CN1847267A - Chitin oligose preparing process - Google Patents
Chitin oligose preparing process Download PDFInfo
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- CN1847267A CN1847267A CN 200610080091 CN200610080091A CN1847267A CN 1847267 A CN1847267 A CN 1847267A CN 200610080091 CN200610080091 CN 200610080091 CN 200610080091 A CN200610080091 A CN 200610080091A CN 1847267 A CN1847267 A CN 1847267A
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Abstract
The present invention relates to chitin oligose preparing process, and is especially enzyme generating fermentation process to degrade chitin colloid for preparing bioactive chitin oligose. The preparation process has simple technological path, effective inhibition of further degradation of bioactive chitin oligose, reuse of degrading enzyme and other advantages, and is suitable for industrial production. The preparation process includes: culturing Aeromonascaviae strain to obtain fermented liquid, centrifugally separating the fermented liquid to obtain enzyme supernatant; preparing chitin colloid; compounding chitin colloid buffer solution in an enzyme reactor and adding abacterial enzyme liquid for enzymolysis to obtain enzymolyzed chitin oligose liquid; four stages of membrane separation to obtain concentrate and spray drying to obtain bioactive chitin oligose product.
Description
Technical field
The present invention relates to a kind of chitin oligo saccharide, especially relate to a kind of method that adopts enzymatic production method degrade chitin colloid for preparing biological activity chitin oligo saccharide.
Background technology
Chitin is with N-acetyl-2-deoxidation-D glucose (GlcNAc) and a spot of 2-is amino and 2-deoxyglucose and β (1,4) linear polymer that forms in succession of glycosidic link, it is that occurring in nature is only second to cellulosic natural high polymer, and chitin is the good substrates of chitinase.Because of it is insoluble in the common solvent, so limited its application.
Because the composition that in existing literature institutes such as chitin, chitosan, oligochitosan and chitin oligo saccharide is definitely referred to is had nothing in common with each other, existing as follows to each used in present patent application nominal definition:
Chitin: claiming chitin again, is the polymkeric substance that 2-acetylaminohydroxyphenylarsonic acid 2 DDGs connect with β-1,4 glycosidic link.
Chitosan: be partially or completely to take off resulting chitosan behind the acetyl by chitin.
Chitin oligo saccharide: be meant that the polymerization degree is 2～10 low-molecular-weight chitin degrading product.
Oligochitosan: be meant that the polymerization degree is 2～10 low-molecular-weight degradation of chitosan product.
More than define referring to following document:
1. Zheng builds celestial being, functional oligose [M], Beijing: Chemical Industry Press, 2004.215.
2. Jiang Ting is big, chitin [M], Beijing: Chemical Industry Press, 2003.1～4.
Zheng Jianxian (Zheng Jianxian, functional oligose [M], Beijing: Chemical Industry Press, 2004.251-266) summarize the present method for preparing chitosan oligomer, can roughly be divided into 5 kinds of chemical degradation method, mechanical degradation method, enzyme hydrolysis method, enzymic synthesis method and gene engineering researches etc.
Chemical degradation method is to adopt acid or oxygenant that chitosan is degraded, can be divided into acid degradation method and oxidation degradation method, this is the main method of present industrial application, but more serious, the wayward reaction end of these methods or environmental pollution, or easily produce by product.
The enzymic synthesis method is to utilize the transglycosylation of some enzymes, the higher oligose of oligose synthesized polymer degree that the catalyzed polymerization degree is lower, and therefore the chitosan oligomer for synthetic high physiologically active is of great importance, and is under test at present.
Gene engineering research be enzyme gene transformation that synthesis of oligonucleotides chitosan process in the organism body is related in engineering bacteria, express production, also still under test.
Enzymolysis process is produced does not have other reaction reagents addings in whole degradation process, do not have other byproduct of reaction and produce, and has demonstrated fully its superiority.At present, the enzyme that utilizes the enzymolysis process hydrolyzing chitosan to produce chitosan oligomer is divided into two classes.One class is to adopt specificity enzymic hydrolysis chitin or chitosan, and the specificity enzyme comprises chitoanase, chitin enzyme and N,O-Diacetylmuramidase.Another kind of is to adopt non-specificity enzymic hydrolysis, has now found that the various enzymes that can be used for chitosan/oligochitosan production in the enzyme hydrolysis method have kind more than 30 (SKJAK-BRAEK, G.; ANTHONSEN, T.; SANDFORD, P., Chitin and chitosan-sources, chemistry, biochemistry, physical properties and aplications, Carbohydrate Research, 1995,268:143-149).
Application number provides the industrialized preparing process of a kind of low chitose and oligochitosan for 01126457.8 application for a patent for invention, adopts prozyme that chitosan is degraded and obtains low chitose and oligochitosan.
Application number provides a kind of preparation method and application of chitin oligosaccharide for 03112619.7 application for a patent for invention, form prozyme with non-specificity enzyme and prepare chitin oligosaccharide, but be still waiting to develop cheapness, enzyme and suitable reactive system to be to realize large-scale industrial production efficiently.
The specificity lytic enzyme of chitosan shows great affinity to chitosan, and enzyme dosage is little, and the viscosity degradation speed of solution is also quite big in the hydrolysis.Therefore very fast to the chitosan speed that is hydrolyzed with the specificity enzyme, effect is better.Enzymic degradation chitosan mild condition, degradation process and degraded product molecular weight distribution all are easy to control, and the yield of the oligose of high-polymerization degree is than acid hydrolysis height, in addition in the enzymatic hydrolysis, the Production by Enzymes process does not pollute environment, is more satisfactory chitin oligo saccharide/oligochitosan production method.Chitoanase and chitin enzyme can't mass production, therefore use to be restricted.The method that adopts specificity lytic enzyme enzymolysis process to prepare chitin oligo saccharide is not appeared in the newspapers so far.
The applicant provides the chitinase system of a kind of efficient chitin degrading bacterium Aeromonas caviae and generation thereof in for 200410005111.8 application for a patent for invention at application number, wherein culture Aeromonas caviae (Aeromonas caviaeQ2) is preserved in Chinese typical culture collection center on December 14th, 2003 by the applicant, and deposit number is CCTCC NO:M203097.
Summary of the invention
--acquisition of----high vigor enzyme and chitin colloid prepare this two big bottleneck in a large number to the objective of the invention is to the general difficult point that runs in the domestic and international chitin oligo saccharide production; provide a kind of operational path simple; can effectively suppress the further degraded under the enzyme effect of active chitin oligo saccharide; degrading enzyme is reusable; the service efficiency height of enzyme can obtain the product of different polymerization degree as required, pollutes few; the separation efficiency height helps the chitin oligose preparing process that large-scale industrial is produced.
The technical solution used in the present invention is that a strain of adopting the applicant to filter out from the ocean can be induced the bacterial strain Aeromonas caviae (Aeromonas caviaeQ2) that produces high reactivity chitinase system for this reason, obtain to secrete highly active enzyme without inducing with gene manipulation techniques again and be, the enzymic degradation chitin colloid is combined with membrane sepn, make this enzyme tie up to the chitin oligo saccharide that obtains solubility behind the hydrolysis chitin colloid.
Concrete steps of the present invention are:
1) strain culturing and enzyme liquid preparation
(Aeromonas caviaeQ2) cultivates in substratum with the bacterial strain Aeromonas caviae; The zymocyte liquid centrifugation that then cultivation is obtained obtains fermented liquid supernatant; Remove remaining thalline and other solid substances in the enzyme liquid with ultrafiltration process again, it is standby that separation obtains enzyme liquid.The component of described substratum and precentagewise content thereof are:
Glycerine 1%～10%
Yeast extract 0.5%～5%
Corn steep liquor 1%～5%
Soybean cake powder 1%～10%
NaCl 0.5％～3％
Yu Weishui.
Culture temperature is 25～37 ℃, and air flow is 5～200l/min, and incubation time is 10～40h.
2) preparation chitin colloid
(1), chitin is mixed with mineral acid, make the chitin acid solution, by weight with volume proportion, chitin (Kg): mineral acid (L)=1: (8～20), preferred chitin (Kg): mineral acid (L)=1: 10; Described dense mineral acid is a kind of in phosphoric acid, sulfuric acid or the hydrochloric acid etc., and phosphoric acid concentration is 60%～85%, and sulfuric acid concentration is 60%～98%, and concentration of hydrochloric acid is 20%～35%;
(2), the chitin acid solution is added drop-wise in water or the alkaline solution, make its colloidal precipitation, become Colloidal fluid; Described alkali lye is selected from a kind of in ammoniacal liquor, potash, the soda etc.; Described potash is selected from a kind of in potassium hydroxide, salt of wormwood, the saleratus etc., and described soda is selected from a kind of in sodium hydroxide, yellow soda ash, the sodium bicarbonate etc.;
(3), chitin glue is separated with the inorganic salt fertilizer that contains the chitin oligosaccharide;
(4), the chitin glue after will separating washes with water, promptly gets chitin colloid.
3) enzyme digestion reaction
In the enzyme reaction jar, chitin colloid is suspended in the damping fluid that concentration is 20mM, being mixed with final concentration is the chitin colloid solution of chitin dry weight 0.1%～10%, the steam sterilizing postcooling is to room temperature, and the chitinase liquid aseptic delivery that step 1) is obtained is to the intravital chitin colloid solution of the enzyme reaction jar of the bacterium of going out, and the chitinase liquid phase is 1～10mU/ml to the consumption of chitin colloid, temperature of reaction is 10～60 ℃, pH is 3.0～8.9, and insulation gets the chitin oligo saccharide enzymolysis solution.
4) one-level membrane sepn
The solid phase component content for the treatment of chitin colloid in the chitin oligo saccharide enzymolysis solution that step 3) obtains was reduced to 0.05%～2% o'clock, the chitin oligo saccharide enzymolysis solution is pumped into the microfiltration membrane tripping device from the enzyme reaction jar, pressure control range is 0.15～0.8MPa, temperature controlling range is 10～50 ℃, remove the materials such as suspended substance, chitin colloid and partial organic substances in the enzymolysis solution, the part that does not see through is returned the enzyme reaction jar and is continued to participate in enzymolysis, seeing through liquid is active chitin oligo saccharide filtrate, enters secondary membrane sepn batch can through pipeline.
5) secondary membrane sepn
The secondary membrane sepn is selected the ultra-filtration membrane tripping device for use, with pressure is impellent, utilize the selective permeability of macromolecule membrane, rely on certain pressure reduction and flow velocity at normal temperatures, make low molecular weight substances such as active chitin oligo saccharide see through film less than membrane pore size, and make component materials such as high-molecular weight protein, amino acid, polypeptide and enzyme by effectively catching, the working pressure span of control is 0.3～1.5MPa, and temperature controlling range is 10～50 ℃, and the isolated molecule weight range is 2,000～20,000Da.The active chitin oligo saccharide that contains that sees through ultra-filtration membrane enters the further separation and purification that three grades of membrane sepn batch cans carry out product through liquid.
6) three grades of membrane sepn
Three grades of membrane sepn adopt the nanofiltration membrane separation device, the molecular weight cut-off of membrane sepn is 800～2,000Da, the working pressure span of control is 0.5～5.0MPa, pH is 2.5～7.5, to obtain active chitin oligo saccharide and oligosaccharide mixture product after the concentrated solution spraying drying of holding back, see through liquid and enter level Four membrane sepn batch can.
7) level Four membrane sepn
The level Four membrane sepn adopts the nanofiltration membrane separation device, isolating molecular weight cut-off is 100～300Da, the working pressure span of control is 0.5～5.0MPa, pH is 2.5～7.5, active chitin oligo saccharide product will be obtained after the concentrated solution spraying drying of holding back, see through liquid and mainly contain monose, disaccharides and water, can be used as byproduct.
In step 1), cultivate the zymocyte liquid that obtains and to carry out centrifugation with continuous flow centrifuge.
In step 2) in (2), in chitin acid solution and alkali lye mixing N-process, be preferably under the ultrasonic wave and carried out, be about to the chitin acid solution and be added drop-wise in the ultrasonic cleaner.
In step 2) in (3), described separation can be selected filtration, press filtration or method such as centrifugal for use, in filtration, press filtration or centrifugal process, preferably heats, and temperature is 20～60 ℃.Or in utilizing and the time the timely processing of exothermic phenomenon also can the people for heating.Make the salt dissolving that is attached in the chitin colloid for 20～60 ℃ with warm water, also make the mineral manure concentration that contains chitin oligo saccharide of washing higher, reduce the cost of these fertilizer of transportation.
In step 3), described damping fluid is the Sodium phosphate dibasic of pH 5.8～8.0--phosphate sodium dihydrogen buffer solution (hereinafter to be referred as PB), pH is acetate-sodium acetate buffer of 3.6～5.8, and pH is that 3.0～6.6 citric acid-sodium citrate buffer solution or pH are 7.1～8.9 Tris-hydrochloride buffer.
In step 4), described microfiltration membrane is selected from tubular membrane, Ultra-flow flat sheet membrane or hollow-fibre membrane etc., and the microfiltration membrane tripping device is with the tubular membrane best results, and the separation molecular weight cut-off that microfiltration membrane is held back suspended particulate matter is 10,000～750,000Da.
In step 7), the described liquid that sees through can concentrate to such an extent that contain the byproduct of chitin monose, disaccharides through the level V membrane reverse osmosis.
Described microfiltration membrane, ultra-filtration membrane, nanofiltration membrane can be polymer material film, or inorganic material film, or the blend film of polymer material film and inorganic material film or composite membrane; Described polymer material film, or inorganic material film, or the blend film of polymer material film and inorganic material film or composite membrane have film-forming properties, all can be selected from the organic fluorine material film, organochlorine material membrane, polysulfone membrane, poly-fragrant inkstone film and material modified film thereof or the like.Polyester film for example, the plain film of polycarbonate fibre, polyamide membrane, polypropylene screen, poly tetrafluoroethylene, polychloroethylene film, the polysulfones cellulose acetate membrane, aromatic polyamide membrane, poly-croak piperazine class film, sulfonated polyether amine film, cellulose membrane, the modified-cellulose film, polyaryletherketone film or polymethylation polysulfones polyetherketone film, perhaps polyester film, the plain film of polycarbonate fibre, polyamide membrane, polypropylene screen, poly tetrafluoroethylene, polychloroethylene film, the polysulfones cellulose acetate membrane, aromatic polyamide membrane, poly-croak piperazine class film, sulfonated polyether amine film, cellulose membrane, the modified-cellulose film, the blend membrane or the complexes membrane of polyaryletherketone film and polymethylation polysulfones polyetherketone film.Inorganic material film can be porous ceramics film, porous glass film or porous metal film etc.
Enzyme digestion reaction of the present invention and membrane separation plant are mutual coupling connection, and operating method can be operate continuously, also can be periodical operation; The raw material additional way can be a fed-batch, also can be continuous feeding.Fed-batch be membrane separation apparatus discharge a certain amount of after, in the enzyme reaction jar, drip stock liquid, but add speed must with leach speed and equate.Being connected together becomes a semi-enclosed recycle system with enzyme reaction jar and various filter (comprising ultrafiltration, nanofiltration etc.), and the enzyme liberating reaction was carried out simultaneously with separating of chitin oligo saccharide, boosts productivity.This process is film cyclophorase reactor just.Simply say, exactly enzymolysis solution in the enzyme reaction jar is returned the enzyme reaction jar by membrane separation apparatus, take out the process of product simultaneously.Product leaves the enzyme reaction jar along with seeing through liquid in this process, but the bed material continuous supplementation.
--acquisition, the low-cost chitin colloid of----high vigor specificity chitinase prepares this two big bottleneck in a large number to the present invention is directed to the general difficult point that runs in the domestic and international chitin oligo saccharide production, a strain of adopting the applicant to filter out from the ocean can be induced the bacterial strain Aeromonas caviae (Aeromonas caviaeQ2) that produces high reactivity chitinase system, obtain to secrete highly active enzyme without inducing with gene manipulation techniques again and be, the enzymic degradation chitin colloid is combined with membrane sepn, make this enzyme tie up to the chitin oligo saccharide that obtains solubility behind the hydrolysis chitin colloid.Its operational path is simple, can effectively suppress the further degraded under the enzyme effect of active chitin oligo saccharide, degrading enzyme can be reused continuously, and the service efficiency height of enzyme can be as required by the film in the different apertures of selection and the chitin oligo saccharide product of control manipulation condition acquisition different polymerization degree.Preparation process is simple and direct simultaneously, has reduced the pollution that sepn process causes, has improved separation efficiency.Process can be carried out continuously, helps realizing suitability for industrialized production.
Embodiment
Following examples will the present invention is further illustrated.
Embodiment 1
1, strain culturing and high vigor enzyme liquid preparation
Aeromonas caviae (Aeromonas caviaeQ2) bacterial classification is got an articulating by the inclined-plane to be gone into to be equipped with the 500ml of 100ml nutrient solution and shakes in the bottle, 35 ℃ of shaking culture 24h are shake-flask seed liquid, the shake-flask seed substratum is for containing peptone 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% inserts in the 10L seed fermentation jar of the substratum that contains glycerine 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% and soybean cake powder 7% with 5% inoculum size, in 35 ℃ of fermentation 24h, get seed culture fluid.In the 100L fermentor tank, add culture medium solution, wherein contain glycerine 1%, yeast extract 2.5%, corn steep liquor 2.5%, NaCl 1%, soybean cake powder 5%, in 121 ℃ of sterilization 20min, add seed culture fluid 10L, at 35 ℃, rotating speed is 100～300r/min, and air flow is under the condition of 100l/min, cultivates 24h, the zymocyte liquid that must contain chitinase system, centrifugal through whizzer, clear enzyme solution on the separated and collected obtains aseptic enzyme liquid after the ultrafiltration, after the enzyme activity, carry out the preparation of chitin oligo saccharide in the ratio adding enzyme reaction jar in 10mU/ml after measured.
2. prepare the chitin colloid
Get the commercially available chitin of 10Kg (chitin is good with fine particle, 〉=10 orders), be dissolved in the industrial strong phosphoric acid of 100L (75%), under the normal temperature,, generate the chitin phosphoric acid solution through stirring, the dissolving of 12h.Under agitation gradually the chitin phosphoric acid solution is joined in the industrial strong aqua (～21%) of 220L, make to be neutralized to pH6.5～6.Neutralization back chitin is separated out with glue, and (temperature is 20～60 ℃) press filtration (or centrifugal) while hot makes it to separate with dense ammonium phosphate salt.The chitin glue of gained washes out the salt that remains in the glue with hot water, and this washings joins in the ammonium phosphate solution after the press filtration, is the ammophos that contains soluble chitin oligo saccharide.This glue can be used as the substrate of chitinase (being), should preserve under hygrometric state.
3, chitin oligo saccharide production
(1) in the enzyme reaction jar, chitin colloid is mixed in the 20mM PB damping fluid (pH is 6.0), cumulative volume is 150L, ultimate density is 2%, steam sterilizing.The chitinase of 10mU/ml is joined in the reaction system,, treat that the solid phase composition is reduced to and began to start the one-level membrane sepn at 1.5% o'clock 40 ℃ of stirrings.
(2) the one-level membrane sepn adopts membrane area 7m
2The tubular fibre membrane filter, control pressure 0.50MPa is 20 ℃ of down operations.Its particle diameter scope of holding back suspended particulate matter of mould material is 10,000Da, and the feed liquid that is trapped is returned the enzyme reaction jar and is continued reaction, and being separated to the stoste material liquid volume is 1/2 o'clock, adds the pure water top and is washed till and holds back feed liquid and detect no chitin oligosaccharide compositions and promptly stop.The batch can that changes the secondary membrane separation unit through liquid over to is standby.
(3) start the secondary membrane separation unit, adopt the rolling flat sheet membrane, the molecular weight cut off of film is 5000Da, and control pressure 0.3MPa is 20 ℃ of operations down.The active liquid that sees through of two-stage ultrafiltering membrane sepn directly enters three grades of nanofiltration membrane separation devices, hold back feed liquid and return the batch can of secondary membrane separation unit, when separated material liquid volume is contracted to 1/3 volume, adds pure water top and be washed till and hold back the part feed liquid and do not have the chitin oligosaccharide compositions and detect promptly and stop.It is standby through the batch can that liquid changes three grades of membrane separation units over to contain active chitin oligo saccharide.
(4) start three grades of membrane separation units, with 2 cun rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 1000Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 0.8MPa, pH6.0 holds back feed liquid and returns the batch can of three grades of membrane separation units and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.Concentrate active chitin oligo saccharide and the oligosaccharide mixture of holding back in the batch can of three grades of membrane separation units of collection, again spraying drying.It is standby through the batch can that liquid changes the level Four membrane separation unit over to contain active lower molecular weight chitin oligo saccharide.
(5) start the level Four membrane separation unit, with rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 300Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 0.8MPa, pH6.0 holds back feed liquid and returns the batch can of level Four membrane separation unit and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Collect in the level Four membrane separation unit batch can and concentrate the active lower molecular weight chitin oligo saccharide of holding back, obtain required chitin oligo saccharide product after the spraying drying.
See through liquid and mainly contain monose, disaccharides and water, change other batch can over to as byproduct.
Embodiment 2
1, strain culturing and high vigor enzyme liquid preparation
Aeromonas caviae (Aeromonas caviaeQ2) bacterial classification is got an articulating by the inclined-plane to be gone into to be equipped with the 500ml of 100ml nutrient solution and shakes in the bottle, 35 ℃ of shaking culture 24h are shake-flask seed liquid, the shake-flask seed substratum is for containing peptone 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% inserts in the 10L seed fermentation jar of the substratum that contains glycerine 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% and soybean cake powder 7% with 5% inoculum size, in 35 ℃ of fermentation 24h, get seed culture fluid.In the 100L fermentor tank, add culture medium solution, wherein contain glycerine 1.5%, yeast extract 0.5%, corn steep liquor 1%, NaCl 0.5%, soybean cake powder 10%, in 121 ℃ of sterilization 20min, add seed culture fluid 10L, at 25 ℃, 300r/min, air flow are under the condition of 5l/min, cultivate 40h, the zymocyte liquid that must contain chitinase system, centrifugal through whizzer, clear enzyme solution on the separated and collected obtains aseptic enzyme liquid after the ultrafiltration, after the enzyme activity, carry out the preparation of chitin oligo saccharide in the ratio adding enzyme reaction jar in 1mU/ml after measured.
2. preparation chitin colloid
Get the commercially available chitin of 10Kg (chitin is good with fine particle, 〉=10 orders), be dissolved in the industrial strong phosphoric acid of 80L (85%), under the normal temperature,, generate the chitin phosphoric acid solution through stirring, the dissolving of 12h.Getting industrial strong aqua 200L is incorporated in the 200L pure water, again chitin solution is added in the ammoniacal liquor after the dilution gradually, after being neutralized into pH and being 5～6, the chitin glue of separating out separates chitin glue with filtering centrifuge (be centrifugal basket drier, or tubular type continuous flow centrifuge).The ammonium phosphate solution that collection contains the chitin oligosaccharide is fertilizer/agricultural chemicals.Chitin glue temperature is that 20～60 ℃ of hot water wash out the ammonium phosphate salt that remains in the glue, is product chitin glue.
3, chitin oligo saccharide production
(1) in the enzyme reaction jar, chitin colloid is mixed in 20mM citric acid-sodium citrate buffer solution in (pH is 3.0), cumulative volume is 150L, ultimate density is 0.1%, steam sterilizing.The chitinase of 1mU/ml is joined in the reaction system, and 60 ℃ of stirrings, the solid phase composition is reduced to and started the one-level membrane separation unit at 0.05% o'clock.
(2) the one-level membrane sepn adopts membrane area 1m
2Tubular membrane, control pressure 0.15MPa is 10 ℃ of down operations.Its particle diameter scope of holding back suspended particulate matter of mould material is 100,000Da, and the feed liquid that is trapped is returned the enzyme reaction jar and is continued reaction, and being separated to the stoste material liquid volume is 1/2 o'clock, adds the pure water top and is washed till and holds back feed liquid and detect no chitin oligosaccharide compositions and promptly stop.The batch can that changes the secondary membrane separation unit through liquid over to is standby.
(3) start the secondary membrane separation unit, adopt the rolling flat sheet membrane, the molecular weight cut off of film is 2000Da, and control pressure 0.6MPa is 10 ℃ of operations down.The active liquid that sees through of two-stage ultrafiltering membrane sepn directly enters three grades of nanofiltration membrane separation devices, hold back feed liquid and return the batch can of secondary membrane separation unit, when separated material liquid volume is contracted to 1/3 volume, adds pure water top and be washed till and hold back the part feed liquid and do not have the chitin oligosaccharide compositions and detect promptly and stop.It is standby through the batch can that liquid changes three grades of membrane separation units over to contain active chitin oligo saccharide.
(4) start three grades of membrane separation units, with 2 cun rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 800Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 0.5MPa, pH2.5 holds back feed liquid and returns the batch can of three grades of membrane separation units and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Concentrate active chitin oligo saccharide and the oligosaccharide mixture of holding back in the batch can of three grades of membrane separation units of collection, again spraying drying.
It is standby through the batch can that liquid changes the level Four membrane separation unit over to contain active lower molecular weight chitin oligo saccharide.
(5) start the level Four membrane separation unit, with rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 100Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 0.5MPa, pH2.5 holds back feed liquid and returns the batch can of level Four membrane separation unit and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Collect in the level Four membrane separation unit batch can and concentrate the active lower molecular weight chitin oligo saccharide of holding back, obtain required chitin oligo saccharide product after the spraying drying.
See through liquid and mainly contain monose, disaccharides and water, change other batch can over to as byproduct.
Embodiment 3
1, strain culturing and high vigor enzyme liquid preparation
Aeromonas caviae (Aeromonas caviaeQ2) bacterial classification is got an articulating by the inclined-plane to be gone into to be equipped with the 500ml of 100ml nutrient solution and shakes in the bottle, 37 ℃ of shaking culture 20h are shake-flask seed liquid, the shake-flask seed substratum is for containing peptone 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% inserts in the 10L seed fermentation jar of the substratum that contains glycerine 4%, yeast extract 5%, corn steep liquor 3%, NaCl 1% and soybean cake powder 5% with 5% inoculum size, in 37 ℃ of fermentation 20h, get seed culture fluid.In the 100L fermentor tank, add culture medium solution, wherein contain glycerine 4%, yeast extract 3%, corn steep liquor 2%, NaCl 0.5%, soybean cake powder 3%, in 121 ℃ of sterilization 20min, add seed culture fluid 10L, at 37 ℃, 200r/min, air flow are under the condition of 150l/min, cultivate 20h, the zymocyte liquid that must contain chitinase system, centrifugal through whizzer, clear enzyme solution on the separated and collected obtains aseptic enzyme liquid after the ultrafiltration, after the enzyme activity, carry out the preparation of chitin oligo saccharide in the ratio adding enzyme reaction jar in 4mU/ml after measured.
2. preparation chitin colloid
Get the commercially available chitin of 10Kg (chitin is good with fine particle, 〉=10 orders), be dissolved in the industrial strong phosphoric acid of 150L (60%), in the PE jar of 90 ℃ of insulations, stir 2h, chitin is dissolved in the phosphoric acid, gets the solution 200L that industrial potassium hydroxide is mixed with 5.5M concentration.Under agitation be about 6.5 making it to be neutralized to pH in the chitin solution adding alkali lye lentamente.The chitin colloid of separating out gets chitin glue after separating; Use this glue of a spot of hot wash again, be product (chitin glue), and isolated solution is potassium, the phosphatic manure that contains chitin oligo saccharide.This fertilizer can with the nitrogenous fertilizer fit applications.
3, chitin oligo saccharide production
(1) in the enzyme reaction jar, chitin colloid is mixed in the 20mMTris-hydrochloride buffer (pH is 8.9), cumulative volume is 150L, ultimate density is 4%, steam sterilizing.The chitinase of 4mU/ml is joined in the reaction system, and 10 ℃ of stirrings, the solid phase composition is reduced to and started the one-level membrane separation unit at 0.5% o'clock.
(2) the one-level membrane sepn adopts membrane area 1m
2Tubular membrane, control pressure 0.3MPa is 30 ℃ of down operations.Its particle diameter scope of holding back suspended particulate matter of mould material is 200,000Da, and the feed liquid that is trapped is returned the enzyme reaction jar and is continued reaction, and being separated to the stoste material liquid volume is 1/2 o'clock, adds the pure water top and is washed till and holds back feed liquid and detect no chitin oligosaccharide compositions and promptly stop.The batch can that changes the secondary membrane separation unit through liquid over to is standby.
(3) start the secondary membrane separation unit, adopt the rolling flat sheet membrane, the molecular weight cut off of film is 4000Da, and control pressure 1.0MPa is 30 ℃ of operations down.The active liquid that sees through of two-stage ultrafiltering membrane sepn directly enters three grades of nanofiltration membrane separation devices, hold back feed liquid and return the batch can of secondary membrane separation unit, when separated material liquid volume is contracted to 1/3 volume, adds pure water top and be washed till and hold back the part feed liquid and do not have the chitin oligosaccharide compositions and detect promptly and stop.It is standby through the batch can that liquid changes three grades of membrane separation units over to contain active chitin oligo saccharide.
(4) start three grades of membrane separation units, with 2 cun rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 1500Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 1.5MPa, pH4.0 holds back feed liquid and returns the batch can of three grades of membrane separation units and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Concentrate active chitin oligo saccharide and the oligosaccharide mixture of holding back in the batch can of three grades of membrane separation units of collection, again spraying drying.
It is standby through the batch can that liquid changes the level Four membrane separation unit over to contain active lower molecular weight chitin oligo saccharide.
(5) start the level Four membrane separation unit, with rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 300Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 3MPa, pH4.0 holds back feed liquid and returns the batch can of level Four membrane separation unit and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Collect in the level Four membrane separation unit batch can and concentrate the active lower molecular weight chitin oligo saccharide of holding back, obtain required chitin oligo saccharide product after the spraying drying.
See through liquid and mainly contain monose, disaccharides and water, change other batch can over to as byproduct.
Embodiment 4
1, strain culturing and high vigor enzyme liquid preparation
Aeromonas caviae (Aeromonas caviaeQ2) bacterial classification is got an articulating by the inclined-plane to be gone into to be equipped with the 500ml of 100ml nutrient solution and shakes in the bottle, 28 ℃ of shaking culture 24h are shake-flask seed liquid, the shake-flask seed substratum is for containing peptone 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% inserts in the 10L seed fermentation jar of the substratum that contains glycerine 4%, yeast extract 5%, corn steep liquor 3%, NaCl 1% and soybean cake powder 5% with 5% inoculum size, in 28 ℃ of fermentation 24h, get seed culture fluid.In the 100L fermentor tank, add culture medium solution, wherein contain glycerine 10%, yeast extract 1.5%, corn steep liquor 3%, NaCl 2%, soybean cake powder 1%, in 121 ℃ of sterilization 20min, add seed culture fluid 10L, at 28 ℃, 100r/min, air flow are under the condition of 200l/min, cultivate 30h, the zymocyte liquid that must contain chitinase system, centrifugal through whizzer, clear enzyme solution on the separated and collected obtains aseptic enzyme liquid after the ultrafiltration, after the enzyme activity, carry out the preparation of chitin oligo saccharide in the ratio adding enzyme reaction jar in 5mU/ml after measured.
2. preparation chitin colloid
Similar to Example 1, its difference is that mineral acid adopts the vitriol oil (98%) of 80L, under the normal temperature, through stirring, the dissolving of 12h, generates the chitin sulphuric acid soln.Be preferably under the ultrasonic wave in chitin solution and the sodium hydroxide solution mixing N-process and carry out, be about to the chitin acid solution and be added drop-wise in the ultrasonic cleaner, and heat when press filtration to 40～60 ℃, pH is 6.5.
3, chitin oligo saccharide production
(1) in the enzyme reaction jar, chitin colloid is mixed in acetate-sodium acetate buffer (pH is 4.0), cumulative volume is 150L, ultimate density is 5%, steam sterilizing.The chitinase of 5mU/ml is joined in the reaction system, and 20 ℃ of stirrings, the solid phase composition is reduced to and started the one-level membrane separation unit at 1% o'clock.
(2) the one-level membrane sepn adopts membrane area 1m
2The Ultra-flow flat sheet membrane, control pressure 0.6MPa is 40 ℃ of down operations.Its particle diameter scope of holding back suspended particulate matter of mould material is 400,000Da, and the feed liquid that is trapped is returned the enzyme reaction jar and is continued reaction, and being separated to the stoste material liquid volume is 1/2 o'clock, adds the pure water top and is washed till and holds back feed liquid and detect no chitin oligosaccharide compositions and promptly stop.The batch can that changes the secondary membrane separation unit through liquid over to is standby.
(3) start the secondary membrane separation unit, adopt the rolling flat sheet membrane, the molecular weight cut off of film is 8000Da, and control pressure 1.5MPa is 40 ℃ of operations down.The active liquid that sees through of two-stage ultrafiltering membrane sepn directly enters three grades of nanofiltration membrane separation devices, hold back feed liquid and return the batch can of secondary membrane separation unit, when separated material liquid volume is contracted to 1/3 volume, adds pure water top and be washed till and hold back the part feed liquid and do not have the chitin oligosaccharide compositions and detect promptly and stop.It is standby through the batch can that liquid changes three grades of membrane separation units over to contain active chitin oligo saccharide.
(4) start three grades of membrane separation units, with 2 cun rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 2000Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 3MPa, pH5.0 holds back feed liquid and returns the batch can of three grades of membrane separation units and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Concentrate active chitin oligo saccharide and the oligosaccharide mixture of holding back in the batch can of three grades of membrane separation units of collection, again spraying drying.
It is standby through the batch can that liquid changes the level Four membrane separation unit over to contain active lower molecular weight chitin oligo saccharide.
(5) start the level Four membrane separation unit, with rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 300Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 4MPa, pH5.0 holds back feed liquid and returns the batch can of level Four membrane separation unit and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Collect in the level Four membrane separation unit batch can and concentrate the active lower molecular weight chitin oligo saccharide of holding back, obtain required chitin oligo saccharide product after the spraying drying.
See through liquid and mainly contain monose, disaccharides and water, change other batch can over to as byproduct.
Embodiment 5
1, strain culturing and high vigor enzyme liquid preparation
Aeromonas caviae (Aeromonas caviaeQ2) bacterial classification is got an articulating by the inclined-plane to be gone into to be equipped with the 500ml of 100ml nutrient solution and shakes in the bottle, 30 ℃ of shaking culture 24h are shake-flask seed liquid, the shake-flask seed substratum is for containing peptone 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% inserts in the 10L seed fermentation jar of the substratum that contains glycerine 4%, yeast extract 5%, corn steep liquor 3%, NaCl 1% and soybean cake powder 5% with 5% inoculum size, in 30 ℃ of fermentation 24h, get seed culture fluid.In the 100L fermentor tank, add culture medium solution, wherein contain glycerine 6%, yeast extract 5%, corn steep liquor 4%, NaCl 3%, soybean cake powder 2%, in 121 ℃ of sterilization 20min, add seed culture fluid 10L, at 30 ℃, 250r/min, air flow are under the condition of 30l/min, cultivate 24h, the zymocyte liquid that must contain chitinase system, centrifugal through whizzer, clear enzyme solution on the separated and collected obtains aseptic enzyme liquid after the ultrafiltration, after the enzyme activity, carry out the preparation of chitin oligo saccharide in the ratio adding enzyme reaction jar in 6mU/ml after measured.
2. preparation chitin colloid
Similar to Example 2, its difference is that mineral acid adopts the sulfuric acid (60%) of 180L, in the PE jar of 60 ℃ of insulations, stir 4h, chitin is dissolved in the sulfuric acid, under ultrasonic wave, carry out in chitin solution and the dense solution of potassium carbonate mixing N-process, being about to the chitin acid solution is added drop-wise in the ultrasonic cleaner, being neutralized into pH is 5～6, press filtration, and remaining step is with embodiment 2.
3, chitin oligo saccharide production
(1) in the enzyme reaction jar, chitin colloid is mixed in acetate-sodium acetate buffer (pH is 5.0), cumulative volume is 150L, ultimate density is 10%, steam sterilizing.The chitinase of 6mU/ml is joined in the reaction system, and 30 ℃ of stirrings, the solid phase composition is reduced to and started the one-level membrane separation unit at 2% o'clock.
(2) the one-level membrane sepn adopts membrane area 1m
2The Ultra-flow flat sheet membrane, control pressure 0.8MPa is 50 ℃ of down operations.Its particle diameter scope of holding back suspended particulate matter of mould material is 750,000Da, and the feed liquid that is trapped is returned the enzyme reaction jar and is continued reaction, and being separated to the stoste material liquid volume is 1/2 o'clock, adds the pure water top and is washed till and holds back feed liquid and detect no chitin oligosaccharide compositions and promptly stop.The batch can that changes the secondary membrane separation unit through liquid over to is standby.
(3) start the secondary membrane separation unit, adopt the rolling flat sheet membrane, the molecular weight cut off of film is 20,000Da, and control pressure 1.5MPa is 50 ℃ of operations down.The active liquid that sees through of two-stage ultrafiltering membrane sepn directly enters three grades of nanofiltration membrane separation devices, hold back feed liquid and return the batch can of secondary membrane separation unit, when separated material liquid volume is contracted to 1/3 volume, adds pure water top and be washed till and hold back the part feed liquid and do not have the chitin oligosaccharide compositions and detect promptly and stop.It is standby through the batch can that liquid changes three grades of membrane separation units over to contain active chitin oligo saccharide.
(4) start three grades of membrane separation units, with 2 cun rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 2000Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 5MPa, pH7.5 holds back feed liquid and returns the batch can of three grades of membrane separation units and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Concentrate active chitin oligo saccharide and the oligosaccharide mixture of holding back in the batch can of three grades of membrane separation units of collection, again spraying drying.
It is standby through the batch can that liquid changes the level Four membrane separation unit over to contain active lower molecular weight chitin oligo saccharide.
(5) start the level Four membrane separation unit, with rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 200Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 5MPa, pH7.5 holds back feed liquid and returns the batch can of level Four membrane separation unit and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Collect in the level Four membrane separation unit batch can and concentrate the active lower molecular weight chitin oligo saccharide of holding back, obtain required chitin oligo saccharide product after the spraying drying.
See through liquid and mainly contain monose, disaccharides and water, change other batch can over to as byproduct.
Embodiment 6
1, strain culturing and high vigor enzyme liquid preparation
Aeromonas caviae (Aeromonas caviaeQ2) bacterial classification is got an articulating by the inclined-plane to be gone into to be equipped with the 500ml of 100ml nutrient solution and shakes in the bottle, 37 ℃ of shaking culture 24h are shake-flask seed liquid, the shake-flask seed substratum is for containing peptone 1%, yeast extract 5%, corn steep liquor 5%, NaCl 1% inserts in the 10L seed fermentation jar of the substratum that contains glycerine 4%, yeast extract 5%, corn steep liquor 3%, NaCl 1% and soybean cake powder 5% with 5% inoculum size, in 37 ℃ of fermentation 24h, get seed culture fluid.In the 100L fermentor tank, add culture medium solution, wherein contain glycerine 8%, yeast extract 4%, corn steep liquor 5%, NaCl 1%, soybean cake powder 4%, in 121 ℃ of sterilization 20min, add seed culture fluid 10L, at 37 ℃, 300r/min, air flow are under the condition of 50l/min, cultivate 10h, the zymocyte liquid that must contain chitinase system, centrifugal through whizzer, clear enzyme solution on the separated and collected obtains aseptic enzyme liquid after the ultrafiltration, after the enzyme activity, carry out the preparation of chitin oligo saccharide in the ratio adding enzyme reaction jar in 8mU/ml after measured.
2. preparation chitin colloid
Similar to Example 2, its difference is that mineral acid adopts the sulfuric acid (60%) of 180L, in the PE jar of 60 ℃ of insulations, stir 4h, chitin is dissolved in the sulfuric acid, under ultrasonic wave, carry out in chitin solution and the dense solution of potassium carbonate mixing N-process, being about to the chitin acid solution is added drop-wise in the ultrasonic cleaner, being neutralized into pH is 5～6, press filtration, and remaining step is with embodiment 2.
3, chitin oligo saccharide production
(1) in the enzyme reaction jar, chitin colloid is mixed in the PB damping fluid (pH is 7.0), cumulative volume is 150L, ultimate density is 8%, steam sterilizing.The chitinase of 8mU/ml is joined in the reaction system, and 30 ℃ of stirrings, the solid phase composition is reduced to and started the one-level membrane separation unit at 1% o'clock.
(2) the one-level membrane sepn adopts membrane area 1m
2Tubular membrane, control pressure 0.8MPa is 20 ℃ of down operations.Its particle diameter scope of holding back suspended particulate matter of mould material is 600,000Da, and the feed liquid that is trapped is returned the enzyme reaction jar and is continued reaction, and being separated to the stoste material liquid volume is 1/2 o'clock, adds the pure water top and is washed till and holds back feed liquid and detect no chitin oligosaccharide compositions and promptly stop.The batch can that changes the secondary membrane separation unit through liquid over to is standby.
(3) start the secondary membrane separation unit, adopt the rolling flat sheet membrane, the molecular weight cut off of film is 15,000Da, and control pressure 0.8MPa is 20 ℃ of operations down.The active liquid that sees through of two-stage ultrafiltering membrane sepn directly enters three grades of nanofiltration membrane separation devices, hold back feed liquid and return the batch can of secondary membrane separation unit, when separated material liquid volume is contracted to 1/3 volume, adds pure water top and be washed till and hold back the part feed liquid and do not have the chitin oligosaccharide compositions and detect promptly and stop.It is standby through the batch can that liquid changes three grades of membrane separation units over to contain active chitin oligo saccharide.
(4) start three grades of membrane separation units, with 2 cun rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 1000Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 4MPa, pH7.5 holds back feed liquid and returns the batch can of three grades of membrane separation units and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Concentrate active chitin oligo saccharide and the oligosaccharide mixture of holding back in the batch can of three grades of membrane separation units of collection, again spraying drying.
It is standby through the batch can that liquid changes the level Four membrane separation unit over to contain active lower molecular weight chitin oligo saccharide.
(5) start the level Four membrane separation unit, with rolling flat sheet membrane nanofiltration device, the molecular weight cut off of film is 300Da, and the rejection of film is 80%～95%, membrane area 1.7m
2, control pressure 1.5MPa, pH7.5 holds back feed liquid and returns the batch can of level Four membrane separation unit and enter circulation and separate, and stops when the very few fourth monosaccharide component of the feed liquid that circulates detects.
Collect in the level Four membrane separation unit batch can and concentrate the active lower molecular weight chitin oligo saccharide of holding back, obtain required chitin oligo saccharide product after the spraying drying.
See through liquid and mainly contain monose, disaccharides and water, change other batch can over to as byproduct.
Embodiment 7
Similar to Example 1, its difference is:
(1) the enzyme liquid and preparation method thereof is with embodiment 1.
(2) preparation chitin colloid: similar to Example 2, its difference is that mineral acid adopts the concentrated hydrochloric acid (hydrochloric amount 〉=35%) of 120L, in the PE jar of 40 ℃ of insulations, stir 2h, chitin is dissolved in the hydrochloric acid, chitin solution and dense potassium bicarbonate solution mixing N-process can be placed in the groove of ultrasonic cleaner and carry out, and remaining step is with embodiment 2.
(3) in the enzyme reaction jar, chitin colloid is blended in citric acid-sodium citrate buffer solution (pH is 6.0), and cumulative volume is 150L, and ultimate density is 1%, the dosage of chitinase is to 1mU/ml, 40 ℃ of down reactions, the chitin oligo saccharide enzymolysis solution, treat in this chitin oligo saccharide enzymolysis solution that the solid phase composition was reduced in the chitin oligo saccharide at 0.5% o'clock, start the one-level membrane separation unit, microfiltration membrane is selected from the tubular fibre membrane filter, and its particle diameter scope of holding back suspended particulate matter of mould material is 20,000Da.
Embodiment 8
Similar to Example 1, its difference is:
(1) the enzyme liquid and preparation method thereof is with embodiment 2.
(2) preparation chitin colloid: similar to Example 2, its difference is that mineral acid adopts the hydrochloric acid (hydrochloric amount 〉=20%) of 200L, in the PE jar of 60 ℃ of insulations, stir 4h, chitin is dissolved in the hydrochloric acid, can carry out under ultrasonic wave in chitin solution and the strong aqua mixing N-process, remaining step is with embodiment 2.
(3) in the enzyme reaction jar, chitin colloid is blended in acetate-sodium acetate buffer (pH is 5.0), and cumulative volume is 150L, and ultimate density is 10%, the dosage of chitinase is to 5mU/ml, 35 ℃ of down reactions, the chitin oligo saccharide enzymolysis solution, treat in this chitin oligo saccharide enzymolysis solution that the solid phase composition was reduced in the chitin oligo saccharide at 1% o'clock, start the one-level membrane separation unit, microfiltration membrane is selected from tubular membrane, and its particle diameter scope of holding back suspended particulate matter of mould material is 100,000Da.
Embodiment 9
Similar to Example 1, its difference is:
(1) the enzyme liquid and preparation method thereof is with embodiment 3.
(2) in the enzyme reaction jar, chitin colloid is blended in the Tris-hydrochloride buffer (pH8.9), and cumulative volume is 150L, and ultimate density is 10%, the dosage of chitinase is to 10mU/ml, 40 ℃ of down reactions, the chitin oligo saccharide enzymolysis solution, treat in this chitin oligo saccharide enzymolysis solution that the solid phase composition was reduced in the chitin oligo saccharide at 0.5% o'clock, start the one-level membrane separation unit, microfiltration membrane is selected from the Ultra-flow flat sheet membrane, and its particle diameter scope of holding back suspended particulate matter of mould material is 200,000Da.
Embodiment 10
Similar to Example 1, its difference is:
(1) the enzyme liquid and preparation method thereof is with embodiment 4.
(2) in the enzyme reaction jar, chitin colloid is blended in the Tris-hydrochloride buffer (pH7.5), and cumulative volume is 150L, and ultimate density is 5%, the dosage of chitinase is to 10mU/ml, 10 ℃ of down reactions, the chitin oligo saccharide enzymolysis solution, treat in this chitin oligo saccharide enzymolysis solution that the solid phase composition was reduced in the chitin oligo saccharide at 0.5% o'clock, start the one-level membrane separation unit, microfiltration membrane is selected from hollow-fibre membrane, and its particle diameter scope of holding back suspended particulate matter of mould material is 750,000Da.
Embodiment 11
Similar to Example 1, its difference is:
(1) the enzyme liquid and preparation method thereof is with embodiment 5.
(2) in the enzyme reaction jar, chitin colloid is blended in the Tris-hydrochloride buffer (pH8.5), and cumulative volume is 150L, and ultimate density is 10%, the dosage of chitinase is to 10mU/ml, 60 ℃ of down reactions, the chitin oligo saccharide enzymolysis solution, treat in this chitin oligo saccharide enzymolysis solution that the solid phase composition was reduced in the chitin oligo saccharide at 1% o'clock, start the one-level membrane separation unit, microfiltration membrane is selected from hollow-fibre membrane, and its particle diameter scope of holding back suspended particulate matter of mould material is 600,000Da.
Claims (10)
1. chitin oligose preparing process is characterized in that the steps include:
1) strain culturing and enzyme liquid preparation
The bacterial strain Aeromonas caviae is cultivated in substratum; The zymocyte liquid centrifugation that then cultivation is obtained obtains fermented liquid supernatant, removes remaining thalline and other solid substances in the enzyme liquid with ultrafiltration process again, and it is standby that separation obtains enzyme liquid;
2) preparation chitin colloid
(1), chitin is mixed with mineral acid, make the chitin acid solution, by weight with volume proportion, chitin: mineral acid=1: 8～20, wherein chitinous unit is Kg, the unit of mineral acid is L;
(2), the chitin acid solution is added drop-wise in water or the alkaline solution, make its colloidal precipitation, become Colloidal fluid;
(3), chitin glue is separated with the inorganic salt fertilizer that contains the chitin oligosaccharide;
(4), the chitin glue after will separating washes with water, promptly gets chitin colloid;
3) enzyme digestion reaction
In the enzyme reaction jar, chitin colloid is suspended in the damping fluid that concentration is 20mM, being mixed with final concentration is the chitin colloid solution of chitin dry weight 0.1%～10%, the steam sterilizing postcooling is to room temperature, the chitinase liquid aseptic delivery that step 1) is obtained is to the intravital chitin colloid solution of the enzyme reaction jar of the bacterium of going out, insulation gets the chitin oligo saccharide enzymolysis solution;
4) one-level membrane sepn
The solid phase component content for the treatment of chitin colloid in the chitin oligo saccharide enzymolysis solution that step 3) obtains was reduced to 0.05%～2% o'clock, the chitin oligo saccharide enzymolysis solution is pumped into the microfiltration membrane tripping device from the enzyme reaction jar, pressure control range is 0.15～0.8MPa, temperature controlling range is 10～50 ℃, remove suspended substance, chitin colloid and partial organic substances in the enzymolysis solution, the part that does not see through is returned the enzyme reaction jar and is continued to participate in enzymolysis, seeing through liquid is active chitin oligo saccharide filtrate, enters secondary membrane sepn batch can through pipeline;
5) secondary membrane sepn
The secondary membrane sepn is selected the ultra-filtration membrane tripping device for use, with pressure is impellent, the working pressure span of control is 0.3～1.5MPa, temperature controlling range is 10～50 ℃, the isolated molecule weight range is 2,000～20,000Da, the active chitin oligo saccharide that contains that sees through ultra-filtration membrane enters the further separation and purification that three grades of membrane sepn batch cans carry out product through liquid;
6) three grades of membrane sepn
Three grades of membrane sepn adopt the nanofiltration membrane separation device, the molecular weight cut-off of membrane sepn is 800～2,000Da, the working pressure span of control is 0.5～5.0MPa, pH is 2.5～7.5, to obtain active chitin oligo saccharide and oligosaccharide mixture product after the concentrated solution spraying drying of holding back, see through liquid and enter level Four membrane sepn batch can;
7) level Four membrane sepn
The level Four membrane sepn adopts the nanofiltration membrane separation device, isolating molecular weight cut-off is 100～300Da, the working pressure span of control is 0.5～5.0MPa, pH is 2.5～7.5, active chitin oligo saccharide product will be obtained after the concentrated solution spraying drying of holding back, see through liquid and mainly contain monose, disaccharides and water, as byproduct.
2. chitin oligose preparing process as claimed in claim 1 is characterized in that in step 1), cultivates the zymocyte liquid that obtains and carries out centrifugation with continuous flow centrifuge, and the component of described substratum and precentagewise content thereof are:
Glycerine 1%～10%
Yeast extract 0.5%～5%
Corn steep liquor 1%～5%
Soybean cake powder 1%～10%
NaCl 0.5～3％
Yu Weishui;
Culture temperature is 25～37 ℃, and air flow is 5～200l/min, and incubation time is 10～40h.
3. chitin oligose preparing process as claimed in claim 1 is characterized in that in step 2) (1) in, chitin is mixed with dense mineral acid, make the chitin acid solution, by weight with volume proportion, chitin: mineral acid=1: 10; Described mineral acid is a kind of in phosphoric acid, sulfuric acid or the hydrochloric acid, and phosphoric acid concentration is 60%～85%, and sulfuric acid concentration is 60%～98%, and concentration of hydrochloric acid is 20%～35%, and wherein chitinous unit is Kg, and the unit of mineral acid is L.
4. chitin oligose preparing process as claimed in claim 1 is characterized in that in step 2) in (2), in chitin acid solution and alkali lye mixing N-process, having under the ultrasonic wave and carrying out, be about to the chitin acid solution and be added drop-wise in the ultrasonic cleaner; Described alkali lye is selected from a kind of in ammoniacal liquor, potash, the soda; Described potash is selected from a kind of in potassium hydroxide, salt of wormwood, the saleratus, and described soda is selected from a kind of in sodium hydroxide, yellow soda ash, the sodium bicarbonate.
5. chitin oligose preparing process as claimed in claim 1 is characterized in that in step 2) in (3), filtration, press filtration or centrifugal method are selected in described separation for use, heat in filtration, press filtration or centrifugal process, and temperature is 20～60 ℃; Or in utilizing and the time exothermic phenomenon in time handle, with warm water the salt that is attached in the chitin colloid is dissolved.
6. chitin oligose preparing process as claimed in claim 1, it is characterized in that in step 3), described damping fluid is the Sodium phosphate dibasic of pH5.8～8.0 a--phosphate sodium dihydrogen buffer solution, pH is acetate-sodium acetate buffer of 3.6～5.8, and pH is that 3.0～6.6 citric acid-sodium citrate damping fluid or pH are 7.1～8.9 Tris-hydrochloride buffer; The chitinase liquid phase is 1～10mU/ml to the consumption of chitin colloid, and temperature of reaction is 10～60 ℃, and pH is 3.0～8.9.
7. chitin oligose preparing process as claimed in claim 1, it is characterized in that in step 4) described microfiltration membrane is selected from tubular membrane, Ultra-flow flat sheet membrane or hollow-fibre membrane, the separation molecular weight cut-off that microfiltration membrane is held back suspended particulate matter is 10,000～750,000Da.
8. chitin oligose preparing process as claimed in claim 1 is characterized in that in step 7), and the described liquid that sees through concentrates to such an extent that contain the byproduct of chitin monose, disaccharides through the level V membrane reverse osmosis.
9. chitin oligose preparing process as claimed in claim 1 is characterized in that described microfiltration membrane, ultra-filtration membrane, nanofiltration membrane are polymer material film, or inorganic material film, or the blend film of polymer material film and inorganic material film or composite membrane; Described polymer material film, or inorganic material film, or the blend film of polymer material film and inorganic material film or composite membrane have film-forming properties, is selected from the organic fluorine material film, organochlorine material membrane, polysulfone membrane, poly-fragrant inkstone film and material modified film thereof.
10. as claim 1 or 9 described chitin oligose preparing process, it is characterized in that described polymer material film, or inorganic material film, or the blend film of polymer material film and inorganic material film or composite membrane have film-forming properties, be selected from polyester film, the plain film of polycarbonate fibre, polyamide membrane, polypropylene screen, poly tetrafluoroethylene, polychloroethylene film, the polysulfones cellulose acetate membrane, aromatic polyamide membrane, poly-croak piperazine class film, sulfonated polyether amine film, cellulose membrane, the modified-cellulose film, polyaryletherketone film or polymethylation polysulfones polyetherketone film, perhaps polyester film, the plain film of polycarbonate fibre, polyamide membrane, polypropylene screen, poly tetrafluoroethylene, polychloroethylene film, the polysulfones cellulose acetate membrane, aromatic polyamide membrane, poly-croak piperazine class film, sulfonated polyether amine film, cellulose membrane, the modified-cellulose film, the blend membrane or the complexes membrane of polyaryletherketone film and polymethylation polysulfones polyetherketone film; Inorganic material film is porous ceramics film, porous glass film or porous metal film.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1150316C (en) * | 1999-04-06 | 2004-05-19 | 中国科学院微生物研究所 | Chitinase produced by fumigacin |
| CN1417324A (en) * | 2001-11-01 | 2003-05-14 | 国家海洋局第三海洋研究所 | Guinea pig aeromonad as efficient chitin degrading bacterium and produced chitinase system |
| CN1427005A (en) * | 2001-12-21 | 2003-07-02 | 李瑞捷 | Preparation method of chitin oligosaccharide |
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