WO2020015316A1 - 辅酶q10的提取纯化方法及由其制备的辅酶q10 - Google Patents

辅酶q10的提取纯化方法及由其制备的辅酶q10 Download PDF

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WO2020015316A1
WO2020015316A1 PCT/CN2018/123214 CN2018123214W WO2020015316A1 WO 2020015316 A1 WO2020015316 A1 WO 2020015316A1 CN 2018123214 W CN2018123214 W CN 2018123214W WO 2020015316 A1 WO2020015316 A1 WO 2020015316A1
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coenzyme
solvent
ketone
polar organic
organic solvent
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PCT/CN2018/123214
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English (en)
French (fr)
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闵一
陈召峰
张锦阳
涂仕春
胡伟江
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浙江新和成股份有限公司
黑龙江新和成生物科技有限公司
上虞新和成生物化工有限公司
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Publication of WO2020015316A1 publication Critical patent/WO2020015316A1/zh

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C50/00—Quinones
    • C07C50/26—Quinones containing groups having oxygen atoms singly bound to carbon atoms
    • C07C50/28—Quinones containing groups having oxygen atoms singly bound to carbon atoms with monocyclic quinoid structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

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  • the invention relates to the field of biotechnology, in particular to a high-efficiency extraction and purification method for coenzyme Q10 and a coenzyme Q10 prepared by the method.
  • Coenzyme Q10 as an antioxidant, can remove free radicals in the human body, and has the health effects of anti-aging and improving cardiovascular immunity. Therefore, research on its preparation process has high industrial value.
  • Coenzyme Q10 products are obtained through fermentation and extraction.
  • the main manufacturers are Xiamen Jindawei, Japan's KANEKA Company, Shenzhou Biological, Livzon Group, Zhejiang Pharmaceutical and Xinhecheng.
  • Coenzyme Q10 is a fat-soluble substance with poor water solubility, so most of the current extraction processes are mainly based on non-polar solvent extraction, such as petroleum ether, n-hexane.
  • a mixed solvent can also be used.
  • Patent Document 1 discloses A mixed solvent of n-hexane and isopropanol.
  • Patent Document 2 discloses a mixed solvent of acetone and water.
  • the purification processes reported so far have the following problems: unclear impurity distribution, poor silica gel pretreatment process, poor emulsification due to improper process design, and difficult processing of crystallization mother liquor.
  • Patent document 1 US2016 / 0304915A1
  • Patent Document 2 US2007 / 0025976A1
  • Patent Document 3 US9315839B2
  • Patent Document 4 CN103819326A
  • Patent document 5 CN102557912A
  • the present invention provides a method for extracting and purifying Coenzyme Q10, which includes extraction, precipitation, decolorization, and refining steps.
  • the present invention effectively removes the strong polar impurities, polar lipid impurities, pigments, neutral and non-polar lipids in the process of preparing Coenzyme Q10 through special restrictions on the process conditions of the extraction, precipitation, decolorization and refining steps. Impurities, and impurities such as quinone homologues.
  • a method for extracting and purifying Coenzyme Q10 including the following steps:
  • Coenzyme Q10 cells are extracted.
  • the extraction system is a combination of non-polar organic solvents, polar organic solvents and water;
  • step ii) mixing the extract obtained in step i) with a ketone solvent to precipitate and remove impurities;
  • step iii) decoloring the processed material after removing impurities obtained in step ii) by adsorbent adsorption or crystallization;
  • step iv) performing chromatographic purification on the decolored matter obtained in step iii) to obtain coenzyme Q10 after extraction and purification.
  • the volume ratio of the non-polar organic solvent, the polar organic solvent and water in the extraction system of step i) of the method of the present invention is 6 to 10: 2 to 6: 1, preferably 7 to 9: 2 to 4: 1.
  • the extraction leads to delamination. After removing the separated oil phase from the solvent, a coenzyme Q10 extract is obtained.
  • the non-polar organic solvent in step i) of the method of the present invention is at least one of cyclohexane, diethyl ether, isopropyl ether, isooctane, n-hexane or petroleum ether, preferably cyclohexane, n-hexane or petroleum ether.
  • the polar organic solvent is at least one of acetone, acetonitrile, ethyl acetate, methanol, ethanol, or isopropanol, preferably acetone, acetonitrile, or ethyl acetate, and the water is water in the bacteria or / And added water.
  • the volume-mass ratio of the extraction system and the coenzyme Q10 bacterial cells in step i) of the present invention is expressed in mL / g as 10 to 20: 1, preferably 10 to 18: 1, and more preferably 10 to 15: 1.
  • the volume-mass ratio of the ketone solvent in step ii) of the method of the present invention and the coenzyme Q10 extract obtained in step i) is expressed in mL / g as 1-30: 1, preferably 1-20: 1, and more preferably 5 to 10: 1, the ketone solvent is at least one of acetone, methyl ethyl ketone, 2-pentyl ketone, or methyl isobutyl ketone, and preferably acetone or methyl ethyl ketone.
  • the adsorbent in step iii) of the method of the present invention is at least one of activated clay, diatomaceous earth or activated carbon, preferably activated clay; the amount of the adsorbent is 0.1 to 100 times, preferably 0.1 to 40 times, the mass of coenzyme Q10. , More preferably 0.3 to 5 times.
  • the crystal decolorization in step iii) of the method of the present invention uses a mixed system composed of an alcohol solvent and a ketone solvent, and the volume ratio of the alcohol solvent to the ketone solvent in the mixed system is 0.1 to 9: 1, preferably 0.5 to 7: 1, more preferably 2 to 6: 1, the alcohol solvent is at least one of methanol, ethanol or isopropanol, preferably ethanol, and the ketone solvent is acetone, methyl ethyl ketone, 2 -At least one of amyl ketone or methyl isobutyl ketone, preferably acetone or methyl ethyl ketone.
  • the processed material after removing impurities described in step ii) of the method of the present invention may be a filtrate containing coenzyme Q10 obtained by filtering the impurities precipitated in step ii), or a filtrate containing coenzyme Q10 obtained by filtering the impurities precipitated in step ii). After the solvent was removed from the filtrate, a filtrate concentrate was obtained.
  • the volume-mass ratio of the mixed system used for crystallization in step iii) of the method of the present invention and coenzyme Q10 is expressed in mL / g as 5 to 35: 1, preferably 10 to 30: 1, and more preferably 15 to 22: 1
  • the temperature of the crystal is -10 to 25 ° C.
  • Step iv) of the method of the present invention comprises: dissolving the decolored material (ie, the crude Coenzyme Q10) obtained in step iii) in a non-polar solvent, loading the sample, purifying through a chromatography column, using a non-polar organic solvent and A mixed solvent consisting of a polar organic solvent was used as an eluent to obtain coenzyme Q10.
  • the non-polar organic solvent is at least one of petroleum ether, diethyl ether, n-hexane or isooctane, preferably petroleum ether or n-hexane
  • the polar organic solvent is isopropyl alcohol.
  • the stationary phase in the chromatography column is silica gel, and the mobile phase is A mixed solvent of a non-polar organic solvent and a polar organic solvent.
  • the yield of coenzyme Q10 prepared according to the method of the present invention can reach more than 98.5%, and the purity can reach more than 99.8%.
  • the extraction and purification method of the present invention can significantly and effectively remove a variety of impurities, including bacterial pigments, non-polar lipids, neutral lipids, polar lipids and quinone homologues, and the loss of the target product coenzyme Q10 is small. , The total yield can be maintained above 98.5%, and the purity can reach above 99.8%;
  • the extraction and purification method of the present invention can also solve the emulsification problem in the alkaline washing step in the prior art.
  • the above impurities can be effectively removed, and coenzymes in bacteria can be effectively removed.
  • Q10 was efficiently extracted and purified.
  • the method of the invention has simple steps, low cost, competitive advantages, and is suitable for industrialized production.
  • FIG. 1 is a liquid chromatogram of a coenzyme Q10 product separated and purified by using the method of the present invention, wherein the chromatographic peak of coenzyme Q10 is at about 9.8 min.
  • Coenzyme Q10 which is an extraction raw material in the method of the present invention, can be derived from any fermentation bacteria. Bacteria produced by fermentation of coenzyme Q10 conventionally used in the art can be used in the present invention.
  • the fermentation bacteria may be selected from Rhodospirillum, such as Rhodospirillum rubrum, Rhodothalassium salexigens, Rhodospirillum salsalinarum, and the like; Rhodopseudomonas, such as Rhodopseudanonas palustris, Rhodopseudomonas acidophila, Rhodopseudomonas sulfoviridis, Rhodopseudomonas ), Rhodopseudomonas rutila, Rhodopseudomonas blastica, etc .; Rhodobacter, such as Rhodopseudomonas sphaeroides, Rhodobacter capsulatus Rh
  • the coenzyme Q10 bacterial cells described in step i) of the method of the present invention may be wet bacterial cells or dried bacterial cells; preferably, the bacterial cells may be subjected to a simple wall breaking operation, such as grinding or milling.
  • the extraction system used in step i) of the method of the present invention is not limited without affecting the effect of the present invention, as long as it is a non-polar organic solvent, a mixed solvent of organic solvent and water, and the extraction solution can be delaminated, for example An oil phase and an aqueous phase are formed, and coenzyme Q10 can be dissolved in the oil phase.
  • the extraction liquid was separated into layers, the upper layer was an oil phase containing coenzyme Q10, and the lower layer was an aqueous phase containing bacterial residue.
  • an extract containing coenzyme Q10 can be obtained.
  • the non-polar organic solvent is at least one of cyclohexane, diethyl ether, isopropyl ether, isooctane, n-hexane or petroleum ether, and the polar organic solvent is acetone, acetonitrile, ethyl acetate, methanol, ethanol.
  • the ratio of the non-polar organic solvent, the polar organic solvent and water in the extraction system does not affect the effect of the present invention, as long as the extraction solution can be separated into layers, for example, to form
  • the oil phase and the water phase may be used, and coenzyme Q10 may be dissolved in the oil phase.
  • the volume ratio of the non-polar organic solvent, the polar organic solvent and water is 6 to 10: 2 to 6: 1, and more preferably 7 to 9: 2 to 4: 1.
  • the method of the present invention does not specifically limit the use ratio of the extraction system and the coenzyme Q10 bacterial cells, and the volume-mass ratio of the mixed solvent and the coenzyme Q10 bacterial cells is preferably 10-20: 1, more preferably 10-18: 1, It is preferably 10 to 15: 1.
  • the ketone solvent used in step ii) of the method of the present invention is not limited without affecting the effect of the present invention, as long as it can dissolve coenzyme Q10, but it does not dissolve or hardly dissolve fat-soluble polar lipid impurities, and then coenzyme Q10 can be separated from this kind of impurities. Fat-soluble polar lipid impurities are precipitated because they are insoluble in ketone solvents, and are removed after simple filtration to further purify the coenzyme Q10 product.
  • the ketone solvent is preferably at least one of acetone, methyl ethyl ketone, 2-pentyl ketone, or methyl isobutyl ketone.
  • the mass ratio of the ketone solvent to the extract obtained in step i) is also different.
  • the effect of the present invention is not limited, but is preferably 1 to 30: 1, more preferably 1 to 20: 1, and most preferably 5 to 10: 1.
  • Step iii) of the method of the present invention can decolorize the adsorbent, and the adsorbent used is not limited without affecting the effect of the present invention, as long as it can selectively adsorb and remove pigment impurities, thereby effectively separating coenzyme Q10 And pigment impurities.
  • the adsorbent is preferably at least one of activated clay, diatomaceous earth, or activated carbon. Further, it is preferably activated clay.
  • the disadvantage of decolorization of activated carbon is that it will cause irreversible adsorption of the product, resulting in loss of yield, but adsorption of activated white clay, diatomite or a combination thereof can solve this problem well.
  • Step iii) can further remove the pigments extracted with the coenzyme Q10.
  • the adsorption method may use methods such as chromatography and decolorization, and filtering and decoloring after mixing.
  • the amount of the adsorbent is not limited without affecting the effect of the present invention.
  • the amount of the adsorbent obtained is obtained by filtering the coenzyme Q10 contained in the filtrate obtained after filtering the impurities precipitated in step ii) or concentrating the filtrate obtained in step ii).
  • the amount of the adsorbent used is 0.1 to 100 times the mass of Coenzyme Q10, more preferably 0.1 to 40 times, and most preferably 0.3 to 5 times.
  • Step iii) of the method of the present invention may also be decolorization of crystals.
  • the mixed system composed of an alcohol solvent and a ketone solvent is not limited without affecting the effect of the present invention, as long as Coenzyme Q10 is slightly soluble in the alcohol solvent , And the pigment is easily soluble in ketone solvents, which can effectively separate coenzyme Q10 and pigment.
  • the alcohol solvent is methanol, ethanol or isopropanol
  • the ketone solvent is acetone, methyl ethyl ketone, 2-pentyl ketone or methyl isobutyl ketone.
  • the volume ratio of the alcohol-based solvent to the ketone-based solvent in the mixed system is not limited without affecting the effects of the present invention.
  • the volume ratio of the alcohol-based solvent to the ketone-based solvent is preferably 0.1 to 9: 1, and more preferably 0.5. ⁇ 7: 1, and most preferably 2 ⁇ 6: 1.
  • the processed material after removing impurities may be a filtrate containing coenzyme Q10 obtained by filtering the impurities precipitated in step ii), or a solution obtained by desolving the filtrate containing coenzyme Q10 obtained by filtering the impurities precipitated in step ii).
  • the filtrate was concentrated.
  • the volume-mass ratio of the mixed system used for the crystal decolorization and the coenzyme Q10 is not limited without affecting the effect of the present invention, and is preferably 5 to 35: 1, more preferably 10 to 30: 1, and most preferably 15 ⁇ 22: 1.
  • the crystallization temperature is not limited without affecting the effect of the present invention, and is preferably -10 to 25 ° C, more preferably -8 to 15 ° C, and most preferably -5 to 5 ° C.
  • Step iv) of the method of the present invention is a process of removing neutral and non-polar lipids and quinone homologues using chromatography techniques.
  • Conventional chromatography techniques in the art can be used in the present invention.
  • a silica gel layer is used.
  • Analytical method Dissolve the coenzyme Q10 obtained in step iii) in a non-polar solvent, load the sample, add it to a silica-filled chromatography column, and use a non-polar organic solvent and a polar solvent to form a mixed solvent as the eluent; Solution to obtain further purified Coenzyme Q10 product.
  • the non-polar solvent, non-polar organic solvent and polar organic solvent used in the above step iv) are not limited without affecting the effect of the present invention.
  • the non-polar solvent used for dissolving the coenzyme Q10 obtained in the dissolution step iii) can be used as long as it can achieve the purpose of dissolving the coenzyme Q10.
  • Conventional non-polar solvents in the art can be used, such as petroleum ether, ether, n-hexane, isooctane Alkanes, etc.
  • the non-polar organic solvent used for elution is preferably at least one of petroleum ether, diethyl ether, n-hexane or isooctane, and the polar organic solvent is preferably isopropyl alcohol, ethanol, 1,4-dioxane, At least one of ethyl acetate and acetone.
  • the volume ratio of the non-polar organic solvent to the polar organic solvent is not limited without affecting the effect of the present invention. The volume ratio is preferably 10 to 100: 1, more preferably 20 to 80: 1, and most preferably It is 30 to 70: 1.
  • a silica gel chromatography column is used in step iv) of the method of the present invention, and there is no limitation on the specifications of the silica gel, as long as the present invention can be realized.
  • the silica gel has a specific surface area of 300 to 800 m 2 / g and a pore size of 6 to 12 nm.
  • the detection of the coenzyme Q10 content involved in each step of the method of the present invention described above can be performed by any method, preferably, using high performance liquid chromatography (HPLC) detection, and the HPLC test conditions are as follows:
  • Draw a standard curve take pure coenzyme Q10 of different quality in a volumetric flask, dissolve in ethanol to make up the volume, and use high-performance liquid detection to draw a standard curve.
  • Detection of coenzyme Q10 content take a quantitative test object, dissolve in ethanol to make up the volume, and use high-performance liquid detection to calculate the external standard content of coenzyme Q10 in the test object.
  • the chromatographic peak of Coenzyme Q10 is at about 9.8 min.
  • the content of Coenzyme Q10 was measured to be 38.9% and the yield was 99.8%.
  • the lower layer of the methanol / water solution containing bacteria residue is subjected to sedimentation, filtration and solid-liquid separation, and the solvent can be recovered and reused. ii) precipitation
  • the coenzyme Q10 extract obtained in the extraction step was melted in a flask, and 500 mL of industrial methyl ethyl ketone was added. Mechanical stirring at room temperature for 30min, suction filtration, washing 3 times with methyl ethyl ketone, the washing liquid was filtered together, the filtrate was removed by rotary evaporation to remove the solvent, and weighed to obtain 55.83g of the treated product after removing impurities. The content of coenzyme Q10 was 69.5 %, And the yield was 99.6%.
  • Figure 1 shows the spectrum of the final Coenzyme Q10 sample detected by HPLC, where the peak position of Coenzyme Q10 is at 9.8 min.
  • the steps of coenzyme Q10 extraction and purification steps i) and ii) were performed in the same manner as in Example 1.
  • the coenzyme Q10 extract obtained after step i) extraction was 98.54 g, the coenzyme Q10 content was 39.5%, and the yield was 99.8%, the impurity-removed processed material obtained after the precipitation in step ii) was 56.48 g, the content of coenzyme Q10 was 68.7%, and the yield was 99.7%.
  • the processed material after the impurities are removed is subjected to an adsorption decoloring operation in the following step iii).
  • step iv) of Example 1 the obtained decolored matter was subjected to chromatography purification to obtain 38.53 g of a coenzyme Q10 product.
  • the purity of the coenzyme Q10 was measured to be 99.8% and the yield was 99.7%.
  • the total yield was 98.6%.
  • Coenzyme Q10 was extracted in the same manner as in step i) of Example 1, except that a mixed solvent having a volume ratio of cyclohexane: ethyl acetate: water of 8: 5: 1 (v / v / v) was used.
  • the coenzyme Q10 extract obtained after separating the oil and water phases and rotary evaporation was 100.01 g.
  • the coenzyme Q10 content was measured to be 38.8%, and the yield was 99.5%.
  • Coenzyme Q10 was extracted in the same manner as in step i) of Example 1, and 102.65 g of coenzyme Q10 extract was obtained. The content of coenzyme Q10 was determined to be 37.8%, and the yield was 99.5%.
  • a precipitation operation was performed in the same manner as in step ii) of Example 1, except that 2-butyl ketone was used instead of industrial methyl ethyl ketone. After suction filtration and rotary evaporation, 54.74 g of the processed material after removing impurities was obtained. The content of coenzyme Q10 was measured to be 70.6%, and the yield was 99.6%.
  • step i) and step ii) of Example 2 Extraction and precipitation operations were performed in the same manner as in step i) and step ii) of Example 2 to obtain 56.11 g of a treated product after removing impurities, and the content of coenzyme Q10 was measured to be 69.3% and the yield was 99.6%.
  • the adsorption-decoloration operation of step iii) of Example 2 was performed on the treated product after removing impurities, except that diatomite was used instead of activated white clay. After suction filtration and rotary evaporation, 47.08 g of decolored matter was obtained.
  • the content of coenzyme Q10 was 82.1%, and the yield was 99.4%.
  • Extraction and precipitation operations were performed in the same manner as in step i) and step ii) of Example 2 to obtain 55.87 g of a treated product after removing impurities, and the content of coenzyme Q10 was measured to be 69.4%, and the yield was 99.5%.
  • This impurity-removed processed material was subjected to the adsorption decolorization operation of step iii) of Example 2, except that activated carbon was used instead of activated clay. After suction filtration and rotary evaporation, 47.01 g of crude coenzyme Q10 was obtained. The content of coenzyme Q10 was measured to be 81.8%, and the yield was 99.2%.
  • Extraction and precipitation operations were performed in the same manner as in step i) and step ii) of Example 1 to obtain 56.15 g of a treated product after removing impurities, and the content of coenzyme Q10 was determined to be 69.2% with a yield of 99.7%.
  • the main eluate was combined with rotary evaporation to remove the solvent and weighed to obtain 38.51 g of Coenzyme Q10 product.
  • the purity of Coenzyme Q10 was measured to be 99.8%, the yield was 99.3%, and the total yield was 98.5%.
  • the extraction operation was performed in the same manner as in step i) of Example 1, and 98.59 g of Coenzyme Q10 extract was obtained.
  • the content of Coenzyme Q10 was determined to be 39.4%, and the yield was 99.6%.
  • the obtained extract was precipitated in the same manner as step ii) of Example 1.
  • 490.6 g of a coenzyme Q10-containing filtrate was obtained.
  • the content of the coenzyme Q10 was determined to be 7.9%, and the yield was 99.8%.
  • the obtained filtrate was subjected to crystal decolorization in step iii) of Example 1 to obtain 46.51 g of a decolorized substance, the content of coenzyme Q10 was 83.0%, and the yield was 99.6%.
  • step iv) of Example 1 was subjected to chromatography purification in step iv) of Example 1 to obtain 38.53 g of a coenzyme Q10 product, with a coenzyme Q10 content of 99.8%, a yield of 99.6% and a total yield of 98.6%.
  • Coenzyme Q10 was extracted in the same manner as step i) of Example 1, except that the volume ratio of n-hexane: methanol: water was 5: 3: 1 (v / v / v). After separating the oil and water phases and rotary evaporation, 98.75 g of Coenzyme Q10 extract was obtained. The content of Coenzyme Q10 was measured to be 32.0%, and the yield was 81.0%.
  • Coenzyme Q10 was extracted in the same manner as step i) of Example 1, except that the volume ratio of cyclohexane: ethanol: water was 7: 1: 1 (v / v / v). After separating the oil and water phases and rotary evaporation, 99.17 g of Coenzyme Q10 extract was obtained. The content of Coenzyme Q10 was measured to be 31.5% and the yield was 80.1%.
  • Coenzyme Q10 extraction was performed in the same manner as in step i) of Example 1 to obtain 101.02 g of coenzyme Q10 extract.
  • the content of coenzyme Q10 was determined to be 38.5% and the yield was 99.7%.
  • Extraction and precipitation operations were performed in the same manner as in step i) and step ii) of Example 1 to obtain 56.05 g of a treated product after removing impurities, and the content of coenzyme Q10 was measured to be 69.3% with a yield of 99.7%.
  • This processed product after removing impurities was subjected to crystal decoloration in the same manner as in step iii) of Example 1, except that ethanol was used instead of the mixed solvent of acetone and ethanol. After crystallization, suction filtration, washing with the mixed solvent three times, and drying to obtain Coenzyme Q10 48.90g, the content of Coenzyme Q10 was measured to be 70.7%, and the yield was 89.0%.
  • Extraction and precipitation operations were performed in the same manner as in step i) and step ii) of Example 1 to obtain 57.11 g of a treated product after removing impurities, and the content of coenzyme Q10 was determined to be 67.8%, and the yield was 99.8%.
  • This processed product after removing impurities was subjected to crystal decolorization in the same manner as in step iii) of Example 1, except that isopropyl alcohol was used instead of the mixed solvent of acetone and ethanol. After the crystallization was completed, suction filtration, washing with the mixed solvent three times, and drying yielded 47.18 g of coenzyme Q10. The content of coenzyme Q10 was measured to be 72.3%, and the yield was 88.1%.
  • Extraction and precipitation operations were performed in the same manner as in step i) and step ii) of Example 1, and 56.07 g of the treated product after removing impurities were obtained.
  • the content of coenzyme Q10 was determined to be 69.2%, and the yield was 99.6%.
  • This treated product after removing impurities was subjected to crystal decolorization in the same manner as in step iii) of Example 1, except that the volume ratio of the mixed solvent V butanone : V ethanol was 1:12. After the crystallization, suction filtration was performed, the mixed solvent was washed three times, and 46.51 g of coenzyme Q10 was dried to obtain a coenzyme Q10 content of 79.0% and a yield of 94.7%.
  • step i) 780 g of Coenzyme Q10 bacteria (5.0% Coenzyme Q10 content and 2.1% Moisture content) were taken, and the extraction operation of step i) was performed in the same manner as in Example 1 to obtain 100.11 g of Coenzyme Q10 extract with a Coenzyme Q10 content of 38.8 %, And the yield was 99.6%.
  • 500 mL of n-hexane was added to the extract to dissolve it, 500 mL of water was added, and the mixture was stirred at room temperature.
  • 100 mL of a NaOH solution (1.5 mol / L) was added dropwise to the extract for saponification.
  • the organic 1701072 phase was further washed with 120 mL of water in three times to obtain an organic phase containing coenzyme Q10.
  • the solvent was evaporated in a rotary evaporator at 40 ° C to obtain coenzyme Q10 50.20 g.
  • the content of coenzyme Q10 was measured and the yield was 68.8%. It was 88.9%, and the total yield was 88.6%.
  • step i) Take 780 g of coenzyme Q10 bacteria (5.5% of coenzyme Q10 and 3% of water content), and perform the extraction operation of step i) in the same manner as in Example 1 to obtain 103.31 g of coenzyme Q10 extract with a coenzyme Q10 content of 41.3 %, Yield is 99.5%.
  • the crystallization operation of step iii) is directly performed.
  • the temperature was reduced again at a cooling rate of 1 °C / 3min, lowered to -5 °C, and kept for 3h; suction filtration, mixed solvent washing three times, and drying to obtain Coenzyme Q10 75.60 g, the content of coenzyme Q10 was 50.0%, the yield was 88.6%, and the total yield was 88.1%.
  • the content of coenzyme Q10 in the crystallization mother liquor is 6%, which is very difficult to handle.
  • step i) Take 780 g of Coenzyme Q10 bacteria (Coenzyme Q10 content is 5.3%, moisture content is 2.7%), and perform the extraction operation of step i) in the same manner as in Example 1 to obtain 104.29 g of Coenzyme Q10 extract with a Coenzyme Q10 content of 39.4 %, The yield was 99.4%.
  • the extract is then directly subjected to chromatography purification in step iv).
  • the specific steps are: using the glass chromatography column of Example 1, weighing the obtained coenzyme Q10 extract, adding petroleum ether and heating to dissolve it completely, and cooling to room temperature Load. Elution was performed using a petroleum ether solution containing 1.5% ethyl acetate as the eluent.
  • the elution flow rate was 7.2 mL / min.
  • the eluents were connected in stages. The main eluents were combined.
  • the solvent was removed by rotary evaporation and weighed. 45.24g of Coenzyme Q10 product was obtained.
  • the content of Coenzyme Q10 was measured to be 85.2%, the yield was 93.8%, and the total yield was 93.2%.
  • the process of passing through the column was very slow, and a large amount of impurities were adsorbed on the column, which blocked the packing.
  • Examples 1 and 3 and Comparative Examples 1 and 2 both use the combination of the non-polar organic solvent, the polar organic solution, and water of the present invention.
  • the solvent formulations used in Comparative Examples 1 and 2 are The ratio is not within the scope of the present invention, so the generated oil phase and water phase are not significantly separated, and the coenzyme Q10 cannot be fully integrated into the oil phase, resulting in a small amount of coenzyme Q10 in the oil phase and a reduced yield.
  • the mixed solvents of Examples 1 and 3 have a proper distribution ratio for each group, and the oil phase and the water phase are obvious. A large amount of coenzyme Q10 is extracted from the upper oil phase, and the bacteria residue and some polar impurities are separated into the lower water. In contrast, the yield of Coenzyme Q10 was significantly higher than that of Comparative Examples 1 and 2.
  • Comparative Example 3 uses n-hexane as a solvent to dissolve the coenzyme Q10 extract obtained in step i). Three layers of oil layer, emulsified layer and water layer are observed. The emulsified layer is clear and abundant. Coenzyme Q10 and some impurities coexist in the oil. The phase and the emulsified layer cannot be effectively separated.
  • Example 1 of the present application uses methyl ethyl ketone as a solvent to precipitate the coenzyme Q10 extract obtained in step i) to remove strong polar impurities, effectively reducing the emulsification effect.
  • Examples 2, 5 and 6 activated clay, diatomaceous earth and activated carbon were used as adsorbents for decolorization respectively.
  • the experimental results show that the activated clay has the best selective adsorption and the highest yield after decolorization.
  • the yield of coenzyme Q10 after the decolorization of activated carbon is the lowest, probably because activated carbon will cause irreversible adsorption of the product, resulting in a loss of yield.
  • Activated white clay and diatomaceous earth can solve this problem well, and the pigments extracted with coenzyme Q10 in step iii) can be selectively removed by adsorption.
  • Examples 7 to 9 and Comparative Examples 4 to 6 examined the decolorization of crystals.
  • the experimental results show that Comparative Examples 4 and 5 have the worst decoloring effect using ethanol or isopropanol alone, and the yield after crystallization is also not ideal.
  • Comparative Example 6 also uses methyl ethyl ketone and ethanol as a mixed solvent, the solvent ratio is not within the scope of the present invention, and therefore, the yield after crystallization is not high compared to Example 1.
  • Comparative Examples 7 to 9 only include part of the steps of the method of the present invention, and the extraction rate or purity of Coenzyme Q10 is not as good as that of Example 1 of the present application.
  • Comparative Example 7 adopts an alkaline washing process, which causes a large amount of emulsifiable impurities in the fat-soluble impurities to be severely emulsified, taking away part of the coenzyme Q10, and reducing the yield.
  • Comparative Example 8 was directly crystallized and purified after extraction without further processing of impurities in advance, such as the process of removing fat-soluble polar impurities insoluble in ketone solvents by the precipitation method of the present invention, so the impurity content in the crystallization mother liquor is high and the composition is complex , Resulting in a very low yield of product recovered from the mother liquor.
  • Comparative Example 9 was purified by direct chromatography through a column after extraction. Due to the large amount of impurities contained in the loading solution, some impurities were irreversibly adsorbed on the silica gel. The plugging of the silica gel resulted in a low number of application of the silica gel, and the yield and purity of coenzyme Q10 also decreased.
  • Examples 1, 2, 9, and 10 were all operated according to steps i) to iv) of the method of the present invention, and the conditions of each step were met, so all the ideal separation and purification effects were obtained, and the purity of coenzyme Q10 could reach above 99.8% The total yield can be kept above 98.5%.
  • the extraction and purification method of the invention can effectively remove strong polar impurities, polar lipid impurities, pigments, neutral and non-polar lipid impurities, and impurities such as quinone homologues during the preparation of coenzyme Q10.
  • the product coenzyme Q10 The total yield can be kept above 98.5%, and the purity can reach above 99.8%.
  • the method of the present invention does not have the emulsification problem in the alkaline washing step in the prior art, the operation steps are few and simple, the reagents and equipment used are conventional, the cost is low, and it has competitive advantages, which is very suitable for industrial production.

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Abstract

公开了辅酶Q10的提取纯化方法及由其制备的辅酶Q10。该方法从辅酶Q10菌体中提取纯化辅酶Q10，包括提取、沉淀、脱色和层析精制步骤，针对性地去除了细菌色素、非极性脂类、中性脂类、极性脂类以及醌类同系物等杂质，操作步骤简单、产品损失少，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。

Description

辅酶Q10的提取纯化方法及由其制备的辅酶Q10 技术领域
本发明涉及生物技术领域，具体涉及一种辅酶Q10的高效提取纯化方法及由其制备的辅酶Q10。
背景技术
辅酶Q10作为抗氧化剂在人体内可去除自由基，具有抗衰老和提高心血管免疫力的保健作用，因此对其制备过程的研究具有较高的工业价值。
目前辅酶Q10产品通过发酵提取的方式获得，主要的生产商有厦门金达威、日本KANEKA公司、神州生物、丽珠集团、浙江医药和新和成等企业。辅酶Q10是脂溶性物质，水溶性差，所以目前的大部分提取工艺以非极性溶剂提取为主，例如石油醚，正己烷，当然为了提取完全，也可使用混合溶剂，例如专利文献1公开了正己烷和异丙醇的混合溶剂，专利文献2公开了丙酮和水的混合溶剂等；在提取出辅酶Q10的同时，其他脂溶性杂质也会一并提取出，包括细菌色素、非极性脂类、中性脂类、极性脂类以及醌类同系物；同时，随辅酶Q10一并提取出的这些脂溶性杂质的含量会随着提取溶剂极性的增大而增多，有时杂质的含量要远大于提取出的辅酶Q10的含量，这给分离纯化带来更大的挑战。
已报道的文献中较多的工艺采用提取后直接层析过柱进行纯化，例如专利文献3和4，这种做法几乎不可能实现工业化，原因在于大量杂质会不可逆吸附于硅胶上，造成层析硅胶的活性位点被占据，套用次数低而导致成本上升；另一种做法是提取后直接进行结晶纯化，该方法会导致结晶母液中杂质含量高且成分复杂，不利于母液中产品的回收；还有报道将提取后的辅酶Q10粗品采用碱水洗的工艺进行纯化，例如专利文献5，其缺点在于脂溶性杂质中含有大量可乳化杂质，在操作过程中会造成严重的乳化而带走产品，降低 收率。
综上所述，目前已报道的纯化工艺存在以下问题：杂质分布不明确、硅胶层析前处理工艺效果差、工艺设计不当导致乳化现象、结晶母液复杂处理困难等。
现有技术文献：
专利文献1：US2016/0304915A1
专利文献2：US2007/0025976A1
专利文献3：US9315839B2
专利文献4：CN103819326A
专利文献5：CN102557912A
发明内容
发明要解决的问题
为解决以上技术问题，本发明提出了一种辅酶Q10的提取纯化方法，包括提取、沉淀、脱色和精制步骤。本发明通过对提取、沉淀、脱色以及精制步骤的工艺条件进行特殊限定，有效去除了辅酶Q10制备过程中存在的强极性杂质、极性脂类杂质、色素、中性和非极性脂类杂质，以及醌类同系物等杂质。
用于解决问题的方案：
一种辅酶Q10的提取纯化方法，具体的，包括如下步骤：
i)对辅酶Q10菌体进行提取，提取体系是非极性有机溶剂、极性有机溶剂和水的组合；
ii)将步骤i)得到的提取物与酮类溶剂混合以沉淀去除杂质；
iii)将步骤ii)得到的去除杂质后的处理物采用吸附剂吸附或结晶进行脱色；以及
iv)将步骤iii)得到的脱色物进行层析精制，得到提取纯化后的辅酶Q10。
本发明方法的步骤i)的提取体系中非极性有机溶剂、极性有机溶剂和水的体积比为6～10:2～6:1，优选为7～9:2～4:1。所述提取导致分层，将分离得到的油相脱除溶剂后，得到辅酶Q10提取物。
本发明方法的步骤i)的非极性有机溶剂为环己烷，乙醚，异丙醚，异辛烷，正己烷或石油醚中的至少一种，优选为环己烷、正己烷或石油醚，所述极性有机溶剂为丙酮，乙腈，乙酸乙酯，甲醇，乙醇或异丙醇中的至少一种，优选为丙酮、乙腈或乙酸乙酯，所述水为所述细菌中的水分或/和添加的水。
本发明步骤i)中提取体系与辅酶Q10菌体的体积质量比以mL/g表示为10～20:1，优选为10～18:1，更优选为10～15:1。
本发明方法的步骤ii)的酮类溶剂和所述步骤i)得到的辅酶Q10提取物的体积质量比以mL/g表示为1～30:1，优选为1～20:1，更优选为5～10:1，所述酮类溶剂为丙酮、丁酮、2-戊基酮或甲基异丁基酮中的至少一种，优选为丙酮或丁酮。
本发明方法的步骤iii)的吸附剂为活性白土、硅藻土或活性炭中的至少一种，优选活性白土；所述吸附剂用量为辅酶Q10质量的0.1～100倍，优选为0.1～40倍，更优选为0.3～5倍。
本发明方法的步骤iii)所述的结晶脱色采用醇类溶剂和酮类溶剂组成的混合体系，所述混合体系中的醇类溶剂与酮类溶剂的体积比为0.1～9:1，优选为0.5～7:1，更优选为2～6:1，所述醇类溶剂为甲醇，乙醇或异丙醇中的至少一种，优选为乙醇，所述酮类溶剂为丙酮、丁酮、2-戊基酮或甲基异丁基酮中的至少一种，优选为丙酮或丁酮。
本发明方法的步骤ii)中所述的去除杂质后的处理物可以为过滤步骤ii)中沉淀的杂质得到的含有辅酶Q10的滤液，或将过滤步骤ii)中沉淀的杂质得到的含有辅酶Q10的滤液脱除溶剂后，得到的滤液浓缩物。
本发明方法的步骤iii)中所述结晶使用的混合体系与辅酶Q10的体积质 量比以mL/g表示为5～35:1，优选为10～30:1，更优选为15～22:1，所述结晶的温度为-10～25℃。
本发明方法的步骤iv)包括：将所述步骤iii)得到的脱色物(即，辅酶Q10粗品)溶解于非极性溶剂，上样，通过层析柱进行纯化，用非极性有机溶剂和极性有机溶剂组成混合溶剂作为洗脱剂，得到辅酶Q10。
本发明方法的步骤iv)中，所述非极性有机溶剂为石油醚，乙醚，正己烷或异辛烷中的至少一种，优选为石油醚或正己烷，所述极性有机溶剂为异丙醇，乙醇，1,4-二氧六环，乙酸乙酯或丙酮中的至少一种，优选为异丙醇或乙酸乙酯，所述层析柱中的固定相为硅胶，流动相为非极性有机溶剂和极性有机溶剂的混合溶剂。
根据本发明方法制备的辅酶Q10的收率可达到98.5％以上，纯度达到99.8％以上。
发明的效果
1)本发明的提取纯化方法可显著而有效地去除多种杂质，包括细菌色素、非极性脂类、中性脂类、极性脂类以及醌类同系物，目标产物辅酶Q10的损失小，总收率可保持在98.5％以上，纯度达到99.8％以上；
2)本发明的提取纯化方法还可解决现有技术中碱洗步骤出现的乳化问题，通过对提取、沉淀、脱色以及精制步骤的工艺条件进行特殊限定，有效去除上述杂质，将细菌中的辅酶Q10高效地提取纯化出来。本发明方法的步骤简单，成本低，具有竞争优势，适合于工业化生产。
附图说明
图1为使用本发明方法分离纯化后的辅酶Q10产物的液相色谱图，其中，辅酶Q10的色谱峰在大约9.8min处。
具体实施方式
本发明方法中的提取原料辅酶Q10可来自于任何发酵细菌，本领域常规使用的辅酶Q10发酵生产的细菌均可用于本发明。所述发酵细菌可选自红螺菌属(Rhodospirillum)，例如深红红螺菌(Rhodospirillum rubrum)、需盐红海菌(Rhodothalassium salexigens)、盐场红螺菌(Rhodospirillum salinarum)等；红假单胞菌属(Rhodopseudomonas)，例如沼泽红假单胞菌(Rhodopseudanonas palustris)、嗜酸红假单胞菌(Rhodopseudomonas acidophila)、绿硫红假单胞菌(Rhodopseudomonas sulfoviridis)、海洋红假单胞菌(Rhodopseudomonas marina)、血色红假单胞菌(Rhodopseudomonas rutila)、生芽红假单胞菌(Rhodopseudomonas blastica)等；红细菌属(Rhodobacter)，例如球形红细菌(Rhodopseudomonas sphaeroides)、荚膜红细菌(Rhodobacter capsulatus)、维氏红细菌(Rhodobacter veldkampii)、嗜硫红细菌(Rhodobacter sulfidophila)、亚德里亚红细菌(Rhodobacter adriaticus)等；红球状菌属(Rhodopila)，例如球形红球形菌(Rhodopila globiformis)等；红微菌属(Rhodomicrobium)，例如万尼氏红微菌(Rhodomicrobium vannielii)等，本发明所使用的发酵细菌不限于此。
本发明方法的步骤i)中所述的辅酶Q10菌体可以为湿菌体或者经过干燥的菌体；优选的，可以将所述菌体进行简单的破壁操作，例如研磨或碾磨。
本发明方法的步骤i)所使用的提取体系在不影响本发明效果的情况下不受限制，只要是非极性有机溶剂、有机溶剂与水形成的混合溶剂，且能够使提取液分层，例如形成油相与水相，而辅酶Q10溶解于油相即可。提取液分层后，上层为包含辅酶Q10的油相，下层为含有菌渣的水相。回收油相中的有机溶剂后，即可得到含辅酶Q10的提取物。优选地，非极性有机溶剂为环己烷，乙醚，异丙醚，异辛烷，正己烷或石油醚中的至少一种，极性有机溶剂为丙酮，乙腈，乙酸乙酯，甲醇，乙醇或异丙醇中的至少一种，水为发酵细菌中的水分或/和添加的水。所述细菌中的水分以细菌烘干前后的失重来计算(水量＝细菌烘前质量-细菌烘后质量)。
在上述步骤i)中，所述提取体系中非极性有机溶剂、极性有机溶剂和水的配比也不影响本发明效果的情况下不受限制，只要能使提取液分层，例如 形成油相与水相，且使辅酶Q10溶解于油相即可。优选地，非极性有机溶剂、极性有机溶剂和水的体积比为6～10:2～6:1，更优选为7～9:2～4:1。本发明方法对所述提取体系与辅酶Q10菌体的使用比例也没有特别限制，优选混合溶剂与辅酶Q10菌体的体积质量比为10～20:1，更优选为10～18:1，最优选为10～15:1。
本发明方法的步骤ii)中所使用的酮类溶剂在不影响本发明效果的情况下不受限制，只要能溶解辅酶Q10，但不溶解或难溶解脂溶性极性脂类杂质，进而将辅酶Q10与该类杂质分离即可。脂溶性极性脂类杂质因不溶于酮类溶剂而沉淀，经过简单过滤后将其除去，从而进一步纯化辅酶Q10产物。所述酮类溶剂优选为丙酮、丁酮、2-戊基酮或甲基异丁基酮中的至少一种，所述酮类溶剂与步骤i)得到的提取物的质量配比也在不影响本发明效果的情况下不受限制，优选为1～30:1，更优选为1～20:1，最优选为5～10:1。
本发明方法的步骤iii)可为吸附剂脱色，所使用的吸附剂在不影响本发明效果的情况下不受限制，只要能对色素类杂质具有选择性吸附去除功能，从而能有效分离辅酶Q10和色素类杂质即可。所述吸附剂优选为活性白土、硅藻土或活性炭中的至少一种。进一步的，优选为活性白土。活性炭脱色的缺点在于，会造成产品不可逆吸附，从而导致收率损失，但是活性白土、硅藻土或其组合吸附则可以很好地解决该问题。步骤iii)可进一步去除随辅酶Q10一起提取出的色素类物质。所述吸附方法可以使用层析色谱脱色、混合后过滤脱色等方法。所述的吸附剂用量在不影响本发明效果的情况下不受限制，优选地，以过滤步骤ii)沉淀的杂质后得到的滤液中含有的辅酶Q10或浓缩步骤ii)的所述滤液后得到的辅酶Q10计，所述吸附剂用量为辅酶Q10质量的0.1～100倍，更优选为0.1～40倍，最优选为0.3～5倍。
本发明方法的步骤iii)还可以为结晶脱色，所使用的醇类溶剂和酮类溶剂组成的混合体系在不影响本发明效果的情况下不受限制，只要辅酶Q10可微溶于醇类溶剂，而色素易溶于酮类溶剂，进而有效分离辅酶Q10和色素即可。优选地，所述醇类溶剂为甲醇，乙醇或异丙醇，所述酮类溶剂为丙酮、丁酮、 2-戊基酮或甲基异丁基酮。所述混合体系中醇类溶剂与酮类溶剂的体积比在不影响本发明效果的情况下不受限制，优选醇类溶剂：酮类溶剂的体积比为0.1～9:1，更优选为0.5～7:1，最优选为2～6:1。
所述的去除杂质后的处理物可以为过滤步骤ii)中沉淀的杂质得到的含有辅酶Q10的滤液，或将过滤步骤ii)中沉淀的杂质得到的含有辅酶Q10的滤液脱溶后，得到的滤液浓缩物。所述的结晶脱色使用的混合体系与辅酶Q10的体积质量比在不影响本发明效果的情况下不受限制，优选为5～35:1，更优选为10～30:1，最优选为15～22:1。
所述结晶温度在不影响本发明效果的情况下不受限制，优选-10～25℃，更优选为-8～15℃，最优选为-5～5℃。
本发明方法的步骤iv)是使用层析技术对中性和非极性脂类以及醌类同系物进行去除的过程，本领域常规的层析技术均可用于本发明，优选地，使用硅胶层析法。将步骤iii)脱色得到的辅酶Q10溶解于非极性溶剂，上样、加入硅胶填充的层析柱中，用非极性有机溶剂和极性溶剂组成混合溶剂为洗脱剂；回收层析得到的溶液，得到进一步纯化的辅酶Q10产品。
上述步骤iv)中所使用的非极性溶剂、非极性有机溶剂和极性有机溶剂在不影响本发明效果的情况下不受限制。用于溶解步骤iii)脱色得到的辅酶Q10的非极性溶剂只要能达到溶解辅酶Q10的目的即可，本领域常规的非极性溶剂均可使用，例如石油醚，乙醚，正己烷，异辛烷等。用于洗脱的非极性有机溶剂优选为石油醚，乙醚，正己烷或异辛烷中的至少一种，极性有机溶剂优选为异丙醇，乙醇，1,4-二氧六环，乙酸乙酯，丙酮中的至少一种。所述的非极性有机溶剂与极性有机溶剂的体积比在不影响本发明效果的情况下不受限制，优选体积比为10～100:1，更优选为20～80:1，最优选为30～70:1。
本发明方法的步骤iv)中使用硅胶层析柱，对于硅胶的规格没有限制，只要可实现本发明即可。优选地，硅胶规格为：比表面积为300～800m 2/g，孔径为6～12nm。
上述本发明方法的各步骤中所涉及的辅酶Q10含量的检测可采用任何方 法，优选地，使用高效液相色谱法(HPLC)检测，HPLC测试条件如下：
色谱柱为C18柱，150mm×4.6mm，流速1mL/min，检测波长275nm，流动相：乙腈/乙醇＝50/50(v/v)。
绘制标准曲线：取不同质量的辅酶Q10纯品于容量瓶中，乙醇溶解定容，用高效液相检测，绘制标准曲线。
辅酶Q10含量的检测：取定量待测物，乙醇溶解定容，用高效液相检测，计算待测物中辅酶Q10的外标含量。辅酶Q10的色谱峰在大约9.8min处。
以下结合实施例对本发明进行详细说明，本发明不限于这些实施例。
实施例1
i)提取
取辅酶Q10菌体780g(辅酶Q10含量为5.0％，水分含量为2.1％)，经简单研磨后，加入正己烷：甲醇：水的体积比为9:3:1(v/v/v)的混合溶剂8L。控温25℃，机械搅拌后静置分层，上层为含有产品的正己烷相，下层为含有菌渣的甲醇/水溶液。分离油水两相后，上层液在40℃下在旋转蒸发仪中蒸发溶剂得到辅酶Q10提取物100.14g，测得辅酶Q10含量为38.9％，收率为99.8％。下层含有菌渣的甲醇/水溶液经过沉降、过滤后进行固液分离，溶剂可回收再使用。ii)沉淀
将提取步骤中获得的辅酶Q10提取物在烧瓶中熔融，加入500mL的工业丁酮。室温下机械搅拌30min，抽滤，用丁酮洗涤3遍，洗涤液一并抽滤，滤液旋转蒸发脱除溶剂、称重，得去除杂质后的处理物55.83g，测得辅酶Q10含量为69.5％，收率为99.6％。
iii)结晶脱色
取沉淀步骤获得的去除杂质后的处理物，加入760mL的混和溶剂(V 丙酮：V 乙醇＝1：4)，于30℃冷阱中搅拌溶解，完全溶解后保温30min；以1℃/3min的速率降温，温度降为20℃，瓶底有大量晶体颗粒析出，保温30min；以1℃/3min的降温速率再次降温，降至-5℃，保温3h；抽滤，混合溶剂洗涤三次，烘干得脱色物46.90g，测得辅酶Q10的含量为82.7％，收率为99.9％。
iv)层析精制
将52.6g 200～300目的层析硅胶装入柱长为45cm、柱外径为5.5cm的玻璃层析柱。在结晶脱色步骤获得的脱色物中加入石油醚加热溶解完全，冷却至室温后上样。用含有1.5％乙酸乙酯的石油醚溶液作为洗脱剂进行洗脱，洗脱流速为7.2mL/min，分段接洗脱液，将主洗脱液合并，旋转蒸发脱除溶剂、称重，得辅酶Q10产品38.76g，测得辅酶Q10纯度为99.8％，收率为99.7％，总收率为99.2％。图1示出了HPLC检测的最终的辅酶Q10样品的谱图，其中，辅酶Q10的出峰位置在9.8min处。
实施例2
以与实施例1相同的方式进行辅酶Q10提取纯化的步骤i)和步骤ii)的操作，其中步骤i)提取后获得的辅酶Q10提取物为98.54g，辅酶Q10含量为39.5％，收率为99.8％，步骤ii)沉淀后获得的去除杂质后的处理物为56.48g，辅酶Q10含量为68.7％，收率为99.7％。将该去除杂质后的处理物进行如下步骤iii)的吸附脱色操作。
首先，将该56.48g去除杂质后的处理物溶于800mL正己烷，然后在φ7cm布氏漏斗底端铺2层滤纸，再取30.86g粉末型活性白土，加正己烷铺在滤纸上，最后在填料上铺1层滤纸。将溶于正己烷的辅酶Q10溶液用布氏漏斗抽滤，直到滴下的液体没有颜色时停止抽滤。将滤液旋转蒸发脱除溶剂、称重，得脱色物46.69g，测得辅酶Q10含量为82.6％，收率为99.4％。
以与实施例1的步骤iv)相同的方式，将所得脱色物进行层析精制，由此获得辅酶Q10产品38.53g，测得辅酶Q10纯度为99.8％，收率为99.7％，上述各步骤的总收率为98.6％。
实施例3
以与实施例1的步骤i)相同的方式提取辅酶Q10，除了使用环己烷：乙酸乙酯：水的体积比为8:5:1(v/v/v)的混合溶剂外。分离油水两相并旋转蒸发后得到的辅酶Q10提取物为100.01g，测得辅酶Q10含量为38.8％，收率为99.5％。
实施例4
以与实施例1的步骤i)相同的方式提取辅酶Q10，获得辅酶Q10提取物102.65g，测定辅酶Q10含量为37.8％，收率为99.5％。接下来以与实施例1的步骤ii)相同的方式进行沉淀操作，除了用2-戊基酮代替工业丁酮外。抽滤并旋转蒸发后得到去除杂质后的处理物54.74g，测得辅酶Q10含量为70.6％，收率为99.6％。
实施例5
以与实施例2的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物56.11g，测得辅酶Q10含量为69.3％，收率为99.6％。对该去除杂质后的处理物进行实施例2的步骤iii)的吸附脱色操作，除了以硅藻土代替活性白土外。抽滤并旋转蒸发后得到脱色物47.08g，测得辅酶Q10含量为82.1％，收率为99.4％。
实施例6
以与实施例2的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物55.87g，测得辅酶Q10含量为69.4％，收率为99.5％。对该去除杂质后的处理物进行实施例2的步骤iii)的吸附脱色操作，除了以活性炭代替活性白土外。抽滤并旋转蒸发后得到辅酶Q10粗品47.01g，测得辅酶Q10含量为81.8％，收率为99.2％。
实施例7
以与实施例1的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物56.15g，测定辅酶Q10含量为69.2％，收率为99.7％。对该去除杂质后的处理物以与实施例1的步骤iii)相同的方式进行结晶脱色，除了加入780mL的混和溶剂(V 丁酮：V 乙醇＝1：1)外。结晶完毕，抽滤，混合溶剂洗涤三次，烘干得脱色物47.05g，测得辅酶Q10含量为82.0％，收率为99.3％。
实施例8
以与实施例1的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物56.01g，测定辅酶Q10含量为69.3％，收率为99.6％。对该 去除杂质后的处理物以与实施例1的步骤iii)相同的方式进行结晶脱色，除了加入850mL的混和溶剂(V 2-戊基酮：V 甲醇＝1：6)外。结晶完毕，抽滤，混合溶剂洗涤三次，烘干得脱色物46.48g，测得辅酶Q10含量为83.0％，收率为99.4％。
实施例9
以与实施例1的步骤i)至步骤iii)相同的方式进行提取、沉淀和结晶操作，获得脱色物46.80g，测定辅酶Q10含量为82.7％，收率为99.7％。以与实施例1的步骤iv)相同的方式进行层析精制，除了以正己烷代替石油醚，用3.0％异丙醇的正己烷溶液代替1.5％乙酸乙酯的石油醚溶液作为洗脱剂以外。将主洗脱液合并旋转蒸发脱除溶剂、称重，得到辅酶Q10产品38.51g，测得辅酶Q10纯度为99.8％，收率为99.3％，总收率为98.5％。
实施例10
以与实施例1的步骤i)相同的方式进行提取操作，获得辅酶Q10提取物98.59g，测定辅酶Q10含量为39.4％，收率为99.6％。将所得提取物以与实施例1的步骤ii)相同的方式进行沉淀，抽滤后得到含辅酶Q10的滤液490.6g，测得辅酶Q10含量为7.9％，收率99.8％。将所得滤液进行实施例1的步骤iii)的结晶脱色，获得脱色物46.51g，辅酶Q10的含量为83.0％，收率为99.6％。进一步地，对所得脱色物进行实施例1的步骤iv)的层析精制，获得辅酶Q10产品38.53g，辅酶Q10含量为99.8％，收率为99.6％，总收率为98.6％。
比较例1
以与实施例1的步骤i)相同的方式提取辅酶Q10，除了正己烷：甲醇：水的体积比为5:3:1(v/v/v)之外。分离油水两相并旋转蒸发后得到辅酶Q10提取物98.75g，测得辅酶Q10含量为32.0％，收率为81.0％。
比较例2
以与实施例1的步骤i)相同的方式提取辅酶Q10，除了环己烷：乙醇：水的体积比为7:1:1(v/v/v)之外。分离油水两相并旋转蒸发后得到辅酶Q10提取物99.17g，测得辅酶Q10含量为31.5％，收率为80.1％。
比较例3
以与实施例1的步骤i)相同的方式进行辅酶Q10的提取，得到101.02g辅酶Q10提取物，测得辅酶Q10含量为38.5％，收率为99.7％。在1000mL烧瓶中加入300ml正己烷溶解，加入300ml水，室温机械搅拌30min，静置1小时分层，可以观察到油层、乳化层和水层，分层后将油层旋转蒸发脱除溶剂、称重，得到辅酶Q10 65.05g，测得辅酶Q10含量为45.5％，收率为76.1％，总收率为75.9％。
比较例4
以与实施例1的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物56.05g，测得辅酶Q10含量为69.3％，收率为99.7％。对该去除杂质后的处理物以与实施例1的步骤iii)相同的方式进行结晶脱色，除了以乙醇代替丙酮与乙醇的混合溶剂外。结晶完毕，抽滤，混合溶剂洗涤三次，烘干得辅酶Q10 48.90g，测得辅酶Q10含量为70.7％，收率为89.0％。
比较例5
以与实施例1的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物57.11g，测定辅酶Q10含量为67.8％，收率为99.8％。对该去除杂质后的处理物以与实施例1的步骤iii)相同的方式进行结晶脱色，除了以异丙醇代替丙酮与乙醇的混合溶剂外。结晶完毕，抽滤，混合溶剂洗涤三次，烘干得辅酶Q10 47.18g，测得辅酶Q10含量为72.3％，收率为88.1％。
比较例6
以与实施例1的步骤i)和步骤ii)相同的方式进行提取和沉淀操作，获得去除杂质后的处理物56.07g，测定辅酶Q10含量为69.2％，收率为99.6％。对该去除杂质后的处理物以与实施例1的步骤iii)相同的方式进行结晶脱色，除了混和溶剂V 丁酮：V 乙醇的体积比为1:12外。结晶完毕抽滤，混合溶剂洗涤三次，烘干得辅酶Q10 46.51g，测得辅酶Q10含量为79.0％，收率为94.7％。
比较例7
取辅酶Q10菌体780g(辅酶Q10含量为5.0％，水分含量为2.1％)，以与实施例1相同的方式进行步骤i)的提取操作，获得100.11g辅酶Q10提取物，辅酶Q10 含量为38.8％，收率为99.6％。向该提取物中加入500mL正己烷使其溶解，再加入500mL水，室温搅拌，往提取液中滴加100mLNaOH溶液(1.5mol/L)进行皂化，静置分层，分液漏斗分离油水两相后，有机1701072相再用120mL水，分三次进行洗涤，得到含辅酶Q10的有机相，40℃下在旋转蒸发仪中蒸发溶剂得到辅酶Q10 50.20g，测得辅酶Q10含量为68.8％，收率为88.9％，总收率为88.6％。
比较例8
取辅酶Q10菌体780g(辅酶Q10含量为5.5％，水分含量为3％)，以与实施例1相同的方式进行步骤i)的提取操作，获得103.31g辅酶Q10提取物，辅酶Q10含量为41.3％，收率为99.5％。直接进行步骤iii)的结晶操作。具体步骤为：将该提取物加入600mL的混和溶剂(V 丙酮：V 乙醇＝2：1)中，于30℃冷阱中搅拌溶解，完全溶解后保温30min；以1℃/3min的速率降温，温度降为20℃，瓶底有大量晶体颗粒析出，保温30min；以1℃/3min的降温速率再次降温，降至-5℃，保温3h；抽滤，混合溶剂洗涤三次，烘干得辅酶Q10 75.60g，测得辅酶Q10的含量为50.0％，收率为88.6％，总收率为88.1％。结晶母液中辅酶Q10的含量有6％，非常难处理。
比较例9
取辅酶Q10菌体780g(辅酶Q10含量为5.3％，水分含量为2.7％)，以与实施例1相同的方式进行步骤i)的提取操作，获得104.29g辅酶Q10提取物，辅酶Q10含量为39.4％，收率为99.4％。然后将该提取物直接进行步骤iv)的层析精制，具体步骤为：使用实施例1的玻璃层析柱，称取获得的辅酶Q10提取物，加入石油醚并加热溶解完全，冷却至室温后上样。用含有1.5％乙酸乙酯的石油醚溶液作为洗脱剂进行洗脱，洗脱流速为7.2mL/min，分段接洗脱液，将主洗脱液合并，旋转蒸发脱除溶剂、称重，得辅酶Q10产品45.24g，测得辅酶Q10含量为85.2％，收率为93.8％，总收率为93.2％，过柱过程非常缓慢，大量杂质吸附在柱子上，堵塞了填料。
由上述结果可以看出，实施例1和3与比较例1和2均使用了本发明的非极 性有机溶剂、极性有机溶液和水的组合，但是，比较例1和2使用的溶剂配比不在本发明范围内，因此所产生的油相与水相分相不显著，辅酶Q10不能充分融入油相中，导致油相中的辅酶Q10的含量少，收率降低。相比之下，实施例1和3的混合溶剂各组分配比适当，油相与水相呈相明显，上层油相萃取出大量的辅酶Q10，将菌渣和部分极性杂质分离到下层水相，辅酶Q10的收率明显高于比较例1和2。
比较例3使用正己烷作为溶剂来溶解步骤i)得到的辅酶Q10提取物，可观察到油层、乳化层和水层三种分层，乳化层清晰且大量存在，辅酶Q10和部分杂质共存于油相和乳化层中，不能有效分离。与其相对，本申请实施例1使用丁酮作为溶剂，对步骤i)得到的辅酶Q10提取物进行沉淀去除强极性杂质，有效减少了乳化影响，油层与水层分界明显，乳化层很薄且不清晰，脂溶性极性杂质不溶于丁酮而沉淀。因此，现有技术中以正己烷等为溶剂进行纯化时，如果遇到水可明显看到乳化现象，最终得到的辅酶Q10纯度及收率较低，使用本发明的方法则可显著提高纯度及收率。
实施例2、5和6分别使用活性白土、硅藻土和活性炭作为吸附剂进行脱色，实验结果显示，活性白土的选择性吸附最好，脱色后的收率最高。相比之下，活性炭脱色后的辅酶Q10收率最低，可能是因为活性炭会造成产品不可逆吸附，从而导致收率损失。活性白土和硅藻土可以很好地解决这个问题，可以选择性地将步骤iii)中随辅酶Q10一起提取出的色素类物质通过吸附而去除。
实施例7～9与比较例4～6对结晶脱色进行了研究。实验结果显示，比较例4和5单独使用乙醇或异丙醇的脱色效果最差，结晶后收率也不理想。比较例6虽然也以丁酮和乙醇作为混合溶剂，但溶剂的配比不在本发明范围内，因此与实施例1相比结晶后的收率不高。
比较例7～9仅包含本发明方法的部分步骤，辅酶Q10的提取率或者纯度均不及本申请实施例1。比较例7采用碱水洗的工艺，导致脂溶性杂质中大量的可乳化杂质严重乳化，带走部分辅酶Q10，使收率降低。比较例8在提取后直 接结晶纯化，未预先进行杂质的进一步处理，例如本发明的通过沉淀法去除不溶于酮类溶剂的脂溶性极性杂质的工序，因此结晶母液中杂质含量高且成分复杂，导致从母液中回收产品的收率很低。比较例9提取后直接层析过柱进行纯化，由于上样液中含有大量杂质，有些杂质会不可逆吸附于硅胶上，堵塞填料导致硅胶的套用次数低，辅酶Q10的收率和纯度也降低。
实施例1、2、9、10均按照本发明方法的步骤i)～iv)进行操作，且满足各步骤的条件，因此均获得了理想的分离纯化效果，辅酶Q10的纯度可达到99.8％以上，总收率可保持在98.5％以上。
产业上的可利用性
本发明的提取纯化方法可有效去除辅酶Q10制备过程中存在的强极性杂质、极性脂类杂质、色素、中性和非极性脂类杂质，以及醌类同系物等杂质，产物辅酶Q10的总收率可保持在98.5％以上，纯度达到99.8％以上。此外，本发明方法不存在现有技术中碱洗步骤出现的乳化问题，操作步骤少且简单，所用试剂和设备皆为常规，成本低且具有竞争优势，非常适合于工业化生产。

Claims (12)

  1. 一种辅酶Q10的提取纯化方法，其包括如下步骤：
    i)对辅酶Q10菌体进行提取，提取体系是非极性有机溶剂、极性有机溶剂和水的组合；
    ii)将步骤i)得到的提取物与酮类溶剂混合以沉淀去除杂质；
    iii)将步骤ii)得到的去除杂质后的处理物采用吸附剂吸附或结晶进行脱色；以及
    iv)将步骤iii)得到的脱色物进行层析精制，得到提取纯化后的辅酶Q10。
  2. 根据权利要求1所述的方法，其特征在于，所述步骤i)的提取体系中非极性有机溶剂、极性有机溶剂和水的体积比为6～10:2～6:1，优选为7～9:2～4:1，所述提取导致分层，将分离得到的油相脱除溶剂后，得到辅酶Q10提取物。
  3. 根据权利要求1或2所述的方法，其特征在于，所述步骤i)中提取体系与辅酶Q10菌体的体积质量比以mL/g表示为10～20:1，优选为10～18:1，更优选为10～15:1。
  4. 根据权利要求2所述的方法，其特征在于，所述非极性有机溶剂为环己烷、乙醚、异丙醚、异辛烷、正己烷或石油醚中的至少一种，优选为环己烷、正己烷或石油醚，所述极性有机溶剂为丙酮、乙腈、乙酸乙酯、甲醇、乙醇和异丙醇中的至少一种，优选为丙酮、乙腈或乙酸乙酯，所述水为所述细菌中的水分或/和添加的水。
  5. 根据权利要求1所述的方法，其特征在于，所述步骤ii)的酮类溶剂和所述步骤i)得到的提取物的体积质量比以mL/g表示为1～30:1，优选为1～20:1，更优选为5～10:1，所述酮类溶剂为丙酮、丁酮、2-戊基酮和甲基异丁基酮中的至少一种，优选为丙酮或丁酮。
  6. 根据权利要求1所述的方法，其特征在于，所述步骤iii)的吸附剂为活性白土、硅藻土和活性炭中的至少一种，优选为活性白土；所述吸附剂用 量为辅酶Q10质量的0.1～100倍，优选为0.1～40倍，更优选为0.3～5倍。
  7. 根据权利要求1所述的方法，其特征在于，所述步骤iii)的结晶脱色采用醇类溶剂和酮类溶剂组成的混合体系，所述混合体系中的醇类溶剂与酮类溶剂的体积比为0.1～9:1，优选为0.5～7:1，更优选为2～6:1，所述醇类溶剂为甲醇、乙醇和异丙醇中的至少一种，优选为乙醇，所述酮类溶剂为丙酮、丁酮、2-戊基酮和甲基异丁基酮中的至少一种，优选为丙酮或丁酮。
  8. 根据权利要求1所述的方法，其特征在于，所述的去除杂质后的处理物为过滤步骤ii)中沉淀的杂质得到的含有辅酶Q10的滤液，或将过滤步骤ii)中沉淀的杂质得到的含有辅酶Q10的滤液脱除溶剂后，得到的滤液浓缩物。
  9. 根据权利要求7或8所述的方法，其特征在于，步骤iii)中所述结晶使用的混合体系与辅酶Q10的体积质量比以mL/g表示为5～35:1，优选为10～30:1，更优选为15～22:1，所述结晶的温度为-10～25℃。
  10. 根据权利要求1-9任一项所述的方法，其特征在于，所述步骤iv)包括：将所述步骤iii)得到的脱色物溶解于非极性溶剂，上样，通过层析柱进行纯化，用非极性有机溶剂和极性有机溶剂组成混合溶剂作为洗脱剂，得到辅酶Q10。
  11. 根据权利要求10所述的方法，其特征在于，所述非极性有机溶剂为石油醚、乙醚、正己烷或异辛烷中的至少一种，优选为石油醚或正己烷，所述极性有机溶剂为异丙醇、乙醇、1,4-二氧六环、乙酸乙酯和丙酮中的至少一种，优选为异丙醇或乙酸乙酯，所述层析柱中的固定相为硅胶，流动相为非极性有机溶剂和极性有机溶剂的混合溶剂。
  12. 根据权利要求1-11任一项所述的方法制备的辅酶Q10，所述辅酶Q10的纯度达到99.8％以上
PCT/CN2018/123214 2018-07-19 2018-12-24 辅酶q10的提取纯化方法及由其制备的辅酶q10 WO2020015316A1 (zh)

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