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WO2019127829A1 - 氧化型辅酶q10的发酵生产方法、及由其制备而得的高含量氧化型辅酶q10 - Google Patents

氧化型辅酶q10的发酵生产方法、及由其制备而得的高含量氧化型辅酶q10 Download PDF

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WO2019127829A1
WO2019127829A1 PCT/CN2018/074822 CN2018074822W WO2019127829A1 WO 2019127829 A1 WO2019127829 A1 WO 2019127829A1 CN 2018074822 W CN2018074822 W CN 2018074822W WO 2019127829 A1 WO2019127829 A1 WO 2019127829A1
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Prior art keywords
fermentation
coenzyme
acid
controlled
liquid
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PCT/CN2018/074822
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English (en)
French (fr)
Inventor
袁慎峰
于洪巍
陈召峰
朱永强
闵一
李永
胡柏剡
邱贵生
于凯
Original Assignee
浙江新和成股份有限公司
浙江大学
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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Priority to JP2019525838A priority Critical patent/JP6931879B2/ja
Priority to DE112018006584.7T priority patent/DE112018006584T5/de
Priority to AU2018319219A priority patent/AU2018319219B2/en
Priority to US16/016,813 priority patent/US10774350B2/en
Priority to DKPA201970421A priority patent/DK180437B1/en
Publication of WO2019127829A1 publication Critical patent/WO2019127829A1/zh

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

Definitions

  • the present application relates to the field of microbial fermentation, and in particular to a high content (for example, more than 70%, 80% or more) of a production strain (for example, Rhodobacter sphaeroides) by regulating an Oxidation-Reduction Potential (ORP). Or 90% or more) a fermentation method of oxidized coenzyme Q10.
  • a high content for example, more than 70%, 80% or more
  • a production strain for example, Rhodobacter sphaeroides
  • ORP Oxidation-Reduction Potential
  • Coenzyme Q10 (CoQ10) is also known as ubiquinone, terpene oxime, and its chemical name is 2,3-dimethoxy-5-methyl-6-non-p-pentylene phenylhydrazine.
  • the biological activity of Coenzyme Q10 is derived from the redox properties of its anthracene ring and the physicochemical properties of its side chain. It is a natural antioxidant and cellular metabolic activator produced by the cell itself, which has antioxidant activity, eliminates free radicals, and enhances immunity. Force, anti-aging and other functions. It is widely used in the treatment of various diseases such as heart disease, cancer, diabetes, acute and chronic hepatitis, Parkinson's disease, etc., and also has many applications in food, cosmetics and anti-aging health care products.
  • Microbial fermentation is the main production method of coenzyme Q10.
  • the production of Coenzyme Q10 by microbial fermentation has great competitive advantages in terms of product quality and safety, and is suitable for large-scale industrial production.
  • the fermentation stage of microbial production of coenzyme Q10 is generally divided into the following two stages: 1) the stage of growth of bacteria (also known as the stage of microbial growth and reproduction), in which it is generally necessary to maintain sufficient oxygen supply and nutrients for rapid growth of microorganisms.
  • the synthesis of the metabolite coenzyme Q10 is initiated quickly; 2) the coenzyme Q10 synthesis accumulation stage (sometimes called the synthesis stage), at this stage, the fermentation bacteria rapidly consume oxygen, in the fermentation broth The dissolved oxygen is generally at a lower value, and the fermenting microorganism is in an oxygen-restricted state, at which time the metabolite coenzyme Q10 is rapidly synthesized and accumulated.
  • the stage of synthesis and accumulation according to the change of the titer of coenzyme Q10 in the fermentation broth, it is usually divided into the early stage (the increase curve of the coenzyme Q10 titer remains high) and the medium term (the coenzyme Q10 titer increase curve slows down, but Still maintains a clear increase trend) and later (coenzyme Q10 titer increase curve tends to balance, with a slight increase in fermentation time).
  • the time interval between the early, middle, and late stages of the coenzyme Q10 synthesis accumulation phase is about 10-20 hours.
  • a phased oxygen supply control strategy is adopted to regulate the fermentation process, and high oxygen supply is adopted during the growth stage and the pre-synthesis accumulation stage of the fermentation process to promote rapid growth of the cells and rapid synthesis of coenzyme Q10.
  • the oxygen supply is reduced in stages to maintain a higher coenzyme Q10 than the production rate, reducing the consumption of glucose on the substrate;
  • the change of oxygen supply mode will surely get the best physiological state of the production bacteria and reduce the cost of coenzyme Q10 production.
  • the patent CN105420417A proposes a synergistic control of the fermentation process of coenzyme Q10 by adjusting the oxygen consumption rate (dissolved oxygen) and the conductivity (addition of nutrient rate); the patent CN104561154A adjusts the process parameters based on the shape of the bacteria in the fermentation process; Patent CN103509729B enhances the ability of microorganisms to synthesize coenzyme Q10 by modifying red-global bacteria.
  • the coenzyme Q10 produced is a mixture of oxidized coenzyme Q10 and reduced coenzyme Q10, and the proportion of reduced coenzyme Q10 is relatively high.
  • the process described in the patent US Pat. No. 7,910,340 B2 after the end of the fermentation, the reduced coenzyme Q10 content of the coenzyme Q10 produced by the microorganism is above 70%.
  • any type of coenzyme Q10 can function as an electron transporter and has a related physiological function.
  • the market demand for oxidized coenzyme Q10 has increased in recent years.
  • the object of the present invention is to solve the problems of low ratio of oxidized coenzyme Q10 produced by the microbial fermentation method in the prior art, complicated post-treatment process, and the like, and provide a production strain (for example, a ball by controlling the ORP of the fermentation liquid).
  • Rhodobacter sphaeroides produces a high-yield oxidized coenzyme Q10 fermentation production method.
  • the present application relates to the following fermentation production method of oxidized coenzyme Q10:
  • a fermentation production method of oxidized coenzyme Q10 wherein the redox potential ORP of the fermentation broth is controlled to be -50 to 300 mV in the fermentation process of the production strain, and it is preferable to control the redox potential ORP of the fermentation liquid to be 50 to 200 mV.
  • the redox potential ORP of the fermentation liquid is controlled by at least one of: controlling dissolved oxygen of the fermentation liquid, and controlling pH of the fermentation liquid; preferably, the fermentation liquid is controlled
  • the manner of dissolving oxygen is combined with the manner of controlling the pH of the fermentation broth.
  • the dissolved oxygen in the fermentation liquid is controlled by at least one of the following methods: controlling the stirring volume input per unit volume of the fermenter, controlling the air intake flow rate per unit volume of the fermentation liquid, and controlling the fermenter Internal pressure; preferably two or more of the above modes are combined to control dissolved oxygen in the fermentation broth.
  • the fermenter has a stirring input power per unit volume of 0.25 to 0.50 kw/m 3 , the air intake flow rate per unit volume of the fermentation liquid is 1.0 to 15.0 vvm, and/or the fermenter
  • the internal pressure is 0.05 to 0.3 MPa; preferably, the fermentation tank has a stirring input power per unit volume of 0.30 to 0.40 kw/m 3 , and the air intake flow rate per unit volume of the fermentation liquid is 5.0 to 8.0 vvm, and/or
  • the internal pressure of the fermenter is 0.08 to 0.15 MPa.
  • the pH of the fermentation liquid is controlled by controlling the pH of the fermentation liquid to 3.5 to 6.0; preferably, the fermentation is controlled by controlling the pH of the fermentation liquid to 4.0 to 5.0.
  • the pH of the liquid preferably, the pH of the fermentation broth is controlled by adding an acid or adding a base; further preferably, the fermentation broth is controlled by adding the acid or the base in stages or continuously. pH.
  • the acid is an organic acid or an inorganic acid
  • the base is an organic base or an inorganic base
  • the acid is phosphoric acid, hydrochloric acid, sulfuric acid, lactic acid, propionic acid, citric acid.
  • one or more of oxalic acid, and/or preferably the base is one or more of ammonia water, sodium hydroxide, and liquid ammonia; more preferably, the acid is phosphoric acid, lactic acid, Or citric acid, and/or the base is aqueous ammonia or liquid ammonia.
  • the ORP of the fermentation broth is controlled during the synthesis and accumulation phase of the coenzyme Q10 during the fermentation; preferably, the ORP of the fermentation broth is controlled in the middle and late stages of the synthesis and accumulation phase of the coenzyme Q10 during the fermentation; and preferably, during the fermentation process
  • the ORP of the fermentation broth was controlled in the later stage of the synthesis of the coenzyme Q10.
  • the conductivity of the fermentation liquid is controlled in the fermentation process to be 5.0 to 30.0 ms/cm; preferably, in the growth stage of the bacteria, the control oxygen consumption rate is 30 to 150 mmol/(L ⁇ Between h), and controlling the conductivity of the fermentation broth to be between 5.0 and 30.0 ms/cm; and preferably, in the stage of synthesis and accumulation of coenzyme Q10, controlling the oxygen consumption rate to be 60-120 mmol/(L ⁇ h) And controlling the conductivity of the fermentation broth between 8.0 and 15.0 ms/cm.
  • the production strain is Rhodobacter sphaeroides; preferably, the Rhodobacter sphaeroides is a naturally-selected strain, a strain selected by physical or chemical mutagenesis, or a genetic engineering method. More preferably, the Rhodobacter sphaeroides is a Rhodobacter sphaeroides strain deposited under the accession number CGMCC No. 5997, a Rhodobacter sphaeroides strain deposited under the accession number CGMCC No. 5998, or a Rhodobacter sphaeroides deposited under the accession number CGMCC No. 5999 Strain.
  • the coenzyme Q10 is a high-content oxidized coenzyme Q10; and preferably, the content of the oxidized coenzyme Q10 of the high-content oxidized coenzyme Q10 is 96% or more, more preferably 97% or more, and most preferably More than 99%.
  • the present application also relates to coenzyme Q10 prepared by the above method, wherein the coenzyme Q10 is a high content of oxidized coenzyme Q10; and preferably, the content of the oxidized coenzyme Q10 of the high content oxidized coenzyme Q10 is 96% or more. It is more preferably 97% or more, and most preferably 99% or more.
  • coenzyme Q10 prepared by the above method, wherein the coenzyme Q10 is used for preparing food, functional nutritious food, special health food, nutritional supplement, nutrient, animal medicine, beverage, feed, cosmetics, medicine, Pharmacy, preventive medicine.
  • the present application provides a fermentative production method of high-content oxidized coenzyme Q10, which has at least the following effects:
  • the oxidized coenzyme Q10 content of the coenzyme Q10 produced by the microorganism can reach more than 96%, and the product composition is relatively simple, which is more convenient for post-treatment; the oxidized coenzyme Q10 is more stable than the reduced coenzyme Q10.
  • the content of the oxidized coenzyme Q10 is less degraded in the living body than the coenzyme Q10 obtained by the prior art fermentation.
  • the fermentation process of the present application is highly potent.
  • Figure 1 shows a process map of electron transfer in a erythropoietin.
  • the present application provides a high-yield oxidized coenzyme Q10 fermentation production method, which is to produce a high content of oxidized coenzyme Q10 by controlling the redox potential ORP of the fermentation broth during fermentation.
  • the ORP is controlled to -50 to 300 mV, and the ORP of the fermentation broth is preferably controlled to 50 to 200 mV.
  • the strain suitable for use in the method of the present invention is not particularly limited, and may be an existing production strain for producing coenzyme Q10, or may be engineered by a conventional method or genetically engineered.
  • the strain for fermentative production of coenzyme Q10 is Rhodobacter sphaeroides.
  • Rhodobacter sphaeroides is a photosynthetic bacterium belonging to the genus Rhodobacter sphaeroides.
  • the Rhodobacter sphaeroides is a naturally-selected strain, a strain selected by physical or chemical mutagenesis, or a genetically engineered strain.
  • the strain for fermentative production of Coenzyme Q10 is a Rhodobacter sphaeroides strain of the accession number CGMCC No. 5997; or the Rhodobacter sphaeroides strain of the deposit number CGMCC No. 5998; or the bulb of the deposit number CGMCC No. 5999 Rhodococcus strain.
  • the ORP of the fermentation broth is generally controlled between -150 and -300 mV. Due to the physiological action of the bacteria itself, the coenzyme Q10 produced exists in an oxidized and reduced form, wherein the reduced form accounts for about 70%.
  • the present application provides a method for producing a oxidized coenzyme Q10 by a fermenting microorganism by controlling the ORP of the fermentation broth. In the present application, the ORP is controlled to -50 to 300 mV, and the proportion of the oxidized coenzyme Q10 produced by the fermenting bacteria is over 96%. The mechanism of the method is shown in Figure 1.
  • the cycle is used for electron transfer to provide energy for the metabolism of red-like bacteria.
  • the reducing power provided by the reduced NADH is one of the necessary conditions for the conversion of the oxidized coenzyme Q10 into the reduced coenzyme Q10 in the cell.
  • the environment of the fermentation broth will greatly affect the cells
  • the cycle in which the redox potential ORP of the fermentation broth is a key indicator.
  • the ORP value of the fermentation broth is between -150 and -300 mv, that is, when the fermentation broth is under reducing conditions, the coenzyme Q10 produced by the erythropoiesis is mostly reduced coenzyme Q10.
  • the oxidative activity in the fermentation broth environment is enhanced, which leads to the inhibition of the conversion of NAD + to NADH, and the coenzyme Q10 produced by the erythropoiesis bacteria lacks sufficient reducing power conditions provided by NADH.
  • the conversion of oxidized coenzyme Q10 to reduced coenzyme Q10 is also greatly affected, and the final expression is that oxidized coenzyme Q10 is abundantly produced in cells.
  • the above-mentioned fermentative production method of high-content oxidized coenzyme Q10 is suitable for the whole process of microbial fermentation.
  • the above method is used in the stage of synthesis and accumulation of coenzyme Q10.
  • the ORP of the fermentation broth is controlled in the middle and late stages of the synthesis and accumulation phase of the coenzyme Q10 during the fermentation process. More preferably, the ORP of the fermentation broth is controlled later in the synthesis phase of the coenzyme Q10 during fermentation.
  • the ORP value of the fermentation liquid controls the ORP value of the fermentation liquid by controlling the dissolved oxygen or pH of the fermentation liquid during the fermentation process, or synergistically controlling the dissolved oxygen and pH of the fermentation liquid.
  • the ORP value of the fermentation broth is controlled by synergistically controlling the dissolved oxygen and pH of the fermentation broth.
  • the change in dissolved oxygen concentration during fermentation is a dynamic balance between the rate of oxygen supply and the rate of oxygen consumption.
  • the oxygen supply in the fermentation liquid is increased by controlling the stirring power input per unit volume of the fermenter, controlling the air flow rate per unit volume of the fermentation liquid, or controlling the internal pressure of the fermenter, or randomly combining the three methods.
  • the dissolved oxygen (dissolved oxygen) in the fermentation broth makes the ORP value of the fermentation liquid reach -50 to 300 mv.
  • the stirring volume input power per unit volume of the fermenter is controlled to be 0.25 to 0.50 kw/m 3
  • the air intake flow rate per unit volume of the fermentation liquid is controlled to be 1.0 to 15.0 vvm (where vvm means air volume/fermentation liquid)
  • the internal pressure of the fermenter is controlled to 0.05 to 0.3 MPa; more preferably, the fermenter is stirred per unit volume.
  • the power control is 0.30 ⁇ 0.40kw/m 3
  • the air intake flow rate per unit volume of fermentation broth is 5.0-8.0 vvm
  • the internal pressure of the fermenter is controlled to 0.08-0.15 MPa.
  • the pH of the fermentation broth it is also preferred to control the pH of the fermentation broth so that the ORP value of the fermentation broth reaches -50 to 300 mv.
  • the pH can be controlled to 3.5 to 6.0, more preferably, the pH is controlled to 4.0 to 5.0; and preferably by adding an acid or a base;
  • the acid can be a conventional acid conventionally used for adjusting the pH of the fermentation broth
  • it is one or more selected from the group consisting of phosphoric acid, hydrochloric acid, sulfuric acid, lactic acid, propionic acid, citric acid, and oxalic acid
  • the base may be a conventional base conventionally used for adjusting the pH of the fermentation liquid, preferably ammonia water or hydroxide.
  • the acid is phosphoric acid, lactic acid, or citric acid
  • the alkali is ammonia water or liquid ammonia.
  • the pH of the fermentation broth can be controlled in stages or continuously with an acid or a base.
  • the conductivity of the fermentation broth is controlled to be 5.0 to 30.0 ms/cm to maintain the nutrient supply of the fermenting bacteria.
  • the conductivity of the fermentation broth is controlled by the feed medium.
  • the medium to be used in the present invention is not particularly limited, and may be various conventional mediums containing a carbon source, a nitrogen source, a phosphorus source, and a micronutrient.
  • the feed medium is as follows - 8 to 12 g of yeast powder per liter of feed liquid, 5 to 10 g of ammonium sulfate, 1 to 2 g of magnesium sulfate, 3 to 6 g of sodium chloride, and 2 to 4 g of potassium dihydrogen phosphate. , dipotassium hydrogen phosphate 2 ⁇ 4g, calcium chloride 1-2g, biotin 0.013 ⁇ 0.025g, pH 7.0.
  • the temperature in the fermentation process is not particularly limited as long as the effects of the present application can be prevented. From the viewpoint that the production strain can produce more oxidized coenzyme Q10, it is preferred to control the temperature to 25 to 35 °C.
  • the seed culture stage prior to the fermentation production of Coenzyme Q10 can be cultured using conventional means in the art, preferably in the culture mode of the reference CN105483171A.
  • a medium having a concentration of Fe 2+ of 0.1 to 0.5 mol/L is used, and the Rhodobacter sphaeroides strain is sequentially screened by resuscitation and seed culture to obtain fermented seeds.
  • the seed medium formulation used in the method of the present invention is not particularly limited, and may be various conventional media containing a carbon source, a nitrogen source, a phosphorus source, and a micronutrient.
  • the seed culture medium is the following formulation disclosed in CN105483171A—in addition to 0.1 to 0.5 mol Fe 2+ per liter of the medium, the yeast medium contains: 1 g of yeast powder, 1 g of ammonium chloride, 1 g of sodium chloride, and 0.0028 of ferric citrate. g, potassium dihydrogen phosphate 0.6 g, dipotassium hydrogen phosphate 0.9 g, magnesium sulfate 0.25 g, calcium chloride 0.1 g, pH was adjusted to 7.0.
  • the cell growth stage of the Rhodococcus-like bacteria in the fermentation process preferably including the pre- and/or the intermediate phase of the Coenzyme Q10 synthesis accumulation stage, can be carried out by the conventional means in the art in combination with the technical solution of the present application, preferably, the reference patent CN105420417A, using online control of oxygen consumption rate and conductivity in conjunction with the technical solution of the present application.
  • the oxygen consumption rate is adjusted by the stirring speed and the air flow rate
  • the conductivity is adjusted by means of fed-feed or batch feeding.
  • the oxygen consumption rate is controlled between 30 and 150 mmol/(L ⁇ h) during the growth phase of the erythropoiesis bacteria, and the conductivity is stabilized between 5.0 and 30.0 ms/cm; during the synthesis and accumulation phase of the coenzyme Q10
  • the oxygen consumption rate is between 60 and 120 mmol/(L ⁇ h), and the electrical conductivity is stable between 8.0 and 15.0 ms/cm.
  • the medium used is a medium containing a carbon source, a nitrogen source, a phosphorus source, and a micronutrient as is conventional in the art.
  • yeast powder per liter of medium 3 g of ammonium chloride, 2.8 g of sodium chloride, 0.005 g of ferric citrate, 0.6 g of potassium dihydrogen phosphate, 0.9 g of dipotassium hydrogen phosphate, 12.55 g of magnesium sulfate, calcium chloride. 0.1 g, pH 7.0.
  • Rhodobacter sphaeroides strains with the accession number CGMCC No. 5997, CGMCC No. 5998, or CGMCC No. 5999.
  • Pre-fermentation culture Wash the cultured bevel with sterile water to prepare a bacterial suspension with a concentration of 10 8 to 10 9 cells per ml; inoculate the prepared bacterial suspension 2 ml into the mother bottle medium for seed
  • the culture medium was 100 ml, 32 ° C, and 180 rpm, and cultured for 28-30 hours.
  • Seed medium 1 g yeast powder, 1 g NH 4 Cl, 1 g sodium chloride, 2.8 mg ferric citrate, 0.6 g KH 2 PO 4 , 0.9 g K 2 HPO 4 , 0.25 g MgSO 4 , 0.1 g CaCl 2 , 0.5 ⁇ g biotin, pH 7.0.
  • the inoculum amount for inoculating the Rhodobacter sphaeroides strain obtained by the above seed culture into the fermenter may be a conventional content in the art, for example, 10 to 300 ml, preferably 25 to 200 ml, and more preferably 50 to 100 ml.
  • the inoculum can be adjusted as needed.
  • Retention time Reduced coenzyme Q10 was 13.5 min, and oxidized coenzyme Q10 was 22.0 min.
  • the rhodococcus-like bacterial strain CGMCC No. 5998 obtained from seed culture was inoculated with 50 ml into a 5 L fermentor containing fermentation medium to start fermentation, and the oxygen supply conditions for controlling the fermenter were: air intake per unit volume of fermentation broth
  • the flow control is 1.0vvm
  • the stirring input power per unit volume is controlled to 0.25kw/m 3
  • the internal pressure of the fermenter is controlled to 0.1MPa
  • the pH of the fermentation liquid is controlled by the addition of liquid ammonia.
  • the fermentation medium is: 8 g of yeast powder per liter of medium, 3 g of ammonium chloride, 2.8 g of sodium chloride, 0.005 g of ferric citrate, 0.6 g of potassium dihydrogen phosphate, 0.9 g of dipotassium hydrogen phosphate, and 12.55 g of magnesium sulfate.
  • the calcium chloride was 0.1 g, and the pH was adjusted to 7.0.
  • the conductivity of the whole fermentation process was controlled by the feed medium to be about 12 ms/cm.
  • the feed medium contains 8 g of yeast powder per liter of feed liquid, 5 g of ammonium sulfate, 1 g of magnesium sulfate, 3 g of sodium chloride, 2 g of potassium dihydrogen phosphate, 2 g of dipotassium hydrogen phosphate, 1 g of calcium chloride, and biotin. 0.013 g, pH was adjusted to 7.0.
  • the ORP value of the fermentation broth was measured to be -35 mv, and part of the fermentation broth was taken and extracted under an inert gas atmosphere (refer to the above-mentioned potency measurement and content determination), and the oxidized coenzyme Q10 and the reduced type in the cells were used.
  • the content ratio of coenzyme Q10 was 96.5:3.5.
  • the high oxygen supply amount is always maintained, and the high stirring input power and the tank pressure are maintained, so that the dissolved oxygen in the fermentation liquid is continuously maintained at a high value, and the ORP value of the fermentation liquid is always at -35 mv.
  • the microorganisms in the fermentation broth have been in a state of growth and reproduction. According to the test results, it can be confirmed that when the ORP value of the fermentation liquid is maintained at a certain value or more, the fermenting microorganisms in the growth stage of the cells produce a high-oxidized coenzyme Q10.
  • the Rhodobacter sphaeroides strain CGMCC No. 5999 obtained from seed culture was inoculated into a 5 L fermenter containing fermentation medium, and fermentation was started (the air intake flow rate per unit volume of the fermenter was controlled to 0.45 vvm, The unit volume stirring input power is controlled to be 0.1kw/m 3 , the tank pressure is 0.02MPa), the fermentation liquid conductivity is 12ms/cm, and the pH value is controlled to be about 7.0.
  • the fermentation medium is: 8 g of yeast powder per liter of medium, 3 g of ammonium chloride, 2.8 g of sodium chloride, 0.005 g of ferric citrate, 0.6 g of potassium dihydrogen phosphate, 0.9 g of dipotassium hydrogen phosphate, and 12.55 g of magnesium sulfate.
  • the calcium chloride was 0.1 g, and the pH was adjusted to 7.0.
  • the feed medium contains 10 g of yeast powder per liter of feed liquid, 8 g of ammonium sulfate, 1.5 g of magnesium sulfate, 5 g of sodium chloride, 3 g of potassium dihydrogen phosphate, 3 g of dipotassium hydrogen phosphate, 1 g of calcium chloride, and biotin 0.020. g, pH is adjusted to 7.0.
  • the fermentation broth After detecting that the dissolved oxygen in the fermentation broth is no longer decreased, slowly increase the pH of the fermentation broth to about 4.0 by adding a certain amount of phosphoric acid within 1 hour, and add liquid ammonia to keep the pH stable at about 4.0, and the fermentation tank has a volume of fermentation broth air.
  • the inlet flow rate, the stirring input power per unit volume, and the tank pressure remained unchanged.
  • the conductivity of the fermentation broth was 12 ms/cm.
  • the ORP value of the fermentation broth was between 58 and 135 mv.
  • the fermentation process enters the stage of synthesis and accumulation of the coenzyme Q10.
  • pH adjustment was carried out in the early stage of the synthesis and accumulation phase of coenzyme Q10. The final test results show that controlling the ORP value by adjusting the pH value enables the fermenting microorganism to efficiently produce high-oxidized coenzyme Q10 in the early stage of coenzyme Q10 synthesis and accumulation.
  • Rhodobacter sphaeroides strain CGMCC No. 5997 obtained from seed culture was inoculated into a 10 L fermentor containing fermentation medium, and fermentation was started (the air intake flow rate per unit volume of fermentation broth was 0.6 vvm, The unit volume stirring input power is controlled to be 0.1kw/m 3 , the tank pressure is 0.02MPa), the fermentation liquid conductivity is 12ms/cm, and the pH value is controlled to be about 7.0;
  • the fermentation medium is: 8 g of yeast powder per liter of medium, 3 g of ammonium chloride, 2.8 g of sodium chloride, 0.005 g of ferric citrate, 0.6 g of potassium dihydrogen phosphate, 0.9 g of dipotassium hydrogen phosphate, and 12.55 g of magnesium sulfate.
  • the calcium chloride was 0.1 g, and the pH was adjusted to 7.0.
  • the feed medium contains 12 g of yeast powder per liter of feed liquid, 10 g of ammonium sulfate, 2 g of magnesium sulfate, 6 g of sodium chloride, 4 g of potassium dihydrogen phosphate, 4 g of dipotassium hydrogen phosphate, 2 g of calcium chloride, and 0.025 g of biotin.
  • the pH was adjusted to 7.0.
  • the unit volume of the fermenter After detecting that the dissolved oxygen in the fermentation broth is no longer decreased, slowly add a certain amount of phosphoric acid to adjust the pH of the fermentation broth to about 5.0 within 1 hour, and add liquid ammonia to keep the pH stable at about 5.0, and at the same time, the unit volume of the fermenter
  • the air inlet flow rate of the fermentation liquid is controlled to be 5.0 vvm
  • the stirring input power per unit volume is controlled to be 0.3 kw/m 3
  • the tank pressure is 0.08 MPa.
  • the ORP value of the fermentation broth was maintained between 100 and 210 mV.
  • the ORP value of the fermentation broth is controlled by cooperatively controlling the oxygen supply conditions of the fermentation broth and the pH of the fermentation broth.
  • the present embodiment is regulated during the synthesis and accumulation phase of the coenzyme Q10 in the fermentation process, and the ratio of the produced oxidized coenzyme Q10 is 99.1%.
  • the fermentation medium is: 8 g of yeast powder per liter of medium, 3 g of ammonium chloride, 2.8 g of sodium chloride, 0.005 g of ferric citrate, 0.6 g of potassium dihydrogen phosphate, 0.9 g of dipotassium hydrogen phosphate, and 12.55 g of magnesium sulfate.
  • the calcium chloride was 0.1 g, and the pH was adjusted to 7.0.
  • the feed medium contains 12 g of yeast powder per liter of feed liquid, 10 g of ammonium sulfate, 2 g of magnesium sulfate, 6 g of sodium chloride, 4 g of potassium dihydrogen phosphate, 4 g of dipotassium hydrogen phosphate, 2 g of calcium chloride, and 0.025 g of biotin.
  • the pH was adjusted to 7.0.
  • the oxygen consumption rate is maintained at about 70 mmol/(L ⁇ h)
  • the conductivity of the fermentation broth is controlled to 12 ms/cm
  • the pH control is about 6.0.
  • the fermentation is continued, and the fermentation is in the early stage of the coenzyme Q10 synthesis and accumulation stage.
  • the air intake flow rate per unit volume of fermentation tank is controlled to be 6.0vvm
  • the stirring input power per unit volume is controlled to 0.2kw/m 3
  • the tank pressure is 0.1MPa.
  • the pH value is adjusted to about 3.5 in about 2h.
  • the conductivity of the fermentation broth was controlled at 12 ms/cm, and the fermentation was continued; after stabilization, the ORP value of the fermentation broth was maintained between 100 and 200 mv.
  • the ORP value of the fermentation broth is controlled in the late stage of the synthesis and accumulation stage of the coenzyme Q10 in the fermentation process, and the effective price of the oxidized coenzyme Q10 is also increased, and the titer of the fermented microorganism is also pleasingly improved.
  • the fermentation production method of the oxidized coenzyme Q10 of the present application can control the redox potential ORP of the fermentation liquid so that the content of the oxidized coenzyme Q10 in the coenzyme Q10 produced by the microorganism can reach 96% or more, which is convenient for post-treatment.
  • the high content of oxidized coenzyme Q10 produced is stable relative to reduced coenzyme Q10, which can be used for preparing foods, functional nutritious foods, special health foods, nutritional supplements, nutraceuticals, animal herbs, beverages, feeds, cosmetics, pharmaceuticals. , medicines, preventive medicines.

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Abstract

一种氧化型辅酶Q10的发酵生产方法、及由其制备而得的高含量氧化型辅酶Q10，其特征在于，在生产菌株的发酵过程中控制发酵液的氧化还原电势ORP为-50~300mV，优选控制发酵液的氧化还原电势ORP为50~200mV。该方法使得微生物产出的辅酶Q10中氧化型辅酶Q10含量可达96%以上。

Description

氧化型辅酶Q10的发酵生产方法、及由其制备而得的高含量氧化型辅酶Q10 技术领域
本申请涉及微生物发酵领域，具体涉及一种通过调控氧化还原电势ORP(Oxidation-Reduction Potential)，使生产菌株(如，类球红细菌Rhodobacter sphaeroides)生产高含量(例如，70％以上、80％以上、或90％以上)氧化型辅酶Q10的发酵方法。
背景技术
辅酶Q10(CoQ10)又名泛醌、癸烯醌，化学名称为2，3-二甲氧基-5-甲基-6-癸异戊烯苯醌。辅酶Q10的生物活性来自于其醌环的氧化还原特性及其侧链的理化性质，它是细胞自身产生的天然抗氧化剂和细胞代谢激活剂，其具有抗氧化性、消除自由基、提高机体免疫力、抗衰老等功能。临床上广泛应用于各类心脏病、癌症、糖尿病、急慢性肝炎、帕金森症等疾病的治疗，而且在食品、化妆品及抗衰老保健品方面也有很多的应用。
微生物发酵法是是目前辅酶Q10的主要生产方式。利用微生物发酵法生产辅酶Q10，无论是从产品的质量和安全性方面，都有较大的竞争优势，适用于大规模的工业化生产。
微生物生产辅酶Q10的发酵阶段一般分为以下两个阶段：1)菌体生长阶段(也称为，微生物生长繁殖阶段)，在此阶段，一般需要保持足够的供氧和营养，使微生物快速生长繁殖达到生产所需的菌浓，同时，代谢产物辅酶Q10的合成快速启动；2)辅酶Q10合成积累阶段(有时也称为，合成阶段)，在此阶段，发酵细菌快速耗氧，发酵液中溶氧一般处于较低值，发酵微生物处于氧限制状态，此时代谢产物辅酶Q10快速合成积累。在合成积累阶段阶段，按照发酵液中辅酶Q10的效价变化来区分，通常分为前期(辅酶Q10效价保持较高的增涨曲线)、中期(辅酶Q10效价增涨曲线放缓，但仍保持明显的增涨趋势)及后期(辅酶Q10效价增涨曲线趋于平衡，随发酵时间略微增涨)。通常地，辅酶Q10合成积累阶段的前期、中期、及后期之间的时间间隔为10-20小时左右。
正如CN102876743B所公开的，采取分阶段的供氧控制策略来调控发酵 过程，在发酵过程的菌体生长阶段和前期的合成积累阶段采取高供氧，促进菌体的快速生长和辅酶Q10合成的快速启动，当菌体生长进入稳定期(菌体不再呈现明显的净增长)后，分阶段降低供氧以维持较高的辅酶Q10比生产速率，降低对底物葡萄糖的消耗；这种分阶段的供氧模式改变必将得到最佳的生产菌生理特性状态，降低辅酶Q10生产的成本。
在微生物发酵生产辅酶Q10过程中，本领域人员通常会通过调节发酵液的菌种、溶氧、温度、压力、培养基、补加营养素的速率等影响因子，以达到高产辅酶Q10的目的。如专利CN105420417A提出通过调节氧消耗速率(溶氧)和电导率(补加营养素速率)协同控制辅酶Q10的发酵过程；专利CN104561154A则以发酵过程中的菌体形状为判断依据，来调节工艺参数；专利CN103509729B通过改造类红球细菌来提高微生物合成辅酶Q10的能力。这些工艺共同的特点是生产出的辅酶Q10都是氧化型辅酶Q10和还原型辅酶Q10的混合物，且还原型辅酶Q10的比例要相对高一些。特别是专利US7910340B2所述的工艺，在发酵结束后，微生物产出的辅酶Q10中还原型辅酶Q10含量在70％以上。
由于氧化型辅酶Q10和还原型辅酶Q10在细胞内能够相互转换，所以无论哪种类型的辅酶Q10都能够作为电子传递体，起到相关的生理功能。同时，由于氧化型辅酶Q10相对稳定，更易于保存，近些年氧化型辅酶Q10的市场需求量与日俱增。
在上述专利CN105420417A、CN104561154A、CN103509729B中都未报道对微生物产出的辅酶Q10产品做专门的处理，以使还原型辅酶Q10转化为氧化型辅酶Q10。虽然专利US7910340B2中提出可以在后处理过程中采取氧化的手段将还原型辅酶Q10大部分转化为氧化型辅酶Q10，但其后处理工序复杂，成本较高。现有技术中也未有报道关于使用微生物发酵法直接产出高含量氧化型辅酶Q10的方法。
发明内容
技术问题
本发明的目的在于解决现有技术中微生物发酵法产出的氧化型辅酶Q10比例不高，后处理工序复杂等问题，提供了一种通过控制发酵液的ORP，使 生产菌株(如，类球红细菌Rhodobacter sphaeroides)生产高含量氧化型辅酶Q10的发酵生产方法。
解决方案
本申请涉及以下的氧化型辅酶Q10的发酵生产方法：
一种氧化型辅酶Q10的发酵生产方法，其中，在生产菌株的发酵过程中控制发酵液的氧化还原电势ORP为-50～300mV，优选控制发酵液的氧化还原电势ORP为50～200mV。
上述的发酵生产方法中，通过下述方式的至少一种来控制发酵液的氧化还原电势ORP：控制所述发酵液的溶氧、和控制所述发酵液的pH；优选将控制所述发酵液的溶氧的方式、与控制所述发酵液的pH的方式结合。
上述的发酵生产方法中，通过下述方式的至少一种来控制所述发酵液中的溶氧：控制发酵罐的单位体积搅拌输入功率、控制单位体积发酵液空气进气流量、和控制发酵罐的内部压力；优选将上述方式的两种以上结合来控制所述发酵液中的溶氧。
上述的发酵生产方法中，所述发酵罐的单位体积搅拌输入功率为0.25～0.50kw/m 3、所述单位体积发酵液空气进气流量为1.0～15.0vvm、和/或所述发酵罐的内部压力为0.05～0.3MPa；优选地，所述发酵罐的单位体积搅拌输入功率为0.30～0.40kw/m 3，所述单位体积发酵液空气进气流量为5.0～8.0vvm，和/或所述发酵罐的内部压力为0.08～0.15MPa。
上述的发酵生产方法中，通过将所述发酵液的pH控制为3.5～6.0来控制所述发酵液的pH；优选地，通过将所述发酵液的pH控制为4.0～5.0来控制所述发酵液的pH；还优选地，通过加入酸或加入碱的方式来控制所述发酵液的pH；进一步优选地，通过分阶段或持续加入所述酸或所述碱的方式来控制所述发酵液的pH。
上述的发酵生产方法中，所述酸为有机酸或无机酸、和/或所述碱为有机碱或无机碱；优选地，所述酸为磷酸、盐酸、硫酸、乳酸、丙酸、柠檬酸、和草酸中的一种或两种以上，和/或优选所述碱为氨水、氢氧化钠、和液氨中的一种或两种以上；更优选地，所述酸为磷酸、乳酸、或柠檬酸，和/或所述碱为氨水、或液氨。
上述的发酵生产方法中，在发酵过程中辅酶Q10合成积累阶段控制发酵 液的ORP；优选地，在发酵过程中辅酶Q10合成积累阶段的中后期控制发酵液的ORP；还优选的，在发酵过程中辅酶Q10合成积累阶段的后期控制发酵液的ORP。
上述的发酵生产方法中，在所述发酵过程中控制所述发酵液的电导率为5.0～30.0ms/cm；优选地，在菌体生长阶段，控制氧消耗速率在30～150mmol/(L·h)之间，并控制所述发酵液的电导率在5.0～30.0ms/cm之间；还优选地，在辅酶Q10合成积累阶段，控制氧消耗速率在60～120mmol/(L·h)之间，并控制所述发酵液的电导率在8.0～15.0ms/cm之间。
上述的发酵生产方法中，所述生产菌株为类球红细菌Rhodobacter sphaeroides；优选所述类球红细菌为自然选育的菌株、经物理或化学诱变方法选育的菌株、或者基因工程方法改造的菌株；更优选所述类球红细菌为保藏编号CGMCC No.5997的类球红细菌菌株、保藏编号CGMCC No.5998的类球红细菌菌株、或保藏编号CGMCC No.5999的类球红细菌菌株。
上述的发酵生产方法中，所述辅酶Q10为高含量氧化型辅酶Q10；并优选所述高含量氧化型辅酶Q10的氧化型辅酶Q10的含量为96％以上，更优选97％以上，最优选为99％以上。
本申请还涉及通过上述方法制备而得的辅酶Q10，其中，所述辅酶Q10为高含量氧化型辅酶Q10；并优选所述高含量氧化型辅酶Q10的氧化型辅酶Q10的含量为96％以上，更优选97％以上，最优选为99％以上。
在上述方法制备而得的辅酶Q10中，其中，所述辅酶Q10用于制备食品、功能性营养食品、特殊的健康食品、营养增补剂、营养品、动物药材、饮料、饲料、化妆品、药品、药剂、预防性药物。
有益效果
本申请提供了一种高含量氧化型辅酶Q10的发酵生产方法，其至少具有以下效果：
通过控制发酵液氧化还原电势ORP使得微生物产出的辅酶Q10中氧化型辅酶Q10含量可达到96％以上，产物构成相对单一，更方便于后处理；氧化型辅酶Q10较还原型辅酶Q10稳定，高含量氧化型辅酶Q10相较于现有技术发酵生产得到的辅酶Q10在生物体内被降解的少。此外，本申请发酵方法效价高。
附图说明
图1表示了类红球细菌体内的电子传递的过程图。
具体实施方式
本申请提供了一种高含量氧化型辅酶Q10的发酵生产方法，是通过控制发酵过程中发酵液氧化还原电势ORP，生产高含量氧化型辅酶Q10。将ORP控制至-50～300mV，优选地控制发酵液的ORP至50～200mV。
适用于本发明方法的菌株，没有特别限制，可以是现有的生产辅酶Q10的生产菌株，也可以用常规方法改造或应用基因工程方法改造的工程菌。
优选所述用于发酵生产辅酶Q10的菌株为类球红细菌(Rhodobacter sphaeroides)。类球红细菌为属于红细菌属(Rhodobacter)球形种(sphaeroides)的光合细菌。
更优选所述类球红细菌为自然选育的菌株、经物理或化学诱变方法选育的菌株、或者基因工程方法改造的菌株。进一步优选的，用于发酵生产辅酶Q10的菌株为保藏编号CGMCC No.5997的类球红细菌菌株；或保藏编号CGMCC No.5998的类球红细菌菌株；或保藏编号CGMCC No.5999的类球红细菌菌株。
现有技术中，在辅酶Q10的合成积累阶段，为保持辅酶Q10较高的产出率，一般会控制发酵液的ORP在-150～-300mV之间。而由于细菌本身的生理作用，产生的辅酶Q10以氧化型和还原型混合形式存在，其中还原型占到了70％左右。本申请提供了一种通过控制发酵液的ORP，使发酵微生物产出氧化型辅酶Q10的方法。在本申请中，控制ORP至-50～300mV，发酵细菌产出氧化型辅酶Q10占比达到96％以上。所述方法的机理如图1所示。在类红球细菌体内存在着
Figure PCTCN2018074822-appb-000001
的循环来进行电子传递，为类红球细菌的代谢提供能量。其中还原态的NADH提供的还原力是细胞体内氧化型辅酶Q10转化为还原型辅酶Q10的必要条件之一。发酵液的环境会极大地影响细胞中
Figure PCTCN2018074822-appb-000002
的循环，其中发酵液的氧化还原电位ORP是关键指标。一般情况下，发酵液的ORP值处于-150～-300mv，也就是发酵液处于还原性条件下时，类红球细菌中产出的辅酶Q10大部分为还原型辅酶Q10。通过提高发酵 液中ORP，使发酵液环境中的氧化性增强，这将导致NAD +转化为NADH的过程被抑制，类红球细菌产出的辅酶Q10在缺少了足够的NADH提供的还原力条件下，氧化型辅酶Q10向还原型辅酶Q10的转化也受到极大的影响，最终表现为氧化型辅酶Q10在细胞中大量生成。
上述的高含量氧化型辅酶Q10的发酵生产方法适用于微生物发酵全过程。作为优选的，上述方法在辅酶Q10合成积累阶段进行使用。进一步的，在发酵过程中辅酶Q10合成积累阶段的中后期控制发酵液的ORP。更为优选的，在发酵过程中辅酶Q10合成积累阶段的后期控制发酵液的ORP。
上述方法中，发酵液的ORP值在发酵过程中通过控制发酵液溶氧、或pH、或者协同控制发酵液溶氧和pH来控制发酵液的ORP值。作为优选的，发酵液的ORP值通过协同控制发酵液溶氧和pH进行控制。
发酵过程中溶解氧浓度的变化是供氧速率和耗氧速率之间的一个动态平衡。本申请中，通过控制发酵罐单位体积搅拌输入功率、控制单位体积发酵液空气流量、或控制发酵罐内部压力、或者将这三种方式随机组合的方式来提高发酵液中的供氧，从而提高发酵液中的溶氧(溶解氧)，使发酵液的ORP值达到-50～300mv。其中，优选将发酵罐单位体积搅拌输入功率控制为0.25～0.50kw/m 3，单位体积发酵液空气进气流量控制为1.0～15.0vvm(其中，vvm意指空气体积(air volume)/发酵液体积(culture volume)/min(分钟)，即，每分钟通气量与发酵罐内实际发酵液体积的比值)，发酵罐内部压力控制为0.05～0.3MPa；更优选的，发酵罐单位体积搅拌输入功率控制为0.30～0.40kw/m 3，单位体积发酵液空气进气流量控制为5.0～8.0vvm，发酵罐内部压力控制为0.08～0.15MPa。
本申请中，还优选控制发酵液pH来使发酵液的ORP值达到-50～300mv。例如，可将pH控制为3.5～6.0，更优选的，pH控制为4.0～5.0；并优选通过加酸或加碱的方式进行；所述酸可为常规用于调整发酵液的pH的常规酸，优选为磷酸、盐酸、硫酸、乳酸、丙酸、柠檬酸、草酸中的一种或两种以上；所述碱可为常规用于调整发酵液的pH的常规碱，优选为氨水、氢氧化钠、液氨中的一种或两种以上；作为优选的，所述酸为磷酸、乳酸、柠檬酸，所述碱为氨水、液氨。为了避免对发酵菌体造成剧烈的影响，可采用分阶段或持续加酸或碱的方式控制发酵液的pH。
上述的方法中，控制发酵液电导率为5.0～30.0ms/cm，以保持发酵细菌的营养供给。其中，发酵液电导率是通过补料培养基控制的。用于本发明的培养基没有特别限制，可以是各种常规的含有碳源、氮源、磷源和微量营养素的培养基。例如所述补料培养基为如下配方—每升补料液中含酵母粉8～12g，硫酸铵5～10g，硫酸镁1～2g，氯化钠3～6g，磷酸二氢钾2～4g，磷酸氢二钾2～4g，氯化钙1～2g，生物素0.013～0.025g，pH值7.0。
上述的发酵方法中，对于发酵过程中的温度没有特别的限制，只要能够不影响本申请的效果即可。从生产菌株能够产生更多的氧化型辅酶Q10的角度，优选将温度控制为25～35℃。
在本申请中，发酵生产辅酶Q10前的种子培养阶段可以使用本领域常规手段培养，优选的参照专利CN105483171A的培养方式。具体的，在种子培养阶段使用Fe 2+的浓度为0.1～0.5mol/L的培养基，类红球细菌菌株依次经复苏和种子培养基扩培后筛选得到发酵种子。用于本发明方法中种子培养基配方没有特别限制，可以是各种常规的含有碳源、氮源、磷源和微量营养素的培养基。例如，所述种子培养基为CN105483171A公开的如下配方—每升培养基中除了0.1～0.5mol Fe 2+外，还包含：酵母粉1g、氯化铵1g、氯化钠1g、柠檬酸铁0.0028g、磷酸二氢钾0.6g、磷酸氢二钾0.9g、硫酸镁0.25g、氯化钙0.1g，pH值调节为7.0。
在本申请中，发酵过程中类红球细菌的菌体生长阶段，优选的还包括辅酶Q10合成积累阶段的前期和/或中期可用本领域常规手段结合本申请技术方案进行，优选的，参照专利CN105420417A，利用在线控制氧消耗速率和电导率结合本申请技术方案进行。其中，氧消耗速率通过搅拌转速和空气流量进行调节，所述电导率通过流加补料或分批补料的方式进行调节。具体的，在类红球细菌菌体生长阶段控制氧消耗速率在30～150mmol/(L·h)之间，同时电导率稳定在5.0～30.0ms/cm之间；在辅酶Q10合成积累阶段控制氧消耗速率在60～120mmol/(L·h)之间，同时电导率稳定在8.0～15.0ms/cm之间。在发酵阶段，使用的培养基为本领域常规的含有碳源、氮源、磷源和微量营养素的培养基。例如，每升培养基中酵母粉8g，氯化铵3g，氯化钠2.8g，柠檬酸铁0.005g，磷酸二氢钾0.6g，磷酸氢二钾0.9g，硫酸镁12.55g，氯化钙0.1g，pH值7.0。
类球红细菌的示例
保藏编号：CGMCC No.5997、CGMCC No.5998、CGMCC No.5999；保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；保藏单位地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所；保藏日期：2012年4月13日；公开的专利文献：CN105420417(A)；公开日期：2016-03-23。
此外，上述三种菌株还在CN105483171A(2016-04-13)中公开；CGMCC No.5998还在CN103509729 A(2014-01-15)中公开。
以下采用具体实施例的方式进一步详细地说明本发明，然而，本发明不限于以下所述的实施例。
实施例
菌种及发酵前培养
菌种：类球红细菌(Rhodobacter sphaeroides)，保藏编号为CGMCC No.5997、CGMCC No.5998、或CGMCC No.5999的菌株。
发酵前培养：用无菌水洗涤培养好的斜面，制成菌浓为10 8～10 9个细胞每毫升的菌悬液；将制好的菌悬液接种2ml到母瓶培养基中进行种子培养，其中的培养基为100ml，32℃，转速180rpm，培养28-30小时。
种子培养基(g/L):1g酵母粉、1g NH 4Cl、1g氯化钠、2.8mg柠檬酸铁、0.6g KH 2PO 4、0.9g K 2HPO 4、0.25g MgSO 4、0.1g CaCl 2、0.5μg生物素、pH7.0。
将经过上述种子培养所得的类红球细菌菌株接种到发酵罐中的接种量可为本领域中的常规含量，例如，10-300ml、优选25-200ml，还优选50-100ml。可根据需求对该接种量进行调整。
效价测定：样品制备：氮气氛围下，取发酵液1ml至10ml离心管中，加入180μl的1mol/l的HCl，混合均匀，静置3～5min，然后放置92℃水浴下加热30min；离心去上清，加入8ml浸提液(乙酸乙酯：乙醇＝5：3)，浸提2h，HPLC反相测试。高效液相色谱条件：C18柱：150nm*4.6mm，流动相为甲醇：异丙醇＝75：25(以体积计)，流量1.00ml/min，检测波长：275nm，进样量40μl。保留时间12min。
氧化型辅酶Q10的含量测定：样品制备：氮气氛围下，取发酵液1ml至10ml离心管中，加入180μl的1mol/l的HCl，混合均匀，静置3～5min，然后放置92℃水浴下加热30min；离心去上清，加入8ml浸提液(乙酸乙酯：乙醇 ＝5：3)，浸提2h，HPLC反相测试。高效液相色谱条件：柱：YMC-Pack4.6*250mm，流动相为甲醇/正己烷＝85：15(以体积计)，流量1mL/min，检测波长：275nm，进样量40μl。保留时间：还原型辅酶Q10为13.5min，氧化型辅酶Q10为22.0min。
实施例1
30℃下，将由种子培养所得的类红球细菌菌株CGMCC No.5998接种50ml到装有发酵培养基的5L发酵罐中，开始发酵，控制发酵罐供氧条件为：单位体积发酵液空气进气流量控制为1.0vvm，单位体积搅拌输入功率控制为0.25kw/m 3，发酵罐内部压力控制为0.1MPa；并流加液氨控制发酵液pH值为7.0左右。
发酵培养基为：每升培养基中含酵母粉8g、氯化铵3g、氯化钠2.8g、柠檬酸铁0.005g、磷酸二氢钾0.6g、磷酸氢二钾0.9g、硫酸镁12.55g、氯化钙0.1g，pH值调节为7.0。
通过补料培养基控制整个发酵过程电导率为12ms/cm左右。所述补料培养基为每升补料液中含酵母粉8g、硫酸铵5g、硫酸镁1g、氯化钠3g、磷酸二氢钾2g、磷酸氢二钾2g、氯化钙1g、生物素0.013g，pH值调节为7.0。
持续发酵15小时后，检测发酵液ORP值为-35mv，取部分发酵液，惰性气体气氛下提取并检测(参照上述的效价测定和含量测定的内容)，细胞中氧化型辅酶Q10与还原型辅酶Q10的含量比为96.5:3.5。
在本实施例中，始终保持了较高的供氧量，并维持较高的搅拌输入功率和罐压，使发酵液中溶氧持续保持在较高的数值，发酵液ORP值一直在-35mv以上，发酵液中微生物一直处于生长繁殖状态。根据检测结果，可以确认，当发酵液ORP值维持一定数值以上时，菌体生长阶段下的发酵微生物产出的是高含量氧化型的辅酶Q10。
实施例2
30℃下，将由种子培养所得的类红球细菌菌株CGMCC No.5999接种40ml到装有发酵培养基的5L发酵罐中，开始发酵(发酵罐单位体积发酵液空气进气流量控制为0.45vvm，单位体积搅拌输入功率控制为0.1kw/m 3，罐压0.02MPa)，发酵液电导率为12ms/cm，pH值控制为7.0左右。
发酵培养基为：每升培养基中含酵母粉8g、氯化铵3g、氯化钠2.8g、柠 檬酸铁0.005g、磷酸二氢钾0.6g、磷酸氢二钾0.9g、硫酸镁12.55g、氯化钙0.1g，pH值调节为7.0。
补料培养基为每升补料液中含酵母粉10g、硫酸铵8g、硫酸镁1.5g、氯化钠5g、磷酸二氢钾3g、磷酸氢二钾3g、氯化钙1g、生物素0.020g，pH值调节为7.0。
检测发酵液中溶氧不再下降后，1小时内缓慢持续通入一定量的磷酸调节发酵液pH至4.0左右，流加液氨，使pH保持稳定在4.0左右，发酵罐单位体积发酵液空气进气流量、单位体积搅拌输入功率、罐压保持不变，发酵液电导率为12ms/cm；pH稳定后，检测发酵液ORP值为在58～135mv之间。
10小时后，取部分发酵液，惰性气体气氛下提取并检测，细胞中氧化型辅酶Q10与还原型辅酶Q10的含量比为97.3:2.7。
在本实施例中，在发酵液中溶氧不再下降后，发酵过程进入辅酶Q10合成积累阶段。在辅酶Q10合成积累阶段前期，进行了pH值调节。最终检测结果证明，通过调节pH值来控制ORP值能使得发酵微生物在辅酶Q10合成积累阶段前期有效产出高含量氧化型的辅酶Q10。
实施例3
30℃下，将由种子培养所得的类红球细菌菌株CGMCC No.5997接种90ml到装有发酵培养基的10L发酵罐中，开始发酵(发酵罐单位体积发酵液空气进气流量控制为0.6vvm，单位体积搅拌输入功率控制为0.1kw/m 3，罐压0.02MPa)，发酵液电导率为12ms/cm，pH值控制为7.0左右；
发酵培养基为：每升培养基中含酵母粉8g、氯化铵3g、氯化钠2.8g、柠檬酸铁0.005g、磷酸二氢钾0.6g、磷酸氢二钾0.9g、硫酸镁12.55g、氯化钙0.1g，pH值调节为7.0。
补料培养基为每升补料液中含酵母粉12g、硫酸铵10g、硫酸镁2g、氯化钠6g、磷酸二氢钾4g、磷酸氢二钾4g、氯化钙2g、生物素0.025g，pH值调节为7.0。
检测发酵液中溶氧不再下降后，1小时内缓慢持续通入一定量的磷酸调节发酵液pH至5.0左右，流加液氨，使pH保持稳定在5.0左右，同时，将发酵罐单位体积发酵液空气进气流量控制为5.0vvm，单位体积搅拌输入功率控制为0.3kw/m 3，罐压0.08MPa。稳定后，发酵液ORP值维持在100～210mV之间。
10小时后，取部分发酵液，惰性气体气氛下提取并检测，细胞中氧化型辅酶Q10与还原型辅酶Q10的含量比为99.1:0.9。
本实施例中，通过协同控制发酵液供氧条件和发酵液pH值来控制发酵液ORP值。同样的，本实施例是在发酵过程辅酶Q10合成积累阶段进行的调控，产出的氧化辅酶Q10占比达到99.1％。
实施例4
1)30℃下，将由种子培养所得的类红球细菌菌株CGMCC No.5999接种120ml到装有发酵培养基的10L发酵罐中，开始发酵(发酵罐单位体积发酵液空气进气流量控制为0.4vvm，单位体积搅拌输入功率控制为0.1kw/m 3，罐压0.02MPa)，控制氧消耗速率为50mmol/(L·h)，发酵液电导率为12ms/cm，pH值控制为7.0左右。
发酵培养基为：每升培养基中含酵母粉8g、氯化铵3g、氯化钠2.8g、柠檬酸铁0.005g、磷酸二氢钾0.6g、磷酸氢二钾0.9g、硫酸镁12.55g、氯化钙0.1g，pH值调节为7.0。
补料培养基为每升补料液中含酵母粉12g、硫酸铵10g、硫酸镁2g、氯化钠6g、磷酸二氢钾4g、磷酸氢二钾4g、氯化钙2g、生物素0.025g，pH值调节为7.0。
2)15小时后，增加供氧(发酵罐单位体积发酵液空气进气流量控制为0.6vvm，单位体积搅拌输入功率控制为0.2kw/m 3，罐压0.04MPa)，氧消耗速率上升到70mmol/(L·h)后保持平稳，发酵液电导率为12ms/cm，pH值控制为7.0，继续发酵，此时发酵处于菌体生长阶段。
3)20小时后，再次增加供氧(发酵罐单位体积发酵液空气进气流量控制为0.8vvm，单位体积搅拌输入功率控制为0.2kw/m 3，罐压0.05MPa)，氧消耗速率上升到90mmol/(L·h)后保持平稳，发酵液电导率为12ms/cm，pH值控制为7.0，继续发酵，此时发酵处于菌体生长阶段。
4)10小时后，氧消耗速率维持在70mmol/(L·h)左右，发酵液电导率控制为12ms/cm，pH控制为6.0左右，继续发酵，此时发酵处于辅酶Q10合成积累阶段前期。
5)20小时后，此时发酵效价增涨趋于平衡，发酵进入辅酶Q10合成积累阶段后期。控制发酵罐单位体积发酵液空气进气流量控制为6.0vvm，单位体 积搅拌输入功率控制为0.2kw/m 3，罐压0.1MPa，通过持续加入磷酸，在2h左右将pH值调节到3.5左右，控制发酵液电导率为12ms/cm，继续发酵；稳定后发酵液ORP值维持在100～200mv之间。
6)15小时后，停止发酵，取部分发酵液，惰性气体气氛下提取检测，效价为3182mg/L，氧化型辅酶Q10：还原型辅酶Q10为99.3:0.7。
在本实施例中，在发酵过程辅酶Q10合成积累阶段后期来控制发酵液ORP值，在有效产出高含量氧化型辅酶Q10的同时，发酵微生物的效价也有令人可喜的提升。
实用性
本申请的氧化型辅酶Q10的发酵生产方法，通过控制发酵液氧化还原电势ORP使得微生物产出的辅酶Q10中氧化型辅酶Q10含量可达到96％以上，便于后处理。
产出的高含量氧化型辅酶Q10相对于还原型辅酶Q10稳定，其可用于制备食品、功能性营养食品、特殊的健康食品、营养增补剂、营养品、动物药材、饮料、饲料、化妆品、药品、药剂、预防性药物。

Claims (12)

  1. 一种氧化型辅酶Q10的发酵生产方法，其特征在于，在生产菌株的发酵过程中控制发酵液的氧化还原电势ORP为-50～300mV，优选控制发酵液的氧化还原电势ORP为50～200mV。
  2. 根据权利要求1所述的发酵生产方法，其中，通过下述方式的至少一种来控制发酵液的氧化还原电势ORP：调节所述发酵液的溶氧、和控制所述发酵液的pH；优选将控制所述发酵液的溶氧的方式、与控制所述发酵液的pH的方式结合。
  3. 根据权利要求2所述的发酵生产方法，其中，通过下述方式的至少一种来控制所述发酵液中的溶氧：控制发酵罐的单位体积搅拌输入功率、控制单位体积发酵液空气进气流量、和控制发酵罐的内部压力；优选将上述方式的两种以上结合来控制所述发酵液中的溶氧。
  4. 根据权利要求3所述的发酵生产方法，其中，所述发酵罐的单位体积搅拌输入功率为0.25～0.50kw/m 3、所述单位体积发酵液空气进气流量为1.0～15.0vvm、和/或所述发酵罐的内部压力为0.05～0.3MPa；优选地，所述发酵罐的单位体积搅拌输入功率为0.30～0.40kw/m 3，所述单位体积发酵液空气进气流量为5.0～8.0vvm，和/或所述发酵罐的内部压力为0.08～0.15MPa。
  5. 根据权利要求2-4任一项所述的发酵生产方法，其中，通过将所述发酵液的pH控制为3.5～6.0来控制所述发酵液的pH；优选地，通过将所述发酵液的pH控制为4.0～5.0来控制所述发酵液的pH；还优选地，通过加入酸或加入碱的方式来控制所述发酵液的pH；进一步优选地，通过分阶段或持续加入所述酸或所述碱的方式来控制所述发酵液的pH。
  6. 根据权利要求5所述的发酵生产方法，其中，所述酸为有机酸或无机酸、和/或所述碱为有机碱或无机碱；优选地，所述酸为磷酸、盐酸、硫酸、乳酸、丙酸、柠檬酸、和草酸中的一种或两种以上，和/或优选所述碱为氨水、氢氧化钠、和液氨中的一种或两种以上；更优选地，所述酸为磷酸、乳酸、或柠檬酸，和/或所述碱为氨水、或液氨。
  7. 根据权利要求1-6中任一项所述的发酵生产方法，其中，在发酵过程中辅酶Q10合成积累阶段控制发酵液的ORP；优选地，在发酵过程中辅酶Q10合成积累阶段的中后期控制发酵液的ORP；还优选的，在发酵过程中辅酶Q10合成积累阶段的后期控制发酵液的ORP。
  8. 根据权利要求1-7中任一项所述的发酵生产方法，其中，在所述发酵过程中控制所述发酵液的电导率为5.0～30.0ms/cm；优选地，在菌体生长阶段，控制氧消耗速率在30～150mmol/(L·h)之间，并控制所述发酵液的电导率在5.0～30.0ms/cm之间；还优选地，在辅酶Q10合成积累阶段，控制氧消耗速率在60～120mmol/(L·h)之间，并控制所述发酵液的电导率在8.0～15.0ms/cm之间。
  9. 根据权利要求1-8中任一项所述的发酵生产方法，其中，所述生产菌株为类球红细菌Rhodobacter sphaeroides；优选所述类球红细菌为自然选育的菌株、经物理或化学诱变方法选育的菌株、或者基因工程方法改造的菌株；更优选所述类球红细菌为保藏编号CGMCC No.5997的类球红细菌菌株、保藏编号CGMCC No.5998的类球红细菌菌株、或保藏编号CGMCC No.5999的类球红细菌菌株。
  10. 根据权利要求1-9中任一项所述的发酵生产方法，其中，所述辅酶Q10为高含量氧化型辅酶Q10；并优选所述高含量氧化型辅酶Q10的氧化型辅酶Q10的含量为96％以上，更优选97％以上，最优选为99％以上。
  11. 根据权利要求1-10任一项所述的方法制备而得的辅酶Q10，其中，所述辅酶Q10为高含量氧化型辅酶Q10；并优选所述高含量氧化型辅酶Q10的氧化型辅酶Q10的含量为96％以上，更优选97％以上，最优选为99％以上。
  12. 根据权利要求11所述的方法制备而得的辅酶Q10，其中，所述辅酶Q10用于制备食品、功能性营养食品、特殊的健康食品、营养增补剂、营养品、动物药材、饮料、饲料、化妆品、药品、药剂、预防性药物。
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