CN111286500B - Coenzyme Q production by combining plasma action with oxygen limitation 10 Method (2) - Google Patents
Coenzyme Q production by combining plasma action with oxygen limitation 10 Method (2) Download PDFInfo
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Abstract
The invention discloses a method for producing coenzyme Q by combining plasma action with oxygen limitation 10 The method of (1), the method comprising: uniformly covering the bacterial suspension on the surface of a slide glass, then placing the slide glass in an ARTP (ARTP induced fluorescence) mutagenizing instrument for carrying out mutagenizing treatment and elution to obtain the mutagenized bacterial suspension; diluting the mutagenized bacterial suspension under aseptic conditions, coating the diluted bacterial suspension on a plate containing a plate culture medium added with anhydrous sodium sulfite, and culturing to obtain a plate with single colony; picking single colonies from the flat plate with the single colonies growing on the flat plate, placing the single colonies into a 24-hole plate, adding a seed culture medium, covering a cover, and culturing to obtain a seed solution; production of coenzyme Q by fermentation 10 The step (2). The invention combines the plasma effect with the oxygen limitation model, can obtain and screen rhodobacter sphaeroides production strains with improved specific growth rate, oxygen affinity and product synthesis rate, and effectively improves coenzyme Q 10 The level of fermentation.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a method for producing coenzyme Q by combining plasma action with oxygen limitation 10 The method of (1).
Background
Coenzyme Q 10 (Coenzyme Q 10 ) Is a fat-soluble compound widely present on the cell membrane of organisms, also known as Ubiquinone (Ubiquinone), which acts as an electron carrier on the mitochondrial respiratory chain and is responsible for the transfer of electrons from complex I (or complex II) to complex III. The research shows that the coenzyme Q 10 Has physiological functions of resisting oxidation, eliminating in vivo free radicals and promoting ATP synthesis, is used as medicine and food additive for treating cardiovascular diseases, has gradually increased market demand in the last two decades, and is used for producing coenzyme Q by microbiological method 10 Has great development potential.
Rhodobacter sphaeroides (Rhodobacter sphaeroides) is a coenzyme Q commonly used in recent years 10 The strain is produced, and many researchers start from the aspects of mutation breeding, gene modification, metabolic engineering and the like, so that the yield of the strain is improved. Fuzhanglong and the like use the composite resistance of the strain to (vitamin K3+ sodium azide + p-hydroxybenzoic acid) as a screening marker, and perform chemical mutagenesis on rhodobacter sphaeroides by using nitrosoguanidine to successfully screen out a high-yield strain with stable heredity. Zhu et al, by overexpressing the transcriptional regulator ppsR of the photosynthesis gene in rhodobacter sphaeroides, and catalytic GGCRtE synthesized from PP and enhanced coenzyme Q 10 The yield of the modified strain RspPE is improved to 73.2mg/L by the supply of isoprene side chain precursor GGPP in biosynthesis, which is 47 percent higher than that of wild strains. For a large number of mutant strains obtained by mutagenesis, proper and efficient screening pressure needs to be selected for rapid breeding, and at present, most of the mutagenesis breeding adopts precursor, antibiotic or structural analogue as the selection pressure for screening.
Research finds that in rhodobacter sphaeroides fermentation processes, oxygen supply is always a key factor influencing bacterial growth and product synthesis in the fermentation process. The high oxygen consumption rate can ensure the rapid growth of thalli and the improvement of the product synthesis rate, but the consumption of power energy is larger in the production process, and the production cost is increased.
Therefore, it is highly desirable to provide a method for efficiently producing coenzyme Q 10 The method can rapidly obtain and screen out the bacterial strain with improved specific growth rate, oxygen affinity and product synthesis rate, thereby realizing the improvement of coenzyme Q 10 The purpose of the yield.
Disclosure of Invention
The invention aims to provide a method for producing coenzyme Q by combining plasma action with oxygen limitation 10 The method can obtain rhodobacter sphaeroides production strains with higher growth rate, oxygen affinity and product synthesis rate, and effectively improves coenzyme Q 10 The production efficiency is improved.
In order to achieve the purpose, the invention adopts the following technical scheme.
The invention provides a method for producing coenzyme Q by combining plasma action with oxygen limitation 10 The method of (a), the method comprising: a step of ARTP mutagenesis; oxygen limitation screening coenzyme Q 10 A step of highly producing strains; production of coenzyme Q by fermentation 10 A step (2); wherein the content of the first and second substances,
the step of ARTP mutagenesis comprises:
uniformly covering a proper amount of rhodobacter sphaeroides bacterial suspension on the surface of a slide, then placing the slide in an ARTP (argon-induced fluorescence) mutagen, carrying out mutagenesis treatment by taking inert gas as working gas, and eluting to obtain mutagenized bacterial suspension;
the oxygen limitation screening coenzyme Q 10 A step of highly producing a strain comprising:
diluting the mutagenized bacterial suspension under an aseptic condition, coating the diluted bacterial suspension on an oxygen limitation plate containing a plate culture medium added with 0.2-0.4 g/L anhydrous sodium sulfite, and carrying out primary culture to obtain a plate with a single colony;
and selecting the single colony with full appearance and proper size from the flat plate with the single colony, placing the single colony in a 24-hole plate, adding a seed culture medium, covering a cover, and performing secondary culture to obtain a seed solution.
Further, the working conditions of the mutagenesis treatment are as follows: mutagenizing for 15-30 seconds under the condition of a gas carrying capacity of 10SLM (SLM means liter per minute under the standard condition).
Further, the time for mutagenesis was 25 seconds.
Further, the addition amount of the anhydrous sodium sulfite is preferably 0.4 g/L.
Further, the inert gas is helium.
Furthermore, the purity of the helium is more than 99.99%.
Further, the addition amount of the anhydrous sodium sulfite in the plate medium is 0.4 g/L.
Further, the first culturing is as follows: culturing at 32 ℃ for 6-7 days in a dark place.
Further, coenzyme Q is selected in the oxygen limitation 10 In the step of producing the strain with high yield, the liquid loading volume of the seed culture medium is 2 mL.
Further, the second culturing is as follows: culturing at 32 deg.C under the condition of oscillation rate of 220r/min for 20 h.
Further, the fermentation produces coenzyme Q 10 Comprises the following steps:
further, inoculating the seed liquid into a 24-pore plate filled with a fermentation culture medium for fermentation culture to obtain fermentation liquid.
Further, the inoculation amount of the seed solution is 16%.
The fermentation culture comprises the following steps: culturing at 32 deg.C under condition of oscillation rate of 220r/min under dark condition for 48 h.
Further, the plating medium includes: yeast extract, dipotassium phosphate, sodium chloride, ferrous sulfate, magnesium sulfate and agar powder; the pH value is 7.0 to 7.2.
Further, the seed medium comprises: ammonium sulfate, yeast extract, sodium glutamate, corn steep liquor dry powder, glucose, dipotassium hydrogen phosphate, ferrous sulfate, magnesium sulfate, sodium chloride and calcium carbonate; the pH value is 7.1-7.2.
Further, the fermentation medium comprises: ammonium sulfate, sodium glutamate, corn steep liquor dry powder, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, sodium chloride, calcium chloride, glucose and calcium carbonate 10 g/L; the pH value is 6.5-6.6.
In one embodiment of the present invention, the plate medium includes: 15g/L of yeast extract, 1g/L of dipotassium hydrogen phosphate, 2g/L of sodium chloride, 0.1g/L of ferrous sulfate, 0.15g/L of magnesium sulfate and 20g/L of agar powder; the pH value before sterilization is 7.0-7.2.
In an embodiment of the present invention, the seed culture medium includes: 3g/L of ammonium sulfate, 8g/L of yeast extract, 0.8g/L of sodium glutamate, 0.748g/L of corn steep liquor dry powder, 10g/L of glucose, 1.5g/L of dipotassium hydrogen phosphate, 0.25g/L of ferrous sulfate, 1.8g/L of magnesium sulfate, 2g/L of sodium chloride and 8g/L of calcium carbonate; the pH value before digestion is 7.1-7.2.
In one embodiment of the present invention, the fermentation medium comprises: 3g/L of ammonium sulfate, 3.5g/L of sodium glutamate, 4.2g/L of corn steep liquor dry powder, 1.6g/L of monopotassium phosphate, 0.8g/L of ferrous sulfate, 10g/L of magnesium sulfate, 3g/L of sodium chloride, 0.07g/L of calcium chloride, 32.5g/L of glucose and 10g/L of calcium carbonate; the pH value before sterilization is 6.5-6.6.
In the invention, the elution is carried out for 1min by placing the chip on a vortex oscillator to shake violently, so that the bacteria liquid on the slide glass is completely eluted.
In the present invention, the oxygen limitation screening coenzyme Q 10 In the step of producing strains with high yield, the dilution is to dilute the bacterial suspension to 10 -4 And (4) doubling.
In the invention, the bacterial suspension can be obtained by adopting the following culture method: under the aseptic condition, 8-10 rhodobacter sphaeroides colonies which are full in appearance and moderate in size are picked, placed into 10mL of aseptic normal saline, and fully scattered to prepare bacterial suspension.
In the present invention, Rhodobacter sphaeroides (Rhodobacter sphaeroides) J-1 was provided by the inner Mongolia Jindawei pharmaceutical Co., Ltd as a starting strain. The Rhodobacter sphaeroides (Rhodobacter sphaeroides) is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 2010 at 12 th and 21 th, and the preservation center number is CGMCC No. 4497.
In the invention, an atmospheric room temperature plasma mutagenic instrument ARTP-II S type (Beijing Si Qingyuan biotechnology company): performing ARTP mutagenesis;
in the present invention, high performance liquid chromatography (Agilent Technologies inc. Agilent 1100 series): off-gas mass spectrometer (Extrel MAX300-LG USA): o in tail gas 2 、CO 2 Detecting; coenzyme Q 10 Detecting the concentration;
in the present invention, the glucose meter SBA-50B (biological research institute of academy of sciences, Shandong province): detecting residual sugar in the fermentation liquor;
in the present invention, 721 visible spectrophotometer (shanghai prism technology ltd): and (5) detecting the bacterial concentration.
In the present invention, the raw materials used are all commercially available products unless otherwise specified.
In one embodiment of the invention, the coenzyme Q in the fermentation liquor is detected by adopting a standard curve drawing method 10 The contents are as follows:
drawing a standard curve, comprising the following steps:
accurately weighing 0.1g of coenzyme Q 10 The standard substance (accurate to 0.0002g) is placed in a 100mL volumetric flask, absolute ethyl alcohol is used for metering to a scale mark, 1g/L of standard substance solution is prepared, then the standard substance solution is diluted to 10, 20, 30, 40 and 50mg/L by the absolute ethyl alcohol, and the standard substance solution is filtered by a 0.22 mu m organic phase filter head and is analyzed by a high-performance liquid chromatograph.
Mobile phase: ethanol: methanol 1: 1, chromatographic conditions: the detection wavelength is 275nm, the flow rate is 1.1mL/min, the sample injection amount is 20 mu L, and the column temperature is 35 ℃.
A chromatographic column: hypersil ODS C18(150 mm. times.4.6 mm,5 μm). And drawing a standard curve by taking the peak area as a vertical coordinate and the concentration of the standard substance as a horizontal coordinate.
② coenzyme Q in the fermentation liquor 10 And (3) detection of titer:
after fermentation is finished, sucking 0.5mL of fermentation liquid by using an 8-pore pipette, transferring the fermentation liquid to a position corresponding to another pore plate, adding 50 mu L of hydrochloric acid solution, slightly shaking the pore plate, adding 1mL of anhydrous acetone and 100 mu L of 30% hydrogen peroxide, adding 3.35mL of anhydrous ethanol, covering a cover, and performing ultrasonic extraction for 45 min; filtering with 0.22 μm filter head, sucking 1mL, placing in a liquid phase vial for liquid phase detection, and calculating the titer of the fermentation liquid according to a standard curve.
In an embodiment of the present invention, the following detection method may be adopted as the detection method for the bacterial concentration in the fermentation liquid:
1) the bacterial concentration in the fermentation liquor is measured by a turbidimetry method: sampling 5ml fermentation liquor, diluting to proper gradient, vibrating uniformly, and measuring OD at 700nm wavelength by using spectrophotometer 700 Thus obtaining the bacterial concentrate.
2) After 70h fermentation, the bacterial concentration was characterized by dry weight: and (3) putting the quantitative filter paper into an oven at 105 ℃ for baking for 2 hours, taking out the quantitative filter paper and accurately weighing the mass of the filter paper. And (3) taking 5ml of fermentation liquor to perform suction filtration on filter paper, washing the precipitate for three times by using deionized water, placing the precipitate in an oven to be dried to constant weight to obtain the mass of the precipitate, and dividing the mass by the sampling volume to obtain the dry weight (g/L) of the thallus.
In an embodiment of the present invention, the detection of residual sugar in fermentation broth is as follows: and centrifuging the fermentation liquor at the rotating speed of 12000 r/min for 10min, taking the supernatant, diluting the supernatant with deionized water to a proper gradient, and detecting the supernatant by using a glucose determinator.
In one embodiment of the invention, on-line Oxygen Uptake Rate (OUR) detection: detecting tail gas by adopting a mass spectrometer in the fermentation process, collecting data by using Biostar software, and calculating on line to obtain the oxygen uptake rate OUR, wherein the specific calculation formula is as follows:
wherein, F in Is the inlet gas flow (mmol/L) and V is the gas flowVolume of fermentation broth (L), C inert,in ，C O2,in ， C O2,out And C CO2,out Respectively representing the volume fractions of inert gas and oxygen in the inlet gas and oxygen and carbon dioxide in the tail gas, P in Indicating intake air pressure (Pa), t in Is the temperature (. degree. C.) and h is the relative humidity (%) of the intake air.
The invention has the beneficial effects that:
the invention utilizes ARTP to mutate rhodobacter sphaeroides, combines an oxygen restriction model to screen, and can effectively screen out a high-yield strain of coenzyme Q10. According to the invention based on coenzyme Q 10 The synthesis characteristic of the product is that a method for reducing oxygen in a growth environment and constructing an oxygen limitation model is firstly adopted, and the oxygen limitation model is combined for screening, so that the coenzyme Q is rapidly screened out 10 To achieve the purpose of improving Q 10 The purpose of the yield of (2).
In the invention, the ARTP adopts the working conditions of normal temperature and normal pressure, the operation is simple, convenient and efficient, the damage and lethal effect of non-teratogenic denaturation treatment are effectively reduced, and the mutation efficiency is high.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the lethality in test example 1 of the present invention.
FIG. 2 is a colony morphology of rhodobacter sphaeroides in example 3 of the present invention; wherein, FIG. 2a is the colony morphology of rhodobacter sphaeroides in example 11; FIG. 2b is a colony morphology of rhodobacter sphaeroides in example 12.
FIG. 3 is a graph showing the titer results of the selected strains in example 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without inventive exercise, are within the scope of the present invention.
Example 1
This example provides a method for producing coenzyme Q by combining the plasma action with oxygen limitation 10 The method comprises the following steps: a step of ARTP mutagenesis; oxygen limitation screening coenzyme Q 10 A step of highly producing strains; production of coenzyme Q by fermentation 10 A step (2); wherein the content of the first and second substances,
the step of ARTP mutagenesis comprises:
uniformly covering the surface of a slide with the bacterial suspension of rhodobacter sphaeroides, then placing the slide in an ARTP mutagenesis instrument, carrying out mutagenesis treatment by taking inert gas as working gas, and eluting to obtain the mutagenized bacterial suspension;
the oxygen limitation screening coenzyme Q 10 A step of highly producing a strain comprising:
diluting the mutagenized bacterial suspension under aseptic conditions, coating the diluted bacterial suspension on an oxygen limitation plate containing a plate culture medium added with 0.2-0.4 g/L anhydrous sodium sulfite, and performing primary culture to obtain a plate with a single colony;
and (3) picking the single colony from the flat plate with the single colony, placing the single colony in a 24-well plate, adding a seed culture medium, covering a cover, and performing secondary culture to obtain a seed solution.
The mutagenesis treatment specifically comprises the following steps: and (3) carrying out mutagenesis for 15-30 seconds under the condition of a gas carrying capacity of 10SLM (SLM means liter per minute under a standard condition).
Example 2
This example provides a method for producing coenzyme Q by combining the plasma action with oxygen limitation 10 The method comprises the following steps: a step of ARTP mutagenesis; oxygen limitation screening coenzyme Q 10 A step of highly producing strains; production of coenzyme Q by fermentation 10 A step (2); wherein, the first and the second end of the pipe are connected with each other,
the step of ARTP mutagenesis:
uniformly covering a proper amount of rhodobacter sphaeroides bacterial suspension on the surface of a slide glass, then placing the slide glass in an ARTP mutagenesis instrument, carrying out mutagenesis for 25s under the condition of a gas carrying capacity of 10SLM (SLM refers to liter per minute under a standard condition) by taking inert gas as working gas, and eluting to obtain the mutagenized bacterial suspension;
the oxygen limitation screening coenzyme Q 10 The steps of producing strains with high yield are as follows:
diluting and coating the mutagenized bacterial suspension under the aseptic condition, growing on an oxygen-limited plate containing a plate culture medium added with 0.4g/L anhydrous sodium sulfite, and culturing at 32 ℃ in a dark place for 6-7 days to obtain a plate with a single colony;
and (3) selecting a single colony which is full in appearance and moderate in size from the flat plate with the single colony, placing the single colony in a 24-well plate, adding a seed culture medium (the liquid loading volume is 2mL), covering a cover, and culturing for 20 hours at 32 ℃ under the oscillation (220r/min) condition to obtain a seed solution.
The fermentation produces coenzyme Q 10 The steps of (1):
and (3) inoculating the seed solution into a 24-pore plate filled with a fermentation culture medium by using an 8-pore pipette according to the inoculation amount of 16%, and performing fermentation culture for 48 hours at 32 ℃ in a dark place under oscillation (220r/min) to obtain the fermentation liquid.
Example 3
This example provides a method for producing coenzyme Q by combining the plasma action with oxygen limitation 10 The method comprises the following steps: a step of ARTP mutagenesis; oxygen limitation screening coenzyme Q 10 A step of highly producing strains; production of coenzyme Q by fermentation 10 A step (2); wherein the content of the first and second substances,
the step of ARTP mutagenesis comprises:
uniformly covering the surface of a slide with the bacterial suspension of rhodobacter sphaeroides, then placing the slide in an ARTP mutagenesis instrument, carrying out mutagenesis treatment by taking inert gas as working gas, and eluting to obtain the mutagenized bacterial suspension;
the mutagenesis treatment specifically comprises the following steps: mutagenesis was performed for 25 seconds at a 10SLM loading (SLM meaning liters per minute under standard conditions).
And (3) coating the mutagenized bacterial suspension on a normal plate to grow to obtain a plate with a single colony.
Examples 4 to 10
Examples 4 to 10 differ from example 3 only in that: the mutagenesis time in examples 4 to 10 was 0, 10, 15, 20, 30, 35 and 40s, respectively, and the other conditions were completely the same.
Test example 1
The strains in examples 3 to 10 of the present invention were tested for the lethality of rhodobacter sphaeroides after ARTP mutagenesis, respectively:
are respectively provided withTo pairThe plate with single colony in example 3-example 10 was counted and the test results were recorded to obtain the lethality curve of ARTP mutagenesis, which is shown in FIG. 1.
FIG. 1 is a graph showing lethality in test example 1 of the present invention; therefore, the mutation rate of rhodobacter sphaeroides can be improved by the mutagenesis method.
Test example 2
In this test example, the growth of rhodobacter sphaeroides (starting strain not subjected to ARTP mutagenesis) in a culture solution to which anhydrous sodium sulfite was added at various concentrations was observed, and the results are shown in Table 1.
TABLE 1
| Anhydrous sodium sulfite (g/L) | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 |
| CFU | +++ | ++ | + | +/- | - |
From table 1, it can be seen that: rhodobacter sphaeroides which is not mutagenized by ARTP in the present invention has poor viability in anhydrous sodium sulfite, and grows aseptically when the concentration of anhydrous sodium sulfite reaches 0.4 g/L.
Examples 11 to 12
Example 11 and example 12 are different from example 2 only in that: the amounts of the anhydrous sodium sulfite added in examples 11 and 12 were 0g/L and 0.2g/L, respectively, and the other conditions were completely the same.
Test example 3
The results of observing the rhodobacter sphaeroides on the single colony-grown plate in examples 11 and 12 of the present invention are shown in FIG. 2.
FIG. 2 is a colony morphology of rhodobacter sphaeroides in test example 3 of the present invention; wherein, FIG. 2a is the colony morphology of rhodobacter sphaeroides in example 11; FIG. 2b is a colony morphology of rhodobacter sphaeroides in example 12.
From fig. 2, it can be found that:
(see figure 2a) the diameter of the colony grown on the original plate without the anhydrous sodium sulfite is larger, when the original plate is used for passage in the early stage, the bacterial strain can degenerate to cause the colony to turn yellow, and the coenzyme Q is generated after the fermentation of the degenerate colony 10 The yield of (a) is very low; (see FIG. 2b) while the colonies growing on the oxygen limited plates turned darker greenish black, with smaller diameter and clean edges, and the probability of yellow colonies was greatly reduced.
Thus, coenzyme Q can be obtained by the method of the invention 10 Is highProducing strain to realize coenzyme Q 10 The yield is improved.
Test example 4
In this test example, the fermentation liquid obtained in example 2 of the present invention was subjected to HPLC analysis to observe the mutagenesis of rhodobacter sphaeroides, as shown in FIG. 3.
FIG. 3 is a graph showing the titer results of the selected strains in example 2 of the present invention.
As is clear from FIG. 3, 506 strains of coenzyme Q were co-selected in example 2 of the present invention 10 High yield of bacteria.
Test example 5
This test example separately examined physiological metabolic characteristic parameters of the high-producing strain selected in the present invention (coenzyme Q obtained from test example 4) and the starting strain of rhodobacter sphaeroides 10 Selecting strains with improved titer from high-yielding strains):
high-yield strains and starting strains are subjected to conventional fermentation culture in a 5L reactor, parameters in the process are collected, test results are recorded, and the parameters of the fermentation process of the starting strains and the high-yield strains are shown in Table 2.
TABLE 2
| Parameter(s) | Unit of | Rhodobacter sphaeroides J-1 | High-yield bacterium |
| Potency of the drug max | mg/L | 652.69±26 | 770.06±16 |
| DCW max | g/L | 51.2±1.4 | 52.9±1.1 |
| μ max | h -1 | 0.0863±0.0032 | 0.0922±0.0045 |
| Y P/X | mg/gDCW | 13.60±0.17 | 15.75±0.22 |
| Y P/S | mg/g | 2.41±0.09 | 2.84±0.12 |
| Q O2,max | mmol/g/h | 4.20±0.42 | 5.19±0.60 |
| Specific productivity max | mg/g/h | 0.2097±0.0094 | 0.2143±0.0087 |
According to the culture results in Table 2, it was shown that the high-producing strain grew fast in the early stage of fermentation and accumulated more biomassThe product synthesis is started at the material amount, stronger oxygen affinity is shown at the middle and later stages of fermentation, the oxygen consumption rate is obviously maintained at a higher level, and finally the coenzyme Q 10 The titer of the strain is improved by 18.0 percent compared with the original strain, and reaches 770.1 mg/L.
In summary, the invention combines the room temperature and pressure plasma (ARTP) mutagenesis with the oxygen limitation screening model constructed by anhydrous sodium sulfite, and establishes the coenzyme Q 10 A24-pore plate high-flux rapid culture technology for producing the bacterial strain is used for breeding the oxygen-tolerance high-performance mutant strain of Rhodobacter sphaeroides (Rhodobacter sphaeroides).
The invention adopts the plasma effect combined with the oxygen limitation model, and can well breed the coenzyme Q 10 High-yield strain, and screening out mutant strain with strong affinity to oxygen to effectively increase coenzyme Q 10 And (4) fermentation level.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Coenzyme Q production by combining plasma action with oxygen limitation 10 The method of (a), wherein the method comprises: a step of ARTP mutagenesis; oxygen limitation screening coenzyme Q 10 A step of highly producing strains; production of coenzyme Q by fermentation 10 A step (2); wherein the content of the first and second substances,
the step of ARTP mutagenesis comprises:
uniformly covering the surface of a slide with the bacterial suspension of rhodobacter sphaeroides, then placing the slide in an ARTP mutagenesis instrument, carrying out mutagenesis treatment by taking inert gas as working gas, and eluting to obtain the mutagenized bacterial suspension;
the oxygen limitation screening coenzyme Q 10 A step of highly producing a strain comprising:
diluting the mutagenized bacterial suspension under an aseptic condition, coating the diluted bacterial suspension on an oxygen limitation plate containing a plate culture medium added with 0.2-0.4 g/L anhydrous sodium sulfite, and carrying out primary culture to obtain a plate with a single colony;
and (3) picking a single colony from the flat plate with the single colony, placing the single colony in a 24-well plate, adding a seed culture medium, covering a cover, and performing secondary culture to obtain a seed solution.
2. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (1), wherein the mutagenesis treatment is specifically: carrying out mutagenesis for 15-30 seconds under the gas carrying capacity of 10 SLM; SLM refers to liters per minute under standard conditions.
3. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (1), wherein the first culturing is: culturing for 6-7 days at 32 ℃ in a dark place.
4. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (1), wherein coenzyme Q is screened for oxygen limitation 10 In the step of producing the strain with high yield, the liquid loading volume of the seed culture medium is 2 mL.
5. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (3), wherein the second culturing is: culturing at 32 deg.C and 220r/min for 20 hr to obtain seed solution.
6. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (2), wherein the fermentation produces coenzyme Q 10 The method comprises the following steps:
inoculating the seed liquid into a 24-pore plate filled with a fermentation culture medium according to the inoculation amount of 16% for fermentation culture to obtain a fermentation liquid.
7. Plasma action in combination with oxygen limitation according to claim 6Production of coenzyme Q 10 The method of (2), wherein the fermentation culture is: culturing at 32 deg.C and 220r/min under dark condition for 48 h.
8. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (1), wherein said plating medium comprises: yeast extract, dipotassium hydrogen phosphate, sodium chloride, ferrous sulfate, magnesium sulfate and agar powder; the pH value before sterilization is 7.0-7.2.
9. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 1 10 The method of (3), wherein said seed medium comprises: ammonium sulfate, yeast extract, sodium glutamate, corn steep liquor dry powder, glucose, dipotassium hydrogen phosphate, ferrous sulfate, magnesium sulfate, sodium chloride and calcium carbonate; the pH value before digestion is 7.1-7.2.
10. Production of coenzyme Q by plasma action in combination with oxygen limitation according to claim 6 10 The method of (2), wherein the fermentation medium comprises: ammonium sulfate, sodium glutamate, corn steep liquor dry powder, monopotassium phosphate, ferrous sulfate, magnesium sulfate, sodium chloride, calcium chloride, glucose and calcium carbonate; the pH value before sterilization is 6.5-6.6.
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| CN101333509A (en) * | 2008-07-07 | 2008-12-31 | 天辰神舟实业有限公司 | Rhodobacter sphaeroides mutant of coenzyme Q10 and culturing method |
| CN105925519A (en) * | 2016-05-06 | 2016-09-07 | 苏州华赛生物工程技术有限公司 | Method for reducing or eliminating by-product D in coenzyme Q10 producing strain SZ, coenzyme Q10 high-yield strain and application thereof |
| CN109762757A (en) * | 2018-12-25 | 2019-05-17 | 神舟生物科技有限责任公司 | A kind of high yield Co-Q10 hydrogenlike silicon ion and its mutagenic and breeding and application |
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| CN101333509A (en) * | 2008-07-07 | 2008-12-31 | 天辰神舟实业有限公司 | Rhodobacter sphaeroides mutant of coenzyme Q10 and culturing method |
| CN105925519A (en) * | 2016-05-06 | 2016-09-07 | 苏州华赛生物工程技术有限公司 | Method for reducing or eliminating by-product D in coenzyme Q10 producing strain SZ, coenzyme Q10 high-yield strain and application thereof |
| CN109762757A (en) * | 2018-12-25 | 2019-05-17 | 神舟生物科技有限责任公司 | A kind of high yield Co-Q10 hydrogenlike silicon ion and its mutagenic and breeding and application |
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| Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides;Nguyen Ba Kien et al.;《J Ind Microbiol Biotechnol》;20200227;全文 * |
| Improvement of Coenzyme Q10 Production: Mutagenesis Induced by High Hydrostatic Pressure Treatment and Optimization of Fermentation Conditions;Yahong Yuan et al.;《Journal of Biomedicine and Biotechnology》;20121231;全文 * |
| Mutagenesis of Rhodobacter sphaeroides using atmospheric and room temperature plasma treatment for efficient production of coenzyme Q10;Rong-Song Zou et al.;《Journal of Bioscience and Bioengineering》;20190129;全文 * |
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