CN1978636B - Process for extracting beta-mannanase utilizing Erwinia - Google Patents
Process for extracting beta-mannanase utilizing Erwinia Download PDFInfo
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Abstract
This invention involves the technology of extraction of beta-mannan enzyme from Erwinia bacilli in the industry of biological agents. The technical program includes original species activation, primary seed cultivation, secondary seed cultivation, fermentation, proteinase inhibitor, crude separation, refining of concentrated efficient bacilli and mannan-liquid, formulas, vacuum packaging technology and condensed efficient liquid agent and mannan enzyme products. This invention uses centralized preparation agent program to reduce technical difficulty of bio-degumming and biological pulping process, and can reduce the production cost by extracting cell pellets and secretions from the same broth. The efficient concentrated liquid agent, produced by this invention technical programs, is suitable for bio-degumming of crudefiber plants, biological pulping of grasses and biological glycosylated processing of herbaceous fiber raw materials biopulping alcohol; mannan enzymes are widely used in food, medicine, fodder, paper, textiles, petroleum mining industry.
Description
One, technical field
The present invention relates to same fermented liquid prepares microbial inoculum and zymin simultaneously by shunting technology.Microbial inoculum is mainly used in the extraction of draft fiber with refining, comprises the bast-fibre biological degumming of textile industry, the bio-pulping of paper industry and the biological saccharification of draft fibrous material of Alcohol Production industry; 'beta '-mannase finds broad application in many-sides such as food, medicine, feed, papermaking, textile printing and dyeing, oil production and biotechnology research.
Two, background technology
(1) domestic and international current situation and trend
1, exploitation draft fibre resources is a global evolution trend
Follow the shortage of the oil and the forest reserves and human life quality's raising, the textile and paper industry is being risen the upsurge of draft fibre resources development and use both at home and abroad.The draft fiber has been developed multiple product innovations such as building materials, high-grade paper pulp, automobile component, finishing material, degradable plastic sheeting for farm use in recent years except as the textile raw material, develops enormously potential.At present, flax main product states such as Russia, Egypt are stepping up the development of flax industry; Developed countries such as the U.S., Japan, France, Belgium, Italy are developing the bluish dogbane industry; State such as Indonesia, Malaysia introduces a fine variety ramie one after another; Tropic countries such as Philippines, Brazil, New Zealand are being developed the new purposes of bast-fibres such as sisal, pineapple fibre energetically; India, Bangladesh are stepping up the development of jute dress ornament, have basically formed the situation of " supplementing cotton with fiber crops " and " with careless Dai Mu ".
2, microbial fermentation is that the draft fiber extracts and the purified developing direction
The draft textile plant that maybe may develop in the world relates to 19 sections 37 and belongs to more than 200 kinds, and its fiber is wrapped in the cane or blade of plant more.These natural herb fibrous materials not only comprise the nonbonding mould assembly material that a large amount of sugar degrees are extremely low, the epidermis very high as the keratinization degree, and contain 20%-30% complicated component and with the non-glucide of Mierocrystalline cellulose bonding, as pectin, xylogen etc., be difficult to adopt the simple method saccharification.Aspect extraction draft Mierocrystalline cellulose, traditional natural bubble system method exist the cycle long, be not suitable for scale operation, polluted air, influence problem such as water industry development, conventional chemical comes unstuck or pulping process exist to consume a large amount of industrial chemicals and problems such as the energy, damage fiber production and quality, serious environment pollution, has restricted the development that the draft fiber development utilizes industry.For this reason, extensively carry out the research of " biological degumming " and " bio-pulping " technology both at home and abroad.Because complicated component, the sound construction of non-cellulose material in the draft fibrous materials such as crudefiber crop, and the difficult people's will to the greatest extent of the kind of production by biological enzyme and quantity, to such an extent as to the application cost of zymin is too high and do not know that because of the mechanism of degraded non-cellulose treatment effect is undesirable, the function of bacteria preparation is not strong and fermentation period is long partially, so, reach 80 years although carry out the biological process extraction with the research history made from extra care bast-fibre, but really reach the achievement of producing realistic scale, external report is also very rare.Trace it to its cause, exactly at the composition of draft fibrous material non-cellulose materials such as crudefiber crop and constructional feature and take the work of genetic engineering means orderly improvement bacterial classification to do very little.
3, the purposes of mannase is extensive day by day
'beta '-mannase (β-1,4-D-mannanohydrolase), be that a class can hydrolysis contain β-1, the restriction endonuclease of the mannosans of 4-D-seminose glycosidic bond (comprising mannosans, polygalactomannan, konjac glucomanna, gala konjac glucomanna etc.) belongs to the hemicellulose enzyme.Along with to the exploitation of nature hemicellulose resource and the discovery of mannooligo saccharide pharmaceutical use, the research and development of 'beta '-mannase has entered a new upsurge, and it finds broad application in many-sides such as food, feed, medicine, papermaking, textile printing and dyeing, oil production and biotechnology research.Microorganism is the important source that produces 'beta '-mannase.Reported that the common monoid that extracts 'beta '-mannase comprises genus bacillus, pseudomonas, the vibrios in the bacterium, the aspergillus in the fungi, wood are mould, yeast, mould, shuttle born of the same parents bacterium, pore fungus, sclerotinite and actinomycetic streptomycete etc.
(2) technical foundation of the present invention's formation
1, China has taken the lead in breaking through the technical barrier of biological degumming and bio-pulping
Hemp Inst., China Academy of Agricultural Sciences specialized in bast-fibre biological degumming and bio-pulping technical study in continuous 35 years, rich experience and data have been accumulated, large quantities of professional technique talents have been brought up, select the bacterial classification that strain more than 100 has specific function, research has formed 14 and has been in domestic and international top standard's scientific and technological achievement, has taken the lead in breaking through the technical barrier of bast-fibre biological degumming and careless class bio-pulping.Simultaneously, having created Ministry of Agriculture's crudefiber crop genetic improvement and engineered microbes emphasis open laboratory, national crudefiber crop comes unstuck and technical innovation platform such as slurrying microbial strains preservation center, Hunan Province's crudefiber crop genetic breeding and ramie product biological processing key lab, crudefiber crop processing biotechnological formulation pilot plant.
2, the relevant patented technology that forms before the present patent application
Hemp Inst., China Academy of Agricultural Sciences is as the patentee, and the patent of invention of having obtained the authorization comprises: (1) combined bacterial-chemical process for hemp degumming, the patent No.: ZL85103481; (2) the biological degumming of ramie process synthesis is controlled useless method and apparatus, the patent No.: ZL90105510.7; (3) biological degumming of ramie Technology and equipment, the patent No.: ZL95112564.8; (4) the draft fibre factory comes unstuck or slurrying high-effect bacterial preparation technology, the patent No.: ZL02108820.9.The patent of invention of having applied for (being contained in careful patent) comprising: (1) a kind of bluish dogbane biotechnological formulation preparation technology of coming unstuck, application number: 01145354.0; (2) a kind of high-efficiency clean kenaf bast bio-pulping process, the application Chinese invention patent is also announced publication number: CN1451815A; (3) Herba Poae Sphondylodis under the batch production condition/kenaf bast bio-pulping process, the application Chinese invention patent is also announced publication number: CN1517486A; (4) efficient low-consume clean wheat straw bio-pulping process, the application Chinese invention patent has also entered the substantive examination stage, application number: 200410046963.1.
3, genetic engineering means improvement Erwinia is obtained remarkable break-throughs
Obtain national inventing patent--on the basis of biological degumming of ramie Technology and equipment, adopt genetic engineering means that the T85-260 bacterial strain is improved, obtained an Erwinia dissociant CXJZ95-198 who non-cellulose degraded in the draft fibrous material is had high efficiency and broad spectrum.
Recent research result shows that the high-effect bacterial of this bacterial strain being made the alternative existing patented technology preparation of liquid microbial inoculum is very good in the effect that factory uses; The work of 'beta '-mannase enzyme reaches 99.4U/ml in the mensuration 7.5h fermented liquid, to extract fermented liquid that the CXJZ microbial inoculum time-division flows out through ultrafiltration → saltout → processing of organic solvent separations → column chromatography purification supervisor, obtain the 'beta '-mannase sample, the activity of purifying 'beta '-mannase sample reaches 1490U/ml, therefore, formed and adopted international advanced key processing unit, prepared the technology of coming unstuck simultaneously with slurrying high-effect bacterial and 'beta '-mannase with the shunting of CXJZ bacterial strain fermentation liquor.This technology has not only reduced come unstuck production cost with the slurrying microbial inoculum of draft fibrous material batch production, and shows that utilizing same fermented liquid to prepare mannase has active high, obvious advantages such as cost is low, steady sources, extraction are convenient.
4, compare with the capability and performance of domestic and international similar technical products
(1) the draft fibrous material comes unstuck or the efficient liquid microbial inoculum of slurrying
Both at home and abroad about crudefiber crop come unstuck biotechnological formulation the research report seldom, India Bhattacharyya S.K. (1992) has reported with 4 kinds of bacterium mixed cultures and had handled jute 7 days that Bangladesh Mohiuddin G.Shamsul Haque (1998) has reported that utilizing fungi to cross culture (crude enzyme liquid) processing hard bark (incomplete base portion comes unstuck) can reach the purpose of coming unstuck in 5 days.The draft fibrous material comes unstuck or the slurrying high-effect bacterial only need be handled 7h and can reach and come unstuck or the purpose of slurrying, time much shorter not only, and also it is also much more to handle raw material type, and promptly this project product has significantly " high efficiency " and " broad spectrum " advantage.
(2) mannase preparation
Many far away from amylase, cellulase, zytase about the research report of mannase both at home and abroad, the report that especially drops into suitability for industrialized production still less.
This project of table 1 mannase preparation performance reports that with representative the result relatively both at home and abroad
| Sequence number | The mannosans enzyme source | Fermentation period | Fermentation broth enzyme is lived | Refining enzyme is lived |
| l | Erwinia | 7h | 170u／ml | 2000u／ml |
| ck-1 | Pichia yeast genetic engineering bacteria (HBM047) | 62h | l?000u／ml | |
| ck-2 | Alkaliphilic bacillus | 46h | 500u／ml | |
| ck-3 | Aspergillus?niger | 96h | 150u／ml | |
| ck-4 | Bacilluss．1icheniformis | 36h | 260u／ml |
The mannase preparation that this project is developed is the vigor height not only, and fermentation period is extremely short, and promptly production cost reduces significantly.
5, the quasi-solution practical problems of determining
(1) the centralized preparation microbial inoculum is to reduce the technical difficulty of implementing biological degumming and bio-pulping process
Research in 1985 form first-generation ramie biological degumming technology one by one combined bacterial-chemical process for hemp degumming once applied in 5 tame enterprises.Causing one of reason of stopping production afterwards is because enterprise is difficult to grasp the strain preparation technology, and especially the technical problem of spawn degeneration or variation is difficult to solve.In fact, the successful precedent that does not have biological degumming technology to be used for scale operation abroad can be used for reference, and domestic crudefiber crop is come unstuck and careless class slurrying enterprise still adopts chemical process mostly, therefore, implement biological degumming and bio-pulping process, strain preparation is not a restrictive factor unavoidably.The present invention adopts the centralized preparation microbial inoculum to offer enterprise and uses the technical difficulty that can reduce enterprise implement biological degumming and bio-pulping process significantly.
(2) same broth extraction thalline and secretory product thereof are to reduce the product tooling cost
The research of seed selection mannase superior strain starting both at home and abroad than later, effect is fewer, so, although the market outlook of mannase are fine, to such an extent as to owing to tooling cost is more slow than higher industry development.Simultaneously, with respect to industries such as weaving, papermaking, crudefiber crop is come unstuck and careless class slurrying is a roughing process of extracting fiber from the draft fibrous material, if the high-effect bacterial production cost is too high, must cause finished product to lack the competitive power of market value aspect.The present invention is according to the market requirement and existing rare resources, adopts international advanced key processing unit to shunt same broth extraction thalline and secretory product thereof, can reduce come unstuck tooling cost with careless class slurrying special bacteria agent and mannase of crudefiber crop significantly.
Three, summary of the invention
(1) overall technical architecture
The present invention adopts international advanced key processing unit, as automatic control fermentor tank, ultrafiltration, column chromatography device etc., after the fermented liquid of Erwinia dissociant CXJZ95-198 shunted, is refined into efficient liquid microbial inoculum and mannase respectively.Technical scheme comprises original seed activation, first order seed, secondary seed, fermentation, proteolytic enzyme inhibition, roughing out, micro-filtration, ultrafiltration, saltouts, dissolved, chromatography, washing, prescription, vacuum-packed operation and concentrate efficient liquid microbial inoculum and mannosans enzyme product.
(2) concrete summary of the invention
1, original seed activation
The original seed activation is got the CXJZ95-198 colonies typical and is inoculated into common glucose nutrient broth medium, obtains kind of a daughter bacteria liquid at 120r/min-180r/min, 33 ℃-35 ℃ following shaking culture 5.0h-5.5h.
2, first order seed is cultivated
To activate CXJZ95-198 kind daughter bacteria liquid and be inoculated into the laboratory with in the ventilating/stirring fermentor tank, substratum disposes with tap water, prescription is: the concentration of glucose, Rhizoma amorphophalli powder, peptone, extractum carnis, NaCl is followed successively by 0.5%, 0.2%, 0.5%, 0.5%, 0.5%, nature pH value, inoculum size 2%, 31 ℃-35 ℃ of temperature, air flow is 0.2dm
3/ L-0.8dm
3/ L, stir speed (S.S.) is 180r/min-260r/min, incubation time 5-7h.
3, secondary seed is cultivated
The first order seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.0%, 0.2%, 0.05%, 0.05%, 0.05%, natural pH value, and inoculum size 2%, 31 ℃-35 ℃ of temperature, air flow is 0.2dm
3/ L-0.8dm
3/ L, stir speed (S.S.) is 180r/min-260r/min, incubation time 5-7h.
4, fermentation
The secondary seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.5%, 0.3%, 0.05%, 0.05%, 0.05%, natural pH value, and 31 ℃-35 ℃ of temperature, air flow is 0.4dm
3/ L-1.2dm
3/ L, stir speed (S.S.) is 180r/min-220r/min, inoculum size 5%, fermentation 7-8h is more than the fermented liquid mannosans enzyme activity 180U/ml.
5, proteolytic enzyme suppresses
In fermented liquid, add an amount of proteinase inhibitor during fermentation ends, to stop fermentation and protection extracellular enzyme activity.
6, roughing out
Sophisticated fermented liquid adopts 5 μ m coarse filtration films to carry out solid-liquid separation, discards the solid substance greater than 5 μ m.
7, micro-filtration
With 0.2 μ m microfiltration membrane the liquid phase shunting thing less than 5 μ m is further separated, and concentrate doubly greater than 0.2 μ m shunting thing 10-15.
8, washing
The physiological saline that adopts an amount of 8.5g/L until no soluble saccharide material, obtains to concentrate Black Liquor with Efficient Bacteria liquid to carrying out repetitive scrubbing greater than 0.2 μ m solid substance.
(above 7-8 is collectively referred to as " it is refining to concentrate Black Liquor with Efficient Bacteria liquid ")
9, ultrafiltration
Adopt the film bag of different molecular weight 100kD-10kD that the liquid phase shunting thing less than 0.2 μ m is carried out ultrafiltration and concentration, cycles of concentration is 15-25 times.
10, saltout
Adopt the ammonium sulfate precipitation method that molecular weight is separated acquisition mannase liquid at the protein of 100kD-10kD, the scope of ammonium sulfate saturation ratio is: 45%-70%.
11, dissolved
Adopt acetone precipitation to be further purified the ammonium sulfate precipitation thing, acetone to the proportional range of mannase liquid is: 1.2: 1-1.8: 1.
12, chromatography
Adopt Sephadex G-100 gel chromatography column to be further purified the mannase liquid that acetone precipitation obtains, finally obtain the pure mannase liquid of chromatography.
(above 9-12 is collectively referred to as " mannase liquid is refining ")
13, prescription
Adding an amount of conventional preservatives, additive is protected concentrating Black Liquor with Efficient Bacteria liquid and the pure mannase liquid of chromatography.
14, vacuum packaging
Concentrated Black Liquor with Efficient Bacteria liquid and the pure mannase liquid of chromatography behind the prescription are sub-packed in corresponding container, add a cover and the plastics vacuum packaging bag of packing into, adopt Vacuum Packaging Machine to bleed, seal.
15, concentrate efficient liquid microbial inoculum
Concentrate efficient liquid microbial inoculum and be pistac liquid, tasteless, the viable bacteria amount reaches 10
10Cfu/ml is applicable to that ramie, bluish dogbane, flax, kendir, jute carry out biological degumming and Herba Poae Sphondylodis, kenaf bast, wheat straw, offal and carry out the biological saccharification complete processing that bio-pulping and draft fibrous material are produced alcohol.
16, mannase
The concentrated liquid mannase is brown or tan liquid, has the special odor of acid, and enzyme activity is more than 2000U/ml, and bacteria containing amount≤10000cfu/ml is widely used in industrial circles such as food, medicine, feed, papermaking, weaving, oil production.
(3) innovative point of the present invention
Take the lead in selecting the Erwinia dissociant CXJZ95-198 that draft fibrous material non-cellulose mass degradation is had high efficiency and broad spectrum at home and abroad, further investigation has formed the patent of invention of a series of places leading level in the world and has identified achievement thus.This project further utilizes this bacterial strain to produce mannase preparation, there is no report at home and abroad.The vigor of mannase preparation improves 1 times than the product that derives from engineering strain and drop into large-scale production.Simultaneously, the fermentation period that bacterial strain uses therefor produces enzyme only needs about 7h, and this does not at home and abroad all appear in the newspapers.Make with extra care out microbial inoculum and zymin respectively after adopting international advanced key processing unit that same fermented liquid is shunted, not only on the microbiological industry history, start the precedent of same broth extraction thalline and secretory product two series products thereof, and promoted the competitive power of microniological proudcts aspect production cost significantly.Main innovate point of the present invention comprises first order seed cultivation, secondary seed cultivation, fermentation, roughing out, concentrate that Black Liquor with Efficient Bacteria liquid is refining, mannase liquid process for refining link and concentrate efficient liquid microbial inoculum and mannosans enzyme product, and concrete intension is:
1, first order seed is cultivated
To activate CXJZ95-198 kind daughter bacteria liquid and be inoculated into the laboratory with in the ventilating/stirring fermentor tank, substratum disposes with tap water, prescription is: the concentration of glucose, Rhizoma amorphophalli powder, peptone, extractum carnis, NaCl is followed successively by 0.5%, 0.2%, 0.5%, 0.5%, 0.5%, nature pH value, inoculum size 2%, 31 ℃-35 ℃ of temperature, air flow is 0.2dm
3/ L-0.8dm
3/ L, stir speed (S.S.) is 180r/min-260r/min, incubation time 5-7h.
2, secondary seed is cultivated
The first order seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.0%, 0.2%, 0.05%, 0.05%, 0.05%, natural pH value, and inoculum size 2%, 31 ℃-35 ℃ of temperature, air flow is 0.2dm
3/ L-0.8dm
3/ L, stir speed (S.S.) is 180r/min-260r/min, incubation time 5-7h.
3, fermentation
The secondary seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.5%, 0.3%, 0.05%, 0.05%, 0.05%, natural pH value, and 31 ℃-35 ℃ of temperature, air flow is 0.4dm
3/ L-1.2dm
3/ L, stir speed (S.S.) is 180r/min-220r/min, inoculum size 5%, fermentation 7-8h is more than the fermented liquid mannosans enzyme activity 180U/ml.
4, roughing out
Sophisticated fermented liquid employing 5u slightly considers film and carries out solid-liquid separation, discards the solid substance greater than 5u.
5, concentrating Black Liquor with Efficient Bacteria liquid makes with extra care
With 0.2 μ m microfiltration membrane the liquid phase shunting thing less than 5 μ m is further separated, and concentrate doubly greater than 0.2 μ m shunting thing 10-15; The physiological saline that adopts an amount of 8.5g/L until no soluble saccharide material, obtains to concentrate Black Liquor with Efficient Bacteria liquid to carrying out repetitive scrubbing greater than 0.2 μ m solid substance.
6, mannase liquid is refining
Adopt the film bag of different molecular weight 100kD-10kD that the liquid phase shunting thing less than 0.2 μ m is carried out ultrafiltration and concentration, cycles of concentration is 15-25 times; Adopt the ammonium sulfate precipitation method that molecular weight is separated acquisition mannase liquid at the protein of 100kD-10kD, the scope of ammonium sulfate saturation ratio is: 45%-70%; Adopt acetone precipitation to be further purified the ammonium sulfate precipitation thing, acetone to the proportional range of mannase liquid is: 1.2: 1-1.8: 1 adopts Sephadex G-100 gel chromatography column to be further purified the mannase liquid that acetone precipitation obtains, and finally obtains the pure mannase liquid of chromatography.
7, concentrate efficient liquid microbial inoculum
Concentrate efficient liquid microbial inoculum and be pistac liquid, tasteless, the viable bacteria amount reaches 10
10Cfu/ml is applicable to that ramie, bluish dogbane, flax, kendir, jute carry out biological degumming and Herba Poae Sphondylodis, kenaf bast, wheat straw, offal and carry out the biological saccharification complete processing that bio-pulping and draft fibrous material are produced alcohol.
8, mannase
The concentrated liquid mannase is brown or tan liquid, has the special odor of acid, and enzyme activity is more than 2000U/ml, and bacteria containing amount≤10000cfu/ml is widely used in industrial circles such as food, medicine, feed, papermaking, weaving, oil production.
Four, description of drawings
The 1--original seed; The 2--activation; The 3--first order seed; The 4--secondary seed; The 5--fermentation; 6--proteolytic enzyme suppresses; The 7--roughing out; The 8--micro-filtration; The 9--ultrafiltration; 10--saltouts; The 11--dissolved; The 12--chromatography; The 13--washing; The 14--prescription; The 15--vacuum packaging; The 16--microbial inoculum; The 17--mannase.
Five, embodiment
(1) technical scheme
Processing steps (operation) such as the technical scheme that the present invention relates to comprises original seed activation, seed culture, fermentation, proteolytic enzyme inhibition, roughing out, micro-filtration, ultrafiltration, saltouts, dissolved, column chromatography, prescription, packing.
1, original seed activation
The original seed activation is got the CXJZ95-198 colonies typical and is inoculated into common glucose nutrient broth medium, obtains kind of a daughter bacteria liquid at 120r/min-180r/min, 33 ℃-35 ℃ following shaking culture 5.0h-5.5h.
2, first order seed is cultivated
To activate CXJZ95-198 kind daughter bacteria liquid and be inoculated into the laboratory with in the ventilating/stirring fermentor tank, substratum disposes with tap water, prescription is: the concentration of glucose, Rhizoma amorphophalli powder, peptone, extractum carnis, NaCl is followed successively by 0.5%, 0.2%, 0.5%, 0.5%, 0.5%, nature pH value, inoculum size 2%, 31 ℃-35 ℃ of temperature, air flow is 0.2dm
3/ L-0.8dm
3/ L, stir speed (S.S.) is 180r/min-260r/min, incubation time 5-7h.
3, secondary seed is cultivated
The first order seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.0%, 0.2%, 0.05%, 0.05%, 0.05%, natural pH value, and inoculum size 2%, 31 ℃-35 ℃ of temperature, air flow is 0.2dm
3/ L-0.8dm
3/ L, stir speed (S.S.) is 180r/min-260r/min, incubation time 5-7h.
4, fermentation
The secondary seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.5%, 0.3%, 0.05%, 0.05%, 0.05%, natural pH value, and 31 ℃-35 ℃ of temperature, air flow is 0.4dm
3/ L-1.2dm
3/ L, stir speed (S.S.) is 180r/min-220r/min, inoculum size 5%, fermentation 7-8h is more than the fermented liquid mannosans enzyme activity 180U/ml.
5, proteolytic enzyme suppresses
In fermented liquid, add an amount of proteinase inhibitor during fermentation ends, to stop fermentation and protection extracellular enzyme activity.
6, roughing out
Sophisticated fermented liquid adopts 5 μ m coarse filtration films to carry out solid-liquid separation, discards the solid substance greater than 5 μ m.
7, micro-filtration
With 0.2 μ m microfiltration membrane the liquid phase shunting thing less than 5 μ m is further separated, and concentrate doubly greater than 0.2 μ m shunting thing 10-15.
8, washing
The physiological saline that adopts an amount of 8.5g/L until no soluble saccharide material, obtains to concentrate Black Liquor with Efficient Bacteria liquid to carrying out repetitive scrubbing greater than 0.2 μ m solid substance.
9, ultrafiltration
Adopt the film bag of different molecular weight 100kD-10kD that the liquid phase shunting thing less than 0.2 μ m is carried out ultrafiltration and concentration, cycles of concentration is 15-25 times.
10, saltout
Adopt the ammonium sulfate precipitation method that molecular weight is separated acquisition mannase liquid at the protein of 100kD-10kD, the scope of ammonium sulfate saturation ratio is: 45%-70%.
11, dissolved
Adopt acetone precipitation to be further purified the ammonium sulfate precipitation thing, acetone to the proportional range of mannase liquid is: 1.2: 1-1.8: 1.
12, chromatography
Adopt Sephadex G-100 gel chromatography column to be further purified the mannase liquid that acetone precipitation obtains, finally obtain the pure mannase liquid of chromatography.
13, prescription
Adding an amount of conventional preservatives, additive is protected concentrating Black Liquor with Efficient Bacteria liquid and the pure mannase liquid of chromatography.
14, vacuum packaging
Concentrated Black Liquor with Efficient Bacteria liquid and the pure mannase liquid of chromatography behind the prescription are sub-packed in corresponding container, add a cover and the plastics vacuum packaging bag of packing into, adopt Vacuum Packaging Machine to bleed, seal.
(2) experimental size and place
Present embodiment carries out under laboratory condition, and fermentation is the 12L/ jar in batches; Experiment place: Hemp Inst., China Academy of Agricultural Sciences.
(3) implementation result
1, the draft fibrous material comes unstuck or the concentrated efficient liquid microbial inoculum of slurrying
Employing 16L controls fermentor tank automatically and technique scheme is tested more than 200 time, produces efficient liquid microbial inoculum 200kg nearly, and promptly every jar of fermented liquid (12L) can be produced efficient liquid microbial inoculum 1kg.The product basic specification is as shown in table 2.These microbial inoculums are used for ramie under the batch production condition, flax, the test of bluish dogbane biological degumming in a large number except the bio-pulping technical study that is used for kenaf bast, wheat straw in the laboratory.Batch production application test result demonstration is come unstuck with conventional chemical or pulping process compares, and 1. the caustic soda consumption reduces more than 70%; 2. power consumption is saved more than 20%; 3. screened yield improves 5 percentage points; 4. industrial comprehensive wastewater water quality is single, directly enters bio-oxidation and handles and be easy to up to standardly, and cost of sewage disposal saves about 60%.
The basic specification of table 2 microbial inoculum
| Product hierarchy | Outward appearance | Smell | Viable bacteria amount (cfu/ml) | Viable bacteria storage rate (%) |
| The top grade product | Pistac liquid | Tasteless | 10 10 | 100 |
| |
10 10 | 95 |
2, mannase
Utilize the liquid state shunting thing of producing efficient liquid microbial inoculum to carry out the refining research of mannase, test-results shows that in the CXJZ95-198 bacterial strain 7h fermented liquid, the mannosans enzyme activity reaches more than the 180u/ml; Adopt technique scheme to make with extra care out mannase from the liquid state shunting thing of producing efficient liquid microbial inoculum, production efficiency is 500ml mannase/10L fermented liquid.The product basic specification is as shown in table 2.
The basic specification of table 2 mannase preparation
| Product hierarchy | Outward appearance | Smell | Enzyme activity (u/ml) | Vigor storage rate (%) | Contain bacterium (cfu/ml) |
| The top grade product | Brown or tan liquid | Special odor with acid, free from extraneous odour | 2000 | 100 | ≤10000 |
| Salable product | 2000 | 95 | ≤50000 |
Claims (3)
1. technology of utilizing Erwinia dissociant CXJZ95-198 to extract 'beta '-mannase may further comprise the steps:
(1) first order seed is cultivated: Erwinia dissociant CXJZ95-198 activation bacterium liquid is inoculated into the laboratory with in the ventilating/stirring fermentor tank, substratum disposes with tap water, prescription is glucose, Rhizoma amorphophalli powder, peptone, extractum carnis, NaCl, concentration is followed successively by 0.5%, 0.2%, 0.5%, 0.5%, 0.5%, nature pH value, inoculum size 2%, 31 ℃～35 ℃ of temperature, air flow is 0.2dm
3/ L～0.8dm
3/ L, stir speed (S.S.) is 180r/min～260r/min, incubation time 5～7h;
(2) secondary seed is cultivated: the first order seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.0%, 0.2%, 0.05%, 0.05%, 0.05%, natural pH value, and inoculum size 2%, 31 ℃～35 ℃ of temperature, air flow is 0.2dm
3/ L～0.8dm
3/ L, stir speed (S.S.) is 180r/min～260r/min, incubation time 5～7h;
(3) fermentation: the secondary seed nutrient solution is inoculated in the common ventilating/stirring fermentor tank, and substratum disposes with tap water, and prescription is: soybean cake powder, Rhizoma amorphophalli powder, NH
4H
2PO
4, K
2HPO
4, MgSO
47H
2The concentration of O is followed successively by 1.5%, 0.3%, 0.05%, 0.05%, 0.05%, natural pH value, and 31 ℃～35 ℃ of temperature, air flow is 0.4dm
3/ L～1.2dm
3/ L, stir speed (S.S.) is 180r/min～220r/min, inoculum size 5%, fermentation 7～8h is more than the fermented liquid beta-mannase enzyme activity 180U/ml;
(4) roughing out: adopt 5 μ m coarse filtration films to carry out solid-liquid separation sophisticated fermented liquid, discard solid substance greater than 5 μ m;
(5) concentrating Black Liquor with Efficient Bacteria liquid makes with extra care: with 0.2 μ m microfiltration membrane the liquid phase shunting thing less than 5 μ m is further separated, and concentrate greater than 10～15 times of 0.2 μ m shunting things; The physiological saline that adopts an amount of 8.5g/L until no soluble saccharide material, obtains to concentrate Black Liquor with Efficient Bacteria liquid to carrying out repetitive scrubbing greater than 0.2 μ m solid substance;
(6) 'beta '-mannase liquid is refining: adopt the film bag of different molecular weight 100kD～10kD that the liquid phase shunting thing less than 0.2 μ m is carried out ultrafiltration and concentration, cycles of concentration is 15～25 times; Adopt the ammonium sulfate precipitation method that molecular weight is separated acquisition 'beta '-mannase liquid at the protein of 100kD～10kD, the scope of ammonium sulfate saturation ratio is 45%～70%; Adopt acetone precipitation to be further purified the ammonium sulfate precipitation thing, acetone is 1.2: 1～1.8: 1 to the proportional range of 'beta '-mannase liquid; Adopt the SephadexG-100 gel chromatography column to be further purified mannase liquid, finally obtain the pure 'beta '-mannase liquid of chromatography.
2. a kind of technology of utilizing Erwinia dissociant CXJZ95-198 to extract 'beta '-mannase as claimed in claim 1, described concentrated Black Liquor with Efficient Bacteria liquid is pistac liquid, and is tasteless, and the viable bacteria amount reaches 10
10Cfu/ml.
3. a kind of technology of utilizing Erwinia dissociant CXJZ95-198 to extract 'beta '-mannase as claimed in claim 1, the pure 'beta '-mannase liquid of described chromatography is brown or brown liquid, special odor with acid, enzyme activity more than 2000U/ml, bacteria containing amount≤10000cfu/ml.
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| CN2005100324976A CN1978636B (en) | 2005-12-05 | 2005-12-05 | Process for extracting beta-mannanase utilizing Erwinia |
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| CN1978636B true CN1978636B (en) | 2011-09-21 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266096A (en) * | 1999-03-05 | 2000-09-13 | 中国科学院微生物研究所 | Method for producing alkaline 'beta'-mannase |
| WO2004113538A1 (en) * | 2003-06-26 | 2004-12-29 | Ctc Bio, Inc. | Gene coding mannanase and recombinant mannanase expressed from transformant thereof |
-
2005
- 2005-12-05 CN CN2005100324976A patent/CN1978636B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266096A (en) * | 1999-03-05 | 2000-09-13 | 中国科学院微生物研究所 | Method for producing alkaline 'beta'-mannase |
| WO2004113538A1 (en) * | 2003-06-26 | 2004-12-29 | Ctc Bio, Inc. | Gene coding mannanase and recombinant mannanase expressed from transformant thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李宝坤，等.CXJZ95-198菌株在不同碳源中产β-甘露聚糖酶的规律研究.中国麻业27 1.2005,27(1),37-40. |
| 李宝坤,等.CXJZ95-198菌株在不同碳源中产β-甘露聚糖酶的规律研究.中国麻业27 1.2005,27(1),37-40. * |
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