CN107446128A - A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique - Google Patents
A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique Download PDFInfo
- Publication number
- CN107446128A CN107446128A CN201710713149.8A CN201710713149A CN107446128A CN 107446128 A CN107446128 A CN 107446128A CN 201710713149 A CN201710713149 A CN 201710713149A CN 107446128 A CN107446128 A CN 107446128A
- Authority
- CN
- China
- Prior art keywords
- polyglutamic acid
- carrying
- freeze
- permeate
- pga
- 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/14—Ultrafiltration; Microfiltration
- B01D61/145—Ultrafiltration
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/46—Post-polymerisation treatment
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Water Supply & Treatment (AREA)
- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The method that hyperfiltration technique carries out being classified separation to the polyglutamic acid of different molecular weight is the present invention relates to the use of, is comprised the following steps, polyglutamic acid zymotic fluid obtains polyglutamic acid solution after degerming and decolourize；The concentration of polyglutamic acid solution is adjusted, ultrafiltration is carried out with 10kDa milipore filters, operating pressure is 0.4 0.5MPa, and it is 47 times to add water elution number, and trapped fluid A is freeze-dried, obtains γ PGA 1；It will transmit through liquid A and dilute 13 times, ultrafiltration is carried out to permeate A with 5kDa milipore filters, operating pressure is 0.5 0.6MPa, and it is 68 times to add water elution number, and trapped fluid B is freeze-dried, obtains γ PGA 2；Liquid freeze-drying is will transmit through, obtains γ PGA 3.The present invention utilizes hyperfiltration technique, by controlling milipore filter, ultrafiltration pressure and washing steps to carry out classification separation to the polyglutamic acid of different molecular weight, avoids the use of organic solvent, improves extraction effect.
Description
Technical field
The invention belongs to technical field of membrane separation, and in particular to a kind of that classification point is carried out to polyglutamic acid using hyperfiltration technique
From method.
Background technology
Gamma-polyglutamic acid（poly-γ-glutamic acid, γ-PGA）It is a kind of boiomacromolecule carrier material, with
Bacillus subtilis is prepared by fermentation technique, and its finished product is a kind of white, odourless taste pulverulent solids.With science and technology
Progressive and industrial expansion, thing followed problem of environmental pollution is also increasing, should ' ' clear water is blue or green again by Kingsoft Yin Shan '
Mountain ' it has been the universal common recognition of people, therefore each field is more likely to development and application environment friendly material.And γ-poly- paddy
Propylhomoserin is increasingly paid close attention to as a kind of biodegradable Green Polymer Material by people.Because it has the water suction of brilliance special
Property, and no matter in inside of human body, or its final catabolite is glutamic acid in external environment, to human body without
Toxic side effect, environment is not polluted, therefore gamma-polyglutamic acid all shows superiority in many fields.Its strand
In active very strong-COOH group, many available functions are found to have, such as water imbibition, moisture retention and absorption heavy metal
Deng, while monomer glutamic acid belongs to amino acid, and any pollution will not be caused after degraded, is environmentally friendly material.Polyglutamic acid
Due to containing more carboxyl and active group, therefore it has stronger water-retaining property and slow release, and the property of these two aspects makes it
Agriculturally can preferably it play a role.Research shows that low molecular polyglutamic acid is more notable in the upper effect of agricultural application.
Membrane separation technique refers to the biased sample comprising different-grain diameter molecule when passing through pellicle, different size of particle diameter
Molecule realizes the technology of the separation of selectivity, and principle is to be selected liquid to be separated by pressure, concentration, potential difference etc.
Property pass through membrane aperture, reach the purpose of separation, membrane separation technique have efficiently, amorphousness become, low energy consumption and it is simple to operate easily
In the control the advantages that, be widely used, be such as used to handle industrial water, food, water for beverages purification, refined, weaving, pharmacy,
The every field such as fermentation.
Gamma-polyglutamic acid is formed by D- type glutamic acid monomers and the condensation of L-type glutamic acid monomer（With amido link between monomer
Connection）, it is a kind of linear molecule.Because the degree of polymerization of glutamic acid monomer is different, the gamma-polyglutamic acid relative molecular mass of formation
As little as 100,000, high energy reaches 2,000,000, and span is very big, and its relative average molecular mass is about 1.23 × 104.Average molecular matter
The size of amount and the purposes of gamma-polyglutamic acid are closely related, and relative molecular mass is especially small（Less than 20000）It is best suitable for being applied to
In agricultural, and it is then ineffective used in other field.And in contrast, as sewage disposal in terms of gamma-polyglutamic acid
It is required that relative molecular mass is especially big（More than 1500000), otherwise the effect of flocculated impurities can have a greatly reduced quality.Therefore have left relative
Molecular mass discusses the property of gamma-polyglutamic acid, almost not in all senses, so how to efficiently separate different average molecular matter
Weight gamma-polyglutamic acid, it is the current technical issues that need to address to control its relative molecular mass.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, there is provided one kind is divided polyglutamic acid using hyperfiltration technique
The method of level separation.The present invention utilizes hyperfiltration technique, by controlling different milipore filters and different ultrafiltration pressures and elution
Number, classification separation is carried out to the polyglutamic acid of different molecular weight using deionized water completely, avoids the use of organic solvent,
Extraction effect is improved, it is energy-saving, beneficial to the realization of industrialized production.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique, comprises the following steps：
(1) polyglutamic acid zymotic fluid obtains polyglutamic acid solution after degerming and decolourize；
(2) concentration for adjusting polyglutamic acid solution is 10-15 g/L, and ultrafiltration, operating pressure 0.4- are carried out with 10kDa milipore filters
0.5MPa, it is 4-7 times to add water elution number, can effectively be divided macromolecular polyglutamic acid and free glutamic acid, pigment etc.
From preservation trapped fluid A and permeate A, trapped fluid A is freeze-dried, obtains white γ-PGA-1 samples respectively, and it is corresponding poly-
Glutamic acid mean molecule quantity is 1-2 × 103kDa；
(3) it will transmit through liquid A and dilute 1-3 times, continue to carry out permeate A ultrafiltration, operating pressure 0.5- with 5kDa milipore filters
0.6MPa, it is 6-8 times to add water elution number, and the polyglutamic acid and free glutamic acid of different molecular magnitude effectively can be divided
From preservation trapped fluid B and permeate B, trapped fluid B is freeze-dried, obtains white γ-PGA-2 samples respectively, and it is corresponding poly-
Glutamic acid mean molecule quantity is 100-120kDa；Liquid freeze-drying is will transmit through, obtains white γ-PGA-3 samples, it is corresponding poly-
Glutamic acid mean molecule quantity is 30-40kDa.
When carrying out ultrafiltration, feed temperature and operating pressure can produce certain influence to the operating efficiency of milipore filter.With
The increase of pressure, some macromoleculars enter fenestra, aggravate the pollution of film, and membrane flux reduces.The rise of temperature can make fenestra
Footpath increases, and reduces the resistance through film, but temperature is too high, can have damage to film.
Water elution number is added to have considerable influence to polyglutamic acid purity, it is miscellaneous through repeatedly adding water elution to remove small molecule
Matter, sugar and free glutamic acid, after more than 6 times plus water elution number ultrafiltration, the Testing index of the pigment of wash-off, ion and total reducing sugar
Change is little, tends towards stability, and illustrates that ultrafiltration removal of impurities has been completed.This experiment purpose is separating-purifying polyglutamic to greatest extent
Acid, small molecular weight impurity, sugar and free glutamic acid are removed, while ensure polyglutamic acid transmitance.
During the polyglutamic acid separation classification of different molecular weight, not using organic solvent, membrane separation process is utilized
The removal of impurity is gone, polyglutamic acid is retained, the method has that cost is low, technique is simple, separative efficiency is high, energy-saving and environmental protection etc.
Feature, obtained product purity is up to more than 90%, and the influence to the structural and functional characteristic of product is minimum, is advantageous to widen
The application of polyglutamic acid, ensure that safety and green of the polyglutamic acid in fields such as cosmetics, food, agricultural applications use.
Using the chemical method decolorization adsorption pigment such as ion exchange resin and the removal of impurity is gone, the method is few suitable for dosage, body
The sample that the viscosity of system is small and pigment content is low decolourizes, if sample viscosity is larger and pigment content is more, easily causes resin dirt
Dye, regeneration treatment is difficult, and has a certain degree of destruction to the structure of polyglutamic acid；Ultrafiltration membrane technique utilizes and sieves principle,
Refer to when sample flows through film surface, due to the effect of pressure, particle diameter will be trapped more than the material of membrane aperture in solution,
Grain size can then pass through membrane module when being less than membrane aperture, and so as to reach the effect of separation, saturating past liquid is permeate, is cut
That stays turns into concentrate.
As preferable technical scheme：
Preferably, solution temperature when described polyglutamic acid solution, permeate A carry out ultrafiltration is 20-30 DEG C.
Preferably, when the trapped fluid A, trapped fluid B and permeate B are freeze-dried, sublimation drying used
It is 18h.
Preferably, the polyglutamic acid zymotic fluid is the lichen bacillus ferments culture to numbering CGMCC3336, is obtained
Zymotic fluid.
Preferably, the degerming method of the polyglutamic acid zymotic fluid is：
(1) thalline is removed
Zymotic fluid pH is adjusted to 3.0, viscosity is reduced to original 1/6, adds fermentating liquid volume 2%-3% diatomite, above-mentioned
2%-3% refers to quality percent by volume；Then with 800 mesh filter clothes, plate-frame filtering removes thalline, 0.22MPa pressure, and flow velocity 30~
50mL/min；
Preferably, the discoloration method of the polyglutamic acid zymotic fluid is：1%~2% activated carbon is added, normal temperature is carried out and stirs at a slow speed
Mix, decolourize 90~120min, filters, to the basic clear, colorless of liquid；Once decolourized, obtained again according to above-mentioned decolorization condition
Clarify the less polyglutamic acid solution of impurity.
Preferably, used during described plus water elution water for deionized water.
The preparation method of polyglutamic acid zymotic fluid is：
First, actication of culture：
Numbering CGMCC3336 strain is cultivated into 16 hours in 37 DEG C in culture medium slant, prepares the inclined-plane of maturation
Seed.
2nd, seed culture：
Inclined-plane seed after activation is accessed in 500ml triangular flasks of the ring equipped with 50ml liquid seed culture mediums, 37 DEG C,
220rpm, 16 hours of culture to exponential phase.
3rd, fermented and cultured：
Seed culture fluid is accessed in fermentation culture, inoculum concentration 10%, 72h, fermentation tank 10L are cultivated under 220rpm, fill liquid
Measure as 60%, added when residual sugar is down to 5g/L before fed-batch medium to fermentation ends 4 it is small when, flow acceleration 1ml/min.Under
Tank γ-PGA yield is 30g/L~45g/L.
Culture medium employed in the preparation of polyglutamic acid zymotic fluid is prepared：
（1）Slant medium：Dusty yeast 5.0, tryptone 10, NaCl 5.0, agar 20, said components unit are g/L,
pH7.0±0.1.121 DEG C of high pressure steam sterilization 20min.
(2) seed culture medium：Yeast extract 7.0, tryptone 10, glucose 30, K2HPO4·H2O 0.5, MgSO4·6H2O
0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
（3）Fermentation medium：Glucose 80, monosodium glutamate 80, yeast extract 20, NH4NO3 4.1、NaCl 10、MgSO4·6H2O
0.5、CaCl2·6H2O 1.0、FeCl3·6H2O 0.01, said components unit are g/L, PH7.0.121 DEG C of high pressure steam sterilizations
20min。
Fed-batch medium composition：Glucose 900, NH4NO3 60、CaCl2·6H2O 20、FeCl3·7H2O 0.15, it is above-mentioned
Component unit is g/L, 7.0,121 DEG C of high pressure steam sterilization 20min of PH.
Beneficial effect：
Had the invention provides a kind of using membrane separation technique using deionized water to the gamma-polyglutamic acid of different molecular weight
Effect separation, avoids the use of organic solvent, improves extraction effect, energy-saving, beneficial to the realization of industrialized production.
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 for carrying out being classified separation to polyglutamic acid using hyperfiltration technique, comprises the following steps：
（1）Polyglutamic acid zymotic fluid obtains polyglutamic acid solution after degerming and decolourize；The preparation method of polyglutamic acid zymotic fluid
For：
1. actication of culture：
Numbering CGMCC3336 strain is cultivated into 16 hours in 37 DEG C in culture medium slant, prepares the inclined-plane of maturation
Seed.
2. seed culture：
Inclined-plane seed after activation is accessed in 500ml triangular flasks of the ring equipped with 50ml liquid seed culture mediums, 37 DEG C,
220rpm, 16 hours of culture to exponential phase.
3. fermented and cultured：
Seed culture fluid is accessed in fermentation culture, inoculum concentration 10%, 72h, fermentation tank 10L are cultivated under 220rpm, fill liquid
Measure as 60%, added when residual sugar is down to 5g/L before fed-batch medium to fermentation ends 4 it is small when, flow acceleration 1ml/min.Under
Tank γ-PGA yield is 40g/L.
Culture medium employed in the preparation of polyglutamic acid zymotic fluid is prepared：
Slant medium：Dusty yeast 5.0, tryptone 10, NaCl 5.0, agar 20, said components unit are g/L, pH7.0
±0.1.121 DEG C of high pressure steam sterilization 20min.
Seed culture medium：Yeast extract 7.0, tryptone 10, glucose 30, K2HPO4·H2O 0.5, MgSO4·6H2O
0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
Fermentation medium：Glucose 80, monosodium glutamate 80, yeast extract 20, NH4NO3 4.1、NaCl 10、MgSO4·6H2O
0.5、CaCl2·6H2O 1.0、FeCl3·6H2O 0.01, said components unit are g/L, PH7.0.121 DEG C of high pressure steam sterilizations
20min。
Fed-batch medium composition：Glucose 900, NH4NO3 60、CaCl2·6H2O 20、FeCl3·7H2O 0.15, it is above-mentioned
Component unit is g/L, 7.0,121 DEG C of high pressure steam sterilization 20min of PH.
The degerming method of polyglutamic acid zymotic fluid is：
Zymotic fluid pH is adjusted to 3.0, viscosity is reduced to original 1/6, adds the silicon of fermentating liquid volume quality percent by volume 2%
Diatomaceous earth, above-mentioned 2%-3% refer to；Then thalline, 0.22MPa pressure, flow velocity 30mL/min are removed with 800 mesh filter clothes, plate-frame filtering；
The discoloration method of polyglutamic acid zymotic fluid is：
1% activated carbon is added, normal temperature is carried out and mixes slowly, decolouring 90min, filter, to the basic clear, colorless of liquid；Then will be de-
Color liquid crosses NF membrane after diluting 2~3 times, and polyglutamic acid solution is made.
（2）The concentration for adjusting polyglutamic acid solution is 10g/L, and ultrafiltration is carried out with 10kDa milipore filters, polyglutamic acid solution
Temperature is 25 DEG C, operating pressure 0.4MPa, and it is 4 times to add water elution number, preserves trapped fluid A and permeate A respectively, will retain
Liquid A is freeze-dried 18h, obtains white γ-PGA-1 samples, its corresponding polyglutamic acid mean molecule quantity is 1 × 103kDa；
（3）It will transmit through liquid A and dilute 1 times, continue to carry out permeate A ultrafiltration with 5kDa milipore filters, permeate A temperature is 20 DEG C
Operating pressure is 0.5MPa, and it is 6 times to add water elution number, preserves trapped fluid B and permeate B respectively, trapped fluid B is freeze-dried
18h, white γ-PGA-2 samples are obtained, its corresponding polyglutamic acid mean molecule quantity is 100kDa；It will transmit through liquid freeze-drying
18h, white γ-PGA-3 samples are obtained, its corresponding polyglutamic acid mean molecule quantity is 30kDa.
Embodiment 2
A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique, comprises the following steps：
（1）Polyglutamic acid zymotic fluid obtains polyglutamic acid solution after degerming and decolourize；The preparation method of polyglutamic acid zymotic fluid
For：
1. actication of culture：
Numbering CGMCC3336 strain is cultivated into 16 hours in 37 DEG C in culture medium slant, prepares the inclined-plane of maturation
Seed.
2. seed culture：
Inclined-plane seed after activation is accessed in 500ml triangular flasks of the ring equipped with 50ml liquid seed culture mediums, 37 DEG C,
220rpm, 16 hours of culture to exponential phase.
3. fermented and cultured：
Seed culture fluid is accessed in fermentation culture, inoculum concentration 10%, 72h, fermentation tank 10L are cultivated under 220rpm, fill liquid
Measure as 60%, added when residual sugar is down to 5g/L before fed-batch medium to fermentation ends 4 it is small when, flow acceleration 1ml/min.Under
Tank γ-PGA yield is 35g/L.
Culture medium employed in the preparation of polyglutamic acid zymotic fluid is prepared：
Slant medium：Dusty yeast 5.0, tryptone 10, NaCl 5.0, agar 20, said components unit are g/L, pH7.0
±0.1.121 DEG C of high pressure steam sterilization 20min.
Seed culture medium：Yeast extract 7.0, tryptone 10, glucose 30, K2HPO4·H2O 0.5, MgSO4·6H2O
0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
Fermentation medium：Glucose 80, monosodium glutamate 80, yeast extract 20, NH4NO3 4.1、NaCl 10、MgSO4·6H2O
0.5、CaCl2·6H2O 1.0、FeCl3·6H2O0.01, said components unit are g/L, PH7.0.121 DEG C of high pressure steam sterilizations
20min。
Fed-batch medium composition：Glucose 900, NH4NO3 60、CaCl2·6H2O 20、FeCl3·7H2O 0.15, it is above-mentioned
Component unit is g/L, 7.0,121 DEG C of high pressure steam sterilization 20min of PH.
The degerming method of polyglutamic acid zymotic fluid is：
Zymotic fluid pH is adjusted to 3.0, viscosity is reduced to original 1/6, adds quality percent by volume as fermentating liquid volume 3%
Diatomite；Then thalline, 0.22MPa pressure, flow velocity 50mL/min are removed with 800 mesh filter clothes, plate-frame filtering；
The discoloration method of polyglutamic acid zymotic fluid is：
2% activated carbon is added, normal temperature is carried out and mixes slowly, decolouring 120min, filter, to the basic clear, colorless of liquid；Then will
Destainer crosses NF membrane after diluting 3 times, and polyglutamic acid solution is made.
（2）The concentration for adjusting polyglutamic acid solution is 12 g/L, and ultrafiltration, polyglutamic acid solution are carried out with 10kDa milipore filters
Temperature be 30 DEG C, operating pressure 0.5MPa, add water elution number be 6 times, respectively preserve trapped fluid A and permeate A, will cut
Stay liquid A be freeze-dried 18h, obtain white γ-PGA-1 samples, its corresponding polyglutamic acid mean molecule quantity be 1-2 ×
103kDa；
（3）Will transmit through liquid A and dilute 2 times, continue to carry out permeate A ultrafiltration with 5kDa milipore filters, permeate A for 30 DEG C, grasp
It is 0.6MPa to make pressure, and it is 8 times to add water elution number, preserves trapped fluid B and permeate B respectively, trapped fluid B is freeze-dried
18h, white γ-PGA-2 samples are obtained, its corresponding polyglutamic acid mean molecule quantity is 120kDa；It will transmit through liquid freeze-drying
18h, white γ-PGA-3 samples are obtained, its corresponding polyglutamic acid mean molecule quantity is 35kDa.
Embodiment 3
A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique, comprises the following steps：
（1）Polyglutamic acid zymotic fluid obtains polyglutamic acid solution after degerming and decolourize；The preparation method of polyglutamic acid zymotic fluid
For：
1. actication of culture：
Numbering CGMCC3336 strain is cultivated into 16 hours in 37 DEG C in culture medium slant, prepares the inclined-plane of maturation
Seed.
2. seed culture：
Inclined-plane seed after activation is accessed in 500ml triangular flasks of the ring equipped with 50ml liquid seed culture mediums, 37 DEG C,
220rpm, 16 hours of culture to exponential phase.
3. fermented and cultured：
Seed culture fluid is accessed in fermentation culture, inoculum concentration 10%, 72h, fermentation tank 10L are cultivated under 220rpm, fill liquid
Measure as 60%, added when residual sugar is down to 5g/L before fed-batch medium to fermentation ends 4 it is small when, flow acceleration 1ml/min.Under
Tank γ-PGA yield is 38g/L.
Culture medium employed in the preparation of polyglutamic acid zymotic fluid is prepared：
Slant medium：Dusty yeast 5.0, tryptone 10, NaCl 5.0, agar 20, said components unit are g/L, pH7.0
±0.1.121 DEG C of high pressure steam sterilization 20min.
Seed culture medium：Yeast extract 7.0, tryptone 10, glucose 30, K2HPO4·H2O 0.5, MgSO4·6H2O
0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
Fermentation medium：Glucose 80, monosodium glutamate 80, yeast extract 20, NH4NO3 4.1、NaCl 10、MgSO4·6H2O
0.5、CaCl2·6H2O 1.0、FeCl3·6H2O0.01, said components unit are g/L, PH7.0.121 DEG C of high pressure steam sterilizations
20min。
Fed-batch medium composition：Glucose 900, NH4NO3 60、CaCl2·6H2O 20、FeCl3·7H2O 0.15, it is above-mentioned
Component unit is g/L, 7.0,121 DEG C of high pressure steam sterilization 20min of PH.
The degerming method of polyglutamic acid zymotic fluid is：
Zymotic fluid pH is adjusted to 3.0, viscosity is reduced to original 1/6, adds the diatomite of fermentating liquid volume 2.5%, above-mentioned
2%-3% refers to quality percent by volume；Then with 800 mesh filter clothes, plate-frame filtering removes thalline, 0.22MPa pressure, and flow velocity 30~
50mL/min；
The discoloration method of polyglutamic acid zymotic fluid is：
1%~2% activated carbon is added, normal temperature is carried out and mixes slowly, decolouring 100min, filter, to the basic clear, colorless of liquid；So
NF membrane is crossed after destainer is diluted into 2.5 times afterwards, polyglutamic acid solution is made.
（2）The concentration for adjusting polyglutamic acid solution is 15 g/L, and ultrafiltration, polyglutamic acid solution are carried out with 10kDa milipore filters
Temperature be 25 DEG C, operating pressure 0.45MPa, add water elution number be 5 times, respectively preserve trapped fluid A and permeate A, will
Trapped fluid A be freeze-dried 18h, obtain white γ-PGA-1 samples, its corresponding polyglutamic acid mean molecule quantity be 1.3 ×
103kDa；
（3）It will transmit through liquid A and dilute 3 times, continue to carry out permeate A ultrafiltration with 5kDa milipore filters, permeate A temperature is 30
DEG C, operating pressure 0.55MPa, it is 7 times to add water elution number, preserves trapped fluid B and permeate B respectively, trapped fluid B is freezed
18h is dried, obtains white γ-PGA-2 samples, its corresponding polyglutamic acid mean molecule quantity is 110kDa；It will transmit through liquid freezing
18h is dried, obtains white γ-PGA-3 samples, its corresponding polyglutamic acid mean molecule quantity is 40kDa.
Claims (7)
1. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique, it is characterized in that, comprise the following steps：
Polyglutamic acid zymotic fluid obtains polyglutamic acid solution after degerming and decolourize；
The concentration for adjusting polyglutamic acid solution is 10-15 g/L, and ultrafiltration, operating pressure 0.4- are carried out with 10kDa milipore filters
0.5MPa, it is 4-7 times to add water elution number, preserves trapped fluid A and permeate A respectively, trapped fluid A is freeze-dried, obtained white
Color γ-PGA-1 samples, its corresponding polyglutamic acid mean molecule quantity are 1-2 × 103kDa；
It will transmit through liquid A and dilute 1-3 times, continue to carry out permeate A ultrafiltration, operating pressure 0.5- with 5kDa milipore filters
0.6MPa, it is 6-8 times to add water elution number, preserves trapped fluid B and permeate B respectively, trapped fluid B is freeze-dried, obtained white
Color γ-PGA-2 samples, its corresponding polyglutamic acid mean molecule quantity are 100-120kDa；Liquid freeze-drying is will transmit through, is obtained white
Color γ-PGA-3 samples, its corresponding polyglutamic acid mean molecule quantity are 30-40kDa.
2. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique according to claim 1, its feature
It is, solution temperature when described polyglutamic acid solution, permeate A carry out ultrafiltration is 20-30 DEG C.
3. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique according to claim 1, its feature
It is, when the trapped fluid A, trapped fluid B and permeate B are freeze-dried, sublimation drying used is 18h.
4. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique according to claim 1, its feature
It is, the polyglutamic acid zymotic fluid is the lichen bacillus ferments culture to numbering CGMCC3336, obtained zymotic fluid.
5. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique according to claim 1, its feature
It is, the degerming method of the polyglutamic acid zymotic fluid is：
Zymotic fluid pH is adjusted to 3.0, viscosity is reduced to original 1/6, adds fermentating liquid volume 2%-3% diatomite, above-mentioned
For quality percent by volume；Then thalline, 0.22MPa pressure, 30~50mL/min of flow velocity are removed with 800 mesh filter clothes, plate-frame filtering.
6. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique according to claim 1, its feature
It is, the discoloration method of the polyglutamic acid zymotic fluid is：1%~2% activated carbon is added, normal temperature is carried out and mixes slowly, decolourize
90~120min, filter, to the basic clear, colorless of liquid；Once decolourized again according to above-mentioned decolorization condition, obtain clarifying miscellaneous
The less polyglutamic acid solution of matter.
7. a kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique according to claim 1, its feature
Be, use during described plus water elution water for deionized water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710713149.8A CN107446128A (en) | 2017-08-18 | 2017-08-18 | A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710713149.8A CN107446128A (en) | 2017-08-18 | 2017-08-18 | A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107446128A true CN107446128A (en) | 2017-12-08 |
Family
ID=60491515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710713149.8A Pending CN107446128A (en) | 2017-08-18 | 2017-08-18 | A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107446128A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108606299A (en) * | 2018-05-08 | 2018-10-02 | 刘柏海 | A method of producing monosodium glutamate using multistage sterilization technique |
| CN111003874A (en) * | 2019-10-25 | 2020-04-14 | 温州大学 | Sewage tail water grading treatment method for soluble organic matter pollution |
| CN117720722A (en) * | 2023-12-19 | 2024-03-19 | 山东福瑞达生物科技有限公司 | Preparation method and application of oligoglutamic acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN103613753A (en) * | 2013-11-14 | 2014-03-05 | 天津北洋百川生物技术有限公司 | Method for separating and purifying polyglutamic acid by using additive-free organic solvent |
-
2017
- 2017-08-18 CN CN201710713149.8A patent/CN107446128A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN103613753A (en) * | 2013-11-14 | 2014-03-05 | 天津北洋百川生物技术有限公司 | Method for separating and purifying polyglutamic acid by using additive-free organic solvent |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108606299A (en) * | 2018-05-08 | 2018-10-02 | 刘柏海 | A method of producing monosodium glutamate using multistage sterilization technique |
| CN111003874A (en) * | 2019-10-25 | 2020-04-14 | 温州大学 | Sewage tail water grading treatment method for soluble organic matter pollution |
| CN117720722A (en) * | 2023-12-19 | 2024-03-19 | 山东福瑞达生物科技有限公司 | Preparation method and application of oligoglutamic acid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Chitosan-based materials: Preparation, modification and application | |
| CN107446128A (en) | A kind of method for carrying out being classified separation to polyglutamic acid using hyperfiltration technique | |
| US20180127462A1 (en) | Method for extracting epsilon-polylysine and its hydrochloride salt from fermentation broth | |
| CN107349787B (en) | Preparation method of forward osmosis membrane added with aminated graphene quantum dots, prepared forward osmosis membrane and application of forward osmosis membrane | |
| CN101487034A (en) | Preparation f beta-poly malic acid and salt thereof | |
| CN106111065A (en) | A kind of modified activated carbon absorbent and its preparation method and application | |
| CN106749743A (en) | A kind of celestial grass Polyose extraction system and extraction process | |
| CN1106448C (en) | Tech. and process for extracting pullulan from fermentation liquor | |
| CN113735991A (en) | Process for extracting pullulan from high-viscosity fermentation liquor | |
| CN110372858A (en) | A kind of method of polyglutamic acid extraction and purification | |
| CN103613753B (en) | A kind of method without adding organic separated from solvent purification polyglutamic acid | |
| Nitayaphat | Utilization of chitosan/bamboo charcoal composite as reactive dye adsorbent | |
| CN103965064A (en) | Method for extraction of L-alanine from L-alanine fermentation broth | |
| CN105274179A (en) | Process for extracting L-isoleucine | |
| CN104066747A (en) | Process of purification of teicoplanin | |
| CN110437290A (en) | A kind of steviol glycoside extracting and developing and purification process | |
| CN107056966A (en) | A kind of process for purification of liquaemin | |
| CN101077942A (en) | Method for refining powdered catechu dye by film separation technique and dyeing method thereof | |
| Pandit et al. | Chitosan biobased materials in textile industry | |
| CN103772086A (en) | Pretreatment process for preparing fractions of marine microorganism small molecule metabolites | |
| CN105363039B (en) | A kind of sea grass polysaccharide microcapsules and preparation method thereof | |
| CN110042129B (en) | Preparation method of agricultural gamma-polyglutamic acid | |
| CN110106212B (en) | Preparation method of cosmetic-grade gamma-polyglutamic acid | |
| CN104549178A (en) | Adsorbing material for azide method coupling gelatin and preparation method of adsorbing material | |
| CN101798356A (en) | Method for separating and extracting natural tea polysaccharide |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171208 |