CN108740997A - A kind of preparation method of protease chitosan microball - Google Patents
A kind of preparation method of protease chitosan microball Download PDFInfo
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- CN108740997A CN108740997A CN201810829618.7A CN201810829618A CN108740997A CN 108740997 A CN108740997 A CN 108740997A CN 201810829618 A CN201810829618 A CN 201810829618A CN 108740997 A CN108740997 A CN 108740997A
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- Prior art keywords
- protease
- chitosan
- chitosan microball
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- microball
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 84
- 239000011806 microball Substances 0.000 title claims abstract description 57
- 239000004365 Protease Substances 0.000 title claims abstract description 50
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 49
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000007711 solidification Methods 0.000 claims abstract description 16
- 230000008023 solidification Effects 0.000 claims abstract description 16
- 239000011259 mixed solution Substances 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 41
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000003431 cross linking reagent Substances 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 7
- 150000003863 ammonium salts Chemical class 0.000 claims description 7
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 235000019257 ammonium acetate Nutrition 0.000 claims description 6
- 229940043376 ammonium acetate Drugs 0.000 claims description 6
- 239000001099 ammonium carbonate Substances 0.000 claims description 6
- 235000012501 ammonium carbonate Nutrition 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 4
- IBFYXTRXDNAPMM-BVTMAQQCSA-N Geniposide Chemical group O([C@@H]1OC=C([C@@H]2[C@H]1C(=CC2)CO)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O IBFYXTRXDNAPMM-BVTMAQQCSA-N 0.000 claims description 4
- IBFYXTRXDNAPMM-FZEIBHLUSA-N Geniposide Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)[C@H]2[C@@H]1CC=C2CO IBFYXTRXDNAPMM-FZEIBHLUSA-N 0.000 claims description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 4
- VGLLGNISLBPZNL-RBUKDIBWSA-N arborescoside Natural products O=C(OC)C=1[C@@H]2C([C@H](O[C@H]3[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O3)OC=1)=C(CO)CC2 VGLLGNISLBPZNL-RBUKDIBWSA-N 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 229920002085 Dialdehyde starch Polymers 0.000 claims description 3
- 229910021389 graphene Inorganic materials 0.000 claims description 3
- 239000005543 nano-size silicon particle Substances 0.000 claims description 3
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical compound [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 claims description 3
- 235000012239 silicon dioxide Nutrition 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 239000003607 modifier Substances 0.000 claims description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 239000011805 ball Substances 0.000 claims 1
- 235000013601 eggs Nutrition 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 claims 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 claims 1
- 229910001948 sodium oxide Inorganic materials 0.000 claims 1
- 235000019419 proteases Nutrition 0.000 abstract description 31
- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 235000013305 food Nutrition 0.000 abstract description 9
- 102000018120 Recombinases Human genes 0.000 abstract description 7
- 108010091086 Recombinases Proteins 0.000 abstract description 7
- 102000035195 Peptidases Human genes 0.000 abstract description 6
- 230000003197 catalytic effect Effects 0.000 abstract description 6
- 235000019833 protease Nutrition 0.000 abstract description 6
- 238000004132 cross linking Methods 0.000 abstract description 5
- 238000007796 conventional method Methods 0.000 abstract description 3
- 238000003860 storage Methods 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 28
- 108090000790 Enzymes Proteins 0.000 description 28
- 229940088598 enzyme Drugs 0.000 description 28
- 230000000694 effects Effects 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 108090000526 Papain Proteins 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 11
- 102000002322 Egg Proteins Human genes 0.000 description 11
- 108010000912 Egg Proteins Proteins 0.000 description 11
- 235000014103 egg white Nutrition 0.000 description 11
- 210000000969 egg white Anatomy 0.000 description 11
- 230000009514 concussion Effects 0.000 description 9
- 235000019441 ethanol Nutrition 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 7
- 238000009792 diffusion process Methods 0.000 description 7
- 235000011121 sodium hydroxide Nutrition 0.000 description 7
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 6
- 239000004088 foaming agent Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 235000019834 papain Nutrition 0.000 description 5
- 229940055729 papain Drugs 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000004005 microsphere Substances 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 239000004971 Cross linker Substances 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 239000008236 heating water Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000004408 titanium dioxide Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108010004032 Bromelains Proteins 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- 235000019835 bromelain Nutrition 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 2
- 229940012466 egg shell membrane Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 108010059881 Lactase Proteins 0.000 description 1
- 101710089165 Protein white Proteins 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229940116108 lactase Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 230000002522 swelling effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/015—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Microbiology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of preparation method of protease chitosan microball, it includes that prepared by chitosan/modifying agent mixed solution, solidification, crosslinking, protease are fixed.Prepared by the present invention is a kind of mutual flow-through macropore chitosan microball of immobilization proteinase, the efficiency hydrolyzed compared to protease chitosan microball catalytic proteins prepared by conventional method increases 200%, it can be digested with efficient catalytic food, while the thermal stability of the immobilization proteinase, pH stability, storage stability etc. are above resolvase.
Description
Technical field
The present invention relates to a kind of preparation methods of protease chitosan microball.
Background technology
Protease is widely used in food-processing industry, holds both at home and abroad generally using addition floating preteins enzyme at present
Being vulnerable to environment influences deactivation, and environmental stability is poor, and not reproducible utilization.In order to improve the practical application of protease
Effect, many novel enzyme immobilization technologies are studied and report, such as using nano material as carrier, can effectively improve enzyme
Immobilization content and enzyme activity.But these novel nano-materials are difficult to be separated and recovered in food system, carrier residual is easy
Cause potential safety problem, so the fixed enzyme vector that can be applied to food processing is very limited.Large scale microballoon is one
The fixed enzyme vector that kind is easily isolated, can be advantageously applied to food service industry, microsphere immobilized glycosidase, lactase etc. are
Through being used in food processing, but all it is reaction substrate small molecule in these food systems, immobilization proteinase can not also
Realize the application in food.
Currently, protease is efficiently immobilized in the problem that world wide is still urgently to be resolved hurrily, because of the catalysis of protease
Substrate is macromolecular, and substrate has the problems such as diffusion is slow, and mass transfer is poor in the reaction system, so the hair of albumen enzyme immobilization technology
Exhibition is constantly subjected to limit.Some patented technologies prepare chitosan microball using glutaraldehyde as cross linker, for bromelain
Immobilization.But the chitosan microball three-dimensional network prepared by this method is fine and close, the free space of enzyme molecule is small, protein bottom
Object diffusion rate is low, therefore catalytic performance is poor.
Invention content
The object of the present invention is to provide a kind of preparation method of protease chitosan microball, the controllable hole which has
The mutual flow-through macropore of diameter is suitble to proteins diffusion, obtained immobilization proteinase to have very high catalytic activity, can efficiently urge
Helping digestion product digest.
To achieve the above object, method provided by the invention includes the following steps：
1) chitosan, ammonium salt, modifying agent are added in weak acid solution, it is 1-8%, amounts of ammonium salt that chitosan content, which is made,
The mixed solution for being 0.2-6% for 0.01-0.2mol/L, modifier content；
2) mixed solution is added drop-wise in the solidification liquid containing carbonate, so that chitosan is cured reaction, precipitation is washed
Microballoon is obtained after washing；
3) microballoon is added in cross-linking agent solution, microballoon is made to crosslink reaction, washed, it is poly- to obtain mutual flow-through macropore shell
Sugared microballoon；
4) after dissolving protease, mutual flow-through macropore chitosan microball is then added, is shaken under cryogenic conditions, makes protease
Be carried on mutual flow-through macropore chitosan microball to get.
Preferably, the weak acid is acetic acid, carbonic acid, citric acid, and the weight concentration of the weak acid solution is 0.1-5%.
Preferably, the modifying agent is membranes of fowl eggshells powder or nano silicon dioxide or nano-titanium dioxide or nano-graphite
Alkene.
Preferably, the ammonium salt is ammonium acetate or ammonium carbonate.
Preferably, the solidification liquid is the 10-40% ethanol solutions containing sodium hydroxide；The sodium hydroxide it is a concentration of
100-200g/L；
Preferably, a concentration of 0.001-0.1mol/L of the carbonate in solidification liquid.
Preferably, the crosslinking agent is Geniposide or glutaraldehyde or dialdehyde starch.
Preferably, the mass ratio of the protease and mutual flow-through macropore chitosan microball is 5-100mg：2g.
Protease chitosan microball prepared by the present invention has the following advantages：
1) during preparing chitosan microball, ammonium salt and carbonic acid are added in chitosan solution and solidification liquid respectively
Salt is as pore-foaming agent, and during microballoon is formed, both pore-foaming agents occur chemical reaction and generate ammonia and titanium dioxide respectively
Carbon gas, two kinds of gases from the inside and outside impact microsphere matrices of microballoon, form the hole of micron level, and hole respectively
Diameter is uniform in size, and intercommunity is good.It is formed by the diffusion that duct is conducive to protein molecule, to increase protein and albumen
The effective reaction space of enzyme, while being conducive to the mass transfer of enzymolysis product, it avoids clogging.Pass through adding for two kinds of pore-foaming agents of control
Dosage, can be with adjustment aperture size.
2) cross-linking reaction is carried out to chitosan microball, it is therefore an objective to make the mutual unicom of the hole on microballoon, be substrate protein
Diffusion mass transfer provides more sufficient space, while can also reduce the swelling action of chitosan microball, further increases microballoon
Mechanical performance provides sufficient covalent bond site for immobilised enzymes, plays the role of linking arm.And add in chitosan microball
The modifying agent entered can improve the mechanical performance and durability of microballoon.
3) what prepared by the present invention is a kind of mutual flow-through macropore chitosan microball of immobilization proteinase, is prepared compared to conventional method
Protease chitosan microball catalytic proteins hydrolysis efficiency increase 200%, can digest with efficient catalytic food, simultaneously this
Thermal stability, pH stability, storage stability of immobilization proteinase etc. are above resolvase.
4) present invention also have many advantages, such as high mechanical strength, be easy to degrade and bio-compatible it is good, and low production cost,
It is easy to operate, preparation process is environmentally protective, can be used for immobilized papain, bromelain, trypsase, rotten albumen
Enzyme, neutral proteinase and alkali protease etc..
Description of the drawings
Fig. 1 is the surface sweeping electron microscope of protease chitosan microball prepared by embodiment 1-4.
Fig. 2 is the electrophoretogram that protease chitosan microball prepared by embodiment 1-4 digests 100% egg white solution.
Fig. 3 is the thermal adaptability of protease chitosan microball and resolvase prepared by embodiment 1.
Fig. 4 is the pH adaptability of protease chitosan microball and resolvase prepared by embodiment 2.
The opposite enzyme activity that the protease chitosan microball that Fig. 5 is prepared by embodiment 3 retains after recycling repeatedly.
Fig. 6 is after protease chitosan microball prepared by embodiment 4 handles different time with resolvase under the conditions of 60 DEG C
The opposite enzyme activity retained.
Specific implementation mode
In order to preferably explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
Present disclosure is not limited solely to following embodiment.
Embodiment 1
1) prepared by chitosan/modifying agent mixed solution
Chitosan, ammonium acetate, nano-titanium dioxide (grain size 50nm) are added in acetic acid solution (2%, w/w), stirred,
Be made chitosan content be 6% (w/w), ammonium acetate content be 0.01mol/L, nanometer titanium dioxide Ti content is 0.5% (w/w)
Mixed solution.
2) cure
It weighs 100g sodium hydroxides to be dissolved in 1L 20% (v/v) ethanol solution, 0.001mol sodium carbonate is then added, stirs
Dissolving is mixed, solidification liquid is obtained；The mixed solution that step 1) obtains is added drop-wise to syringe in solidification liquid, 45 DEG C of heating water baths,
Make chitosan solidify to form microballoon, stands 5h and sediment water and ethyl alcohol washes clean are obtained into chitosan microball.
3) it is crosslinked
By the glutaraldehyde cross-linking agent solution soluble in water that 0.01mol/L is made, chitosan microball is then immersed in crosslinking
In agent solution, 45 DEG C of water-bath concussion reaction 4h are clean by washing of precipitate with water after reaction, obtain mutual flow-through macropore chitosan
Microballoon.
4) protease is fixed
The Papain enzyme aqueous solution for preparing a concentration of 5mg/ml takes the mutual flow-through macropore chitosan microballs of 2g to be added to 5ml
In Papain enzyme aqueous solution, then concussion reaction 8h spends water washing, until being detected without protein in cleaning solution, i.e.,
Obtain protease chitosan microball.
Embodiment 2
1) prepared by chitosan/modifying agent mixed solution
Chitosan, ammonium acetate, nano-graphene (grain size 50nm) are added in acetic acid solution (3%, w/w), stirred, system
It is molten at the mixing that chitosan content is 5% (w/w), ammonium acetate content is 0.03mol/L, nano-graphene content is 1% (w/w)
Liquid.
2) cure
It weighs 110g sodium hydroxides to be dissolved in 1L 22% (v/v) ethanol solution, 0.005mol sodium carbonate is then added, stirs
Dissolving is mixed, solidification liquid is obtained；The mixed solution that step 1) obtains is added drop-wise to syringe in solidification liquid, 50 DEG C of heating water baths,
Make chitosan solidify to form microballoon, stands 5h and sediment water and ethyl alcohol washes clean are obtained into chitosan microball.
3) it is crosslinked
By the dialdehyde starch cross-linking agent solution soluble in water that 0.015mol/L is made, chitosan microball is then immersed in friendship
Join in agent solution, 50 DEG C of water-bath concussion reaction 4.5h are clean by washing of precipitate with water after reaction, obtain mutual flow-through macropore shell
Glycan microballoon.
4) protease is fixed
The Papain enzyme aqueous solution for preparing a concentration of 10mg/ml takes the mutual flow-through macropore chitosan microballs of 2g to be added to 5ml
In Papain enzyme aqueous solution, then concussion reaction 9h spends water washing, until being detected without protein in cleaning solution, i.e.,
Obtain protease chitosan microball.
Embodiment 3
1) prepared by chitosan/modifying agent mixed solution
Chitosan, ammonium carbonate, nano silicon dioxide (grain size 50nm) are added in lactic acid solution (0.5%, w/w), stirred
It mixes, it is 2% (w/w) that chitosan content, which is made, as 4% (w/w), ammonium carbonate content 0.06mol/L, nanometer titanium dioxide silicone content
Mixed solution.
2) cure
It weighs 120g sodium hydroxides to be dissolved in 1L 24% (v/v) ethanol solution, 0.01mol potassium carbonate, stirring is then added
Dissolving, obtains solidification liquid；The mixed solution that step 1) obtains is added drop-wise to syringe in solidification liquid, 55 DEG C of heating water baths make
Chitosan solidifies to form microballoon, stands 5h and sediment water and ethyl alcohol washes clean are obtained chitosan microball.
3) it is crosslinked
By the Geniposide cross-linking agent solution soluble in water that 0.02mol/L is made, chitosan microball is then immersed in crosslinking
In agent solution, 55 DEG C of water-bath concussion reaction 5h are clean by washing of precipitate with water after reaction, obtain mutual flow-through macropore chitosan
Microballoon.
4) protease is fixed
The Papain enzyme aqueous solution for preparing a concentration of 15mg/ml takes the mutual flow-through macropore chitosan microballs of 2g to be added to 5ml
In Papain enzyme aqueous solution, then concussion reaction 10h spends water washing, until being detected without protein in cleaning solution, i.e.,
Obtain protease chitosan microball.
Embodiment 4
1) prepared by chitosan/modifying agent mixed solution
Chitosan, ammonium carbonate, eggshell membrane powder (8000 mesh) are added in citric acid solution (1%, w/w), stirred, system
It is 3.5% (w/w), ammonium carbonate content 0.09mol/L, the mixing that eggshell membrane powder content is 3% (w/w) at chitosan content
Solution.
2) cure
It weighs 130g sodium hydroxides to be dissolved in 1L 26% (v/v) ethanol solution, 0.015mol saleratus is then added,
Stirring and dissolving obtains solidification liquid；The mixed solution that step 1) obtains is added drop-wise to syringe in solidification liquid, 60 DEG C of water-baths add
Heat makes chitosan solidify to form microballoon, stands 5h and sediment water and ethyl alcohol washes clean are obtained chitosan microball.
3) it is crosslinked
By the Geniposide cross-linking agent solution soluble in water that 0.025mol/L is made, chitosan microball is then immersed in crosslinking
In agent solution, 60 DEG C of water-bath concussion reaction 5.5h are clean by washing of precipitate with water after reaction, and it is poly- to obtain mutual flow-through macropore shell
Sugared microballoon.
4) protease is fixed
The Papain enzyme aqueous solution for preparing a concentration of 20mg/ml takes the mutual flow-through macropore chitosan microballs of 2g to be added to 5ml
In papain solution, then concussion reaction 12h spends water washing, until cleaning solution in without protein be detected to get
Protease chitosan microball.
Test example
1, enzyme activity determination method：
Enzyme-activity unit is catalysis casein hydrolysis production 1 per minute under certain condition (this experiment is 37 DEG C, pH 7.0)
Papain enzyme amount needed for μ g tyrosine is 1 enzyme activity unit.Traditional chitosan microball refers to that be not added with pore-foaming agent made
Standby chitosan microball, the control as the mutual chitin microspheric immobilized enzyme of flow-through macropore.
Table 1 illustrates the enzyme activity of the protease chitosan microball prepared by embodiment 1-4 and opposite enzyme activity.It can be with from result
It obtains, the activity of the microballoon papain prepared by embodiment 1 to 4 is significantly larger than the microballoon of conventional method preparation, specific enzyme activity
It improves close to 3 times.This is mainly attributed to large scale duct and good intercommunity, and sufficient reaction is provided for catalysis reaction
Space, while having ensured the smooth diffusion of enzymolysis substrate and product.
The enzyme activity of 1 embodiment 1-4 of table and opposite enzyme activity
2, internal microstructure is observed：
Fig. 1 illustrates the mutual flow-through macropore chitosan microball prepared by embodiment 1-4 and amplifies 40 under scanning electron microscope respectively
Again, 300 times and 6000 times of shape characteristic, it can be seen from the figure that prepared microballoon has multi-stage pore structure.It is put into 40
Times and 300 times of electron microscope be observed that the primary macropore of 10-100 μm of diameter runs through microballoon, can be in 6000 times of figure
Find out there is the secondary aperture of abundant 1-10 μm to be interspersed on macropore hole wall, each rank pore size is uniform, mutual unicom,
Sufficient space is provided for the diffusion mass transfer of substrate protein white matter and product.Influence pore size because be known as pore-foaming agent concentration,
Crosslinker concentration, reaction temperature etc., from embodiment 1 to 4, since pore-foaming agent and crosslinker concentration increase, primary macropore diameter by
Decrescence small, secondary aperture but gradually increases.
3, immobilised enzymes digests egg white solution：
100ml egg white solutions are taken, it is uniform to it using magnetic stirrer 6h, then use NaOH solution and HCl solution
Adjust pH to 7.0.The papain chitosan microball prepared by 2g embodiments 1-4 is taken respectively, is each added to 20ml egg white
In solution, 30min is reacted at 50 DEG C using water-bath concussion (120rpm), then takes the egg white solution after enzyme digestion reaction respectively, it is dilute
It releases debita spissitudo and carries out protein electrophorese experiment.The results are shown in Figure 2, and " egg white " refers to the egg white sample without enzymolysis processing in figure
Product, " M " refer to Marker samples, and " 1-4 " number respectively represents the egg white obtained by immobilised enzymes enzymolysis egg white solution prepared by embodiment 1-4
Sample.The not only rich in protein of 100% egg white solution, molecular weight distribution is extensive, and viscosity is very big.From figure it is found that 4 kinds
In the egg white solution of mutual flow-through macropore chitin microspheric immobilized papain catalyzing hydrolysis, the protein of macromolecule is all quick
Degradation, illustrates that they can have good application effect in food processing.
4, the zymologic property of immobilised enzymes
Fig. 3-6 is the thermal adaptability of the immobilised enzymes and resolvase prepared by embodiment 1-4, pH adaptability, repeats profit
With the comparison of rate and temperature tolerance, the results showed that, the performance of immobilised enzymes is apparently higher than resolvase, with bigger temperature and
PH accommodations reuse the right activity retained close to 50% of 10 successors, and at 60 DEG C after processing, enzyme activity is significantly higher than trip
From enzyme.
Claims (8)
1. a kind of preparation method of protease chitosan microball, it is characterised in that include the following steps：
1) chitosan, ammonium salt, modifying agent are added in weak acid solution, are made that chitosan content is 1-8%, amounts of ammonium salt is
0.01-0.2mol/L, the mixed solution that modifier content is 0.2-6%；
2) mixed solution is added drop-wise in the solidification liquid containing carbonate, so that chitosan is cured reaction, after washing of precipitate
Obtain microballoon；
3) microballoon is added in cross-linking agent solution, microballoon is made to crosslink reaction, washed, it is micro- to obtain mutual flow-through macropore chitosan
Ball；
4) after dissolving protease, mutual flow-through macropore chitosan microball is then added, is shaken under cryogenic conditions, protease is made to load
In on mutual flow-through macropore chitosan microball to get.
2. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The weak acid is acetic acid or carbon
The weight concentration of acid or citric acid, the weak acid solution is 0.1-5%.
3. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The modifying agent is birds, beasts and eggs shell
Film powder or nano silicon dioxide or nano-titanium dioxide or nano-graphene.
4. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The ammonium salt be ammonium acetate or
Ammonium carbonate.
5. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The solidification liquid is to contain hydrogen
The 10-40% ethanol solutions of sodium oxide molybdena；A concentration of 100-200g/L of the sodium hydroxide.
6. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The carbonate is in solidification liquid
In a concentration of 0.001-0.1mol/L.
7. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The crosslinking agent is Geniposide
Or glutaraldehyde or dialdehyde starch.
8. the preparation method of protease chitosan microball as described in claim 1, it is characterised in that：The protease and mutual flow-through
The mass ratio of macropore chitosan microball is 5-100mg：2g.
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