CN103224965A - Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same - Google Patents
Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same Download PDFInfo
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Abstract
The invention discloses a method for producing pyrroloquinoline quinine through microbial fermentation and a fermentation medium used in the method for producing the pyrroloquinoline quinine through the microbial fermentation. An oxygen control fermentation culture method is used for culturing methylotrophic bacteria, a carbon source contained in the fermentation medium is methyl alcohol, and a nitrogen source contained in the fermentation medium is ammonium sulfate, a dissolved oxygen level is controlled by stages to be 30%-50% of saturated dissolved oxygen in the fermentation process, pH is controlled to be 5.5-6.0 through fed-batch, the fed-batch is achieved through a pH-nitrogen source joint control technology, the methylotrophic bacteria are cultured for six days in a 7.5L fermentation tank, and eventually the cell density (OD 600) exceeds 10, yield of PQQ reaches 2g/L, and production intensity of the PQQ is 333.33mg/L/day. The method for producing the pyrroloquinoline quinine through the microbial fermentation is simple in operation, automatic control and continuous fermentation can be achieved easily, and a solid foundation for industrially producing the pyrroloquinoline quinine through the methylotrophic bacteria is laid.
Description
Technical field
The present invention relates to the microbial fermentation technology research field, be specifically related to the method for a kind of microbial fermentation High-efficient Production pyrroloquinoline quinone (PQQ) and used fermention medium.
Background technology
Pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ) be the coenzyme of the 3rd class oxydo-reductase, also be to be called as a kind of new VITAMIN, has different physiological roles, generate, regulate the free radical level in control liver injury and liver poisoning, promotion nerve growth factor, there is important medical value aspects such as Antiradiation injury, cardiovascular disorder and diabetes.
At present PQQ mainly is a chemical synthesis, and microorganism is synthetic to have cleaning low-carbon (LC), gentle advantage such as controlled, with low cost, is a kind of mode of production that has potentiality.The principal element that influences microbial fermentation production PQQ large-scale application has: it is few that (1) is suitable for industrial microorganism; (2) it is long that the PQQ of bibliographical information produces the bacterial strain production cycle, and productive rate is not high; (3) PQQ is not the secondary metabolite of microorganism as a kind of coenzyme, and its synthetic regulation and control are restricted by the strictness of culture condition and metabolic regulation.
Summary of the invention
In order to address the above problem, the purpose of this invention is to provide a kind of cultural method and fermention medium that the PQQ resultant quantity is increased, grope the control method that an effective microbial fermentation is produced PQQ, realize utilizing the purpose of methylotrophic bacteria High-efficient Production PQQ.
Another object of the present invention provides the fermention medium of the described PQQ of a kind of fermentative preparation.
In order to realize purpose of the present invention, the invention provides a kind of method of microbial fermentation production pyrroloquinoline quinone, it comprises the steps:
(1) activated spawn: original strain methylotrophic bacteria Methylovorus sp.MP688 is inoculated in the solid medium, under the condition that is fit to the original strain growth, cultivates, get the activatory bacterial classification;
(2) culture of seed liquid: the bacterial classification inoculation that step (1) gained is activated is cultivated under the condition that is fit to thalli growth in liquid nutrient medium, seed culture fluid;
(3) ferment tank: step (2) gained seed culture fluid is inoculated in the fermentor tank that contains fermention medium in proportion; Homo(io)thermism in the fermentation culture process, simultaneously, the pH of fermentor tank, dissolved oxygen and mixing speed adopt two stage control strategies; The nitrogenous source feed supplement adopts pH-nitrogenous source joint control mode to carry out, and the carbon source feed supplement adopts the fed-batch mode to carry out;
(4) the results fermented liquid gets pyrroloquinoline quinone.
Further, the invention provides following optimized technical scheme:
Wherein, described methylotrophic bacteria Methylovorus sp.MP688 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 20th, 2010, and its preserving number is CGMCCNo.4096.(patent No. that details see also Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A application is 201010548511.9 patent of invention).
Preferably, culture temperature is 30～35 ℃ described in the step (1), and incubation time is 2～3 days; Described solid medium is to add agar powder and carbon source methyl alcohol on the basis of minimum medium, the concentration of described agar powder in described film solid media is 1～2g/L, the concentration of described methyl alcohol in described film solid media is 7.5～15g/L, wherein, the component of described minimum medium and proportioning are as follows:
Minimum medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 1.4g，
Na
2HPO
4·12H
2O 3g，
Ironic citrate 10-30mg,
CaCl
2 10-30mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-2mg，
Folic acid 0.02-0.05mg,
Vitamin H 0.02-0.05mg,
Riboflavin 0.02-0.05mg,
Nicotinic acid 0.02-0.05mg,
Thioctic Acid 0.02-0.05mg.
Preferably, step (2) concrete steps are: the bacterial classification that step (1) gained activates is chosen single bacterium colony, be inoculated into 30～35 ℃ of shaking table vibrations in the 5ml liquid nutrient medium, rotating speed is 200～250 rev/mins, cultivated 2～3 days, get the aforementioned bacterium liquid of 1～2ml then and be seeded to the numerous cultivation of continuation expansion under the same conditions in the triangular flask that contains the 100ml liquid nutrient medium, until obtaining strain cell density (OD
600) be 1.2～1.5 seed liquor; Wherein, described liquid nutrient medium is to add carbon source methyl alcohol on the basis of minimum medium, and the concentration in the described methyl alcohol liquid medium within is 7.5～15g/L.Wherein, the component of described minimum medium and proportioning are as follows:
Minimum medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 1.4g，
Na
2HPO
4·12H
2O 3g，
Ironic citrate 10-30mg,
CaCl
2 10-30mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-2mg，
Folic acid 0.02-0.05mg,
Vitamin H 0.02-0.05mg,
Riboflavin 0.02-0.05mg,
Nicotinic acid 0.02-0.05mg,
Thioctic Acid 0.02-0.05mg.
Preferably, the volume ratio of seed culture fluid and fermention medium is 1:50～1:10 described in the step (3); Described fermention medium, concrete composition and proportioning are as follows:
Fermention medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 2.1-2.8g，
Na
2HPO
4·12H
2O 4.5-6g，
Ironic citrate 30-80mg,
CaCl
2 0.05-0.25mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-1mg，
Folic acid 0.02-0.05mg.
Preferably, leavening temperature is 30～35 ℃ described in the step (3), and fermentation time is 6～8 days.
Preferably, being specially of described two stage control strategies:
(1) initial stage: the initial stage air flow that ferments after the inoculation is controlled at 0.02～5vvm, does not control dissolved oxygen, and rotating speed is controlled at 200～300rpm, and after initial pH value was adjusted into 6.5-7.0, the pH value was no longer controlled;
(2) later stage: along with bacterial growth, the pH value descends, and treats that the pH value drops at 5.5～6.0 o'clock, adds ammoniacal liquor by the pump feedback flow pH is controlled at this level;
Treat that dissolved oxygen in the fermentor tank drops to 30% o'clock of saturated dissolved oxygen, by the dissolved oxygen strategy related dissolved oxygen is controlled at 30%～50% level with rotating speed and air flow, be specially: preferentially adjust rotating speed, described speed range is 200～800rpm, when rotating speed reaches upper limit 800rpm, adjust air flow, described air flow scope is 0.2～5vvm.
Preferably, carbon source is a methyl alcohol described in the step (3), and described nitrogenous source is one or both in ammonium sulfate, the ammoniacal liquor; Described carbon source starting point concentration is 7.5～15g/L, nitrogenous source starting point concentration 7.5～15g/L; The total concn of carbon source is controlled in the 15g/L in the fermenting process, and the total concn of ammonium sulfate is controlled in the 3g/L;
Described pH-nitrogenous source joint control mode is carried out the nitrogenous source feed supplement for start nitrogenous source feed supplement program when fermented liquid pH value drops to 6.0 along with bacterial growth by fed-batch mode, and described nitrogenous source feed supplement liquid is ammoniacal liquor or the ammonium sulfate of 0.10～0.25g/ml;
Growth velocity of on-line monitoring bacterium and nitrogenous source feed supplement discharge curve in case growth velocity of bacterium or nitrogenous source feed rate had been compared reduction with a last time period, add carbon source methyl alcohol by the fed-batch mode, and the feed supplement amount is that every liter of fermented liquid adds 5～15g methyl alcohol; Described methyl alcohol is industrial methanol or analytical pure.
Preferably, the fermented liquid of step (4) is according to patents " extracting method of pyrroloquinoline quinone " such as the Zhao Yong of Wuhan University virtue preparations pyrroloquinoline quinone product (license number be CN1138776C).The pyrroloquinoline quinone Determination on content is with reference to people document Enzymatic determination of pyrroloquinoline quinone using crude membranes from Escherichia coli in 1987 such as Geiger O..
Further, the invention provides a kind of pyrroloquinoline quinone, it is to be obtained by aforesaid method production.
Further, the invention provides a kind of fermention medium, concrete composition and proportioning are as follows:
Fermention medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 2.1-2.8g，
Na
2HPO
4·12H
2O 4.5-6g，
Ironic citrate 30-80mg,
CaCl
2 0.05-0.25mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-1mg，
Folic acid 0.02-0.05mg.
Further, the invention provides the application of above-mentioned fermention medium in the production pyrroloquinoline quinone.
Beneficial effect of the present invention is as follows:
The present invention adopts fermention medium, helps methylotrophic bacteria synthetic pyrroloquinoline quinone rapidly and efficiently, and component is inorganic salt, does not have complicated organism, and is therefore with low cost, is fit to industry's enlarging production.
Homo(io)thermism in the fermentation culture process of the present invention, the pH of fermentor tank, dissolved oxygen and mixing speed adopt two stage control strategies; By restriction dissolved oxygen (30-50%) and control weak acid environment (pH5.5-6.0), rate of bacterial growth is maintained lower level (be lower than 2OD
600/ 24 hours), coordinate synthetic two processes of bacterial growth and PQQ, make the more approach that flows to synthetic PQQ of carbon source metabolism stream, thereby obtain the PQQ of high yield; Simultaneously, carry out the nitrogenous source feed supplement, carry out the carbon source feed supplement, make the fermenting process optimum by the fed-batch mode by pH-nitrogenous source joint control mode.The present invention cultivates 6 days final cell density (OD in the automatic fermentor tank of 7.5L
600) surpassing 10, PQQ output has reached 2g/L, and the PQQ production intensity is 333.33mg/L/ days, and output and production intensity surpass the maximum of existing bibliographical information, and have shortened fermentation period; Simultaneously, the present invention is simple to operate, is convenient to realize controlling automatically and continuously fermenting, and has established solid basis for using methylotrophic bacteria suitability for industrialized production PQQ.
Description of drawings
The result of Fig. 1 fermentative production PQQ of the present invention.
Among the figure: ● the expression growth curve of bacteria, ■ represents the PQQ resultant curve.
Embodiment
Introduce the present invention in detail below in conjunction with accompanying drawing and embodiment thereof.But protection scope of the present invention is not limited to following example, should comprise the full content in claims.
Employed medicine in following examples, available from Chemical Reagent Co., Ltd., Sinopharm Group, the medicine rank is an analytical pure; The instrument that uses among the embodiment is the conventional instrument of biology laboratory, and used fermentor tank model is BIOSTAR Bplus, Germany.
The activation of embodiment 1 methylotrophic bacteria Methylovorus sp.MP688
Methylovorus sp.MP688 is preserved in the Chinese common micro-organisms CGMCC4096 of DSMZ, is the bacterial strain that a strain PQQ output reaches production level.Rule on the solid medium flat board with original strain (glycerine preservation or lyophilised state), cultivated 2 days for 30 ℃; Choose single bacterium colony, be inoculated in the 5ml liquid nutrient medium, 30 ℃, 200 rev/mins of shaking tables are cultivated 48 hours as activated seed.Described solid medium is to add agar powder and carbon source methyl alcohol on the basis of minimum medium, and the concentration of described agar powder in described film solid media is 1g/L; Described liquid nutrient medium is to add carbon source methyl alcohol on the basis of minimum medium; The concentration of wherein said methyl alcohol in film solid media or liquid nutrient medium is 10g/L; The component and the proportioning of described minimum medium are as follows:
Minimum medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 1.4g，
Na
2HPO
4·12H
2O3g，
Ironic citrate 30mg,
CaCl
2 30mg，
MnCl
2·4H
2O 5mg，
ZnSO
4·7H
2O 5mg，
CuSO
4·5H
2O 0.5mg，
Folic acid 0.02mg,
Vitamin H 0.02mg,
Riboflavin 0.05mg,
Nicotinic acid 0.05mg,
Thioctic Acid 0.05mg.
The preparation of embodiment 2 seed liquor
Get the bacterium liquid 2ml that embodiment 1 obtains, and be seeded to the triangular flask that contains the 100ml liquid nutrient medium, 30 ℃, 200 rev/mins of shaking tables were cultivated 36 hours, and similarity condition activates 1-2 time down, makes final fine and closely woven degree (OD
600) be 1.2～1.5, make seed liquor.Used liquid nutrient medium is with embodiment 1.
Embodiment 3 carries out PQQ production instance A in the 5L automatic fermenter
With the seed liquor of embodiment 2, be inoculated in the automatic fermentor tank of 7.5L according to the ratio of 1:20.Inoculation secondary fermentation initial stage air flow is controlled at 1vvm, and mixing speed is controlled at 300rpm, and temperature is controlled at 0 ℃, and initial pH value is made as 7.0; Described carbon source methyl alcohol starting point concentration is 7.5g/L, nitrogenous source ammonium sulfate initial concentration 15g/L; Described fermention medium, concrete composition and proportioning are as follows:
Fermention medium (in 1L)
MgSO
4·7H
2O 0.4g，
(NH
4)
2SO
4 3g，
KH
2PO
4 2.8g，
Na
2HPO
4·12H
2O 6g，
Ironic citrate 60mg,
CaCl
2 0.05mg，
MnCl
2·4H
2O 5mg，
ZnSO
4·7H
2O 5mg，
CuSO
4·5H
2O 0.5mg，
Folic acid 0.02mg.
Along with bacterial growth, the pH value descends, and waits to drop at 6.0 o'clock, adds ammoniacal liquor by the pump feedback flow pH is controlled at this level; Treat that dissolved oxygen in the fermentor tank drops to 40% o'clock of saturated dissolved oxygen, by the dissolved oxygen strategy related dissolved oxygen is controlled at 40% level with mixing speed and air flow, mixing speed adjustment preferential (the 800rpm upper limit), adjust air flow when rotating speed reaches the upper limit, the air flow maximum is set to 5vvm.Adopt pH-nitrogenous source joint control technology to realize the feed supplement of nitrogenous source ammoniacal liquor, feed supplement liquid concentration is 0.15g/ml; The growth velocity of on-line monitoring bacterium when the growth velocity curve in the bacterium 12 hours and when growth velocity reduced in preceding 12 hours, adds carbon source methyl alcohol by the fed-batch mode, and the feed supplement amount is that every liter of fermented liquid adds 10g methyl alcohol; Described carbon source feed supplement liquid is specially industrial methanol or analytical pure.Per 12 hour records and calculating cell density (OD
600) and PQQ output.Under this fermentation condition, through 6 days cultivation, final cell density (OD
600) can meet or exceed 10, PQQ output meets or exceeds 2g/L, and the PQQ production intensity was at least 333.33mg/L/ days, and concrete outcome can be referring to Fig. 1.
Form and proportioning with the seed liquor of embodiment 2 and the fermention medium among the embodiment 3, be inoculated in the automatic fermentor tank of 7.5L according to the ratio of 1:50.The initial stage air flow that ferments after the inoculation is controlled at 1vvm, and mixing speed is controlled at 300rpm, and temperature is controlled at 30 ℃, no longer control after initial pH value is made as 6.5; Described carbon source methyl alcohol starting point concentration is 7.5g/L, nitrogenous source ammonium sulfate starting point concentration 15g/L.
Along with bacterial growth, the pH value descends, and waits to drop at 5.8 o'clock, adds ammoniacal liquor by the pump feedback flow pH is controlled at this level; Treat that dissolved oxygen in the fermentor tank drops to 30% o'clock of saturated dissolved oxygen, by the dissolved oxygen strategy related dissolved oxygen is controlled at 30% level with mixing speed and air flow, mixing speed adjustment preferential (the 800rpm upper limit), adjust air flow when rotating speed reaches the upper limit, the air flow maximum is set to 5vvm.Adopt pH-nitrogenous source joint control technology to realize the feed supplement of nitrogenous source ammoniacal liquor, feed supplement liquid concentration is 0.15g/ml; The growth velocity of on-line monitoring bacterium when the growth velocity curve in the bacterium 12 hours and when growth velocity reduced in preceding 12 hours, adds carbon source methyl alcohol by the fed-batch mode, and the feed supplement amount is that every liter of fermented liquid adds 10g methyl alcohol; Described carbon source feed supplement liquid is specially industrial methanol or analytical pure.Per 12 hour records and calculating cell density (OD
600) and PQQ output.Under this fermentation condition, through 6 days cultivation, final cell density (OD
600) can meet or exceed 10, PQQ output meets or exceeds 1g/L, and the PQQ production intensity was at least 166.67mg/L/ days.
Embodiment 5 carries out PQQ production instance C in the 5L automatic fermenter
With the seed liquor of embodiment 2 and whole parameter controls of the fermenting process among the embodiment 3, change used fermention medium, concrete composition and proportioning are as follows:
MgSO
4·7H
2O 1g，
(NH
4)
2SO
4 5g，
KH
2PO
4 2.1g，
Na
2HPO
4·12H
2O 4.5g，
Ironic citrate 80mg,
CaCl
2 0.1mg，
MnCl
2·4H
2O 5mg，
ZnSO
4·7H
2O 5mg，
CuSO
4·5H
2O 0.5mg，
Folic acid 0.05mg.
Under this fermentation condition, through 6 days cultivation, final cell density (OD
600) can meet or exceed 10, PQQ output meets or exceeds 1.5g/L, and the PQQ production intensity was at least 250mg/L/ days.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the method for a microbial fermentation production pyrroloquinoline quinone is characterized in that, comprises the steps:
(1) activated spawn: original strain methylotrophic bacteria Methylovorus sp.MP688 is inoculated in the solid medium, under the condition that is fit to the original strain growth, cultivates, get the activatory bacterial classification;
(2) culture of seed liquid: the bacterial classification inoculation that step (1) gained is activated is cultivated under the condition that is fit to thalli growth in liquid nutrient medium, seed culture fluid;
(3) ferment tank: step (2) gained seed culture fluid is inoculated in the fermentor tank that contains fermention medium in proportion; Homo(io)thermism in the fermentation culture process, simultaneously, the pH of fermentor tank, dissolved oxygen and mixing speed adopt two stage control strategies; The nitrogenous source feed supplement adopts pH-nitrogenous source joint control mode to carry out, and the carbon source feed supplement adopts the fed-batch mode to carry out;
(4) the results fermented liquid gets pyrroloquinoline quinone.
2. the method for claim 1 is characterized in that, culture temperature is 30～35 ℃ described in the step (1), and incubation time is 2～3 days; Described solid medium is to add agar powder and carbon source methyl alcohol on the basis of minimum medium, the concentration of described agar powder in described film solid media is 1～2g/L, the concentration of described methyl alcohol in described film solid media is 7.5～15g/L, wherein, the component of described minimum medium and proportioning are as follows:
Minimum medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 1.4g，
Na
2HPO
4·12H
2O 3g，
Ironic citrate 10-30mg,
CaCl
2 10-30mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-2mg，
Folic acid 0.02-0.05mg,
Vitamin H 0.02-0.05mg,
Riboflavin 0.02-0.05mg,
Nicotinic acid 0.02-0.05mg,
Thioctic Acid 0.02-0.05mg.
3. the method for claim 1, it is characterized in that, step (2) concrete steps are: the bacterial classification that step (1) gained activates is chosen single bacterium colony, be inoculated into 30～35 ℃ of shaking table vibrations in the 5ml liquid nutrient medium, rotating speed is 200-250 rev/min, cultivated 2～3 days, and got the aforementioned bacterium liquid of 1～2ml then and be seeded to the numerous cultivation of continuation expansion under the same conditions in the triangular flask that contains the 100ml liquid nutrient medium, until obtaining strain cell density (OD
600) be 1.2～1.5 seed liquor; Wherein, described liquid nutrient medium is to add carbon source methyl alcohol on the basis of minimum medium, and the concentration in the described methyl alcohol liquid medium within is 7.5～15g/L.
4. method according to claim 1 is characterized in that, the volume ratio of seed culture fluid and fermention medium is 1:50～1:10 described in the step (3); Described fermention medium, concrete composition and proportioning are as follows:
Fermention medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 2.1-2.8g，
Na
2HPO
4·12H
2O 4.5-6g，
Ironic citrate 30-80mg,
CaCl
2 0.05-0.25mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-1mg，
Folic acid 0.02-0.05mg.
5. method according to claim 1 is characterized in that, leavening temperature is 30～35 ℃ described in the step (3), and fermentation time is 6～8 days.
6. method according to claim 1 is characterized in that, being specially of two stage control strategies described in the step (3):
(1) initial stage: the initial stage air flow that ferments after the inoculation is controlled at 0.02～5vvm, does not control dissolved oxygen, and rotating speed is controlled at 200～300rpm, and after initial pH value was adjusted into 6.5-7.0, the pH value was no longer controlled;
(2) later stage: along with bacterial growth, the pH value descends, and treats that the pH value drops at 5.5～6.0 o'clock, adds ammoniacal liquor or ammonium sulfate is controlled at this level with pH by the pump feedback flow;
Treat that dissolved oxygen in the fermentor tank drops to 30% o'clock of saturated dissolved oxygen, by the dissolved oxygen strategy related dissolved oxygen is controlled at 30%～50% level with rotating speed and air flow, be specially: preferentially adjust rotating speed, described speed range is 200～800rpm, when rotating speed reaches upper limit 800rpm, adjust air flow, described air flow scope is 0.2～5vvm.
7. method according to claim 1 is characterized in that, carbon source is a methyl alcohol described in the step (3), and described nitrogenous source is one or both in ammonium sulfate, the ammoniacal liquor; Described carbon source starting point concentration is 7.5～15g/L, nitrogenous source starting point concentration 7.5～15g/L; The total concn of carbon source is controlled in the 15g/L in the fermenting process, and the total concn of ammonium sulfate is controlled in the 3g/L;
Described pH-nitrogenous source joint control mode is for starting nitrogenous source feed supplement program when fermented liquid pH value drops to 6.0 along with bacterial growth, carry out the nitrogenous source feed supplement by fed-batch mode, carry out the nitrogenous source feed supplement by flow feeding liquid, described nitrogenous source feed supplement liquid is ammoniacal liquor or the ammonium sulfate of 0.10-0.25g/ml;
Growth velocity of on-line monitoring bacterium and nitrogenous source feed supplement discharge curve in case growth velocity of bacterium or nitrogenous source feed rate had been compared reduction with a last time period, add carbon source methyl alcohol by the fed-batch mode, and the feed supplement amount is that every liter of fermented liquid adds 5～15g methyl alcohol; Described methyl alcohol is industrial methanol or analytical pure.
8. a pyrroloquinoline quinone is characterized in that, it is to be prepared by any described method production of claim 1～7.
9. a fermention medium is characterized in that, the concrete composition and the proportioning of this substratum are as follows:
Fermention medium (in 1L)
MgSO
4·7H
2O 0.4-1g，
(NH
4)
2SO
4 3-5g，
KH
2PO
4 2.1-2.8g，
Na
2HPO
4·12H
2O 4.5-6g，
Ironic citrate 30-80mg,
CaCl
2 0.05-0.25mg，
MnCl
2·4H
2O 5-10mg，
ZnSO
4·7H
2O 5-10mg，
CuSO
4·5H
2O 0.5-1mg，
Folic acid 0.02-0.05mg.
10. the application of the described substratum of claim 9 in producing the preparation pyrroloquinoline quinone.
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| CN109628509A (en) * | 2018-11-22 | 2019-04-16 | 北大方正集团有限公司 | The method of semicontinuous fermentation process production pyrroloquinoline quinone |
| CN110195032A (en) * | 2019-05-28 | 2019-09-03 | 浙江工商大学 | Hyphomicrobium LXL-PQ-409 and its application |
| CN111303248A (en) * | 2020-03-02 | 2020-06-19 | 丽珠集团福州福兴医药有限公司 | Material supplementing method for improving teicoplanin fermentation yield |
| CN112143683A (en) * | 2020-09-30 | 2020-12-29 | 安徽新熙盟生物科技有限公司 | Culture medium and method for improving vitamin PQQ conversion rate by using same |
| CN113337432A (en) * | 2021-06-04 | 2021-09-03 | 华熙生物科技股份有限公司 | Methylophagus for producing pyrroloquinoline quinone and application thereof |
| CN113913448A (en) * | 2021-07-23 | 2022-01-11 | 中国人民解放军军事科学院军事医学研究院 | Method for increasing yield of methylotrophic bacteria pyrroloquinoline quinone and application |
| CN113981021A (en) * | 2021-05-21 | 2022-01-28 | 江苏一鸣生物科技有限公司 | Method for synthesizing PQQ through fermentation of paracoccus ammoniaphila |
| CN114134187A (en) * | 2021-11-19 | 2022-03-04 | 江西诚志生物工程有限公司 | Process for producing pyrroloquinoline quinone disodium salt by fermentation method |
| CN115747275A (en) * | 2022-07-29 | 2023-03-07 | 郑州尼采生物科技有限公司 | Method for increasing yield of pyrroloquinoline quinone fermented by methylobacterium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109628509A (en) * | 2018-11-22 | 2019-04-16 | 北大方正集团有限公司 | The method of semicontinuous fermentation process production pyrroloquinoline quinone |
| CN109628509B (en) * | 2018-11-22 | 2022-05-10 | 北大方正集团有限公司 | Method for producing pyrroloquinoline quinone by semi-continuous fermentation process |
| CN110195032A (en) * | 2019-05-28 | 2019-09-03 | 浙江工商大学 | Hyphomicrobium LXL-PQ-409 and its application |
| CN111303248B (en) * | 2020-03-02 | 2022-04-12 | 丽珠集团福州福兴医药有限公司 | Material supplementing method for improving teicoplanin fermentation yield |
| CN111303248A (en) * | 2020-03-02 | 2020-06-19 | 丽珠集团福州福兴医药有限公司 | Material supplementing method for improving teicoplanin fermentation yield |
| CN112143683A (en) * | 2020-09-30 | 2020-12-29 | 安徽新熙盟生物科技有限公司 | Culture medium and method for improving vitamin PQQ conversion rate by using same |
| CN112143683B (en) * | 2020-09-30 | 2023-09-29 | 安徽新熙盟生物科技有限公司 | Culture medium and method for improving conversion rate of vitamin PQQ by using culture medium |
| CN113981021A (en) * | 2021-05-21 | 2022-01-28 | 江苏一鸣生物科技有限公司 | Method for synthesizing PQQ through fermentation of paracoccus ammoniaphila |
| CN113981021B (en) * | 2021-05-21 | 2023-09-26 | 江苏一鸣生物科技有限公司 | Method for synthesizing PQQ through paracoccus ammoniaphiles fermentation |
| CN113337432A (en) * | 2021-06-04 | 2021-09-03 | 华熙生物科技股份有限公司 | Methylophagus for producing pyrroloquinoline quinone and application thereof |
| CN113913448A (en) * | 2021-07-23 | 2022-01-11 | 中国人民解放军军事科学院军事医学研究院 | Method for increasing yield of methylotrophic bacteria pyrroloquinoline quinone and application |
| CN113913448B (en) * | 2021-07-23 | 2023-10-20 | 中国人民解放军军事科学院军事医学研究院 | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application |
| CN114134187A (en) * | 2021-11-19 | 2022-03-04 | 江西诚志生物工程有限公司 | Process for producing pyrroloquinoline quinone disodium salt by fermentation method |
| CN115747275A (en) * | 2022-07-29 | 2023-03-07 | 郑州尼采生物科技有限公司 | Method for increasing yield of pyrroloquinoline quinone fermented by methylobacterium |
| CN115747275B (en) * | 2022-07-29 | 2023-11-28 | 郑州尼采生物科技有限公司 | Method for improving yield of pyrroloquinoline quinone by fermenting with bacillus methylobacterium |
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