CN103665371A - Method for refining polyglutamic acid in biologic fermentation broth by using ultrafiltration and nanofiltration techniques - Google Patents
Method for refining polyglutamic acid in biologic fermentation broth by using ultrafiltration and nanofiltration techniques Download PDFInfo
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Abstract
The invention discloses a method for refining polyglutamic acid in biologic fermentation broth by using ultrafiltration and nanofiltration techniques, which belongs to the technique field of biologic synthesis and purification. Aiming at the distribution characteristics of target extracts and molecular weights of impurities in the fermentation broth, the method is used for effectively separating gamma-PGA of different molecular weights with the combination of modern film separation techniques, and the method mainly comprises the following steps: reducing the viscosity of kieselguhr, filtering and sterilizing, heating to be combined with ultrafiltration decontamination proteins and macromolecule active organisms, removing metal ions and impurities with positive charges by using a cation exchange resin, decoloring, subsequently removing micromolecule and ionic impurities in a nanofiltration mode, performing ultrafiltration and extraction at different stages so as to obtain refined gamma-PGA of different molecular weights. The extraction yield is kept greater than 92%, which is higher than the highest yield of 90.6% reported in documents, high market pertinence is achieved, production can be performed according to demands, and the economic benefits are professionally improved as gamma-PGA of different molecular weights are refined with the combination of ultrafiltration and nanofiltration techniques.
Description
Technical field:
The invention belongs to biological process synthesizes 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 bioabsorbable polymer material of many uses and that can degrade and just seem particularly important.It is the synthetic a kind of extracellular high molecular polymerization compound of some genus bacillus (Bacillus) that γ-poly-glutaric acid (γ-Polyglutamicacid) is write a Chinese character in simplified form γ-PGA, it take L-glutamic acid as only monomer, be by L mono-L-glutamic acid (L-Glu), D mono-L-glutamic acid (D-Glu) by amido linkage in conjunction with a kind of peptide molecule forming, 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 that has potentiality, utilizes hydrogel that γ-PGA makes not only to retain the fundamental characteristics of ortho-water gelatinous material, also has the characteristics such as structural controllability, high-hydroscopicity, higher mechanical strength, biodegradability.Particularly by fermentation of bacillus, produce the γ-PGA obtaining, on its molecular chain, there is the side chain carboxyl group that a large amount of activity are higher, there is good high-hydroscopicity (1:3500, m/v), biocompatibility, biological degradability, the advantage such as nontoxic, can be used as that cosmetics additive, water-holding agent, High hydrophilous resin, heavy metal absorbent, foodstuff additive, medical carrier, tissue engineering material etc. are widely used in makeup, food, agricultural, medicine, synthon and the field such as film.
Since the nineties in 20th century, Japan, some developed countries such as the U.S. are very active to the research of the preparation of γ-PGA and application, the technology of Production by Microorganism Fermentation γ-PGA is also in the prostatitis in the world, with Ajincomoto Co., Inc, the performance of the state other unit that He Guangqi university of Meiji Seika Kaisba company is representative to γ-PGA, synthesize and should be used as more in depth research, 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, to γ-PGA, the research of producing of going into operation in a large number is also short of in China's Mainland, especially in sticky fermented liquid, concerning molecular weight γ-PGA within the specific limits, extract the efficiency of γ-PGA and only have 40～50%, cause a large amount of γ-PGA 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, can make the product application of different relative molecular masses in various different field.If γ-PGA molecular weight of 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 with inferior.
Adopt the pKa value of determination of acid-basetitration sequestered γ-PGA, obtain the pKa=2.23 of γ-PGA, this value is unanimous on the whole with the pKa value of the α-carboxyl of L-glutamic acid.Utilize TGA and DSC to carry out the analysis of thermal properties, show that its heat decomposition temperature is 235.9 ℃, fusing point is 223.5 ℃.After γ-PGA acid hydrolysis, with GITC, do derivatization reagent, HPLC records the ratio of D-Glu and Pidolidone in γ-PGA and is stabilized in D:L=3:2.Generally speaking, the molecular-weight average of the γ-PGA being produced by genus bacillus (Mw) is 10
4-l0
6between, in this patent, with the molecular weight (Mw) of laboratory biological fermentation process output γ-PGA, be distributed in 5 * 10
4-10
6between.
Membrane separation technique utilize film to the selection perviousness of each component in mixture come separated, extract and concentrated object product, membrane sepn process is carried out at normal temperatures, without phase transformation, energy consumption is low, equipment is simple, convenient operation and control, is applied in a plurality of fields such as chemical industry, medicine, light industry, food, weaving, electronics, metallurgy.The membrane separation technique that is applicable to fermentation liquor treatment has micro-filtration, ultrafiltration, nanofiltration and reverse osmosis.Micro-filtration is held back particle more than 0.01～10um, 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 hold back the macromolecular substance such as virus, protein, enzyme, polysaccharide; Reverse osmosis only allows solvent molecule to pass through, and the small-molecule substances such as salt, amino acid are also trapped; Nanofiltration membrane mean pore size 2nm, holding back component can be little of microbiotic, synthetic drug, dyestuff, disaccharides etc., allows the small-molecule substances such as water, inorganic salt, organism to pass through, and 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 in asymmetric membrane, play crown_interception is fine and close surperficial cortex, its thickness is 0.1～15um only, subcutaneous, there are porous network structure and a supporting layer, supporting layer thickness, at 50～250um, had so both guaranteed the effectively catching to macromolecular substance, and while reducing lock out operation again, film is to resistance.Improve membrane flux, also there is certain physical strength.
γ-PGA is a kind of extracellular products, and its fermented liquid is very glutinous, removes thalline and is difficult to, and be not suitable for industrial large-scale application by general centrifuging.In order to reduce, to remove the energy expenditure of thalline and save the consumption of organic solvent for precipitating, DO.J.H etc. are optimized former technique, have proposed the method for high efficiency separation γ-PGA a kind of.The method is comprised of two steps: first adjust PH to 3.0 to make reduced viscosity, then bactofugation body, make like this centrifugal energy reduce to original 17%; Then to going bacterium liquid to pull back to PH5.0, with hollow-fibre membrane, filter, make fermented liquid supernatant by original 20g/L, be concentrated into 60g/L, extract required ethanol consumption and be reduced to originally 1/4, reduced the cost of extraction γ-PGA.But this method because do not remove other impurity, so Interception process to the having relatively high expectations of tunica fibrosa, and bactofugation is difficult to amplify industrial production.The method > > of patent < < separation and Extraction gamma-polyglutamic acid-from fermented liquid, in application number 201010188232.6, mention, with ultra-filtration membrane, remove small molecules pigment and impurity, but in extraction process, repeatedly regulate PH, can introduce a large amount of ions, with a small amount of method of protein of protease hydrolyzed, introduce too impurity, do not fully utilize film and extract, be not suitable for amplifying and produce.A kind of separation purification method > > of patent < < gamma-polyglutamic acid-, in application number 201210396366.6, mention, after gac and diatomite suction filtration, directly be used for ultrafiltration and concentration, this processing mode is easy to cause stifled film phenomenon in industrial amplification, and wash film program, make extraction step loaded down with trivial details, this technique should not be amplified industrial production.The extracting method > > of a patent < < gamma-polyglutamic acid-, in application number 201010177020.8, mention, after enzymolysis protein, with 2～3 times of ethanol, carry out alcohol precipitation, by ultrafiltration and concentration method, carrying out removal of impurities, last freeze-drying, in this technique, uses a large amount of ethanol, increase the danger of industrial application, also do not mention according to γ-PGA molecular weight is different and effectively extracting.Bibliographical information, γ-the PGA that extracts different molecular weight changes fermentation condition to produce the approaching γ-PGA of molecular weight as far as possible, but in actual production technique, γ-PGA molecular weight is larger, be difficult to accurately control aborning γ-PGA molecular weight, often output γ-PGA distributes in certain molecular weight ranges, and in traditional extraction technique, do not have to relate in same batch fermentation liquid and accurately extract γ-PGA according to molecular weight difference, therefore, in extraction process, the reduction of fermentation broth viscosity, simultaneously in later separation how according to molecular weight difference and high efficiency extraction γ-PGA, reduce costs is the subject matter of puzzlement Bacillus licheniformis (B.licheniformis) fermentation preparation γ-PGA always, need to pay much attention to.
Summary of the invention
The object of this invention is to provide a kind of from γ-PGA fermented liquid the method for high efficiency extraction γ-PGA, 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 method > > of 200910228297 patent of invention < < producing gamma-polyglutamic acid by inert carrier solid state fermentation method, the fermentation process adopting is a kind of efficient fermentation process of the said firm's invention, specifically see patent of invention < < a large amount of method > > who produces 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) that produces gamma-polyglutamic acid-is distributed in 5 * 10
4-5 * 10
5between.The present invention is directed to the distribution character of target extract and impurity molecule amount in fermented liquid, in conjunction with Modern Membrane Technology, the method of high efficiency extraction γ-PGA a kind of is provided, make γ-PGA total recovery be greater than 92%, higher than the highest level 90.6% of document announcement at present, and the γ-PGA of different molecular weight is carried out to effective separation, 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 reduced the use of organic solvent, made cost more than 30%, also reduced risk level simultaneously.
A method of utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, comprises the steps:
One, fermented liquid preparation: the lichen bacillus ferments of numbering CGMCC3336 is cultivated, obtained fermented liquid;
Two, broth extraction
(1) except thalline
Fermented liquid PH is adjusted to 3.0, and reduced viscosity, to original 1/6, adds 2%-3% (m:v) diatomite, 800 order filter clothes, and Plate Filtration is except thalline, 0.22MPa pressure, flow velocity 30～50ml/min.
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6～7, be heated to 90 ℃ of insulation 20min, make protein denaturation, remove heat denatured protein, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, can remove most of metal ion and positively charged impurity, after ion-exchange finishes, readjustment PH to 7, introduces the follow-up nanofiltration of a small amount of ion meeting and removes.
(4) decolouring
Add 1%～2% gac, carry out normal temperature low rate mixing, decolouring 90～120min, suction filtration, liquid is clarified colourless substantially, and decolorizing effect is remarkable.
(5) except small molecules amino acid, ionic impurity
2～3 times of rear nanofiltration membrane of crossing of fermented liquid dilution, after testing, can remove the small molecular weight impurities such as Most amino-acids, polypeptide, oligosaccharides, salt ion.
(6) ultrafiltration grading extraction
Adopt 50,000～500,000 ultra-filtration membranes to carry out pressurized circulation ultrafiltration and concentration to the clear liquid of above-mentioned processing, γ-PGA that 0.1um Middle hollow fiber membrane molecular weight 150,000 is upper and lower, γ-PGA that 500kDa ultra-filtration membrane isolated molecule amount 350,000 is upper and lower.Obtain respectively γ-PGA clear liquid of the different clear liquid of molecular weight: molecular weight <15 ten thousand; Molecular weight is at γ-PGA clear liquid of 150,000～350,000, γ-PGA clear liquid that molecular weight is greater than 350,000.Molecular weight is less than γ-PGA of 150,000 to carry out 5kDa ultrafiltration and concentration and further removes small molecular weight impurity; Molecular weight is greater than γ-PGA clear liquid of 150,000 and carries out 2～3 volumes times alcohol precipitation, and throw out is dissolved in the water of 1～2 times of original volume.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 ℃ in culture medium slant, prepared ripe inclined-plane seed.
Two, seed culture:
Inclined-plane seed access one ring after activation is equipped with in the 500ml triangular flask of 50ml liquid seed culture medium, and 37 ℃, 220rpm, cultivates 16 hours to logarithmic phase.
Three, fermentation culture:
Seed culture fluid is accessed in fermentation culture, and inoculum size is to cultivate 72h under 10%, 220rpm, and fermentor tank is 10L, and liquid amount is 60%, adds fed-batch medium to fermentation ends first 4 hours when residual sugar is down to 5g/L, and 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 reduced viscosity, to original 1/6, adds 2%-3% (m:v) diatomite, 800 order filter clothes, and Plate Filtration is except thalline, 0.22MPa pressure, flow velocity 30～50ml/min.
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6～7 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, make protein denaturation, remove heat denatured protein, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, can remove most of metal ion and positively charged impurity, after ion-exchange finishes, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove.
(4) decolouring
Add 1%～2% gac, carry out normal temperature low rate mixing, decolouring 90～120min, suction filtration, liquid is clarified colourless substantially, and decolorizing effect is remarkable.
(5) except small molecules amino acid, ionic impurity
2～3 times of rear nanofiltration membrane of crossing of fermented liquid dilution, after testing, can remove the small molecular weight impurities such as Most amino-acids, polypeptide, oligosaccharides, salt ion.
(6) ultrafiltration grading extraction
Adopt 50,000～500,000 ultra-filtration membranes to carry out pressurized circulation ultrafiltration and concentration to the clear liquid of above-mentioned processing, γ-PGA that 0.1um Middle hollow fiber membrane molecular weight 150,000 is upper and lower, γ-PGA that 500kDa ultra-filtration membrane isolated molecule amount 350,000 is upper and lower.Obtain respectively γ-PGA clear liquid of the different clear liquid of molecular weight: molecular weight <15 ten thousand; Molecular weight is at γ-PGA clear liquid of 150,000～350,000, γ-PGA clear liquid that molecular weight is greater than 350,000.Molecular weight is less than γ-PGA of 150,000 to carry out 5kDa ultrafiltration and concentration and further removes small molecular weight impurity; Molecular weight is greater than γ-PGA clear liquid of 150,000 and carries out 2～3 volumes times alcohol precipitation, and throw out is dissolved in the water of 1～2 times of original volume.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 ℃ 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 ℃ 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 ℃ 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 ℃ of high pressure steam sterilization 20min.
Beneficial effect:
The present invention is compared with prior art: first, adopt the degerming of Plate Filtration method, overcome traditional centrifuging and removed the problem of thalline difficulty, effectively reduce in fermentative Production γ-PGA process, cost aspect degerming, greatly improved 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; The 3rd, the γ-PGA of the refining different molecular weight of ultrafiltration nanofiltration combination technology, market specific aim is stronger, can accomplish to produce as required, with specialization, has improved economic benefit; The 4th, greatly reduced the use of organic solvent, made cost more than 30%, also reduced risk level simultaneously; The 5th, adopt spray drying technology, the γ-PGA obtaining is white powder, than the easy realization of freeze-drying, saves the energy, and more efficient, mode of appearance is also better; The 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 ℃ 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 ℃ 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 ℃ 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 ℃ of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 ℃ in culture medium slant, prepared ripe inclined-plane seed.
Three, seed culture:
Inclined-plane seed access one after activation is encircled to the 500ml triangular flask of 50ml liquid seed culture medium, connect altogether 14 bottles, 37 ℃, 220rpm, cultivates 16 hours to logarithmic phase.
Four, fermentation culture:
By in the seed culture fluid access fermentation culture of having grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, when residual sugar is down to 5g/L in fermenting process, add fed-batch medium to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss as 5.9L, and 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, adds the diatomite of 2% quality volume percent, 800 order filter clothes, 0.22MP pressure, Plate Filtration is except thalline, flow velocity 36ml/min.
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6.7 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 9%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove.
(4) decolouring
Add 1% gac, carry out normal temperature low rate mixing, decolouring 90min, suction filtration, liquid is clarified colourless substantially, and 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, and flow velocity 15ml/min obtains 44.4% and sees through liquid and 50% one-level trapped fluid.Will be through liquid through 50kDa ultra-filtration membrane, 0.1MP filtering under pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 1.8L of seeing through, after testing, γ-PGA content is 40g/L, molecular-weight average is in 120,000 left and right, and spraying is dried and 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 to ultrafiltration through 500kDa ultra-filtration membrane, flow velocity 4ml/min, collect 50% and see through liquid and 44% through liquid, detect γ-PGA content and be respectively 45g/L and 43g/L, molecular-weight average is about 280,000 and 450,000, add respectively 2.5 times of volume ethanol to carry out alcohol precipitation, flocculation part is redissolved respectively to 2.5L, spray respectively dry, obtaining molecular-weight average and be respectively γ-PGA white powder of 280,000 and 450,000, is respectively 99.2g and 90.4g.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 ℃ 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 ℃ 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 ℃ 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 ℃ of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 ℃ in culture medium slant, prepared ripe inclined-plane seed.
Three, seed culture:
Inclined-plane seed access one after activation is encircled to the 500ml triangular flask of 50ml liquid seed culture medium, connect altogether 14 bottles, 37 ℃, rotating speed 220rpm, cultivates 16 hours to logarithmic phase.
Four, fermentation culture:
By in the seed culture fluid access fermentation culture of having grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, when residual sugar is down to 5g/L in fermenting process, add fed-batch medium 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, adds the diatomite of 2.5% quality volume percent, 800 order filter clothes, 0.22MP pressure, Plate Filtration is except thalline, flow velocity 34ml/min.
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6.7 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 14%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove.
(4) decolouring
Add 1.5% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, liquid is clarified colourless substantially, and 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, and flow velocity 18ml/min obtains 49% and sees through liquid and 44% one-level trapped fluid.To cross ultra-filtration membrane (50kDa) through liquid, 0.1MP pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 2.7L of seeing through, after testing, γ-PGA content is 42g/L, molecular-weight average is in 110,000 left and right, and spraying is dried and 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 to 500kDa ultra-filtration membrane and carry out ultrafiltration, flow velocity 5ml/min, collects 44% and sees through liquid and 42% through liquid, detects γ-PGA content and is respectively 45g/L and 40g/L, molecular-weight average is about 280,000 and 400,000, add respectively 3 times of volume ethanol to carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, spray respectively dry, 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 ℃ 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 ℃ 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 ℃ 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 ℃ of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 ℃ in culture medium slant, prepared ripe inclined-plane seed.
Three, seed culture:
Inclined-plane seed access one after activation is encircled to the 500ml triangular flask of 50ml liquid seed culture medium, connect altogether 14 bottles, 37 ℃, rotating speed 220rpm, cultivates 16 hours to logarithmic phase.
Four, fermentation culture:
By in the seed culture fluid access fermentation culture of having grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, when residual sugar is down to 5g/L in fermenting process, add fed-batch medium to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss as 5.9L, and 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, adding quality volume percent is 2.5% diatomite, 800 order filter clothes, and 0.22MP pressure, Plate Filtration is except thalline, flow velocity 36ml/min.
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH7.0 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 12%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove.
(4) decolouring
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, liquid is clarified colourless substantially, and 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, and flow velocity 20ml/min obtains 58% and sees through liquid and 41% one-level trapped fluid.To cross ultra-filtration membrane (50kDa) through liquid, 0.1MP pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 2.9L of seeing through, after testing, γ-PGA content is 40g/L, molecular-weight average is in 120,000 left and right, and spraying is dried and 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 to 500kDa ultra-filtration membrane and carry out ultrafiltration, flow velocity 5ml/min, collect 42% and see through liquid and 55% through liquid, detect γ-PGA content and be respectively 36g/L and 35.5g/L, molecular-weight average is about 250,000 and 450,000, add respectively 3 times of volume ethanol to carry out alcohol precipitation, seeing through flocculation part in liquid redissolves to original volume, not seeing through liquid alcohol precipitation flocculation part redissolves to 1.5 times of original volumes, spray respectively dry, 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 ℃ 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 ℃ 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 ℃ 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 ℃ of high pressure steam sterilization 20min.
Two, actication of culture:
The bacterial classification of numbering CGMCC3336 is cultivated 16 hours in 37 ℃ in culture medium slant, prepared ripe inclined-plane seed.
Three, seed culture:
Inclined-plane seed access one after activation is encircled to the 500ml triangular flask of 50ml liquid seed culture medium, connect altogether 14 bottles, 37 ℃, rotating speed 220rpm, cultivates 16 hours to logarithmic phase.
Four, fermentation culture:
By in the seed culture fluid access fermentation culture of having grown, inoculum size is 10%, rotating speed 220rpm, cultivate 72h, fermentor tank is 10L, and liquid amount is 6L, when residual sugar is down to 5g/L in fermenting process, add fed-batch medium to fermentation ends first 4 hours, flow acceleration is 1ml/min.Lower tank amasss as 5.9L, and 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, adding quality volume percent is 3% diatomite, 800 order filter clothes, and 0.22MP pressure, Plate Filtration is except thalline, flow velocity 32ml/min.
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH7.2 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 7%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove.
(4) decolouring
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, liquid is clarified colourless substantially, and 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, obtains 61% and sees through liquid and 35% one-level trapped fluid.To cross ultra-filtration membrane (50kDa) through liquid, 0.1MP pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 3.2L of seeing through, after testing, concentrated is 32g/L through γ-PGA content in liquid, molecular-weight average is in 100,000 left and right, and spraying is dried and 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 to 500kDa ultra-filtration membrane and carry out ultrafiltration, flow velocity 6ml/min, collects 67% and sees through liquid and 28% through liquid, detects γ-PGA content and is respectively 38g/L and 40g/L, molecular-weight average is respectively 230,000 and 380,000, add respectively 3 times of volume ethanol to carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, spray respectively dry, obtain γ-PGA white powder, quality is respectively 75.3g and 35.8g.Total recovery is 94.3%.
Claims (7)
1. a method of utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, comprises the steps:
Fermented liquid preparation:
The lichen bacillus ferments of numbering CGMCC3336 is cultivated, obtained fermented liquid;
Broth extraction:
(1) except thalline
Fermented liquid PH is adjusted to 3.0, and reduced viscosity, to 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 is except thalline, 0.22MPa pressure, flow velocity 30～50ml/min;
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6～7, be heated to 90 ℃ of insulation 20min, make protein denaturation, remove heat denatured protein, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above foreign protein;
(3) Zeo-karb is except metal ion and some positively charged impurity
Addition by fermentating liquid volume 30% adds Zeo-karb, under room temperature, stirs, and carries out Static Adsorption impurity 30min, and after ion-exchange finishes, readjustment PH to 7, introduces the follow-up nanofiltration of a small amount of ion meeting and remove;
(4) decolouring
Add 1%～2% gac, carry out normal temperature low rate mixing, decolouring 90～120min, suction filtration, clarifies colourless to liquid substantially;
(5) except small molecules amino acid, ionic impurity
2～3 times of rear nanofiltration membrane of crossing of fermented liquid dilution;
(6) ultrafiltration grading extraction
Adopt 50,000～500,000 ultra-filtration membranes to carry out pressurized circulation ultrafiltration and concentration to the clear liquid of above-mentioned processing, γ-PGA that 0.1um Middle hollow fiber membrane molecular weight 150,000 is upper and lower, γ-PGA that 500kDa ultra-filtration membrane isolated molecule amount 350,000 is upper and lower; Obtain respectively γ-PGA clear liquid of the different clear liquid of molecular weight: molecular weight <15 ten thousand; Molecular weight is at γ-PGA clear liquid of 150,000～350,000, γ-PGA clear liquid that molecular weight is greater than 350,000; Molecular weight is less than γ-PGA of 150,000 to carry out 5kDa ultrafiltration and concentration and further removes small molecular weight impurity; Molecular weight is greater than γ-PGA clear liquid of 150,000 and carries out 2～3 volumes times alcohol precipitation, and throw out is dissolved in the water of 1～2 times of original volume; By above-mentioned solution spray-dried separately, obtain refining γ-PGA.
2. the method for utilizing according to claim 1 polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid, comprises the steps:
Actication of culture:
The Bacillus licheniformis of numbering CGMCC3336 is cultivated 16 hours in 37 ℃ in culture medium slant, prepared ripe inclined-plane seed;
Seed culture:
Inclined-plane seed access one ring after activation is equipped with in the 500ml triangular flask of 50ml liquid seed culture medium, and 37 ℃, 220rpm, cultivates 16 hours to logarithmic phase;
Fermentation culture:
Seed culture fluid is accessed in fermentation culture, and inoculum size is to cultivate 72h under 10%, 220rpm, and fermentor tank is 10L, and liquid amount is 60%, adds fed-batch medium to fermentation ends first 4 hours when residual sugar is down to 5g/L, and 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 reduced viscosity, to original 1/6, adds the diatomite of 2%-3% quality volume percent, 800 order filter clothes, and Plate Filtration is except thalline, 0.22MPa pressure, flow velocity 30～50ml/min;
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6～7 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, make protein denaturation, remove heat denatured protein, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, can remove most of metal ion and positively charged impurity, after ion-exchange finishes, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove;
(4) decolouring
Add 1%～2% gac, carry out normal temperature low rate mixing, decolouring 90～120min, suction filtration, liquid is clarified colourless substantially, and decolorizing effect is remarkable;
(5) except small molecules amino acid, ionic impurity
2～3 times of rear nanofiltration membrane of crossing of fermented liquid dilution, after testing, can remove the small molecular weight impurities such as Most amino-acids, polypeptide, oligosaccharides, salt ion;
(6) ultrafiltration grading extraction
Adopt 50,000～500,000 ultra-filtration membranes to carry out pressurized circulation ultrafiltration and concentration to the clear liquid of above-mentioned processing, γ-PGA that 0.1um Middle hollow fiber membrane molecular weight 150,000 is upper and lower, γ-PGA that 500kDa ultra-filtration membrane isolated molecule amount 350,000 is upper and lower; Obtain respectively γ-PGA clear liquid of the different clear liquid of molecular weight: molecular weight <15 ten thousand; Molecular weight is at γ-PGA clear liquid of 150,000～350,000, γ-PGA clear liquid that molecular weight is greater than 350,000; Molecular weight is less than γ-PGA of 150,000 to carry out 5kDa ultrafiltration and concentration and further removes small molecular weight impurity; Molecular weight is greater than γ-PGA clear liquid of 150,000 and carries out 2～3 volumes times alcohol precipitation, and throw out is dissolved in the water of 1～2 times of original volume; By above-mentioned solution spray-dried separately, obtain refining γ-PGA.
3. according to the method for utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid described in claim 1 or 2, 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, adds the diatomite of 2% quality volume percent, 800 order filter clothes, 0.22MPa pressure, Plate Filtration is except thalline, flow velocity 36ml/min;
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6.7 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 9%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove;
(4) decolouring
Add 1% gac, carry out normal temperature low rate mixing, decolouring 90min, suction filtration, liquid is clarified colourless substantially, and 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, and flow velocity 15ml/min obtains 44.4% and sees through liquid and 50% one-level trapped fluid; Will be through liquid through 50kDa ultra-filtration membrane, 0.1MP filtering under pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 1.8L of seeing through, after testing, γ-PGA content is 40g/L, molecular-weight average is in 120,000 left and right, and spraying is dried and 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 to ultrafiltration through 500kDa ultra-filtration membrane, flow velocity 4ml/min, collect 50% and see through liquid and 44% through liquid, detect γ-PGA content and be respectively 45g/L and 43g/L, molecular-weight average is about 280,000 and 450,000, add respectively 2.5 times of volume ethanol to carry out alcohol precipitation, flocculation part is redissolved respectively to 2.5L, spray respectively dry, obtain molecular-weight average and be respectively γ-PGA white powder of 280,000 and 450,000.
4. according to the method for utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid described in claim 1 or 2, 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, adds the diatomite of 2.5% quality volume percent, 800 order filter clothes, 0.22MPa pressure, Plate Filtration is except thalline, flow velocity 34ml/min;
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH6.7 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 14%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove;
(4) decolouring
Add 1.5% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, liquid is clarified colourless substantially, and 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, and flow velocity 18ml/min obtains 49% and sees through liquid and 44% one-level trapped fluid; To cross ultra-filtration membrane (50kDa) through liquid, 0.1MP pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 2.7L of seeing through, after testing, γ-PGA content is 42g/L, molecular-weight average is in 110,000 left and right, and spraying is dried and 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 to 500kDa ultra-filtration membrane and carry out ultrafiltration, flow velocity 5ml/min, collect 44% and see through liquid and 42% through liquid, detect γ-PGA content and be respectively 45g/L and 40g/L, molecular-weight average is about 280,000 and 400,000, add respectively 3 times of volume ethanol to carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, spray respectively dry, obtain γ-PGA white powder.
5. according to the method for utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid described in claim 1 or 2, 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, adding quality volume percent is 2.5% diatomite, 800 order filter clothes, and 0.22MPa pressure, Plate Filtration is except thalline, flow velocity 36ml/min;
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH7.0 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 12%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove;
(4) decolouring
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, liquid is clarified colourless substantially, and 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, and flow velocity 20ml/min obtains 58% and sees through liquid and 41% one-level trapped fluid; To cross ultra-filtration membrane (50kDa) through liquid, 0.1MP pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 2.9L of seeing through, after testing, γ-PGA content is 40g/L, molecular-weight average is in 120,000 left and right, and spraying is dried and 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 to 500kDa ultra-filtration membrane and carry out ultrafiltration, flow velocity 5ml/min, collect 42% and see through liquid and 55% through liquid, detect γ-PGA content and be respectively 36g/L and 35.5g/L, molecular-weight average is about 250,000 and 450,000, add respectively 3 times of volume ethanol to carry out alcohol precipitation, seeing through flocculation part in liquid redissolves to original volume, not seeing through liquid alcohol precipitation flocculation part redissolves to 1.5 times of original volumes, spray respectively dry, obtain γ-PGA white powder.
6. according to the method for utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid described in claim 1 or 2, 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, adding quality volume percent is 3% diatomite, 800 order filter clothes, and 0.22MPa pressure, Plate Filtration is except thalline, flow velocity 32ml/min;
(2) except foreign protein and macromole acitve organic matter
Remove the fermented liquid of thalline and adjust PH7.2 with sodium hydroxide, be heated to 90 ℃ of insulation 20min, be cooled to room temperature, cross 0.45um hollow-fibre membrane and carry out ultrafiltration, remove 80% above 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, under room temperature, stir, carry out Static Adsorption impurity 30min, after testing, metal ion is reduced to 7%, removes the positively charged impurity of part, after ion-exchange finishes simultaneously, with 6M sodium hydroxide readjustment PH to 7, introduce the follow-up nanofiltration of a small amount of ion meeting and remove;
(4) decolouring
Add 1.2% gac, carry out normal temperature low rate mixing, decolouring 120min, suction filtration, liquid is clarified colourless substantially, and 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, obtains 61% and sees through liquid and 35% one-level trapped fluid; To cross ultra-filtration membrane (50kDa) through liquid, 0.1MP pressure, to seeing through liquid, concentrate the organism of also again removing ion and molecular weight simultaneously, discard second entrapment liquid, obtain the concentrated liquid 3.2L of seeing through, after testing, concentrated is 32g/L through γ-PGA content in liquid, molecular-weight average is in 100,000 left and right, and spraying is dried and 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 to 500kDa ultra-filtration membrane and carry out ultrafiltration, flow velocity 6ml/min, collect 67% and see through liquid and 28% through liquid, detect γ-PGA content and be respectively 38g/L and 40g/L, molecular-weight average is respectively 230,000 and 380,000, add respectively 3 times of volume ethanol to carry out alcohol precipitation, flocculation part is redissolved respectively to original volume, spray respectively dry, obtain γ-PGA white powder.
7. according to the method for utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid described in claim 1 or 2, 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 ℃ 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 ℃ 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 ℃ 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 ℃ of high pressure steam sterilization 20min.
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| CN106589350A (en) * | 2016-11-10 | 2017-04-26 | 北京赛诺膜技术有限公司 | Method for refining small molecule polyglutamic acid by using ultra-filtration technology |
| CN107198219A (en) * | 2017-05-02 | 2017-09-26 | 上海纳德生物科技有限公司 | A kind of preparation method of the food additives of the polyglutamic acid containing γ |
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| CN111936523A (en) * | 2018-04-04 | 2020-11-13 | 科莱恩国际有限公司 | Methods for purifying complex biological compositions |
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| US11350652B2 (en) | 2014-09-10 | 2022-06-07 | International Dehydrated Foods, Inc. | High protein curd composition |
| US11388910B2 (en) | 2014-04-28 | 2022-07-19 | International Dehydrated Foods, Inc. | Process for preparing a collagen-rich composition |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999025864A2 (en) * | 1997-11-18 | 1999-05-27 | Worcester Polytechnic Institute | HIGH MOLECULAR WEIGHT η-POLY(GLUTAMIC ACID) |
| 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 |
-
2013
- 2013-11-14 CN CN201310574779.3A patent/CN103665371B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999025864A2 (en) * | 1997-11-18 | 1999-05-27 | Worcester Polytechnic Institute | HIGH MOLECULAR WEIGHT η-POLY(GLUTAMIC ACID) |
| 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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| US10694767B2 (en) * | 2014-04-28 | 2020-06-30 | International Dehydrated Foods, Inc. | Process for preparing a pumpable broth composition |
| US11388910B2 (en) | 2014-04-28 | 2022-07-19 | International Dehydrated Foods, Inc. | Process for preparing a collagen-rich composition |
| US11350652B2 (en) | 2014-09-10 | 2022-06-07 | International Dehydrated Foods, Inc. | High protein curd composition |
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| WO2017218827A1 (en) * | 2016-06-15 | 2017-12-21 | International Dehydrated Foods, Inc. | Process for preparing a pumpable broth composition |
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| CN112167260A (en) * | 2020-11-03 | 2021-01-05 | 黄河三角洲京博化工研究院有限公司 | Rapid preparation method of polyglutamic acid for pesticide adjuvant |
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| CN117050021A (en) * | 2023-10-13 | 2023-11-14 | 北京绿色康成生物技术有限公司 | Method for separating and extracting tetrahydropyrimidine from 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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