CN101538593A - Method for coupling production of Gamma-polyglutamic acid by technologies of microbial fermentation and membrane separation - Google Patents
Method for coupling production of Gamma-polyglutamic acid by technologies of microbial fermentation and membrane separation Download PDFInfo
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Abstract
The invention discloses a method for coupling production of Gamma-polyglutamic acid by technologies of microbial fermentation and membrane separation. Aiming at solving the difficulties that the viscosity of fermentation liquor is continuously increased due to the product, namely polyglutamic acid, so as to have serious influence on the cell growth and dissolved oxygen supply in the fermentation process, the invention proposes a method for coupling the fermentation and separation. The method comprises the following steps of: strain activation, seed culture, fermentation culture, micro-membrane filtration, ultrafiltration concentration and feed supplementing. The method can lead the viscosity of the fermentation liquor to be maintained to lower level, thus being beneficial to increasing dissolved oxygen in a fermentation tank, reducing the inhibiting effect of high viscosity of the product to the cell growth, improving the yield of Gamma-PGA and fermentation yield, reducing the production cost and being beneficial to post treatment of the fermentation liquor and refining of the product. The method has mild operation condition, good stability, fastness, high efficiency, simple equipment and low investment, has common instructive significance to the production of high-viscosity biological polymer by microorganism fermentation and has wide application prospect in the industrial community.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to the method for a kind of microbial fermentation and membrane separation technique coupling production of Gamma-polyglutamic acid.
Background technology
(γ-PGA) is the outer polypeptide of a kind of born of the same parents that is produced by multiple bacillus (Bacillus species) to gamma-polyglutamic acid-, and it is the main ingredient of certain micro-organisms pod membrane.γ-PGA is a kind of water-soluble and biodegradable material that is formed by connecting by γ-amido linkage by D-type or L-type L-glutamic acid, and it is made up of about 5000 L-glutamic acid monomer usually, and relative molecular mass is generally 100,000～1,000,000.Have the higher side chain carboxyl group of a large amount of activity on the molecular chain of γ-PGA, have high moisture retention and water-absorbent (1: 3500, m/v).Microorganism synthetic γ-PGA can be decomposed by soil bacteria excretory lytic enzyme, so its environmentally safe, be the green bio product, have the physics and chemistry and the biological characteristics of many uniquenesses such as splendid biodegradability, film-forming properties, one-tenth fibering, plasticity-, cohesiveness, moisture retention.
Because γ-PGA has good biocompatibility, biological degradability and nontoxic pollution-free, make biological medicine material that its absorbs as a kind of ideal degradable in vivo, be widely used as medicine slow/controlled release material, the retaining wetting Agent for Printing Inks of soil, sand ground, the water-setting agent and the high strength fibre of food.
Pay attention to environmental protection, emphasizing today of Sustainable development; this favor that is subjected to people by biosynthetic degradable type functional materials; just little by little be applied to the antifreeze and fresh-keeping of medical manufacturing, food-processing and vegetables, fruit, sea-food; also but Application and Development is a kind of Multifucntional biological products with very big exploitation value and bright prospects in many fields such as cosmetic industry, tobacco, leather production and plant seed protections.The γ of Japan-PGA research and development are positioned at prostatitis, the world, and Ajincomoto Co., Inc has successfully developed the biological production of γ-PGA, and product just progressively is applied to agricultural, chemical industry, medicine and other fields.
Present γ-PGA mainly contains microbe fermentation method and obtains, main production bacterial classification comprises Bacillus anthracis (Bacillus anthracis), amylomyces (Bacillus mesentericus), subtilis (Bacillus.subtilis) and bacillus natto (Bacillus natto) etc.
Obtain full-bodied fermented liquid by microbial fermentation, available organic solvent precipitation method, chemical precipitation method and the membrane sepn precipitator method obtain γ-PGA.Organic solvent deposit is meant and utilizes method centrifugal or the cohesion thalline to remove thalline in the fermented liquid, adds lower alcohols (as methyl alcohol, ethanol) the precipitable γ of obtaining-PGA in supernatant liquor, obtains white crystals through lyophilize.And chemical precipitation is to replace lower alcohols salt precipitation γ-PGA with copper/saturated copper sulphate and sodium chloride solution.Also can take the membrane sepn precipitator method to full-bodied fermented liquid.The precipitation drying can obtain white γ-PGA crude product.After dissolving crude product is in water, the centrifugal insoluble impurities of removing, the method for employing dialysis or electrodialysis desalination obtains the aqueous solution of γ-PGA, after lyophilize, can obtain high purity γ-PGA.
Before to fermented liquid precipitate and separate polyglutamic acid, need remove the thalline in the fermentation of bacillus subtilis liquid.A step is not only in the thalline separation influences the key operation of product separation effect, and,, will produce degraded to the γ-PGA in the fermented liquid as untimely separation owing to contain the degrading enzyme of polyglutamic acid in the fermented liquid in the thalline, thereby cause the decline of fermented liquid viscosity, influence product yield.The separation of cell has methods such as gravity settling, centrifugation, pressure filtration, membrane sepn usually in the fermented liquid.The gravity settling overlong time can cause a large amount of degradeds of γ-PGA.Modal in the centrifugation is differential centrifugation, but γ-PGA fermented liquid viscosity is very high, in order to reach the ideal separating effect, needs very high centrifugal force, and energy consumption is higher, is difficult to use in suitability for industrialized production.The pressure reduction of pressure filtration can not surpass 0.6Mpa usually, but for full-bodied fermented liquid, the resistance of filter cake causes the violent increase of pressure reduction, therefore before operation, to carry out pre-treatment to fermented liquid, add flocculating aids etc. as heating, in feed liquid, in the sepn process of polyglutamic acid fermented liquid, because the product polyglutamic acid has the ability of very strong absorption thalline, causes filtering separation degerming efficient that tangible reduction is arranged.In biological product separated, micro-filtration (MF) was widely used in cell, bacterium separates with atomic, and the magnitude range of target substance is 0.01 μ m～10 μ m.With the static pressure difference is impellent, utilizes " screening " effect of film to separate, and its effect is equivalent to " filtration ".Because porosity accounts for 70%～80% of cumulative volume, so resistance is very little, filtration velocity is very fast.
Microfiltration process is mainly used in separates macromole, colloidal particle, protein and other particulates, their separating mechanism be according to the physical and chemical performance of the physical and chemical performance of molecule or particulate, the film that uses different with their interaction (as big or small, shape and electrical property) realize isolating.The micro-filtration separating process generally experiences following several stages: 1. filter the starting stage, the particle bigger than membrane pore size is trapped within the surface of film, and the particle littler than membrane pore size enters and pass through fenestra, the some of them particle has reduced the effective diameter of fenestra owing to the effect of various power is adsorbed in the fenestra; 2. filter mid-term stage, particulate begins to form cake layer on the film surface, when the fenestra internal adsorption is tending towards saturated gradually; 3. filter later stage, along with multiparticulates more is trapped on the film surface, it is saturated that the fenestra internal adsorption also is tending towards, and particulate begins to stop up fenestra, and membrane flux is tended towards stability, and constantly descends then.
γ after the micro-filtration degerming-PGA fermented liquid will have dilution to a certain degree, consume a large amount of precipitation agents if directly carry out the precipitation defection, and production cost significantly improves, and can cause organic solvent to pollute.Fermented liquid after the degerming is concentrated with the ultrafiltration instrument, can reduce the usage quantity of precipitation agent in the later separation, help reducing cost, the protection environment.The dilution ultrafiltration can also realize the decolouring of fermented liquid, and the quality of target product and outward appearance are improved.Ultrafiltration is a kind of sieve aperture sepn process, at static pressure difference is under the effect of impellent, and solvent and little solute particle see through film to low-tension side from highly compressed feed liquid side in the stock liquid, is commonly referred to as light liquid or sees through liquid, and macroparticle component tunicle stops, and their concentration in the surplus liquid of filter is increased.According to such separating mechanism, the principal element that ultra-filtration membrane has selective meter's surface layer is to form the hole with a certain size and shape.Ultrafiltration is mainly used in and separates macromolecular cpd (protein, nucleic acid polymers, starch, natural gum, enzyme etc.) from liquid phase substance, colloidal dispersion (clay, pigment, the mineral material, emulsion particle, microorganism), emulsion (lubricating grease-washing composition and oil-in-water emulsions), thus reach concentrating, separate, purify and purifying of some solution that contains polymer substance (protein, enzyme, virus).
The production of γ-PGA and exploitation mainly are limited to laboratory level and pilot scale level at present.The key step that the laboratory prepares γ-PGA on a small scale comprises: microbial transformation, degerming concentrates, decolouring, precipitation, drying.Obtaining white crystals after the drying only is the thick product of γ-PGA, also needs to be further purified.Earlier the thick product of γ-PGA is dissolved in the distilled water, centrifugally removes undissolved impurity, adopt dialysis or electrodialytic method desalination then, then γ-PGA the aqueous solution that obtains is carried out vacuum lyophilization, get final product refining γ-PGA.The condition of regulating and controlling microbial fermentation is to improve the key of γ-PGA output, traditional production method, γ in the fermented liquid-PGA is accumulation constantly, cause the continuous rising of viscosity, resistance to mass transfer strengthens, the rapid decline of dissolved oxygen, thalline death, and cause the activity of γ-PGA synthetic enzyme to descend, stagnating even downtrending appears in the accumulation of γ-PGA in the fermented liquid, and output is difficult to further improve.And each unitary operation all is relatively independent in the Laboratory Production preparation process, and the production time prolongs, and production efficiency reduces, and cost increases.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the method for a kind of microbial fermentation and membrane separation technique coupling production of Gamma-polyglutamic acid is provided.
The method of microbial fermentation and membrane separation technique coupling production of Gamma-polyglutamic acid comprises the steps:
(1) actication of culture: from freezing glycerine pipe, get 10 microlitre bacterium liquid, coat isolation medium after the dilution, 37 ℃, cultivate 12h;
(2) seed culture: the bacterial classification inoculation after will activating is cultivated 12h, the subtilis seed culture medium: extractum carnis 5g/L, peptone 10g/L, NaCl 5g/L, pH6.5 under 37 ℃ of rotating speed 200rpm conditions in the seed bottle that contains seed culture medium;
(3) fermentation culture: in the bio-reactor, subtilis fermentation culture in fermention medium obtains being rich in the fermented liquid of gamma-polyglutamic acid-, fermentation of bacillus subtilis substratum: glucose 60g/L, peptone 60g/L, Sodium Glutamate 80g/L, NaCl 10g/L, MgSO47H2O 1.0g/L, CaCl21.0g/L, pH6.5;
(4) micro-filtrate membrane filtration: subtilis is fermentation culture in bio-reactor, when fermented liquid viscosity and cell density increase to 30～40mPa.min, open microfiltration systems, fermented liquid enters micro-filtration and filters, trapped fluid returns in the bio-reactor, and filtered solution enters concentration basin;
(5) ultrafiltration and concentration: fluid accumulation to the 2～2.5L in the concentration basin, the unlatching ultrafiltration system concentrates and decolours, and trapped fluid returns concentration basin and forms concentrated solution, sees through liquid and turns back in the bio-reactor.
(6) feed supplement: sterilising medium pumps in the fermentor tank with 1.0ml/min, the ability of keeping the cell growth and synthesizing polyglutamic acid.
The microfiltration membrane material that uses in the described micro-filtration comprises organic film material, inorganic material film or composite membrane; Organic film material is: polyethylene, polypropylene, polyvinylidene difluoride (PVDF) or tetrafluoroethylene; Inorganic material film is: porous ceramics, sintered glass or aluminum oxide; Composite membrane is inorganics filled polymeric film, and polymer/inorganic supports composite membrane or inorganic/organic assorted poly-film.The aperture of microfiltration membrane is 0.8 μ m, 1.0 μ m or 1.2 μ m.Micro-filtration membrane module is flat, tubular type, rolling or tubular fibre formula.The micro-filtrate membrane filtration operational condition is: 30～40 ℃ of temperature, pH6.0～7.0, pressure 0.2～0.5MPa.The ultra-filtration membrane material that uses in the ultrafiltration system comprises organic film material or inorganic material film; Organic film material is: cellulose acetate, polysulfones, polysulfonamides, SPSF, polypropylene nitrile, polyvinyl chloride, polyvinylidene difluoride (PVDF), polyethersulfone or polyetherketone; Inorganic material film is: pottery, sintered glass or aluminum oxide.The ultra-filtration membrane molecular weight that dams is 1-10 ten thousand.Hyperfiltration membrane assembly is flat, tubular type, rolling or tubular fibre formula.The ultrafiltration and concentration operational condition is: 30～40 ℃ of temperature, pH6.0～7.0, pressure 0.2～0.5MPa.
The present invention compares with existing polyglutamic acid fermentation technique, have the following advantages and outstanding effect: the high viscosity character that the present invention is directed to polyglutamic acid, when fermentation of bacillus subtilis is produced polyglutamic acid, utilizing membrane separation technique that the polyglutamic acid in the fermented liquid is in time separated concentrates, make the viscosity of fermented liquid maintain lower level, help increasing the dissolved oxygen in the fermentor tank, reduce the restraining effect of full-bodied fermented liquid to thalli growth, make the activity of subtilis gamma-polyglutamic acid-synthetase series maintain higher level all the time, further improve the output of gamma-polyglutamic acid-.And, the present invention is based on microbial fermentation and membrane separation technique is coupled, realized the operate continuously that microbe transformation method is produced gamma-polyglutamic acid-, help the refining of the aftertreatment of fermented liquid and target product, improved production efficiency, reduced production cost.This method operational condition gentleness, good stability, rapidly and efficiently, equipment is simple, and energy consumption is low, can be applied to the suitability for industrialized production of polyglutamic acid, microbial fermentation is prepared full-bodied product have general directive significance.
Description of drawings
Accompanying drawing is the apparatus structure synoptic diagram of microbial fermentation and membrane separation technique coupling production of Gamma-polyglutamic acid, 1-1 wherein, and 1-2,1-3 are peristaltic pump; The Glass Containers of 2-1 for sterilizing; 2-2 is a concentration basin; 3 is bio-reactor; 4 is the micro-filtration filtering system; 5 is ultrafiltration system.
Embodiment
1. micro-filtrate membrane filtration degerming process condition is optimized
(1) optimization of micro-filtrate membrane filtration degerming process operation temperature:
The fermented liquid viscosity that is rich in γ-PGA reduces with the rising of temperature, therefore can suitably improve temperature reducing the fermented liquid viscosity, thereby improve permeation flux.But the too high meeting of temperature causes the decomposition of γ-PGA, causes product yield to descend, and also will consider microfiltration membrane temperature tolerance range simultaneously.Comprehensive above situation is investigated the influence of interior each temperature of 30～60 ℃ of temperature ranges to permeation flux and degerming rate.
(2) optimization of micro-filtrate membrane filtration degerming process operation pressure reduction:
Micro-filtrate membrane filtration is impellent with pressure reduction, so the selection of pressure reduction has remarkable influence to the filtration sterilization process, this paper has investigated that (different pressure reduction are to the influence of permeation flux and degerming rate in 0.2～0.5MPa) in pressure differential range.
Optimum result shows that along with the increase of temperature, permeation flux improves simultaneously, but turnover occurred at 40 ℃, when promptly temperature is higher than 40 ℃, permeation flux with the increase of temperature improve not obvious.Along with the rising of temperature, the degerming rate also improves constantly, but locates to occur weight break point at 40 ℃, when temperature is higher than 40 ℃, along with the rising degerming rate raising of temperature is not obvious.And operation pressure reduction is to the almost not influence of degerming rate.Take all factors into consideration above-mentioned parameters such as permeation flux, degerming rate and concentrated solution flow velocity, we determine that operating temperature range is 30～40 ℃, and operation pressure reduction is 0.2～0.5MPa in scope, and the degerming rate reaches as high as more than 97%.
2. ultrafiltration and concentration and decolorization condition optimizing
(1) optimization of ultrafiltration and concentration and decolorization service temperature:
Adopt and the similar method of micro-filtrate membrane filtration, select under the differing temps (30 ℃, 37 ℃, 40 ℃, 50 ℃, 60 ℃) influence permeation flux.
(2) optimization of ultrafiltration and concentration and decolorization operation pressure reduction:
Adopt and the similar method of micro-filtrate membrane filtration, (0.4MPa is 0.5MPa) to the influence of permeation flux for 0.2MPa, 0.3MPa to select several different pressure differences.
Optimum result shows that permeation flux then enters a plateau with the rising increase earlier of pressure reduction, and when promptly pressure reduction was greater than 0.3MPa, permeation flux changed with pressure reduction hardly.Along with temperature raises, the trend that permeation flux increases weakens, and when temperature during greater than 40 ℃, improves not obvious that the temperature permeation flux increases again.Take all factors into consideration above factor, ultrafiltration instrument operational condition is as follows: 30～40 ℃ of temperature, pressure reduction 0.2～0.5MPa.
Embodiment 1:
In conjunction with shown in Figure 1, utilize subtilis (Bacillus subtilis ZJU-7, culture presevation number: CGMCCNo.1250, open in 200410010509.0 patent documentation) to produce the step for preparing γ-PGA specific as follows for fermentation and membrane separation technique coupling:
(1) actication of culture: from freezing glycerine pipe, get 10 μ l bacterium liquid, coat isolation medium after the dilution, cultivate 12h for 37 ℃.Isolation medium: extractum carnis 5g/L, peptone 10g/L, NaCl 5g/L, agar 15g/L, pH7.0.
(2) seed culture: the bacterial classification inoculation after will activating shakes in the bottle to the 250mL/1000mL that the 30mL/250mL seed culture medium is housed, and in 200rpm, cultivates 12h for 37 ℃.Seed culture medium is specially the subtilis seed culture medium: extractum carnis 5g/L, peptone 10g/L, NaCl 5g/L, pH6.5.
(3) fermentor cultivation: fermentor tank 3 liquid amounts 70%, A.T.C and pH, 115 ℃ of sterilization 30min, inoculum size 5% (v/v), the hydrochloric acid with 25% strong aqua and 4mol/L in the fermenting process is controlled pH=6.50 ± 0.02, and leavening temperature is 37 ℃.Fermention medium is specially the fermentation of bacillus subtilis substratum: glucose 60g/L, peptone 60g/L, Sodium Glutamate 80g/L, NaCl 10g/L, MgSO47H2O1.0g/L, CaCl2 1.0g/L, pH6.5, rotating speed are 400～600rpm, and air flow is 0.3～0.6vvm.
(4) micro-filtrate membrane filtration: after the fermentation beginning, γ-PGA constantly accumulates, and the fermented liquid viscosity constantly increases.When viscosity reaches 30mPa.min, open pump 1-2, flow rate pump is 10ml/min, and the part fermentation culture is pumped into micro-filtrate membrane filtration system 4, microfiltration membrane is a cellulose acetate film, and the aperture is 1.2 μ m, and the film bag is the plate surface film, 37 ℃ of service temperatures, pressure 0.2MPa, pH6.5.Trapped fluid returns in the fermentor tank, and filtered solution enters concentration basin 2-2.The degerming rate reaches 97%.
(5) the ultrafiltration instrument concentrates and decolouring: the degerming filtered solution volume in concentration basin reaches 2L, opens pump 1-3, and flow rate pump is 10ml/min, and filtered solution is pumped into ultrafiltration instrument 5, concentrates and decolours.Ultra-filtration membrane is a polyvinylidene fluoride film, and molecular weight cut-off is 100KD, and the film bag is a rolled film, 37 ℃ of service temperatures, pressure 0.3MPa, pH6.5.Trapped fluid returns concentration basin 2-2, does not contain macromolecular filtered solution and returns in the fermentor tank 3.The viscosity of fermentation cylinder for fermentation liquid maintains 30～40mPa.min all the time, and the fermented liquid concentration ratio that contains γ-PGA is 5.
(6) container 2-1 is equipped with the fresh culture of sterilization, pumps in the fermentor tank with the speed of 1.0ml/min by pump 1-1, for the growth of cell and metabolism provide necessary nutrition.Fresh culture: glucose 60g/L, peptone 60g/L, Sodium Glutamate 80g/L, NaCl 10g/L, MgSO47H2O 1.0g/L, CaCl2 1.0g/L, pH6.5,115 ℃ of sterilization 30min.
Microfiltration membrane system, ultrafiltration instrument and membrane module SF-SA match luxuriant and rich with fragrance membrane separation technique company limited available from Hangzhou.
Utilize this method to produce γ-PGA, after the fermentation ends, the accumulation volume of γ-PGA reaches 90g/L, separates a coupled batch fermentation and compares with not adopting, and output has improved 55%.
Embodiment 2:
In conjunction with shown in Figure 1, utilize subtilis (Bacillus subtilis ZJU-7, culture presevation number: it is specific as follows that CGMCC No.1250) step for preparing γ-PGA is produced in the coupling of fermentation and membrane separation technique:
(1) actication of culture: with implementing example 1.
(2) seed culture: with implementing example 1.
(3) fermentor cultivation: with implementing example 1.
(4) micro-filtrate membrane filtration: after the fermentation beginning, γ-PGA constantly accumulates, and the fermented liquid viscosity constantly increases.When viscosity reaches 30mPa.min, open pump 1-2, flow rate pump is 10ml/min, and the part fermentation culture is pumped into micro-filtrate membrane filtration system 4, microfiltration membrane is a polyethersulfone, and the aperture is 1.0 μ m, and the film bag is the plate surface film, 37 ℃ of service temperatures, pressure 0.5MPa, pH6.5.Trapped fluid returns in the fermentor tank, and filtered solution enters concentration basin 2-2.The degerming rate reaches 99%.
(5) with implementing example 1.
(6) with implementing example 1.
Microfiltration membrane system, ultrafiltration instrument and membrane module SF-SA match luxuriant and rich with fragrance membrane separation technique company limited available from Hangzhou.
Utilize this method to produce γ-PGA, after the fermentation ends, the accumulation volume of γ-PGA reaches 92g/L, separates a coupled batch fermentation and compares with not adopting, and output has improved 59%.
Embodiment 3:
In conjunction with shown in Figure 1, utilize subtilis (Bacillus subtilis ZJU-7, culture presevation number: it is specific as follows that CGMCC No.1250) step for preparing γ-PGA is produced in the coupling of fermentation and membrane separation technique:
(1) actication of culture: with implementing example 1.
(2) seed culture: with implementing example 1.
(3) fermentor cultivation: with implementing example 1.
(4) micro-filtrate membrane filtration: with implementing example 2.
(5) the ultrafiltration instrument concentrates and decolouring: the degerming filtered solution volume in concentration basin reaches 2L, opens pump 1-3, and flow rate pump is 10ml/min, and filtered solution is pumped into ultrafiltration instrument 5, concentrates and decolours.Ultra-filtration membrane is a hollow-fibre membrane, and molecular weight cut-off is 150KD, and the film bag is a rolled film, 37 ℃ of service temperatures, pressure 0.5MPa, pH6.5.Trapped fluid returns concentration basin 2-2, does not contain macromolecular filtered solution and returns in the fermentor tank 3.The viscosity of fermentation cylinder for fermentation liquid maintains 30～40mPa.min all the time, and the fermented liquid concentration ratio that contains γ-PGA is 6.
(6) with implementing example 1.
Microfiltration membrane system, ultrafiltration instrument and membrane module SF-SA match luxuriant and rich with fragrance membrane separation technique company limited available from Hangzhou.
Utilize this method to produce γ-PGA, after the fermentation ends, the accumulation volume of γ-PGA reaches 87g/L, separates a coupled batch fermentation and compares with not adopting, and output has improved 50%.
Embodiment 4:
In conjunction with shown in Figure 1, utilize subtilis (Bacillus subtilis ZJU-7, culture presevation number: it is specific as follows that CGMCC No.1250) step for preparing γ-PGA is produced in the coupling of fermentation and membrane separation technique:
(1) actication of culture: with implementing example 1.
(2) seed culture: with implementing example 1.
(3) fermentor cultivation: with implementing example 1.
(4) micro-filtrate membrane filtration: after the fermentation beginning, γ-PGA constantly accumulates, and the fermented liquid viscosity constantly increases.When viscosity reaches 30mPa.min, open pump 1-2, flow rate pump is 10ml/min, and the part fermentation culture is pumped into micro-filtrate membrane filtration system 4, and microfiltration membrane is a ceramic membrane, and the aperture is 0.8 μ m, and membrane module is a rolling, 37 ℃ of service temperatures, pressure 0.4MPa, pH6.5.Trapped fluid returns in the fermentor tank, and filtered solution enters concentration basin 2-2.The permeation flux of ceramic membrane is far above organic membrane, and the degerming rate reaches 95%, a little less than organic membrane.
(5) the ultrafiltration instrument concentrates and decolouring: the degerming filtered solution volume in concentration basin reaches 2.5L, opens pump 1-3, and flow rate pump is 10ml/min, and filtered solution is pumped into ultrafiltration instrument 5, concentrates and decolours.Ultra-filtration membrane is a hollow-fibre membrane, and molecular weight cut-off is 150KD, and the film bag is a rolled film, 37 ℃ of service temperatures, pressure 0.3MPa, pH6.5.Trapped fluid returns concentration basin 2-2, does not contain macromolecular filtered solution and returns in the fermentor tank 3.The viscosity of fermentation cylinder for fermentation liquid maintains 30～40mPa.min all the time, and the fermented liquid concentration ratio that contains γ-PGA is 6.
(6) with implementing example 1.
Microfiltration membrane system is available from of a specified duration my the film Science and Technology Ltd. in Nanjing, and ultrafiltration instrument and membrane module SF-SA match luxuriant and rich with fragrance membrane separation technique company limited available from Hangzhou.
Utilize this method to produce γ-PGA, after the fermentation ends, the accumulation volume of γ-PGA reaches 90g/L, separates a coupled batch fermentation and compares with not adopting, and output has improved 55%.
Embodiment 5:
In conjunction with shown in Figure 1, utilize subtilis (Bacillus subtilis ZJU-7, culture presevation number: it is specific as follows that CGMCC No.1250) step for preparing γ-PGA is produced in the coupling of fermentation and membrane separation technique:
(1) actication of culture: with implementing example 1.
(2) seed culture: with implementing example 1.
(3) fermentor cultivation: with implementing example 1.
(4) micro-filtrate membrane filtration: with implementing example 4.
(5) the ultrafiltration instrument concentrates and decolouring: the degerming filtered solution volume in concentration basin reaches 2L, opens pump 1-3, and flow rate pump is 10ml/min, and filtered solution is pumped into ultrafiltration instrument 5, concentrates and decolours.Ultra-filtration membrane is a hollow-fibre membrane, and molecular weight cut-off is 500KD, and the film bag is a rolled film, 37 ℃ of service temperatures, pressure 0.3MPa, pH6.5.Trapped fluid returns concentration basin 2-2, does not contain macromolecular filtered solution and returns in the fermentor tank 3.The viscosity of fermentation cylinder for fermentation liquid maintains 30～40mPa.min all the time, and the fermented liquid concentration ratio that contains γ-PGA is 7.
(6) with implementing example 1.
Microfiltration membrane system is available from of a specified duration my the film Science and Technology Ltd. in Nanjing, and ultrafiltration instrument and membrane module SF-SA match luxuriant and rich with fragrance membrane separation technique company limited available from Hangzhou.
Utilize this method to produce γ-PGA, after the fermentation ends, the accumulation volume of γ-PGA reaches 84g/L, separates a coupled batch fermentation and compares with not adopting, and output has improved 43%.
The present invention is coupled microbial fermentation and the membrane separation technique in γ-PGA production technique, realized the operate continuously that microbe transformation method is produced gamma-polyglutamic acid-, help the aftertreatment of fermented liquid and making with extra care of target product, improved production efficiency, reduced production cost.This method operational condition gentleness, good stability, rapidly and efficiently, equipment is simple, drops into lowly, microbial fermentation is prepared full-bodied product have general directive significance, has broad application prospects in industry member.
Claims (9)
1, the method for a kind of microbial fermentation and membrane separation technique coupling production of Gamma-polyglutamic acid is characterized in that comprising the steps:
(1) actication of culture: from freezing glycerine pipe, get 10 microlitre bacterium liquid, coat isolation medium after the dilution, 37 ℃, cultivate 12h;
(2) seed culture: the bacterial classification inoculation after will activating is cultivated 12h, the subtilis seed culture medium: extractum carnis 5g/L, peptone 10g/L, NaCl 5g/L, pH6.5 under 37 ℃ of rotating speed 200rpm conditions in the seed bottle that contains seed culture medium;
(3) fermentation culture: in the bio-reactor, subtilis fermentation culture in fermention medium obtains being rich in the fermented liquid of gamma-polyglutamic acid-, fermentation of bacillus subtilis substratum: glucose 60g/L, peptone 60g/L, Sodium Glutamate 80g/L, NaCl 10g/L, MgSO47H2O 1.0g/L, CaCl21.0g/L, pH6.5;
(4) micro-filtrate membrane filtration: subtilis is fermentation culture in bio-reactor, when fermented liquid viscosity and cell density increase to 30～40mPa.min, open microfiltration systems, fermented liquid enters micro-filtration and filters, trapped fluid returns in the bio-reactor, and filtered solution enters concentration basin;
(5) ultrafiltration and concentration: fluid accumulation to the 2～2.5L in the concentration basin, the unlatching ultrafiltration system concentrates and decolours, and trapped fluid returns concentration basin and forms concentrated solution, sees through liquid and turns back in the bio-reactor.
(6) feed supplement: sterilising medium pumps in the fermentor tank with the speed of 1.0ml/min, the ability of keeping the cell growth and synthesizing polyglutamic acid.
2, the method for claim 1 is characterized in that the microfiltration membrane material that uses in the described micro-filtration comprises organic film material, inorganic material film or composite membrane; Organic film material is: polyethylene, polypropylene, polyvinylidene difluoride (PVDF) or tetrafluoroethylene; Inorganic material film is: porous ceramics, sintered glass or aluminum oxide; Composite membrane is inorganics filled polymeric film, and polymer/inorganic supports composite membrane or inorganic/organic assorted poly-film.
3, method as claimed in claim 2, the aperture that it is characterized in that described microfiltration membrane are 0.8 μ m, 1.0 μ m or 1.2 μ m.
4, the method for claim 1 is characterized in that the micro-filtration membrane module that uses in the described micro-filtration is flat, tubular type, rolling or tubular fibre formula.
5, the method for claim 1 is characterized in that described micro-filtrate membrane filtration operational condition is: 30～40 ℃ of temperature, pH6.0～7.0, pressure 0.2～0.5MPa.
6, the method for claim 1 is characterized in that the ultra-filtration membrane material that uses in the described ultrafiltration system comprises organic film material or inorganic material film; Organic film material is: cellulose acetate, polysulfones, polysulfonamides, SPSF, polypropylene nitrile, polyvinyl chloride, polyvinylidene difluoride (PVDF), polyethersulfone or polyetherketone; Inorganic material film is: pottery, sintered glass or aluminum oxide.
7, method as claimed in claim 6 is characterized in that the employed ultra-filtration membrane molecular weight that dams is 1-10 ten thousand.
8, the method for claim 1 is characterized in that the hyperfiltration membrane assembly that uses in the described ultrafiltration system is flat, tubular type, rolling or tubular fibre formula.
9, the method for claim 1 is characterized in that the ultrafiltration and concentration operational condition is: 30～40 ℃ of temperature, pH6.0～7.0, pressure 0.2～0.5MPa.
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| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN102382865A (en) * | 2011-10-27 | 2012-03-21 | 天津北洋百川生物技术有限公司 | Method for producing gamma-polyglutamic acid through refluxing process |
| CN102533884A (en) * | 2011-12-30 | 2012-07-04 | 宝鸡阜丰生物科技有限公司 | Method for cleanly producing glutamic acid, gamma-polyglutamic acid and organic fertilizer |
| CN103497890A (en) * | 2013-09-26 | 2014-01-08 | 北京华都诗华生物制品有限公司 | Perfusion culture system and method for bacteria fermentation tank |
| CN103665371A (en) * | 2013-11-14 | 2014-03-26 | 天津北洋百川生物技术有限公司 | Method for refining polyglutamic acid in biologic fermentation broth by using ultrafiltration and nanofiltration techniques |
| CN105543314A (en) * | 2016-03-06 | 2016-05-04 | 河北工业大学 | Method for producing polymyxin E through fermentation and foam separation coupling |
| CN105821100A (en) * | 2016-01-25 | 2016-08-03 | 南京工业大学 | Process for realizing high yield of polyoxin by continuous fermentation and separation coupling |
| CN109609408A (en) * | 2018-12-27 | 2019-04-12 | 黄河三角洲京博化工研究院有限公司 | One plant of gamma-polyglutamic acid superior strain and the method for preparing gamma-polyglutamic acid is carried out liquid fermentation using the bacterial strain |
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| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN101948785B (en) * | 2010-08-31 | 2012-06-13 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN102382865A (en) * | 2011-10-27 | 2012-03-21 | 天津北洋百川生物技术有限公司 | Method for producing gamma-polyglutamic acid through refluxing process |
| CN102533884A (en) * | 2011-12-30 | 2012-07-04 | 宝鸡阜丰生物科技有限公司 | Method for cleanly producing glutamic acid, gamma-polyglutamic acid and organic fertilizer |
| CN102533884B (en) * | 2011-12-30 | 2014-01-22 | 宝鸡阜丰生物科技有限公司 | Method for cleanly producing glutamic acid, gamma-polyglutamic acid and organic fertilizer |
| CN103497890A (en) * | 2013-09-26 | 2014-01-08 | 北京华都诗华生物制品有限公司 | Perfusion culture system and method for bacteria fermentation tank |
| CN103665371A (en) * | 2013-11-14 | 2014-03-26 | 天津北洋百川生物技术有限公司 | Method for refining polyglutamic acid in biologic fermentation broth by using ultrafiltration and nanofiltration techniques |
| CN103665371B (en) * | 2013-11-14 | 2016-03-02 | 天津北洋百川生物技术有限公司 | A kind of method utilizing polyglutamic acid in ultrafiltration nanofiltration refined biological fermented liquid |
| CN105821100A (en) * | 2016-01-25 | 2016-08-03 | 南京工业大学 | Process for realizing high yield of polyoxin by continuous fermentation and separation coupling |
| CN105543314A (en) * | 2016-03-06 | 2016-05-04 | 河北工业大学 | Method for producing polymyxin E through fermentation and foam separation coupling |
| CN109609408A (en) * | 2018-12-27 | 2019-04-12 | 黄河三角洲京博化工研究院有限公司 | One plant of gamma-polyglutamic acid superior strain and the method for preparing gamma-polyglutamic acid is carried out liquid fermentation using the bacterial strain |
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