CN105861410A - Method for improving clostridium butyricum growth efficiency - Google Patents

Method for improving clostridium butyricum growth efficiency Download PDF

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CN105861410A
CN105861410A CN201610356765.8A CN201610356765A CN105861410A CN 105861410 A CN105861410 A CN 105861410A CN 201610356765 A CN201610356765 A CN 201610356765A CN 105861410 A CN105861410 A CN 105861410A
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butyric acid
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acid bacteria
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CN105861410B (en
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王志
夏会丽
陈雄
李冬生
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Hubei University of Technology
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Abstract

The invention discloses a method for improving the clostridium butyricum growth efficiency, and belongs to the field of food fermentation. The invention provides the method for improving the clostridium butyricum growth efficiency by reducing generation of acidic products in the fermentation process and promoting ethanol synthesis, and the method particularly comprises the step that phenylalanine solution feeding is conducted in a sterile and anaerobic mode at constant speed when three stage fermentation of clostridium butyricum is conducted for 0.8 h till 4 h to 6 h before fermentation is completed, wherein the total concentration of acetaldehyde ranges from 1.5 mmol/L to 5.0 mmol/L. According to the method for improving the clostridium butyricum growth efficiency, acetaldehyde molecules serve as metabolic regulation molecules for use; by means of addition of the acetaldehyde molecules, the oxidation efficiency of NADH is promoted, the synthesis efficiency of ethanol is significantly enhanced, the generation efficiency of acetic acid and butyric acid is significantly reduced, the growth efficiency of the clostridium butyricum is further improved, and an excellent technical effect is produced. The method for improving the clostridium butyricum growth efficiency has the advantages that the materials are easy to purchase, the operation controllability is high, and the method is suitable for clostridium butyricum fermentation production.

Description

A kind of method improving butyric acid bacteria growth efficiency
Technical field
The invention belongs to field of food fermentation, be specifically related to a kind of method improving butyric acid bacteria growth efficiency.
Background technology
Except as important functional microorganism in addition to beans class, Pickles, wine brewing process play a significant role, butyric acid bacteria (Clostridium butyricum,Clostridium butyrium) as people with or animal new probiotic bacterium have adjustment intestinal colony balance, promote intestinal beneficial flora propagation, strengthen immunity, prophylaxis of tumours occur function.Additionally, the prebiotic product such as material such as vitamin B group, vitamin K that butyric acid bacteria produces at intestinal, with health role to body.The main metabolites of butyric acid bacteria is butanoic acid, and regeneration and reparation to gut epithelium tissue have very important significance.Butyric acid bacteria is typical anaerobic spore-bearing bacilli, produces the materials such as butanoic acid, acetic acid, ethanol in sweat the most in a large number.Although butyric acid bacteria has certain acid resistance, but these acid molecules accumulate in a large number and can produce its growth, metabolism serious acid stress inhibitory action, thus reduce its growth efficiency.Therefore, release or alleviate the inhibitory action that itself is grown by butyric acid bacteria acidic metabolite (acetic acid, butanoic acid etc.) targetedly, it is achieved the High Density Cultivation of Clostridium butyricum, it has also become the key technology of research and development Clostridium butyricum microbial ecological agent.
By sweat adds alkaline matter such as: sodium hydroxide, ammonia, sodium bicarbonate, sodium carbonate etc. can neutralize the organic acid that bacterial metabolism produces, maintain pH stable in setting value.As: the method that documents 1(employing fed-batch fermentation method prepares active clostridium butyrium agent, patent of invention CN201110287802.1,2011) disclose the method filling into ammonia control pH in butyric acid bacteria fermentation；Documents 2(butyric acid bacteria fermentation medium and preparation method thereof and butyric acid bacteria cultivation and fermentation method, patent of invention CN201410089743.0,2014) disclose that to control the initial pH of fermentation medium with alkali be 7.4-7.5 etc.；The method that documents 3(continuous fermentation method produces butyric acid bacteria preparation, patent of invention CN201210135950.6,2012) disclose a kind of method that continuous fermentation produces butyric acid bacteria, control the initial pH6.0-7.5 of fermentation with alkali；The separation of Clostridium butyricum and characteristic research thereof in the pit mud of documents 4(wine cellar, Xie Shugui, microbiology is circulated a notice of, and 2007,34 (6): 1047-1051) determine the suitableeest initial pH of Clostridium butyricum and add sodium carbonate liquor control ph 6.5 by stream.
Obviously, documents 1,2,3,4 is all without reference to butyric acid bacteria acid, alcohol metabolic characteristic and alkyd Metabolic stress during the fermentation and the content of regulation and control, but uses common technological means alkaline matter to neutralize.Although the pH that prior art can control yeasting is suitable for butyric acid bacteria growth, but if butyric acid bacteria cell physiological metabolism pressure under anaerobic can be alleviated, the angle aoxidized from DPNH (NADH) regulates and controls, then the efficiency that butyric acid bacteria may be made to grow improves further.
The NADH under anaerobic produced by glycolytic pathway (EMP) during cell growth metabolism, can through dehydrogenase catalyzed reaction (such as: ethanol dehydrogenase, butanol dehydrogenase) or by generate butyryl CoA aoxidize, and by EMP Embden Meyerbof Parnas pathway, acetic acid synthesize and butanoic acid synthesis acquisition ATP meet growth demand.But, acetic acid, the accumulation of butanoic acid also can increase the weight of the metabolism burden of cell, cause acid stress effect.The physiological metabolism affecting cell that this will necessarily be serious and the activity of cell, and cause aqtocytolysis.And generate ethanol type organic matter, then can weaken acidosis effect.Therefore, if the NADH oxidation efficiency through ethanol route of synthesis can under anaerobic be increased, then cytoactive may be made to be maintained, and promote cell growth further.Select suitable hydrogen acceptor may realize above-mentioned purpose as metabolic regulation molecule.
Acetaldehyde, as building heterocycle ring system reagent, is usually used in organic chemical industry's industry, also serves as the blender of food service industry fruit essence.But, acetaldehyde molecule is not applied to anaerobe as metabolic factor---the report of the New function of butyric acid bacteria incubation, that is: acetaldehyde as metabolic regulation molecule, is strengthened the formation efficiency of ethanol by the activity promoting butyric acid bacteria intracellular ethanol dehydrogenase, is also possible to alleviate the report of acidosis effect the New function of raising butyric acid bacteria cell growth efficiency further.
Summary of the invention
Present invention aim at providing a kind of new method improving butyric acid bacteria growth efficiency, it is a kind of generation by reducing sweat acid product, promotes the synthesis method that improves butyric acid bacteria growth efficiency of ethanol.
The object of the invention is achieved through the following technical solutions:
A kind of method improving butyric acid bacteria growth efficiency, adds acetaldehyde for the stream when butyric acid bacteria ferments；Being preferably: when butyric acid bacteria fermentation 0-8 h, start aseptic, anaerobic stream and add acetaldehyde solution to 4-6 h before fermentation ends, acetaldehyde total concentration is 1.5-5.0 mmol/L, and the amount that i.e. every liter fermentation broth stream adds acetaldehyde is 1.5-5.0 mmol.Described aseptic, anaerobic stream is added acetaldehyde solution and is realized by the method comprised the steps: logical for glass two blue top capping material bottle is transformed into threeway indigo plant top capping material bottle, and wherein two lead to take over filter, and threeway connects feed supplement syringe needle by silica gel tube.After sterilizing; being connected on a filter of threeway indigo plant top capping material bottle by pipe fitting by bottled nitrogen, filtered sterile nitrogen is passed through at the bottom of feed supplement bottle bottle, after bubbling displaced air; the syringe needle of sterilizing penetrates the material-feeding port of fermentation cover under flame is protected, and carries out acetaldehyde solution feed supplement by peristaltic pump.Described fermentation is preferably three grade fermemtation, and the time of three grade fermemtation is preferably 24 h.
Preferably, being formulated as follows of the culture medium that the described method improving butyric acid bacteria growth efficiency is used: glucose 16.6 g/L, yeast powder 7.2 g/L, calcium carbonate 4.7 g/L, peptone 25 g/L, dipotassium hydrogen phosphate 0.5 g/L, Magnesium sulfate heptahydrate 0.8 g/L, manganese sulfate monohydrate 0.02 g/L, pH7.3；Anaerobism pipe slant medium need to it is possible to additionally incorporate agar, agar concentration 20 g/L.After deoxygenation standby through 0.1 MPa steam sterilization 20 min.
It is furthermore preferred that the described method improving butyric acid bacteria growth efficiency, comprise the steps:
(1) seed amplification culture and the collection closing bottle seed cell
By aseptic, oxygen free operation requirement, pick out cell to freshly prepd anaerobism pipe slant medium from butyric acid bacteria strain lyophilizing pipe.Cultivate after 12-16 h for 36-38 DEG C, by aseptic, oxygen free operation requirement, add that 10 mL are aseptic, anaerobic water prepares anaerobism pipe seed liquor.
120 ML anaerobism bottled culture medium 30 mL, in the ratio (v/v) of 1-2% by butyric acid bacteria (Clostridium butyrium) anaerobism pipe seed liquor accesses in anaerobism bottle fluid medium, 36-38 DEG C, 100 r/min cultivate 12-14 h on shaking table and obtain anaerobism bottle seed liquor.By aseptic, oxygen free operation requirement, carry out anaerobism bottle seed liquor closing bottle operation, obtain closing bottle seed liquor.
(2) secondary seed is cultivated
Equipped with culture medium secondary seed tank sterilizing (10 L), nitrogen displacement deoxygenation after; the lower bottle seed liquor of closing of flame protection accesses secondary seed tank; it is carried out as follows secondary seed fermentation culture: cultivation temperature 36-38 DEG C; rotating speed is 100 r/min; it is 0.1 vvm that nitrogen flow controls, and tank pressure controls as 0.03-0.04 MPa, after anaerobism protects 3 h, stops logical nitrogen, fermentation culture 12-14 After h, obtain secondary seed solution.
(3) three grade fermemtation
After three grade fermemtation tank (100 L) sterilizing of culture medium, nitrogen displacement deoxygenation, by secondary seed solution with inoculum concentration (v/v) subcultivation of 1-2% to three grade fermemtation tank, it is carried out as follows three grade fermemtation: fermentation temperature 36-38 DEG C, rotating speed 100 R/min, nitrogen flow controls to be 0.1vvm, and tank pressure controls as 0.03-0.04 MPa, after anaerobism protects 3 h, stops logical nitrogen.After fermentation culture 24 h, put tank.When 0-8 h is cultivated in three grade fermemtation, starting aseptic, anaerobic at the uniform velocity stream and add and fill into acetaldehyde solution to 4-6 h before fermentation ends, making acetaldehyde total concentration is 1.5-5.0 mmol/L.Preferred: when 3 h is cultivated in three grade fermemtation, start aseptic, anaerobic at the uniform velocity stream and add and fill into acetaldehyde solution to 4 h before fermentation ends, acetaldehyde total concentration is 2.0 mmol/L.
Aerobic feed supplement Technology application is to anaerobic fermentation process, in addition it is also necessary to overcome more technical difficulties, including the holding (100 L general stirred fermentor system) of the anaerobic environment of the logical blue top capping material bottle connector of feed liquid and two.The present invention is in order to ensure that aseptic, anaerobic stream adds acetaldehyde solution, transformed on the basis of common two logical (filtrated air bottle inlet path and feed liquid bottle outlet path) glass indigo plant lid bottles, stainless steel part punching, welding is led to by two, pick out threeway, obtain threeway glass indigo plant top capping material bottle, wherein two lead to take over filter (filtering accuracy 0.22 μm), and threeway connects feed supplement syringe needle by silica gel tube.After sterilizing; bottled nitrogen is connected on a filter of threeway glass indigo plant top capping material bottle by pipe fitting; filtered sterile nitrogen is passed directly at the bottom of feed supplement bottle bottle; the second logical spilling bottle that slight bubbling displaced air 30 min(gas directly connects filter from threeway glass indigo plant top capping material bottle is outer) after; open the syringe needle wrapping up sterilizing being connected to threeway through silica gel tube, under flame protection, penetrate tank deck material-feeding port.Acetaldehyde solution feed supplement is carried out on request by peristaltic pump.
Compared with prior art the invention have the advantages that and significantly improve: operating by the present invention, not using acetaldehyde molecule as organic chemical industry's reagent and food service industry deodorant tune, but use as metabolic regulation molecule.The present invention has that material is easily purchased, operation controllability is high, be applicable to the feature of fermenting and producing.The combined coefficient (when putting tank, concentration of alcohol adds 11.7-29.7%) added the oxidation efficiency promoting NADH, significantly enhance ethanol of acetaldehyde molecule.And, also create beat all effect: the interpolation of acetaldehyde molecule makes acetic acid, the formation efficiency of butanoic acid significantly reduces, when putting tank, reduce 28.7-42.1% and 22.2-57.8% respectively.So, in butyric acid bacteria anaerobic fermentation process, the acidosis effect produced cell due to the accumulation of acetic acid and butanoic acid is alleviated the most effectively, and promote the maintenance of cytoactive, further increase the growth efficiency of butyric acid bacteria, when putting tank, butyric acid bacteria viable count improves 15.1-28.1%, creates fabulous technique effect.This is the most different from prior art.
Detailed description of the invention
Below in conjunction with embodiment, the present invention done further detailed description, but embodiments of the present invention are not limited to this.
Implement Example 1
(1) preparation of culture medium
Glucose 20 g/L, peptone 30 g/L, yeast powder 20 g/L, dipotassium hydrogen phosphate 0.5 g/L, Magnesium sulfate heptahydrate 0.5 g/L, precipitated calcium carbonate 1 g/L, manganese sulfate monohydrate 0.02 g/L, pH7.3；Anaerobism pipe slant medium need to it is possible to additionally incorporate agar, agar concentration 20；pH 7.3.Through 0.1 after deoxygenation Min is standby for MPa steam sterilization 20.
(2) seed amplification culture and the collection closing bottle seed cell
By aseptic, oxygen free operation requirement, pick out cell to freshly prepd anaerobism pipe slant medium from butyric acid bacteria strain lyophilizing pipe.Cultivate after 12 h for 37 DEG C, by aseptic, oxygen free operation requirement, add that 10 mL are aseptic, anaerobic water prepares anaerobism pipe seed liquor.
120 ML anaerobism bottled culture medium 30 mL, the ratio (v/v) in 1% by butyric acid bacteria (Clostridium butyrium) anaerobism pipe seed liquor accesses in anaerobism bottle fluid medium, 37 DEG C, 100 r/min cultivate 12 h on shaking table and obtain anaerobism bottle seed liquor.By aseptic, oxygen free operation requirement, carry out anaerobism bottle seed liquor closing bottle operation, obtain closing bottle seed liquor.
(3) secondary seed is cultivated
Equipped with culture medium secondary seed tank sterilizing (10 L), nitrogen displacement deoxygenation after; secondary seed tank is accessed closing bottle seed liquor in the ratio (v/v) of 1% under flame protection; it is carried out as follows secondary seed fermentation culture: cultivation temperature 37 DEG C; rotating speed is 100 r/min; it is 0.1 vvm that nitrogen flow controls, and tank pressure controls as 0.03-0.04 MPa, after anaerobism protects 3 h, stops logical nitrogen, after fermentation culture 12 h, obtains secondary seed solution.
(4) three grade fermemtation
Equipped with culture medium three grade fermemtation tank (100 L) sterilizing, nitrogen displacement deoxygenation after; by secondary seed solution with 1% inoculum concentration (v/v) subcultivation to three grade fermemtation tank; it is carried out as follows three grade fermemtation: fermentation temperature 37 DEG C; rotating speed 100 r/min; nitrogen flow controls as 0.1vvm; tank pressure controls as 0.03-0.04 MPa, after anaerobism protects 3 h, the logical nitrogen of stopping.After fermentation culture 24 h, put tank.
Using the general seed culture medium of above-mentioned butyric acid bacteria and fermentation medium, in a manner described anaerobic fermentation 24 h, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 2.6 × 108CFU/mL, the concentration of alcohol in fermentation liquid is 89 mg/L, acetic acid concentration is 251 mg/L, butyric acid density is 4.5 g/L.
Implement Example 2
(1) preparation of culture medium
Glucose 16.6 g/L, yeast powder 7.2 g/L, calcium carbonate 4.7 g/L, peptone 25 g/L, dipotassium hydrogen phosphate 0.5 g/L, Magnesium sulfate heptahydrate 0.8 g/L, manganese sulfate monohydrate 0.02 g/L, pH7.3；Anaerobism pipe slant medium need to it is possible to additionally incorporate agar, agar concentration 20 g/L.After deoxygenation standby through 0.1 MPa steam sterilization 20 min.
(2) seed amplification culture and the collection closing bottle seed cell
By aseptic, oxygen free operation requirement, pick out cell to freshly prepd anaerobism pipe slant medium from butyric acid bacteria strain lyophilizing pipe.Cultivate after 12 h for 37 DEG C, by aseptic, oxygen free operation requirement, add that 10 mL are aseptic, anaerobic water prepares anaerobism pipe seed liquor.
120 ML anaerobism bottled culture medium 30 mL, the ratio (v/v) in 1% by butyric acid bacteria (Clostridium butyrium) anaerobism pipe seed liquor accesses in anaerobism bottle fluid medium, 37 DEG C, 100 r/min cultivate 12 h on shaking table and obtain anaerobism bottle seed liquor.By aseptic, oxygen free operation requirement, carry out anaerobism bottle seed liquor closing bottle operation, obtain closing bottle seed liquor.
(3) secondary seed is cultivated
Equipped with culture medium secondary seed tank sterilizing (10 L), nitrogen displacement deoxygenation after; secondary seed tank is accessed closing bottle seed liquor in the ratio (v/v) of 1% under flame protection; it is carried out as follows secondary seed fermentation culture: cultivation temperature 37 DEG C; rotating speed is 100 r/min; it is 0.1 vvm that nitrogen flow controls, and tank pressure controls as 0.03-0.04 MPa, after anaerobism protects 3 h, stops logical nitrogen, after fermentation culture 12 h, obtains secondary seed solution.
(4) three grade fermemtation
Equipped with culture medium three grade fermemtation tank (100 L) sterilizing, nitrogen displacement deoxygenation after; by secondary seed solution with 1% inoculum concentration (v/v) subcultivation to three grade fermemtation tank; it is carried out as follows three grade fermemtation: fermentation temperature 37 DEG C; rotating speed 100 r/min; nitrogen flow controls as 0.1vvm; tank pressure controls as 0.03-0.04 MPa, after anaerobism protects 3 h, the logical nitrogen of stopping.After fermentation culture 24 h, put tank.
Using above-mentioned preferred culture medium, in a manner described anaerobic fermentation 24 h, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 5.7 × 108CFU/mL, improves 2.2 times than embodiment 1.And, the concentration of alcohol in fermentation liquid is 111 mg/L, improves 24.7% than embodiment 1.Acetic acid concentration in fermentation liquid is 188 mg/L, reduces 75% than embodiment 1.Butyric acid density in fermentation liquid is 2.7 g/L, reduces 60% than embodiment 1.Achieve beat all result.It addition, glucose, yeast powder and peptone reduce 17%, 64% and 17% than the concentration used by embodiment 1 respectively used by the preferred culture medium formula that used of embodiment 2, save the cost of raw material greatly.
Embodiment 3
(1) preparation of culture medium: with embodiment 2.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 2.
(3) secondary seed is cultivated: with embodiment 2.
(4) three grade fermemtation: in addition to adding regulatory factor, remaining condition of three grade fermemtation is with embodiment 2.
Regulatory factor is added: three grade fermemtation is cultivated when starting, and aseptic, anaerobic at the uniform velocity stream adds acetaldehyde solution (concentration is 0.2 mol/L) to 4 h before fermentation ends, and acetaldehyde total concentration is 3.0 mmol/L.
Anaerobic fermentation 24 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 6.56 × 108CFU/mL, improves 15.1% than embodiment 2；Concentration of alcohol in fermentation liquid is 124 mg/L, improves 11.7% than embodiment 2；Acetic acid concentration is 132 mg/L, reduces 29.8% than embodiment 2；Butyric acid density is 2.1 g/L, reduces 22.2% than embodiment 2.
Embodiment 4
(1) preparation of culture medium: with embodiment 2.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 2.
(3) secondary seed is cultivated: with embodiment 2.
(4) three grade fermemtation: in addition to adding regulatory factor, remaining condition of three grade fermemtation is with embodiment 2.
Regulatory factor is added: when three grade fermemtation cultivation cycle is 6 h, starts aseptic, anaerobic and at the uniform velocity fills into acetaldehyde solution (concentration is 0.2 mol/L) to 4 h before fermentation ends, and acetaldehyde total concentration is 1.5 mmol/L.
Anaerobic fermentation 24 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 6.91 × 108CFU/mL, improves 21.2% than embodiment 2；Concentration of alcohol in fermentation liquid is 130 mg/L, improves 17.1% than embodiment 2；Acetic acid concentration is 134 mg/L, reduces 28.7% than embodiment 2；Butyric acid density is 1.83 g/L, reduces 32.2% than embodiment 2.
Embodiment 5
(1) preparation of culture medium: with embodiment 2.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 2.
(3) secondary seed is cultivated: with embodiment 2.
(4) three grade fermemtation: in addition to adding regulatory factor, remaining condition of three grade fermemtation is with embodiment 2.
Regulatory factor is added: when three grade fermemtation cultivation cycle is 3 h, starts aseptic, anaerobic and at the uniform velocity fills into acetaldehyde solution (concentration is 0.2 mol/L) to 4 h before fermentation ends, and acetaldehyde total concentration is 2.0 mmol/L.
Anaerobic fermentation 24 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 7.3 × 108CFU/mL, improves 28.1% than embodiment 1；Concentration of alcohol in fermentation liquid is 144 mg/L, improves 29.7% than embodiment 2；Acetic acid concentration is 109 mg/L, reduces 42.1% than embodiment 2；Butyric acid density is 1.14 g/L, reduces 57.8% than embodiment 2.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify; all should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (6)

1. the method improving butyric acid bacteria growth efficiency, it is characterised in that: described method is: at butyric acid bacteria fermentation 0-8h Time, starting aseptic, anaerobic at the uniform velocity stream and add acetaldehyde solution to 4-6h before fermentation ends, acetaldehyde total concentration is 1.5-5.0mmol/L；
Described aseptic, anaerobic stream is added acetaldehyde solution and is realized by the method comprised the steps: by logical for glass two blue top capping material bottle Being transformed into threeway indigo plant top capping material bottle, wherein two lead to take over filter, and threeway connects feed supplement syringe needle by silica gel tube；After sterilizing, Being connected on a filter of threeway indigo plant top capping material bottle by pipe fitting by bottled nitrogen, filtered sterile nitrogen is passed through feed supplement bottle The bottle end, after displaced air, the syringe needle of sterilizing penetrates material-feeding port under flame is protected, and carries out acetaldehyde solution feed supplement by peristaltic pump.
The method of raising butyric acid bacteria growth efficiency the most according to claim 1, it is characterised in that: described fermentation is three grades Fermentation, the time of three grade fermemtation is 24h.
The method of raising butyric acid bacteria growth efficiency the most according to claim 1, it is characterised in that: butyric acid bacteria fermentation is used Being formulated as follows of culture medium: glucose 16.6g/L, yeast powder 7.2g/L, calcium carbonate 4.7g/L, peptone 25g/L, phosphorus Acid hydrogen dipotassium 0.5g/L, Magnesium sulfate heptahydrate 0.8g/L, manganese sulfate monohydrate 0.02g/L, pH7.3；Anaerobism pipe slant medium needs another Outer addition agar, agar concentration 20g/L；Sterilizing after deoxygenation.
The method of raising butyric acid bacteria growth efficiency the most according to claim 1, it is characterised in that: comprise the steps:
(1) seed amplification culture and the collection closing bottle seed cell
By aseptic, oxygen free operation requirement, pick out cell to anaerobism pipe slant medium from butyric acid bacteria strain lyophilizing pipe；36-38 DEG C of training After supporting 12-16h, by aseptic, oxygen free operation requirement, addition 10mL is aseptic, anaerobic water prepares anaerobism pipe seed liquor；
Bottled culture medium 30mL of 120mL anaerobism, accesses anaerobism bottle liquid in the ratio of 1-2% by butyric acid bacteria anaerobism pipe seed liquor and trains Support in base, 36-38 DEG C, 100r/min cultivates 12-14h on shaking table and obtain anaerobism bottle seed liquor；Want by aseptic, oxygen free operation Ask, carry out anaerobism bottle seed liquor closing bottle operation, obtain closing bottle seed liquor；
(2) secondary seed is cultivated
After the secondary seed tank sterilizing of culture medium, nitrogen displacement deoxygenation, the lower bottle seed liquor of closing of flame protection accesses two grades of kinds Sub-tank, is carried out as follows secondary seed fermentation culture: cultivation temperature 36-38 DEG C, rotating speed is 100r/min, nitrogen flow Controlling is 0.1vvm, and tank pressure controls to be 0.03-0.04MPa, after anaerobism protection 3h, stops leading to nitrogen, fermentation culture 12-14h After, obtain secondary seed solution；
(3) three grade fermemtation
After the three grade fermemtation tank sterilizing of culture medium, nitrogen displacement deoxygenation, by secondary seed solution with the inoculum concentration subcultivation of 1-2% extremely Three grade fermemtation tank, is carried out as follows three grade fermemtation: fermentation temperature 36-38 DEG C, rotating speed 100r/min, nitrogen flow controls For 0.1vvm, tank pressure controls as 0.03-0.04MPa, after anaerobism protection 3h, the logical nitrogen of stopping；After fermentation culture 24h, put Tank；
When 0-8h is cultivated in three grade fermemtation, start aseptic, anaerobic at the uniform velocity stream and add and fill into acetaldehyde solution to 4-6h before fermentation ends, make Acetaldehyde total concentration is 1.5-5.0mmol/L.
The method of raising butyric acid bacteria growth efficiency the most according to claim 4, it is characterised in that: described secondary seed tank For 10L fermentation tank, described three grade fermemtation tank is 100L fermentation tank.
The method of raising butyric acid bacteria growth efficiency the most according to claim 4, it is characterised in that: cultivate 3h in three grade fermemtation Time, start aseptic, anaerobic and at the uniform velocity fill into acetaldehyde solution to 4h, the total final concentration of 2.0mmol/L of acetaldehyde before fermentation ends.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109706203A (en) * 2018-12-18 2019-05-03 贾召鹏 A kind of preparation method of high yield hyaluronic acid

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
夏会丽等: "NADH氧化途径迁移促进丁酸梭状芽孢杆菌的生长", 《食品与发酵工业》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109706203A (en) * 2018-12-18 2019-05-03 贾召鹏 A kind of preparation method of high yield hyaluronic acid

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