CN107619841A - A kind of fermentation technique of bacillus licheniformis high density fermentation coupling γ polyglutamic acid conversions - Google Patents
A kind of fermentation technique of bacillus licheniformis high density fermentation coupling γ polyglutamic acid conversions Download PDFInfo
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- CN107619841A CN107619841A CN201610573907.6A CN201610573907A CN107619841A CN 107619841 A CN107619841 A CN 107619841A CN 201610573907 A CN201610573907 A CN 201610573907A CN 107619841 A CN107619841 A CN 107619841A
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- polyglutamic acid
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Abstract
Liquid high-density fermentation is carried out the invention discloses a kind of bacillus licheniformis and with the strain, while passes through glutamic acid feed supplement, the fermentation technique of coupling γ polyglutamic acid conversions.Its main flow has solid plate to spread cultivation, prepared by shake-flask seed liquid, fermentor liquid high density fermentation and γ polyglutamic acids convert.Its advantage is that cost is cheap, and flow is simple, easy to operate, and the thalline gemma and γ polyglutamic acids obtained has very high application value in agricultural industry.
Description
Technical field
The present invention relates to a kind of bacillus and the method fermented with the bacterium and convert gamma-polyglutamic acid, and in particular to one
Plant bacillus licheniformis and carry out high density liquid fermentation and the method for carrying out gamma-polyglutamic acid conversion with the bacterium.
Background technology
Bacillus（Bacillus）It is the superior microorganism population of generally existing in soil micro-ecosystem.The fungus strain all has
Very high anti-adversity ability and antibacterial action, the biological control available for a variety of soil-borne diseases.Bacillus by successfully colonize to
Plant rhizosphere, body surface or in vivo, and the nutriment in pathogen competitive environment, while antibacterial material is secreted so as to suppress cause of disease
Bacteria growing, its metabolite can also induce plant defense system to resist disease fungus invasion jointly, reach the biology of plant disease
Prevention effect.
Bacillus licheniformis（Bacillus lincheniformis）It is a kind of aerobic, production gemma, shaft-like gram sun
Property bacterium, it is nontoxic to people and animals, a variety of antibiotic and biology enzyme can be produced, there is broad spectrum antibiotic activity and extremely strong degeneration-resistant energy
Power.The bacterium can adjust flora imbalance and reach therapeutic purposes, and body can be promoted to produce antibacterial substance, kill pathogenic bacteria.Energy
Resistant activity material is produced, and there is unique biology take oxygen mechanism, the growth and breeding of pathogenic bacteria can be suppressed.
Gamma-polyglutamic acid（poly-γ-glutamic acid）It is to be bonded by Pidolidone and D-Glu by acid amides
A kind of peptide molecule formed is closed, there is thickening, emulsification, film forming, moisturizing, flocculation, bonding, nontoxic, water-soluble, biodegradable etc.
Performance, suitable for fields such as agricultural, food, cosmetics, biomedicine, environmental protection.Particularly in agriculture field, γ-PGA can
Using the slow-released carrier as agricultural chemicals, in fertilizer, Insecticides (tech) ＆ Herbicides (tech), pest repellant etc. in use, add appropriate γ-
PGA salt can extend these medicines and use effect in the residence time on effective object surface and the action time of active component, raising
Fruit, reduce the usage amount of fertilizer and pesticide.γ-PGA itself can also be used as new liquid Fertilizer application.There are document report, γ-poly-
Nitrogen and phosphorus in the degradable soil of glutamic acid, so as to strengthen the utilization rate of nitrogenous fertilizer and phosphate fertilizer.
In summary, bacillus licheniformis has good biocontrol effect, and its metabolite γ-PGA can be used as agricultural
The sustained release agent of chemicals, itself can also be used as liquid fertilizer.The two is combined, and can improve crops and agricultural chemicals biological and ecological methods to prevent plant disease, pests, and erosion
Ability, extend the action time of fertilizer and pesticide and reduce usage amount, reduce destruction of the agricultural chemicals to ecological environment.
The content of the invention
The present invention is intended to provide a kind of carrying out the same of bacillus licheniformis high density liquid fermentation efficiently at low cost
When, obtain the fermentation process of the gamma-polyglutamic acid of comparable concentrations.
Technical scheme is as follows：
The liquid fermentation stage is divided into two stages, and the first stage is the high density viable bacteria fermentation stage, and second stage is polyglutamic acid
Transformation stage.
The polyglutamic acid transformation stage is lived with high density viable bacteria fermentation stage bacterium reaches 1 × 1010It is used as during more than cfu/mL
Initial point；Into after the transformation stage, by 10~60 g/L conversion precursor（One kind or its mixture in glutamic acid, sodium glutamate）
With 10~60 g/L nutrient matrix（Glucose, sucrose, bean cake powder, beancake powder, corn steep liquor or its mixture）Batch feeding, and
Zymotic fluid pH is maintained between 6.0~6.5, to accelerate the conversion of gamma-polyglutamic acid, until fermentation broth viscosity is not further added by；Mend
After the completion of material, continue culture 24 hours according to original parameter, to gemma rate >=90%, terminate fermentation.
The present invention has advantages below：
1）The present invention provides a kind of method of bacillus licheniformis high density liquid fermentation, and this method is simple to operate, and cost is cheap,
There is very high operability, the related request of industrialized production can be met.
2）The present invention provide it is a kind of in the bacillus licheniformis liquid fermentation middle and later periods, by converting precursor and nutrient matrix
Feed operation stimulates the conversion of gamma-polyglutamic acid, thalli growth and the coupling of gamma-polyglutamic acid conversion is realized, so as to obtain
While high density bacillus licheniformis bacterium solution, the gamma-polyglutamic acid of comparable concentrations is obtained.
3）For the overall fermentation time of the present invention at 48~72 hours or so, fermentation period was short, and fermenting speed is fast, saves a large amount of people
Power material resources, while bacillus licheniformis bacterium amount is larger, gamma-polyglutamic acid concentration is considerable, relatively low to operation level requirement, has work
Industry Development volue.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
The various changes carried out on the premise of invention spirit and scope to the material component in these embodiments and dosage or change
Belong to protection scope of the present invention.
The numeral being formulated in following examples typicallys represent w/v without unit, and unit is g/L.
Embodiment 1
In the triangular flask for 5 L that the bacillus licheniformis access for taking inclined-plane to preserve is equipped with liquid seed culture medium, liquid amount 20%,
Incubated 12 hours at 37 DEG C, the rpm of rotating speed 200；The composition of the fluid nutrient medium, in terms of bulking value, unit is g/
L, wherein glucose 5, bean cake powder 5, farina 10, manganese sulfate 0.2, pH value 7.5；
Seed liquor is transferred after sterilizing（121 DEG C, 30 minutes of sterilising conditions）Fermentation tank in, carry out liquid fermentation, inoculum concentration
5%, liquid amount 70%, incubated 12 hours, rotating speed 300rpm at 37 DEG C, initial 1.0, the pH controls of ventilating ratio are 5.0~8.0
Between；The composition of the fermentation medium, in terms of bulking value, unit g/L, wherein glucose 10, beancake powder 5, corn steep liquor 2,
Manganese sulfate 0.2, sodium chloride 1, dipotassium hydrogen phosphate 1 and defoamer 2, pH value 7.5；
After fermentation tank culture 12 hours, with blood counting chamber count zymotic fluid viable bacteria density, bacterium live >=100 hundred million/mL when, by 20 paddy
Propylhomoserin sodium, 20 glucose carry out feed supplement, stimulate the conversion of thalli growth and gamma-polyglutamic acid, until fermentation broth viscosity no longer increases
Add.After the completion of feed supplement, according to original parameter continue culture 24 hours, pH control between 6.0~8.0, cultivate to gemma rate >=
90%, stop fermentation.
By national standard counting method, thalline fermentation unit is 1.12 × 1010cfu/ml.100 ml zymotic fluids are taken, pass through centrifugation
Thalline is removed, the cold ethanol of 3 times of volumes is added into supernatant（4℃）, centrifuge after refrigerated overnight and the supernatant that inclines, will precipitate
It is re-dissolved in isometric deionized water, is detected after hydrolysis through paper chromatography, gamma-polyglutamic acid content is 11.32 g/L.
Wherein described paper chromatography solvent for use system is n-butanol：80% formic acid（V/V）：Water=15：3：2, developer
For 0.5 % ninhydrin acetone solns, through 0.1 % copper sulphate（CuSO4·5H2O）：75% ethanol（V/V）=2：After 38 solution elution
Absorbance is determined in 520 nm, the content of gamma-polyglutamic acid is calculated by glutamic acid standard curve.
Embodiment 2
With example 1, farina therein is changed into the cornstarch of identical mass concentration, feed phase glucose changes
For the sucrose of identical mass concentration.
After fermentation ends, analysis result shows that bacillus licheniformis fermentation unit is 1.01 × 10 in zymotic fluid10Cfu/ml,
The content of gamma-polyglutamic acid is 12.69 g/L.
Embodiment 3
With embodiment 1, the sodium glutamate of feed phase is changed into the Pidolidone of equivalent.
After fermentation ends, analysis result shows that bacillus licheniformis fermentation unit is 1.21 × 10 in zymotic fluid10cfu /
Ml, the content of gamma-polyglutamic acid is 12.38 g/L.
Embodiment 4
With embodiment 1, the sodium glutamate of feed phase and glucose dosage are changed into 25g/L.
After fermentation ends, analysis result shows that bacillus licheniformis fermentation unit is 1.11 × 10 in zymotic fluid10cfu /
Ml, the content of gamma-polyglutamic acid is 10.4 g/L.
Embodiment 5
With embodiment 1, when by the starting point selection of feed phase being that zymotic fluid viable bacteria density reaches 12,000,000,000/mL, sodium glutamate and
Glucose dosage changes into 700g.
After fermentation ends, analysis result shows that bacillus licheniformis fermentation unit is 1.35 × 10 in zymotic fluid10cfu /
Ml, the content of gamma-polyglutamic acid is 15.66 g/L.
Embodiment 6
With embodiment 1, the sodium glutamate of feed phase is changed into the Pidolidone of equivalent, glucose is changed into 1:1 mass
The sucrose of ratio：Bean cake powder.
After fermentation ends, analysis result shows that bacillus licheniformis fermentation unit is 1.15 × 10 in zymotic fluid10cfu /
Ml, the content of gamma-polyglutamic acid is 13.82 g/L.
Claims (4)
1. a kind of method that bacillus licheniformis high density liquid ferments and is coupled gamma-polyglutamic acid conversion, it is characterised in that：Liquid
Body fermentation is divided into two stages, and the first stage is the high density viable bacteria fermentation stage, and second stage is the polyglutamic acid transformation stage.
2. method as claimed in claim 1, it is characterised in that：The gamma-polyglutamic acid transformation stage is with the high density viable bacteria fermentation stage
Bacterium, which lives, reaches starting points of 10,000,000,000 more than the cfu/mL as the transformation stage；
Method as claimed in claim 1, it is characterised in that：The gamma-polyglutamic acid transformation stage, by 10-60 g/L conversion precursor
With 10-60 g/L nutrient matrix batch feeding, accelerate the conversion of gamma-polyglutamic acid, until fermentation broth viscosity is not further added by；Mend
After the completion of material, continue culture 24 hours according to original parameter, to gemma rate >=90%, terminate fermentation.
3. gamma-polyglutamic acid transformation stage feed process as claimed in claim 3, it is characterised in that：Described conversion precursor
For one or more of mixtures in glutamic acid, sodium glutamate；
Gamma-polyglutamic acid transformation stage feed process as claimed in claim 3, it is characterised in that：Described nutrient matrix is
Glucose, sucrose, bean cake powder, beancake powder, corn steep liquor or its mixture.
4. gamma-polyglutamic acid transformation stage feed process as claimed in claim 3, it is characterised in that：Zymotic fluid pH value maintains
6.0~6.5.
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Cited By (1)
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| CN109182405A (en) * | 2018-10-15 | 2019-01-11 | 天津科技大学 | A method of γ-polyglutamic acid is produced using lichens bud pole bacterium solid state fermentation |
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| CN102367431A (en) * | 2011-11-11 | 2012-03-07 | 中国科学院过程工程研究所 | Bacillus licheniformis and applications thereof |
| CN103205388A (en) * | 2013-05-08 | 2013-07-17 | 东莞市保得生物工程有限公司 | Method for producing viable bacteria of bacillus licheniformis by high-density solid fermentation |
| CN103820508A (en) * | 2014-01-27 | 2014-05-28 | 广东省微生物研究所 | Application of rare earth element in production of gamma-polyglutamic acid by bacillus licheniformis fermentation |
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| CN102367431A (en) * | 2011-11-11 | 2012-03-07 | 中国科学院过程工程研究所 | Bacillus licheniformis and applications thereof |
| CN103205388A (en) * | 2013-05-08 | 2013-07-17 | 东莞市保得生物工程有限公司 | Method for producing viable bacteria of bacillus licheniformis by high-density solid fermentation |
| CN103820508A (en) * | 2014-01-27 | 2014-05-28 | 广东省微生物研究所 | Application of rare earth element in production of gamma-polyglutamic acid by bacillus licheniformis fermentation |
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