CN108949846A - A kind of method that high density fermentation improves PQQ yield - Google Patents
A kind of method that high density fermentation improves PQQ yield Download PDFInfo
- Publication number
- CN108949846A CN108949846A CN201810866397.0A CN201810866397A CN108949846A CN 108949846 A CN108949846 A CN 108949846A CN 201810866397 A CN201810866397 A CN 201810866397A CN 108949846 A CN108949846 A CN 108949846A
- Authority
- CN
- China
- Prior art keywords
- tank
- fermentation
- pqq
- high density
- fermentor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of methods that high density fermentation improves PQQ yield, are applied to PQQ industrialized production using high cell density fermentation technology and obtain the fermentation liquid of the PQQ of high-content by seed tank culture and fermentation tank culture.It has the advantage that: the method that high density fermentation of the invention improves PQQ yield, greatly improved PQQ metabolite content and shorten fermentation period, reduces production cost and improve production overall efficiency and benefit, improve PQQ yield；By the proportion for optimizing compost, have adjusted feed supplement formula, tank pressure, revolving speed has been turned up, ventilation quantity increases reaction rate, it is ensured that oxygen dissolving value, shorten the methods of fermentation period, so that obtained fermentation strain cell density significantly improves as 25-35g/L, the average fermentation period is 5-6 days, and the fermentation level of average PQQ is 2.0-2.5g/L.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of method that high density fermentation improves PQQ yield.
Background technique
The scientific name of PQQ is a kind of new prothetic group pyrroloquinoline quinone, has treatment heart disease, and neurogenic disease protects liver
It is dirty, the effects of maintaining functions of mitochondria.
In prokaryotes, plant and mammal, it is all widely present pyrroloquinoline quinone, it is not only the auxiliary of many enzymes
Base is responsible for the function of transmitting electronics, proton and chemical group in enzymatic reaction, can also stimulate the growth of microorganism, plant
The sprouting of pollen promotes the growth of plant.
The biological function of PQQ mainly has: stimulation microorganism, plant, animal and human body cell fast-growth；Remove body
Interior extra free radical；Nerve growth factor is stimulated to generate；There is protective effect etc. to cardiac muscle of mammal cell.Its effect:
The important growth factor of body；Body Free Radical Level is adjusted, body is protected；Anti-oxidative damage；Enhance mitochondrial function；Solution
Poison；Skin aging is prevented, has attenuation to melanin pigmentation disorder；Prevent and treat Parkinson's disease；Senile dementia prevention and cure etc..
The industrialization of PQQ, scale it is still necessary to want the regular hour, but its food, light industry, agricultural and in terms of all
It has broad application prospects.As PQQ is listed in a kind of novel vitamin, the relevant research of function becomes major related institute
The research hotspot in school, the huge applications prospect in terms of medicine, food and agricultural cause pursuing for personages of various circles of society.Future
Market conservative estimation is on 100,000,000,000 yuan.Currently, PQQ is used for major scientific research institutions, industry mainly as test reagent at home
Be mainly obtained by chemical synthesis and the microorganism normal fermentation method of PQQ product are made, and there are high production cost, yields
It is low, the problems such as comprehensive production efficiency is low, causes the price of current PQQ high, although wide market, still fail extensive
Promotion and application.
High cell density fermentation (high cell density fermentation, HCDF) is nearest ten or twenty year development
Get up, Fungal biodiversity and target product ratio are improved by application certain culture technique and equipment, obtains hypermetabolism production
The novel fermentation technology of object.
High cell density fermentation reports that the field of application has at present: first is that utilizing works bacterium produces various interferon, growth
The factor, biological enzyme, peptides, polysaccharide isoreactivity ingredient；Second is that application high cell density fermentation technology in the food industry is used for
The culture of living cells；Third is that for saccharomycete production alcohol and utilizing spirulina cells production spirulina etc..Due to being limited to send out
Ferment technology and equipment not yet has the report in terms of PQQ industry mass production at present.
Summary of the invention
Of the existing technology in order to solve the problems, such as, the present invention provides the sides that a kind of high density fermentation improves PQQ yield
Method, which shorten fermentation periods, improve PQQ yield.
The object of the present invention is to provide a kind of methods that high density fermentation improves PQQ yield.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, the high density fermentation
Improve PQQ yield method the following steps are included:
(1) actication of culture: Hyphomicrobium strain is accessed into slant medium, mature slant strains are obtained after culture；
(2) plant the preparation of mother liquor: the mature slant strains for taking step (1) to prepare are inoculated in seed culture medium, shaking table training
It supports, obtains kind of a mother liquor；
(3) seed tank culture: 1.5-2m is used3The 50-60% of pure water to seed tank volume is added in seeding tank, calculates
All seed tank culture material in addition to anhydrous methanol are added in required seed tank culture material while stirring, are completely dissolved and divide to raw material
It with the 60-65% of pure water constant volume to seed tank volume after dissipating uniformly, is uniformly mixed, adjusts pH to 7.0, it is cold after seeding tank sterilizing
But to 25-35 DEG C, the anhydrous methanol through being sterile filtered is added, is uniformly mixed, kind mother liquor access kind obtained in step (2)
In sub- tank, control tank presses 0.08-0.10MPa, and 30-35 DEG C of tank temperature, air mass flow 0.8-1.0VVM, pH 6.8-7.0, ventilation is stirred
Culture 42-50 hours is mixed, seed tank culture bacterium solution is formed；
Every 1.5m3Seeding tank be added the seed tank culture material include following weight raw material:
(4) fermentor initial formulation: 20m is used3The 30-40% of pure water to fermenter volume is added in the above fermentor,
Fermentation tank culture material is added while stirring, with pure water constant volume to fermenter volume after raw material is completely dissolved and is uniformly dispersed
40-50% is uniformly mixed, and is adjusted pH to 7.0, is cooled to 25-35 DEG C after fermentor sterilizing, by 10-20% inoculative proportion, uses
The seed tank culture bacterium solution that filtrated air obtains step (3) is pressed into fermentor from seeding tank, controls fermentor initial pressure
0.08-0.10MPa, initial flow 0.8-1VVM, initial mixing speed 110-130rpm/min, 30 DEG C of initial temperature；From seed
It is brought into tank in fermentor not using complete anhydrous methanol as the initial concentration of methanol in fermentor；
Every 20m3The fermentation tank culture material that fermentor is added includes the raw material of following weight:
(5) fermented and cultured and feed supplement: before fermentation, pure water and glycerol are added in feed supplement tank, is added after sterilizing through sterile
The anhydrous methanol of filtering is uniformly mixed and forms feed supplement；In fermentation process, when the concentration of methanol in fermentor drops to 1000-
When 1500ppm, start feed supplement, the concentration that methanol and glycerol are total in fermentor is made to remain 1000-1500ppm, side feed supplement
Side fermented and cultured 5-6 days obtains the high-content fermentation liquid of PPQ.
PPG2000 is the common polyethers defoaming agent of biological fermentation industry, has good high-temperature stability and defoaming energy
Power, to biological hypha nontoxicity.
Fossil water refers to and is added to minerals based food additive on the basis of pure water and manufactured.In the present invention
Fossil water raw material are as follows:
Preparation method is, by said sequence, under agitation, sequentially adds into the ingredient beaker equipped with pure water
To being completely dissolved, all dissolutions and after mixing are filtered rear spare noon raw material with 0.22 μm of sterilised membrane filter.
Seeding tank culture transferring condition: for microscopy without miscellaneous bacteria, thallus is full, and dyeing is deep, value >=4.0 OD.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (3)
In step (4), it is all made of 25% ammonium hydroxide after being sterile filtered and adjusts pH.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (3),
The seeding tank sterilizing is specially to sterilize when total steam pressure is greater than 0.3MPa into vapour reality tank, controls temperature 121-125
DEG C, pressure 0.10-0.12MPa, time 35-50min, volume is the 60-80% of seed tank volume after cooling.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (4),
The fermentor sterilizing is specially to be greater than in the case of 0.4MPa to sterilize into vapour reality tank using total steam pressure, controls temperature 121-
125 DEG C, pressure 0.10-0.12MPa, time 30-45min.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (5),
In fermentation process, cultivation temperature is 32-34 DEG C, and adjusting pH is 6.5-7.0, and control tank pressure is 0.08-0.20MPa, speed of agitator
For 110-200rpm/min, controls dissolved oxygen and be not less than 30%.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield further adjusts pH
Specifically: (the 1 to 2nd day) adjusting pH is 6.8 during logarithmic growth, and producing (the 3-6 days) anti-phase adjusting pH is 7.0.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (5),
The volume ratio of anhydrous methanol and glycerol is 18-20:5-3 in feed supplement tank.The total concentration of methanol and glycerol is always 40- in feed supplement tank
60%.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (5),
In fermentation process, feed rate is 30-50 liter per hour, should specifically be kept according to the concentration of methanol and glycerol in control fermentor
In standard feed supplement into fermentor of 1000-1500PPM.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (5),
In fermentation process, defoaming agent is added, liquid level foam is controlled, prevents from escaping liquid.The defoaming agent is PPG2000, PPG2000
Concentration be 20%, every 20m31.5-5.0kg is added in fermentor.
The method that the high density fermentation of specific embodiment according to the present invention improves PQQ yield, wherein in step (5),
In fermentation process, oxygen dissolving value is controlled specifically: turn by adjusting tank pressure, ventilation quantity, the oxygen content being passed through in air and stirring
Speed, come oxygen dissolving value in the whole process that ensures to ferment not less than 30%.General oxygen dissolving value is 30-40%, and oxygen content is in fermentation whole process
25-35%.The ventilation quantity for adjusting fermentor is 1:1.0-2.0, is passed into fermentor after pure oxygen sterilizing is added in air,
The oxygen content being passed through in air is set to reach 25-35%；Revolving speed is 110-200rpm/min.
PQQ yield is improved in order to implement high density fermentation, the present invention also adjusts Zymolysis Equipment, and scheme is as follows:
(1) increase stirring motor: the stirring motor in fermentor is changed to 55KW by original 35KW, to guarantee to stir function
Rate；
(2) increase paddle size: former paddle diameter is 1:3.2-3.4 with tank diameter ratio, is now changed to 1:2.5-
2.8, it ensure that power of agitator, stirring intensity and dissolved oxygen rate；
(3) increase the heat exchange area of fermentor: primary fermentation tank heat exchange area is 1.0-1.2:1 with fermenter volume ratio,
Be changed to 1.5-2.0:1, increase heat exchange area, ensure that cooling water in time in fermentor biological metabolism heat and machinery stir
It mixes heat to take away rapidly, meets requirement of the zymotechnique to temperature；
(4) tank bottom has installed air and liquid mixer additional, increases fermentor dissolved oxygen efficiency；
(5) fermentor has installed the data sampling sensors such as temperature, speed of agitator, defoaming, DO, PH, exhaust and control system additional
System accurately calculates the indirect parameters such as online OUR, CER, RQ, KLa, bacterium be dense.Living cells sensor, electronics are smelt simultaneously, same to position
Plain C13The technologies such as metabolic flux analysis are applied to fermentor.
The invention has the benefit that the method that high density fermentation of the invention improves PQQ yield, creative using is high
Cell density fermentation technology is applied to PQQ industrialized production, and PQQ metabolite content greatly improved and shorten fermentation period,
It reduces production cost and improves production overall efficiency and benefit, improve PQQ yield, be widely popularized PQQ and benefit the society
It is possibly realized.
The method that high density fermentation of the invention improves PQQ yield, optimizes the proportion of compost, provides abundance for strain
Nutrition so that Hyphomicrobium grows fine, vitality is vigorous, and cell density increases, mycelia metabolic capability greatly enhances；
Have adjusted feed supplement formula, joined anhydrous methanol and glycerol, and have adjusted proportion so that formula in carbon-nitrogen ratio more
Rationally, control mycelia own growth and product catabolism rate are more advantageous to；
Tank pressure, revolving speed, ventilation quantity and the oxygen content for being passed through filtrated air has been turned up, ensure that high density production mycelia to oxygen
The demand of gas improves the yield of PQQ, shortens fermentation period；
Implement high density fermentation of the invention improve PQQ yield method post-fermentation strain cell density significantly improve for
25-35g/L, average fermentation period are only 5-6 days, and the fermentation level of average PQQ is 2.0-2.5g/L, realize and are greatly shortened
Fermentation period and PQQ production cost is reduced, improve PQQ comprehensive production efficiency, is conducive to subsequent extracted and refine, reduce waste water
Discharge, be good for the environment and other effects.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiment belongs to the range that the present invention is protected.
Embodiment 1
Present embodiments provide a kind of method that high density fermentation improves PQQ yield, comprising the following steps:
(1) actication of culture: accessing slant medium for Hyphomicrobium strain, and 30 DEG C are cultivated 5 days, and mature inclined-plane bacterium is obtained
Kind；
(2) plant the preparation of mother liquor: the mature slant strains for taking step (1) to prepare are inoculated in seed culture medium, and 31 DEG C are shaken
Bed culture 30 hours, obtains kind of a mother liquor；
(3) seed tank culture: 1.5m is used3Pure water is added to 1.0m in seeding tank3, calculated by the 80% of seed tank volume
All composts in addition to anhydrous methanol are added in required compost while stirring, with pure after raw material is completely dissolved and is uniformly dispersed
1.2m of the water purification constant volume to seed tank volume3, it is uniformly mixed, adjusts pH to 7.0, be cooled to 30 DEG C after seeding tank sterilizing, be added
Anhydrous methanol through being sterile filtered is uniformly mixed, and by 0.1% inoculative proportion, access kind mother liquor, control tank presses 0.10MPa, tank temperature
It 30 DEG C, air mass flow 0.9VVM, pH 7.0, divulges information stir culture 45 hours, forms seed tank culture bacterium solution；Seeding tank culture transferring
Condition: for microscopy without miscellaneous bacteria, thallus is full, and dyeing is deep, and OD value is 4.2；
Compost includes following raw material:
(4) fermentor initial formulation: 20m is used3Pure water is added to 7m in fermentor3, compost is added while stirring, to
Raw material be completely dissolved and be uniformly dispersed after with pure water constant volume arrive fermenter volume 40-50%, be uniformly mixed, adjusting pH to
7.0, it is cooled to 25-35 DEG C after fermentor sterilizing, by 10% inoculative proportion, the seeding tank that step (3) is obtained with filtrated air
It cultivates bacterium solution and is pressed into fermentor from seeding tank, control fermentor initial pressure 0.08-0.10MPa, initial flow 0.8-1VVM,
Initial mixing speed 110-130rpm/min, 30 DEG C of initial temperature；The culture in culture material formula and seeding tank in fermentor
Material formula is not identical；Fermentation tank culture material are as follows:
(5) fermented and cultured and feed supplement: being added pure water and glycerol 300kg in feed supplement tank, is added after sterilizing through sterile mistake
The anhydrous methanol 2000kg of filter, pure water constant volume to 4500L are uniformly mixed and form feed supplement, are then 1500ppm to methanol concentration
When start feed supplement, the feed supplement is slowly added to fermentor, makes methanol and the glycerol melting concn in fermentor be always
1500ppm, fermented and cultured 5.5 days in feed supplement obtain the high-content fermentation liquid of PPQ.
Embodiment 2
Present embodiments provide a kind of method that high density fermentation improves PQQ yield, comprising the following steps:
(1) actication of culture: accessing slant medium for Hyphomicrobium strain, and 32 DEG C are cultivated 4 days, and mature inclined-plane bacterium is obtained
Kind；
(2) plant the preparation of mother liquor: the mature slant strains for taking step (1) to prepare are inoculated in seed culture medium, and 30 DEG C are shaken
Bed culture 30 hours, obtains kind of a mother liquor；
(3) seed tank culture: 1.5m is used3Pure water is added to 1.0m in seeding tank3, it is added in addition to methanol while stirring
All composts are uniformly mixed with pure water constant volume to the 80% of seed tank volume after raw material is completely dissolved and is uniformly dispersed,
PH to 7.0, seeding tank sterilizing, when total steam pressure is greater than 0.3MPa into vapour are adjusted with 25% ammonium hydroxide after aseptic filtration
Real tank sterilizing, controls 125 DEG C of temperature, pressure 0.10MPa, time 35min, and volume is seed tank volume after being cooled to 25 DEG C
1.4m3, the anhydrous methanol through being sterile filtered is added, is uniformly mixed, by 0.15% inoculative proportion, access kind mother liquor controls tank pressure
0.10MPa 30 DEG C of tank temperature, air mass flow 1.0VVM, pH 7.0, divulges information stir culture 50 hours, forms seed tank culture bacterium
Liquid；Seeding tank culture transferring condition: for microscopy without miscellaneous bacteria, thallus is full, and dyeing is deep, and OD value is 4.0；
The compost includes the raw material of following weight:
(4) fermentor initial formulation: 20m is used3Pure water is added to 7m in fermentor3, compost is added while stirring, to
Raw material uses pure water constant volume to the 50% of fermenter volume after being completely dissolved and being uniformly dispersed, be uniformly mixed, after aseptic filtration
25% ammonium hydroxide adjust pH to 7.0, fermentor sterilizing, using total steam pressure be greater than 0.4MPa in the case of into vapour reality tank sterilize,
Control 125 DEG C of temperature, pressure 0.12MPa, time 45min, after be cooled to 35 DEG C；
The culture material formula in culture material formula and seeding tank in fermentor is not identical；Fermentation tank culture material are as follows:
First by the sterilizing of seed transferring pipeline, the sterilizing of culture transferring pipeline, sterilizing are carried out when total steam pressure is greater than 0.3MPa
Pressure 0.30MPa, sterilization time 60min；By 20% inoculative proportion, the seed tank culture that step (3) is obtained with filtrated air
Bacterium solution is pressed into fermentor from seeding tank, controls fermentor initial pressure 0.10MPa, initial flow 1VVM, initial mixing speed
130rpm/min, 30 DEG C of initial temperature；
(5) fermented and cultured and feed supplement: being added pure water and glycerol 300kg in feed supplement tank, is added after sterilizing through sterile mistake
The anhydrous methanol 1800kg of filter is uniformly mixed constant volume to 4500L and forms feed supplement, then starts when methanol concentration is 1000ppm
The feed supplement is slowly added to fermentor by feed supplement, fermented and cultured 5 days in feed supplement, and feed rate is 30-50 liter per hour, is made
Methanol and glycerol mixing remain that concentration is 1000ppm in fermentor, and control dissolved oxygen is 30%, control in fermentation process, training
Supporting temperature is 34 DEG C, adjusts pH, wherein it is 6.8 that pH is adjusted during logarithmic growth, and producing anti-phase to adjust pH be 7.0 is 7.0, control
Tank pressure is 0.20MPa, speed of agitator 110rpm/min, also needs in fermentation process that the PPG2000 that concentration is 20% is added, adds
Dosage is 2.0kg as defoaming agent, is controlled liquid level foam, prevents from running liquid, obtains the high-content fermentation liquid of PPQ.Control
Dissolved oxygen maintains dissolved oxygen in fermentation whole process to be not less than 30% specifically by tank pressure, ventilation quantity and speed of agitator is adjusted.Fermentation is whole
Interior oxygen content is 35%, ventilation quantity 1:2.0, and pure oxygen is added in ventilation opening, makes the content of oxygen in air for being passed through fermentor
Reach 35%.
Embodiment 3
Present embodiments provide a kind of method that high density fermentation improves PQQ yield, comprising the following steps: (1) strain is living
Change: Hyphomicrobium strain is accessed into slant medium, 28 DEG C are cultivated 6 days, and mature slant strains are obtained；
(2) plant the preparation of mother liquor: the mature slant strains for taking step (1) to prepare are inoculated in seed culture medium, and 32 DEG C are shaken
Bed culture 24 hours, obtains kind of a mother liquor；
(3) seed tank culture: using 1.5 seeding tanks, and pure water is added to 0.9m3, calculated by the 80% of seed tank volume
All composts in addition to methanol are added in required compost while stirring, and pure water is used after raw material is completely dissolved and is uniformly dispersed
Constant volume is uniformly mixed to the 80% of seed tank volume, adjusts pH to 7.0 with 25% ammonium hydroxide after aseptic filtration, and seeding tank sterilizes,
It sterilizes when total steam pressure is greater than 0.3MPa into vapour reality tank, controls 121 DEG C of temperature, pressure 0.12MPa, the time
50min, volume is the 1.45m of seed tank volume after being cooled to 35 DEG C3, the anhydrous methanol through being sterile filtered is added, is uniformly mixed,
By 0.2% inoculative proportion, access kind mother liquor, control tank presses 0.08MPa, 30 DEG C of tank temperature, air mass flow 0.8VVM, pH 7.0, leads to
Wind stir culture 42 hours, form seed tank culture bacterium solution；Seeding tank culture transferring condition: for microscopy without miscellaneous bacteria, thallus is full, dyeing
Deep, OD value is 4.0；
The compost includes the raw material of following weight:
(4) fermentor initial formulation: 20m is used3Pure water is added to 7m in the above fermentor3, by fermenter volume
80% calculates required compost, and compost is added while stirring, pure water constant volume is used after raw material is completely dissolved and is uniformly dispersed
It to the 40% of fermenter volume, is uniformly mixed, adjusts pH to 7.0 with 25% ammonium hydroxide after aseptic filtration, fermentor sterilizing uses
Total steam pressure sterilizes in the case of being greater than 0.4MPa into vapour reality tank, controls 121 DEG C of temperature, pressure 0.10MPa, time 30min, after
It is cooled to 25 DEG C；
The culture material formula in culture material formula and seeding tank in fermentor is not identical；Compost in fermentor are as follows:
First by the sterilizing of seed transferring pipeline, the sterilizing of culture transferring pipeline, sterilizing are carried out when total steam pressure is greater than 0.3MPa
Pressure 0.2MPa, sterilization time 60min；By 15% inoculative proportion, the seed tank culture bacterium that step (3) is obtained with filtrated air
Liquid is pressed into fermentor from seeding tank, controls fermentor initial pressure 0.08MPa, initial flow 0.8VVM, initial mixing speed
110rpm/min, 30 DEG C of initial temperature；Culture material formula in fermentor is identical as the culture material formula in seeding tank；
(5) fermented and cultured and feed supplement: being added pure water and glycerol 500kg in feed supplement tank, is added after sterilizing through sterile mistake
The anhydrous methanol 1800kg of filter is uniformly mixed constant volume and opens then when methanol concentration drops to 1200ppm to 4500L formation feed supplement
The feed supplement is flowed slowly into fermentor by beginning feed supplement, and making methanol and glycerol melting concn in fermentor is always 1200ppm, side
Feed supplement side fermented and cultured 6 days, feed rate are 30-50 liter per hour, are adjusted according to DO value, need the control DO value to be after feed supplement
Between 30-40%, in fermentation process, cultivation temperature is 32 DEG C, adjusts pH, wherein it is 6.8 that pH is adjusted during logarithmic growth, is produced
It is 7.0 that the anti-phase, which adjusts pH, and control tank pressure is 0.08MPa, speed of agitator 200rpm/min, and control dissolved oxygen is not less than 30%, hair
It also needs that the PPG2000 that concentration is 20% is added during ferment, additive amount is 1.2kg as defoaming agent, controls liquid level foam
System prevents from running liquid, obtains the high-content fermentation liquid of PPQ.It controls dissolved oxygen and turns specifically by tank pressure, ventilation quantity and stirring is adjusted
Speed maintains dissolved oxygen in fermentation whole process to be not less than 30%.Oxygen content is 25%, ventilation quantity 1:1.0 in fermentation whole process, and is divulged information
Mouth is passed through after pure oxygen sterilizing is added, and the oxygen content being passed through in fermentor is made to reach 25%.
PQQ yield is improved in order to implement high density fermentation, the present invention also adjusts Zymolysis Equipment, and scheme is as follows:
(1) increase stirring motor: the stirring motor in fermentor is changed to 55KW by original 35KW, to guarantee to stir function
Rate；
(2) increase paddle size: former paddle diameter is 1:3.2-3.4 with tank diameter ratio, is now changed to 1:2.5-
2.8, it ensure that power of agitator, stirring intensity and dissolved oxygen rate；
(3) increase the heat exchange area of fermentor: primary fermentation tank heat exchange area is 1.0-1.2:1 with fermenter volume ratio,
Be changed to 1.5-2.0:1, increase heat exchange area, ensure that cooling water in time in fermentor biological metabolism heat and machinery stir
It mixes heat to take away rapidly, meets requirement of the zymotechnique to temperature；
(4) tank bottom has installed air and liquid mixer additional, increases fermentor dissolved oxygen efficiency；
(5) fermentor has installed the data sampling sensors such as temperature, speed of agitator, defoaming, DO, PH, exhaust and control system additional
System accurately calculates the indirect parameters such as online OUR, CER, RQ, KLa, bacterium be dense.Living cells sensor, electronics are smelt simultaneously, same to position
Plain C13The technologies such as metabolic flux analysis are applied to fermentor.
High density fermentation of the invention improves the method for PQQ yield compared with common fermentation processes:
(1) normal fermentation
Fermentation period: 8-12 days general
Cell density: generally 5g-10g/L
Fermentation level: generally in 0.5-1.0g/L, individually in 1.0-1.2g/L, experimental level 2.0g/L, but work are had been reported that
Industry mass production, which has no, to be had been reported that.
(2) high density fermentation of the invention
Fermentation period: 5-6 days general
Cell density: generally 25-35g/L
Fermentation level: 2.0-2.5g/L
It is 25-35g/L that high density fermentation of the invention, which implements post-fermentation strain cell density, and the average fermentation period is 5-6
It, the fermentation level of average PQQ is 2.0-2.5g/L, it realizes and fermentation period is greatly shortened and reduces PQQ production cost,
Improve PQQ comprehensive production efficiency.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of method that high density fermentation improves PQQ yield, which is characterized in that the high density fermentation improves PQQ yield
Method the following steps are included:
(1) actication of culture: Hyphomicrobium strain is accessed into slant medium, mature slant strains are obtained after culture；
(2) plant the preparation of mother liquor: the mature slant strains for taking step (1) to prepare are inoculated in seed culture medium, and shaking table culture obtains
To kind of a mother liquor；
(3) seed tank culture: 1.5-2m is used3The 50-60% of pure water to seed tank volume is added in seeding tank, calculates required kind
All seed tank culture material in addition to anhydrous methanol are added in sub- tank compost while stirring, are completely dissolved and are uniformly dispersed to raw material
It afterwards with the 60-65% of pure water constant volume to seed tank volume, is uniformly mixed, adjusts pH to 7.0, be cooled to after seeding tank sterilizing
25-35 DEG C, the anhydrous methanol through being sterile filtered is added, is uniformly mixed, kind mother liquor obtained in step (2) is accessed seeding tank
Interior, control tank presses 0.08-0.10MPa, and 30-35 DEG C of tank temperature, air mass flow 0.8-1.0VVM, pH 6.8-7.0, ventilation stirring is trained
It supports 42-50 hours, forms seed tank culture bacterium solution；
Every 1.5m3Seeding tank be added the seed tank culture material include following weight raw material:
(4) fermentor initial formulation: 20m is used3The 30-40% of pure water to fermenter volume is added, while stirring in the above fermentor
It mixes side and fermentation tank culture material is added, with the 40- of pure water constant volume to fermenter volume after raw material is completely dissolved and is uniformly dispersed
50%, it is uniformly mixed, adjusts pH to 7.0, be cooled to 25-35 DEG C after fermentor sterilizing, by 10-20% inoculative proportion, use is sterile
The seed tank culture bacterium solution that air obtains step (3) is pressed into fermentor from seeding tank, controls fermentor initial pressure 0.08-
0.10MPa, initial flow 0.8-1VVM, initial mixing speed 110-130rpm/min, 30 DEG C of initial temperature；
Every 20m3The fermentation tank culture material that fermentor is added includes the raw material of following weight:
(5) fermented and cultured and feed supplement: before fermentation, pure water and glycerol are added in feed supplement tank, is added after sterilizing through being sterile filtered
Anhydrous methanol, be uniformly mixed form feed supplement；In fermentation process, when the concentration of methanol in fermentor drops to 1000-1500ppm
When, start feed supplement, so that the concentration that methanol and glycerol are total in fermentor is remained 1000-1500ppm, training of fermenting in feed supplement
It supports 5-6 days, obtains the high-content fermentation liquid of PPQ.
2. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that step (3) neutralizes step
Suddenly it in (4), is all made of 25% ammonium hydroxide after being sterile filtered and adjusts pH.
3. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (3), institute
Stating seeding tank sterilizing is specially to sterilize when total steam pressure is greater than 0.3MPa into vapour reality tank, controls 121-125 DEG C of temperature,
Pressure 0.10-0.12MPa, time 35-50min.
4. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (4), institute
Stating fermentor sterilizing is specially to be greater than in the case of 0.4MPa to sterilize into vapour reality tank using total steam pressure, controls temperature 121-125
DEG C, pressure 0.10-0.12MPa, time 30-45min.
5. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (5), hair
During ferment, cultivation temperature is 32-34 DEG C, and adjusting pH is 6.5-7.0, and control tank pressure is 0.08-0.20MPa, and speed of agitator is
110-200rpm/min, control oxygen dissolving value are not less than 30%.
6. the method that high density fermentation according to claim 5 improves PQQ yield, which is characterized in that adjust pH specifically:
It is 6.8 that pH is adjusted during logarithmic growth, and producing anti-phase adjusting pH is 7.0.
7. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (5), mend
The volume ratio of anhydrous methanol and glycerol is 18-20:5-3 in batch can.
8. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (5), hair
During ferment, feed rate is 30-50 liter per hour.
9. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (5), hair
During ferment, defoaming agent is added, liquid level foam is controlled, prevents from running liquid.
10. the method that high density fermentation according to claim 1 improves PQQ yield, which is characterized in that in step (5), hair
During ferment, oxygen dissolving value is controlled specifically: by adjusting tank pressure, ventilation quantity, the oxygen content and speed of agitator being passed through in air,
Ensure that oxygen dissolving value is not less than 30% in fermentation whole process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810866397.0A CN108949846A (en) | 2018-08-01 | 2018-08-01 | A kind of method that high density fermentation improves PQQ yield |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810866397.0A CN108949846A (en) | 2018-08-01 | 2018-08-01 | A kind of method that high density fermentation improves PQQ yield |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108949846A true CN108949846A (en) | 2018-12-07 |
Family
ID=64465687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810866397.0A Pending CN108949846A (en) | 2018-08-01 | 2018-08-01 | A kind of method that high density fermentation improves PQQ yield |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108949846A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823185A (en) * | 2018-06-25 | 2018-11-16 | 安徽新熙盟生物科技有限公司 | The cultural method of high enzyme activity fermentation liquid and the method for extracting acidproof alpha-amylase |
| CN112375792A (en) * | 2020-11-25 | 2021-02-19 | 内蒙古拜克生物有限公司 | Method for producing PQQ by biological method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000015827A2 (en) * | 1998-09-11 | 2000-03-23 | Archer-Daniels-Midland Company | Bacterial strains for the production of 2-keto-l-gulonic acid |
| RU2011108196A (en) * | 2011-03-03 | 2012-09-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR PRODUCING PYRROLOCINOLINOquinone (PQQ) USING THE GENUS BACTERIA Methylobacterium OR Hyphomicrobium |
| CN103740780A (en) * | 2013-12-20 | 2014-04-23 | 浙江大学 | Method for synthesizing pyrroloquinoline quinone through lactobacillus fermentation |
| CN104328155A (en) * | 2014-11-20 | 2015-02-04 | 郑州轻工业学院 | Method and application for producing pyrroloquinoline quinone by using gluconobacter oxydans |
| CN106282044A (en) * | 2015-05-20 | 2017-01-04 | 浙江海正药业股份有限公司 | A kind of Hyphomicrobium and the preparation method of pyrroloquinoline quinone |
| CN107384984A (en) * | 2017-09-05 | 2017-11-24 | 安徽曦萌生物工程有限公司 | A kind of method that fermentation method improves citrin QQ yield |
-
2018
- 2018-08-01 CN CN201810866397.0A patent/CN108949846A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000015827A2 (en) * | 1998-09-11 | 2000-03-23 | Archer-Daniels-Midland Company | Bacterial strains for the production of 2-keto-l-gulonic acid |
| RU2011108196A (en) * | 2011-03-03 | 2012-09-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR PRODUCING PYRROLOCINOLINOquinone (PQQ) USING THE GENUS BACTERIA Methylobacterium OR Hyphomicrobium |
| CN103740780A (en) * | 2013-12-20 | 2014-04-23 | 浙江大学 | Method for synthesizing pyrroloquinoline quinone through lactobacillus fermentation |
| CN104328155A (en) * | 2014-11-20 | 2015-02-04 | 郑州轻工业学院 | Method and application for producing pyrroloquinoline quinone by using gluconobacter oxydans |
| CN106282044A (en) * | 2015-05-20 | 2017-01-04 | 浙江海正药业股份有限公司 | A kind of Hyphomicrobium and the preparation method of pyrroloquinoline quinone |
| CN107384984A (en) * | 2017-09-05 | 2017-11-24 | 安徽曦萌生物工程有限公司 | A kind of method that fermentation method improves citrin QQ yield |
Non-Patent Citations (4)
| Title |
|---|
| TEIZI URAKAMI等: "Production of Pyrroloquinoline Quinone by Using Methanol-Utilizing Bacteria", 《APPL ENVIRON MICROBIOL.》 * |
| 周怡雯等: "新辅酶吡咯喹啉醌研究进展", 《中国生化药物杂志》 * |
| 尹芳等: "甲醇利用型细菌发酵生产吡咯喹啉醌的培养条件", 《华东理工大学学报》 * |
| 黄莉娟: "高细胞密度发酵技术的研究进展", 《安徽农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823185A (en) * | 2018-06-25 | 2018-11-16 | 安徽新熙盟生物科技有限公司 | The cultural method of high enzyme activity fermentation liquid and the method for extracting acidproof alpha-amylase |
| CN112375792A (en) * | 2020-11-25 | 2021-02-19 | 内蒙古拜克生物有限公司 | Method for producing PQQ by biological method |
| CN112375792B (en) * | 2020-11-25 | 2022-05-17 | 内蒙古拜克生物有限公司 | Method for producing PQQ by biological method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102260638B (en) | Pit mud functional bacteria and preparation method thereof | |
| CN106978465B (en) | Fermentation method for improving fermentation yield of total triterpenoids in inonotus obliquus | |
| US10378028B2 (en) | System for hydrogen production under limited aerobic conditions | |
| CN106434830A (en) | Method for improving 2-keto-L-gluconic acid fermentation efficiency | |
| CN108949846A (en) | A kind of method that high density fermentation improves PQQ yield | |
| CN103173507A (en) | Production technology for fermentatively producing sodium hyaluronate by utilizing bacterium | |
| CN110408545A (en) | A kind of purplish red aspergillus kind and the method and product that Hongqu powder (red colouring agent) is prepared by it | |
| CN101407761A (en) | Liquid inocula composed of yeast fused strain, Geotrichum candidum Link and Rhizopus, and preparation and use thereof | |
| CN101195801A (en) | Glossy ganoderma polyoses producing equipment and technique | |
| CN102864082B (en) | Method for culturing citric acid fermenting seeds and method for preparing citric acid by fermenting | |
| CN101451110A (en) | Construction method of genetic engineering bacterial strain capable of producing low diacetyl beer | |
| CN208857315U (en) | A kind of plant cell and organ Liquid Culture bag | |
| CN110438052A (en) | The clostridium butyricum of one plant of high-yield of 1,3-propanediol and a kind of sequence inoculation fermentation technique | |
| CN105420286A (en) | Alcohol production method | |
| CN101659970B (en) | Method for circularly treating avermectins waste ferment water and pleurin waste ferment water | |
| CN107699595A (en) | A kind of method of Production by Microorganism Fermentation L hydroxyprolines | |
| CN107988294A (en) | Adjust the zymotechnique that temperature improves recombination human source collagen production level | |
| CN104277977B (en) | A kind of preparation method of aspergillus niger spore suspension and the storage method of aspergillus niger spore | |
| CN107988293A (en) | Adjust the zymotechnique that pressure improves recombination human source collagen production level | |
| CN110129205A (en) | A kind of Pleurotus nebrodensis liquid spawn culture medium and Pleurotus nebrodensis liquid spawn preparation method | |
| CN109321624A (en) | Enzymolysis process for preparing active collagen peptide from fresh pigskin | |
| CN108048511A (en) | Adjust the zymotechnique that pH promotes recombination human source collagen production level | |
| CN101953267A (en) | Method for culturing hepialus armoricanus oberthur cordyceps sinensis | |
| CN112956372B (en) | Method for cultivating lucid ganoderma | |
| CN102154129A (en) | Rhodosporidium paludigenum for degrading gossypol and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181207 |
|
| RJ01 | Rejection of invention patent application after publication |