CN113151244B - pH-responsive enzyme-loaded starch granule with controllable enzyme distribution position and preparation method thereof - Google Patents
pH-responsive enzyme-loaded starch granule with controllable enzyme distribution position and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a pH-responsive enzyme-loaded starch granule with controllable enzyme distribution position and a preparation method thereof. According to the invention, starch is gelatinized, then is subjected to free radical grafting reaction with a cross-linking agent and a compound with pH responsiveness, then is subjected to ethanol granulation to obtain pH responsiveness starch nanoparticles, and finally is mixed with a lipase solution for reaction to obtain pH responsiveness enzyme-loaded starch particles with controllable enzyme distribution positions. The pH responsive starch enzyme-carrying particle with controllable enzyme distribution position realizes interface catalysis in emulsion, can realize recycling of a catalyst by adjusting pH, and realizes high-efficiency catalysis by distributing the enzyme on the particle surface.
Description
Technical Field
The invention belongs to the field of enzyme catalysis, and particularly relates to a pH-responsive enzyme-loaded starch granule with controllable enzyme distribution position, and a preparation method thereof.
Background
In the catalytic process of the Pickering emulsion system, as a carrier of enzyme, a high polymer material is favored with the characteristic of green environmental protection, in order to enable the high polymer material to meet the requirement of immobilized enzyme and have some special properties, the high polymer material is modified by a chemical method, so that some chemical reagents are inevitably used to damage the environment to a certain extent, starch as a high polymer material has the advantages of green environmental protection, degradability and the like, and abundant hydroxyl groups on a starch molecular chain provide possibility for starch molecules to have some special properties.
The separation and recovery of the catalyst after the catalysis of the Pickering emulsion is difficult, the most common mode is to separate the oil and water of the emulsion through high-speed centrifugation to achieve the purpose of demulsification, then the catalyst is taken out for recycling, the process is not only serious in energy consumption but also tedious, some catalyst can be recovered by a magnet through the fact that carrier particles have magnetism, but the problem that the particles have magnetism is not easy, the development of the magnetic particles is restricted, and the development of the catalyst recovery of an emulsion catalysis system is also restricted.
The carrier immobilized enzyme is used, and is usually bound by covalent bonds or physical bonds, and the two ways have advantages and disadvantages respectively. The purpose of the immobilized enzyme is mainly to facilitate the recycling of the enzyme and realize the high-efficiency catalysis of the enzyme, the distribution mode of the enzyme on the particles is one of the main factors determining the catalytic effect, if the enzyme exists on the surface of the particles, the contact between a substrate and the enzyme is facilitated for the general interface catalytic reaction, and when the enzyme exists in the particles, the large mass transfer resistance can be generated, which is not favorable for the high-efficiency catalysis of the enzyme.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a preparation method of pH-responsive enzyme-loaded starch granules with controllable enzyme distribution positions.
According to the invention, the gelatinized starch is grafted with the cross-linking agent and has a pH responsive group, so that starch molecules have pH responsiveness, then the starch paste is granulated by ethanol, and the obtained nano-particles can stably form Pickering emulsion. The invention prepares the enzyme-loaded particle with high catalytic activity and highly concentrated enzyme distribution on the surface of the nanoparticle, thereby efficiently utilizing the enzyme and having important guiding significance for researching the advantages and the industrialized prospect of the enzyme combination mode and the distribution mode.
Another object of the present invention is to provide a pH-responsive enzyme-loaded starch granule with controllable enzyme distribution position prepared by the above method.
The purpose of the invention is realized by the following technical scheme:
a preparation method of pH-responsive enzyme-loaded starch granules with controllable enzyme distribution positions comprises the following steps:
(1) Gelatinizing starch, performing free radical grafting reaction on the starch, a cross-linking agent and a compound with pH responsiveness, and performing ethanol granulation to obtain pH responsiveness starch nanoparticles;
(2) And mixing the pH-responsive starch nanoparticles with a lipase solution for reaction, and performing centrifugal washing to obtain the pH-responsive enzyme-loaded starch particles with controllable enzyme distribution positions.
Preferably, the starch gelatinization in the step (1) refers to gelatinization for 30 minutes at 95 ℃ after mixing the starch and water according to the proportion of 30-50 mg/ml.
Preferably, the proportion of the starch, the cross-linking agent and the compound with pH responsiveness in the step (1) is (3-5) g: (0.1-0.3) g: (2-6) ml.
Preferably, the crosslinking agent in the step (1) is at least one of N, N-methylene-bisacrylamide and hexamethylene-bisacrylamide; the compound with pH responsiveness is at least one of dimethylaminoethyl methacrylate, ethylene glycol methacrylate and diethylaminoethyl methacrylate.
Preferably, the pH value of the free radical grafting reaction in the step (1) is 8-10, the temperature is 60-80 ℃, and the time is 1-3 h.
Preferably, the free radical grafting reaction in the step (1) is performed under the action of a catalyst, the catalyst is ammonium persulfate and sodium bisulfite, and the mass ratio of the starch to the ammonium persulfate to the sodium bisulfite is (3-5) g: (30 to 90) mg: (20-60) mg.
Preferably, the free radical grafting reaction in the step (1) is carried out under the protection of nitrogen or inert gas; and after the free radical grafting reaction is finished, carrying out centrifugal washing on the product mixed solution to obtain a product, wherein the centrifugal speed is 3000-5000 rpm, the centrifugal time is 3-5 min, and the washing times are 3-5 times.
Preferably, the ethanol granulation in the step (1) refers to that the product is mixed according to a volume ratio of 1: 20-40 is dripped into ethanol, and then is stirred at 800-1200 r/min to obtain the mixed liquid of the nano particles.
More preferably, the dropping rate is 0.005 to 0.02mL/s.
More preferably, the obtained nanoparticle mixed solution is centrifugally washed to obtain the pH-responsive starch nanoparticles, wherein the centrifugal speed is 3000-5000 rpm, the centrifugal time is 3-5 min, and the washing times are 3-5 times.
Preferably, the concentration of the lipase solution in the step (2) is 2-10 mg/mL, the pH is 6-8, and the solvent is phosphate buffer solution; the concentration of the phosphate buffer solution is 0.01-0.03 mol/L. More preferably 4 to 6mg/ml.
Preferably, the mass ratio of the pH-responsive starch nanoparticles to the lipase in the step (2) is 1g: 40-200 mg.
Preferably, the temperature of the mixing reaction in the step (2) is 20-40 ℃ and the time is 1-3 h.
Preferably, in the centrifugal washing in the step (2), the centrifugal speed is 3000-5000 rpm, the centrifugal time is 3-5 min, and the washing times are 5-15.
Preferably, the preparation method of the pH-responsive enzyme-loaded starch granule with the controllable enzyme distribution position comprises the following steps:
(1) Gelatinizing starch, adding ammonium persulfate and sodium bisulfite under the protection of nitrogen or inert gas, adding a cross-linking agent and a compound with pH responsiveness, performing grafting reaction for 1-3 h at the temperature of 60-80 ℃, performing centrifugal washing, performing ethanol granulation, and performing centrifugal washing to obtain pH responsiveness starch nanoparticles;
(2) Mixing the pH-responsive starch nanoparticles with a lipase solution with the concentration of 2-10 mg/mL and the pH of 6-8 at 20-40 ℃ for reaction for 1-3 h, and carrying out centrifugal washing to obtain the pH-responsive enzyme-loaded starch particles with controllable enzyme distribution positions.
The pH-responsive enzyme-loaded starch granules with controllable enzyme distribution positions are prepared by the method.
According to the pH-responsive enzyme-loaded starch granule with controllable enzyme distribution positions, the base material is obtained by starch grafting modification granulation, the pH responsiveness is realized, the lipase is adsorbed on the outer surface, the catalytic activity is certain, the enzyme amount with different concentrations is added, the granules with different enzyme distribution structures are prepared, and the high-efficiency catalysis of the enzyme is realized.
The invention is based on that the glucose unit of the starch contains rich hydroxyl, namely under mild conditions, the hydroxyl on the starch can be modified and grafted with some groups, so that the starch has some special properties. Therefore, the pH responsiveness is imparted to the starch by grafting a pH responsive group to the starch by means of a radical reaction, and then the immobilization of the lipase enzyme is realized by the hydrogen bond adsorption in an aqueous solution after the obtained granules are granulated with ethanol.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1) The preparation of the enzyme-loaded starch granules does not need a complex process, has mild conditions and simple and convenient operation, and greatly keeps the activity of the enzyme.
2) The pH responsive starch enzyme-carrying particles with controllable enzyme distribution positions are utilized to realize interface catalysis in emulsion, the catalyst can be recycled by adjusting pH, and the enzyme is distributed on the particle surface to realize high-efficiency catalysis.
Drawings
FIG. 1 shows the difference in enzyme activity between particles with different enzyme loading rates.
FIG. 2 shows the enzyme distribution positions of the particles with different enzyme loading rates.
FIG. 3 is a comparison of catalytic effect of particles with different enzyme loading rates compared to silica and calcium carbonate particles at the same enzyme loading rate.
FIG. 4 is a pH stability test of the enzyme-loaded starch granules and free enzyme in example 3.
FIG. 5 is a temperature stability test of the enzyme-loaded starch granules and free enzyme of example 3.
FIG. 6 is a pH-responsiveness test of the starch enzyme-loaded particles in example 3 to form a Pickering emulsion.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The raw materials, reagents and the like which are not indicated for manufacturers are all conventional products which can be obtained by commercial purchase.
Example 1
(1) 100mL of 30mg/mL corn starch aqueous solution is placed into a four-necked flask, the temperature is raised to 95 ℃, the mixture is stirred for 30 minutes, the obtained gelatinized starch is cooled, nitrogen is added for 30 minutes, 3mL of ammonium persulfate (10 mg/mL) aqueous solution and 2mL of sodium bisulfite (10 mg/mL) aqueous solution are added, and the mixture is kept at 60 ℃ for 10 minutes. Adjust pH =8, add crosslinker 0.1gn n, n-methylenebisacrylamide and pH responsive group 2mL dimethylaminoethyl methacrylate, hold for 1 hour. The mixture was washed by centrifugation with water at 3000rpm for 3min for 3 washes.
Ethanol granulation: and (3) sucking 1mL of precipitate, adding the precipitate into 20mL of absolute ethyl alcohol at the speed of 0.02mL per 2s one drop, stirring at 800rpm, and after granulation is successful, centrifuging at 3000rpm for 3min for 3 times, and washing for 3 times to obtain the pH-responsive starch nanoparticles.
(2) Enzyme grafting: adding 1g of pH-responsive starch nanoparticles into 20ml of lipase phosphate buffer solution (pH =7, phosphate concentration is 0.02 mol/L), the concentration of lipase is 2mg/ml, the temperature is 20 ℃, pH =6, enzyme mixing is carried out for 1h,3000rpm, the centrifugation time is 3min, and the washing times are 5 times until protein is not contained in supernatant, so as to obtain the pH-responsive starch enzyme-loaded particles with controllable enzyme distribution positions.
Example 2
(1) 100mL of a 40mg/mL corn starch aqueous solution was placed in a four-necked flask, heated to 95 ℃ and stirred for 30 minutes, and after the resulting gelatinized starch was cooled down, nitrogen was purged with 30 minutes, 3mL of an ammonium persulfate (20 mg/mL) aqueous solution and 2mL of a sodium bisulfite (20 mg/mL) aqueous solution were added, and the mixture was held at 70 ℃ for 10 minutes. The pH was adjusted to =9, 0.2gN, N-methylenebisacrylamide as a crosslinking agent and 4mL of dimethylaminoethyl methacrylate as a pH responsive group were added, and the mixture was held for 2 hours. The mixture was washed by centrifugation with water at 4000rpm for 4min for 4 washes.
Ethanol granulation: sucking 1mL of precipitate, adding the precipitate into 30mL of absolute ethyl alcohol at the speed of 0.02mL per 2s drop, stirring at 1000 revolutions, and after granulation is successful, centrifuging at 4000rpm for 4min, and washing for 4 times to obtain the pH-responsive starch nanoparticles.
(2) Enzyme grafting: adding 1g of pH-responsive starch nanoparticles to 20ml of a lipase phosphate buffer solution (pH =7, phosphate concentration of 0.02 mol/L), lipase concentration of 4mg/ml, temperature of 30 ℃, pH =7, enzyme mixing of 2h,4000rpm, centrifugation time of 4min, and washing times of 10 times until protein is not contained in the supernatant, so as to obtain the pH-responsive starch-loaded enzyme particles with controllable enzyme distribution positions.
Example 3
(1) 100mL of a 50mg/mL corn starch aqueous solution was placed in a four-necked flask, heated to 95 ℃ and stirred for 30 minutes, and after the resulting gelatinized starch was cooled down, nitrogen was purged with 30 minutes, 3mL of an ammonium persulfate (30 mg/mL) aqueous solution and 2mL of a sodium bisulfite (30 mg/mL) aqueous solution were added, and the mixture was held at 80 ℃ for 10 minutes. Adjust pH =10, add crosslinker 0.3g n, n-methylenebisacrylamide and pH-responsive group 6mL dimethylaminoethyl methacrylate, and hold for 3 hours. The mixture was washed by centrifugation with water at 5000rpm for 5min for 5 washes.
Ethanol granulation: and (3) sucking 1mL of precipitate, adding the precipitate into 40mL of absolute ethyl alcohol at the speed of 0.02mL per 2s one drop, stirring at 1200 rpm, and after granulation is successful, centrifuging at 5000rpm for 5min for 5 times, and washing for 5 times to obtain the pH-responsive starch nanoparticles.
(2) Enzyme grafting: adding 1g of pH-responsive starch nanoparticles into 20ml of lipase phosphate buffer solution (pH =7, phosphate concentration is 0.02 mol/L), the concentration of lipase is 6mg/ml, the temperature is 40 ℃, pH =8, enzyme mixing is carried out for 3h,5000rpm, the centrifugation time is 5min, and the washing times are 15 times until protein is not contained in supernatant, so as to obtain the pH-responsive starch enzyme-loaded particles with controllable enzyme distribution positions.
Example 4
In this example, the concentration of lipase in step (2) was 8mg/ml, and other steps and conditions were the same as those in example 1.
Example 5
In this example, the concentration of lipase in step (2) was 10mg/ml, and other steps and conditions were the same as those in example 1.
Determination of Material Properties
(1) Determination of enzyme Activity
The enzyme activity is measured as follows:
solution A:30.0mg of p-nitrophenylpalmitate was dissolved in 10.0mL of isopropanol (8.89 mmol/L).
Solution B: tris-HCl buffer (50 mmol/L) at pH 8.0.
The enzyme activity determination method of the immobilized enzyme comprises the following steps: adding 1mL of A into 9mL of B, shaking uniformly, preheating for 5min at 37 ℃ in a shaking table, adding 0.1g of immobilized enzyme (obtained in the step (2) in the embodiment), shaking uniformly, reacting for 10min at 37 ℃, finally adding 50.0mL of 95% ethanol water solution (ethanol accounts for 95% of volume) to terminate the reaction, centrifuging the obtained mixed solution for 10min at 4000r/min, taking supernatant, measuring absorbance at 410nm, and repeating the three times to obtain an average value. 1 unit of enzyme activity is defined as: at 37 ℃, the enzyme amount required for producing 1.0 mu moL of p-nitrophenol by catalyzing and hydrolyzing p-nitrophenylpalmitate (p-NPP) with immobilized enzyme within 1min is 1 enzyme activity unit (U).
As can be seen from FIG. 1, the pH-responsive starch enzyme-carrying granular materials of examples 1 to 3 differ in their properties due to the change in the preparation process. But overall, the enzyme activity is higher, indicating better catalysis. Examples 4 to 5 show that the enzyme activity decreases with the increase of the loading rate, which indicates that the catalytic effect of a part of enzymes is not fully exerted, and the interface catalytic efficiency decreases.
(2) Determination of CLSM
The laser confocal measurement is as follows:
to determine how the enzyme is distributed on the enzyme-carrying particles, the lipase was first stained with FITC, samples were prepared from the stained lipase according to the procedure, and the samples were observed under a confocal laser microscope.
As can be seen from fig. 2, due to the change of the preparation process, examples 1 to 5 show different distribution structures, and as the enzyme loading rate increases, the enzyme permeates from outside to inside, which is shown in the fact that the enzyme is firstly distributed on the outer surface of the granule, then gradually permeates into the granule, the center of the granule is changed from black to green, and finally the whole granule is filled with the enzyme, the fluorescence intensity curve also shows the phenomenon that the surface of the granule is strong and the inside of the granule is weak, and the fluorescence intensity in the granule is slowly raised, mainly because the granule obtained by ethanol granulation is mixed with the enzyme, the enzyme molecules firstly contact the surface of the granule, when the enzyme concentration is low, the enzyme molecules do not permeate into the inside, a layer of enzyme molecule is formed on the surface of the granule, and as the enzyme concentration increases, the enzyme molecules begin to permeate into the granule, and gradually fill the whole granule; this explains why the enzyme activity of the granules decreases at high loading rates because a part of the enzyme penetrates into the granules to form a large mass transfer resistance between the substrate and the enzyme, and the catalytic efficiency of the enzyme inside decreases compared to that of the enzyme on the surface, so that the enzyme activity gradually decreases. And at a lower loading rate, the enzyme molecules are distributed on the surface of the particles and can be fully utilized so as to show higher enzyme activity.
(3) Determination of the conversion of the product
The conversion rate is determined as follows:
aqueous solutions (5 mL), n-butanol (4 mL) and 140ul of vinyl acetate, each containing silica-loaded particles, calcium carbonate-loaded particles (enzyme loading same as in the examples) and enzyme-loaded starch particles prepared in the examples, were added to a clean glass vial at room temperature, followed by a small amount of NaOH solution (1 moL L) -1 ) The pH value is adjusted to 9 or more. The Pickering emulsion was homogenized using an ULTRATURRAX T25 homogenizer from IKA, germany, at a stirring speed of 15000rpm for a homogenization time of 2 minutes. The reaction was stirred at 100rpm for a period of time at room temperature. After the reaction was completed, 0.06ml of hydrochloric acid (0.5 moL L) was added to the emulsion -1 ) Breaking the emulsion. After complete phase separation, the resulting upper oil phase was carefully separated by decantation and the yield was determined by liquid chromatography.
As can be seen from FIG. 3, the conversion rates of examples 1-5 were always higher than those of the silica-supported enzyme particles and the calcium carbonate-supported enzyme particles under the same enzyme loading, mainly because the enzyme-supported particulate materials obtained in the examples can highly concentrate enzymes on the particle surface to realize high-efficiency catalysis, while the silica-supported enzyme particles and the calcium carbonate-supported enzyme particles have no such performance and show lower catalytic effects.
(4) Stability test
Ability to resist pH
In order to investigate the pH stability of the pH-responsive enzyme-loaded starch granules and free enzyme in which the distribution position of the enzyme is controllable in example 3, the recovery rates of the enzyme activity at pH 3.0 to 10.0 were examined, respectively, and the results were compared. After incubating the pH-responsive enzyme-loaded starch granules with controllable enzyme distribution position of example 3 and the free enzyme in a buffer solution with pH of 3-10.0 at 25 ℃ for 24h, the enzyme activities of the pH-responsive enzyme-loaded starch granules with controllable enzyme distribution position of example 3 and the free enzyme were determined as described above. The initial enzyme activity was defined as 100%, and the relative recovery of enzyme activity was calculated for example 3 and the free enzyme under pH conditions.
As shown in fig. 4, the recovery rates of the free lipase and the enzyme of example 3 reach the maximum at pH =8.0, and the recovery rate of the enzyme of example 3 is much greater than that of the free lipase in the whole pH range. From the trend, example 3 is more gradual than free lipase, which shows that example 3 has stronger pH stability and higher acid and alkali resistance compared with free lipase. In a broader pH range (4.0-10), example 3 showed stronger enzymatic activity (> 50% relative activity), whereas the activity of free lipase decreased rapidly as the pH was away from 8.0. This phenomenon is due to the change in the structure of the enzyme active center resulting from the change in the microenvironment provided by the starch carrier, making the enzyme "rigid" a possible cause of this phenomenon.
Capability of resisting temperature
In order to investigate the thermostability of the pH-responsive enzyme-loaded starch granules with controllable enzyme distribution positions of example 3, the recovery rates of enzyme activity of example 3 at a temperature of 20 to 90 ℃ were respectively investigated and compared with that of free lipase. After incubating example 3 and the free enzyme at 20-90 ℃ for 24h, respectively, the enzyme activities of example 3 and the free enzyme were determined as described above. The initial enzyme activity was defined as 100%, and the relative recovery rates of enzyme activity of example 3 and the free enzyme under the corresponding temperature conditions were calculated.
As shown in fig. 5, the recovery rates of the free lipase and the enzyme activity of example 3 are significantly reduced in the whole temperature range, but the recovery rate of the enzyme activity of example 3 is much greater than that of the free lipase, which fully indicates that example 3 has higher thermal stability than the free lipase. The main reason is that the multipoint combination of the starch carrier and the active group in the enzyme protein can further stabilize the active center structure of the enzyme protein, can effectively prevent the protein from unfolding, and can maintain the space conformation required by enzyme catalysis, thereby effectively maintaining the catalytic activity of the immobilized enzyme.
(5) Test of pH responsiveness
To investigate the pH responsiveness of the enzyme-loaded starch granules with controllable enzyme distribution site of example 3, an aqueous solution (5 mL) and n-butanol (4 mL) each containing the granules of example 3 were added to a clean glass bottle at room temperature, followed by a small amount of NaOH solution (1 moL L-1) to adjust the pH to 9 or more. The Pickering emulsion was formed by using an ULTRATURRAX T25 homogenizer of IKA, germany, with a stirring speed of 15000rpm and a homogenization time of 2 minutes, and after the emulsion was left to stand for 24 hours, the morphology of the emulsion was observed under a microscope, and then 0.06ml of hydrochloric acid (0.5 moL L) was added to the emulsion -1 ) After the emulsion broke within 30min, naOH solution (1 moL L) was added -1 ) And after the emulsion is formed again and placed for 24 hours, observing the appearance of the emulsion under a microscope. This was repeated eight times for one cycle.
As shown in FIG. 6, after the eighth time, it can be seen that the pH remained very sensitive and the formed emulsion was uniformly stable, mainly because the tertiary amine group on the (DMAEMA) group was protonated under acidic conditions to impart hydrophilicity to the particles, which resulted in the breaking of Pickering emulsion, and when base was added to the solution, the tertiary amine group lost H + Deprotonation occurs and the particles are therefore hydrophobic, which stabilizes the emulsion, forming a stable Pickering emulsion.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. A preparation method of pH-responsive enzyme-loaded starch granules with controllable enzyme distribution positions is characterized by comprising the following steps:
(1) Gelatinizing starch, performing free radical grafting reaction on the starch, a cross-linking agent and a compound with pH responsiveness, and performing ethanol granulation to obtain pH responsiveness starch nanoparticles;
(2) Mixing the pH-responsive starch nanoparticles with a lipase solution for reaction, and carrying out centrifugal washing to obtain pH-responsive enzyme-loaded starch particles with controllable enzyme distribution positions;
the concentration of the lipase solution in the step (2) is 2-6 mg/mL;
the cross-linking agent in the step (1) is at least one of N, N-methylene bisacrylamide and hexamethylene bisacrylamide; the compound with pH responsiveness is at least one of dimethylaminoethyl methacrylate, ethylene glycol methacrylate and diethylaminoethyl methacrylate;
the pH value of the lipase solution in the step (2) is 6-8, and the solvent is phosphate buffer solution; the mass ratio of the pH-responsive starch nanoparticles to the lipase in the step (2) is 1g: 40-120 mg;
the temperature of the mixing reaction in the step (2) is 20-40 ℃, and the time is 1-3 h;
the proportion of the starch, the cross-linking agent and the compound with pH responsiveness in the step (1) is (3-5) g: (0.1-0.3) g: (2-6) ml; the pH value of the free radical grafting reaction in the step (1) is 8-10, the temperature is 60-80 ℃, and the time is 1-3 h;
the free radical grafting reaction in the step (1) is carried out under the action of a catalyst, the catalyst is ammonium persulfate and sodium bisulfite, and the mass ratio of the starch to the ammonium persulfate to the sodium bisulfite is (3-5) g: (30 to 90) mg: (20-60) mg;
the ethanol granulation in the step (1) refers to that the product is prepared by mixing the raw materials according to the volume ratio of 1: 20-40 is dripped into ethanol, and then is stirred at 800-1200 r/min to obtain nano-particle mixed liquid; the dropping speed is 0.005-0.02 mL/s;
and (2) carrying out the free radical grafting reaction in the step (1) under the protection of nitrogen or inert gas.
2. The method for preparing the pH-responsive enzyme-loaded starch granule with controllable enzyme distribution position according to claim 1, wherein the starch gelatinization in the step (1) refers to gelatinization at 95 ℃ for 30 minutes after mixing starch and water according to a ratio of 30-50 mg/ml.
3. The method for preparing the pH-responsive enzyme-loaded starch granules with controllable enzyme distribution positions according to claim 1, wherein after the free radical grafting reaction in the step (1) is finished, the product mixed solution is subjected to centrifugal washing to obtain a product, wherein the centrifugal speed is 3000-5000 rpm, the centrifugal time is 3-5 min, and the washing times are 3-5 times; in the centrifugal washing in the step (2), the centrifugal speed is 3000-5000 rpm, the centrifugal time is 3-5 min, and the washing times are 5-15.
4. A pH-responsive enzyme-loaded starch granule having a controlled enzyme distribution according to any one of claims 1 to 3.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010226966A (en) * | 2009-03-25 | 2010-10-14 | Ishikawa Prefecture | Starch granule or cellulose powder immobilized lipase, and method for producing fat or fatty oil reaction product |
| CN110358759A (en) * | 2019-06-25 | 2019-10-22 | 华南协同创新研究院 | A kind of Pickering lotion and preparation method thereof for encapsulating lipase |
| CN112029758A (en) * | 2020-08-12 | 2020-12-04 | 华南理工大学 | Multi-enzyme immobilization material and preparation method and application thereof |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010226966A (en) * | 2009-03-25 | 2010-10-14 | Ishikawa Prefecture | Starch granule or cellulose powder immobilized lipase, and method for producing fat or fatty oil reaction product |
| CN110358759A (en) * | 2019-06-25 | 2019-10-22 | 华南协同创新研究院 | A kind of Pickering lotion and preparation method thereof for encapsulating lipase |
| CN112029758A (en) * | 2020-08-12 | 2020-12-04 | 华南理工大学 | Multi-enzyme immobilization material and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 基于Pickering乳液模板微反应器的构建及其催化性能研究;齐亮;《中国优秀博硕士学位论文全文数据库(博士) 工程科技I辑 (月刊)》;20200115;第I页第1-2段、第28页第1-2段、第29页倒数第2段、第46-49页、第53页第1-4段 * |
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