CN105483171A - Production method for increasing industrial output of coenzyme Q10 - Google Patents
Production method for increasing industrial output of coenzyme Q10 Download PDFInfo
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- CN105483171A CN105483171A CN201511031083.1A CN201511031083A CN105483171A CN 105483171 A CN105483171 A CN 105483171A CN 201511031083 A CN201511031083 A CN 201511031083A CN 105483171 A CN105483171 A CN 105483171A
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Abstract
The invention relates to a production method for increasing the industrial output of coenzyme Q10. Strains with the preservation number of CGMCC No.5997 or CGMCC No.5998 or CGMCC No.5999 are sequentially subjected to resuscitation and expanding culture in a seed medium to obtain fermentation seeds through screening, after the fermentation seeds are subjected to expanding culture, a fermentation medium is inoculated with the fermentation seeds to conduct fermentation culture so as to obtain coenzyme Q10, and the concentration of Fe2+ in the seed medium is 0.1-0.5 mol/L. The technical problem that the industrial output of an existing fermentation method for producing coenzyme Q10 is not large is solved.
Description
Technical field
The present invention relates to microorganism field, be specifically related to one and can promote that hydrogenlike silicon ion Rhodobactersphaeroides produces ubiquinone
10the peculiar cultural method of strain activity and output.
Background technology
Ubiquinone
10(CoenzymeQ
10) also known as ubiquinone (Ubiquinone, abbreviation UQ), being that one is present in natural fat-soluble quinones, is hydrogen carrier important on electron transport chain in organism.In human body cell, participate in energy manufacture and activation, be that prevention of arterial sclerosis forms the most effective anti-oxidant composition.Ubiquinone
10the nutrition of energy human activin cell and cellular energy, have improve body immunity, strengthen anti-oxidant, delay senility and strengthen the functions such as human activity, medically be widely used in cardiovascular system diseases, extensively use it for dietary supplements and foodstuff additive both at home and abroad.
Ubiquinone
10preparation method mainly contain three kinds: animal vegetable tissue cytapheresis, chemical synthesis and microbe fermentation method.Animal and plant cells extraction method due to production efficiency lower, technique fall behind, substantially need not in suitability for industrialized production at present.Such as, publication number is that the Chinese invention patent application of CN102391093A discloses and a kind ofly from tobacco, extracts ubiquinone
10method, its technical process is: by the tobacco sale that has been separated with nutrient solution or tobacco suspension culture cell tissue block and 75 ~ 85% ethanol-petroleum ether solution mixing homogenate 5 ~ 10 minutes, homogenate terminates rear above-mentioned homogenate mixtures and filters, and namely stratification obtains ubiquinone after upper solution is concentrated
10crude extract.
Ubiquinone is produced by chemical synthesis
10, such as, publication number is that the Chinese invention patent application of CN1931818 discloses a kind of synthesizing coenzyme Q
10method.The method is with ubiquinone
0the isodecyl alcohol deca-isopentyl alcohol that quinhydrones and content are not less than 95% is raw material, and after dehydration, through trifluoromethanesulfonic acid and metal salt catalyst thereof in oxygen-free environment, condensation reaction under room temperature condition, obtains ubiquinone
10quinhydrones.Chemical synthesis productive rate is relatively low, and is the mixture of cis and trans isomers with the allylation reagents that solanesol obtains, and activity is not strong, and need to be separated, therefore the application of this method is restricted.
Microbe fermentation method is the focus of global development in recent years, is considered to the most promising mode of production.Utilize Production by Microorganism Fermentation ubiquinone
10, be no matter the quality from product and security aspect, have larger competitive edge, be applicable to large-scale industrial production.
Such as publication number is that the Chinese invention patent application of CN101024849 discloses a kind of ubiquinone
10fermentation by Photosynthetic Bacteria method: utilize DNA recombinant technology to build, screening high yield ubiquinone
10photosynthetic bacterium strain, meanwhile, adopts chemical substance or ultraviolet mutagenesis, utilizes carotenogenesis inhibitor and growth inhibitor screening high yield ubiquinone
10photosynthetic bacterium mutant, realizes high yield ubiquinone
10the seed selection of bacterial strain and cultivation.Publication number is that the Chinese invention patent application of CN1884562 discloses a kind of production ubiquinone
10method, adopt fermentation process to produce, the bacterial classification that adopts of fermenting is Agrobacterium LQ
10.
Existing microbe fermentation method especially adopts during engineering bacteria all exists ubiquinone
10the problem that industrial output is not high.
Summary of the invention
The invention provides a kind of raising ubiquinone
10the production method of industrial output, solves existing fermentative Production ubiquinone
10the technical problem that industrial output is not high.
A kind of raising ubiquinone
10the production method of industrial output, comprise: by preserving number be CGMCCNo.5997, CGMCCNo.5998 or CGMCCNo.5999 bacterial strain successively through recovery and seed culture medium spread cultivation after screening obtain ferment-seeded, be seeded in fermention medium after gained ferment-seeded is spread cultivation and carry out fermentation culture, obtain ubiquinone
10, Fe in seed culture medium
2+concentration be 0.1 ~ 0.5mol/L.
Preserving number is the bacterial strain of CGMCCNo.5997 is the red ball bacterium of class, and Latin name is rhodobactersphaeroides, called after NHU-ZUB bacterial strain.
The bacterial strain of CGMCCNo.5998 is the red ball bacterium of class, and Latin name is rhodobactersphaeroides, called after NHU-ZDD bacterial strain.
The bacterial strain of CGMCCNo.5999 is the red ball bacterium of class, and Latin name is rhodobactersphaeroides, called after NHU-ZAA bacterial strain.
Above bacterial strain is the bacterial strain that applicant is also survived in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms.
In fermentable is produced, bacterial classification excellent is the key of fermentative production output height by the gross, and before bacterial strain accesses large tank, select outstanding bacterial strain to be the guarantee of high yield, bacterial screening is the important means improving strain quality, stablize fermentation level.And suitable growing environment is the guarantee that bacterial strain grows fast, giving the demand of bacterial strain abundance, could allow bacterial strain not by the restriction of short-board effect, is farthest the service of producing.
Ubiquinone
10be hydrogen carrier important on electron transport chain in organism, thus strengthen electron transport chain intensity and be conducive to ubiquinone
10accumulation.Wherein, iron ion is the important prothetic group of oxyphorase in iron-sulphur protein in electron transport chain, cytopigment and organism.
Find in researchist's research of the present invention, in current published report, the micro-promotor that iron ion is used as substratum uses, and rarely seen utilization in substratum adds iron concentration to screen the bacterial classification being suitable for industrial production and using, and is especially applicable to the bacterial classification after genetic engineering modified.
The present invention is at maintenance ubiquinone
10under the prerequisite of original quality of fermenting, according to ubiquinone
10produce the physiological property of bacterium, by a large amount of flask process optimization experiment, effectively to improve strain activity and ubiquinone
10for the purpose of output, set up a kind of effective and feasible cultivation screening method, have found one effectively can improve ubiquinone
10produce the fermentation culture method of bacterial strain microbial activity and production output, and achieve expansion aborning, effectively improve production output.
Preferably, Fe in described seed culture medium
2+concentration be 0.1 ~ 0.3mol/L; Still more preferably, Fe in seed culture medium
2+concentration be 0.3mol/L.
Further preferably, the culture condition spread cultivation in seed culture medium is: 30 ~ 32 DEG C of lucifuges cultivate 22 ~ 24h.Sterilising temp 121 DEG C, sterilization time 20min.
Preferably, described recovery is carried out successively in plate culture medium and slant medium, Fe in plate culture medium and seed culture medium
2+concentration be 0.1 ~ 0.5mol/L.
Further preferably, Fe in described plate culture medium
2+concentration be 0.1 ~ 0.3mol/L; Still more preferably, Fe in described plate culture medium
2+concentration be 0.3mol/L.
Further preferably, Fe in described slant medium
2+concentration be 0.1 ~ 0.3mol/L; Still more preferably, Fe in described slant medium
2+concentration be 0.3mol/L.
Further preferably, the culture condition in slant medium is that 30 ~ 32 DEG C of lucifuges cultivate 60 ~ 80h.Sterilising temp 121 DEG C, sterilization time 20min.
Preferably, Fe
2+add in substratum with at least one compound form in iron(ic) chloride, ferrous sulfate and ironic citrate.
More preferably, Fe in plate culture medium, slant medium and seed culture medium
2+concentration be 0.1 ~ 0.3mol/L; Most preferably, Fe in plate culture medium, slant medium and seed culture medium
2+concentration be 0.3mol/L.
The present invention's plate culture medium used and slant medium are that class rhodococcus commonly uses substratum, comprise the conventional ingredients such as carbon source, nitrogenous source, inorganic salt, VITAMIN.
Preferably, plate culture medium (every 100mL) composition is: glucose 0.3g, yeast extract 0.8g, cobalt chloride 0.003g, sodium-chlor 0.2g, manganous sulfate 0.0001g, dipotassium hydrogen phosphate 0.13g, magnesium sulfate 0.025g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, agar powder 1.5g, then the Fe adding 0.1 ~ 0.5mol/L
2+.PH is 7.2, and sterilising conditions can be 118 DEG C, 10min.
The composition (every 100mL) of slant medium is: glucose 0.3g, yeast extract 0.8g, cobalt chloride 0.003g, sodium-chlor 0.2g, manganous sulfate 0.0001g, dipotassium hydrogen phosphate 0.13g, magnesium sulfate 0.025g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, agar powder 1.2g; Add the Fe of 0.1 ~ 0.5mol/L again
2+.PH is 7.2, and sterilising conditions can be 118 DEG C, 10min.
Seed culture medium (every 100mL) composition is: ammonium sulfate 0.25g, corn steep liquor 0.05g, glucose 0.3g, yeast extract 0.14g, cobalt chloride 0.003g, sodium-chlor 0.2g, manganous sulfate 0.0001g, dipotassium hydrogen phosphate 0.05g, potassium primary phosphate 0.05g, magnesium sulfate 0.1g, calcium carbonate 0.8g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, then the Fe adding 0.1 ~ 0.5mol/L
2+.PH is 7.2, and sterilising conditions can be 118 DEG C, 10min.
Bacterial screening process of the present invention specifically comprises the following steps:
1) primary dcreening operation (recovery) of bacterial classification: adopt dull and stereotyped single bacterium colony to choose spot inoculation small test tube slant medium, after cultivating for some time, inoculation fermentation shaking flask test sample screens;
2) the multiple sieve (spreading cultivation) of bacterial classification: the result obtained from primary dcreening operation selects outstanding bacterial strain, freeze pipe connects little female bottle and then connects fermentation flask test sample and sieve again;
3) preservation of bacterial classification: painting eggplant bottle inclined-plane is carried out to the bacterial strain that multiple sieve obtains, adds glycerol stocks after cultivation;
4) production and application of bacterial strain: the large female bottle of outstanding inoculation obtained by multiple sieve, plants as one grade fermemtation mother after cultivation, is seeded in fermentor tank and cultivates after spreading cultivation.
In screening step (1) and (2), fermentation flask incubation time is that 31 DEG C of lucifuges cultivate 48h, sterilising temp 121 DEG C, sterilization time 20min.
In screening step (3), the eggplant bottle slant culture time is that 31 DEG C of lucifuges cultivate 3 days, sterilising temp 121 DEG C, sterilization time 20min.
Detecting fermention medium used of tiring in above-mentioned screening process is that bacterial strain uses therefor of the present invention commonly uses fermention medium, comprises the conventional ingredients such as carbon source, nitrogenous source, inorganic salt, VITAMIN.
Preferably, described fermention medium (every 100mL): ammonium sulfate 0.3g, glucose 4g, monosodium glutamate 0.3g, cobalt chloride 0.005g, sodium-chlor 0.28g, calcium carbonate 0.6g, dipotassium hydrogen phosphate 0.15g, magnesium sulfate 0.63g, ferrous sulfate 0.12g, corn steep liquor 0.4g vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, pH is 7.2, and sterilising conditions can be 118 DEG C, 10min.
The fermentation culture conditions detected when tiring is: 26 ~ 34 DEG C, 200rpm ~ 250rpm lucifuge cultivates 48 ~ 96 hours, extract fermented liquid and detect and tires.The object stirred is fermenting process oxygen supply, and adjustment rotating speed is to reach adjustment for the object of oxygen concn, and the oxygen supply on the basis of bacterial strain screening of the present invention again in combining with fermentation process, can improve ubiquinone further
10output.
The spread cultivation process of bacterial classification when production and application obtained is screened as follows in the present invention:
(1) slant culture: received by the glycerine pipe of preservation on eggplant type bottle inclined-plane, cultivates 3 days at 31 DEG C.
(2) seed culture: wash cultured inclined-plane with sterilized water, make 10
8~ 10
9the bacteria suspension of individual cells/ml, pipettes in the seed bottle of 10ml to loading amount 500ml/1000ml, 30 DEG C, cultivate under the condition of 180 ~ 250rpm and obtain seed liquor in 22-26 hour.
Above-mentioned steps (2) gained seed liquor directly can be seeded to fermentation cylinder for fermentation and cultivate, inoculum size 10%, in fermentation, the composition of fermention medium is preferably: described fermentation medium components is: glucose 1.8-2.5g/L, yeast extract paste 7.5 ~ 8.5g/L, sodium-chlor 2.5 ~ 3g/L, dipotassium hydrogen phosphate 0.5 ~ 0.8g/L, magnesium sulfate 0.2 ~ 0.3g/L, ammonium sulfate 2.5 ~ 3.5g/L, pH are 6.5 ~ 7.0.
More preferably: glucose 1.8-2.5g/L, yeast extract paste 8g/L, sodium-chlor 2.8g/L, dipotassium hydrogen phosphate 0.6g/L, magnesium sulfate 0.25g/L, ammonium sulfate 3g/L, pH are 6.8.In fermentor tank, culture condition is: culture temperature 29-33 DEG C, tank internal pressure 0.03 ~ 0.05Mpa, air flow quantity 5 ~ 6L/min, mixing speed be 500-700rpm.
Glucose is added continuously according to thalli growth situation adjusting process parameter and beginning in fermenting process.
The present invention is first at ubiquinone
10have employed distinctive screening and culturing method in whole fermentative production flow process, and use the different compounds of the iron adding suitable concn to improve the various substratum of strain activity and output, preferably add iron concentration 0.1 ~ 0.5mol/L.Iron is the necessary element of Growth of Cells, adds the normal growth that iron ion can promote cell in right amount.Iron ion is the important prothetic group of oxyphorase in iron-sulphur protein in electron transport chain, cytopigment and organism.In production in the past, inadequate to the concern of iron ion, often just add the compound of Single Iron in the medium on a small quantity, limit the quick growth of cell, the present invention passes through the compound that lot of experiment validation adds the iron of different sorts and proportioning in the medium, can improve strain growth activity and produce ubiquinone
10ability.
As can be seen here, the present invention compares former zymotechnique and possesses following advantage: 1. the growth vigor of bacterium cell is strong, and tolerance is strong, easily produces in fermentor tank; 2. physiological status is stablized, and keeps stable throughput; 3. producing strain is stable also slowly can improve output, in the survival of the fittest, ensure throughput.
The present invention is first at ubiquinone
10the substratum of the chemical combination state of adding the different iron of suitable concn is used to carry out screening and cultivate the fermentation culture method simultaneously carried out in fermentative production, preferred interpolation iron concentration 0.1 ~ 0.5mol/L, and in being applied to pilot scale and trying greatly to ferment, obtain desirable fermentation results.
Accompanying drawing explanation
Fig. 1 be add different iron compound to ubiquinone
10synthesis affects comparison diagram.
Fig. 2 is that different ferrous sulfate concentration is to ubiquinone
10synthesis affects comparison diagram.
Fig. 3 is for adding iron ion at different rotating speeds to ubiquinone
10synthesis affects comparison diagram.
Embodiment
(1) plate culture medium basic ingredient (every 100mL): glucose 0.3g, yeast extract 0.8g, cobalt chloride 0.003g, sodium-chlor 0.2g, manganous sulfate 0.0001g, dipotassium hydrogen phosphate 0.13g, magnesium sulfate 0.025g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, agar powder 1.5g, pH is 7.2, and sterilising conditions can be 118 DEG C, 10min.
(2) slant medium basic ingredient (every 100mL): glucose 0.3g, yeast extract 0.8g, cobalt chloride 0.003g, sodium-chlor 0.2g, manganous sulfate 0.0001g, dipotassium hydrogen phosphate 0.13g, magnesium sulfate 0.025g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, agar powder 1.2g, pH is 7.2, and sterilising conditions can be 118 DEG C, 10min.
(3) seed culture medium basic ingredient (every 100mL): ammonium sulfate 0.25g, corn steep liquor 0.05g, glucose 0.3g, yeast extract 0.14g, cobalt chloride 0.003g, sodium-chlor 0.2g, manganous sulfate 0.0001g, dipotassium hydrogen phosphate 0.05g, potassium primary phosphate 0.05g, magnesium sulfate 0.1g, calcium carbonate 0.8g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, pH are 7.2, sterilising conditions can be 118 DEG C, 10min.
(4) fermention medium (every 100mL): ammonium sulfate 0.3g, glucose 4g, monosodium glutamate 0.3g, cobalt chloride 0.005g, sodium-chlor 0.28g, calcium carbonate 0.6g, dipotassium hydrogen phosphate 0.15g, magnesium sulfate 0.63g, corn steep liquor 0.4g, FeSO
40.12g, vitaminB10 .1ug, vitamin K 0.1ug, vitamin A 0.15ug, pH are 7.2, and sterilising conditions can be 118 DEG C, 10min.
(5) fermentation tank culture medium: glucose 1.8-2.5g/L, yeast extract paste 8g/L, sodium-chlor 2.8g/L, dipotassium hydrogen phosphate 0.6g/L, magnesium sulfate 0.25g/L, ammonium sulfate 3g/L, pH are 6.8, and sterilising conditions can be 118 DEG C, 10min.
Below provide fermented liquid object product ubiquinone
10adopt Liquid Detection condition:
Chromatographic column: C18,4.6*150mm, 5um;
Determined wavelength: 275nm;
Moving phase: ethanol: methyl alcohol=1:1;
Flow velocity: 1.5ml/min;
Column temperature: 35 DEG C.
Embodiment 1: add the different compound of iron and do not add shaking flask ubiquinone
10the comparison of fermentation.
Shaking flask primary dcreening operation breeding fermentation flow process: dull and stereotyped single bacterium colony → small test tube inclined-plane → fermentation flask → Liquid Detection Q
10to tire → glycerine pipe.Detailed process is as follows:
Select to cultivate about 7 days single bacterium colonies on flat board, correspondence is chosen in small test tube inclined-plane, wash with 4mL after 31 DEG C of lucifuges cultivate 3 days, draw the 250mL shaking flask of 1mL bacterium liquid to dress 50ml fermention medium, 31 DEG C, 230r/min lucifuge cultivates 48h, extracts fermented liquid detection and tires, and residue bacterium liquid Refrigerator store is stand-by.Same volume 40% glycerine conservation is added by recording result outstanding bacterial strain bacterium liquid.
Shaking flask sieves breeding fermentation flow process again: primary dcreening operation glycerine pipe → little female bottle → fermentation flask → Liquid Detection Q
10tire → eggplant bottle inclined-plane spread cultivation and for the production of.Detailed process is as follows:
Choose the 250mL shaking flask that primary dcreening operation obtains glycerine pipe access dress 50ml seed culture medium, 31 DEG C, 230r/min lucifuge cultivates 23h, draw the 250mL shaking flask of 1mL bacterium liquid to dress 50ml fermention medium, 31 DEG C, 230r/min lucifuge cultivates 48h, 96h, extracts fermented liquid detection respectively and tires.Select outstanding bacterial strain freeze pipe to be coated with eggplant bottle inclined-plane to spread cultivation, the application of the inoculation spread cultivation out sub-bottle is large produces.
The compound adding 0.1mol/L iron in dull and stereotyped, inclined-plane and seed culture medium in the shake flat experiment Comparative result do not added as table 1, table 1: the different compound adding iron and the fermentation results do not added.
Result shows: the compound adding iron in substratum embodies following advantage:
1. dull and stereotyped single colony growth is fast, and bacterium colony color and luster is bright and new full;
2. in seed bottle, the growth of bacterial strain Individual Size is fast, and form is mellow and full, and overall bacterium is dense higher;
3. in fermentation flask, strain growth is very fast, tires higher.More excellent to add ferrous sulfate.
Embodiment 2: the ferrous sulfate concentration shaking flask ubiquinone adding different gradient in substratum
10the comparison of fermentation.
Shaking flask primary dcreening operation breeding fermentation flow process: dull and stereotyped single bacterium colony → small test tube inclined-plane → fermentation flask → Liquid Detection Q
10to tire → glycerine pipe.Detailed process is as follows:
Select to cultivate about 7 days single bacterium colonies on flat board, correspondence is chosen in small test tube inclined-plane, 31 DEG C of lucifuges are cultivated after 3 days with under 4mL washing, draw the 250mL shaking flask of 1mL bacterium liquid to dress 50ml fermention medium, 31 DEG C, 230r/min lucifuge cultivates 48h, extracts fermented liquid detection and tires, and residue bacterium liquid Refrigerator store is stand-by.Same volume 40% glycerine conservation is added by recording result outstanding bacterial strain bacterium liquid.
Shaking flask sieves breeding fermentation flow process again: primary dcreening operation glycerine pipe → little female bottle → fermentation flask → Liquid Detection Q
10tire → eggplant bottle inclined-plane spread cultivation and for the production of.Detailed process is as follows:
Choose the 250mL shaking flask that primary dcreening operation obtains glycerine pipe access dress 50ml seed culture medium, 31 DEG C, 230r/min lucifuge cultivates 23h, draw the 250mL shaking flask of 1mL bacterium liquid to dress 50ml fermention medium, 31 DEG C, 230r/min lucifuge cultivates 48h, 96h, extracts fermented liquid detection respectively and tires.Select outstanding bacterial strain freeze pipe to be coated with eggplant bottle inclined-plane to spread cultivation, the application of the inoculation spread cultivation out sub-bottle is large produces.
In dull and stereotyped, inclined-plane and seed culture medium, add the ferrous sulfate concentration of different gradient, screen the bacterial classification that obtains at shake flat experiment Comparative result as table 2:
Table 2: add different gradient ferrous sulfate fermenting experiment comparing result:
Result shows, add ferrous sulfate concentration 0.1 ~ 0.5mol/L seed selection in flat board and seed culture medium after, effect is particularly evident.
Embodiment 3: the shake flask fermentation Coenzyme Q10 99.0 after adding iron ion under different oxygen supply condition compares.
Shaking flask primary dcreening operation breeding fermentation flow process: dull and stereotyped single bacterium colony → small test tube inclined-plane → fermentation flask → Liquid Detection Q
10to tire → glycerine pipe.Detailed process is as follows:
Select to cultivate about 7 days single bacterium colonies on flat board, correspondence is chosen in small test tube inclined-plane, 31 DEG C of lucifuges are cultivated after 3 days with under 4mL washing, draw the 250mL shaking flask of 1mL bacterium liquid to dress 50ml fermention medium, 31 DEG C, 230r/min lucifuge cultivates 48h, extracts fermented liquid detection and tires, and residue bacterium liquid Refrigerator store is stand-by.Same volume 40% glycerine conservation is added by recording result outstanding bacterial strain bacterium liquid.
Shaking flask sieves breeding fermentation flow process again: primary dcreening operation glycerine pipe → little female bottle → fermentation flask → Liquid Detection Q
10tire → eggplant bottle inclined-plane spread cultivation and for the production of.Detailed process is as follows:
Choose the 250mL shaking flask that primary dcreening operation obtains glycerine pipe access dress 50ml seed culture medium, 31 DEG C, 230r/min lucifuge cultivates 23h, draw the 250mL shaking flask of 1mL bacterium liquid to dress 50ml fermention medium, 31 DEG C, 230r/min lucifuge cultivates 48h, 96h, extracts fermented liquid detection respectively and tires.Select outstanding bacterial strain freeze pipe to be coated with eggplant bottle inclined-plane to spread cultivation, the application of the inoculation spread cultivation out sub-bottle is large produces.
Seed after the seed culture medium passing through to add the dull and stereotyped seed selection of 0.3mol/L ferrous sulfate and with the addition of 0.3mol/L ferrous sulfate spreads cultivation, receives in fermentation shake flask, under equal conditions, under different rotating speeds, and ubiquinone under more different oxygen supply condition
10ferment effect.
Table 3: shaking flask ubiquinone under different rotating speeds condition
10fermentation results
Result shows, under increasing oxygen supply condition, adds iron ion and better can promote ubiquinone
10synthesis.
Embodiment 4: at the ferment tank Coenzyme Q10 99.0 results contrast of different strains after interpolation iron ion.
Flow process investigated by fermentor tank: glycerine pipe → slant culture → female bottle seed → fermentor tank, and idiographic flow is as follows;
(1) slant culture: received by the glycerine pipe of preservation on eggplant type bottle inclined-plane, cultivates 3 days at 31 DEG C.
(2) seed culture: wash cultured inclined-plane with sterilized water, make 10
8~ 10
9the bacteria suspension of individual cells/ml, pipettes in the seed bottle of 10ml to loading amount 500ml/1000ml, 30 DEG C, cultivate under the condition of 180 ~ 250rpm and obtain seed liquor in 22-26 hour.
(3) fermentation culture: step (2) gained seed liquor is inoculated into 10 liters of fermentor tanks, inoculum size 10%, culture temperature 29-33 DEG C, tank internal pressure 0.03 ~ 0.05Mpa, control by stages strategy is taked in oxygen supply, initial mixing speed 500rpm, air flow quantity 6L/min after inoculation, after inoculation, along with growth lag phase terminates, thalline starts to grow into logarithmic phase fast, and OUR rises fast, 24 hours, and maintain 30-50mmol/Lh, rotating speed 500-700rpm, in tank, residual glucose controls between 0.5-2.0%, cultivates 110 hours.Glucose is added continuously according to thalli growth situation adjusting process parameter and beginning in process.
The fermentation results situation of relatively adding and not being added between different strains in inclined-plane, kind Combined inkbottle:
Result shows, in slant medium and female bottle substratum, increase iron ion, under identical fermentation condition, and ubiquinone
10output all has the growth of certain amplitude.
Claims (10)
1. one kind is improved ubiquinone
10the production method of industrial output, comprise: by preserving number be CGMCCNo.5997, CGMCCNo.5998 or CGMCCNo.5999 bacterial strain successively through recovery and seed culture medium spread cultivation after screening obtain ferment-seeded, be seeded in fermention medium after gained ferment-seeded is spread cultivation and carry out fermentation culture, obtain ubiquinone
10, it is characterized in that, Fe in seed culture medium
2+concentration be 0.1 ~ 0.5mol/L.
2. production method according to claim 1, is characterized in that, Fe in described seed culture medium
2+concentration be 0.1 ~ 0.3mol/L.
3. production method according to claim 1, is characterized in that, the culture condition spread cultivation in seed culture medium is: 30 ~ 32 DEG C of lucifuges cultivate 22 ~ 24h.
4. production method according to claim 1, is characterized in that, described recovery is carried out successively in plate culture medium and slant medium, Fe in plate culture medium and seed culture medium
2+concentration be 0.1 ~ 0.5mol/L.
5. production method according to claim 4, is characterized in that, Fe in described plate culture medium
2+concentration be 0.1 ~ 0.3mol/L.
6. production method according to claim 4, is characterized in that, Fe in described slant medium
2+concentration be 0.1 ~ 0.3mol/L.
7. production method according to claim 4, is characterized in that, the culture condition in slant medium is that 30 ~ 32 DEG C of lucifuges cultivate 60 ~ 80h.
8. the production method according to claim 1 or 4, is characterized in that, Fe
2+add in substratum with at least one compound form in iron(ic) chloride, ferrous sulfate and ironic citrate.
9. production method according to claim 1, it is characterized in that, the composition of described fermention medium is: glucose 1.8-2.5g/L, yeast extract paste 7.5 ~ 8.5g/L, sodium-chlor 2.5 ~ 3g/L, dipotassium hydrogen phosphate 0.5 ~ 0.8g/L, magnesium sulfate 0.2 ~ 0.3g/L, ammonium sulfate 2.5 ~ 3.5g/L, pH are 6.5 ~ 7.0.
10. production method according to claim 1, is characterized in that, during fermentation culture in fermentor tank culture temperature 29-33 DEG C, tank internal pressure 0.03 ~ 0.05Mpa, air flow quantity 5 ~ 6L/min, mixing speed be 500-700rpm.
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