CN107418949A - A kind of carrier material fixed for cellulase and preparation method thereof - Google Patents
A kind of carrier material fixed for cellulase and preparation method thereof Download PDFInfo
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- CN107418949A CN107418949A CN201710639092.1A CN201710639092A CN107418949A CN 107418949 A CN107418949 A CN 107418949A CN 201710639092 A CN201710639092 A CN 201710639092A CN 107418949 A CN107418949 A CN 107418949A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
- B01J13/043—Drying and spraying
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
Abstract
The invention discloses a kind of carrier material fixed for cellulase and preparation method thereof, comprise the following steps:Obtained polyacrylonitrile complex microsphere is added in DMAC, after soaking 4 6h, after addition lithium chloride is well mixed, add chitin and catalyst, after well mixed, nitrogen is passed through, is heated after 2 4h are stirred under normal temperature, temperature is controlled at 100 120 DEG C, the 24h of stirring reaction 18, after being down to room temperature, after washes of absolute alcohol, cleaned again with deionized water, vacuumizing and drying.The carrier material mechanical strength of the application is excellent, and stability is preferable, stronger with the adhesion of enzyme, and enzyme molecule is not easy to come off from carrier in use, and service life is longer.Present invention also provides the application method of the carrier material fixed for cellulase.
Description
Technical field
The present invention relates to enzymic preparation field, more particularly to a kind of carrier material fixed for cellulase and its preparation
Method.
Background technology
Cellulase is a kind of multi-component compound enzyme system, mainly including circumscribed 1,4 beta-glucanase, inscribe beta glucan
Enzyme and beta-glucosidase, by each component enzyme synergy by cellulose degradation into glucose.Each component of cellulase is big
Part is glycoprotein, and the glycosyl in structure has certain protective effect from protease hydrolytic to cellulase.Separate sources
Cellulase Molecules structure and folding mode are different, and their general character is generally by three parts structure composition:Spherical
Catalytic activity area with catalysis, the land with cellulose-binding function and connect their one end height glycosyl
The peptide acid of change.Current research thinks that cellulosic material will be digested effectively, needs cellulase being adsorbed in solid first
On substrate, substrate cleavage is then being hydrolyzed into cell-oligosaccharide until glucose into small fragment.
Enzyme immobilizatio is that enzyme is incorporated on solid material or is limited in certain space, remains to maintain vigour and special
Property, and a kind of technology that can be recycled.The preparation method of immobilised enzymes mainly has four kinds:Investment, cross-linking method, absorption
Method and covalent coupling method.Investment, which refers to be embedded in enzyme molecule in the three-D space structure of porous polymer materials, consolidates enzyme
Fixedization, this method preparation technology is simple, and influence of the formation condition gently and to enzymatic activity is relatively low, but the pore size of carrier
Diffusion that can be to each material in enzymatic reaction has an impact.Cross-linking method is to react to be formed altogether with amino acid residue using crosslinking agent
Valence link, enzyme molecule is set to be incorporated into network structure, so as to reach the effect of enzyme immobilization.Cross-linking method can make immobilised enzymes
Stability improves, and service life extends, but covalent cross-linking reduces the reaction site of enzyme, so as to destroy the activity of enzyme.Absorption
Method refers to that resolvase is incorporated on carrier by electrostatic interaction or hydrogen bond etc. to prepare the method for immobilised enzymes, and this method has can
The advantages such as inverse property, simplification and enzyme activity height, but because the interaction of enzyme and carrier is weaker, be easy to come off.Covalent bond
Method is that enzyme is fixed on suitable carrier by the method for Covalent bonding together, and enzyme produces chemical action with carrier, and adhesion is strong,
Enzyme slip is low, has preferable stability and recycles performance, but reacts fierce, easily loses enzyme activity.The above four
Kind method respectively has advantage and disadvantage, a variety of methods can be used to be used in mixed way to improve the use quality of immobilised enzymes.
Chinese patent CN201310674347.x discloses a kind of preparation method of cellulase immobilization carrier, including such as
Lower step:(1) charing of cellulose, cellulose is carbonized under the conditions of 400 DEG C -500 DEG C of inert gas shielding;(2) carrier
Modification, in the cellulose after charing, add ionic liquid and inorganic agent, stir, isothermal holding obtains cellulose carrier
Solution;(3) carrier drying is molded, and solvent is added in cellulose carrier solution, and the washing that stirs removes impurity, Ran Hougan
Cellulase immobilization carrier is made in dry, vacuum outgas.The invention has that carrier surface area is big, adsorption site is more, can be steady with enzyme
Surely the advantages that combining.
The content of the invention
It is an object of the invention to provide a kind of carrier material fixed for cellulase and preparation method thereof, the carrier material
Expect that mechanical strength is excellent, stability is preferable, stronger with the adhesion of enzyme, and enzyme molecule is not easy to take off from carrier in use
Fall, service life is longer.
It is a further object of the present invention to provide the application method that this is used for the carrier material that cellulase is fixed.
To achieve the above object, the present invention uses following technical scheme:
A kind of preparation method for the carrier material fixed for cellulase, comprises the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 20-45min, Zhi Hou
The mixture that glacial acetic acid and absolute ethyl alcohol are added dropwise in process is stirred vigorously, continues to stir 4-5h after being added dropwise to complete, stands 60- afterwards
72h, drying, Nano-meter SiO_2 is made after grinding2/Fe3O4Composite;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, ultrasonic disperse 1-2h, add
Silane coupler is uniformly mixed, and is sprayed afterwards, and polyacrylonitrile complex microsphere is made;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 4-6h, adds lithium chloride and mix
After closing uniformly, chitin and catalyst are added, after being well mixed, nitrogen is passed through, is heated after stirring 2-4h under normal temperature, control temperature
At 100-120 DEG C, stirring reaction 18-24h, after being down to room temperature, clean, take out true after washes of absolute alcohol, then with deionized water
Sky drying.
Preferably, nanometer Fe in the step (1)3O4The use quality of particle and the volume ratio of silester are 30-45g/
L。
Preferably, polyacrylonitrile and Nano-meter SiO_2 in the step (2)2/Fe3O4The mass ratio of composite is 4:1.
Preferably, the particle diameter of polyacrylonitrile complex microsphere prepared by the step (2) is 5 μm -7 μm.
Preferably, the usage amount mass ratio 5 of polyacrylonitrile complex microsphere and chitin in the step (3):28.
Preferably, catalyst is metallic sodium in the step (3).
A kind of carrier material fixed for cellulase, is prepared by above method.
A kind of immobilized cellulase, using it is described for cellulase fix carrier material as carrier, using glutaraldehyde as
Crosslinking agent is prepared.
The invention has the advantages that
The application is with nanometer Fe3O4Nano-meter SiO_2 is made for core2/Fe3O4Composite, composite tool are magnetic same
When there is more aperture, higher specific surface area, be a kind of mesoporous material of the magnetic carried.By Nano-meter SiO_22/Fe3O4It is multiple
Condensation material is the polyacrylonitrile complex microsphere that filler is prepared into, and the specific surface area of complex microsphere can be effectively improved, so as to carry
The high contact surface of polyacrylonitrile complex microsphere and cellulase, is relatively beneficial to improve load capacity of the cellulase on carrier,
The load fastness of cellulase is improved, it is difficult for drop-off in use, improve organic efficiency.
Embodiment
In order to be better understood from the present invention, below by embodiment, the present invention is further described, and embodiment is served only for solving
The present invention is released, any restriction will not be formed to the present invention.
Embodiment 1
A kind of preparation method for the carrier material fixed for cellulase, comprises the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 20min, nanometer Fe3O4
The use quality of particle and the volume ratio of silester are 30g/L, and glacial acetic acid and anhydrous is added dropwise in process is stirred vigorously afterwards
The mixture of ethanol, continue to stir 4h after being added dropwise to complete, stand 60h afterwards, dry, Nano-meter SiO_2 is made after grinding2/Fe3O4It is multiple
Condensation material;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, polyacrylonitrile and nanometer
SiO2/Fe3O4The mass ratio of composite is 4:1, ultrasonic disperse 1h, add silane coupler and be uniformly mixed, carry out afterwards
Spraying, polyacrylonitrile complex microsphere is made, the particle diameter of polyacrylonitrile complex microsphere is 5 μm -7 μm;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 4h, adds lithium chloride mixing
After uniformly, the usage amount mass ratio 5 of chitin and catalyst metals sodium, polyacrylonitrile complex microsphere and chitin is added:28, mix
After closing uniformly, nitrogen being passed through, is heated after stirring 2h under normal temperature, control temperature is at 100 DEG C, stirring reaction 18h, after being down to room temperature,
Cleaned after washes of absolute alcohol, then with deionized water, vacuumizing and drying.
Using the carrier material fixed for cellulase as carrier, immobilization fibre is prepared with glutaraldehyde as cross linker
Plain enzyme.
After tested, the immobilized cellulase prepared after 20 secondary responses its enzyme activity remain to keep original enzyme activity
The 80% of power.
Embodiment 2
A kind of preparation method for the carrier material fixed for cellulase, comprises the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 45min, nanometer Fe3O4
The use quality of particle and the volume ratio of silester are 45g/L, and glacial acetic acid and anhydrous is added dropwise in process is stirred vigorously afterwards
The mixture of ethanol, continue to stir 5h after being added dropwise to complete, stand 72h afterwards, dry, Nano-meter SiO_2 is made after grinding2/Fe3O4It is multiple
Condensation material;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, polyacrylonitrile and nanometer
SiO2/Fe3O4The mass ratio of composite is 4:1, ultrasonic disperse 2h, add silane coupler and be uniformly mixed, carry out afterwards
Spraying, polyacrylonitrile complex microsphere is made, the particle diameter of polyacrylonitrile complex microsphere is 5 μm -7 μm;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 6h, adds lithium chloride mixing
After uniformly, the usage amount mass ratio 5 of chitin and catalyst metals sodium, polyacrylonitrile complex microsphere and chitin is added:28, mix
After closing uniformly, nitrogen being passed through, is heated after stirring 4h under normal temperature, control temperature is at 120 DEG C, stirring reaction 24h, after being down to room temperature,
Cleaned after washes of absolute alcohol, then with deionized water, vacuumizing and drying.
Using the carrier material fixed for cellulase as carrier, immobilization fibre is prepared with glutaraldehyde as cross linker
Plain enzyme.
After tested, the immobilized cellulase prepared after 20 secondary responses its enzyme activity remain to keep original enzyme activity
The 82% of power.
Embodiment 3
A kind of preparation method for the carrier material fixed for cellulase, comprises the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 20min, nanometer Fe3O4
The use quality of particle and the volume ratio of silester are 45g/L, and glacial acetic acid and anhydrous is added dropwise in process is stirred vigorously afterwards
The mixture of ethanol, continue to stir 4h after being added dropwise to complete, stand 72h afterwards, dry, Nano-meter SiO_2 is made after grinding2/Fe3O4It is multiple
Condensation material;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, polyacrylonitrile and nanometer
SiO2/Fe3O4The mass ratio of composite is 4:1, ultrasonic disperse 1h, add silane coupler and be uniformly mixed, carry out afterwards
Spraying, polyacrylonitrile complex microsphere is made, the particle diameter of polyacrylonitrile complex microsphere is 5 μm -7 μm;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 6h, adds lithium chloride mixing
After uniformly, the usage amount mass ratio 5 of chitin and catalyst metals sodium, polyacrylonitrile complex microsphere and chitin is added:28, mix
After closing uniformly, nitrogen being passed through, is heated after stirring 2h under normal temperature, control temperature is at 120 DEG C, stirring reaction 18h, after being down to room temperature,
Cleaned after washes of absolute alcohol, then with deionized water, vacuumizing and drying.
Using the carrier material fixed for cellulase as carrier, immobilization fibre is prepared with glutaraldehyde as cross linker
Plain enzyme.
After tested, the immobilized cellulase prepared after 20 secondary responses its enzyme activity remain to keep original enzyme activity
The 89% of power.
Embodiment 4
A kind of preparation method for the carrier material fixed for cellulase, comprises the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 45min, nanometer Fe3O4
The use quality of particle and the volume ratio of silester are 30g/L, and glacial acetic acid and anhydrous is added dropwise in process is stirred vigorously afterwards
The mixture of ethanol, continue to stir 5h after being added dropwise to complete, stand 60h afterwards, dry, Nano-meter SiO_2 is made after grinding2/Fe3O4It is multiple
Condensation material;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, polyacrylonitrile and nanometer
SiO2/Fe3O4The mass ratio of composite is 4:1, ultrasonic disperse 2h, add silane coupler and be uniformly mixed, carry out afterwards
Spraying, polyacrylonitrile complex microsphere is made, the particle diameter of polyacrylonitrile complex microsphere is 5 μm -7 μm;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 4h, adds lithium chloride mixing
After uniformly, the usage amount mass ratio 5 of chitin and catalyst metals sodium, polyacrylonitrile complex microsphere and chitin is added:28, mix
After closing uniformly, nitrogen being passed through, is heated after stirring 4h under normal temperature, control temperature is at 100 DEG C, stirring reaction 24h, after being down to room temperature,
Cleaned after washes of absolute alcohol, then with deionized water, vacuumizing and drying.
Using the carrier material fixed for cellulase as carrier, immobilization fibre is prepared with glutaraldehyde as cross linker
Plain enzyme.
After tested, the immobilized cellulase prepared after 20 secondary responses its enzyme activity remain to keep original enzyme activity
The 88% of power.
Embodiment 5
A kind of preparation method for the carrier material fixed for cellulase, comprises the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 30min, nanometer Fe3O4
The use quality of particle and the volume ratio of silester are 40g/L, and glacial acetic acid and anhydrous is added dropwise in process is stirred vigorously afterwards
The mixture of ethanol, continue to stir 5h after being added dropwise to complete, stand 66h afterwards, dry, Nano-meter SiO_2 is made after grinding2/Fe3O4It is multiple
Condensation material;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, polyacrylonitrile and nanometer
SiO2/Fe3O4The mass ratio of composite is 4:1, ultrasonic disperse 2h, add silane coupler and be uniformly mixed, carry out afterwards
Spraying, polyacrylonitrile complex microsphere is made, the particle diameter of polyacrylonitrile complex microsphere is 5 μm -7 μm;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 5h, adds lithium chloride mixing
After uniformly, the usage amount mass ratio 5 of chitin and catalyst metals sodium, polyacrylonitrile complex microsphere and chitin is added:28, mix
After closing uniformly, nitrogen being passed through, is heated after stirring 3h under normal temperature, control temperature is at 110 DEG C, stirring reaction 20h, after being down to room temperature,
Cleaned after washes of absolute alcohol, then with deionized water, vacuumizing and drying.
Using the carrier material fixed for cellulase as carrier, immobilization fibre is prepared with glutaraldehyde as cross linker
Plain enzyme.
After tested, the immobilized cellulase prepared after 20 secondary responses its enzyme activity remain to keep original enzyme activity
The 85% of power.
Claims (8)
1. a kind of preparation method for the carrier material fixed for cellulase, it is characterised in that comprise the following steps:
(1) silester is dissolved in ethanol solution, adds nanometer Fe3O4Particle ultrasonic disperse 20-45min, afterwards violent
The mixture of glacial acetic acid and absolute ethyl alcohol is added dropwise in whipping process, continues to stir 4-5h after being added dropwise to complete, stands 60-72h afterwards,
Drying, Nano-meter SiO_2 is made after grinding2/Fe3O4Composite;
(2) polyacrylonitrile is dissolved in DMSO, adds Nano-meter SiO_22/Fe3O4Composite, ultrasonic disperse 1-2h, add silane
Coupling agent is uniformly mixed, and is sprayed afterwards, and polyacrylonitrile complex microsphere is made;
(3) polyacrylonitrile complex microsphere made from step (2) is added in DMAC, after soaking 4-6h, it is equal adds lithium chloride mixing
After even, chitin and catalyst are added, after being well mixed, nitrogen is passed through, is heated after stirring 2-4h under normal temperature, control temperature exists
100-120 DEG C, stirring reaction 18-24h, after being down to room temperature, clean, vacuumize after washes of absolute alcohol, then with deionized water
Drying.
2. the preparation method of the carrier material according to claim 1 fixed for cellulase, it is characterised in that:It is described
Nanometer Fe in step (1)3O4The use quality of particle and the volume ratio of silester are 30-45g/L.
3. the preparation method of the carrier material according to claim 1 fixed for cellulase, it is characterised in that:It is described
Polyacrylonitrile and Nano-meter SiO_2 in step (2)2/Fe3O4The mass ratio of composite is 4:1.
4. the preparation method of the carrier material according to claim 1 fixed for cellulase, it is characterised in that:It is described
The particle diameter of polyacrylonitrile complex microsphere prepared by step (2) is 5 μm -7 μm.
5. the preparation method of the carrier material according to claim 1 fixed for cellulase, it is characterised in that:It is described
The usage amount mass ratio 5 of polyacrylonitrile complex microsphere and chitin in step (3):28.
6. the preparation method of the carrier material according to claim 1 fixed for cellulase, it is characterised in that:It is described
Catalyst is metallic sodium in step (3).
A kind of 7. carrier material fixed for cellulase, it is characterised in that:As the method described in claim any one of 1-6
It is prepared.
A kind of 8. immobilized cellulase, it is characterised in that:With the carrier material for being used for cellulase and fixing described in claim 7
Expect for carrier, to be prepared with glutaraldehyde as cross linker.
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CN109315795A (en) * | 2018-11-27 | 2019-02-12 | 湖北文理学院 | A kind of method of enzyme process joint infra-red drying removal walnut kernel pellicle |
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