乙醇和水将微球彻底清洗干净，在50～60℃下烘干，用80目标准筛将粘结在一起微球筛去，即可得到单分散的碳化硅陶瓷微球生胚，2) Add vacuum pump oil and Tween 20 to the above-mentioned gelatinous nano-silicon carbide paddle, stir at 700r/min for 60min, then add saturated calcium sulfate solution and continue stirring for 15min, then add absolute ethanol, and stir for 100min. Let stand for 70min. After the liquid level is stratified, pour off the upper pump oil, collect the silicon carbide microspheres at the bottom of the beaker, thoroughly clean the microspheres with 50% ethanol and water, and dry them at 50-60°C. Use an 80-mesh standard sieve to sieve the bonded microspheres to obtain monodisperse silicon carbide ceramic microsphere green embryos.
上述各物质用量为：M 纳米碳化硅粉末:M 真空泵油:M 吐温20:V 饱和硫酸钙溶液:V 无水乙醇＝1kg:4kg:0.5kg:4.5L:17L； Above-mentioned each material consumption is: M nano silicon carbide powder : M vacuum pump oil : M Tween 20 : V saturated calcium sulfate solution : V absolute ethanol =1kg: 4kg: 0.5kg: 4.5L: 17L;
3)采用高温马弗炉对碳化硅陶瓷微球生胚进行烧结，具体为：首先以10℃/min的速度将温度升至200℃，保温30min；接着以10℃/min的速度将温度升至300℃，保温30min，最后以5℃/min的速度将温度升至500℃，保温20min；自然冷却到室温即可得到多孔碳化硅微球；3) Use a high temperature muffle furnace to sinter the silicon carbide ceramic microsphere green embryos, specifically: first, the temperature is raised to 200°C at a speed of 10°C/min, and the temperature is kept for 30 minutes; then the temperature is raised at a speed of 10°C/min. to 300°C, hold for 30min, and finally raise the temperature to 500°C at a rate of 5°C/min, hold for 20min; naturally cool to room temperature to obtain porous silicon carbide microspheres;
4)将上述多孔碳化硅微球浸泡在15％聚苯乙烯-乙酸丁酯(乙酸丁酯为溶剂)溶液中，在无菌条件，90～100℃下保温80min，之后使其缓慢冷却至 室温，这样，多孔碳化硅微球中就充满了聚苯乙烯溶液；待聚苯乙烯凝胶凝固后，取出沉积在底部的碳化硅微球，加纯化水进行研磨，加纯化水进行研磨，通过标准筛筛选和流化床去除聚苯乙烯碎片和破碎的碳化硅碎片，即可得到内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料，待聚苯乙烯凝胶凝固后，4) Soak the above-mentioned porous silicon carbide microspheres in a 15% polystyrene-butyl acetate (butyl acetate is a solvent) solution, keep warm at 90-100° C. for 80 min under sterile conditions, and then slowly cool it to room temperature , in this way, the porous silicon carbide microspheres are filled with polystyrene solution; after the polystyrene gel is solidified, take out the silicon carbide microspheres deposited at the bottom, add purified water for grinding, add purified water for grinding, and pass the standard Screening and fluidized bed removal of polystyrene fragments and broken silicon carbide fragments, the porous silicon carbide microsphere composite filled with polystyrene gel can be obtained. After the polystyrene gel is solidified,
上述各物质用量为：The dosage of each of the above substances is:
M 多孔碳化硅微球:V 10～20％聚苯乙烯-乙酸丁酯溶液＝1kg:4L。 M Porous silicon carbide microspheres : V 10-20% polystyrene-butyl acetate solution =1kg:4L.
本发明在层析过程中使用了内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料作为填充剂，该填充剂能够有效分离辅酶Q10及杂质，并且层析周期较短，同硅胶填充剂(周期在18h以上)比较，缩短了周期，例如可以缩短7h。The present invention uses a porous silicon carbide microsphere composite filled with polystyrene gel as a filler in the chromatography process. The filler can effectively separate coenzyme Q10 and impurities, and the chromatography cycle is short, which is similar to the silica filler. (The cycle is more than 18h), the cycle is shortened, for example, it can be shortened by 7h.
实施例1Example 1
辅酶Q10发酵液100m 3，发酵单位2980mg/L，总量为298kg。 Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2980mg/L, and the total amount is 298kg.
辅酶Q10发酵液经板框过滤得到湿菌渣①23.2吨，加入甲醇4640L，搅拌并静置10min，过滤得到湿菌渣②。The coenzyme Q10 fermentation broth was filtered by plate and frame to obtain 23.2 tons of wet bacterial residue ①, 4640 L of methanol was added, stirred and allowed to stand for 10 min, and filtered to obtain wet bacterial residue ②.
湿菌渣②中加入一次水13920L，并加入亚硫酸钠2.32kg，搅拌并静置20min，过滤得到湿菌渣③，经闪蒸干燥，得到固体菌渣4135kg，水分含量为1.9％。Add 13920L of primary water to the wet fungus residue ②, add 2.32 kg of sodium sulfite, stir and let stand for 20 minutes, filter to obtain wet fungal residue ③, and flash dry to obtain 4135 kg of solid fungal residue with a moisture content of 1.9%.
在固体菌渣中加入乙二醇-丁醚16540L，搅拌并静置180min，过滤后得到滤液。Add 16540L of ethylene glycol-butyl ether to the solid bacterial residue, stir and let stand for 180min, and filter to obtain a filtrate.
在所得滤液中加入纯化水132320L，在30min内快速降温至5℃，搅拌并静置120min，过滤后得到固体辅酶Q10 294kg，收率98％。In the obtained filtrate, 132,320 L of purified water was added, rapidly cooled to 5° C. within 30 min, stirred and allowed to stand for 120 min, and 294 kg of solid coenzyme Q10 was obtained after filtration, with a yield of 98%.
在固体辅酶Q10加入醋酸丁酯1460L溶解，浓度控制在20％。Add 1460L of butyl acetate to solid coenzyme Q10 to dissolve, and the concentration is controlled at 20%.
层析设备中，玻璃柱直径0.5米，高2.5米，加入填充剂596kg。将经过醋酸丁酯处理的层析柱内加入辅酶Q10醋酸丁酯溶液，以醋酸丁酯为流动相，流速为3L/min。收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液。In the chromatography equipment, the diameter of the glass column is 0.5 meters and the height is 2.5 meters, and 596kg of filler is added. Coenzyme Q10 butyl acetate solution was added to the chromatography column treated with butyl acetate, butyl acetate was used as the mobile phase, and the flow rate was 3 L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
辅酶Q10醋酸丁酯溶液经减压浓缩得到辅酶Q10固体290kg，收率是98.6％。The coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 290 kg of solid coenzyme Q10, and the yield was 98.6%.
辅酶Q10固体中加入纯化水1420L，水洗60min，过滤后进行双锥干燥，得到纯品辅酶Q10 289.7kg。1420L of purified water was added to the solid coenzyme Q10, washed with water for 60min, filtered and dried in a double cone to obtain 289.7kg of pure coenzyme Q10.
经测算，总收率：97.2％，成品有效含量98.4％。After calculation, the total yield is 97.2%, and the effective content of the finished product is 98.4%.
实施例2Example 2
辅酶Q10发酵液100m 3，发酵单位2763mg/L，总量为276kg。 Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2763mg/L, and the total amount is 276kg.
辅酶Q10发酵液经板框过滤得到湿菌渣①22.8吨，加入甲醇6840L，搅拌并静置30min，过滤得到湿菌渣②。The coenzyme Q10 fermentation broth was filtered through the plate and frame to obtain 22.8 tons of wet bacterial residue ①, 6840 L of methanol was added, stirred and allowed to stand for 30 minutes, and filtered to obtain wet bacterial residue ②.
在湿菌渣②中加入一次水15960L，并加入亚硫酸钠2.74kg，搅拌并静置30min，过滤得到湿菌渣③，经闪蒸干燥，得到固体菌渣4024kg，水分含量为2.1％。Add 15960L of primary water to the wet fungus residue ②, add 2.74 kg of sodium sulfite, stir and let stand for 30min, filter to obtain wet fungal residue ③, and flash dry to obtain 4024 kg of solid fungal residue with a moisture content of 2.1%.
在上述固体菌渣中加入乙二醇-丁醚20120L，搅拌并静置190min，过滤后得到滤液。Add ethylene glycol-butyl ether 20120L to the above-mentioned solid bacterial residue, stir and let stand for 190min, and filter to obtain a filtrate.
在所得滤液中加入纯化水181080L，在30min内快速降温至4℃，搅拌并静置130min，过滤后得到固体辅酶Q10 272kg，收率98.5％。181080L of purified water was added to the obtained filtrate, rapidly cooled to 4°C in 30min, stirred and left to stand for 130min, and 272kg of solid coenzyme Q10 was obtained after filtration, with a yield of 98.5%.
在固体辅酶Q10加入醋酸丁酯1232L溶解，浓度控制在22％。Add 1232L of butyl acetate to solid coenzyme Q10 to dissolve, and the concentration is controlled at 22%.
玻璃柱直径0.5米，高2.6米，加入填充剂678kg，将经过醋酸丁酯处理的层析柱内加入辅酶Q10醋酸丁酯溶液，以醋酸丁酯为流动相，流速为3.5L/min。收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液。The glass column has a diameter of 0.5 meters and a height of 2.6 meters. Add 678 kg of filler, and add coenzyme Q10 butyl acetate solution to the column treated with butyl acetate. Use butyl acetate as the mobile phase and the flow rate is 3.5 L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
将辅酶Q10醋酸丁酯溶液经减压浓缩得到辅酶Q10固体268.6kg，收率是98.7％。The coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 268.6 kg of coenzyme Q10 solid, and the yield was 98.7%.
在辅酶Q10固体中加入纯化水1600L，水洗70min，过滤后进行双锥干燥，得到纯品辅酶Q10 268.5kg。1600L of purified water was added to the solid coenzyme Q10, washed with water for 70min, filtered and dried in a double cone to obtain 268.5kg of pure coenzyme Q10.
经测算，总收率：97.3％，成品有效含量98.5％。After calculation, the total yield is 97.3%, and the effective content of the finished product is 98.5%.
实施例3Example 3
辅酶Q10发酵液100m 3，发酵单位2860mg/L，总量为286kg。 Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2860mg/L, and the total amount is 286kg.
辅酶Q10发酵液经板框过滤得到湿菌渣①23.1吨，加入甲醇9240L，搅拌并静置40min，过滤得到湿菌渣②。Coenzyme Q10 fermentation broth was filtered by plate and frame to obtain 23.1 tons of wet bacterial residue ①, 9240 L of methanol was added, stirred and allowed to stand for 40 min, and filtered to obtain wet bacterial residue ②.
在湿菌渣②中加入一次水18480L，并加入亚硫酸钠3.47kg，搅拌并静置40min，过滤得到湿菌渣③，经闪蒸干燥，得到固体菌渣4052kg，水分含量为2.2％。Add 18480L of water to the wet fungus residue ②, add 3.47 kg of sodium sulfite, stir and let stand for 40min, filter to obtain wet fungal residue ③, and flash dry to obtain 4052 kg of solid fungal residue with a moisture content of 2.2%.
在所得固体菌渣中加入乙二醇-丁醚24312L，搅拌并静置200min，过滤后得到滤液。24312L of ethylene glycol-butyl ether was added to the obtained solid bacterial residue, stirred and allowed to stand for 200min, and filtered to obtain a filtrate.
在滤液中加入纯化水243120L，在30min内快速降温至3℃，搅拌并静置140min，过滤后得到固体辅酶Q10 282kg，收率98.6％。243120L of purified water was added to the filtrate, rapidly cooled to 3°C within 30min, stirred and left to stand for 140min, and 282kg of solid coenzyme Q10 was obtained after filtration, with a yield of 98.6%.
在固体辅酶Q10加入醋酸丁酯1128L溶解，浓度控制在25％。Add 1128L of butyl acetate to solid coenzyme Q10 to dissolve, and the concentration is controlled at 25%.
玻璃柱直径0.5米，高2.8米，加入填充剂715kg，将经过醋酸丁酯处理的层析柱内加入辅酶Q10醋酸丁酯溶液，以醋酸丁酯为流动相，流速为4L/min。收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液。The glass column has a diameter of 0.5 meters and a height of 2.8 meters. 715 kg of filler is added. Coenzyme Q10 butyl acetate solution is added to the chromatography column treated with butyl acetate. The mobile phase is butyl acetate, and the flow rate is 4 L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
将所得辅酶Q10醋酸丁酯溶液经减压浓缩得到辅酶Q10固体278.6kg，收率是98.8％。The obtained coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 278.6 kg of coenzyme Q10 solid, with a yield of 98.8%.
在辅酶Q10固体中加入纯化水2230L，水洗80min，过滤后进行双锥干燥，得到纯品辅酶Q10 278.5kg。2230L of purified water was added to the solid coenzyme Q10, washed with water for 80min, filtered and dried in a double cone to obtain 278.5kg of pure coenzyme Q10.
经测算，总收率：97.4％，成品有效含量98.7％。After calculation, the total yield is 97.4%, and the effective content of the finished product is 98.7%.
实施例4Example 4
辅酶Q10发酵液100m 3，发酵单位2900mg/L，总量为290kg。 Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2900mg/L, and the total amount is 290kg.
辅酶Q10发酵液经板框过滤得到湿菌渣①23.3吨，加入甲醇11650L，搅拌并静置50min，过滤得到湿菌渣②。The coenzyme Q10 fermentation broth was filtered through the plate and frame to obtain 23.3 tons of wet bacterial residue ①, 11650 L of methanol was added, stirred and allowed to stand for 50 minutes, and filtered to obtain wet bacterial residue ②.
在湿菌渣②中加入一次水20970L，并加入亚硫酸钠4.19kg，搅拌并静置50min，过滤得到湿菌渣③，经闪蒸干燥，得到固体菌渣4101kg，水分含量为2.4％。Add 20970L of primary water to the wet mushroom residue ②, add 4.19kg of sodium sulfite, stir and let stand for 50min, filter to obtain wet mushroom residue ③, and flash dry to obtain 4101kg of solid bacteria residue with a moisture content of 2.4%.
在上述固体菌渣中加入乙二醇-丁醚28707L，搅拌并静置210min，过滤后得到滤液。Add 28707L of ethylene glycol-butyl ether to the above-mentioned solid bacterial residue, stir and let stand for 210min, and filter to obtain a filtrate.
在滤液中加入纯化水315777L，在30min内快速降温至2℃，搅拌并静置150min，过滤后得到固体辅酶Q10 286kg，收率98.5％。315777L of purified water was added to the filtrate, rapidly cooled to 2°C within 30min, stirred and allowed to stand for 150min, and 286kg of solid coenzyme Q10 was obtained after filtration, with a yield of 98.5%.
在固体辅酶Q10加入醋酸丁酯1020L溶解，浓度控制在28％。Add 1020L of butyl acetate to solid coenzyme Q10 to dissolve, and the concentration is controlled at 28%.
玻璃柱直径0.5米，高2.9米，加入填充剂1001kg，将经过醋酸丁酯处理的层析柱内加入辅酶Q10醋酸丁酯溶液，以醋酸丁酯为流动相，流速为4.5L/min。收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液。The glass column has a diameter of 0.5 meters and a height of 2.9 meters. Add 1001kg of filler, and add coenzyme Q10 butyl acetate solution to the column treated with butyl acetate. Use butyl acetate as the mobile phase and the flow rate is 4.5L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
在所得辅酶Q10醋酸丁酯溶液经减压浓缩得到辅酶Q10固体282.3kg，收率是98.7％。The obtained coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 282.3 kg of coenzyme Q10 solid, and the yield was 98.7%.
在辅酶Q10固体中加入纯化水2500L，水洗90min，过滤后进行双锥干燥，得到纯品辅酶Q10 282.2kg。2500L of purified water was added to the solid coenzyme Q10, washed with water for 90min, filtered and dried in a double cone to obtain 282.2kg of pure coenzyme Q10.
经测算，总收率：97.3％，成品有效含量98.5％。After calculation, the total yield is 97.3%, and the effective content of the finished product is 98.5%.
实施例5Example 5
辅酶Q10发酵液100m 3，发酵单位2751mg/L，总量为275.1kg。 Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2751mg/L, and the total amount is 275.1kg.
辅酶Q10发酵液经板框过滤得到湿菌渣①22.1吨，加入甲醇16506L，搅拌并静置60min，过滤得到湿菌渣②。The coenzyme Q10 fermentation broth was filtered through the plate and frame to obtain 22.1 tons of wet bacterial residue ①, 16506 L of methanol was added, stirred and allowed to stand for 60 min, and filtered to obtain wet bacterial residue ②.
在湿菌渣②中加入一次水22100L，并加入亚硫酸钠4.42kg，搅拌并静置60min，过滤得到湿菌渣③，经闪蒸干燥，得到固体菌渣4085kg，水分含量为2.0％。Add 22100L of primary water to the wet fungus residue ②, add 4.42 kg of sodium sulfite, stir and let stand for 60min, filter to obtain wet fungal residue ③, and flash dry to obtain 4085 kg of solid fungal residue with a moisture content of 2.0%.
在菌渣中加入乙二醇-丁醚32680L，搅拌并静置220min，过滤后得到滤液。Add 32680L of ethylene glycol-butyl ether to the bacterial residue, stir and let stand for 220min, and filter to obtain a filtrate.
滤液中加入纯化水392160L，在30min内快速降温至0℃，搅拌并静置160min，过滤后得到固体辅酶Q10 271kg，收率98.4％。392160L of purified water was added to the filtrate, rapidly cooled to 0°C within 30min, stirred and allowed to stand for 160min, and 271kg of solid coenzyme Q10 was obtained after filtration, with a yield of 98.4%.
在上述固体辅酶Q10加入醋酸丁酯903L溶解，浓度控制在30％。Add 903L of butyl acetate to the above solid coenzyme Q10 to dissolve, and the concentration is controlled at 30%.
玻璃柱直径0.5米，高3米，加入填充剂1100kg，将经过醋酸丁酯处理的层析柱内加入辅酶Q10醋酸丁酯溶液，以醋酸丁酯为流动相，流速为5L/min。收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液。The glass column has a diameter of 0.5 meters and a height of 3 meters. Add 1100kg of filler, and add coenzyme Q10 butyl acetate solution to the chromatography column treated with butyl acetate. Use butyl acetate as the mobile phase and the flow rate is 5L/min. The yellow or orange-yellow liquid was collected to obtain a coenzyme Q10 butyl acetate solution.
将辅酶Q10醋酸丁酯溶液经减压浓缩得到辅酶Q10固体267.2kg，收率是98.6％。The coenzyme Q10 butyl acetate solution was concentrated under reduced pressure to obtain 267.2 kg of coenzyme Q10 solid, and the yield was 98.6%.
在辅酶Q10固体中加入纯化水2500L，水洗90min，过滤后进行双锥干燥，得到纯品辅酶Q10 267.1kg。2500L of purified water was added to the solid coenzyme Q10, washed with water for 90min, filtered and dried in a double cone to obtain 267.1kg of pure coenzyme Q10.
经测算，总收率：97.1％，成品有效含量98.4％。After calculation, the total yield is 97.1%, and the effective content of the finished product is 98.4%.
对比例1Comparative Example 1
辅酶Q10发酵液100m 3，发酵单位2834mg/L，辅酶Q10总量为283.4kg。 The coenzyme Q10 fermentation broth is 100m 3 , the fermentation unit is 2834mg/L, and the total amount of coenzyme Q10 is 283.4kg.
发酵液采用乙酸乙酯-甲醇溶剂浸提、蒸干后得到辅酶Q10粗产品278.9kg，收率是98.4％。辅酶Q10粗产品中加入非极性溶剂正己烷，使辅酶Q10溶解。The fermentation broth was extracted with ethyl acetate-methanol solvent and evaporated to dryness to obtain 278.9 kg of crude coenzyme Q10 product, and the yield was 98.4%. The non-polar solvent n-hexane was added to the crude product of coenzyme Q10 to dissolve coenzyme Q10.
采用小孔径陶瓷膜对得到的溶液进行过滤，去除不溶性杂质和大分子量杂质；本步骤中，小孔径陶瓷膜平均孔径1-100nm，优选5-10nm，将一些悬浮颗粒以及大分子的杂质滤除。The obtained solution is filtered with a small pore size ceramic membrane to remove insoluble impurities and large molecular weight impurities; in this step, the average pore size of the small pore size ceramic membrane is 1-100 nm, preferably 5-10 nm, to filter out some suspended particles and macromolecular impurities .
在所得滤液中加入碱液，进行皂化反应；碱液是氢氧化钠、浓度是5wt％， 碱液的加入量是滤液的0.4倍，温度控制在24℃。Add lye to the obtained filtrate to carry out saponification reaction; the lye is sodium hydroxide, the concentration is 5wt%, the amount of lye added is 0.4 times that of the filtrate, and the temperature is controlled at 24°C.
在皂化反应液中依次加入甲醇、乙醇、水，并且同时采用陶瓷微滤膜进行过滤处理，陶瓷微滤膜的浓缩液减压蒸馏除溶剂之后，得到辅酶Q10；加入的甲醇和乙醇的总体积量控制在皂化反应液体积5倍，加水水洗量控制在皂化反应液体积的1倍。采用经过了疏水改性处理微滤膜，微滤膜表面的水滴接触角的范围是100-160°。Methanol, ethanol and water were added to the saponification reaction solution in turn, and at the same time, a ceramic microfiltration membrane was used for filtration treatment. After the concentrated solution of the ceramic microfiltration membrane was distilled under reduced pressure to remove the solvent, coenzyme Q10 was obtained; the total volume of methanol and ethanol added was The amount is controlled at 5 times the volume of the saponification reaction solution, and the amount of washing with water is controlled at 1 time the volume of the saponification reaction solution. The microfiltration membrane has been hydrophobically modified, and the water droplet contact angle on the surface of the microfiltration membrane is in the range of 100-160°.
辅酶Q10成品258kg，经检测含量90.4％，收率是91.0％。The finished product of coenzyme Q10 is 258kg, and the detected content is 90.4%, and the yield is 91.0%.
对比例2Comparative Example 2
发酵菌体1000kg，辅酶Q10总量为71.2kg。连续逆流超声提取(提取剂5％乙酸乙酯-95％正己烷)，提取液进入到动态循环低温蒸发浓缩器中蒸发浓缩，浓缩温度35℃，真空度-0.085MPa，得到辅酶Q10浸膏68.2kg，测的浸膏中辅酶Q10含量65.72kg，提取收率92.3％，辅酶Q10粗品含量为87.5％。The fermented cell is 1000kg, and the total amount of coenzyme Q10 is 71.2kg. Continuous countercurrent ultrasonic extraction (extractant 5% ethyl acetate-95% n-hexane), the extract enters into a dynamic circulation low-temperature evaporative concentrator for evaporation and concentration, the concentration temperature is 35 ° C, and the vacuum degree is -0.085MPa to obtain coenzyme Q10 extract 68.2 kg, the measured coenzyme Q10 content in the extract was 65.72kg, the extraction yield was 92.3%, and the crude coenzyme Q10 content was 87.5%.
对比例3Comparative Example 3
取辅酶Q10发酵液1m 3(发酵单位2900mg/L)，进陶瓷膜微滤膜(孔径0.1um)过滤，进料温度25℃，透析比1:1.5，浓缩倍数1.8倍，得到膜浆231L。 Take 1m 3 of coenzyme Q10 fermentation broth (fermentation unit 2900mg/L), filter it through a ceramic membrane microfiltration membrane (pore size 0.1um), feed temperature 25°C, dialysis ratio 1:1.5, concentration ratio 1.8 times, and obtain 231L of membrane slurry.
将以上膜浆，经喷雾干燥，控制进风温度140～160℃，出风温度60～70℃，得到辅酶Q10粗品3.1kg，有效含量87.9％，收率91.3％。The above membrane slurry is spray-dried, and the inlet air temperature is controlled at 140-160° C. and the outlet air temperature is 60-70° C. to obtain 3.1 kg of crude coenzyme Q10 with an effective content of 87.9% and a yield of 91.3%.
粗品中加入粗品重量3倍体积的丙酮，加入0.5‰(w/v)沸石，缓慢升温至49℃，回流2.0hr后，趁热经陶瓷滤芯过滤器过滤(孔径0.5um)，滤渣再重复浸提一次，浸提条件不变。收集浸提液，转入分相罐中，加入15％(w/v)NaCL饱和液，温度28℃，搅拌30min，静置20min，收集上层清液，减压蒸馏，得到辅酶Q10提取浓缩物3.8kg，有效含量66.11％，收率92.2％。Add 3 times the volume of acetone to the crude product, add 0.5‰(w/v) zeolite, slowly heat up to 49°C, reflux for 2.0hr, filter through a ceramic filter (pore size 0.5um) while hot, and repeat the immersion of the filter residue. Once mentioned, the extraction conditions remain unchanged. Collect the extract, transfer it to a phase separation tank, add 15% (w/v) NaCl saturated solution, temperature at 28°C, stir for 30min, let stand for 20min, collect the supernatant, and distill under reduced pressure to obtain coenzyme Q10 extract concentrate 3.8kg, effective content 66.11%, yield 92.2%.
将以上丙酮浸提浓缩物加入石油醚溶解，加量为辅酶Q10浓缩物3.0倍体积，再加入等体积水，搅拌10min，静置，分水，收集石油醚层，进硅胶柱进行纯化分离，洗脱液为石油醚，进料速度以2BV/h，洗脱速度以1.5BV/h，进料温度25℃，收集含辅酶Q10段洗脱液，减压蒸馏，得到纯化辅酶Q10提取浓缩物2.8kg，有效含量80.5％，收率88％。The above acetone leaching concentrate was added into petroleum ether to dissolve, and the added amount was 3.0 times the volume of the coenzyme Q10 concentrate, then an equal volume of water was added, stirred for 10 min, allowed to stand, separated from water, collected the petroleum ether layer, and put into a silica gel column for purification and separation, The eluent is petroleum ether, the feed rate is 2BV/h, the elution rate is 1.5BV/h, and the feed temperature is 25°C. The eluate containing coenzyme Q10 is collected and distilled under reduced pressure to obtain purified coenzyme Q10 extract concentrate 2.8kg, the effective content is 80.5%, and the yield is 88%.
将以上减压蒸馏浓缩洗脱液得到的纯化辅酶Q10浓缩物，加入其重量2倍体积无水乙醇，加热至48℃，搅拌溶解，趁热过滤；搅拌下，将过滤液缓慢降温至28℃，加入0.02‰w/v辅酶Q10晶种，加入去离子水至析出晶体，继 续加入去离子水，总加水量为纯化辅酶Q10浓缩物重量3倍(978ml)，温度控制20℃，保温9.0hr，过滤，湿晶体经减压干燥，得到辅酶Q10纯品2.2kg，含量98.0％，总收率75.8％。The purified coenzyme Q10 concentrate obtained by concentrating the eluent by distillation under reduced pressure was added with 2 times the volume of absolute ethanol by weight, heated to 48°C, stirred to dissolve, and filtered while hot; under stirring, the filtrate was slowly cooled to 28°C , add 0.02‰w/v coenzyme Q10 seed crystals, add deionized water until crystals are precipitated, continue to add deionized water, the total amount of water added is 3 times the weight of purified coenzyme Q10 concentrate (978ml), the temperature is controlled at 20°C, and the temperature is kept for 9.0hr. , filtered, and the wet crystals were dried under reduced pressure to obtain 2.2 kg of pure coenzyme Q10 with a content of 98.0% and a total yield of 75.8%.
对比例4Comparative Example 4
称取菌渣(其中，辅酶Q10质量百分比为2.6％)1000g(0.06kg辅酶Q10)，湿法装填入渗漉柱(Φ20.0×35cm)内，装填均匀后其堆积体积为1300mL，10℃恒温浸泡2h，使其充分溶胀，开启渗漉柱出口阀，并同时从柱顶连续加入正己烷，控制流速2.5～3.0mL/min，直至收集的渗漉液体积为1250mL。按照以上步骤重复操作1次，得到渗漉液体积为2480mL，辅酶Q10含量0.023kg/L，收率是95.1％。Weigh 1000g (0.06kg of coenzyme Q10) of bacterial residue (wherein, the mass percentage of coenzyme Q10 is 2.6%), and fill it into a percolation column (Φ20.0×35cm) by wet method. Soak at constant temperature for 2 hours to fully swell, open the outlet valve of the percolation column, and continuously add n-hexane from the top of the column at the same time, and control the flow rate of 2.5-3.0 mL/min until the volume of the collected permeate is 1250 mL. According to the above steps, the operation was repeated once, and the volume of the permeate obtained was 2480 mL, the content of coenzyme Q10 was 0.023 kg/L, and the yield was 95.1%.
将渗漉液浓缩除去溶剂，加正己烷溶解，配制成固形物浓度300mg/mL的溶液。进行三级错流萃取，每级加入等量N,N-二甲基甲酰胺作萃取剂。萃取结束，萃取总收率是89.6％The permeate was concentrated to remove the solvent, and dissolved in n-hexane to prepare a solution with a solid concentration of 300 mg/mL. Carry out three-stage cross-flow extraction, and add equal amount of N,N-dimethylformamide to each stage as extractant. At the end of extraction, the total extraction yield is 89.6%
溶液冷却结晶24h后过滤，并用适量冷乙醇洗涤。充分抽干后，于30℃真空干燥6h，得0.044kg黄色辅酶Q10精品，纯度为98.0％，以菌渣中辅酶Q10为基准，整个工艺辅酶Q10的收率为74.2％。The solution was cooled and crystallized for 24 hours, filtered, and washed with an appropriate amount of cold ethanol. After being fully drained, vacuum dried at 30°C for 6 hours to obtain 0.044kg of yellow coenzyme Q10 fine product with a purity of 98.0%. Based on the coenzyme Q10 in the bacterial residue, the yield of coenzyme Q10 in the whole process was 74.2%.
对比例5Comparative Example 5
辅酶Q10发酵液100m 3，发酵单位2843mg/L，总量为2.84kg。 Coenzyme Q10 fermentation broth is 100m 3 , fermentation unit is 2843mg/L, and the total amount is 2.84kg.
发酵液经固液分离，烘干后将菌渣进行粉碎，得到菌渣4.2吨，与正己烷按质量比1∶2混合，混匀后搅拌浸提，温度55℃，浸提1.5h，经微滤膜过滤，收集提取液和截留物；所述微滤膜为无机陶瓷膜，截留分子量为2000Da，过滤温度为40℃；The fermentation broth was separated from solid and liquid, dried and pulverized the bacterial residue to obtain 4.2 tons of bacterial residue, which was mixed with n-hexane at a mass ratio of 1:2, and then stirred and leached at a temperature of 55°C for 1.5 hours. The microfiltration membrane is filtered to collect the extract and the retentate; the microfiltration membrane is an inorganic ceramic membrane, the molecular weight cut-off is 2000Da, and the filtration temperature is 40°C;
得到的提取液和碱醇溶液(1L溶液中含氯化钠80g、氢氧化钠20g、甲醇80mL，余量为水)按体积比1∶2混合，振荡5min，静置分层，取有机相，在温度55℃，真空度0.04Mpa旋转蒸发浓缩，所得浓缩液用正己烷重溶；The obtained extract and alkali-alcohol solution (containing 80 g of sodium chloride, 20 g of sodium hydroxide, and 80 mL of methanol in 1 L of solution, and the remainder is water) were mixed in a volume ratio of 1:2, shaken for 5 min, left to stand for stratification, and the organic phase was taken. , concentrated by rotary evaporation at a temperature of 55°C and a vacuum of 0.04Mpa, and the obtained concentrated solution was redissolved in n-hexane;
所得液体进行硅胶柱层析，采用含3％(V/V)异丙醚的正己烷溶液进行洗脱，洗脱流速为每小时1倍柱体积，洗脱至无明显黄色为止；然后将所得辅酶Q10洗脱液在温度55℃，真空度0.04Mpa条件下减压浓缩；将所得浓缩液溶解于无水乙醇中，0℃下冷藏过夜，抽真空过滤得辅酶Q10胶体；最后在温度2℃条件下真空干燥，即得纯品辅酶Q102.4kg，纯度为98.01％；收率86％。The obtained liquid was subjected to silica gel column chromatography, eluted with a n-hexane solution containing 3% (V/V) isopropyl ether, and the elution flow rate was 1 column volume per hour, and the elution was carried out until there was no obvious yellow color; then the obtained The coenzyme Q10 eluate was concentrated under reduced pressure at a temperature of 55 °C and a vacuum of 0.04 Mpa; the obtained concentrated solution was dissolved in absolute ethanol, refrigerated at 0 °C overnight, and vacuum filtered to obtain coenzyme Q10 colloid; finally at a temperature of 2 °C Under vacuum drying conditions, 2.4 kg of pure coenzyme Q10 was obtained, the purity was 98.01%, and the yield was 86%.

Claims (9)

  1. 一种从辅酶Q10发酵液中提取辅酶Q10的方法，其特征在于，包括如下步骤：A method for extracting coenzyme Q10 from coenzyme Q10 fermentation broth, comprising the steps of:
    (1)预处理(1) Preprocessing
    将辅酶Q10发酵液用板框过滤，在得到的湿菌渣①中加入甲醇，搅拌、静置后过滤，所得湿菌渣②中加入水和亚硫酸钠，再次搅拌、静置后过滤，所得湿菌渣③经闪蒸干燥得到固体菌渣；The coenzyme Q10 fermentation broth is filtered with a plate and frame, methanol is added to the obtained wet mushroom residue ①, stirred and allowed to stand, and then filtered, water and sodium sulfite are added to the obtained wet mushroom residue ②, stirred again, and filtered after standing, and the obtained wet bacteria Slag ③ is obtained by flash drying to obtain solid bacterial slag;
    (2)浸提(2) leaching
    在上述固体菌渣中加入乙二醇-丁醚，搅拌、静置后过滤；Add ethylene glycol-butyl ether to the above-mentioned solid bacterial residue, stir and filter after standing;
    (3)结晶(3) Crystallization
    在步骤(2)所得滤液中加入水，在30min内快速降温至0～5℃，搅拌、静置后过滤得到固体辅酶Q10；Water is added to the filtrate obtained in step (2), the temperature is rapidly lowered to 0 to 5° C. within 30 minutes, stirred and left to stand, and then filtered to obtain solid coenzyme Q10;
    (4)溶解、层析(4) Dissolution and chromatography
    用醋酸丁酯将上述固体辅酶Q10溶解，然后加入到层析设备中，以醋酸丁酯为流动相，在流速3～5L/min条件下进行层析，收集显示黄色或橙黄色的液体，得到辅酶Q10醋酸丁酯溶液；The above solid coenzyme Q10 is dissolved with butyl acetate, and then added to a chromatography device, using butyl acetate as a mobile phase, and performing chromatography under the condition of a flow rate of 3 to 5 L/min, collecting a yellow or orange-yellow liquid to obtain Coenzyme Q10 butyl acetate solution;
    (5)减压浓缩、水洗和干燥(5) Concentration under reduced pressure, washing with water and drying
    将辅酶Q10醋酸丁酯溶液经减压浓缩、水洗、过滤、干燥后即可得到辅酶Q10。The coenzyme Q10 can be obtained by concentrating the coenzyme Q10 butyl acetate solution under reduced pressure, washing with water, filtering and drying.
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中，所述甲醇用量按照V 甲醇:M 湿菌渣①＝2～6L:10kg比例添加；所述水用量按照V 水:M 湿菌渣①＝6～10L:10kg比例添加，亚硫酸钠用量按照M 亚硫酸钠:M 湿菌渣①＝1～2g:10kg比例添加；所述静置时间控制在20～60min。 method according to claim 1, is characterized in that, in step (1), described methanol consumption is added according to V methanol : M wet fungus residue① =2～6L:10kg ratio; Described water consumption is added according to V water : M Wet mushroom residue① =6～10L:10kg ratio is added, and sodium sulfite consumption is added according to M sodium sulfite :M wet mushroom residue① =1～2g:10kg ratio; Described standing time is controlled at 20～60min.
  3. 根据权利要求1或2所述的方法，其特征在于，步骤(2)中，所述乙二醇-丁醚用量按照V 乙二醇-丁醚:M 固体菌渣＝4～8L:1kg；所述静置时间控制在180～220min。 method according to claim 1 and 2, is characterized in that, in step (2), described ethylene glycol-butyl ether consumption is according to V ethylene glycol-butyl ether : M solid bacterial residue =4～8L: 1kg; The standing time is controlled at 180-220 min.
  4. 根据权利要求1-3任一项所述的方法，其特征在于，步骤(3)中，所述水用量按照V 乙二醇-丁醚:V 纯化水＝1L:8～12L；所述静置时间控制在120～160min。 The method according to any one of claims 1-3, wherein in step (3), the water consumption is according to V ethylene glycol-butyl ether : V purified water =1L:8～12L; The setting time is controlled at 120-160min.
  5. 根据权利要求1-4任一项所述的方法，其特征在于，步骤(4)中，所述固体辅酶Q10的溶解度控制在20～30％。The method according to any one of claims 1-4, wherein in step (4), the solubility of the solid coenzyme Q10 is controlled at 20-30%.
  6. 根据权利要求1-5任一项所述的方法，其特征在于，步骤(4)中，所述层析设备中的玻璃柱高径比为5～6:1，填充剂为内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料，其用量为M 填充剂:M 辅酶Q10发酵液＝2～4kg:1kg。 The method according to any one of claims 1-5, characterized in that, in step (4), the height-diameter ratio of the glass column in the chromatography equipment is 5-6:1, and the filler is polyphenylene filled internally. For the porous silicon carbide microsphere composite material of ethylene gel, the dosage is M filler : M coenzyme Q10 fermentation broth =2-4kg:1kg.
  7. 根据权利要求6所述的方法，其特征在于，所述内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料的制备工艺为：The method according to claim 6, wherein the preparation process of the porous silicon carbide microsphere composite material filled with polystyrene gel is:
    1)将PEG2000、古龙酸溶于去离子水中，在磁力搅拌器缓慢搅拌状态下分3～5次将总加入量80％的纳米碳化硅粉末加入，混合均匀后，继续加入质量浓度为5％的聚氨酯溶液，继续搅拌，再缓慢加入总加入量剩余20％纳米氧化硅粉末，持续搅拌120～150min，即可得到凝胶状纳米碳化硅料浆，1) Dissolve PEG2000 and gulonic acid in deionized water, add nano-silicon carbide powder with a total amount of 80% in 3 to 5 times under the slow stirring state of a magnetic stirrer, and after mixing evenly, continue to add a mass concentration of 5%. The polyurethane solution was continuously stirred, and then slowly added the remaining 20% nano-silicon oxide powder in the total amount, and continued stirring for 120-150 min to obtain a gel-like nano-silicon carbide slurry.
    上述各物质用量为：The dosage of each of the above substances is:
    M PEG2000:M 古龙酸:M 纳米碳化硅粉末:V 5％的聚氨酯溶液＝3kg:1～2kg:5～6kg:14～15L； M PEG2000 : M coronic acid : M nano silicon carbide powder : V 5% polyurethane solution =3kg: 1～2kg: 5～6kg: 14～15L;
    2)在上述凝胶状纳米碳化硅料桨中加入真空泵油和吐温20，在700r/min的转速下搅拌40～60min，后加入饱和硫酸钙溶液继续搅拌10～20min，再加入无水乙醇，搅拌80～100min后静置60～80min，待液面分层后，倾去上层泵油，收集烧杯底部的碳化硅微球，用浓度为40～50％乙醇和水将微球彻底清洗干净，在50～60℃下烘干，用80目标准筛将粘结在一起微球筛去，即可得到单分散的碳化硅陶瓷微球生胚，2) Add vacuum pump oil and Tween 20 to the above-mentioned gelatinous nano-silicon carbide paddle, stir at a speed of 700r/min for 40-60min, then add saturated calcium sulfate solution and continue stirring for 10-20min, and then add absolute ethanol After stirring for 80-100min, let stand for 60-80min. After the liquid level is stratified, pour off the upper pump oil, collect the silicon carbide microspheres at the bottom of the beaker, and thoroughly clean the microspheres with 40-50% ethanol and water. , drying at 50-60 °C, and sieving the bonded microspheres with an 80-mesh standard sieve to obtain monodisperse silicon carbide ceramic microsphere green embryos.
    上述各物质用量为：M 纳米碳化硅粉末:M 真空泵油:M 吐温20:V 饱和硫酸钙溶液:V 无水乙醇＝1kg:3～4kg：0.5～0.6kg：4～5L：15～20L； The dosage of each above-mentioned substance is: M nano silicon carbide powder : M vacuum pump oil : M Tween 20 : V saturated calcium sulfate solution : V absolute ethanol =1kg: 3～4kg: 0.5～0.6kg: 4～5L: 15～20L ;
    3)采用高温马弗炉对碳化硅陶瓷微球生胚进行烧结，具体为：首先以10℃/min的速度将温度升至200℃，保温30min；接着以10℃/min的速度将温度升至300℃，保温30min，最后以5℃/min的速度将温度升至500℃，保温20min；自然冷却到室温即可得到多孔碳化硅微球；3) Use a high temperature muffle furnace to sinter the silicon carbide ceramic microsphere green embryos, specifically: first, the temperature is raised to 200°C at a speed of 10°C/min, and the temperature is kept for 30 minutes; then the temperature is raised at a speed of 10°C/min. to 300°C, hold for 30min, and finally raise the temperature to 500°C at a rate of 5°C/min, hold for 20min; naturally cool to room temperature to obtain porous silicon carbide microspheres;
    4)将上述多孔碳化硅微球浸泡在10～20％聚苯乙烯-乙酸丁酯溶液中，在无菌条件，90～100℃下保温60～80min，之后使其缓慢冷却至室温，待聚苯乙烯凝胶凝固后，加纯化水进行研磨，筛去碎片即可得到内部填充聚苯乙烯凝胶的多孔碳化硅微球复合料，4) Soak the above-mentioned porous silicon carbide microspheres in a 10-20% polystyrene-butyl acetate solution, keep the temperature at 90-100° C. for 60-80 min under sterile conditions, and then slowly cool it to room temperature. After the styrene gel is solidified, add purified water for grinding, and sieve off the fragments to obtain a porous silicon carbide microsphere composite filled with polystyrene gel.
    上述各物质用量为：The dosage of each of the above substances is:
    M 多孔碳化硅微球:V 10～20％聚苯乙烯-乙酸丁酯溶液＝1kg:3～5L。 M Porous silicon carbide microspheres : V 10～20% polystyrene-butyl acetate solution =1kg:3～5L.
  8. 根据权利要求1-7任一项所述的方法，其特征在于，步骤(5)中， 所述减压浓缩中，控制温度90～100℃，压力-0.1～0MPa。The method according to any one of claims 1-7, characterized in that, in step (5), in the concentration under reduced pressure, the temperature is controlled to be 90-100° C. and the pressure to be -0.1-0 MPa.
  9. 根据权利要求1-8任一项所述的方法，其特征在于，步骤(5)中，所述水洗是指用纯化水水洗1次，用量是辅酶Q10固体重量的5～10倍；所述干燥是指采用双锥回转真空干燥机干燥14～15h，干燥温度30～40℃，真空压力为-0.02～-0.08MPa，运行频率为8～15Hz。The method according to any one of claims 1-8, characterized in that, in step (5), the washing with purified water refers to washing once with purified water, and the dosage is 5 to 10 times the solid weight of coenzyme Q10; the Drying refers to using a double-cone rotary vacuum dryer to dry for 14-15 hours, the drying temperature is 30-40°C, the vacuum pressure is -0.02--0.08MPa, and the operating frequency is 8-15Hz.
PCT/CN2021/115304 2020-09-21 2021-08-30 Method for extracting coenzyme q10 from fermentation broth of coenzyme q10 WO2022057603A1 (en)

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CN112174796B (en) * 2020-09-21 2023-02-14 宁夏泰胜生物科技有限公司 Method for extracting coenzyme Q10 from coenzyme Q10 fermentation liquor
CN116063254A (en) * 2021-10-30 2023-05-05 宁夏泰胜生物科技有限公司 Method for purifying crude orlistat
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