CN108740997A - A kind of preparation method of protease chitosan microball - Google Patents

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

A kind of preparation method of protease chitosan microsphere

technical field

The invention relates to a preparation method of protease chitosan microspheres.

Background technique

Protease is widely used in the food processing industry. At present, free protease is commonly used at home and abroad, which is easily denatured and inactivated by the environment, has poor environmental stability, and cannot be reused. In order to improve the practical application effect of protease, many new enzyme immobilization technologies have been studied and reported. For example, using nanomaterials as carriers can effectively improve the immobilized content and activity of enzymes. However, these new nanomaterials are difficult to separate and recover in the food system, and carrier residues are likely to cause potential safety problems, so the immobilized enzyme carriers that can be applied to food processing are very limited. Large-sized microspheres are an easy-to-separate immobilized enzyme carrier, which can be well used in the food industry. Microsphere-immobilized glycosidase, lactase, etc. have been used in food processing, but these food systems are all reaction Substrate small molecule, immobilized protease has not been able to realize the application in food.

At present, the efficient immobilization of protease is still a difficult problem to be solved worldwide, because the catalytic substrate of protease is a macromolecule, and the substrate has problems such as slow diffusion and poor mass transfer in the reaction system, so the development of protease immobilization technology has been restricted. Some patented technologies use glutaraldehyde as a cross-linking agent to prepare chitosan microspheres for the immobilization of bromelain. However, the three-dimensional network of chitosan microspheres prepared by this method is dense, the free space of enzyme molecules is small, and the diffusion rate of protein substrate is low, so the catalytic performance is poor.

Contents of the invention

The purpose of the present invention is to provide a preparation method of protease chitosan microspheres, which have intercommunicating macropores with adjustable pore diameters, which are suitable for protein diffusion, and the obtained immobilized proteases have high catalytic activity and can Efficiently catalyzes enzymatic hydrolysis of food.

To achieve the above object, the method provided by the invention comprises the following steps:

1) Add chitosan, ammonium salt, and modifier to the weak acid solution to make chitosan content of 1-8%, ammonium salt content of 0.01-0.2mol/L, modifier content of 0.2-6 % mixed solution;

2) adding the mixed solution dropwise to the coagulation solution containing carbonate, so that the chitosan undergoes a solidification reaction, and the precipitate is washed to obtain microspheres;

3) adding the microspheres to the crosslinking agent solution, causing the microspheres to undergo a crosslinking reaction, and washing to obtain interconnected macroporous chitosan microspheres;

4) After dissolving the protease, then adding intercommunicating macroporous chitosan microspheres, shaking under low temperature conditions, so that the protease is loaded on the intercommunicating macroporous chitosan microspheres, and the product is obtained.

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 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 coagulation solution is a 10-40% ethanol solution containing sodium hydroxide; the concentration of the sodium hydroxide is 100-200g/L;

Preferably, the concentration of the carbonate in the coagulation solution is 0.001-0.1 mol/L.

Preferably, the crosslinking agent is genipin or glutaraldehyde or dialdehyde starch.

Preferably, the mass ratio of the protease to the interconnected macroporous chitosan microspheres is 5-100mg: 2g.

The protease chitosan microsphere prepared by the present invention has the following advantages:

1) In the process of preparing chitosan microspheres, ammonium salt and carbonate were added as porogens in chitosan solution and coagulation solution, and in the process of microsphere formation, these two porogens A chemical reaction occurs to generate ammonia gas and carbon dioxide gas respectively. The two gases impact the microsphere matrix from the inside and outside of the microsphere respectively, forming micron-scale pores with uniform pore size and good intercommunication. The formed pores are conducive to the diffusion of protein molecules, thereby increasing the effective reaction space between proteins and proteases, and at the same time facilitating the mass transfer of enzymatic hydrolysis products to avoid blockage. By controlling the addition amount of the two porogens, the pore size can also be adjusted.

2) Carry out cross-linking reaction on chitosan microspheres, the purpose is to make the pores on the microspheres communicate with each other, provide more sufficient space for the diffusion and mass transfer of substrate proteins, and at the same time reduce the swelling of chitosan microspheres function, further increase the mechanical properties of the microspheres, provide sufficient covalent binding sites for immobilized enzymes, and play the role of tethers. The modifier added in the chitosan microspheres can improve the mechanical properties and durability of the microspheres.

3) The present invention prepares a kind of immobilized protease intercommunicating macroporous chitosan microspheres. Compared with the protease chitosan microspheres prepared by traditional methods, the efficiency of catalyzing protein hydrolysis increases by 200%, which can efficiently catalyze food enzymolysis , and the heat stability, pH stability, storage stability, etc. of the immobilized protease are all higher than the free enzyme.

4) The present invention also has the advantages of high mechanical strength, easy degradation and good biocompatibility, and the production cost is low, the operation is simple, and the preparation process is green and environmentally friendly. It can be used for immobilizing papain, bromelain, trypsin, chymotrypsin, and protease and alkaline protease, etc.

Description of drawings

Fig. 1 is the scanning electron micrograph of the protease chitosan microsphere that embodiment 1-4 prepares.

Fig. 2 is the electrophoresis graph of enzymatic hydrolysis of 100% egg white liquid prepared by protease chitosan microspheres prepared in Example 1-4.

Fig. 3 is the temperature adaptability of protease chitosan microspheres prepared in Example 1 and free enzyme.

Fig. 4 is the pH adaptability of protease chitosan microspheres prepared in Example 2 and free enzyme.

Fig. 5 shows the relative enzymatic activity retained by the protease chitosan microspheres prepared in Example 3 after being reused many times.

Fig. 6 shows the relative enzymatic activity retained after the protease chitosan microspheres prepared in Example 4 and the free enzyme were treated at 60°C for different periods of time.

Detailed ways

In order to better explain the present invention, the main content of the present invention is further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

Example 1

1) Preparation of chitosan/modifier mixed solution

Chitosan, ammonium acetate, nano-titanium dioxide (particle diameter 50nm) are added in the acetic acid solution (2%, w/w), stir, make chitosan content be 6% (w/w), ammonium acetate content be A mixed solution of 0.01mol/L and 0.5% (w/w) content of nano-titanium dioxide.

2) curing

Weigh 100g of sodium hydroxide and dissolve it in 1L of 20% (v/v) ethanol solution, then add 0.001mol of sodium carbonate, stir and dissolve to obtain a coagulation solution; add the mixed solution obtained in step 1) dropwise into the coagulation solution with a syringe , heated in a water bath at 45°C to solidify the chitosan to form microspheres, let stand for 5 hours, and wash the precipitate with water and ethanol to obtain chitosan microspheres.

3) Cross-linking

Dissolve glutaraldehyde in water to make a 0.01mol/L cross-linking agent solution, then soak the chitosan microspheres in the cross-linking agent solution, shake and react in a water bath at 45°C for 4 hours, and wash the precipitate with water after the reaction. Interconnected macroporous chitosan microspheres were obtained.

4) Protease immobilization

Prepare a papain aqueous solution with a concentration of 5 mg/ml, add 2 g of interconnected macroporous chitosan microspheres to 5 ml of papain aqueous solution, shake and react for 8 hours, and then wash with dewatered until no protein is detected in the washing solution. Protease chitosan microspheres are obtained.

Example 2

1) Preparation of chitosan/modifier mixed solution

Chitosan, ammonium acetate, nano-graphene (particle diameter 50nm) are added in the acetic acid solution (3%, w/w), stir, make chitosan content be 5% (w/w), ammonium acetate content It is a mixed solution of 0.03mol/L and a nano-graphene content of 1% (w/w).

2) curing

Weigh 110g of sodium hydroxide and dissolve it in 1L of 22% (v/v) ethanol solution, then add 0.005mol of sodium carbonate, stir and dissolve to obtain a coagulation solution; the mixed solution obtained in step 1) is added dropwise to the coagulation solution with a syringe , heated in a water bath at 50°C to solidify the chitosan to form microspheres, let stand for 5 hours, wash the precipitate with water and ethanol, and obtain chitosan microspheres.

3) Cross-linking

Dissolve dialdehyde starch in water to make a 0.015mol/L cross-linking agent solution, then soak the chitosan microspheres in the cross-linking agent solution, shake and react in a water bath at 50°C for 4.5 hours, and wash the precipitate with water after the reaction , to obtain interconnected macroporous chitosan microspheres.

4) Protease immobilization

Prepare a papain aqueous solution with a concentration of 10 mg/ml, add 2 g of interconnected macroporous chitosan microspheres to 5 ml of papain aqueous solution, shake and react for 9 hours, and then wash with dewatered until no protein is detected in the washing solution. Protease chitosan microspheres are obtained.

Example 3

1) Preparation of chitosan/modifier mixed solution

Chitosan, ammonium carbonate, nano silicon dioxide (particle diameter 50nm) are added in the lactic acid solution (0.5%, w/w), stir, make chitosan content be 4% (w/w), ammonium carbonate A mixed solution with a content of 0.06mol/L and a content of 2% (w/w) of nano silicon dioxide.

2) curing

Weigh 120g of sodium hydroxide and dissolve it in 1L of 24% (v/v) ethanol solution, then add 0.01mol potassium carbonate, stir and dissolve to obtain a coagulation solution; the mixed solution obtained in step 1) is added dropwise to the coagulation solution with a syringe , heated in a water bath at 55°C to solidify the chitosan to form microspheres, let it stand for 5 hours, and wash the precipitate with water and ethanol to obtain chitosan microspheres.

3) Cross-linking

Dissolve genipin in water to make a 0.02mol/L cross-linking agent solution, then soak the chitosan microspheres in the cross-linking agent solution, shake and react in a water bath at 55°C for 5 hours, and wash the precipitate with water after the reaction. Interconnected macroporous chitosan microspheres were obtained.

4) Protease immobilization

Prepare a papain aqueous solution with a concentration of 15 mg/ml, add 2 g of interconnected macroporous chitosan microspheres to 5 ml of papain aqueous solution, shake and react for 10 h, and then wash with dewatered until no protein is detected in the washing solution. Protease chitosan microspheres are obtained.

Example 4

1) Preparation of chitosan/modifier mixed solution

Chitosan, ammonium carbonate, egg shell membrane powder (8000 mesh) are added in the citric acid solution (1%, w/w), stir, make chitosan content be 3.5% (w/w), ammonium carbonate A mixed solution with a content of 0.09mol/L and an egg shell membrane powder content of 3% (w/w).

2) curing

Weigh 130g of sodium hydroxide and dissolve it in 1L of 26% (v/v) ethanol solution, then add 0.015mol potassium bicarbonate, stir and dissolve to obtain a coagulation solution; the mixed solution obtained in step 1) is added dropwise to the coagulation solution with a syringe , heated in a water bath at 60°C to solidify the chitosan to form microspheres, let stand for 5 hours, and wash the precipitate with water and ethanol to obtain chitosan microspheres.

3) Cross-linking

Dissolve genipin in water to make a 0.025mol/L cross-linking agent solution, then soak the chitosan microspheres in the cross-linking agent solution, shake and react in a water bath at 60°C for 5.5 hours, and wash the precipitate with water after the reaction , to obtain interconnected macroporous chitosan microspheres.

4) Protease immobilization

Prepare a papain aqueous solution with a concentration of 20 mg/ml, add 2 g of interconnected macroporous chitosan microspheres to 5 ml of papain solution, shake and react for 12 hours, and then wash with dewatered until no protein is detected in the washing solution. Protease chitosan microspheres are obtained.

Test case

1. Enzyme activity assay method:

The unit of enzyme activity is the amount of papain required to catalyze the hydrolysis of casein to produce 1 μg of tyrosine per minute under certain conditions (37°C, pH 7.0 in this experiment) is 1 enzyme activity unit. Traditional chitosan microspheres refers to chitosan microspheres prepared without adding porogen, which is used as a control for intercommunicating macroporous chitosan microspheres to immobilize enzymes.

Table 1 shows the enzymatic activity and relative enzymatic activity of the protease chitosan microspheres prepared in Examples 1-4. From the results, it can be concluded that the activity of the microspheres papain prepared in Examples 1 to 4 is much higher than that of the microspheres prepared by the traditional method, and the specific enzyme activity is nearly 3 times higher. This is mainly due to the large-sized pores and good intercommunication, which provide sufficient reaction space for catalytic reactions and ensure the smooth diffusion of enzymatic substrates and products.

Enzyme activity and relative enzyme activity of table 1 embodiment 1-4

2. Internal microstructure observation:

Fig. 1 shows the morphological features of the interconnected macroporous chitosan microspheres prepared in Examples 1-4 respectively enlarged by 40 times, 300 times and 6000 times under a scanning electron microscope, as can be seen from the figure, the prepared The microspheres have a multi-level porous structure. Putting it into the electron microscope pictures of 40 times and 300 times, it can be observed that the primary macropores with a diameter of 10-100 μm run through the microspheres. It can be seen in the picture of 6000 times that there are abundant secondary small pores of 1-10 μm on the wall of the macropores. The pores are distributed in a staggered manner, and the pore sizes of each level are uniform and interconnected, providing sufficient space for the diffusion and mass transfer of substrate proteins and products. Factors affecting the pore size include porogen concentration, crosslinking agent concentration, reaction temperature, etc. From Examples 1 to 4, due to the increase in porogen and crosslinking agent concentrations, the primary macropore diameter gradually decreases, while the secondary pore diameter gradually decreases. increase.

3. Immobilized enzyme enzymatic hydrolysis of egg white liquid:

Take 100ml of egg white liquid and stir it with a magnetic stirrer for 6h until it is uniform, then use NaOH solution and HCl solution to adjust the pH to 7.0. Get 2g of the papain-chitosan microspheres prepared in Examples 1-4 respectively, add to 20ml egg white solution respectively, use water bath vibration (120rpm) to react at 50°C for 30min, then take the egg white liquid after enzymolysis reaction respectively , diluted to an appropriate concentration for protein electrophoresis experiments. The results are shown in Figure 2. In the figure, "egg white" refers to the egg white sample without enzymatic hydrolysis treatment, "M" refers to the Marker sample, and numbers "1-4" respectively represent the immobilized enzyme enzymolysis egg white prepared in Examples 1-4. Egg white samples obtained from liquid. 100% egg white liquid is not only rich in protein content, wide molecular weight distribution, but also has a high viscosity. It can be seen from the figure that in the egg white solution catalyzed by papain-catalyzed hydrolysis of the four kinds of interconnected macroporous chitosan microspheres, the proteins with large molecular weights are all degraded rapidly, indicating that they can all have good application effects in food processing.

4. Enzymatic properties of immobilized enzymes

Fig. 3-6 is the comparison of temperature adaptability, pH adaptability, reuse rate and temperature tolerance of the immobilized enzyme prepared in embodiment 1-4 and free enzyme, the result shows that the performance of immobilized enzyme is obviously higher than The free enzyme has a larger temperature and pH adaptability range, and after repeated use for 10 times, it still retains nearly 50% of its activity, and after being treated at 60°C, the enzyme activity is significantly higher than that of the free enzyme.

Claims (8)

1. a preparation method of protease chitosan microspheres, is characterized in that comprising the following steps: 1) Add chitosan, ammonium salt, and modifier to the weak acid solution to make chitosan content of 1-8%, ammonium salt content of 0.01-0.2mol/L, modifier content of 0.2-6 % mixed solution; 2) adding the mixed solution dropwise to the coagulation solution containing carbonate, so that the chitosan undergoes a solidification reaction, and the precipitate is washed to obtain microspheres; 3) adding the microspheres to the crosslinking agent solution, causing the microspheres to undergo a crosslinking reaction, and washing to obtain interconnected macroporous chitosan microspheres; 4) After dissolving the protease, then adding intercommunicating macroporous chitosan microspheres, shaking under low temperature conditions, so that the protease is loaded on the intercommunicating macroporous chitosan microspheres, and the product is obtained. 2. the preparation method of protease chitosan microsphere as claimed in claim 1 is characterized in that: described weak acid is acetic acid or carbonic acid or citric acid, and the weight concentration of described weak acid solution is 0.1-5%. 3. the preparation method of protease chitosan microsphere as claimed in claim 1, is characterized in that: described modifying agent is poultry egg shell membrane powder or nano-silica or nano-titanium dioxide or nano-graphene. 4. the preparation method of protease chitosan microsphere as claimed in claim 1 is characterized in that: described ammonium salt is ammonium acetate or ammonium carbonate. 5. the preparation method of protease chitosan microsphere as claimed in claim 1 is characterized in that: described coagulation liquid is the 10-40% ethanol solution that contains sodium hydroxide; The concentration of described sodium hydroxide is 100-200g /L. 6. the preparation method of protease chitosan microsphere as claimed in claim 1 is characterized in that: the concentration of described carbonate in coagulation liquid is 0.001-0.1mol/L. 7. the preparation method of protease chitosan microsphere as claimed in claim 1 is characterized in that: described linking agent is genipin or glutaraldehyde or dialdehyde starch. 8. The preparation method of protease chitosan microspheres as claimed in claim 1, characterized in that: the mass ratio of the protease to intercommunicating macroporous chitosan microspheres is 5-100mg: 2g.