CN103665371B - A kind of method utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid - Google Patents
A kind of method utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid Download PDFInfo
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Abstract
The invention discloses a kind of method utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, belong to biological process synthesis and purification techniques field.For the distribution character of Objective extraction thing and impurity molecule amount in fermented liquid, in conjunction with Modern Membrane Technology, γ-the PGA of different molecular weight is effectively separated, key step is: after reducing viscosity, diatomite filtration is degerming, add thermal bond ultrafiltration except foreign protein and Large molecule active organism, Zeo-karb is except metal ion and some positively charged impurity, and after decolouring, nanofiltration is except small molecules amino acid, ionic impurity, ultrafiltration grading extraction, obtains the refining γ-PGA of different molecular weight.Extract yield is stabilized in more than 92%, and higher than the highest yield 90.6% of bibliographical information, ultrafiltration nanofiltration combination technology refines the γ-PGA of different molecular weight, and market specific aim is stronger, can accomplish to produce as required, improve economic benefit with specialization.
Description
Technical field:
The invention belongs to biological process synthesis and purification techniques field, particularly a kind of different molecular weight Biodegradable polymer material---the method for polyglutamic acid in separating-purifying bio-fermented liquid.
Background technology:
Day by day severe at environment, resource is day by day deficient today, find a kind of of many uses and the bioabsorbable polymer material that can degrade just seems particularly important.γ-PGA write a Chinese character in simplified form by γ-poly-glutaric acid (γ-Polyglutamicacid) is a kind of extracellular polyphosphazene polymer polymerisable compounds that some genus bacillus (Bacillus) is synthesized, it take L-glutamic acid as only monomer, combined by L mono-L-glutamic acid (L-Glu), D mono-L-glutamic acid (D-Glu) a kind of peptide molecule formed by amido linkage, γ-PGA is decomposed into L-glutamic acid by enzyme in vivo, enter tricarboxylic acid cycle, have no side effect, in physical environment, after γ-PGA thoroughly decomposes or burns, generate carbonic acid gas and water, to environment without any pollution.Therefore, γ-PGA is a kind of macromolecular material of great potential, and the hydrogel utilizing γ-PGA to make not only remains the fundamental characteristics of Common hydrogels material, also has the characteristics such as structural controllability, high-hydroscopicity, higher mechanical strength, biodegradability.γ-the PGA obtained particularly is produced by fermentation of bacillus, its molecular chain there is a large amount of active higher side chain carboxyl group, there is good high-hydroscopicity (1:3500, m/v), biocompatibility, biological degradability, the advantage such as nontoxic, can be used as cosmetics additive, water-holding agent, High hydrophilous resin, heavy metal absorbent, foodstuff additive, medical carrier, tissue engineering material etc. and be widely used in the fields such as makeup, food, agricultural, medicine, synthon and film.
Since the nineties in 20th century, Japan, some developed countries such as the U.S. are very active to the research of the Synthesis and applications of γ-PGA, the technology of Production by Microorganism Fermentation γ-PGA is also in the prostatitis in the world, with Ajincomoto Co., Inc, Meiji Seika Kaisba company and Guang Qi university are that the other unit of state of representative is to the performance of γ-PGA, synthesize and should be used as and more in depth study, γ-PGA has commerical prod, it is relatively less that China is studied in this respect, TaiWan, China also has remarkable progress in the research of γ-PGA fermentative production, China's Mainland to γ-PGA go into operation in a large number produce research be also short of, especially in sticky fermented liquid, concerning molecular weight γ-PGA within the specific limits, the efficiency extracting γ-PGA only has 40 ~ 50%, a large amount of γ-PGA is caused to waste and loss.
γ-PGA is as the biopolymer product of a kind of All Pure Nature, multifunctionality, Biodegradable, and relative molecular mass (Mw) is 10
4-l0
6scope in, the product application of different relative molecular mass can be made in various different field.γ-PGA molecular weight as cosmetics-stage, food grade is 700,000; The molecular weight of pharmaceutical grade is 1,000,000; The molecular weight of sewage disposal level is 1,000,000; The molecular weight 200,000 of soil, plant modifying agent level is such as the following.
Adopt the pKa value of determination of acid-basetitration sequestered γ-PGA, obtain the pKa=2.23 of γ-PGA, the pKa value of the α-carboxyl of this value and L-glutamic acid is unanimous on the whole.Utilize TGA and DSC to carry out the analysis of thermal properties, show that its heat decomposition temperature is 235.9 DEG C, fusing point is 223.5 DEG C.Do derivatization reagent with GITC after γ-PGA acid hydrolysis, the ratio that HPLC records D-Glu and Pidolidone in γ-PGA is stabilized in D:L=3:2.Generally speaking, the molecular-weight average (Mw) of the γ-PGA produced by genus bacillus is 10
4-l0
6between, in this patent, be distributed in 5 × 10 with the molecular weight (Mw) of laboratory biological fermentation method output γ-PGA
4-10
6between.
Membrane separation technique utilizes the selective penetrated property of film to component each in mixture to be separated, extracts and concentrated object product, membrane separating process carries out at normal temperatures, without phase transformation, energy consumption is low, equipment is simple, convenient operation and control, is applied in multiple fields such as chemical industry, medicine, light industry, food, weaving, electronics, metallurgy.The membrane separation technique being applicable to fermentation liquor treatment has micro-filtration, ultrafiltration, nanofiltration and reverse osmosis.Micro-filtration retains the particle of 0.01 ~ more than 10um, as thalline, cell, insolubles etc., and conventional ultrafiltration pretreatment process; Ultra-filtration membrane molecular weight cut-off 5000 ~ 500000, membrane pore size 1 ~ 20nm, can retain the macromolecular substance such as virus, protein, enzyme, polysaccharide; Reverse osmosis only allows solvent molecule to pass through, and the small-molecule substance such as salt, amino acid is also trapped; Nanofiltration membrane mean pore size 2nm, retaining component can be little of microbiotic, synthetic drug, dyestuff, disaccharides etc., and allow the small-molecule substances such as water, inorganic salt, organism to pass through, cutoff performance is between ultrafiltration and reverse osmosis.Mould material can be divided into polymeric membrane, mineral membrane and liquid membrane, and conventional is polymeric membrane, is secondly mineral membrane.Membrane structure can be divided into symmetric membrane and asymmetric membrane, ultrafiltration is generally asymmetric membrane, what play crown_interception in asymmetric membrane is fine and close superficial cortical layers, its thickness only 0.1 ~ 15um, porous network structure and supporting layer is had subcutaneous, supporting layer thickness, at 50 ~ 250um, had so both ensured the effectively catching to macromolecular substance, again reduce lock out operation time film to resistance.Improve membrane flux, be also provided with certain physical strength.
γ-PGA is a kind of extracellular products, and its fermented liquid is very glutinous, removes thalline be difficult to by general centrifuging, and is not suitable for industrial-scale application.In order to reduce the energy expenditure removing thalline and the consumption of organic solvent of saving for precipitating, DO.J.H etc. are optimized former technique, propose a kind of method of high efficiency separation γ-PGA.The method is made up of two steps: first adjust PH to 3.0 that viscosity is reduced, then bactofugation body, makes centrifugal energy reduce to original 17% like this; Then to going bacterium liquid to pull back to PH5.0, filtering with hollow-fibre membrane, making fermented liquid supernatant by original 20g/L, being concentrated into 60g/L, the ethanol consumption needed for extraction is reduced to original 1/4, reduces the cost extracting γ-PGA.But this method, because do not remove other impurity, so the requirement of Interception process to tunica fibrosa is higher, and bactofugation is difficult to amplify industrial production.The patent method of separation and Extraction gamma-polyglutamic acid-" from the fermented liquid ", mention in application number 201010188232.6, with ultra-filtration membrane removing small molecules pigment and impurity, but repeatedly regulate PH in extraction process, a large amount of ion can be introduced, introduce impurity too with a small amount of method of protein of protease hydrolyzed, do not fully utilize film and extract, be not suitable for amplifying and produce.Patent " a kind of separation purification method of gamma-polyglutamic acid-", mention in application number 201210396366.6, after gac and suction filtered through kieselguhr, directly be used for ultrafiltration and concentration, this processing mode is industrially amplified and is easy to cause stifled film phenomenon, and wash film program and make extraction step loaded down with trivial details, this technique should not amplify industrial production.Patent " a kind of extracting method of gamma-polyglutamic acid-", mention in application number 201010177020.8, alcohol precipitation is carried out with 2 ~ 3 times of ethanol after enzymolysis protein, removal of impurities is being carried out by ultrafiltration and concentration method, last freeze-drying, in this technique, uses a large amount of ethanol, increase the danger of industrial application, also do not mention different according to γ-PGA molecular weight and effectively extract.Bibliographical information, γ-the PGA extracting different molecular weight changes fermentation condition to produce the close γ-PGA of molecular weight as far as possible, but in actual production technique, γ-PGA molecular weight is larger, be difficult to accurate control γ-PGA molecular weight aborning, often output γ-PGA distributes in certain molecular weight ranges, and in traditional extraction technique, do not have to relate to and accurately extract γ-PGA according to molecular weight difference in same batch fermentation liquid, therefore, in extraction process, the reduction of fermentation broth viscosity, simultaneously how different according to molecular weight in later separation and high efficiency extraction γ-PGA, reduce costs is the subject matter of puzzlement Bacillus licheniformis (B.licheniformis) fermentation for γ-PGA always, need to pay much attention to.
Summary of the invention
The object of this invention is to provide a kind of method of high efficiency extraction γ-PGA from γ-PGA fermented liquid, bacterial classification used is Bacillus licheniformis, is now preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, numbering CGMCC3336.Specifically see that Tianjin Peiyang Biotrans Biotech Co., Ltd's application number is the patent of invention " method of producing gamma-polyglutamic acid by inert carrier solid state fermentation method " of 200910228297, the fermentation process adopted is the efficient fermentation process of one of the said firm's invention, specifically sees the patent of invention " a kind of method of a large amount of production gamma-polyglutamic acid-" of application number 201110216717.6.In fermented liquid, major impurity is high molecular weight protein (and belonging to heat denatured protein), polysaccharide, pigment and some small molecules amino acid, salt and other small molecules organic impuritys etc. more, and the molecular weight (Mw) producing gamma-polyglutamic acid-is distributed in 5 × 10
4-5 × 10
5between.The present invention is directed to the distribution character of Objective extraction thing and impurity molecule amount in fermented liquid, in conjunction with Modern Membrane Technology, a kind of method of high efficiency extraction γ-PGA is provided, γ-PGA total recovery is made to be greater than 92%, highest level 90.6% is announced higher than current document, and the γ-PGA of different molecular weight is effectively separated, according to molecular weight different application in different field.In traditional extraction process, the usage quantity of alcohol precipitation step ethanol is generally 4 ~ 6 times of material, and the present invention is reduced to 2 ~ 3 times, greatly reduces the use of organic solvent, makes cost reduce more than 30%, also reduce risk level simultaneously.
Utilize a method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, comprise the steps:
One, fermented liquid preparation: the lichen bacillus ferments of numbering CGMCC3336 is cultivated, obtains fermented liquid;
Two, broth extraction
(1) except thalline
Fermented liquid PH is adjusted to 3.0, and viscosity drop is low to moderate original 1/6, adds 2%-3% (m:v) diatomite, 800 order filter clothes, and Plate Filtration removes thalline, 0.22MPa pressure, flow velocity 30 ~ 50ml/min.
(2) except foreign protein and Large molecule active organism
PH6 ~ 7 adjusted by the fermented liquid removing thalline, and be heated to 90 DEG C of insulation 20min, make protein denaturation, removing heat denatured protein, is cooled to room temperature, crosses 0.45um hollow-fibre membrane and carries out ultrafiltration, remove more than 80% foreign protein.
(3) Zeo-karb is except metal ion and some positively charged impurity
By fermentating liquid volume 30%(v:v) addition add Zeo-karb, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, most of metal ion and positively charged impurity can be removed, after ion-exchange terminates, readjustment PH to 7, introducing a small amount of ion subsequent nano-filtration can remove.
(4) decolour
Add 1% ~ 2% gac, carry out normal temperature low rate mixing, decolouring 90 ~ 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable.
(5) except small molecules amino acid, ionic impurity
Fermented liquid dilution 2 ~ 3 times is rear crosses nanofiltration membrane, after testing, can remove the small molecular weight impurities such as Most amino-acids, polypeptide, oligosaccharides, salt ion.
(6) ultrafiltration grading extraction
The clear liquid of 50,000 ~ 500,000 ultra-filtration membranes to above-mentioned process is adopted to carry out pressurized circulation ultrafiltration and concentration, the γ-PGA of 0.1um Middle hollow fiber membrane molecular weight about 150,000, the γ-PGA of 500kDa Ultra filtration membrane molecular weight about 350,000.Obtain the clear liquid that molecular weight is different respectively: the γ-PGA clear liquid of molecular weight <15 ten thousand; Molecular weight 150,000 ~ 350,000 γ-PGA clear liquid, the γ-PGA clear liquid that molecular weight is greater than 350,000.γ-the PGA that molecular weight is less than 150,000 carries out 5kDa ultrafiltration and concentration and removes small molecular weight impurity further; γ-PGA the clear liquid that molecular weight is greater than 150,000 carries out 2 ~ 3 volumes times alcohol precipitation, and precipitate dissolves is in the water of original volume 1 ~ 2 times.By above-mentioned solution spray-dried separately, obtain refining γ-PGA.
As the preferred version of technique scheme, the present invention utilizes the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, comprises the steps:
One, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 DEG C in culture medium slant, the inclined-plane seed that preparation is ripe.
Two, seed culture:
Inclined-plane seed after activation is accessed a ring to be equipped with in the 500ml triangular flask of 50ml liquid seed culture medium, 37 DEG C, 220rpm, cultivates 16 hours to logarithmic phase.
Three, fermentation culture:
Accessed by seed culture fluid in fermentation culture, inoculum size is cultivate 72h under 10%, 220rpm, and fermentor tank is 10L, and liquid amount is 60%, and add fed-batch medium when residual sugar is down to 5g/L to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank γ-PGA output is 30g/L ~ 45g/L.
Four, broth extraction
(1) except thalline
Fermented liquid PH is adjusted to 3.0, and viscosity drop is low to moderate original 1/6, adds 2%-3% (m:v) diatomite, 800 order filter clothes, and Plate Filtration removes thalline, 0.22MPa pressure, flow velocity 30 ~ 50ml/min.
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts PH6 ~ 7, and be heated to 90 DEG C of insulation 20min, make protein denaturation, removing heat denatured protein, is cooled to room temperature, crosses 0.45um hollow-fibre membrane and carries out ultrafiltration, remove more than 80% foreign protein.
(3) Zeo-karb is except metal ion and some positively charged impurity
By fermentating liquid volume 30%(v:v) addition add Zeo-karb, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, most of metal ion and positively charged impurity can be removed, after ion-exchange terminates, with 6M sodium hydroxide readjustment PH to 7, introducing a small amount of ion subsequent nano-filtration can remove.
(4) decolour
Add 1% ~ 2% gac, carry out normal temperature low rate mixing, decolouring 90 ~ 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable.
(5) except small molecules amino acid, ionic impurity
Fermented liquid dilution 2 ~ 3 times is rear crosses nanofiltration membrane, after testing, can remove the small molecular weight impurities such as Most amino-acids, polypeptide, oligosaccharides, salt ion.
(6) ultrafiltration grading extraction
The clear liquid of 50,000 ~ 500,000 ultra-filtration membranes to above-mentioned process is adopted to carry out pressurized circulation ultrafiltration and concentration, the γ-PGA of 0.1um Middle hollow fiber membrane molecular weight about 150,000, the γ-PGA of 500kDa Ultra filtration membrane molecular weight about 350,000.Obtain the clear liquid that molecular weight is different respectively: the γ-PGA clear liquid of molecular weight <15 ten thousand; Molecular weight 150,000 ~ 350,000 γ-PGA clear liquid, the γ-PGA clear liquid that molecular weight is greater than 350,000.γ-the PGA that molecular weight is less than 150,000 carries out 5kDa ultrafiltration and concentration and removes small molecular weight impurity further; γ-PGA the clear liquid that molecular weight is greater than 150,000 carries out 2 ~ 3 volumes times alcohol precipitation, and precipitate dissolves is in the water of original volume 1 ~ 2 times.By above-mentioned solution spray-dried separately, obtain refining γ-PGA.
Substratum compound method is as follows:
(1) slant medium: yeast powder 5.0, Tryptones 10, NaCl5.0, agar 20, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(2) seed culture medium: yeast extract paste 7.0, Tryptones 10, glucose 30, K
2hPO
4h
2o0.5, MgSO
46H
2o0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(3) fermention medium: glucose 80, monosodium glutamate 80, yeast extract paste 20, NH
4nO
34.1, NaCl10, MgSO
46H
2o0.5, CaCl
26H
2o1.0, FeCl
36H
2o0.01, said components unit is g/L, PH7.0.121 DEG C of high pressure steam sterilization 20min.
Feeding culture based component: glucose 900, NH
4nO
360, CaCl
26H
2o20, FeCl
37H
2o0.15, said components unit is g/L, PH7.0,121 DEG C of high pressure steam sterilization 20min.
Beneficial effect:
The present invention is compared with prior art: first, adopt Plate Filtration method degerming, overcome the problem that conventional centrifugal method removes thalline difficulty, effectively reduce in fermentative Production γ-PGA process, cost in degerming, substantially increase bacteria-eliminating efficacy, can effectively be applied in industrialized production; The second, in leaching process, the characteristic different according to fermented liquid material molecule, carries out ion-exchange removal of impurities and membrane filtration removal of impurities, greatly improves extraction effect, energy-saving and cost-reducing, is beneficial to the realization of industrialized production; 3rd, ultrafiltration nanofiltration combination technology refines the γ-PGA of different molecular weight, and market specific aim is stronger, can accomplish to produce as required, improve economic benefit with specialization; 4th, greatly reduce the use of organic solvent, make cost reduce more than 30%, also reduce risk level simultaneously; 5th, adopt spray drying technology, the γ-PGA obtained is white powder, realization easier than freeze-drying, saves the energy, and more efficient, and mode of appearance is also better; 6th, extract yield is stabilized in more than 92%, higher than the highest yield 90.6% of bibliographical information.
Embodiment
Embodiment 1
One, substratum preparation:
(1) slant medium: yeast powder 5.0, Tryptones 10, NaCl5.0, agar 20, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(2) seed culture medium: yeast extract paste 7.0, Tryptones 10, glucose 30, K
2hPO
4h
2o0.5, MgSO
46H
2o0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(3) fermention medium: glucose 80, monosodium glutamate 80, yeast extract paste 20, NH
4nO
34.1, NaCl10, MgSO
46H
2o0.5, CaCl
26H
2o1.0, FeCl
36H
2o0.01, said components unit is g/L, PH7.0.121 DEG C of high pressure steam sterilization 20min.
Feeding culture based component: glucose 900, NH
4nO
360, CaCl
26H
2o20, FeCl
37H
2o0.15, said components unit is g/L, PH7.0,121 DEG C of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 DEG C in culture medium slant, the inclined-plane seed that preparation is ripe.
Three, seed culture:
Inclined-plane seed after activation is accessed a ring in the 500ml triangular flask of 50ml liquid seed culture medium, connects 14 bottles altogether, 37 DEG C, 220rpm, cultivate 16 hours to logarithmic phase.
Four, fermentation culture:
Accessed in fermentation culture by the seed culture fluid grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, add fed-batch medium when residual sugar in fermenting process is down to 5g/L to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss as 5.9L, γ-PGA output is 45g/L.
Five, broth extraction
(1) except thalline: fermented liquid PH is adjusted to 3.2, and viscosity is reduced to 16.9mPas by 83.4mPas, add the diatomite of 2% quality volume percent, 800 order filter clothes, 0.22MP pressure, Plate Filtration removes thalline, flow velocity 36ml/min.
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts PH6.7, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein.
(3) Zeo-karb is except metal ion and some positively charged impurity
By fermentating liquid volume 30%(v:v) addition add Zeo-karb, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 9%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment PH to 7, introducing a small amount of ion subsequent nano-filtration can remove.
(4) decolour
Add 1% gac, carry out normal temperature low rate mixing, decolouring 90min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4.5L.
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01um aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/8, Glu content and is reduced to 1/7, obtains clear liquid 9L.
(6) ultrafiltration grading extraction
Above-mentioned 9L clear liquid, adopts aperture 0.1um hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 15ml/min, obtains 44.4% permeate and 50% one-level trapped fluid.By permeate through 50kDa ultra-filtration membrane, 0.1MP filtering under pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 1.8L, after testing, γ-PGA content is 40g/L, molecular-weight average is about 120,000, and spraying dry obtains γ-PGA white powder 62.1g.
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is carried out ultrafiltration through 500kDa ultra-filtration membrane, flow velocity 4ml/min, collect 50% permeate and 44% non-permeate, detect γ-PGA content and be respectively 45g/L and 43g/L, molecular-weight average is about 280,000 and 450,000, add 2.5 times of volume ethanol respectively and carry out alcohol precipitation, flocculation part is redissolved respectively to 2.5L, carry out spraying dry respectively, obtaining the γ-PGA white powder that molecular-weight average is respectively 280,000 and 450,000, is 99.2g and 90.4g respectively.Total recovery is 92.4%.
Embodiment 2
One, substratum preparation:
(1) slant medium: yeast powder 5.0, Tryptones 10, NaCl5.0, agar 20, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(2) seed culture medium: yeast extract paste 7.0, Tryptones 10, glucose 30, K
2hPO
4h
2o0.5, MgSO
46H
2o0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(3) fermention medium: glucose 80, monosodium glutamate 80, yeast extract paste 20, NH
4nO
34.1, NaCl10, MgSO
46H
2o0.5, CaCl
26H
2o1.0, FeCl
36H
2o0.01, said components unit is g/L, PH7.0.121 DEG C of high pressure steam sterilization 20min.
Feeding culture based component: glucose 900, NH
4nO
360, CaCl
26H
2o20, FeCl
37H
2o0.15, said components unit is g/L, PH7.0,121 DEG C of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 DEG C in culture medium slant, the inclined-plane seed that preparation is ripe.
Three, seed culture:
Inclined-plane seed after activation is accessed a ring in the 500ml triangular flask of 50ml liquid seed culture medium, connects 14 bottles altogether, 37 DEG C, rotating speed 220rpm, cultivate 16 hours to logarithmic phase.
Four, fermentation culture:
Accessed in fermentation culture by the seed culture fluid grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, add fed-batch medium when residual sugar in fermenting process is down to 5g/L to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss 6L, and γ-PGA output is 42g/L.
Five, broth extraction
(1) except thalline: fermented liquid PH is adjusted to 3.0, and viscosity is reduced to 14.3mPas by 76.4mPas, add the diatomite of 2.5% quality volume percent, 800 order filter clothes, 0.22MP pressure, Plate Filtration removes thalline, flow velocity 34ml/min.
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts PH6.7, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein.
(3) Zeo-karb is except metal ion and some positively charged impurity
By fermentating liquid volume 30%(v:v) addition add Zeo-karb, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 14%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment PH to 7, introducing a small amount of ion subsequent nano-filtration can remove.
(4) decolour
Add 1.5% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4L.
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01um aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/7, Glu content and is reduced to 1/7, obtains clear liquid 9L.
(6) ultrafiltration grading extraction
Above-mentioned 9L clear liquid, adopts aperture 0.1um hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 18ml/min, obtains 49% permeate and 44% one-level trapped fluid.Permeate is crossed ultra-filtration membrane (50kDa), 0.1MP pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 2.7L, after testing, γ-PGA content is 42g/L, molecular-weight average is about 110,000, and spraying dry obtains γ-PGA white powder 102.4g.
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is crossed 500kDa ultra-filtration membrane and carries out ultrafiltration, flow velocity 5ml/min, collects 44% permeate and 42% non-permeate, detects γ-PGA content and is respectively 45g/L and 40g/L, molecular-weight average is about 280,000 and 400,000, add 3 times of volume ethanol respectively and carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, carries out spraying dry respectively, obtain γ-PGA white powder, quality is respectively 80.3g and 57.8g.Total recovery is 92.7%.
Embodiment 3
One, substratum preparation:
(1) slant medium: yeast powder 5.0, Tryptones 10, NaCl5.0, agar 20, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(2) seed culture medium: yeast extract paste 7.0, Tryptones 10, glucose 30, K
2hPO
4h
2o0.5, MgSO
46H
2o0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(3) fermention medium: glucose 80, monosodium glutamate 80, yeast extract paste 20, NH
4nO
34.1, NaCl10, MgSO
46H
2o0.5, CaCl
26H
2o1.0, FeCl
36H
2o0.01, said components unit is g/L, PH7.0.121 DEG C of high pressure steam sterilization 20min.
Feeding culture based component: glucose 900, NH
4nO
360, CaCl
26H
2o20, FeCl
37H
2o0.15, said components unit is g/L, PH7.0,121 DEG C of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 DEG C in culture medium slant, the inclined-plane seed that preparation is ripe.
Three, seed culture:
Inclined-plane seed after activation is accessed a ring in the 500ml triangular flask of 50ml liquid seed culture medium, connects 14 bottles altogether, 37 DEG C, rotating speed 220rpm, cultivate 16 hours to logarithmic phase.
Four, fermentation culture:
Accessed in fermentation culture by the seed culture fluid grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, add fed-batch medium when residual sugar in fermenting process is down to 5g/L to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss as 5.9L, γ-PGA output is 38g/L.
Five, broth extraction
(1) except thalline: fermented liquid PH is adjusted to 3.0, and viscosity is reduced to 15.2mPas by 77.2mPas, add the diatomite that quality volume percent is 2.5%, 800 order filter clothes, 0.22MP pressure, Plate Filtration removes thalline, flow velocity 36ml/min.
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts PH7.0, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein.
(3) Zeo-karb is except metal ion and some positively charged impurity
By fermentating liquid volume 30%(v:v) addition add Zeo-karb, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 12%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment PH to 7, introducing a small amount of ion subsequent nano-filtration can remove.
(4) decolour
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4L.
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01um aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/7, Glu content and is reduced to 1/7, obtains clear liquid 9.2L.
(6) ultrafiltration grading extraction
Above-mentioned 9.2L clear liquid, adopts aperture 0.1um hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 20ml/min, obtains 58% permeate and 41% one-level trapped fluid.Permeate is crossed ultra-filtration membrane (50kDa), 0.1MP pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 2.9L, after testing, γ-PGA content is 40g/L, molecular-weight average is about 120,000, and spraying dry obtains γ-PGA white powder 108.2g.
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is crossed 500kDa ultra-filtration membrane and carries out ultrafiltration, flow velocity 5ml/min, collect 42% permeate and 55% non-permeate, detect γ-PGA content and be respectively 36g/L and 35.5g/L, molecular-weight average is about 250,000 and 450,000, add 3 times of volume ethanol respectively and carry out alcohol precipitation, in permeate, flocculation part redissolves to original volume, non-permeate alcohol precipitation flocculation part redissolves to original volume 1.5 times, carry out spraying dry respectively, obtain γ-PGA white powder.Quality is respectively 70.9g and 35.0g.Total recovery is 92.1%.
Embodiment 4
One, substratum preparation:
(1) slant medium: yeast powder 5.0, Tryptones 10, NaCl5.0, agar 20, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(2) seed culture medium: yeast extract paste 7.0, Tryptones 10, glucose 30, K
2hPO
4h
2o0.5, MgSO
46H
2o0.5, said components unit is g/L, pH7.0 ± 0.1.121 DEG C of high pressure steam sterilization 20min.
(3) fermention medium: glucose 80, monosodium glutamate 80, yeast extract paste 20, NH
4nO
34.1, NaCl10, MgSO
46H
2o0.5, CaCl
26H
2o1.0, FeCl
36H
2o0.01, said components unit is g/L, PH7.0.121 DEG C of high pressure steam sterilization 20min.
Feeding culture based component: glucose 900, NH
4nO
360, CaCl
26H
2o20, FeCl
37H
2o0.15, said components unit is g/L, PH7.0,121 DEG C of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 DEG C in culture medium slant, the inclined-plane seed that preparation is ripe.
Three, seed culture:
Inclined-plane seed after activation is accessed a ring in the 500ml triangular flask of 50ml liquid seed culture medium, connects 14 bottles altogether, 37 DEG C, rotating speed 220rpm, cultivate 16 hours to logarithmic phase.
Four, fermentation culture:
Accessed in fermentation culture by the seed culture fluid grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, add fed-batch medium when residual sugar in fermenting process is down to 5g/L to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss as 5.9L, γ-PGA output is 35g/L.
Five, broth extraction
(1) except thalline: fermented liquid PH is adjusted to 3.2, and viscosity is reduced to 18.1mPas by 78.3mPas, add the diatomite that quality volume percent is 3%, 800 order filter clothes, 0.22MP pressure, Plate Filtration removes thalline, flow velocity 32ml/min.
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts PH7.2, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein.
(3) Zeo-karb is except metal ion and some positively charged impurity
By fermentating liquid volume 30%(v:v) addition add Zeo-karb, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 7%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment PH to 7, introducing a small amount of ion subsequent nano-filtration can remove.
(4) decolour
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4.7L.
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01um aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/7, Glu content and is reduced to 1/7, obtains clear liquid 9.5L.
(6) ultrafiltration grading extraction
By above-mentioned 9.5L clear liquid, adopt aperture 0.1um hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 21ml/min, obtain 61% permeate and 35% one-level trapped fluid.Permeate is crossed ultra-filtration membrane (50kDa), 0.1MP pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 3.2L, after testing, in concentrated permeate, γ-PGA content is 32g/L, molecular-weight average is about 100,000, and spraying dry obtains γ-PGA white powder 94.1g.
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is crossed 500kDa ultra-filtration membrane and carries out ultrafiltration, flow velocity 6ml/min, collects 67% permeate and 28% non-permeate, detects γ-PGA content and is respectively 38g/L and 40g/L, molecular-weight average is respectively 230,000 and 380,000, add 3 times of volume ethanol respectively and carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, carries out spraying dry respectively, obtain γ-PGA white powder, quality is respectively 75.3g and 35.8g.Total recovery is 94.3%.
Claims (7)
1. utilize a method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, comprise the steps:
Prepared by fermented liquid:
The lichen bacillus ferments of numbering CGMCC3336 is cultivated, obtains fermented liquid;
Broth extraction:
(1) except thalline
Fermented liquid pH is adjusted to 3.0, and viscosity drop is low to moderate original 1/6, adds the diatomite of fermentating liquid volume 2%-3%, and above-mentioned is quality volume percent; 800 order filter clothes, Plate Filtration removes thalline, 0.22MPa pressure, flow velocity 30 ~ 50ml/min;
(2) except foreign protein and Large molecule active organism
PH6 ~ 7 adjusted by the fermented liquid removing thalline, and be heated to 90 DEG C of insulation 20min, make protein denaturation, removing heat denatured protein, is cooled to room temperature, crosses 0.45 μm of hollow-fibre membrane and carries out ultrafiltration, remove more than 80% foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Add Zeo-karb by the addition of fermentating liquid volume 30%, stirred at ambient temperature, carry out Static Adsorption impurity 30min, after ion-exchange terminates, readjustment pH to 7, introducing a small amount of ion subsequent nano-filtration can remove;
(4) decolour
Add 1% ~ 2% gac, carry out normal temperature low rate mixing, decolouring 90 ~ 120min, suction filtration, to the basic clear, colorless of liquid;
(5) except small molecules amino acid, ionic impurity
Fermented liquid dilution 2 ~ 3 times is rear crosses nanofiltration membrane;
(6) ultrafiltration grading extraction
The clear liquid of 50,000 ~ 500,000 ultra-filtration membranes to above-mentioned process is adopted to carry out pressurized circulation ultrafiltration and concentration, γ-the PGA of 0.1 μm of Middle hollow fiber membrane molecular weight less than 150,000,500kDa Ultra filtration membrane molecular weight is 150,000 ~ 350,000 and γ-the PGA of molecular weight more than 350,000; Obtain the clear liquid that molecular weight is different respectively: the γ-PGA clear liquid of molecular weight <15 ten thousand; Molecular weight 150,000 ~ 350,000 γ-PGA clear liquid, the γ-PGA clear liquid that molecular weight is greater than 350,000; γ-the PGA that molecular weight is less than 150,000 carries out 5kDa ultrafiltration and concentration and removes small molecular weight impurity further; γ-PGA the clear liquid that molecular weight is greater than 150,000 carries out 2 ~ 3 volumes times alcohol precipitation, and precipitate dissolves is in the water of original volume 1 ~ 2 times; By above-mentioned solution spray-dried separately, obtain refining γ-PGA.
2. utilize the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid according to claim 1, comprise the steps:
Actication of culture:
The Bacillus licheniformis of numbering CGMCC3336 is cultivated 16 hours in 37 DEG C in culture medium slant, the inclined-plane seed that preparation is ripe;
Seed culture:
Inclined-plane seed after activation is accessed a ring to be equipped with in the 500ml triangular flask of 50ml liquid seed culture medium, 37 DEG C, 220rpm, cultivates 16 hours to logarithmic phase;
Fermentation culture:
Accessed by seed culture fluid in fermentation culture, inoculum size is cultivate 72h under 10%, 220rpm, and fermentor tank is 10L, and liquid amount is 60%, and add fed-batch medium when residual sugar is down to 5g/L to fermentation ends first 4 hours, flow acceleration is 1ml/min; Lower tank γ-PGA output is 30g/L ~ 45g/L;
Broth extraction:
(1) except thalline
Fermented liquid pH is adjusted to 3.0, and viscosity drop is low to moderate original 1/6, adds the diatomite of 2%-3% quality volume percent, 800 order filter clothes, and Plate Filtration removes thalline, 0.22MPa pressure, flow velocity 30 ~ 50ml/min;
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts pH6 ~ 7, and be heated to 90 DEG C of insulation 20min, make protein denaturation, removing heat denatured protein, is cooled to room temperature, crosses 0.45 μm of hollow-fibre membrane and carries out ultrafiltration, remove more than 80% foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Zeo-karb is added by the addition of fermentating liquid volume 30% (v:v), stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, most of metal ion and positively charged impurity can be removed, after ion-exchange terminates, with 6M sodium hydroxide readjustment pH to 7, introducing a small amount of ion subsequent nano-filtration can remove;
(4) decolour
Add 1% ~ 2% gac, carry out normal temperature low rate mixing, decolouring 90 ~ 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable;
(5) except small molecules amino acid, ionic impurity
Fermented liquid dilution 2 ~ 3 times is rear crosses nanofiltration membrane, after testing, can remove Most amino-acids, polypeptide, oligosaccharides, salt ion small molecular weight impurity;
(6) ultrafiltration grading extraction
The clear liquid of 50,000 ~ 500,000 ultra-filtration membranes to above-mentioned process is adopted to carry out pressurized circulation ultrafiltration and concentration, γ-the PGA of 0.1 μm of Middle hollow fiber membrane molecular weight less than 150,000,500kDa Ultra filtration membrane molecular weight is 150,000 ~ 350,000 and γ-the PGA of molecular weight more than 350,000; Obtain the clear liquid that molecular weight is different respectively: the γ-PGA clear liquid of molecular weight <15 ten thousand; Molecular weight 150,000 ~ 350,000 γ-PGA clear liquid, the γ-PGA clear liquid that molecular weight is greater than 350,000; γ-the PGA that molecular weight is less than 150,000 carries out 5kDa ultrafiltration and concentration and removes small molecular weight impurity further; γ-PGA the clear liquid that molecular weight is greater than 150,000 carries out 2 ~ 3 volumes times alcohol precipitation, and precipitate dissolves is in the water of original volume 1 ~ 2 times; By above-mentioned solution spray-dried separately, obtain refining γ-PGA.
3. according to claim 1 or 2, utilize the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, it is characterized in that, broth extraction step is as follows:
(1) except thalline: fermented liquid pH is adjusted to 3.2, and viscosity is reduced to 16.9mPas by 83.4mPas, add the diatomite of 2% quality volume percent, 800 order filter clothes, 0.22MPa pressure, Plate Filtration removes thalline, flow velocity 36ml/min;
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts pH6.7, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45 μm of hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Zeo-karb is added by the addition of fermentating liquid volume 30% (v:v), stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 9%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment pH to 7, introducing a small amount of ion subsequent nano-filtration can remove;
(4) decolour
Add 1% gac, carry out normal temperature low rate mixing, decolouring 90min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4.5L;
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01 μm of aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/8, Glu content and is reduced to 1/7, obtains clear liquid 9L;
(6) ultrafiltration grading extraction
Above-mentioned 9L clear liquid, adopts 0.1 μm, aperture hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 15ml/min, obtains 44.4% permeate and 50% one-level trapped fluid; By permeate through 50kDa ultra-filtration membrane, 0.1MP filtering under pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 1.8L, after testing, γ-PGA content is 40g/L, molecular-weight average is 120,000, and spraying dry obtains γ-PGA white powder;
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is carried out ultrafiltration through 500kDa ultra-filtration membrane, flow velocity 4ml/min, collect 50% permeate and 44% non-permeate, detect γ-PGA content and be respectively 45g/L and 43g/L, molecular-weight average is 280,000 and 450,000, add 2.5 times of volume ethanol respectively and carry out alcohol precipitation, flocculation part is redissolved respectively to 2.5L, carry out spraying dry respectively, obtain the γ-PGA white powder that molecular-weight average is respectively 280,000 and 450,000.
4. according to claim 1 or 2, utilize the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, it is characterized in that, broth extraction step is as follows:
(1) except thalline: fermented liquid pH is adjusted to 3.0, and viscosity is reduced to 14.3mPas by 76.4mPas, add the diatomite of 2.5% quality volume percent, 800 order filter clothes, 0.22MPa pressure, Plate Filtration removes thalline, flow velocity 34ml/min;
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts pH6.7, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45 μm of hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Zeo-karb is added by the addition of fermentating liquid volume 30% (v:v), stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 14%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment pH to 7, introducing a small amount of ion subsequent nano-filtration can remove;
(4) decolour
Add 1.5% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4L;
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01 μm of aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/7, Glu content and is reduced to 1/7, obtains clear liquid 9L;
(6) ultrafiltration grading extraction
Above-mentioned 9L clear liquid, adopts 0.1 μm, aperture hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 18ml/min, obtains 49% permeate and 44% one-level trapped fluid; Permeate is crossed 50kDa ultra-filtration membrane, 0.1MP pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 2.7L, after testing, γ-PGA content is 42g/L, molecular-weight average is 110,000, and spraying dry obtains γ-PGA white powder;
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is crossed 500kDa ultra-filtration membrane and carries out ultrafiltration, flow velocity 5ml/min, collect 44% permeate and 42% non-permeate, detect γ-PGA content and be respectively 45g/L and 40g/L, molecular-weight average is 280,000 and 400,000, add 3 times of volume ethanol respectively and carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, carries out spraying dry respectively, obtain γ-PGA white powder.
5. according to claim 1 or 2, utilize the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, it is characterized in that, broth extraction step is as follows:
(1) except thalline: fermented liquid pH is adjusted to 3.0, and viscosity is reduced to 15.2mPas by 77.2mPas, add the diatomite that quality volume percent is 2.5%, 800 order filter clothes, 0.22MPa pressure, Plate Filtration removes thalline, flow velocity 36ml/min;
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts pH7.0, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45 μm of hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Zeo-karb is added by the addition of fermentating liquid volume 30% (v:v), stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 12%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment pH to 7, introducing a small amount of ion subsequent nano-filtration can remove;
(4) decolour
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4L;
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01 μm of aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/7, Glu content and is reduced to 1/7, obtains clear liquid 9.2L;
(6) ultrafiltration grading extraction
Above-mentioned 9.2L clear liquid, adopts 0.1 μm, aperture hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 20ml/min, obtains 58% permeate and 41% one-level trapped fluid; Permeate is crossed 50kDa ultra-filtration membrane, 0.1MP pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 2.9L, after testing, γ-PGA content is 40g/L, molecular-weight average is 120,000, and spraying dry obtains γ-PGA white powder;
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is crossed 500kDa ultra-filtration membrane and carries out ultrafiltration, flow velocity 5ml/min, collect 42% permeate and 55% non-permeate, detect γ-PGA content and be respectively 36g/L and 35.5g/L, molecular-weight average is 250,000 and 450,000, add 3 times of volume ethanol respectively and carry out alcohol precipitation, in permeate, flocculation part redissolves to original volume, non-permeate alcohol precipitation flocculation part redissolves to original volume 1.5 times, carry out spraying dry respectively, obtain γ-PGA white powder.
6. according to claim 1 or 2, utilize the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, it is characterized in that, broth extraction step is as follows:
(1) except thalline: fermented liquid pH is adjusted to 3.2, and viscosity is reduced to 18.1mPas by 78.3mPas, add the diatomite that quality volume percent is 3%, 800 order filter clothes, 0.22MPa pressure, Plate Filtration removes thalline, flow velocity 32ml/min;
(2) except foreign protein and Large molecule active organism
The fermented liquid sodium hydroxide removing thalline adjusts pH7.2, is heated to 90 DEG C of insulation 20min, is cooled to room temperature, cross 0.45 μm of hollow-fibre membrane and carry out ultrafiltration, remove more than 80% foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Zeo-karb is added by the addition of fermentating liquid volume 30% (v:v), stirred at ambient temperature, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 7%, removes the positively charged impurity of part, after ion-exchange terminates simultaneously, with 6M sodium hydroxide readjustment pH to 7, introducing a small amount of ion subsequent nano-filtration can remove;
(4) decolour
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, the basic clear, colorless of liquid, decolorizing effect is remarkable, obtains fermented liquid 4.7L;
(5) except small molecules amino acid, ionic impurity
Be diluted to 10L and cross 0.01 μm of aperture nanofiltration membrane, after testing, ionic concn is reduced to 1/7, Glu content and is reduced to 1/7, obtains clear liquid 9.5L;
(6) ultrafiltration grading extraction
By above-mentioned 9.5L clear liquid, adopt 0.1 μm, aperture hollow-fibre membrane to carry out ultrafiltration to it, flow velocity 21ml/min, obtain 61% permeate and 35% one-level trapped fluid; Permeate is crossed 50kDa ultra-filtration membrane, 0.1MP pressure, the concentrated organism also simultaneously again removing ion and molecular weight is carried out to permeate, discard second entrapment liquid, obtain concentrated permeate 3.2L, after testing, in concentrated permeate, γ-PGA content is 32g/L, molecular-weight average is 100,000, and spraying dry obtains γ-PGA white powder;
γ-the PGA of macromolecule is trapped within the one-level trapped fluid of hollow-fibre membrane, one-level trapped fluid is crossed 500kDa ultra-filtration membrane and carries out ultrafiltration, flow velocity 6ml/min, collect 67% permeate and 28% non-permeate, detect γ-PGA content and be respectively 38g/L and 40g/L, molecular-weight average is respectively 230,000 and 380,000, add 3 times of volume ethanol respectively and carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, carries out spraying dry respectively, obtain γ-PGA white powder.
7. according to claim 1 or 2, utilize the method for polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, it is characterized in that, substratum compound method is as follows:
(1) slant medium: yeast powder 5.0, Tryptones 10, NaCl5.0, agar 20, said components unit is g/L, pH7.0 ± 0.1; 121 DEG C of high pressure steam sterilization 20min;
(2) seed culture medium: yeast extract paste 7.0, Tryptones 10, glucose 30, K
2hPO
4h
2o0.5, MgSO
46H
2o0.5, said components unit is g/L, pH7.0 ± 0.1; 121 DEG C of high pressure steam sterilization 20min;
(3) fermention medium: glucose 80, monosodium glutamate 80, yeast extract paste 20, NH
4nO
34.1, NaCl10, MgSO
46H
2o0.5, CaCl
26H
2o1.0, FeCl
36H
2o0.01, said components unit is g/L, pH7.0; 121 DEG C of high pressure steam sterilization 20min;
Feeding culture based component: glucose 900, NH
4nO
360, CaCl
26H
2o20, FeCl
37H
2o0.15, said components unit is g/L, pH7.0,121 DEG C of high pressure steam sterilization 20min.
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| EP3190904A4 (en) | 2014-09-10 | 2018-07-18 | International Dehydrated Foods, Inc. | Process for preparing a pumpable broth composition |
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| CN112480394A (en) * | 2020-12-01 | 2021-03-12 | 广西大学 | Method for separating and purifying ultra-high molecular weight poly-gamma-glutamic acid from high-viscosity fermentation liquor |
| CN117050021B (en) * | 2023-10-13 | 2023-12-15 | 北京绿色康成生物技术有限公司 | Method for separating and extracting tetrahydropyrimidine from fermentation liquor |
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| CN101538593A (en) * | 2009-04-13 | 2009-09-23 | 浙江大学 | Method for coupling production of Gamma-polyglutamic acid by technologies of microbial fermentation and membrane separation |
| CN102154395A (en) * | 2010-12-30 | 2011-08-17 | 天津北洋百川生物技术有限公司 | Method for extracting gamma-polyglutamic acid by inorganic salt/organic solvent coprecipitation effect |
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| CA2310631A1 (en) * | 1997-11-18 | 1999-05-27 | Worcester Polytechnic Institute | High molecular weight .gamma.-poly(glutamic acid) |
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| CN101538593A (en) * | 2009-04-13 | 2009-09-23 | 浙江大学 | Method for coupling production of Gamma-polyglutamic acid by technologies of microbial fermentation and membrane separation |
| CN102154395A (en) * | 2010-12-30 | 2011-08-17 | 天津北洋百川生物技术有限公司 | Method for extracting gamma-polyglutamic acid by inorganic salt/organic solvent coprecipitation effect |
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