WO2008145071A1 - Procédé de production par fermentation de la coenzyme q10 - Google Patents

Procédé de production par fermentation de la coenzyme q10 Download PDF

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Publication number
WO2008145071A1
WO2008145071A1 PCT/CN2008/071143 CN2008071143W WO2008145071A1 WO 2008145071 A1 WO2008145071 A1 WO 2008145071A1 CN 2008071143 W CN2008071143 W CN 2008071143W WO 2008145071 A1 WO2008145071 A1 WO 2008145071A1
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Prior art keywords
coenzyme
cells
fermentation
liter
alcaligenes
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PCT/CN2008/071143
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English (en)
Chinese (zh)
Inventor
Xuejun Chen
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Tianan Biologic Material Co., Ltd. Ningbo
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Application filed by Tianan Biologic Material Co., Ltd. Ningbo filed Critical Tianan Biologic Material Co., Ltd. Ningbo
Publication of WO2008145071A1 publication Critical patent/WO2008145071A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/05—Alcaligenes

Definitions

  • the invention belongs to the field of fermentation engineering.
  • the present invention relates to a novel method of fermentative production of Q10. Background technique
  • Coenzyme Q10 also known as ubiquinone, is a fat-soluble anthraquinone that is widely distributed in nature and is found mainly in the cells of the heart, liver and kidney of yeast, plant leaves, seeds and animals. It binds to mitochondria in cells such as animals, plants, and microorganisms, and acts as a hydrogen donor in the electron transport chain, which is an essential component of the respiratory chain.
  • Coenzyme Q10 has a wide range of applications. A large number of studies have been carried out in Japan, the United States, Switzerland, Italy and other countries. The results showed that coenzyme Q10 has obvious curative effects in cardiovascular diseases, comprehensive treatment of cancer, and prevention of dental diseases. At the same time, effective use in the field of health care products and cosmetics has shown strong demand.
  • the traditional method for producing coenzyme Q10 at home and abroad is a biological extraction method, which is extracted from animal organs such as pig heart and cow heart, and specifically includes, for example, saponification method, solvent extraction method and adsorption chromatography.
  • this method is limited by the content of the product in the raw material, and the cost is difficult to reduce, and is not suitable for modern industrial production.
  • the chemical synthesis method mainly adopts a semi-synthesis method, and the process generally extracts solanesol from tobacco, and then synthesizes coenzyme Q10 from the solanesol and the coenzyme Q10 parent ring 3,4,5-trimethoxytoluene.
  • the mother core 3, 4, 5-trimethyloxytoluene is currently difficult to synthesize on a large scale, the cost is hard to be further greatly reduced.
  • solanesol undergoes cis and trans isomerization, and a mixture of cis and trans isomers is obtained, which requires separation and also increases the synthesis cost.
  • the synthesized cis and trans isomer products are not easily absorbed by the human body.
  • Microbial fermentation is a new coenzyme Q10 production method that has emerged in recent years. Since the microbial cells are easy to grow in large scale, the product is completely in a natural all-trans configuration and has high bioavailability. Moreover, compared with the chemical synthesis method, it is safe and efficient, and there is almost no toxic chemical residue in the product, which is easy to be separated and purified, and has good clinical curative effect, and is the most promising production method.
  • the key technology of this method is the production capacity of coenzyme Q10 producing bacteria and how to extract coenzyme Q10 from the fermenting cells.
  • yeast such as Candida, Chandler, yeast, Cryptococcus, etc.
  • Pseudomonas red bacillus, yellow bacillus
  • Agrobacterium Aspergillus
  • Monascus Photosynthetic bacteria
  • Rhodospirillum Chlorella sp., etc.
  • a first aspect of the present invention provides a method of producing coenzyme Q10, characterized in that the method comprises the following steps:
  • Another aspect of the invention also relates to coenzyme Q10 produced by this method.
  • Another aspect of the invention relates to the use of Alcaligenes faecalis for the production of coenzyme Q10 for high density fermentation.
  • the method of the invention is suitable for the production of coenzyme Q10 by microbial fermentation, the process is simple, the concentration of the fermentation liquid is high, the production cost is low, and the scale production can be realized.
  • the present inventors have found through research that high-density fermentation can be achieved by using a specific strain, that is, Alcalium igenes eutruphus strain, so that coenzyme Q10 can be produced at a relatively low cost.
  • the present invention provides a method of producing coenzyme Q10, characterized in that the method comprises the steps of:
  • Alcaligenes faecalis is a class of Alcaligenes producing bacteria known in the prior art.
  • the strain has been widely For the production of polyhydroxyalkanoates using anaerobic acidified food waste (see, for example, Yi Zuhua et al., "Study on the production of poly- ⁇ -hydroxybutyric acid from the mutant strain of A. faecalis 65-7", Microorganisms Bulletin, 1995, 11 (1); Chen Qi, et al., “Selection of Poly- ⁇ -Hydroxybutyric Acid Strains Produced by Glucose Fermentation", Bulletin of Microbiology, 1994, 21 (6).
  • Alcaligenes faecalis can be used for high-density fermentation production of coenzyme Q10.
  • the Alcaligenes faecalis is selected from Alcaligenes faecalis H16. 65-7 (see, for example, Yi Zuhua et al., "The Alcaligenes faecalis mutant 65" -7 Research on poly- ⁇ -hydroxybutyric acid production, Microbiology Bulletin, 1995 22 (1); Available from Institute of Microbiology, Chinese Academy of Sciences) or Alcaligenes faecalis NCIB11599 (purchased from Institute of Microbiology, Chinese Academy of Sciences) .
  • high-density fermentation and "high-density culture” as used herein are used interchangeably and have the meaning commonly known and recognized by those skilled in the art, gp, by culturing the strain of A. faecalis under certain conditions. It can be made to a higher density (usually the dry weight of the cells is at least 90 g / liter or more). Since the density of the cells in the fermentation liquid per unit volume is increased, the production of the coenzyme Q10 can be increased under the fermentation conditions of the same scale, thereby reducing the production cost.
  • the high density fermentation of the present invention is carried out in a bioreactor.
  • the bioreactor can be suitably selected by one skilled in the art based on conventional skill, such as a mechanically agitated bioreactor with a well-dissolved oxygen supply and a fully enclosed. Of course, other batch or continuous feed bioreactors can also be employed.
  • the bioreactor may have a volume of 5 liters to 200 M 3 .
  • the medium used for the high-density fermentation may be any medium known in the art for culturing Alcaligenes faecalis.
  • the medium suitable for the present invention has the following composition: glucose 15-25 g / liter, ammonium sulfate 1. 6-2. 0 g / liter, dipotassium hydrogen phosphate 1. 4-1 7 g / liter, disodium hydrogen phosphate 8-10 g / liter, magnesium sulfate 1. 5-2. 5 g / liter, yeast extract 2-4 g / liter, peptone 1-3 g / liter, pH 6 8-7. 0.
  • the medium suitable for use in the present invention has the following composition: glucose 20, ammonium sulfate 1. 8, dipotassium hydrogen phosphate 1. 5, disodium hydrogen phosphate 9, magnesium sulfate 2, yeast extract 3 , peptone 2, PH6. 8-7. 0.
  • the composition of the medium can be suitably changed by those skilled in the art based on routine experimentation.
  • the high density fermentation can be achieved in the following manner. First, the alkaline bacterium is inoculated into the medium according to a conventional aseptic operation, and the inoculum amount is preferably 5-15%.
  • the sugar concentration is controlled to 2-2. 5% (W/W), the temperature is controlled to 30 ° C, the air volume is controlled to 0. 3-1 VVM, and the pH is controlled to be 6. 8-7. Fermentation is carried out.
  • the dissolved oxygen concentration is controlled at around 20% (relative to the maximum dissolved oxygen value before inoculation), which can be adjusted by adjusting the stirring speed and the amount of ventilation. Fermentation until the dry weight of the cells reaches 90 g/L or more, more preferably 100 g/L or more, and even more preferably 120 g/L or more.
  • the cell concentration can be determined by measuring the 0D value at 620 nm after the fermentation broth is diluted.
  • the dry weight of the cells can be weighed by centrifuging the fermentation broth, washing twice with water and then drying the cells at 80 ° C for 24 hours.
  • the fermentation time is usually controlled in the range of about 26-40 hours.
  • the separation method may employ various conventional means such as filtration, vacuum filtration, pressure filtration, centrifugation, or a combination thereof.
  • the currently known methods include an alkali saponification method, an alcohol alkali saponification method, an adsorption chromatography method, and the like.
  • a high speed centrifuge such as a butterfly centrifuge, a tube centrifuge, a horizontal screw centrifuge, etc.
  • a high speed centrifuge such as a butterfly centrifuge, a tube centrifuge, a horizontal screw centrifuge, etc.
  • High-speed centrifuges with factors above 3000-4000.
  • other means such as vacuum filtration, pressure filtration or the use of conventional low speed centrifuges.
  • the isolated cells can be directly dried for downstream operations. However, in a preferred embodiment, it is necessary to re-add an appropriate amount of water to the separated cells, and adjust the pH to alkaline with a basic substance (such as sodium hydroxide, potassium hydroxide, liquid ammonia, ammonia, etc.). For example, 9-14, preferably 10-12), is then heated and dried. The heating temperature is preferably controlled between 80 ° C and 10 °C (rC, usually 30-60 minutes). The pH adjustment and heating step are used to facilitate saponification. The pH is outside of 10-12. It may cause the saponification effect to deteriorate or damage the product.
  • a basic substance such as sodium hydroxide, potassium hydroxide, liquid ammonia, ammonia, etc.
  • the heating temperature is preferably controlled between 80 ° C and 10 °C (rC, usually 30-60 minutes).
  • the pH adjustment and heating step are used to facilitate saponification.
  • the pH is outside of 10-12. It may cause the saponification effect to deteriorate or damage
  • Drying the cells may be carried out by means of spray drying, drum drying, air drying or drying, but it should be noted that the temperature of the cells should be controlled below 80 °C to avoid deterioration of product quality.
  • the dried cells obtained above were subjected to extraction under reflux of an organic solvent.
  • the organic solvent may be chloroform, acetone, carbon tetrachloride, petroleum ether or a combination thereof.
  • the reflux extraction time is 30-60 minutes.
  • the filtrate obtained by extraction can be evaporated to dryness under reduced pressure and then extracted with an organic solvent to increase extraction purity.
  • the organic solvent and conditions used in the second extraction may be the same as or different from the first extraction. For example, it may be extracted with a solvent selected from petroleum ether or diethyl ether for 5-10 minutes.
  • the extracted filtrate is added to a chromatography column (e.g., a silica gel column) to collect a yellow liquid.
  • a chromatography column e.g., a silica gel column
  • Those skilled in the art will be able to select suitable chromatographic conditions based on conventional techniques.
  • coenzyme Q10 is a routine method in the art, including, but not limited to, Craven's color experiment, Wiss-Brubacher method, thin layer chromatography, mass spectrometry, spectrophotometry, infrared and ultraviolet absorption spectroscopy, etc. See, for example, Food Research and Development, Vol. 25, No. 2, April 2004; Science Bulletin, Vol. 18, No. 4, July 2002; Fungal Systems, 19 (2): 217-222, 2000). detailed description
  • the A. faecalis H16. 65-7 was inoculated to a medium having the following composition (in grams per liter) at 10%: glucose 20, ammonium sulfate 1. 8, dipotassium hydrogen phosphate 1. 5, hydrogen phosphate Disodium 9, magnesium sulfate 2, yeast extract 3, peptone 2, PH 6. 8-7. 0 ⁇
  • the high-density fermentation is carried out under the following control conditions: sugar concentration 2-2. 5%, temperature 30 ° C, air volume 0. 3-1 VVM, pH control at 6. 8-7. After 30 hours of fermentation, the dry weight of the cells was measured to be 98 g/L.
  • Alcaligenes faecalis H16. 65-7 was fermented and fermented for 36 hours, and the dry weight of the cells was determined to be 125 g/L.
  • Example 1 According to the process described in Example 1, the fermentation of Alcaligenes faecalis H16. 65-7, fermentation for 38 hours, measured The dry weight of the cells was 128 g/L.
  • Fermentation was carried out in accordance with the procedure described in Example 1, except that Alcaligenes oryzae NCIV11599 was used instead of the Alcaligenes faecalis H16. 65-7 (purchased from the Institute of Microbiology, Chinese Academy of Sciences). After 38 hours of fermentation, the dry cell weight was measured to be 109 g/L.
  • Fermentation was carried out in accordance with the procedure described in Example 1, except that Alcaligenes oryzae NCIV11599 was used instead of the Alcaligenes faecalis H16. 65-7 in Example 1. After 29 hours of fermentation, the dry weight of the cells was determined to be 93 g/L.

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  • Engineering & Computer Science (AREA)
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Abstract

L'invention porte sur un procédé de fabrication de la coenzyme Q10, qui comprend les étapes suivantes consistant à : (a) faire fermenter à densité élevée de Alcaligenes eutrophus à une masse sèche de cellules de 90 g/l ou plus ; (b) séparer les cellules à partir du bouillon fermenté ; et (c) extraire la coenzyme Q10 à partir des cellules isolées. L'invention porte également sur l'utilisation de l'Alcaligenes eutrophus pour la production par fermentation à densité élevée de la coenzyme Q10.
PCT/CN2008/071143 2007-05-30 2008-05-30 Procédé de production par fermentation de la coenzyme q10 WO2008145071A1 (fr)

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Application Number Priority Date Filing Date Title
CN200710041442.0 2007-05-30
CN2007100414420A CN101314782B (zh) 2007-05-30 2007-05-30 发酵生产辅酶q10的方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113912480A (zh) * 2021-09-08 2022-01-11 丽江映华生物药业有限公司 一种辅酶q10的提取方法
CN115819211A (zh) * 2022-12-29 2023-03-21 广东润和生物科技有限公司 一种辅酶q10的分离纯化方法

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CN102057991B (zh) * 2009-12-31 2013-11-06 山东三星玉米产业科技有限公司 一种富含辅酶q10的玉米油及其生产方法
CN102391092B (zh) * 2011-11-22 2013-05-01 杭州华东医药集团康润制药有限公司 一种高纯度辅酶q10的规模化制备方法
CN103613494B (zh) * 2013-11-05 2016-02-03 广东医学院 以红曲菌丝体为原料提取辅酶q10的工艺及生产方法
CN103815279B (zh) * 2014-02-08 2015-02-04 福建农林大学 一种富含辅酶q10红曲及其制备方法
CN115011739B (zh) * 2022-08-03 2022-11-01 南京邦康生物技术有限公司 一种益生菌的生产控制方法与系统

Citations (5)

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Publication number Priority date Publication date Assignee Title
JPS5558098A (en) * 1978-10-27 1980-04-30 Ajinomoto Co Inc Production of coenzyme q10
JPS5655197A (en) * 1979-10-08 1981-05-15 Ajinomoto Co Inc Production of coenzyme q10
JPS57129695A (en) * 1981-02-02 1982-08-11 Ajinomoto Co Inc Preparation of coenzyme q10 by fermentation process
JPS58149688A (ja) * 1982-02-19 1983-09-06 Daikin Ind Ltd 補酵素ｑ↓１↓０の製造法
JPS5955192A (ja) * 1982-09-24 1984-03-30 Daikin Ind Ltd 補酵素ｑ１０の製造法

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CN1912098A (zh) * 2005-08-10 2007-02-14 德古萨股份公司 生产辅酶q10的细菌菌种及生产辅酶q10的方法

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
JPS5558098A (en) * 1978-10-27 1980-04-30 Ajinomoto Co Inc Production of coenzyme q10
JPS5655197A (en) * 1979-10-08 1981-05-15 Ajinomoto Co Inc Production of coenzyme q10
JPS57129695A (en) * 1981-02-02 1982-08-11 Ajinomoto Co Inc Preparation of coenzyme q10 by fermentation process
JPS58149688A (ja) * 1982-02-19 1983-09-06 Daikin Ind Ltd 補酵素ｑ↓１↓０の製造法
JPS5955192A (ja) * 1982-09-24 1984-03-30 Daikin Ind Ltd 補酵素ｑ１０の製造法

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GAO X.F. ET AL.: "Advance in Production of Coenzyme Q10 by Fermentation Methods", NAT. PROD. RES. DEV., vol. 18, no. 5, October 2006 (2006-10-01), pages 858 - 862 *
WANG Q.Y. ET AL.: "The Research Advances of Production of Coenzyme Q10 by Microbial Fermentation", CHEMISTRY & BIOENGINEERING, vol. 23, no. 8, August 2006 (2006-08-01), pages 11 - 13 *
YUAN J. ET AL.: "Recent Progress and Ubiquinone-10 Production by Microbial Fermentation", AMINO ACIDS & BIOTIC. RESOURCES, vol. 26, no. 1, 2004, pages 53 - 56 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113912480A (zh) * 2021-09-08 2022-01-11 丽江映华生物药业有限公司 一种辅酶q10的提取方法
CN115819211A (zh) * 2022-12-29 2023-03-21 广东润和生物科技有限公司 一种辅酶q10的分离纯化方法

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