CN-122011202-A - Microbial mineralization preparation method of high-load serum albumin-mineral composite carrier

Abstract

The invention provides a microbial mineralization preparation method of a high-load serum albumin-mineral composite carrier, belonging to the field of intersection of biological medicine and microbial technology. According to the invention, microorganisms with mineralization function, serum albumin and mineralization liquid containing calcium source and urea are taken as a reaction system, under mild conditions, the microorganisms hydrolyze urea to generate carbonate radicals and release the carbonate radicals to the outside, calcium ions are actively transported to the outside through a calcium ion pump, the local microenvironment pH and the ion saturation are regulated, the formation of a calcium carbonate precursor is slowly and continuously driven, and meanwhile, the serum albumin is fully embedded into a mineral precursor to provide nucleation sites and organic templates, and the formation of a serum albumin-amorphous calcium carbonate mineral phase composite carrier is regulated and stabilized, so that the high load and uniform distribution of the serum albumin in the carrier can be realized, and the natural structure of the serum albumin can be effectively maintained.

Inventors

• YAO DUNFAN
• SONG JIA
• YE HAN
• Qing Dazhou
• GAO XINYUAN

Assignees

• 湖北科技学院

Dates

Publication Date

20260512

Application Date

20260210

Claims (10)

1. 1. A method for preparing a high-load serum albumin-mineral composite carrier by microbial mineralization, characterized in that the mineral is an amorphous mineral, comprising the following steps: s1, activating a microorganism bacterial liquid with mineralization to obtain an activated microorganism bacterial liquid; S2, mixing the serum albumin solution with a mineralization solution containing a calcium source and urea to obtain a substrate solution; S3, mixing the activated microorganism bacterial liquid with the substrate solution, and adjusting the pH value to 6.0-10.0 to perform a microorganism-mediated co-mineralization reaction to obtain the serum albumin-amorphous mineral composite carrier, wherein the co-mineralization reaction comprises the following parameters of 28-32 ℃ of reaction temperature, 20-28 h of co-mineralization time and 160-180 rpm of oscillation rate.
2. 2. The method of mineralizing microorganisms on a high-load serum albumin-mineral composite carrier according to claim 1, wherein the bacteria in the microbial liquid comprise at least one of sarcina bardans, bacillus geourea, bacillus sphaericus and bacillus subtilis.
3. 3. The method of mineralizing microorganisms on a high-load serum albumin-mineral composite carrier according to claim 2, wherein the storage number of the bacillus stearothermophilus is ATCC 11859, the storage number of the urea bacillus is CGMCC 1.7272, the storage number of the bacillus sphaericus is CGMCC 1.1359, and the storage number of the bacillus subtilis is ATCC 6633.
4. 4. The method of microbiological mineralization of a high load serum albumin-mineral composite carrier according to claim 1, wherein the final concentration of serum albumin in the substrate solution is 0.5-5 mg/mL, the final concentration of Ca 2+ is 16-24 mmol/mL, and the final concentration of urea is 16-24 mmol/mL.
5. 5. The method of mineralizing microorganisms with high-load serum albumin-mineral composite carrier according to claim 4, wherein the serum albumin in the serum albumin solution comprises at least one of human serum albumin and bovine serum albumin.
6. 6. The method of microbiological mineralization of high load serum albumin-mineral composite carriers according to claim 1, wherein the calcium source is calcium chloride.
7. 7. The method for mineralizing microorganisms on a high-load serum albumin-mineral composite carrier according to claim 1, wherein the volume ratio of the activated microorganism bacterial liquid to the substrate solution is (1-5): 1.
8. 8. The method for mineralizing microorganisms on a high-load serum albumin-mineral composite carrier according to claim 1, wherein the final OD value of the mixed solution of the activated microorganism bacterial liquid and the substrate solution is 1.0-3.0.
9. 9. The method of microbial mineralization of high load serum albumin-mineral composite carriers according to claim 1, characterized in that the serum albumin loading in the serum albumin-mineral composite carrier is >95%.
10. 10. The method of microbiological mineralization of a high load serum albumin-mineral composite carrier according to claim 1, characterized in that the mineral phase in the serum albumin-mineral composite carrier is amorphous calcium carbonate.

Description

Microbial mineralization preparation method of high-load serum albumin-mineral composite carrier Technical Field The invention relates to the crossing field of biological medicine and microbial technology, in particular to a microbial mineralization preparation method of a high-load serum albumin-mineral composite carrier. Background Serum albumin is a natural transport protein in blood, and its main physiological function is to reversibly bind to various endogenous substances and exogenous molecules and then transport to various tissues in the body. The albumin drug delivery system has the advantages of biodegradability, non-immunogenicity, biocompatibility and the like. The prior art in the field of albumin delivery has heretofore been principally desolvation, emulsification, thermogelation, self-assembly, nab techniques. In general, both desolvation and emulsification require the addition of organic solvents and cross-linking agents. While the thermal gelation, self-assembly and Nab techniques do not require cross-linking agents, the self-crosslinking process employed by them alters the conformation and structure of the protein. Among them, the protein loading by dynamic covalent bond-based chemical crosslinking method was up to 51 wt% (Su S, wang Y, du F S, et al Advanced Functional Materials, 2018, 28: 1805287). However, this method essentially relies on covalent modification of proteins, the process of which involves a synthetic cross-linker with multi-step chemical reactions, and has the disadvantages of low long-term biosafety and complex processes. Thus, there is an urgent need to develop improved or partial replacement protein delivery technologies that are non-destructive, highly biosafety over long periods of time, and simple processes. Inspired by biomineralization theory, mineral-based protein delivery technologies are receiving widespread attention for their good biocompatibility and degradability. The technology simulates the biomineralization process in the natural world, and regulates and controls mineral precipitation through non-covalent interactions such as static electricity, hydrogen bonds and the like by utilizing the functional groups of proteins. The process avoids exogenous chemical modification, and maintains the natural conformation and biological activity of the protein to the greatest extent. Among them, amorphous minerals are considered as potential protein carrier materials due to their high specific surface area and adsorption capacity. Literature (Lei J S, zheng Y, meng Y F, et al Advanced Functional Materials, 2022, 32 (26): 2202928.) reports that Amorphous Calcium Carbonate Phosphate (ACCP) carriers constructed by biomimetic mineralization strategies can achieve protein loadings up to 37.2 wt%, significantly higher than most conventional delivery systems (about 10 wt%). However, this biomimetic mineralization is essentially a chemical co-precipitation process, with rapid reactions. Protein loading in amorphous minerals, while it can regulate mineral morphology to some extent and stabilize the amorphous phase, its interaction with minerals is difficult to adequately perform in chemical transient precipitation, resulting in limited regulation of mineral structure and morphology. This severely limits the loading of proteins in amorphous minerals, limiting the use of mineral-based protein carriers. Therefore, development of a mineralization method which is more moderate in reaction, allows proteins to fully participate in and regulate the mineralization process is expected to greatly improve protein loading, but no effective technical method is seen at present. Disclosure of Invention The invention provides a microbial mineralization preparation method of a high-load serum albumin-amorphous mineral composite carrier, which is used for realizing the formation of the serum albumin-amorphous mineral composite carrier and the effective maintenance of the high-efficiency load and the natural structure of the serum albumin. The embodiment of the invention provides a microbial mineralization preparation method of a high-load serum albumin-amorphous mineral composite carrier, which comprises the following steps: s1, activating a microorganism bacterial liquid with mineralization to obtain an activated microorganism bacterial liquid; S2, mixing the serum albumin solution with a mineralization solution containing a calcium source and urea to obtain a substrate solution; S3, mixing the activated microorganism bacterial liquid with the substrate solution, and adjusting the pH value to 6.0-10.0 to perform a microorganism-mediated co-mineralization reaction to obtain the serum albumin-amorphous mineral composite carrier, wherein the co-mineralization reaction comprises the following parameters of 28-32 ℃ of reaction temperature, 20-28 h of co-mineralization time and 160-180 rpm of oscillation rate. Preferably, the bacteria in the microbial liquid comprise at least one of sarcina bardana,