CN107815476A - A kind of method that γ polyglutamic acids are produced using bacillus licheniformis - Google Patents
A kind of method that γ polyglutamic acids are produced using bacillus licheniformis Download PDFInfo
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Abstract
The present invention relates to a kind of method that γ polyglutamic acids are produced using bacillus licheniformis, comprise the following steps：Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium and cultivates activation；The preparation of seed liquor；Inclined-plane seed after activation is inoculated in seed culture medium, the seed liquor of γ polyglutamic acids must be produced；Seed liquor is inoculated in fermentation medium and fermented；Thalline is removed in centrifugation after fermentation ends, and alcohol precipitation separates out polyglutamic acid, dries, prepares dry powder-shaped γ polyglutamic acids.The present invention implements replacement using soy molasses to the glucose in original formula.The yield of γ polyglutamic acids, which can not only be improved, can also simplify production technology, while greatly reduce production cost.
Description
Technical field
The invention belongs to field of agricultural microbial technology, is related to one kind using bacillus licheniformis production gamma-polyglutamic acid
Method.
Background technology
Gamma-polyglutamic acid is a kind of boiomacromolecule carrier material, is in anthrax spore bar earliest by lvanovics et al.
Found in the folder film of bacterium, be bacillus such as bacillus subtilis, bacillus licheniformis etc. produces during vital movement
Raw secondary metabolite, it can currently be prepared in laboratory by the method for biofermentation.With the progress and industry of science and technology
Development, thing followed problem of environmental pollution is also increasing, therefore each field is more likely to development and application environment is friendly
Section bar material.And gamma-polyglutamic acid is also increasingly closed as a kind of biodegradable ' green ' high polymer material by people
Note.Because it has good water absorption character, and no matter in inside of human body, or its final catabolism in external environment
Product is glutamic acid, and human body is had no toxic side effect, environmentally safe, therefore all shows its superiority in many fields.
It is known that it is widely used at cosmetic industry, food service industry, health care industry, agricultural aspect.
Glucose（Glucose）It is a kind of most wide and mostly important monose of distributed in nature, it is a kind of polyhydroxy aldehyde.
Glucose has critical role in field of biology, is the energy source of living cells and metabolic intermediate product, i.e., biological
Main Energy supply material.Plant can produce glucose by photosynthesis.It is taken as most important C sources to have in biofermentation industry
Extremely it is widely applied.Just still made in the currently biological fermentation culture medium of production gamma-polyglutamic acid using simple glucose
For C sources, but production cost is big, in order to reduce production cost, increases the benefit, it is necessary to be entered using cheap C sources to glucose
Row is replaced, but does not find the method for replacing glucose also at present.
The content of the invention
The technical problems to be solved by the invention are in view of the shortcomings of the prior art, there is provided one kind utilizes bacillus licheniformis
The method for producing gamma-polyglutamic acid.The present invention implements replacement using soy molasses to the glucose in original formula.Not only may be used
It can also simplify production technology to improve the yield of gamma-polyglutamic acid, while greatly reduce production cost.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, in 30 DEG C of -37 DEG C of culture 16-
18 hours, so activation 2-3 times, prepare the inclined-plane seed after the activation of maturation；
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain exist on October 14th, 2009
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, its preserving number is CGMCCNo.3336；Bacillus licheniformis in the application
（Bacilluslicheniformis）TKPG091 is the preservation of bacteria strain being disclosed in the prior art.
（2）The preparation of seed liquor；By step（1）Inclined-plane seed after middle activation is inoculated in seed culture medium, 37 DEG C, is shaken
Bed rotating speed 220rpm cultivates 16 hours to exponential phase, obtains the seed liquor of production gamma-polyglutamic acid；
（3）Ferment tank：According to fermentation medium stereometer, by 5% inoculum concentration, by step（2）In seed liquor inoculation
In fermentation medium, 37-38 DEG C of control Preliminary fermentation temperature, tank pressure 0.02-0.05MPa；Ventilation 0.5-1.5vvm；Initially
Zymotic fluid pH value is adjusted to 7.0, cultivates 72 hours；
The fermentation medium component is：Corn steep liquor 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous calcium chloride
1.5g/L, NH4Cl5.52g/L, soy molasses 80g-200g/L, surplus are water；Sterilize 30-40min at 125 DEG C of temperature, obtains
To described fermentation medium；
It is understood that：Soy molasses be production FSPC during, the molten moieties of alcohol, the production after concentration
Product, because of color honey similar with mobility, so be named as soy molasses, it contains abundant dextrose and saccharose and a small amount of
Albumen, and microorganism needs glucide in growth course and albumen removes to synthesize material needed for itself.Simultaneously in poly- paddy
In the fermentation production process of propylhomoserin, excessive concentration then can be more when glucose does C sources synthesis polysaccharide and synthesize polyglutamic acid
Amount can then be reduced.Based on these features, soy molasses are one of best substitutes of glucose.1. it can ensure C sources total amount
The component proportion of glucose is reduced in the case of constant, weakens the synthesis of polysaccharide.2. relative to the single energy of glucose
Supply, it contains more rich energy matter.This 2 reasons are that it can improve the support of yield.
We have found in an experiment：In the fermentation production process of polyglutamic acid, if concentration mistake when doing C sources with glucose
It is high then can be more synthesis polysaccharide and the amount that synthesizes polyglutamic acid can then reduce, while concentration of glucose is too low and is unfavorable for thalline
Growth.Then we have attempted to do C sources with soy molasses, are made reference with the concentration of glucose being formulated always and a soybean is determined
The interval range of molasses has carried out gradient experiment, and experiment proves that soy molasses have very big value.
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out poly- paddy with the ethanol of 4-5 times of supernatant volume, alcohol precipitation
Propylhomoserin, then redissolve after the distilled water of supernatant volume, dry again；Prepare dry powder-shaped gamma-polyglutamic acid.
As preferable technical scheme：
Preferably, the yield of gamma-polyglutamic acid in sample is detected by efficient liquid phase.
Preferably, slant medium composition is tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar
Powder 20g/L.
Preferably, the seed culture medium composition is：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, chlorine
Change potassium 0.5g/L, MgSO4·7H2O 0.5g/L, pH7.2, surplus is water；Sterilize 30-40min at 125 DEG C of temperature, obtains institute
The seed culture medium stated.
Preferably, the pH of the fermentation medium is to use 1~4mol/L sulfuric acid, hydrochloric acid, sodium hydroxide or potassium hydroxide
Aqueous solution regulation.
Preferably, the drying refers to utilizing spray drying process, prepares dry powder-shaped gamma-polyglutamic acid.
Preferably, the molecular weight of the gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
Soy molasses contain original water-soluble in isoflavones, soyabean oligosaccharides, soybean protein, sucrose, monose and soybean
Property mineral element etc., is yeast-leavened best nutriment, it can also be used in the application of other bacterial fermentations.It may further be used to carry
Soybean lsoflavons, soyabean oligosaccharides etc. are taken, are to have feed addictive and trophism, cheap feedstuff concurrently.
The present invention implements replacement using soy molasses to the glucose in original formula.γ-poly- paddy can not only be improved
The yield of propylhomoserin can also simplify production technology, reduce production cost.It also demonstrate that bacillus licheniformis to containing compound carbohydrate simultaneously
Residue also have good utilization ratio, for it is follow-up some using the strain that glucose effect is bad or cost is too high, greatly
Beans molasses can be used as a kind of good substitute.Secondly, by the microscopy to thalline, find to make tank under C sources with soy molasses
When somatic cells it is more mellow and fuller, primarily determine that in soy molasses containing there is the material of certain protective effect to thalline.
Embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, art technology
Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
Embodiment 1
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：Bacillus licheniformis CGMCCNO.3336 original strains are seeded on slant medium, in 37 DEG C of trainings
Support 18 hours, prepare the inclined-plane seed of maturation；
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain exist on October 14th, 2009
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, its preserving number is CGMCCNo.3336；
（2）The preparation of seed liquor：Inclined-plane seed after activation is inoculated in the 500mL triangular flasks equipped with 50mL fluid nutrient mediums
In, 37 DEG C, shaking speed 220rpm cultivates 16 hours to exponential phase, with reference to OD660Value is grown can at 0.3
The seed culture medium forms：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O 0.5g/L, pH7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature.
（3）Ferment tank：By step（2）In seed liquor 1.5L be inoculated in fermentation tank, with 5% inoculum concentration, 20L hairs
Fermenting pot culture medium loading amount is 10L, 38 DEG C of Preliminary fermentation temperature, tank pressure 0.03MPa, ventilation 1vvm；Preliminary fermentation liquid pH
Value is adjusted to 7, cultivates 72 hours.The pH of fermentation medium is adjusted using 1mol/L sulfuric acid, sodium hydrate aqueous solution.Fermentation training
Supporting base component is：Corn steep liquor 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous calcium chloride 1.5g/L,
NH4Cl5.52g/L, soy molasses 115g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtained fermentation medium；
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic acid with the ethanol of 5 times of supernatant volumes, alcohol precipitation,
Then redissolve again after the distilled water of supernatant volume, and constant volume obtains sample after 25mL by hydrolysis process, passes through efficient liquid
Mutually detect its gamma-polyglutamic acid yield is 19g/L. conversion ratios 30% or so.Substitute glucose saving cost 2800RMB per ton
Left and right, dry powder-shaped gamma-polyglutamic acid is prepared using spray drying process.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand
Dalton.
Embodiment 2
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, cultivated 16 hours in 30 DEG C,
Prepare the inclined-plane seed after activation；
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain exist on October 14th, 2009
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, its preserving number is CGMCCNo.3336；
（2）The preparation of seed liquor；Inclined-plane seed after activation is inoculated in the 500mL triangular flasks equipped with 50mL fluid nutrient mediums
In, 37 DEG C, shaking speed 220rpm cultivates 16 hours to exponential phase, can at 0.3 with reference to being grown with OD660 values；Produced
The seed liquor of gamma-polyglutamic acid；
Seed culture medium forms：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O 0.5g/L, pH7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature, obtained seed culture medium.
（3）Ferment tank：According to fermentation medium stereometer, by 5% inoculum concentration, by step（2）In seed liquor
It is inoculated in fermentation medium, 20L ferment tank culture mediums loading amount is 10L, 37 DEG C of control Preliminary fermentation temperature, tank pressure
0.02MPa；Ventilation 0.5vvm；Preliminary fermentation liquid pH value is adjusted to 7；The pH of the fermentation medium be using 4mol/L hydrochloric acid or
Potassium hydroxide aqueous solution regulation.
The fermentation medium component is：Corn steep liquor 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous chlorination
Calcium 1.5g/L, NH4Cl5.52g/L, soy molasses 80g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtains described
Fermentation medium；
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic acid with the ethanol of 4 times of supernatant volumes, alcohol precipitation,
Then redissolve again after the distilled water of supernatant volume, then pure water redissolves and constant volume obtains sample after 25mL by hydrolysis process
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield is poly- paddy yield 25g/L, conversion ratio 35% or so, substitute grape
Sugar saving cost 3000RMB per ton or so；Dry powder-shaped gamma-polyglutamic acid is prepared using spray drying process.Gamma-polyglutamic acid
Molecular weight be 20 ten thousand to 200 ten thousand dalton.
Comparative example 1
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, cultivated 17 hours in 35 DEG C,
Prepare the inclined-plane seed after activation；
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain exist on October 14th, 2009
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, its preserving number is CGMCCNo.3336；
(2) preparation of seed liquor；By step（1）Inclined-plane seed after middle activation is inoculated in equipped with 50mL fluid nutrient mediums
In 500mL triangular flasks, 37 DEG C, shaking speed 220rpm cultivates 16 hours to exponential phase, is grown with reference to OD660 values 0.3
Then can, obtain produce gamma-polyglutamic acid seed liquor；
Seed culture medium forms：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O0.5g/L, pH7.2, surplus are water；Sterilize 35min at 125 DEG C of temperature, obtains described seed culture medium.
（3）Ferment tank：According to fermentation medium stereometer, by 5% inoculum concentration, by step（2）In seed liquor
It is inoculated in fermentation medium, 20L ferment tank culture mediums loading amount is 10L, 38 DEG C of control Preliminary fermentation temperature, tank pressure
0.05MPa；Ventilation 1vvm；Preliminary fermentation liquid pH value is adjusted to 7.0；The pH of the fermentation medium be using 2mol/L hydrochloric acid or
Sodium hydrate aqueous solution regulation.
The fermentation medium component is：Glucose：90g/L, corn steep liquor：25g/L, sodium chloride：15g/L, MgSO4·
7H2O：2g/L, anhydrous calcium chloride：1.5g/L, NH4Cl：5.52g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtains
Fermentation medium；
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic acid with the ethanol of 5 times of supernatant volumes, alcohol precipitation,
Then redissolve again after the distilled water of supernatant volume, then pure water redissolves and constant volume obtains sample after 25mL by hydrolysis process
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield is that poly- paddy yield is that 20g/L. conversion ratios are 30 ﹪ or so.Utilize
Spray drying process prepares dry powder-shaped gamma-polyglutamic acid.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
In contrast from embodiment 1, embodiment 2 with comparative example 1, it can be found that being that C sources substitute grape using soy molasses
Sugar production polyglutamic acid method is feasible, and its poly- paddy yield is not only able to and also part suitable as C sources yield with using glucose
Yield is higher than glucose, wherein particularly evident with 80g/L effects.
Embodiment 3
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, 16- hours are cultivated in 37 DEG C,
Prepare the inclined-plane seed after activation；Slant medium forms:Tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, fine jade
Cosmetics 20g/L.
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain is October 14 in 2009
Day is in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Committee's common micro-organisms center preservation, its preserving number are CGMCCNo.3336；
(2) preparation of seed liquor；By step（1）The middle inclined-plane seed by after activation is inoculated in equipped with 50mL fluid nutrient mediums
In 500mL triangular flasks, 37 DEG C, shaking speed 220rpm cultivates 16 hours to exponential phase, is grown with reference to OD660 values 0.3
Then can, obtain produce gamma-polyglutamic acid seed liquor；
Seed culture medium forms：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O0.5g/L, pH7.2, surplus are water；Sterilize 38min at 125 DEG C of temperature, obtains described seed culture medium.
（3）Ferment tank：According to fermentation medium stereometer, 5% inoculum concentration, by step（2）In seed liquor connect
For kind in fermentation medium, 20L ferment tank culture mediums loading amount is 10L, 38 DEG C of control Preliminary fermentation temperature, tank pressure
0.02MPa；Ventilation 0.5vvm；Preliminary fermentation liquid pH value is adjusted to 7, cultivates 72h；The pH of fermentation medium is to use 3mol/L salt
What acid or potassium hydroxide aqueous solution were adjusted.
The fermentation medium component is：Corn steep liquor 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous chlorination
Calcium 1.5g/L, NH4Cl5.52g/L, soy molasses 100g/L, surplus is water；Sterilize 35min at 125 DEG C of temperature, obtains described
Fermentation medium；
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic acid with the ethanol of 5 times of supernatant volumes, alcohol precipitation,
Then redissolve again after the distilled water of supernatant volume, then pure water redissolves and constant volume obtains sample after 25mL by hydrolysis process
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield is 26g/L；Using spray drying process prepare dry powder-shaped γ-
Polyglutamic acid.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
Embodiment 4
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, cultivated 16 hours in 30 DEG C,
Prepare the inclined-plane seed after activation；Slant medium forms:Tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, fine jade
Cosmetics 20g/L.
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain is October 14 in 2009
Day is in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Committee's common micro-organisms center preservation, its preserving number are CGMCCNo.3336；
(2) preparation of seed liquor；By step（1）The middle inclined-plane seed by after activation is inoculated in equipped with 50mL fluid nutrient mediums
In 500mL triangular flasks, 37 DEG C, shaking speed 220rpm cultivates 16 hours to exponential phase, is grown with reference to OD660 values 0.3
Then can, obtain produce gamma-polyglutamic acid seed liquor；
Seed culture medium forms：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O0.5g/L, pH7.2, surplus are water；Sterilize 38min at 125 DEG C of temperature, obtains described seed culture medium.
（3）Ferment tank：According to fermentation medium stereometer, 5% inoculum concentration, by step（2）In seed liquor connect
For kind in fermentation medium, 20L ferment tank culture mediums loading amount is 10L, 37 DEG C of control Preliminary fermentation temperature, tank pressure
0.05MPa；Ventilation 1.5vvm；Preliminary fermentation liquid pH value is adjusted to 7.0
The fermentation medium component is：Corn steep liquor 20g/L, sodium chloride 10g/L, MgSO4·7H2O 1g/L, anhydrous calcium chloride
2g/L, NH4Cl5.52g/L, soy molasses 95g/L, surplus are water；Sterilize 35min at 125 DEG C of temperature, obtains described hair
Ferment culture medium；
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic acid with the ethanol of 5 times of supernatant volumes, alcohol precipitation,
Then redissolve again after the distilled water of supernatant volume, then pure water redissolves and constant volume obtains sample after 25mL by hydrolysis process
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield is 24g/L；Using spray drying process prepare dry powder-shaped γ-
Polyglutamic acid.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
Embodiment 5
A kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, is comprised the following steps：
（1）Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, cultivated 16 hours in 30 DEG C,
Prepare the inclined-plane seed after activation；Slant medium forms:Tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, fine jade
Cosmetics 20g/L.
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain is October 14 in 2009
Day is in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Committee's common micro-organisms center preservation, its preserving number are CGMCCNo.3336；
(2) preparation of seed liquor；By step（1）The middle inclined-plane seed by after activation is inoculated in equipped with 50mL fluid nutrient mediums
In 500mL triangular flasks, 37 DEG C, shaking speed 220rpm cultivates 16 hours to exponential phase, is grown with reference to OD660 values 0.3
Then can, obtain produce gamma-polyglutamic acid seed liquor；
Seed culture medium forms：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O0.5g/L, pH7.2, surplus are water；Sterilize 38min at 125 DEG C of temperature, obtains described seed culture medium.
（3）Ferment tank：According to fermentation medium stereometer, 5% inoculum concentration, by step（2）In seed liquor connect
For kind in fermentation medium, 20L ferment tank culture mediums loading amount is 10L, 37 DEG C of control Preliminary fermentation temperature, tank pressure
0.03MPa；Ventilation 1vvm；Preliminary fermentation liquid pH value is adjusted to 7.0
The fermentation medium component is：Corn steep liquor 23g/L, sodium chloride 12g/L, MgSO4·7H2O 1.2g/L, anhydrous chlorination
Calcium 1.8g/L, NH4Cl5.52g/L, soy molasses 200g/L, surplus are water；Sterilize 35min at 125 DEG C of temperature, obtains described
Fermentation medium；
Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic acid with the ethanol of 5 times of supernatant volumes, alcohol precipitation, then
Redissolve again after the distilled water of supernatant volume, then pure water redissolves and constant volume obtains sample after 25mL by hydrolysis process.It is logical
Cross efficient liquid phase detect its gamma-polyglutamic acid yield is 18g/L；Dry powder-shaped γ-poly- paddy is prepared using spray drying process
Propylhomoserin.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
Claims (6)
1. a kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis, it is characterized in that, comprise the following steps：
Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, in 30 DEG C of -37 DEG C of culture 16-18
Hour, prepare the inclined-plane seed after activation；
The bacillus licheniformis（Bacilluslicheniformis）TKPG091, the bacterial strain exist on October 14th, 2009
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, its preserving number is CGMCCNo.3336；
（2）The preparation of seed liquor；By step（1）Inclined-plane seed after middle activation is inoculated in seed culture medium, 37 DEG C, and shaking table turns
Fast 220rpm cultivates 16 hours to exponential phase, obtains the seed liquor of production gamma-polyglutamic acid；
（3）Ferment tank：According to fermentation medium stereometer, by 5% inoculum concentration, by step（2）In seed liquor inoculation
In fermentation medium, 37-38 DEG C of control Preliminary fermentation temperature, tank pressure 0.02-0.05MPa；Ventilation 0.5-1.5vvm；Initially
Zymotic fluid pH value is adjusted to 7.0, cultivates 72 hours；
The fermentation medium component is：Corn steep liquor 20g-25g/L, sodium chloride 10g-15g/L, MgSO4·7H2O 1g-2g/L,
Anhydrous calcium chloride 1.5g-2g/L, NH4Cl 5.52g/L, soy molasses 80g-200g/L, surplus are water；Gone out at 125 DEG C of temperature
Bacterium 30-40min, obtain described fermentation medium；
（4）Extraction：Thalline is removed in centrifugation after fermentation ends, then separates out polyglutamic with the ethanol of 4-5 times of supernatant volume, alcohol precipitation
Acid, then redissolve after the distilled water of supernatant volume, dry again；Prepare dry powder-shaped gamma-polyglutamic acid.
2. a kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis according to claim 1, its feature are existed
In, pass through efficient liquid phase detect sample in gamma-polyglutamic acid yield.
3. a kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis according to claim 1, its feature are existed
In the slant medium composition is:Tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar powder 20g/L.
4. a kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis according to claim 1, its feature are existed
In the seed culture medium composition is：Glucose 45g/L, corn steep liquor 10g/L, tryptone 15g/L, potassium chloride 0.5g/L,
MgSO4·7H2O 0.5g/L, pH7.2, surplus is water；Sterilize 30-40min at 125 DEG C of temperature, obtains described seed culture
Base.
5. a kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis according to claim 1, its feature are existed
In the drying refers to utilizing spray drying process, prepares dry powder-shaped gamma-polyglutamic acid.
6. a kind of method that gamma-polyglutamic acid is produced using bacillus licheniformis according to claim 1, its feature are existed
In the molecular weight of the gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841879A (en) * | 2018-07-11 | 2018-11-20 | 天津慧智百川生物工程有限公司 | A method of polymalic acid is produced using Aureobasidium pullulans |
| CN109182404A (en) * | 2018-10-15 | 2019-01-11 | 天津科技大学 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
| CN114456980A (en) * | 2022-02-28 | 2022-05-10 | 天津科技大学 | Gamma-polyglutamic acid high-yield strain and application thereof |
| CN114507697A (en) * | 2021-12-16 | 2022-05-17 | 山东晶辉生物技术有限公司 | Method for synthesizing degradable biological material polyglutamic acid |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB954552A (en) * | 1959-08-07 | 1964-04-08 | Merck & Co Inc | Production of l (+)-glutamic acid |
| CN102329831A (en) * | 2011-06-23 | 2012-01-25 | 天津实发中科百奥工业生物技术有限公司 | Method for producing levan by fermenting soy molasses |
| CN103710404A (en) * | 2013-12-27 | 2014-04-09 | 天津北洋百川生物技术有限公司 | Production method of gamma-polyglutamic acid with high molecular weight |
| CN104694437A (en) * | 2015-03-23 | 2015-06-10 | 领先生物农业股份有限公司 | Bacillus licheniformis and application of bacillus licheniformis in gamma-polyglutamic acid production |
| CN106047780A (en) * | 2016-08-22 | 2016-10-26 | 南京工业大学 | Bacillus amyloliquefaciens and applications of Bacillus amyloliquefaciens in co-production of bacterial cellulose and gamma-polyglutamic acid |
| CN107267411A (en) * | 2017-05-22 | 2017-10-20 | 广西大学 | A kind of non-glutamic acid dependent form producing strains and its fermentation process that γ polyglutamic acids are produced for hot fermentation |
-
2017
- 2017-12-26 CN CN201711431868.7A patent/CN107815476A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB954552A (en) * | 1959-08-07 | 1964-04-08 | Merck & Co Inc | Production of l (+)-glutamic acid |
| CN102329831A (en) * | 2011-06-23 | 2012-01-25 | 天津实发中科百奥工业生物技术有限公司 | Method for producing levan by fermenting soy molasses |
| CN103710404A (en) * | 2013-12-27 | 2014-04-09 | 天津北洋百川生物技术有限公司 | Production method of gamma-polyglutamic acid with high molecular weight |
| CN104694437A (en) * | 2015-03-23 | 2015-06-10 | 领先生物农业股份有限公司 | Bacillus licheniformis and application of bacillus licheniformis in gamma-polyglutamic acid production |
| CN106047780A (en) * | 2016-08-22 | 2016-10-26 | 南京工业大学 | Bacillus amyloliquefaciens and applications of Bacillus amyloliquefaciens in co-production of bacterial cellulose and gamma-polyglutamic acid |
| CN107267411A (en) * | 2017-05-22 | 2017-10-20 | 广西大学 | A kind of non-glutamic acid dependent form producing strains and its fermentation process that γ polyglutamic acids are produced for hot fermentation |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841879A (en) * | 2018-07-11 | 2018-11-20 | 天津慧智百川生物工程有限公司 | A method of polymalic acid is produced using Aureobasidium pullulans |
| CN109182404A (en) * | 2018-10-15 | 2019-01-11 | 天津科技大学 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
| CN114507697A (en) * | 2021-12-16 | 2022-05-17 | 山东晶辉生物技术有限公司 | Method for synthesizing degradable biological material polyglutamic acid |
| CN114507697B (en) * | 2021-12-16 | 2023-09-08 | 山东晶辉生物技术有限公司 | Synthesis method of degradable biological material polyglutamic acid |
| CN114456980A (en) * | 2022-02-28 | 2022-05-10 | 天津科技大学 | Gamma-polyglutamic acid high-yield strain and application thereof |
| CN114456980B (en) * | 2022-02-28 | 2023-08-11 | 天津北洋百川生物技术有限公司 | Gamma-polyglutamic acid high-yield strain and application thereof |
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