CN108740997B - Preparation method of protease chitosan microspheres - Google Patents
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- CN108740997B CN108740997B CN201810829618.7A CN201810829618A CN108740997B CN 108740997 B CN108740997 B CN 108740997B CN 201810829618 A CN201810829618 A CN 201810829618A CN 108740997 B CN108740997 B CN 108740997B
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- 239000004005 microsphere Substances 0.000 title claims abstract description 84
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 83
- 239000004365 Protease Substances 0.000 title claims abstract description 60
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- 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
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- 239000004088 foaming agent Substances 0.000 description 1
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- 229940116108 lactase Drugs 0.000 description 1
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- 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
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- 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
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- Nutrition Science (AREA)
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- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
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- 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 preparation method of protease chitosan microspheres, which comprises the steps of preparation of a chitosan/modifier mixed solution, solidification, crosslinking, protease fixation and the like. Compared with the protease chitosan microspheres prepared by the traditional method, the efficiency of catalyzing protein hydrolysis is increased by 200%, food enzymolysis can be efficiently catalyzed, and the thermal stability, pH stability, storage stability and the like of the immobilized protease are higher than those of free enzyme.
Description
Technical Field
The invention relates to a preparation method of protease chitosan microspheres.
Background
The protease is widely applied to the food processing industry, and currently, the addition of free protease is commonly adopted at home and abroad, so that the protease is easily denatured and inactivated by environmental influence, has poor environmental stability and cannot be repeatedly used. In order to improve the practical application effect of the protease, many novel enzyme immobilization technologies are researched and reported, for example, the immobilized content and the enzyme activity of the enzyme can be effectively improved by using a nano material as a carrier. However, these novel nanomaterials are difficult to separate and recover in food systems, and carrier residues are likely to cause potential safety problems, so that there are very limited immobilized enzyme carriers that can be applied to food processing. The large-size microspheres are easy-to-separate immobilized enzyme carriers and can be well applied to the food industry, microsphere immobilized glycosidase, lactase and the like are applied to food processing, but the food systems are all reaction substrate small molecules, and the immobilized protease cannot be applied to food.
At present, the efficient immobilization of protease still remains a difficult problem to be solved worldwide, and because the catalytic substrate of protease is macromolecule, and the substrate has the problems of slow diffusion, poor mass transfer and the like in a reaction system, the development of the protease immobilization technology is always limited. Some patent technologies use glutaraldehyde as a cross-linking agent to prepare chitosan microspheres for immobilization of bromelain. However, the chitosan microsphere prepared by the method has a compact three-dimensional network, small free space of enzyme molecules and low diffusion rate of protein substrates, so that the catalytic performance is poor.
Disclosure of Invention
The invention aims to provide a preparation method of protease chitosan microspheres, wherein the microspheres have intercommunicated macropores with adjustable pore diameters and are suitable for protein diffusion, and the obtained immobilized protease has high catalytic activity and can efficiently catalyze food enzymolysis.
In order to achieve the above object, the present invention provides a method comprising the steps of:
1) adding chitosan, ammonium salt and modifier into weak acid solution to prepare mixed solution with chitosan content of 1-8%, ammonium salt content of 0.01-0.2mol/L and modifier content of 0.2-6%;
2) dripping the mixed solution into a solidification solution containing carbonate to enable chitosan to perform a solidification reaction, and washing a precipitate to obtain microspheres;
3) adding the microspheres into a cross-linking agent solution to enable the microspheres to have a cross-linking reaction, and washing to obtain intercommunicated macroporous chitosan microspheres;
4) and (3) dissolving the protease, then adding the intercommunicated macroporous chitosan microspheres, and oscillating at low temperature to load the protease on the intercommunicated macroporous chitosan microspheres to obtain the chitosan microsphere.
Preferably, the weak acid is acetic acid, carbonic acid or citric acid, and the weight concentration of the weak acid solution is 0.1-5%.
Preferably, the modifier is poultry eggshell membrane powder or nano silicon dioxide or nano titanium dioxide or nano graphene.
Preferably, the ammonium salt is ammonium acetate or ammonium carbonate.
Preferably, the coagulating liquid is a 10-40% ethanol solution containing sodium hydroxide; the concentration of the sodium hydroxide is 100-200 g/L;
preferably, the concentration of the carbonate in the solidification solution is 0.001-0.1 mol/L.
Preferably, the cross-linking agent is genipin or glutaraldehyde or dialdehyde starch.
Preferably, the mass ratio of the protease to the intercommunicated macroporous chitosan microspheres is 5-100 mg: 2g of the total weight.
The protease chitosan microspheres prepared by the invention have the following advantages:
1) in the process of preparing the chitosan microspheres, ammonium salt and carbonate are respectively added into a chitosan solution and a solidification solution to serve as pore-forming agents, in the process of forming the microspheres, the two pore-forming agents are subjected to chemical reaction to respectively generate ammonia gas and carbon dioxide gas, the two gases respectively impact a microsphere matrix from the inside and the outside of the microspheres to form micron-level holes, and the microspheres are uniform in pore size and good in intercommunity. The formed pore canal is beneficial to the diffusion of protein molecules, thereby increasing the effective reaction space of the protein and the protease, simultaneously being beneficial to the mass transfer of enzymolysis products and avoiding the blockage. The pore size can be adjusted by controlling the addition of the two pore-forming agents.
2) The chitosan microspheres are subjected to crosslinking reaction, so that holes on the microspheres are communicated with each other, a more sufficient space is provided for diffusion mass transfer of substrate protein, the swelling effect of the chitosan microspheres can be reduced, the mechanical property of the microspheres is further improved, sufficient covalent binding sites are provided for immobilized enzyme, and the effect of connecting arms is achieved. The modifier added into the chitosan microsphere can improve the mechanical property and the durability of the microsphere.
3) Compared with the protease chitosan microspheres prepared by the traditional method, the efficiency of catalyzing protein hydrolysis is increased by 200%, food enzymolysis can be efficiently catalyzed, and the thermal stability, pH stability, storage stability and the like of the immobilized protease are higher than those of free enzyme.
4) The invention also has the advantages of high mechanical strength, easy degradation, good biocompatibility and the like, and has low production cost, simple operation and green and environment-friendly preparation process, and can be used for immobilizing papain, bromelain, trypsin, chymotrypsin, neutral protease, alkaline protease and the like.
Drawings
FIG. 1 is a scanning electron micrograph of the protease chitosan microspheres prepared in examples 1-4.
FIG. 2 is an electrophoresis chart of the protease chitosan microspheres prepared in examples 1-4 for enzymolysis of 100% egg white.
FIG. 3 shows the temperature adaptation of the protease chitosan microspheres prepared in example 1 to free enzymes.
FIG. 4 shows the pH adaptation of the protease chitosan microspheres prepared in example 2 to free enzymes.
FIG. 5 shows the relative enzyme activities retained by the protease chitosan microspheres prepared in example 3 after repeated use.
FIG. 6 shows the relative enzyme activities of the protease chitosan microspheres prepared in example 4 and the free enzyme after different treatment times at 60 ℃.
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples.
Example 1
1) Preparation of chitosan/modifier mixed solution
Adding chitosan, ammonium acetate and nano titanium dioxide (particle size is 50nm) into acetic acid solution (2%, w/w), stirring to obtain mixed solution with chitosan content of 6% (w/w), ammonium acetate content of 0.01mol/L and nano titanium dioxide content of 0.5% (w/w).
2) Curing
Weighing 100g of sodium hydroxide, dissolving the sodium hydroxide in 1L of 20% (v/v) ethanol solution, adding 0.001mol of sodium carbonate, and stirring to dissolve the sodium hydroxide to obtain a solidification solution; dropwise adding the mixed solution obtained in the step 1) into the solidification solution by using an injector, heating in a water bath at 45 ℃ to solidify chitosan to form microspheres, standing for 5h, and washing precipitates with water and ethanol to obtain the chitosan microspheres.
3) Cross-linking
Dissolving glutaraldehyde in water to prepare a cross-linking agent solution of 0.01mol/L, then soaking the chitosan microspheres in the cross-linking agent solution, carrying out water bath oscillation reaction for 4 hours at 45 ℃, and washing precipitates with water after the reaction is finished to obtain the intercommunicated macroporous chitosan microspheres.
4) Protease immobilization
Preparing a papain aqueous solution with the concentration of 5mg/ml, adding 2g of intercommunicated macroporous chitosan microspheres into 5ml of the papain aqueous solution, carrying out oscillation reaction for 8 hours, and then washing with water until no protein is detected in a washing solution, thus obtaining the papain chitosan microspheres.
Example 2
1) Preparation of chitosan/modifier mixed solution
Adding chitosan, ammonium acetate and nano graphene (particle size is 50nm) into an acetic acid solution (3 percent, w/w), and stirring to prepare a mixed solution with the chitosan content of 5 percent (w/w), the ammonium acetate content of 0.03mol/L and the nano graphene content of 1 percent (w/w).
2) Curing
Weighing 110g of sodium hydroxide, dissolving the sodium hydroxide in 1L of 22% (v/v) ethanol solution, adding 0.005mol of sodium carbonate, and stirring to dissolve to obtain a solidification solution; dripping the mixed solution obtained in the step 1) into the solidification solution by using an injector, heating in a water bath at 50 ℃ to solidify chitosan to form microspheres, standing for 5h, and washing precipitates with water and ethanol to obtain the chitosan microspheres.
3) Cross-linking
Dissolving dialdehyde starch in water to prepare a cross-linking agent solution of 0.015mol/L, then soaking the chitosan microspheres in the cross-linking agent solution, carrying out water bath oscillation reaction at 50 ℃ for 4.5h, and washing precipitates with water after the reaction is finished to obtain the intercommunicated macroporous chitosan microspheres.
4) Protease immobilization
Preparing a papain aqueous solution with the concentration of 10mg/ml, adding 2g of intercommunicated macroporous chitosan microspheres into 5ml of the papain aqueous solution, carrying out a shaking reaction for 9 hours, and then washing with water until no protein is detected in a washing solution, thus obtaining the papain chitosan microspheres.
Example 3
1) Preparation of chitosan/modifier mixed solution
Adding chitosan, ammonium carbonate and nano-silica (particle size 50nm) into lactic acid solution (0.5%, w/w), stirring to obtain mixed solution with chitosan content of 4% (w/w), ammonium carbonate content of 0.06mol/L and nano-silica content of 2% (w/w).
2) Curing
Weighing 120g of sodium hydroxide, dissolving the sodium hydroxide in 1L of 24% (v/v) ethanol solution, adding 0.01mol of potassium carbonate, and stirring for dissolving to obtain a solidification solution; dropwise adding the mixed solution obtained in the step 1) into the solidification solution by using an injector, heating in a water bath at 55 ℃ to solidify chitosan to form microspheres, standing for 5h, and washing precipitates with water and ethanol to obtain the chitosan microspheres.
3) Cross-linking
Dissolving genipin in water to prepare a 0.02mol/L cross-linking agent solution, then soaking the chitosan microspheres in the cross-linking agent solution, carrying out water bath oscillation reaction at 55 ℃ for 5 hours, and washing precipitates with water after the reaction is finished to obtain the intercommunicated macroporous chitosan microspheres.
4) Protease immobilization
Preparing a papain aqueous solution with the concentration of 15mg/ml, adding 2g of intercommunicated macroporous chitosan microspheres into 5ml of the papain aqueous solution, carrying out a shaking reaction for 10 hours, and then washing with water until no protein is detected in a washing solution, thus obtaining the papain chitosan microspheres.
Example 4
1) Preparation of chitosan/modifier mixed solution
Adding chitosan, ammonium carbonate and eggshell membrane powder (8000 meshes) into citric acid solution (1%, w/w), and stirring to obtain mixed solution with chitosan content of 3.5% (w/w), ammonium carbonate content of 0.09mol/L and eggshell membrane powder content of 3% (w/w).
2) Curing
Weighing 130g of sodium hydroxide, dissolving the sodium hydroxide in 1L of 26% (v/v) ethanol solution, adding 0.015mol of potassium bicarbonate, and stirring for dissolving to obtain a solidification solution; dropwise adding the mixed solution obtained in the step 1) into the solidification solution by using an injector, heating in a water bath at 60 ℃ to solidify chitosan to form microspheres, standing for 5h, and washing precipitates with water and ethanol to obtain the chitosan microspheres.
3) Cross-linking
Dissolving genipin in water to prepare a cross-linking agent solution of 0.025mol/L, then soaking the chitosan microspheres in the cross-linking agent solution, carrying out water bath oscillation reaction at 60 ℃ for 5.5h, and washing precipitates with water after the reaction is finished to obtain the intercommunicated macroporous chitosan microspheres.
4) Protease immobilization
Preparing a papain aqueous solution with the concentration of 20mg/ml, adding 2g of intercommunicated macroporous chitosan microspheres into 5ml of the papain aqueous solution, carrying out a shaking reaction for 12 hours, and then washing with water until no protein is detected in a washing solution, thus obtaining the papain chitosan microspheres.
Test examples
1. The enzyme activity determination method comprises the following steps:
the enzyme activity unit is that the papain amount required for catalyzing hydrolysis of casein to produce 1 mu g of tyrosine per minute is 1 enzyme activity unit under certain conditions (the experiment is at 37 ℃ and the pH value is 7.0). The traditional chitosan microsphere is prepared without adding pore-foaming agent and is used as a control of an intercommunicated macroporous chitosan microsphere immobilized enzyme.
Table 1 shows the enzyme activities and relative enzyme activities of the protease chitosan microspheres prepared in examples 1-4. From the results, it can be seen that the activity of the microsphere papain prepared in examples 1 to 4 is much higher than that of microspheres prepared by the conventional method, and the specific enzyme activity is improved by nearly 3 times. This is mainly due to the large size of the pores and the good interoperability, providing a sufficient reaction space for the catalytic reaction, while ensuring the smooth diffusion of the enzymatic substrate and the product.
TABLE 1 enzyme Activity and relative enzyme Activity of examples 1-4
2. Observation of internal microstructure:
fig. 1 shows the morphological characteristics of the intercommunicated macroporous chitosan microspheres prepared in examples 1-4 amplified 40 times, 300 times and 6000 times respectively under a scanning electron microscope, and it can be seen from the figure that the prepared microspheres have a multistage pore structure. The primary macroporous penetrating microspheres with the diameter of 10-100 mu m can be observed by placing the microspheres at 40 times and 300 times of electron micrographs, and the 6000 times of images show that the walls of the macropores are provided with abundant secondary micropores with the diameter of 1-10 mu m in staggered distribution, the pore diameters of all levels are uniform and communicated with each other, thereby providing sufficient space for the diffusion mass transfer of substrate proteins and products. The pore size is influenced by the concentration of the pore-forming agent, the concentration of the cross-linking agent, the reaction temperature, etc. from example 1 to 4, the pore size of the primary macropore gradually decreases and the pore size of the secondary macropore gradually increases due to the increase of the concentrations of the pore-forming agent and the cross-linking agent.
3. Carrying out enzymolysis on egg white liquid by using immobilized enzyme:
100ml of egg white was taken, stirred for 6h to homogeneity using a magnetic stirrer and subsequently adjusted to pH 7.0 using NaOH solution and HCl solution. 2g of the papain chitosan microspheres prepared in the examples 1 to 4 are respectively added into 20ml of egg white solution, the mixture is reacted for 30min at 50 ℃ by using water bath oscillation (120rpm), and then egg white liquid after the enzymolysis reaction is respectively taken and diluted to proper concentration for protein electrophoresis experiment. As shown in FIG. 2, the term "egg white" refers to a sample of egg white that has not been subjected to enzymatic hydrolysis treatment, the term "M" refers to a Marker sample, and the designations "1" to "4" represent the egg white samples obtained by enzymatic hydrolysis of the egg white with the immobilized enzyme prepared in examples 1 to 4, respectively. The 100% egg white has rich protein content, wide molecular weight distribution and high viscosity. As can be seen from the figure, in the egg white liquid obtained by catalytic hydrolysis of 4 kinds of intercommunicated macroporous chitosan microsphere immobilized papain, proteins with large molecular weight are rapidly degraded, which indicates that the proteins have good application effect in food processing.
4. Enzymatic Properties of the immobilized enzymes
Fig. 3 to 6 are comparisons of temperature adaptability, pH adaptability, recycling rate and temperature tolerance between the immobilized enzyme prepared in examples 1 to 4 and the free enzyme, and the results show that the performance of the immobilized enzyme is significantly higher than that of the free enzyme, the immobilized enzyme has a larger temperature and pH adaptation range, nearly 50% of activity is retained after 10 times of recycling, and the enzyme activity is significantly higher than that of the free enzyme after 60 ℃.
Claims (3)
1. A preparation method of protease chitosan microspheres is characterized by comprising the following steps:
1) adding chitosan, ammonium salt and modifier into weak acid solution with weight concentration of 0.1-5% to prepare mixed solution with chitosan weight content of 1-8%, ammonium salt content of 0.01-0.2mol/L and modifier weight content of 0.2-6%;
2) dripping the mixed solution into a solidification solution containing carbonate to enable chitosan to perform a solidification reaction, and washing a precipitate to obtain microspheres;
3) adding the microspheres into a cross-linking agent solution to enable the microspheres to have a cross-linking reaction, and washing to obtain intercommunicated macroporous chitosan microspheres;
4) after protease is dissolved, adding the intercommunicated macroporous chitosan microspheres, wherein the mass ratio of the protease to the intercommunicated macroporous chitosan microspheres is 5-100 mg: 2g, oscillating at low temperature to load protease on the communicated macroporous chitosan microspheres to obtain the chitosan microsphere,
the ammonium salt is ammonium acetate or ammonium carbonate;
the modifier is poultry eggshell membrane powder or nano silicon dioxide or nano titanium dioxide or nano graphene;
the weak acid is acetic acid or carbonic acid or citric acid;
the carbonate is sodium carbonate or potassium bicarbonate;
the coagulating liquid is 10-40% ethanol solution containing sodium hydroxide; the concentration of the sodium hydroxide is 100-200 g/L.
2. The method for preparing the protease chitosan microspheres of claim 1, wherein: the concentration of the carbonate in the solidification liquid is 0.001-0.1 mol/L.
3. The method for preparing the protease chitosan microspheres of claim 1, wherein: the cross-linking agent is genipin or glutaraldehyde or dialdehyde starch.
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