A preparation method of edible microcarrier based on albumin

Abstract

The present invention discloses a method for preparing an edible microcarrier based on albumin, which belongs to the technical field of albumin, and comprises the following steps: S1: modifying, preparing an albumin aqueous solution with a certain mass concentration and N ‑ethylmaleimide dimethyl sulfoxide solution to obtain a mixed solution; triethylamine is added to the mixed solution, dialyzed for 12 hours after the reaction, and modified albumin is obtained after freeze drying, and stored in a refrigerator at ‑20°C for standby use; S2: assembling, configuring acetic acid ‑ sodium acetate buffer solution, dissolving the modified albumin prepared in S1 in the above-mentioned buffer solution, and obtaining a modified albumin solution; S3: purifying, using a centrifuge to centrifuge the albumin assembly to obtain the assembled albumin particles, washing the microcarrier particles with deionized water, and centrifuging three times to obtain a finished product. The present invention can solve the problems caused by the residual raw materials, cross-linking agents and emulsifiers, and the prepared albumin assembly has good structural stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of albumin, and in particular relates to a method for preparing an edible microcarrier based on albumin. Background Art

[0002] Cell cultured meat is a meat analog produced by culturing animal cells in vitro and combining tissue engineering technology. Compared with traditional animal husbandry, it has many advantages such as reducing resource consumption and greenhouse gas emissions. In the manufacturing process of cell cultured meat, cell proliferation depends on suitable microcarriers.

[0003] In the prior art, microcarriers are mainly synthesized through oil-in-water emulsions: usually, water-soluble raw materials, hydrophobic solvents, crosslinkers and emulsifiers are mixed and stirred at a certain speed to form a uniform emulsion, and then microcarrier particles are obtained through a certain phase separation operation. Although this method can achieve rapid preparation and scale-up production of microcarriers, it also brings a series of defects in raw material selection and emulsifier / crosslinker residues. These defects have a great impact on the use effect of microcarriers and the food safety of cell cultured meat in the later stage, and cannot be widely used in the field of cell culture and food. Summary of the invention

[0004] In order to solve the above technical problems, the inventors have developed a method for preparing edible microcarriers based on albumin based on their rich experience in the field of albumin technology.

[0005] The preparation principle of the present invention is:

[0006] 1. Modification:

[0007] The chemical modification of albumin is based on the click reaction of carbon-carbon double bonds and thiol groups. This reaction has the advantages of simple operation, mild conditions and high yield. The click reaction of carbon-carbon double bonds and thiol groups is a very classic reaction example. It can achieve a reaction efficiency close to 100% in a short time, so it is often used in chemical modification. Albumin is a helical protein containing three homologous α-helical domains. The three domains gather together to form an asymmetric heart-shaped structure. Each domain contains two subdomains, and each subdomain is composed of 6 helical structures. See the attached Figure 1 .

[0008] According to relevant literature reports, the IA subdomain of albumin contains a cysteine ​​residue (Cys), and the IA subdomain is exposed in the entire albumin structure, so the cysteine ​​residue is an easy modification site. Subsequently, due to the presence of a thiol functional group on the cysteine ​​residue, a click chemistry reaction based on the thiol and carbon-carbon double bond was used to modify the cysteine ​​residue. N-Ethylmaleimide is linked to the albumin molecule to obtain modified albumin, see Figure 1 .

[0009] 2. Assembly

[0010] Due to the presence of ethyl on the nitrogen atom, the five-membered ring structure formed by the addition of thiol to the double bond has a large ring tension, and is prone to ring opening reaction under the attack of nucleophilic reagents (such as water); furthermore, the basic principle of the assembly of modified albumin and pure albumin is that the modified albumin contains -NH 2 + -group, so the modified albumin is positively charged on a macroscopic level; the isoelectric point of pure albumin is pI=4.6, and it is negatively charged in a neutral (pH=7.4) environment. Therefore, there is a strong positive and negative electrical attraction between the two substances, and they can be assembled by electrostatic force, which is manifested in the form of granular substances after aggregation on a macroscopic level, namely the obtained microcarriers.

[0011] After the modification of albumin is completed, the modified albumin is dissolved in a weakly acidic aqueous solution (pH=4.6). N The five-membered ring formed by the addition of -ethylmaleimide to the thiol group on albumin is unstable in aqueous solution and is easily hydrolyzed to obtain carboxyl and amide groups. In an acidic environment, the secondary amine structure in the amide group is very easy to protonate to form a positively charged -NH 2 + - Group, see Figure 2 .

[0012] At this time, a PBS aqueous solution of pure albumin (pH = 7.4) was prepared and gradually added to the weakly acidic aqueous solution of the modified albumin to complete the protein assembly through electrostatic interaction. Figure 3 .

[0013] 3. Purification

[0014] After the assembly is completed, the microcarrier particles are obtained by centrifugation using a low-temperature refrigerated high-speed centrifuge, and then the supernatant is discarded, and a certain amount of deionized water is added and vortexed for washing three times. After centrifugation, the final product is obtained by freeze drying.

[0015] Therefore, innovation in the preparation method of microcarriers has become the best way to break through the above technical bottlenecks. Protein assemblies are protein complexes formed by one or more proteins through covalent linkage, intermolecular forces or hydrogen bonds. They are simple to prepare, have no additives and have good biocompatibility. They are mainly used in the field of drug delivery. Protein, as a natural biopolymer, is widely used in the preparation of microcarrier materials. In summary, the preparation of microcarriers using protein assembly methods has theoretically become a new strategy. This strategy not only meets the preparation requirements of cell-cultured meat in terms of raw material selection, but also avoids the food safety risks caused by the addition of emulsifiers / cross-linking agents.

[0016] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing an edible microcarrier based on albumin, comprising the following steps:

[0017] S1: Modification, preparation of albumin aqueous solution with a certain mass concentration and N -ethylmaleimide dimethyl sulfoxide solution, start magnetic stirring, N -ethylmaleimide dimethyl sulfoxide solution is gradually added dropwise to the albumin aqueous solution to obtain a mixed solution; a certain amount of triethylamine is added to the mixed solution, and after reacting at room temperature for 12 hours, 100 mL of deionized water is added to the system, and dialyzed for 12 hours using a dialysis bag with a molecular weight cutoff of 3000 kDa, and the modified albumin is obtained after freeze-drying, and stored in a refrigerator at -20°C for use;

[0018] S2: assembling, preparing a certain amount of acetic acid-sodium acetate buffer solution, controlling the pH value to 4.6, dissolving the modified albumin prepared in S1 in the above buffer solution to obtain a modified albumin solution; starting magnetic stirring, dropping the pure albumin PBS solution into the modified albumin solution, measuring the pH value of the entire system during the dropping process, and stopping the dropping when the pH value is close to 7.0;

[0019] S3: Purification, centrifugation of albumin assembly using a low-temperature refrigerated high-speed centrifuge at a temperature of 4 o C. After the assembled albumin particles are obtained, the microcarrier particles are washed with deionized water and centrifuged, and the operation is repeated three times to obtain the finished product, which is freeze-dried and stored for characterization.

[0020] Furthermore, in S1, the mass concentration of the albumin aqueous solution was 5 mg / mL, and deionized water was used in the preparation process; NThe mass concentration of the -ethylmaleimide dimethyl sulfoxide solution is 5 mg / mL, and the dimethyl sulfoxide used is anhydrous dimethyl sulfoxide; the magnetic stirring speed is 800 rpm; the amount of triethylamine used is 2-3 mL; the cooling rate during the freeze-drying process is 0.5°C / min to 2°C / min, the vacuum degree is 1-5 Pa, and the freeze-drying time is 24 hours.

[0021] Furthermore, in S2, the preparation method of acetic acid-sodium acetate buffer solution is to take 18 g of sodium acetate, add 9.8 mL of acetic acid, and finally dilute to 1 L with water; the mass concentration ratio of the modified albumin solution to the mass concentration ratio of the pure albumin PBS solution is 1:1; the magnetic stirring speed is 1000 rpm / min.

[0022] Furthermore, the low-temperature refrigerated high-speed centrifuge has a rotation speed of 10,000 rpm and a time of 30 minutes.

[0023] Furthermore, in step 2, when the pH value approaches 7.0, the entire system becomes turbid, which is visible to the naked eye. The pH meter used is a Leiji pH meter (model PHS-3G)

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention selects albumin as the raw material and prepares the microcarrier by using a method in which modified albumin is easy to form an assembly under certain physical and chemical conditions, thereby fundamentally avoiding the problems caused by the residues of raw materials, cross-linking agents and emulsifiers;

[0026] 2. Based on the characteristics of the chemical structure of albumin, the present invention artificially modifies albumin and uses the modified albumin theorem conditions to prepare an assembly, which has good structural stability and a particle size that meets the requirements for the use of microcarriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Schematic diagram of albumin domains and artificial modifications;

[0028] Figure 2 : Schematic diagram of the hydrolysis and protonation process of modified albumin;

[0029] Figure 3 : Schematic diagram of the assembly process;

[0030] Figure 4 : Schematic diagram of the comparison of particle size distribution of microcarriers (sample 1 is the particles formed by assembling pure albumin, and sample 2 is the microcarriers obtained by assembling the modified albumin prepared in Examples 1-3);

[0031] Figure 5: Schematic diagram of immunofluorescence staining after microcarrier culture for 48 h (A: unmodified albumin microcarrier; B: microcarrier of Example 3);

[0032] Figure 6 : Schematic diagram of cell proliferation curve. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0034] The present invention provides a method for preparing an edible albumin microcarrier, comprising the following steps:

[0035] Example 1 First, 50 mg of albumin was taken and 10 mL of deionized water was added to prepare an albumin aqueous solution with a mass concentration of 5 mg / mL; N 50 mg of N-ethylmaleimide was added to 10 mL of anhydrous dimethyl sulfoxide to prepare a solution with a mass concentration of 5 mg / mL; the dimethyl sulfoxide solution of N-ethylmaleimide was gradually added dropwise to the albumin aqueous solution at a magnetic stirring speed of 800 rpm, followed by the addition of 2 mL of triethylamine. After reacting at room temperature for 12 hours, 100 mL of deionized water was added to the entire reaction system, and the system was placed in a dialysis bag with a molecular weight cutoff of 3000 kDa for 12 hours. After the dialysis was completed, freeze-dried was performed at a cooling rate of 2°C / min, and the modified albumin was finally placed in -20 o C in the refrigerator and wait for the next step.

[0036] Then, 50 mg of modified albumin was added to 5 mL of acetic acid-sodium acetate buffer solution (pH=4.6) to prepare a modified albumin solution with a mass concentration of 10 mg / mL; secondly, 10 mg / mL of pure albumin PBS solution was prepared. The pure albumin PBS solution was added dropwise to the modified albumin solution at a magnetic stirring speed of 1000 rpm. During the addition process, the pH value of the entire system was continuously measured using a pH meter. The addition was stopped when the pH value of the entire system was close to 7.0. At this time, the entire system could be observed to become turbid with the naked eye.

[0037] Again, the turbid liquid was divided into 1.5 mL enzyme-free sterile centrifuge tubes and centrifuged at 4 oThe assembled albumin particles, i.e., microcarrier particles, were obtained by centrifugation under C conditions. The microcarrier particles were washed and centrifuged with 1 mL of deionized water per tube, and the operation was repeated three times. The white precipitate at the bottom of the tube was the final product, and the excess water was removed by freeze drying and stored at -20 o C in a refrigerator awaiting characterization.

[0038] Example 2 First, 70 mg of albumin was taken and 10 mL of deionized water was added to prepare an albumin aqueous solution with a mass concentration of 7 mg / mL; N 70 mg of N-ethylmaleimide was added with 10 mL of anhydrous dimethyl sulfoxide to prepare a solution with a mass concentration of 7 mg / mL; the dimethyl sulfoxide solution of N-ethylmaleimide was gradually added dropwise to the albumin aqueous solution at a magnetic stirring speed of 800 rpm, followed by the addition of 3 mL of triethylamine. After reacting at room temperature for 12 hours, 100 mL of deionized water was added to the entire reaction system, and the system was placed in a dialysis bag with a molecular weight cutoff of 3000 kDa for 12 hours. After the dialysis was completed, the system was freeze-dried at a cooling rate of 0.5 °C / min. Finally, the modified albumin was placed in a -20 o C in the refrigerator and wait for the next step.

[0039] Then, 50 mg of modified albumin was added to 5 mL of acetic acid-sodium acetate buffer solution (pH=4.6) to prepare a modified albumin solution with a mass concentration of 10 mg / mL; secondly, 10 mg / mL of pure albumin PBS solution was prepared. The pure albumin PBS solution was added dropwise to the modified albumin solution at a magnetic stirring speed of 1000 rpm. During the addition process, the pH value of the entire system was continuously measured using a pH meter. The addition was stopped when the pH value of the entire system was close to 7.0. At this time, the entire system could be observed to become turbid with the naked eye.

[0040] Again, the turbid liquid was divided into 1.5 mL enzyme-free sterile centrifuge tubes and centrifuged at 4 o The assembled albumin particles, i.e., microcarrier particles, were obtained by centrifugation under C conditions. The microcarrier particles were washed and centrifuged with 1 mL of deionized water per tube, and the operation was repeated three times. The white precipitate at the bottom of the tube was the final product, and the excess water was removed by freeze drying and stored at -20 o C in a refrigerator awaiting characterization.

[0041] Example 3 First, 100 mg of albumin was taken and 10 mL of deionized water was added to prepare an albumin aqueous solution with a mass concentration of 10 mg / mL; N100 mg of N-ethylmaleimide was added to 10 mL of anhydrous dimethyl sulfoxide to prepare a solution with a mass concentration of 10 mg / mL; the dimethyl sulfoxide solution of N-ethylmaleimide was gradually added dropwise to the albumin aqueous solution at a magnetic stirring speed of 800 rpm, followed by the addition of 3 mL of triethylamine. After reacting at room temperature for 12 hours, 100 mL of deionized water was added to the entire reaction system, and the system was placed in a dialysis bag with a molecular weight cutoff of 3000 kDa for 12 hours. After the dialysis was completed, freeze-dried was performed at a cooling rate of 1°C / min, and the modified albumin was finally placed in a -20 o C in the refrigerator and wait for the next step.

[0042] Then, 100 mg of modified albumin was added to 10 mL of acetic acid-sodium acetate buffer solution (pH=4.6) to prepare a modified albumin solution with a mass concentration of 10 mg / mL; secondly, 10 mg / mL of pure albumin PBS solution was prepared. The pure albumin PBS solution was added dropwise to the modified albumin solution at a magnetic stirring speed of 1000 rpm. During the addition process, the pH value of the entire system was continuously measured using a pH meter. The addition was stopped when the pH value of the entire system was close to 7.0. At this time, the entire system could be observed to become turbid with the naked eye.

[0043] Again, the turbid liquid was divided into 1.5 mL enzyme-free sterile centrifuge tubes and centrifuged at 4 o The assembled albumin particles, i.e., microcarrier particles, were obtained by centrifugation under C conditions. The microcarrier particles were washed and centrifuged with 1 mL of deionized water per tube, and the operation was repeated three times. The white precipitate at the bottom of the tube was the final product, and the excess water was removed by freeze drying and stored at -20 o C in a refrigerator awaiting characterization.

[0044] Test 1

[0045] In order to verify the advantages of modified albumin in the assembly process of this technical solution, the particles assembled from pure albumin without modification were used as the control group to compare the difference in particle size between the two materials. Figure 4 As shown in the report, the particle size distribution of the two materials was tested using a particle size meter. The results showed that the particle size of the particles assembled by pure albumin was mainly distributed at the nanometer level, which basically could not reach the 200-300 microns required by microcarriers; while the particle size of the particles assembled by modified albumin was all distributed in the range of 200-300 microns, with a narrower distribution. This result verifies that it is feasible to prepare microcarrier particles by assembling modified albumin.

[0046] Test 2

[0047] The cytotoxicity and proliferation of albumin microcarriers in chicken myoblasts were characterized by DAPI immunofluorescence staining. First, Example 3 and pure albumin particle dispersions were prepared, where the concentration of both microcarriers was 2 mg / mL. Then, 1×10 5 The cells were seeded in two 12-well plates at a density of 10 cells per well, and the amount of microcarriers added to each well was 2 mL. After co-culture for 48 h, the cells were fixed with 4% paraformaldehyde solution for 10-15 min and then rinsed with PBS. After fixation, DAPI solution (common concentration is 1 µg / mL or 300 nM) was added and incubated at room temperature for 5-10 min to mark the cell nuclei. The cells were rinsed with PBS and the sealing solution containing anti-fluorescence attenuation agent was added. After sealing, the cells were observed under a fluorescence microscope. The cell nuclei stained with DAPI will emit blue fluorescence under ultraviolet light excitation. The results are shown in Figure 2. Figure 5 shown.

[0048] After 48 hours of co-culture, the cell nuclei showed a relatively uniform distribution through DAPI staining, and the cell nuclei did not show obvious signs of apoptosis or necrosis, such as nuclear fragmentation or irregular fluorescence signals. This result shows that the two albumin microcarriers have good cell compatibility and no obvious cytotoxicity. However, there was adhesion between the pure albumin particles, the structure was relatively loose, and the structural stability was poor. It is speculated that the reason is that the pure albumin particles at the nanoscale have aggregated, forming irregular adhesions. Figure 5 A. The particle size of the microcarrier particles formed by the modified albumin assembly is 200-300 microns, which is consistent with the test results in Experiment 1. The entire microcarrier presents a relatively regular spherical shape. The obvious difference between the two results shows the effectiveness of this technical method.

[0049] Test 3

[0050] Cell proliferation was assessed using the CCK-8 kit. CCK-8 assays were performed on days 1, 2, 3, 4, and 5 to determine cell proliferation in the modified albumin microcarriers. The plates were removed on the corresponding days after culture and 10 µL of CCK-8 solution was added. After 4 hours of incubation, 100 µL of the corresponding solution was removed and finally the absorbance was measured at 450 nm using a multi-detector microplate reader. The results are shown in Figure 6 shown.

[0051] The cells on the microcarriers were digested and collected by trypsin, stained with 0.4% trypan blue dye, and the cell viability was determined by cell counting.

[0052] like Figure 6As shown in the figure, the microcarriers co-cultured with chicken myoblasts (1-5 days) showed a good proliferation state, and the trypan blue dye staining results showed that the cell survival rate was higher than 85% from 1 to 5 days. Therefore, the prepared modified albumin microcarriers have good biocompatibility, and the cells can proliferate normally on the microcarriers.

[0053] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing an edible albumin microcarrier, characterized in that: The following steps are involved: S1: Modification, preparation of albumin aqueous solution with a certain mass concentration and N -ethylmaleimide dimethyl sulfoxide solution, start magnetic stirring, N -ethylmaleimide dimethyl sulfoxide solution is gradually added dropwise to the albumin aqueous solution to obtain a mixed solution; a certain amount of triethylamine is added to the mixed solution, and after reacting at room temperature for 12 hours, 100 mL of deionized water is added to the system, and dialyzed for 12 hours using a dialysis bag with a molecular weight cutoff of 3000 kDa, and the modified albumin is obtained after freeze-drying, and stored in a refrigerator at -20°C for use; S2: assembling, preparing a certain amount of acetic acid-sodium acetate buffer solution, controlling the pH value to 4.6, dissolving the modified albumin prepared in S1 in the above buffer solution to obtain a modified albumin solution; starting magnetic stirring, dropping the pure albumin PBS solution into the modified albumin solution, measuring the pH value of the entire system during the dropping process, and stopping the dropping when the pH value is close to 7.0; S3: Purification, centrifugation of albumin assembly using a low-temperature refrigerated high-speed centrifuge at a temperature of 4 o C. After the assembled albumin particles are obtained, the microcarrier particles are washed with deionized water and centrifuged, and the operation is repeated three times to obtain the finished product, which is freeze-dried and stored for characterization.

2. The method for preparing an edible albumin microcarrier according to claim 1, characterized in that: In S1, the mass concentration of the albumin aqueous solution was 5 mg / mL, and deionized water was used in the preparation process; N The mass concentration of the -ethylmaleimide dimethyl sulfoxide solution is 5 mg / mL, and the dimethyl sulfoxide used is anhydrous dimethyl sulfoxide; the magnetic stirring speed is 800 rpm; the amount of triethylamine used is 2-3 mL; the cooling rate during the freeze-drying process is 0.5°C / min to 2°C / min, the vacuum degree is 1-5 Pa, and the freeze-drying time is 24 hours.

3. The method for preparing an edible albumin microcarrier according to claim 1, characterized in that: In S2, the preparation method of acetic acid-sodium acetate buffer solution is to take 18g of sodium acetate, add 9.8mL of acetic acid, and finally dilute it to 1L with water; the mass concentration ratio of the modified albumin solution to the mass concentration ratio of the pure albumin PBS solution is 1:1; the magnetic stirring speed is 1000rpm / min.

4. The method for preparing an edible albumin microcarrier according to claim 1, characterized in that: The speed of the low-temperature refrigerated high-speed centrifuge is 10000 rpm for 30 minutes.

5. The method for preparing an edible albumin microcarrier according to claim 1, characterized in that: In step 2, when the pH value approaches 7.0, the whole system becomes turbid.

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