CN109182404A - A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments - Google Patents
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments Download PDFInfo
- Publication number
- CN109182404A CN109182404A CN201811196148.1A CN201811196148A CN109182404A CN 109182404 A CN109182404 A CN 109182404A CN 201811196148 A CN201811196148 A CN 201811196148A CN 109182404 A CN109182404 A CN 109182404A
- Authority
- CN
- China
- Prior art keywords
- polyglutamic acid
- gamma
- fermentation
- seed
- stream plus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of methods using stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising the following steps: actication of culture: the original strain of bacillus licheniformis being 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 gamma-polyglutamic acid must be produced；Seed liquor is inoculated in fermentation medium and is fermented, and fermentation time be for 24 hours -66h when, stream plus concentration be 4g/L-6g/L sucrose solution；Thallus is removed in centrifugation after fermentation, and polyglutamic acid is precipitated in alcohol precipitation, dry, prepares dry powder-shaped gamma-polyglutamic acid.The present invention flows during the fermentation plus sucrose, and using cane sugar substitution glucose or the method for assisting glucose, the yield of gamma-polyglutamic acid not only can be improved, moreover it is possible to delay thallus aging, extend fermentation time.
Description
Technical field
The invention belongs to field of agricultural microbial technology, are related to a kind of raw using stream when the lichen bacillus ferments plus sucrose
The method for producing gamma-polyglutamic acid.
Background technique
Gamma-polyglutamic acid is a kind of boiomacromolecule carrier material, is by lvanovics et al. earliest in anthrax spore bar
It is found in the folder film of bacterium, is bacillus such as bacillus subtilis, bacillus licheniformis etc. produces during vital movement
Raw secondary metabolite can currently be prepared in laboratory by the method for biofermentation.With advances in technology with industry
Development, the following problem of environmental pollution is also increasing, therefore each field is more likely to development and application environment is friendly
Profile material.And " green " high molecular material that gamma-polyglutamic acid is biodegradable as one kind, also increasingly by the pass of people
Note.Because it is with good water absorption character, and no matter in inside of human body, or its final catabolism in external environment
Product is glutamic acid, is had no toxic and side effect to human body, no pollution to the environment, therefore all shows its superiority in many fields.
It is known that food service industry, health care industry is widely used in terms of agriculture in cosmetic industry.
Sucrose can be generated by photosynthesis to be distributed widely in plant with chemical synthesis, especially beet, sweet
Content is high in sugarcane and fruit.Sucrose is the principal mode of plant storage, accumulation and transport sugar.It is usually edible white sugar, red
Sugar is all sucrose.Sucrose is formed by a molecule glucose and a molecule fructose dehydrating condensation, soluble easily in water to be relatively insoluble in ethyl alcohol, sweet tea
Taste is only second to fructose.Sucrose is the source common indirect C in fermentation process, and purposes is to be extensively only second to glucose.Such as
Patent CN107619841A patent is just added to sucrose when producing gamma-polyglutamic acid, and the addition for sucrose is as micro-
The source C of biological growth is added, and the addition time is thallus mid log phase, is that thallus supplies energy, concentration phase as the main source C
To larger.But the yield of the gamma-polyglutamic acid of this method production is still lower.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of new stream plus sucrose when utilizing the lichen bacillus ferments
The method for producing gamma-polyglutamic acid.The present invention flows during the fermentation plus sucrose, reduces glucose effect using sucrose and subtracts
Weak glucose is easy the shortcomings that making thallus aging, and the yield of gamma-polyglutamic acid not only can be improved, moreover it is possible to delay thallus aging,
Extend fermentation time.
To achieve the above object, the present invention adopts the following technical scheme:
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising 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, the inclined-plane seed after preparing mature activation；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(2) preparation of seed liquor；Inclined-plane seed after activation in step (1) is inoculated in seed culture medium, 37 DEG C, shaking table turns
Fast 220rpm cultivates 16 hours to logarithmic growth phase, obtains the seed liquor for producing gamma-polyglutamic acid；
(3) ferment tank: according in terms of fermentation medium volume, the seed liquor in step (2) is inoculated in by 5% inoculum concentration
In fermentation medium, 37-38 DEG C of Preliminary fermentation temperature is controlled, tank presses 0.02-0.05MPa；Ventilation quantity 0.5-1.5vvm；Just originate
Zymotic fluid pH value is adjusted to 7.0, cultivates 72 hours；
The fermentation medium composition are as follows: glucose: 80g/L, corn pulp: 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.In fermentation
Between in the 42h during -66h for 24 hours, uniform flow adds sucrose solution, stream dosage is calculated by culture volume, for every liter of 4g-6g；
Sucrose is the source common indirect C in fermentation process, and purposes is to be extensively only second to glucose.And for many lifes
It needs to induce for the microorganism for generating specific protein in object fermentation process, sucrose is cooked the source C advantageously, because of glucose effect
It is low to may cause induced expressing quantity.It therefore is to have one using cane sugar substitution glucose or the method for assisting glucose
Determine feasibility.
We have found in an experiment: finding during doing the source C production gamma-polyglutamic acid using sucrose replacement glucose
Yield is not very high but thallus OD is very high, and by further testing, during the fermentation based on glucose, stream adds sugarcane for discovery
Method supplemented by sugar can preferably produce polyglutamic acid and the thallus maturity period extends.Based on these features, stream plus sucrose are a kind of
Ideal method 1. it can guarantee the lasting supply source thallus C to improve microbial activity to improve metabolism degree.2. phase
DIRECT ENERGY supply for glucose, sucrose are energized in hydrolysis, and energy supply slowly, mildly, can delay thallus aging.In
It is that we have attempted different stream and add concentration, it is found that the effect for being 4g/L-6g/L from the stream of -66h for 24 hours plus concentration is best, stream plus palpus
Uniformly, slowly stream adds.
(4) extract: thallus is removed in centrifugation after fermentation, then with the ethyl alcohol of 4-5 times of supernatant volume, the poly- paddy of alcohol precipitation precipitation
Then propylhomoserin redissolves after the distilled water of supernatant volume again, dry；Prepare dry powder-shaped gamma-polyglutamic acid.
As a preferred technical scheme:
Preferably, pass through the yield of gamma-polyglutamic acid in efficient liquid phase test sample.
Preferably, the slant medium group becomes tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar
Powder 20g/L.
Preferably, the seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/L of tryptone,
Potassium chloride 0.5 g/L, MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 30-40min at 125 DEG C of temperature,
Obtain the seed culture medium.
Preferably, the pH of the fermentation medium is using 1~4mol/L sulfuric acid, hydrochloric acid, sodium hydroxide or potassium hydroxide
What aqueous solution was adjusted.
Preferably, the drying refers to preparing dry powder-shaped gamma-polyglutamic acid using spray drying process.
Preferably, the fermentation medium composition are as follows: glucose: 80g/L, corn pulp: 25g/L, sodium chloride: 15g/
L, MgSO4·7H2O:2g/L, anhydrous calcium chloride: 1.5g/L, NH4Cl:5.52g/L, surplus are water；It sterilizes at 125 DEG C of temperature
40min。
Preferably, the molecular weight of the gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
The sucrose flow acceleration is that per hour, every L stream plus 0.095g-0.143g. (flow the dense of the sucrose solution added
Degree can be configured according to this range)
In the middle and later periods of fermentation, -66h has carried out sucrose stream and has added the present invention for 24 hours.The yield of gamma-polyglutamic acid not only can be improved also
It can delay thallus aging, extend fermentation time.
The utility model has the advantages that
The sucrose addition of the application stimulates thallus as inducer and protective agent: 1. main functions are to improve bacterial metabolism
Vigor delays thallus aging；2. the application sucrose must be maintained under low concentration, the addition of slightly higher concentration can reduce polyglutamic
The yield of acid adds so stream must be carried out, cannot be added portionwise；3. the application sucrose be added the time be the thalli growth maturity period with
Afterwards；4. the application sucrose is added not as the source C of thallus, the source the C concentration of former culture medium is enough.
Specific 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
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Embodiment 1
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising the following steps:
(1) actication of culture: bacillus licheniformis CGMCC NO.3336 original strain is seeded on slant medium, in 37 DEG C
Culture 18 hours prepares mature inclined-plane seed；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(2) the inclined-plane seed after activation the preparation of seed liquor: is inoculated in the 500mL triangular flask equipped with 50mL fluid nutrient medium
In, 37 DEG C, shaking speed 220rpm cultivates 16 hours to logarithmic growth phase, with reference to OD660Value is grown can at 0.3
The seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/L of tryptone, potassium chloride 0.5
G/L, MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature.
(3) ferment tank: being inoculated in fermentor for the seed liquor 1.5L in step (2), with 5% inoculum concentration, 20L hair
Fermenting pot culture medium loading amount is 10L, and 38 DEG C of Preliminary fermentation temperature, tank presses 0.03MPa, ventilation quantity 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
Support base component are as follows: corn pulp 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous calcium chloride 1.5g/L, NH4Cl
5.52g/L, glucose 80g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtained fermentation medium；Sucrose stream adds
Time is in fermentation time in the 42h during -66h for 24 hours, the sucrose flow acceleration is every L stream plus 0.19g. (stream per hour
The concentration of the sucrose solution added can be configured according to this range)
(4) extract: after fermentation centrifugation remove thallus, then use the ethyl alcohol of 5 times of supernatant volumes, alcohol precipitation precipitation polyglutamic acid,
Then it is redissolved after the distilled water of supernatant volume again, and constant volume obtains sample by hydrolysis process after 25mL, passes through efficient liquid
Mutually detect its gamma-polyglutamic acid yield be 21g/L.Dry powder-shaped gamma-polyglutamic acid is prepared using spray drying process.γ-
The molecular weight of polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.Can also delaying thallus aging, (thallus OD turns from original lasting reduction
Slightly go up to stablize), extending fermentation time, (fermentation period can extend to 96h thallus from original 72h and just enter decline
Phase).
Embodiment 2
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising the following steps:
(1) actication of culture: the original strain of bacillus licheniformis is seeded on slant medium, is cultivated 16 hours in 30 DEG C,
Inclined-plane seed after preparation activation；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(2) preparation of seed liquor；Inclined-plane seed after activation is inoculated in the 500mL triangular flask equipped with 50mL fluid nutrient medium
In, it 37 DEG C, shaking speed 220rpm culture 16 hours can at 0.3 with reference to being grown with OD660 value to logarithmic growth phase；It is produced
The seed liquor of gamma-polyglutamic acid；
Seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/L of tryptone, 0.5 g/L of potassium chloride,
MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature, obtained seed culture medium.
(3) ferment tank: according in terms of fermentation medium volume, 5% inoculum concentration connects the seed liquor in step (2)
For kind in fermentation medium, 20L ferment tank culture medium loading amount is 10L, controls 37 DEG C of Preliminary fermentation temperature, tank pressure
0.02MPa；Ventilation quantity 0.5vvm；Preliminary fermentation liquid pH value is adjusted to 7；The pH of the fermentation medium be using 4mol/L hydrochloric acid or
What potassium hydroxide aqueous solution was adjusted.
The fermentation medium component are as follows: corn pulp 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous chlorination
Calcium 1.5g/L, NH4Cl 5.52g/L, glucose 80g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtains described
Fermentation medium；It is in fermentation time between the sucrose stream added-time in the 42h during -66h for 24 hours, the sucrose flow acceleration is
Per hour, every L stream plus 0.143g. (concentration for the sucrose solution that stream adds can be configured according to this range)
(3) extract: after fermentation centrifugation remove thallus, then use the ethyl alcohol of 4 times of supernatant volumes, alcohol precipitation precipitation polyglutamic acid,
Then it is redissolved after the distilled water of supernatant volume again, then pure water redissolves and constant volume obtains sample by hydrolysis process after 25mL
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield be poly- paddy yield 31g/L；It is prepared using spray drying process
Dry powder-shaped gamma-polyglutamic acid.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton, moreover it is possible to delay thallus aging (bacterium
Body OD switchs to stablize from original lasting reduction slightly to go up), extending fermentation time, (fermentation period can prolong from original 72h
Length to 96h thallus just enters decline phase).
Embodiment 3
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising the following steps:
(1) actication of culture: the original strain of bacillus licheniformis is seeded on slant medium, is cultivated 16 hours in 30 DEG C,
Inclined-plane seed after preparation activation；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(4) preparation of seed liquor；Inclined-plane seed after activation is inoculated in the 500mL triangular flask equipped with 50mL fluid nutrient medium
In, it 37 DEG C, shaking speed 220rpm culture 16 hours can at 0.3 with reference to being grown with OD660 value to logarithmic growth phase；It is produced
The seed liquor of gamma-polyglutamic acid；
Seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/L of tryptone, 0.5 g/L of potassium chloride,
MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature, obtained seed culture medium.
(3) ferment tank: according in terms of fermentation medium volume, 5% inoculum concentration connects the seed liquor in step (2)
For kind in fermentation medium, 20L ferment tank culture medium loading amount is 10L, controls 37 DEG C of Preliminary fermentation temperature, tank pressure
0.02MPa；Ventilation quantity 0.5vvm；Preliminary fermentation liquid pH value is adjusted to 7；The pH of the fermentation medium be using 4mol/L hydrochloric acid or
What potassium hydroxide aqueous solution was adjusted.
The fermentation medium component are as follows: corn pulp 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous chlorination
Calcium 1.5g/L, NH4Cl 5.52g/L, glucose 80g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtains described
Fermentation medium；It is in fermentation time between the sucrose stream added-time in the 42h during -66h for 24 hours, the sucrose flow acceleration is
Per hour, every L stream plus 0.095g. (concentration for the sucrose solution that stream adds can be configured according to this range)
(3) extract: after fermentation centrifugation remove thallus, then use the ethyl alcohol of 4 times of supernatant volumes, alcohol precipitation precipitation polyglutamic acid,
Then it is redissolved after the distilled water of supernatant volume again, then pure water redissolves and constant volume obtains sample by hydrolysis process after 25mL
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield be poly- paddy yield 30g/L；It is prepared using spray drying process
Dry powder-shaped gamma-polyglutamic acid.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton, moreover it is possible to delay thallus aging (bacterium
Body OD switchs to stablize from original lasting reduction slightly to go up), extending fermentation time, (fermentation period can prolong from original 72h
Length to 96h thallus just enters decline phase).
Embodiment 4
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising the following steps:
(1) actication of culture: the original strain of bacillus licheniformis is seeded on slant medium, is cultivated 16 hours in 30 DEG C,
Inclined-plane seed after preparation activation；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(2) preparation of seed liquor；Inclined-plane seed after activation is inoculated in the 500mL triangular flask equipped with 50mL fluid nutrient medium
In, it 37 DEG C, shaking speed 220rpm culture 16 hours can at 0.3 with reference to being grown with OD660 value to logarithmic growth phase；It is produced
The seed liquor of gamma-polyglutamic acid；
Seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/L of tryptone, 0.5 g/L of potassium chloride,
MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature, obtained seed culture medium.
(3) ferment tank: according in terms of fermentation medium volume, 5% inoculum concentration connects the seed liquor in step (2)
For kind in fermentation medium, 20L ferment tank culture medium loading amount is 10L, controls 37 DEG C of Preliminary fermentation temperature, tank pressure
0.02MPa；Ventilation quantity 0.5vvm；Preliminary fermentation liquid pH value is adjusted to 7；The pH of the fermentation medium be using 4mol/L hydrochloric acid or
What potassium hydroxide aqueous solution was adjusted.
The fermentation medium component are as follows: corn pulp 25g/L, sodium chloride 15g/L, MgSO4·7H2O 2g/L, anhydrous chlorination
Calcium 1.5g/L, NH4Cl 5.52g/L, glucose 80g/L, surplus are water；Sterilize 40min at 125 DEG C of temperature, obtains described
Fermentation medium；It is in fermentation time between the sucrose stream added-time in the 42h during -66h for 24 hours, the sucrose flow acceleration is
Per hour, every L stream plus 0.071g. (concentration for the sucrose solution that stream adds can be configured according to this range)
(4) extract: after fermentation centrifugation remove thallus, then use the ethyl alcohol of 4 times of supernatant volumes, alcohol precipitation precipitation polyglutamic acid,
Then it is redissolved after the distilled water of supernatant volume again, then pure water redissolves and constant volume obtains sample by hydrolysis process after 25mL
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield be poly- paddy yield 23g/L；It is prepared using spray drying process
Dry powder-shaped gamma-polyglutamic acid.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
Comparative example 1
A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments, comprising the following steps:
(1) actication of culture: the original strain of bacillus licheniformis is seeded on slant medium, is cultivated 17 hours in 35 DEG C,
Inclined-plane seed after preparation activation；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(2) preparation of seed liquor；Inclined-plane seed after activation in step (1) is inoculated in equipped with 50mL fluid nutrient medium
In 500mL triangular flask, 37 DEG C, shaking speed 220rpm cultivates 16 hours to logarithmic growth phase, grows with reference to OD660 value 0.3
Then can, obtain produce gamma-polyglutamic acid seed liquor；
Seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/L of tryptone, 0.5 g/L of potassium chloride,
MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 35min at 125 DEG C of temperature, obtains the seed culture
Base.
(3) ferment tank: according in terms of fermentation medium volume, 5% inoculum concentration connects the seed liquor in step (2)
For kind in fermentation medium, 20L ferment tank culture medium loading amount is 10L, controls 38 DEG C of Preliminary fermentation temperature, tank pressure
0.05MPa；Ventilation quantity 1vvm；Preliminary fermentation liquid pH value is adjusted to 7.0；The pH of the fermentation medium be using 2mol/L hydrochloric acid or
What sodium hydrate aqueous solution was adjusted.
The fermentation medium component are as follows: glucose: 80g/L, corn pulp: 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) extract: after fermentation centrifugation remove thallus, then use the ethyl alcohol of 5 times of supernatant volumes, alcohol precipitation precipitation polyglutamic acid,
Then it is redissolved after the distilled water of supernatant volume again, then pure water redissolves and constant volume obtains sample by hydrolysis process after 25mL
Product.By efficient liquid phase detect its gamma-polyglutamic acid yield be poly- paddy yield is 19g/L.It is prepared using spray drying process
Dry powder-shaped gamma-polyglutamic acid out.The molecular weight of gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.Thallus OD is opened from 36h-72h
Begin persistently to reduce, OD is reduced rapidly into decline phase after 72h.
From embodiment 1, embodiment 2, embodiment 3, in the comparison of embodiment 4 and comparative example 1, it can be found that stream plus sucrose are raw
It is feasible to produce polyglutamic acid method.The cost of stream plus sucrose is removed, profit 20%-35% can be improved in every liter of fermentation liquid.
Claims (8)
1. it is a kind of using when the lichen bacillus ferments stream plus sugar industry gamma-polyglutamic acid method, characterized in that including with
Lower step:
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, the inclined-plane seed after preparation activation；
Bacillus licheniformis (Bacillus licheniformis) TKPG091, the bacterial strain is on October 14th, 2009
It entrusts in the Chinese microorganism strain preservation management of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, deposit number are CGMCC No.3336；
(2) preparation of seed liquor；Inclined-plane seed after activation in step (1) is inoculated in seed culture medium, 37 DEG C, shaking table turns
Fast 220rpm cultivates 16 hours to logarithmic growth phase, obtains the seed liquor for producing gamma-polyglutamic acid；
(3) ferment tank: according in terms of fermentation medium volume, the seed liquor in step (2) is inoculated in by 5% inoculum concentration
In fermentation medium, 37-38 DEG C of Preliminary fermentation temperature is controlled, tank presses 0.02-0.05MPa；Ventilation quantity 0.5-1.5vvm；Just originate
Zymotic fluid pH value is adjusted to 7.0, cultivates 72 hours；In fermentation time in the 42h during -66h for 24 hours, uniform flow adds sucrose solution, stream
Dosage is calculated by culture volume, is every liter of 4g-6g；
(4) extract: thallus is removed in centrifugation after fermentation, then with the ethyl alcohol of 4-5 times of supernatant volume, alcohol precipitation precipitation polyglutamic
Then acid redissolves after the distilled water of supernatant volume again, dry；Prepare dry powder-shaped gamma-polyglutamic acid.
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 2. according to claim 1
Method, which is characterized in that pass through the yield of gamma-polyglutamic acid in efficient liquid phase test sample.
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 3. according to claim 1
Method, which is characterized in that slant medium composition are as follows: tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, fine jade
Cosmetics 20g/L.
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 4. according to claim 1
Method, which is characterized in that the seed culture medium composition are as follows: glucose 45 g/L, corn pulp 10g/L, 15 g/ of tryptone
L, potassium chloride 0.5 g/L, MgSO4·7H20.5 g/L of O, pH 7.2, surplus is water；Sterilize 30- at 125 DEG C of temperature
40min obtains the seed culture medium.
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 5. according to claim 1
Method, which is characterized in that the fermentation medium composition are as follows: glucose: 80g/L, corn pulp: 25g/L, sodium chloride: 15g/L,
MgSO4·7H2O:2g/L, anhydrous calcium chloride: 1.5g/L, NH4Cl:5.52g/L, surplus are water；It sterilizes at 125 DEG C of temperature
40min。
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 6. according to claim 1
Method, which is characterized in that the drying refers to preparing dry powder-shaped gamma-polyglutamic acid using spray drying process.
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 7. according to claim 1
Method, which is characterized in that the molecular weight of the gamma-polyglutamic acid is 20 ten thousand to 200 ten thousand dalton.
A kind of stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments are utilized 8. according to claim 1
Method, which is characterized in that the flow acceleration of sucrose is that every L stream adds 0.095g-0.143g per hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811196148.1A CN109182404A (en) | 2018-10-15 | 2018-10-15 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811196148.1A CN109182404A (en) | 2018-10-15 | 2018-10-15 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109182404A true CN109182404A (en) | 2019-01-11 |
Family
ID=64944549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811196148.1A Pending CN109182404A (en) | 2018-10-15 | 2018-10-15 | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109182404A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988788A (en) * | 2019-04-24 | 2019-07-09 | 武汉骏安生物科技有限公司 | A method of promoting bacillus licheniformis high yield polyglutamic acid sodium |
| CN110938666A (en) * | 2019-11-20 | 2020-03-31 | 上海应用技术大学 | Preparation method of microbial chitosan |
| CN113501734A (en) * | 2021-08-16 | 2021-10-15 | 福建绿安生物农药有限公司 | Gamma-polyglutamic acid and chitosan oligosaccharide combined water-soluble fertilizer |
| CN114456980A (en) * | 2022-02-28 | 2022-05-10 | 天津科技大学 | Gamma-polyglutamic acid high-yield strain and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556859A (en) * | 2000-05-20 | 2004-12-22 | 韩国科学技术院 | A method for manufacturing highly-concentrated polyglutamic acid with additional supply of saccharides |
| CN102586354A (en) * | 2012-03-09 | 2012-07-18 | 浙江德清汇宁生物科技有限公司 | Glutamic acid-independent production method of gamma-polyglutamic acid |
| CN102965311A (en) * | 2012-11-16 | 2013-03-13 | 南京轩凯生物科技有限公司 | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid |
| CN103695485A (en) * | 2013-12-27 | 2014-04-02 | 天津北洋百川生物技术有限公司 | High-efficiency production method of gamma-polyglutamic acid |
| 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 |
| CN104232504A (en) * | 2014-07-10 | 2014-12-24 | 樟树市狮王生物科技有限公司 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
| 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 |
| CN107699594A (en) * | 2017-11-22 | 2018-02-16 | 天津北洋百川生物技术有限公司 | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis |
| CN107815476A (en) * | 2017-12-26 | 2018-03-20 | 天津北洋百川生物技术有限公司 | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis |
-
2018
- 2018-10-15 CN CN201811196148.1A patent/CN109182404A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556859A (en) * | 2000-05-20 | 2004-12-22 | 韩国科学技术院 | A method for manufacturing highly-concentrated polyglutamic acid with additional supply of saccharides |
| CN102586354A (en) * | 2012-03-09 | 2012-07-18 | 浙江德清汇宁生物科技有限公司 | Glutamic acid-independent production method of gamma-polyglutamic acid |
| CN102965311A (en) * | 2012-11-16 | 2013-03-13 | 南京轩凯生物科技有限公司 | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid |
| CN103695485A (en) * | 2013-12-27 | 2014-04-02 | 天津北洋百川生物技术有限公司 | High-efficiency production method of gamma-polyglutamic acid |
| 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 |
| CN104232504A (en) * | 2014-07-10 | 2014-12-24 | 樟树市狮王生物科技有限公司 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
| 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 |
| CN107699594A (en) * | 2017-11-22 | 2018-02-16 | 天津北洋百川生物技术有限公司 | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis |
| CN107815476A (en) * | 2017-12-26 | 2018-03-20 | 天津北洋百川生物技术有限公司 | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988788A (en) * | 2019-04-24 | 2019-07-09 | 武汉骏安生物科技有限公司 | A method of promoting bacillus licheniformis high yield polyglutamic acid sodium |
| CN109988788B (en) * | 2019-04-24 | 2022-11-15 | 武汉骏安生物科技有限公司 | Method for promoting high yield of sodium polyglutamate by bacillus licheniformis |
| CN110938666A (en) * | 2019-11-20 | 2020-03-31 | 上海应用技术大学 | Preparation method of microbial chitosan |
| CN113501734A (en) * | 2021-08-16 | 2021-10-15 | 福建绿安生物农药有限公司 | Gamma-polyglutamic acid and chitosan oligosaccharide combined water-soluble fertilizer |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109182404A (en) | A method of utilizing stream plus sugar industry gamma-polyglutamic acid when the lichen bacillus ferments | |
| CN107699594A (en) | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis | |
| CN103103061B (en) | Preparation method of pure rice-koji added with Chinese medical herbs | |
| CN101792727B (en) | Bacillus coagulans and application thereof in L-sodium lactate preparation | |
| CN102220248B (en) | Bacterial strain for producing PMLA [Poly (Beta-L-malic acid)] and method for producing PMLA by fermentation of bacterial strain | |
| CN102352323B (en) | Ester producing yeast as well as method and application of yeast for producing Xiaoqu fen-flavor seasoning wine | |
| CN105039453A (en) | Method for preparing rice bran polysaccharides with improved oxidation resistance and application of rice bran polysaccharides | |
| CN106883992B (en) | Abnormal pichia yeast bacterium and its application of one plant height production 4- vinyl guaiacol and ethyl acetate | |
| CN108823261A (en) | A kind of Ultra-low molecular weight Dendrobium officinale polysaccharide and its preparation and application | |
| CN107815476A (en) | A kind of method that γ polyglutamic acids are produced using bacillus licheniformis | |
| CN102994395A (en) | Aureobasidium pullulans and application thereof | |
| CN105969673A (en) | Fermentation method for increasing yield of haematochrome and uranidin in monascus purpureus | |
| CN101875910A (en) | Bacillus amyloliquefaciens for producing gamma-polyglutamic acid | |
| CN102391976B (en) | Strain HD924 for producing dihydroxyacetone by microbial fermentation and method | |
| CN110283731A (en) | Mushroom strain and the method that shiitake mushroom hypha is simply prepared using it | |
| CN112094752A (en) | Method for producing ultra-low molecular weight pullulan by fermenting aureobasidium pullulans | |
| CN106987546A (en) | Tetramethylpyrazine bacillus amyloliquefaciens and the special wheat bran purposes of distilled spirit with sesame flavour | |
| CN1935978A (en) | Method for culturing Chinese caterpillar domestic fungus from fresh egg | |
| CN110129225A (en) | γ~polyglutamic acid producing strains and breeding prepare γ~polyglutamic acid method | |
| CN101037701A (en) | Preparation technique of gamma-polyglutamic acid | |
| CN102876592A (en) | Pichia anomala | |
| CN107760732A (en) | A kind of production method of agriculture level γ polyglutamic acids | |
| CN105087737B (en) | It is a kind of to utilize the method and application that jerusalem artichoke is fermenting raw materials production astaxanthin | |
| CN101230337A (en) | Preparation of nitrite reductase and method for preparing nitrite reductase preparation | |
| CN106636252A (en) | Thelephora ganbajun Zang exopolysaccharide, preparation method thereof and application of exopolysaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190111 |
|
| WD01 | Invention patent application deemed withdrawn after publication |