CN105950538A - Method for promoting fermentation and growth of butylic acid bacteria - Google Patents

Method for promoting fermentation and growth of butylic acid bacteria Download PDF

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CN105950538A
CN105950538A CN201610356791.0A CN201610356791A CN105950538A CN 105950538 A CN105950538 A CN 105950538A CN 201610356791 A CN201610356791 A CN 201610356791A CN 105950538 A CN105950538 A CN 105950538A
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acid bacteria
fermentation
butyric acid
tyrosine
anaerobism
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CN105950538B (en
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王志
陈雄
李冬生
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Hubei University of Technology
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    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The invention discloses a method for promoting fermentation and growth of butylic acid bacteria, and belongs to the field of food fermentation. According to the method, fermentation and growth of the butylic acid bacteria are promoted by reducing production of acidic products during fermentation. The method comprises that tyrosine aqueous solution is added through fed-batch in a sterile and anaerobic mode within 1h after the butylic acid bacteria starts fermentation, and the total concentration of tyrosine is 0.0125-0.05g/L. According to the method, the tyrosine is used as metabolic regulation molecules, and the production efficiency of acetic acid and butyric acid is remarkably reduced by addition of tyrosine molecules, thus effectively mitigating acid stress, promoting growth of the butylic acid bacteria, and bringing about a great technical effect. The method has the characteristics of clear components, easily available raw materials, low usage concentration, low material supplement costs, high operation controllability, and applicability to fermentation production of the butylic acid bacteria.

Description

A kind of method promoting butyric acid bacteria Fermentative growth
Technical field
The invention belongs to field of food fermentation, be specifically related to a kind of method promoting butyric acid bacteria Fermentative growth.
Background technology
Butyric acid bacteria (Clostridium butyricum,Clostridium butyrium) as people with or animal new probiotic bacterium there is adjustment intestinal microbial population balance, strengthen immunity, the critical function such as prophylaxis of tumours generation.The nutritional need of butyric acid bacteria is notable to its growth effect, conventional feed supplement nitrogen source is peptone, yeast extract, bean cake, Semen Glycines powder hydrolyzed solution, fish flour etc., as: documents 1(uses the method that fed-batch fermentation method prepares active clostridium butyrium agent, patent of invention CN201110287802.1,2011) disclosing butyric acid bacteria sweat fills into the glucose (carbon source) of 10-20% and the method for the Semen Glycines powder hydrolyzed solution (nitrogen source) of 5-10%；The method that documents 2(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, the nitrogen source wherein filled into is the Semen Glycines powder hydrolyzed solution etc. of 6-8%.Obviously, documents 1 and 2 all without reference to butyric acid bacteria during the fermentation acid metabolic regulation and control content.Complexity, uncertainty and the non-intellectual affected consideration and the organic nitrogen source composition of nitrogen source nutritional need to butyric acid bacteria growth metabolism for butyric acid bacteria, documents 1 and 2 have employed organic nitrogen source (Semen Glycines powder hydrolyzed solution) and carrys out the general nitrogen source demand meeting butyric acid bacteria.But, these organic nitrogen source complicated component, large usage quantities so that production cost is higher, and which kind of composition of organic nitrogen source is most suitable for the unknown of butyric acid bacteria growth metabolism.
Synthesize substantial amounts of butanoic acid during butyric acid bacteria Fermentative growth and acetic acid is the result of its metabolism adaptive Evolutionary.But, acetic acid, the accumulation of butanoic acid also can cause acid stress effect, and cause aqtocytolysis.Therefore, if energy regulating cell acid metabolic, cytoactive may be made to be maintained, and promote cell growth further.Select suitable metabolic regulation molecule may realize above-mentioned purpose.
Tyrosine is a kind of aromatic series polarity a-amino acid containing phenolic hydroxyl group, extracts, be human body non essential amino acid from the hydrolyzed solution containing protein material such as casein or Semen Maydis.Food stage tyrosine on March 15th, 2016 price be 25 yuan/kg(http: //www.21food.cn/quote/113346.html).Pharmaceutically it is used as treatment hyperthyroidism, alleviates vitiligo symptom and antidepressant drug, it is possible to as food additive.It addition, tyrosine is also the primary raw material of a kind of important chemical industry synthesis medicine such as peptide hormone, L-3,4 dihydroxyphenylalanine.
But it does not have tyrosine molecule is applied to anaerobe as metabolic factor---the report of the New function of butyric acid bacteria incubation, it may be assumed that tyrosine is as metabolic molecule regulation and control butyric acid bacteria growth metabolism the report of the New function promoting its Fermentative growth.
Summary of the invention
Present invention aim at providing a kind of new method promoting butyric acid bacteria Fermentative growth, it is a kind of method promoting butyric acid bacteria Fermentative growth by regulation and control butyric acid bacteria acid metabolic.
The object of the invention is achieved through the following technical solutions:
A kind of method promoting butyric acid bacteria Fermentative growth is the interpolation tyrosine when butyric acid bacteria ferments.Being preferably: aseptic within butyric acid bacteria ferments 1 h, anaerobic stream adds tyrosine solution, and tyrosine total concentration is 0.0125-0.05 g/L, the amount that i.e. every liter fermentation broth stream adds tyrosine is 0.0125-0.05 g.It is furthermore preferred that tyrosine total concentration is 0.025 g/L.
Preferably, the described method promoting butyric acid bacteria Fermentative growth, 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 60 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) fermentation culture
After fermentation tank (100 L) sterilizing of culture medium, nitrogen displacement deoxygenation, protect with inoculum concentration (v/v) flame of 1-2% access fermentation tank by closing bottle seed liquor, be carried out as follows fermentation culture: fermentation temperature 36-38 DEG C, rotating speed 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 1 h, the logical nitrogen of stopping.After fermentation culture 12-14 h, put tank.Fermentation starts aseptic within 1 h, anaerobic at the uniform velocity stream and adds and fill into tyrosine solution, and making tyrosine total concentration is 0.0125-0.05 g/L；Preferably, tyrosine total concentration is 0.025 g/L。
The preparation of described culture medium is preferably: glucose 20 g/L, peptone 30 g/L, yeast powder 5 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 g/L；pH 7.3.After deoxygenation standby through 0.1 MPa steam sterilization 20 min.
The present invention not using tyrosine molecule as food additive, treat hyperthyroidism, alleviate vitiligo symptom and antidepressant medicine and the primary raw material as the chemical industry synthesis medicine such as peptide hormone, L-3,4 dihydroxyphenylalanine uses, but the regulatory molecule as butyric acid bacteria growth metabolism uses.
Compared with prior art the invention have the advantages that and significantly improve: adding the Semen Glycines powder hydrolyzed solution of 5-10% compared to documents 1(, total nitrogen source has reached 50-100 g/L), documents 2(add the Semen Glycines powder hydrolyzed solution of 6-8%, total nitrogen source has reached 60-80 g/L) the Semen Glycines powder hydrolyzed solution of complicated component that used and the highest concentration, the present invention has definite ingredients, raw material is easily purchased, the lowest (preferred concentration only has 0.025 to concentration G/L), the most additionally increase production cost and (cultivate 50 m zymometer material with batch, only use 1.25 kg, every batch fermentation only to increase cost 31.25 yuan, about add the 1% of the nitrogen source costs such as Semen Glycines powder hydrolyzed solution), operation controllability is high, be applicable to the feature of fermenting and producing.
The interpolation of tyrosine molecule promotes the Fermentative growth of butyric acid bacteria, and, also create beat all effect: the interpolation of tyrosine molecule makes the acetic acid of every hundred million Hemapoiesis, butanoic acid significantly reduce, reduce 31.6-47.4% and 27.7-41.8% when putting tank respectively, effectively alleviate acid stress, promote the growth of butyric acid bacteria.When putting tank, butyric acid bacteria viable count improves 40-77.3%, 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 5 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 g/L；pH 7.3.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 60 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) fermentation culture
Equipped with culture medium fermentation tank (100 L) sterilizing, nitrogen displacement deoxygenation after; access fermentation tank is protected with inoculum concentration (v/v) flame of 1% by closing bottle seed liquor; it is carried out as follows fermentation culture: fermentation temperature 37 DEG C; rotating speed 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 1 h, stops logical nitrogen.After fermentation culture 14 h, put tank.
Anaerobic fermentation 14 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 3.7 × 108CFU/mL, the acetic acid that in fermentation liquid, every hundred million cells produce is 0.19 mg, and the butanoic acid that every hundred million cells produce is 0.55 mg.
Embodiment 2
(1) preparation of culture medium: with embodiment 1.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 1.
(3) fermentation culture: in addition to adding regulatory factor, remaining condition of fermentation culture is with embodiment 1.
Regulatory factor is added: fermentation is aseptic within starting 1 h, anaerobic at the uniform velocity stream adds tyrosine solution (concentration is 2.5 g/L), and making tyrosine total concentration is 0.0125 g/L.
Anaerobic fermentation 14 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 5.52 × 108CFU/mL, improves 49.2% than embodiment 1；The acetic acid that in fermentation liquid, every hundred million cells produce is 0.13 mg, reduces 31.6% than embodiment 1；The butanoic acid that every hundred million cells produce is 0.4 mg, reduces 27.7% than embodiment 1.
Embodiment 3
(1) preparation of culture medium: with embodiment 1.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 1.
(3) fermentation culture: in addition to adding regulatory factor, remaining condition of fermentation culture is with embodiment 1.
Regulatory factor is added: fermentation is aseptic within starting 1 h, anaerobic stream adds tyrosine solution (concentration is 2.5 g/L), and making tyrosine total concentration is 0.025 g/L.
Anaerobic fermentation 14 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 77.3% than embodiment 1；The acetic acid that in fermentation liquid, every hundred million cells produce is 0.1 mg, reduces 47.4% than embodiment 1；The butanoic acid that every hundred million cells produce is 0.32 mg, reduces 41.8% than embodiment 1.
Embodiment 4
(1) preparation of culture medium: with embodiment 1.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 1.
(3) fermentation culture: in addition to adding regulatory factor, remaining condition of fermentation culture is with embodiment 1.
Regulatory factor is added: fermentation is aseptic within starting 1 h, anaerobic stream adds tyrosine solution (concentration is 2.5 g/L), and making tyrosine total concentration is 0.035 g/L.
Anaerobic fermentation 14 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 5.27 × 108CFU/mL, improves 42.4% than embodiment 1；The acetic acid that in fermentation liquid, every hundred million cells produce is 0.116 mg, reduces 39% than embodiment 1；The butanoic acid that every hundred million cells produce is 0.378 mg, reduces 31.3% than embodiment 1.
Embodiment 5
(1) preparation of culture medium: with embodiment 1.
(2) seed liquor amplification culture is with the collection closing bottle seed cell: with embodiment 1.
(3) fermentation culture: in addition to adding regulatory factor, remaining condition of fermentation culture is with embodiment 1.
Regulatory factor is added: fermentation is aseptic within starting 1 h, anaerobic stream adds tyrosine solution (concentration is 2.5 g/L), and making tyrosine total concentration is 0.05 g/L.
Anaerobic fermentation 14 h in a manner described, cell number (carrying out bacterium colony CFU counting with Hungate rolling pipe counting method) is for 5.17 × 108CFU/mL, improves 39.7% than embodiment 1；The acetic acid that in fermentation liquid, every hundred million cells produce is 0.118 mg, reduces 37.9% than embodiment 1；The butanoic acid that every hundred million cells produce is 0.379 mg, reduces 31.1% than embodiment 1.
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 (7)

1. the method promoting butyric acid bacteria Fermentative growth, it is characterised in that: described method is: add when butyric acid bacteria ferments Tyrosine.
The method of promotion butyric acid bacteria Fermentative growth the most according to claim 1, it is characterised in that: start in butyric acid bacteria fermentation In 1h, aseptic, anaerobic stream adds tyrosine solution, and tyrosine total concentration is 0.0125-0.05g/L.
The method of promotion butyric acid bacteria Fermentative growth the most according to claim 2, it is characterised in that: tyrosine total concentration is 0.025g/L。
The method of promotion butyric acid bacteria Fermentative growth 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 60mL 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) fermentation culture
After the fermentation tank sterilizing of culture medium, nitrogen displacement deoxygenation, protect with the inoculum concentration flame of 1-2% connect closing bottle seed liquor Enter fermentation tank, be carried out as follows fermentation culture: fermentation temperature 36-38 DEG C, rotating speed 100r/min, nitrogen flow control is 0.1vvm, tank pressure controls as 0.03-0.04MPa, after anaerobism protection 1h, the logical nitrogen of stopping；After fermentation culture 12-14h, Put tank；Fermentation starts aseptic within 1h, anaerobic at the uniform velocity stream and adds and fill into tyrosine solution, and making tyrosine total concentration is 0.0125-0.05 g/L。
The method of promotion butyric acid bacteria Fermentative growth the most according to claim 4, it is characterised in that: being prepared as of culture medium: Glucose 20g/L, peptone 30g/L, yeast powder 5g/L, dipotassium hydrogen phosphate 0.5g/L, Magnesium sulfate heptahydrate 0.5g/L, lightweight Calcium carbonate 1g/L, manganese sulfate monohydrate 0.02g/L, pH7.3；Anaerobism pipe slant medium need to it is possible to additionally incorporate agar, agar concentration 20g/L；pH 7.3；After deoxygenation standby through 0.1MPa steam sterilization 20min.
The method of promotion butyric acid bacteria Fermentative growth the most according to claim 4, it is characterised in that: described fermentation tank is 100 L fermentation tank.
The method of promotion butyric acid bacteria Fermentative growth the most according to claim 4, it is characterised in that: stream adds the tyrosine filled into Total concentration is 0.025g/L.
CN201610356791.0A 2016-05-26 2016-05-26 A method of promoting Miyarisan Fermentative growth Active CN105950538B (en)

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