CN105861410B - A method of improving Miyarisan growth efficiency - Google Patents
A method of improving Miyarisan growth efficiency Download PDFInfo
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Abstract
The invention discloses a kind of methods for improving Miyarisan growth efficiency, belong to field of food fermentation.The present invention is a kind of generation by reducing fermentation process acid product, promotes the synthesis of ethyl alcohol come the method that improves Miyarisan growth efficiency, specially in Miyarisan three grade fermemtation 0-8 h, start sterile, anaerobic at the uniform velocity flow plus acetaldehyde solution to fermentation ends before 4-6 h, acetaldehyde total concentration be 1.5-5.0 mmol/L.The present invention is used acetaldehyde molecule as metabolic regulation molecule, the addition of acetaldehyde molecule promotes the oxidation efficiency of NADH, significantly enhances the combined coefficient of ethyl alcohol, but also significantly reduce the formation efficiency of acetic acid, butyric acid, the growth efficiency of Miyarisan is further improved, fabulous technical effect is produced.The present invention has the characteristics that material is easily purchased, operation controllability is high, is suitable for Miyarisan fermenting and producing.
Description
Technical field
The invention belongs to field of food fermentation, and in particular to a method of improve Miyarisan growth efficiency.
Background technique
In addition to as important functional microorganism other than sauce class, pickles, wine brewing process play a significant role, Miyarisan (fourth
Sour clostridium,Clostridium butyrium) balanced as people's use or animal new probiotic bacterium with adjustment intestinal colony, promote
Intestine beneficial bacteria colony proliferation, enhancing are immune, prevent tumorigenic function.In addition, the prebiotic product that Miyarisan is generated in enteron aisle
Such as B family vitamin, vitamin K substance, it is with health role to body.The main metabolites of Miyarisan are butyric acid, to intestines
The regeneration and reparation of road epithelial tissue have very important significance.Miyarisan is typical anaerobic spore-bearing bacilli, in fermentation process
The substances such as butyric acid, acetic acid, ethyl alcohol are largely generated under anaerobic condition.Although Miyarisan has certain acid resistance, these acid molecules
A large amount of accumulation still can grow it, be metabolized the serious acid stress inhibiting effect of generation, to reduce its growth efficiency.Therefore,
The inhibiting effect that Miyarisan acidic metabolite (acetic acid, butyric acid etc.) grows itself is targetedly released or alleviates, it is real
The High Density Cultivation of existing clostridium butyricum, it has also become the key technology of research and development clostridium butyricum probiotics.
By the way that alkaline matter is added such as in fermentation process: sodium hydroxide, ammonium hydroxide, sodium bicarbonate, sodium carbonate can neutralize
The organic acid that bacterial metabolism generates maintains pH to stablize in setting value.Such as: documents 1(prepares junket using fed batch fermentation method
The method of sour bacterium active bacteria formulation, patent of invention CN201110287802.1,2011) it discloses in Miyarisan fermentation and fills into ammonium hydroxide control
The method of pH processed；Documents 2(butyric acid bacteria fermentation culture medium and preparation method thereof and Miyarisan cultivation and fermentation method, invention are special
Sharp CN201410089743.0,2014) disclosing with the starting pH of alkali control fermentation medium is 7.4-7.5 etc.；Documents 3
(a kind of method of continuous fermentation method production butyric acid bacteria preparation, patent of invention CN201210135950.6,2012) discloses one kind
Continuously ferment the method for producing Miyarisan, controls fermentation starting pH6.0-7.5 with alkali；Butyric acid shuttle in the pit mud of the wine cellar documents 4(
The separation and its characteristic research of bacterium, Xie Shugui, microbiology notification, 2007,34 (6): 1047-1051) clostridium butyricum has been determined
Most suitable initial pH and by stream plus sodium carbonate liquor control pH value 6.5.
Obviously, acid, alcohol metabolic characteristic and alcohol of the documents 1,2,3,4 without reference to Miyarisan during the fermentation
The content of acid metabolic pressure and regulation, but neutralized using common technological means with alkaline matter.Although the prior art can
It is suitable for Miyarisan growth to control the pH of yeasting, if but the physiological metabolism of Miyarisan cell under anaerobic can be alleviated
Pressure, the angle aoxidized from reduced Coenzyme I (NADH) are regulated and controled, then the efficiency that Miyarisan may be made to grow is further
It improves.
The NADH generated during cell growth metabolism by glycolytic pathway (EMP) under anaerobic, can be through dehydrogenase
Catalysis reaction (such as: alcohol dehydrogenase, butanol dehydrogenase) is aoxidized by generating butyryl CoA, and passes through EMP Embden Meyerbof Parnas pathway, acetic acid
Synthesis and butyric acid synthesis obtain ATP and meet growth demand.But the metabolism that the accumulation of acetic acid, butyric acid can also aggravate cell is negative
Load, causes acid stress effect.This will necessarily it is serious influence cell physiological metabolism and cell activity, and cause cell from
It is molten.And generate ethyl alcohol type organic matter, then it can weaken acid poisoning effect.Therefore, if NADH warp can be increased under anaerobic
The oxidation efficiency of ethyl alcohol route of synthesis may then be such that cell activity is maintained, and further promote cell growth.Selection is closed
Suitable hydrogen acceptor may realize above-mentioned purpose as metabolic regulation molecule.
Acetaldehyde is usually used in organic chemical industry's industry, also serves as food service industry fruit essence as building heterocycle ring system reagent
Blender.But anaerobe --- the new function of Miyarisan incubation is not applied to it using acetaldehyde molecule as metabolic factor
The report of energy, it may be assumed that acetaldehyde enhances ethyl alcohol by promoting the activity of Miyarisan alcohol dehydrogenase intracellular as metabolic regulation molecule
Formation efficiency, be also possible to alleviate acid poisoning effect and further increase Miyarisan cell growth efficiency new function report.
Summary of the invention
It is a kind of by reducing fermentation it is an object of that present invention to provide a kind of new method for improving Miyarisan growth efficiency
The generation of process acid product, the method for promoting the synthesis of ethyl alcohol to improve Miyarisan growth efficiency.
The object of the invention is achieved through the following technical solutions:
A method of Miyarisan growth efficiency being improved, in Miyarisan fermentation Shi Liujia acetaldehyde；It is preferred that are as follows: in Miyarisan
When fermentation 0-8 h, starts sterile, anaerobic stream and add 4-6 h before acetaldehyde solution to fermentation ends, acetaldehyde total concentration is 1.5-5.0
Mmol/L, i.e. every liter of fermentation broth stream add the amount of acetaldehyde to be 1.5-5.0 mmol.Sterile, the anaerobic stream adds acetaldehyde solution to pass through
The method that includes the following steps is realized: the logical blue top capping material bottle of glass two being transformed into threeway indigo plant top capping material bottle, wherein two lead to and connect
Filter, third cross silicone tube all and connect feed supplement syringe needle.After sterilizing, bottled nitrogen is connected to threeway indigo plant top capping by pipe fitting
On a filter for expecting bottle, filtered sterile nitrogen is passed through feed supplement bottle bottom of bottle, and after being bubbled displaced air, the syringe needle of sterilizing exists
The material-feeding port that fermentation cover is penetrated under flame protection carries out acetaldehyde solution feed supplement by peristaltic pump.The fermentation is preferably three
Grade fermentation, the time of three grade fermemtation is preferably 24 h.
Preferably, the culture medium that the method for the raising Miyarisan growth efficiency is used is formulated as follows: glucose
16.6 g/L, 7.2 g/L of yeast powder, 4.7 g/L of calcium carbonate, 25 g/L of peptone, 0.5 g/L of dipotassium hydrogen phosphate, seven water sulfuric acid
0.8 g/L of magnesium, manganese sulfate monohydrate 0.02 g/L, pH7.3；Anaerobism pipe slant medium need to additionally incorporate agar, agar concentration 20
g/L.It is spare through 0.1 MPa steam sterilization, 20 min after deoxygenation.
It is furthermore preferred that the method for the raising Miyarisan growth efficiency, includes the following steps:
(1) seed expands culture and closes the collection of bottle seed cell
By sterile, oxygen free operation requirement, cell to freshly prepd anaerobism pipe inclined-plane is picked out from Miyarisan strain freeze-drying pipe and is trained
Support base.After 36-38 DEG C of culture 12-16 h, by sterile, oxygen free operation requirement, 10 mL of addition are sterile, anaerobic water prepares anaerobism pipe
Seed liquor.
Bottled 30 mL of culture medium of 120 mL anaerobism, in 1-2% ratio (v/v) by Miyarisan (Clostridium butyrium) anaerobism pipe seed liquor access anaerobism bottle fluid nutrient medium in, 36-38 DEG C, 100 r/min 12- is cultivated on shaking table
14 h obtain anaerobism bottle seed liquor.By sterile, oxygen free operation requirement, anaerobism bottle seed liquor is carried out to close bottle operation, obtains closing bottle
Seed liquor.
(2) secondary seed culture
After secondary seed tank sterilizing (10 L), nitrogen displacement deoxygenation equipped with culture medium, flame protection is lower will to close bottle seed
Liquid accesses secondary seed tank, and secondary seed fermented and cultured is carried out as follows: 36-38 DEG C of cultivation temperature, revolving speed is 100 r/
Min, nitrogen flow control are 0.1 vvm, and tank is voltage-controlled to be made as 0.03-0.04 MPa, after anaerobism protects 3 h, stop logical nitrogen, hair
After ferment culture 12-14 h, secondary seed solution is obtained.
(3) three grade fermemtation
After three grade fermemtation tank (100 L) sterilizing, nitrogen displacement deoxygenation equipped with culture medium, by secondary seed solution with 1-2%'s
Three grade fermemtation is carried out as follows to three grade fermemtation tank in inoculum concentration (v/v) culture transferring: 36-38 DEG C of fermentation temperature, 100 r/ of revolving speed
Min, nitrogen flow control are 0.1vvm, and tank is voltage-controlled to be made as 0.03-0.04 MPa, after anaerobism protects 3 h, stop logical nitrogen.Hair
After 24 h of ferment culture, tank is put.When three grade fermemtation culture 0-8 h, starts sterile, anaerobic and at the uniform velocity flow plus fill into acetaldehyde solution to fermentation
4-6 h, makes acetaldehyde total concentration 1.5-5.0 mmol/L before terminating.It is preferred: when three grade fermemtation 3 h of culture, to start sterile, nothing
Oxygen at the uniform velocity flows plus fills into 4 h before acetaldehyde solution to fermentation ends, and acetaldehyde total concentration is 2.0 mmol/L.
Aerobic feed supplement Technology application is to anaerobic fermentation process, it is also necessary to overcome technical some difficulties, including feed liquid with
And two logical blue top capping material bottle connector anaerobic environment holding (the general stirred fermentor system of 100 L).The present invention is in order to protect
It demonstrate,proves sterile, anaerobic stream and adds acetaldehyde solution, in common two logical (filtrated air bottle inlet access and feed liquid bottle outlet access) glass indigo plant lid bottles
On the basis of be transformed, by two logical stainless steel parts punchings, welding, it is logical to pick out third, obtain threeway glass indigo plant top capping material bottle,
Wherein two lead to and connect filter (0.22 μm of filtering accuracy), and third crosses silicone tube all and connects feed supplement syringe needle.After sterilizing, by bottle
Dress nitrogen is connected on a filter of threeway glass indigo plant top capping material bottle by pipe fitting, and filtered sterile nitrogen is passed directly into
Feed supplement bottle bottom of bottle, the slight 30 min(gas of displaced air that is bubbled connect filter directly from threeway glass indigo plant top capping material bottle
Outside second logical spilling bottle) after, the syringe needle that the logical wrapping sterilizing of third is connected to through silicone tube is opened, flame penetrates tank deck under protecting
Material-feeding port.Acetaldehyde solution feed supplement is carried out as required by peristaltic pump.
Compared with prior art the invention has the advantages that and significant progress: operated by the present invention, not by second
Aldehyde molecule is used as organic chemical industry's reagent and food service industry deodorant tune as metabolic regulation molecule.The present invention has material
Material is easily purchased, operation controllability is high, is suitable for the characteristics of fermenting and producing.The addition of acetaldehyde molecule promotes the oxidation efficiency of NADH, shows
Work enhances the combined coefficient of ethyl alcohol (concentration of alcohol increases 11.7-29.7% when putting tank).Moreover, also creating unexpected
Effect: the addition of acetaldehyde molecule make acetic acid, butyric acid formation efficiency significantly reduce, reduce 28.7-42.1% respectively when putting tank
And 22.2-57.8%.In this way, in Miyarisan anaerobic fermentation process, cell is generated due to the accumulation of acetic acid and butyric acid acid poisoning
Effect is also effectively alleviated, and promotes the maintenance of cell activity, further improves the growth efficiency of Miyarisan, puts tank
When Miyarisan viable count improve 15.1-28.1%, produce fabulous technical effect.This is most apparent with the prior art
It is different.
Specific embodiment
Further detailed description is done to the present invention below with reference to embodiment, embodiments of the present invention are not limited thereto.
Embodiment 1
(1) preparation of culture medium
20 g/L of glucose, 30 g/L of peptone, 20 g/L of yeast powder, 0.5 g/L of dipotassium hydrogen phosphate, epsom salt
0.5 g/L, 1 g/L of precipitated calcium carbonate, manganese sulfate monohydrate 0.02 g/L, pH7.3；Anaerobism pipe slant medium need to additionally incorporate fine jade
Rouge, agar concentration 20；pH 7.3.It is spare through 0.1 MPa steam sterilization, 20 min after deoxygenation.
(2) seed expands culture and closes the collection of bottle seed cell
By sterile, oxygen free operation requirement, cell to freshly prepd anaerobism pipe inclined-plane is picked out from Miyarisan strain freeze-drying pipe and is trained
Support base.After 37 DEG C of 12 h of culture, by sterile, oxygen free operation requirement, 10 mL of addition are sterile, anaerobic water prepares anaerobism pipe seed liquor.
Bottled 30 mL of culture medium of 120 mL anaerobism, in 1% ratio (v/v) by Miyarisan (Clostridium butyrium) in anaerobism pipe seed liquor access anaerobism bottle fluid nutrient medium, 37 DEG C, 100 r/min cultivate 12 h on shaking table and obtain
To anaerobism bottle seed liquor.By sterile, oxygen free operation requirement, anaerobism bottle seed liquor is carried out to close bottle operation, obtains closing bottle seed liquor.
(3) secondary seed culture
After secondary seed tank sterilizing (10 L), nitrogen displacement deoxygenation equipped with culture medium, flame protection is lower will to close bottle seed
Liquid accesses secondary seed tank in 1% ratio (v/v), is carried out as follows secondary seed fermented and cultured: 37 DEG C of cultivation temperature,
Revolving speed is 100 r/min, and nitrogen flow control is 0.1 vvm, and tank is voltage-controlled to be made as 0.03-0.04 MPa, after anaerobism protects 3 h,
Stop logical nitrogen, after 12 h of fermented and cultured, obtains secondary seed solution.
(4) three grade fermemtation
After nitrogen displacement deoxygenation, secondary seed solution is connect with 1% for three grade fermemtation tank (100 L) sterilizing equipped with culture medium
Three grade fermemtation is carried out as follows to three grade fermemtation tank in kind amount (v/v) culture transferring: 37 DEG C of fermentation temperature, 100 r/min of revolving speed,
Nitrogen flow control is 0.1vvm, and tank is voltage-controlled to be made as 0.03-0.04 MPa, after anaerobism protects 3 h, stops logical nitrogen.Fermentation training
After supporting 24 h, tank is put.
Using the general seed culture medium of above-mentioned Miyarisan and fermentation medium, 24 h of anaerobic fermentation in a manner described, cell
Number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is 2.6 × 108CFU/mL, the concentration of alcohol in fermentation liquid are
89 mg/L, acetic acid concentration are 251 mg/L, butyric acid density is 4.5 g/L.
Embodiment 2
(1) preparation of culture medium
16.6 g/L of glucose, 7.2 g/L of yeast powder, 4.7 g/L of calcium carbonate, 25 g/L of peptone, dipotassium hydrogen phosphate 0.5
G/L, 0.8 g/L of epsom salt, manganese sulfate monohydrate 0.02 g/L, pH7.3；Anaerobism pipe slant medium need to additionally incorporate fine jade
Rouge, 20 g/L of agar concentration.It is spare through 0.1 MPa steam sterilization, 20 min after deoxygenation.
(2) seed expands culture and closes the collection of bottle seed cell
By sterile, oxygen free operation requirement, cell to freshly prepd anaerobism pipe inclined-plane is picked out from Miyarisan strain freeze-drying pipe and is trained
Support base.After 37 DEG C of 12 h of culture, by sterile, oxygen free operation requirement, 10 mL of addition are sterile, anaerobic water prepares anaerobism pipe seed liquor.
Bottled 30 mL of culture medium of 120 mL anaerobism, in 1% ratio (v/v) by Miyarisan (Clostridium butyrium) in anaerobism pipe seed liquor access anaerobism bottle fluid nutrient medium, 37 DEG C, 100 r/min cultivate 12 h on shaking table and obtain
To anaerobism bottle seed liquor.By sterile, oxygen free operation requirement, anaerobism bottle seed liquor is carried out to close bottle operation, obtains closing bottle seed liquor.
(3) secondary seed culture
After secondary seed tank sterilizing (10 L), nitrogen displacement deoxygenation equipped with culture medium, flame protection is lower will to close bottle seed
Liquid accesses secondary seed tank in 1% ratio (v/v), is carried out as follows secondary seed fermented and cultured: 37 DEG C of cultivation temperature,
Revolving speed is 100 r/min, and nitrogen flow control is 0.1 vvm, and tank is voltage-controlled to be made as 0.03-0.04 MPa, after anaerobism protects 3 h,
Stop logical nitrogen, after 12 h of fermented and cultured, obtains secondary seed solution.
(4) three grade fermemtation
After nitrogen displacement deoxygenation, secondary seed solution is connect with 1% for three grade fermemtation tank (100 L) sterilizing equipped with culture medium
Three grade fermemtation is carried out as follows to three grade fermemtation tank in kind amount (v/v) culture transferring: 37 DEG C of fermentation temperature, 100 r/min of revolving speed,
Nitrogen flow control is 0.1vvm, and tank is voltage-controlled to be made as 0.03-0.04 MPa, after anaerobism protects 3 h, stops logical nitrogen.Fermentation training
After supporting 24 h, tank is put.
Using above-mentioned preferred culture medium, 24 h of anaerobic fermentation, cell number (are counted in a manner described with Hungate rolling pipe
Method carries out bacterium colony CFU counting) it is 5.7 × 108CFU/mL improves 2.2 times than embodiment 1.Moreover, the ethyl alcohol in fermentation liquid
Concentration is 111 mg/L, improves 24.7% than embodiment 1.Acetic acid concentration in fermentation liquid is 188 mg/L, is dropped than embodiment 1
Low 75%.Butyric acid density in fermentation liquid is 2.7 g/L, reduces 60% than embodiment 1.Achieve unexpected result.
In addition, preferred culture medium used by embodiment 2 is formulated glucose, yeast powder and peptone used respectively than used in embodiment 1
Concentration reduce 17%, 64% and 17%, greatly saved the cost of raw material.
Embodiment 3
(1) preparation of culture medium: with embodiment 2.
(2) seed liquor expands culture and closes the collection of bottle seed cell: with embodiment 2.
(3) secondary seed culture: with embodiment 2.
(4) three grade fermemtation: in addition to adding regulatory factor, remaining condition of three grade fermemtation is the same as embodiment 2.
Regulatory factor is added: when three grade fermemtation culture starts, sterile, anaerobic at the uniform velocity flows plus acetaldehyde solution (concentration 0.2
Mol/L) to 4 h before fermentation ends, acetaldehyde total concentration is 3.0 mmol/L.
24 h of anaerobic fermentation in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) are
6.56×108CFU/mL improves 15.1% than embodiment 2；Concentration of alcohol in fermentation liquid is 124 mg/L, than embodiment 2
Improve 11.7%；Acetic acid concentration is 132 mg/L, reduces 29.8% than embodiment 2；Butyric acid density is 2.1 g/L, than implementing
Example 2 reduces 22.2%.
Embodiment 4
(1) preparation of culture medium: with embodiment 2.
(2) seed liquor expands culture and closes the collection of bottle seed cell: with embodiment 2.
(3) secondary seed culture: with embodiment 2.
(4) three grade fermemtation: in addition to adding regulatory factor, remaining condition of three grade fermemtation is the same as embodiment 2.
Regulatory factor is added: when three grade fermemtation cultivation cycle is 6 h, it is (dense that beginning is sterile, anaerobic at the uniform velocity fills into acetaldehyde solution
Degree is 0.2 mol/L) to 4 h before fermentation ends, acetaldehyde total concentration is 1.5 mmol/L.
24 h of anaerobic fermentation in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) are
6.91×108CFU/mL improves 21.2% than embodiment 2；Concentration of alcohol in fermentation liquid is 130 mg/L, than embodiment 2
Improve 17.1%；Acetic acid concentration is 134 mg/L, reduces 28.7% than embodiment 2；Butyric acid density is 1.83 g/L, than implementing
Example 2 reduces 32.2%.
Embodiment 5
(1) preparation of culture medium: with embodiment 2.
(2) seed liquor expands culture and closes the collection of bottle seed cell: with embodiment 2.
(3) secondary seed culture: with embodiment 2.
(4) three grade fermemtation: in addition to adding regulatory factor, remaining condition of three grade fermemtation is the same as embodiment 2.
Regulatory factor is added: when three grade fermemtation cultivation cycle is 3 h, it is (dense that beginning is sterile, anaerobic at the uniform velocity fills into acetaldehyde solution
Degree is 0.2 mol/L) to 4 h before fermentation ends, acetaldehyde total concentration is 2.0 mmol/L.
24 h of anaerobic fermentation in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) are
7.3×108CFU/mL improves 28.1% than embodiment 1；Concentration of alcohol in fermentation liquid is 144 mg/L, is mentioned than embodiment 2
It is high by 29.7%；Acetic acid concentration is 109 mg/L, reduces 42.1% than embodiment 2；Butyric acid density is 1.14 g/L, compares embodiment
2 reduce 57.8%.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of method for improving Miyarisan growth efficiency, it is characterised in that: the method are as follows: in Miyarisan fermentation 0-6h
When, 4-6h before beginning is sterile, anaerobic at the uniform velocity flows plus acetaldehyde solution to fermentation ends, acetaldehyde total concentration is 1.5-3.0mmol/L；
Sterile, the anaerobic stream adds acetaldehyde solution to pass through the method realization included the following steps: by the logical blue top capping material of glass two
Bottle is transformed into threeway indigo plant top capping material bottle, wherein two lead to and connect filter, third crosses silicone tube all and connects feed supplement syringe needle；Sterilizing
Afterwards, bottled nitrogen is connected on a filter of threeway indigo plant top capping material bottle by pipe fitting, filtered sterile nitrogen is passed through
Feed supplement bottle bottom of bottle, after displaced air, the syringe needle of sterilizing penetrates material-feeding port under the protection of flames, carries out acetaldehyde solution by peristaltic pump
Feed supplement；
The method includes the following steps:
(1) seed expands culture and closes the collection of bottle seed cell
By sterile, oxygen free operation requirement, cell is picked out to anaerobism pipe slant medium from Miyarisan strain freeze-drying pipe；36-38℃
After cultivating 12-16h, by sterile, oxygen free operation requirement, addition 10mL is sterile, anaerobic water prepares anaerobism pipe seed liquor；
The bottled culture medium 30mL of 120mL anaerobism is trained Miyarisan anaerobism pipe seed liquor access anaerobism bottle liquid in the ratio of 1-2%
It supports in base, 36-38 DEG C, 100r/min cultivates 12-14h on shaking table and obtain anaerobism bottle seed liquor；It is wanted by sterile, oxygen free operation
It asks, anaerobism bottle seed liquor is carried out to close bottle operation, obtains closing bottle seed liquor；
(2) secondary seed culture
After the sterilizing of secondary seed tank, nitrogen displacement deoxygenation equipped with culture medium, the lower bottle seed liquor that will close of flame protection accesses second level
Seeding tank, is carried out as follows secondary seed fermented and cultured: 36-38 DEG C of cultivation temperature, revolving speed 100r/min, nitrogen flow
Control is 0.1vvm, and tank is voltage-controlled to be made as 0.03-0.04MPa, after anaerobism protects 3h, stops logical nitrogen, after fermented and cultured 12-14h,
Obtain secondary seed solution；
(3) three grade fermemtation
After the sterilizing of three grade fermemtation tank, nitrogen displacement deoxygenation equipped with culture medium, by secondary seed solution with the inoculum concentration culture transferring of 1-2%
To three grade fermemtation tank, three grade fermemtation is carried out as follows: 36-38 DEG C of fermentation temperature, revolving speed 100r/min, nitrogen flow control
For 0.1vvm, tank is voltage-controlled to be made as 0.03-0.04MPa, after anaerobism protects 3h, stops logical nitrogen；Fermented and cultured for 24 hours after, put tank；
When three grade fermemtation culture 0-6h, starts sterile, anaerobic and at the uniform velocity flow plus fill into 4-6h before acetaldehyde solution to fermentation ends, make second
Aldehyde total concentration is 1.5-3.0mmol/L.
2. the method according to claim 1 for improving Miyarisan growth efficiency, it is characterised in that: the fermentation is three-level
Fermentation, the time of three grade fermemtation are for 24 hours.
3. the method according to claim 1 for improving Miyarisan growth efficiency, it is characterised in that: used in Miyarisan fermentation
Culture medium it is as follows: glucose 16.6g/L, yeast powder 7.2g/L, calcium carbonate 4.7g/L, peptone 25g/L, dipotassium hydrogen phosphate
0.5g/L, epsom salt 0.8g/L and manganese sulfate monohydrate 0.02g/L, pH7.3；Anaerobism pipe slant medium need to additionally incorporate fine jade
Rouge, agar concentration 20g/L；It sterilizes after deoxygenation.
4. the method according to claim 1 for improving Miyarisan growth efficiency, it is characterised in that: the secondary seed tank
For 10L fermentor, the three grade fermemtation tank is 100L fermentor.
5. the method according to claim 1 for improving Miyarisan growth efficiency, it is characterised in that: in three grade fermemtation culture 3h
When, start sterile, anaerobic and at the uniform velocity fills into 4h before acetaldehyde solution to fermentation ends, the total final concentration of 2.0mmol/L of acetaldehyde.
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| CN102660473A (en) * | 2012-05-04 | 2012-09-12 | 湖北绿雪生物产业有限公司 | Method for producing clostridium butyricum preparation by using continuous fermentation method |
| CN105255959A (en) * | 2015-10-20 | 2016-01-20 | 湖北工业大学 | Feed supplementing method capable of promoting fermentation synthesis efficiency of rifamycin SV |
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