Microbial mineralization preparation method of high-loading serum albumin-mineral composite carrier

Abstract

The invention provides a microbial mineralization preparation method of a high-loading serum albumin-mineral composite carrier, and belongs to the crossing field of biological medicine and microbial technology. According to the method, microorganisms with a mineralization function, serum albumin and mineralization liquid containing a calcium source and urea are used as a reaction system, under the mild condition, the microorganisms hydrolyze urea to generate carbonate radicals and release the carbonate radicals to the outside of cells, calcium ions are actively transported to the outside of the cells through a calcium ion pump, the pH and ion saturation of the local microenvironment are adjusted, and the calcium ions in the microenvironment are converted into calcium ions. According to the present invention, the formation of the calcium carbonate precursor is slowly and continuously driven, and the serum albumin is completely embedded into the mineral precursor to provide the nucleation site and the organic template so as to regulate and stabilize the formation of the serum albumin-amorphous calcium carbonate mineral phase composite carrier, such that the high loading and the uniform distribution of the serum albumin in the carrier can be achieved; the natural structure of the serum albumin can be effectively maintained.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of biomedicine and microbial technology, and in particular to a method for preparing a high-load serum albumin-mineral composite carrier by microbial mineralization. Background Technology

[0002] Serum albumin is a natural transport protein in blood, whose main physiological function is to reversibly bind to various endogenous and exogenous molecules and then transport them to various tissues in the body. Albumin drug delivery systems have advantages such as biodegradability, non-immunogenicity, and biocompatibility. To date, existing technologies in the field of albumin delivery mainly include desolvation, emulsification, thermogelation, self-assembly, and Nab technology. Generally speaking, desolvation and emulsification methods require the addition of organic solvents and cross-linking agents. Although thermogelation, self-assembly, and Nab technology do not require cross-linking agents, the self-cross-linking process they employ alters the conformation and structure of the protein. Among these, the chemical cross-linking method based on dynamic covalent bonds has the highest protein loading capacity of 51 wt% (Su S, Wang YY, Du FS, et al. Advanced Functional Materials, 2018, 28:1805287). However, this method essentially relies on the covalent modification of the protein, and its process involves the synthesis of cross-linking agents and multiple chemical reactions, resulting in disadvantages such as low long-term biocompatibility and process complexity. Therefore, there is an urgent need to develop non-destructive protein delivery technologies with high long-term biosafety and simple processes to improve or partially replace these technologies.

[0003] Inspired by the theory of biomineralization, mineral-based protein delivery technology has attracted widespread attention due to its excellent biocompatibility and degradability. This technology simulates the biomineralization process in nature, utilizing the functional groups of proteins themselves to regulate mineral precipitation through non-covalent interactions such as electrostatics and hydrogen bonds. This process avoids exogenous chemical modification, preserving the natural conformation and biological activity of the protein to the greatest extent. Among them, amorphous minerals are regarded as potential protein carrier materials due to their high specific surface area and adsorption capacity. The literature (Lei JS, Zheng Y, Meng YF, et al. Advanced Functional Materials, 2022, 32(26): 2202928.) reported that amorphous calcium carbonate phosphate (ACCP) carriers constructed through a biomimetic mineralization strategy can achieve protein loading, with a maximum protein loading of 37.2 wt%, significantly higher than most traditional delivery systems (about 10 wt%). However, this biomimetic mineralization is essentially a chemical coprecipitation process, and the reaction is rapid. While protein loading in amorphous minerals can regulate mineral morphology and stabilize the amorphous phase to some extent, the interaction between proteins and minerals is difficult to fully occur during transient chemical precipitation, thus limiting their ability to regulate mineral structure and morphology. This severely restricts the amount of protein that can be loaded into amorphous minerals, thus limiting the application of mineral-based protein carriers. Therefore, developing a mineralization method with a more moderate reaction that allows proteins to fully participate in and regulate the mineralization process holds promise for significantly increasing protein loading, but currently, no effective technical method has been found. Summary of the Invention

[0004] This invention provides a microbial mineralization preparation method for a high-load serum albumin-amorphous mineral composite carrier, so as to achieve the formation of the serum albumin-amorphous mineral composite carrier and the efficient loading and effective maintenance of the natural structure of serum albumin.

[0005] This invention provides a method for the microbial mineralization preparation of a high-load serum albumin-amorphous mineral composite carrier, the method comprising the following steps: S1. Activate the microbial liquid with mineralizing effect to obtain activated microbial liquid; S2. Mix the serum albumin solution with a mineralizing solution containing a calcium source and urea to obtain a substrate solution; S3. The activated microbial culture solution is mixed with the substrate solution, and the pH value is adjusted to 6.0-10.0 to carry out a microbial-mediated co-mineralization reaction to obtain a serum albumin-amorphous mineral composite carrier. The co-mineralization reaction includes the following parameters: reaction temperature of 28-32℃, co-mineralization time of 20-28 h, and oscillation rate of 160-180 rpm.

[0006] Preferably, the bacteria in the microbial solution include at least one of Bacillus pasteurellii, Bacillus urealyticum, Bacillus spheroides, and Bacillus subtilis.

[0007] Preferably, the preservation number of the *Bacillus pasteurellii* is ATCC 11859, the preservation number of the *Bacillus urealyticum* is CGMCC 1.7272, the preservation number of the *Bacillus spheroidosa* is CGMCC 1.1359, and the preservation number of the *Bacillus subtilis* is ATCC 6633.

[0008] Preferably, in the substrate solution, the final concentration of serum albumin is 0.5–5 mg / mL, and Ca... 2+ The final concentration of the urea is 16–24 mmol / mL.

[0009] Preferably, the serum albumin in the serum albumin solution includes at least one of human serum albumin and bovine serum albumin.

[0010] Preferably, the calcium source is calcium chloride.

[0011] Preferably, the volume ratio of the activated microbial culture to the substrate solution is (1-5):1.

[0012] Preferably, the final OD value of the mixture of the activated microbial inoculum and the substrate solution is 1.0 to 3.0.

[0013] Preferably, the serum albumin loading in the serum albumin-amorphous mineral composite carrier is >95%.

[0014] Preferably, the mineral phase in the serum albumin-amorphous mineral composite carrier is amorphous calcium carbonate.

[0015] The technical solutions provided in the embodiments of the present invention have the following advantages compared with the prior art: This invention provides a microbial mineralization method for preparing a high-load serum albumin-amorphous mineral composite carrier. This method achieves the formation of the serum albumin-amorphous mineral composite carrier through microbial mineralization, simultaneously achieving high-efficiency loading of serum albumin and effective maintenance of its natural structure. The method first activates a mineralizing microbial culture to enhance its metabolic activity. Then, a serum albumin solution is mixed with a mineralization solution containing a calcium source and urea to prepare a substrate solution, providing the necessary mineralizing ions and protein template. Next, the activated microbial culture is mixed with the substrate solution, the pH is adjusted to 6.0–10.0, and a co-mineralization reaction is carried out under mild reaction conditions (temperature 28–32°C, time 20–28 h, oscillation rate 160–180 rpm). During co-mineralization, microorganisms hydrolyze urea to produce carbonate ions, which are released extracellularly. Calcium ions are actively transported extracellularly via calcium ion pumps, regulating the local microenvironment's pH and ion saturation. This slowly and continuously drives the formation of calcium carbonate precursors. Simultaneously, serum albumin is fully embedded in the mineral precursors, providing nucleation sites and organic templates, regulating and stabilizing the formation of the serum albumin-amorphous calcium carbonate mineral phase composite carrier. This mechanism allows serum albumin molecules to be fully and uniformly embedded in the gradually forming amorphous mineral network, achieving high loading and uniform distribution. Furthermore, because the microbial-driven mineralization process is slow and under mild conditions, it avoids the damage to the protein structure caused by rapid precipitation or chemical cross-linking, thus effectively maintaining the native conformation and biological activity of serum albumin. Therefore, this invention, through the synergy of microbial metabolic regulation and protein template action, achieves controllable construction of the composite carrier, efficient protein loading, and complete structural preservation. Attached Figure Description

[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 These are X-ray diffraction patterns provided in Embodiment 1 and Comparative Example 1 of the present invention; Figure 2 This is a scanning electron microscope image of the mineralization products without the addition of human serum albumin, provided in Comparative Example 1 of the present invention. Figure 3 This is a scanning electron microscope image of the human serum albumin-amorphous mineral composite carrier provided in Example 1 of the present invention; Figure 4 The image shows circular dichroisms of human serum albumin before and after co-mineralization loading, as provided in Example 1 of this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a method for the microbial mineralization preparation of a high-load serum albumin-amorphous mineral composite carrier, the method comprising the following steps: S1. Activate the microbial liquid with mineralizing effect to obtain activated microbial liquid; S2. Mix the serum albumin solution with a mineralizing solution containing a calcium source and urea to obtain a substrate solution; S3. The activated microbial culture solution is mixed with the substrate solution, and the pH value is adjusted to 6.0-10.0 to carry out a microbial-mediated co-mineralization reaction to obtain a serum albumin-amorphous mineral composite carrier. The co-mineralization reaction includes the following parameters: reaction temperature of 28-32℃, co-mineralization time of 20-28 h, and oscillation rate of 160-180 rpm.

[0021] pH 6.0–10.0: Within this range, serum albumin has a negatively charged surface, which is beneficial for its use as a template to react with Ca. 2+ Interactions; at the same time, the pH environment is also suitable for the metabolic activities and urease function of the microorganisms used.

[0022] Temperature 28~32℃: This is the optimal growth temperature range for the microorganisms used, which can maintain their high metabolic activity while avoiding excessively high temperatures that could lead to protein denaturation or microbial inactivation.

[0023] Time 20–28 h: Provides a sufficiently long window period to allow microbial metabolism, ion release, protein intercalation, and mineral precursor assembly to proceed fully and slowly, which is key to forming a stable amorphous phase and achieving high loading.

[0024] Oscillation rate 160-180 rpm: to maintain system homogeneity, promote mass transfer, and avoid excessive shear force that could damage protein structure or microbial cells.

[0025] In some embodiments, the bacteria in the microbial culture include at least one of Bacillus pasteurellii, Bacillus urealyticum, Bacillus spheroides, and Bacillus subtilis.

[0026] In some embodiments, the preservation number of the *Bacillus pasteurellii* is ATCC 11859, the preservation number of the *Bacillus urealyticum* is CGMCC 1.7272, the preservation number of the *Bacillus spheroidosa* is CGMCC 1.1359, and the preservation number of the *Bacillus subtilis* is ATCC 6633.

[0027] In some embodiments, the activation in step S1 uses bacterial culture prepared by inoculating bacteria in ATCC 1376 NH4-YE microbial culture medium at an inoculation rate of 2% to 5% (V / V) and activating it in a shaker at a constant temperature of 28 to 37°C and a rotation speed of 170 to 220 rpm / min for 22 to 30 h.

[0028] In some embodiments, the final concentration of serum albumin in the substrate solution is 0.5–5 mg / mL, Ca 2+ The final concentration of the urea is 16–24 mmol / mL.

[0029] Final serum albumin concentration (0.5–5 mg / mL): Within this range, sufficient protein molecules can be provided as mineralization nucleation sites and structural templates, while avoiding excessive protein aggregation that could affect its uniform embedding and loading efficiency in minerals.

[0030] Final calcium ion concentration (16–24 mmol / L) and final urea concentration (16–24 mmol / L): These concentrations are matched to provide a balanced calcium intake. 2+ and CO3 2- The source allows microorganisms to slowly and continuously create a supersaturated state in the local microenvironment, driving the formation of calcium carbonate precursors, while preventing excessively high ion concentrations from causing rapid homogeneous precipitation and the formation of crystalline phases.

[0031] In some embodiments, the serum albumin in the serum albumin solution includes at least one of human serum albumin and bovine serum albumin.

[0032] It should be noted that serum albumins (such as human serum albumin HSA and bovine serum albumin BSA) have a low isoelectric point (approximately 4.7–4.8). Under the aforementioned reaction pH conditions, their molecular surface is enriched with negative charges, allowing them to serve as highly efficient anionic polymer templates, attracting calcium ions (Ca). 2+This invention introduces microorganisms with metabolic activities such as urease to continuously and controllably regulate the ion saturation and pH of the local microenvironment under mild conditions, enabling serum albumin to be fully and uniformly embedded in the synchronously formed amorphous calcium carbonate mineral phase. This strategy, based on the synergy between the inherent physicochemical properties of the protein and the microbial mineralization process, is key to achieving extremely high loading levels of serum albumin.

[0033] In some embodiments, the calcium source is calcium chloride.

[0034] Calcium chloride can provide readily soluble and stable calcium ions, and chloride ions have little interference with the mineralization process and protein structure.

[0035] In some embodiments, the volume ratio of the activated microbial culture to the substrate solution is (1-5):1.

[0036] In some embodiments, the final OD value of the mixture of the activated microbial culture and the substrate solution is 1.0 to 3.0.

[0037] The volume ratio of bacterial culture to substrate solution (1:1 to 5:1) and the final OD value of the mixture (1.0 to 3.0) together ensure that there are appropriate numbers of active microorganisms in the reaction system. Too few bacteria will result in insufficient mineralization driving force; too many bacteria may affect the homogeneity of the mineral-protein complex structure due to excessively rapid metabolism or steric hindrance. This OD range is the ideal bacterial quantity for maintaining a slow and continuous mineralization process.

[0038] In some embodiments, the serum albumin loading in the serum albumin-amorphous mineral composite carrier is >95%.

[0039] It should be noted that the serum albumin loading is calculated using the following formula: .

[0040] In some embodiments, the mineral phase in the serum albumin-amorphous mineral composite carrier is amorphous calcium carbonate.

[0041] Overall, compared with the prior art, the beneficial effects of the above-described technical solutions conceived by this invention are as follows: (1) Addressing the technical shortcomings or improvement needs of existing technologies where biomimetic mineralization reactions are too rapid, resulting in low protein loading and difficulty in protein fully participating in and regulating mineral phases and morphology. This invention utilizes, but is not limited to, microorganisms with mineralization functions, serum albumin, and a mineralization solution containing calcium sources and urea to conduct a co-mineralization reaction under mild conditions. Microorganisms slowly and continuously regulate the local microenvironment through their urease and carbonic anhydrase, driving the formation of calcium carbonate precursors; simultaneously, serum albumin molecules, as nucleation sites, fully embed and regulate the mineralization process, inhibiting crystal growth and phase transitions, thereby stabilizing calcium carbonate in an amorphous form and forming a high-load, structurally uniform serum albumin-amorphous mineral composite carrier. As a mild, high-loading, and biocompatible method for preparing serum protein carriers, this invention can improve and partially replace carrier preparation processes based on chemical modification or biomimetic mineralization, and has significant application potential and broad industrialization prospects in the field of protein delivery.

[0042] (2) Traditional biomimetic mineralization is a chemical co-precipitation process. The rapid reaction results in insufficient interaction time between protein molecules and mineral precursors, making it difficult to control the mineral phase, structure, and morphology, and resulting in low protein loading. By using microbial co-mineralization, adding microorganisms with mineralization functions, serum albumin, and mineralization solutions containing calcium sources and urea can not only control the mineral phase, structure, and morphology, but also significantly increase the protein loading in the carrier. The mechanism is as follows: 1) Microorganisms produce CO3 through the hydrolysis of urea by their urease and the action of carbonic anhydrase. 2- And released into the extracellular space, Ca 2+ It is actively transported to the extracellular space via calcium ion pumps, where it forms CaO near the bacteria. 2+ With CO3 2- The supersaturated microenvironment, and the regulation of local microenvironment pH, thereby driving Ca2+. 2+ With CO3 2- 1) Mineral precursors are generated slowly and continuously; 2) During this slow and continuous process, serum albumin molecules are fully and uniformly embedded in the mineral precursors, achieving a fully uniform distribution of serum albumin in the mineral matrix; 3) Serum albumin, on the one hand, acts as a nucleation site, and on the other hand, interacts with Ca through its surface functional groups (such as carboxyl groups). 2+ The electrostatic effect guides mineral deposition, while the steric hindrance effect of its macromolecules can effectively inhibit the growth and crystallization transformation of minerals, regulate and stabilize the formation of serum albumin-amorphous mineral complex carriers, and ultimately significantly increase the serum albumin loading and effectively maintain the natural structure of serum albumin.

[0043] (3) By synergistically combining the low isoelectric point of serum albumin with the microbial mineralization process, targeted and efficient loading is achieved. This invention utilizes the negatively charged surface of serum albumin (such as HSA and BSA) at the pH of the reaction system, enabling it to generate strong electrostatic interactions with the cationic mineralization precursors produced by microbial metabolism, thereby being efficiently captured and embedded in the growing amorphous mineral phase. This design based on the inherent physicochemical properties of proteins is the key to achieving extremely high loading capacity (24 h loading capacity > 95%) of serum albumin in this invention.

[0044] (4) The serum albumin-amorphous mineral composite carrier prepared by the present invention has the following beneficial effects: In terms of chemical properties, serum albumin is used as raw material, which is non-toxic and has good biocompatibility. The mineralization process does not introduce exogenous chemical cross-linking agents, avoiding the risks of organic solvent residue and chemical modification. The composite carrier is biodegradable and environmentally friendly. In terms of physical properties, the composite carrier has the high specific surface area unique to amorphous mineral phase, which is conducive to the uniform loading of a large number of serum albumin molecules. In terms of economic feasibility, the microbial co-mineralization synthesis is adopted, the reaction conditions are mild, the reaction process is simple, the cost of the strains and raw materials used is low, and it has the feasibility of large-scale production.

[0045] (5) The microbial co-mineralization reaction process used in this invention is simple. The loading of serum albumin in the mineral precursor can be completed in a single reaction system without the need for step-by-step loading or post-modification of serum albumin. Compared with the multi-step chemical modification commonly used in the prior art, the operation steps of this invention are greatly simplified, the reaction conditions are mild and controllable, and it is more in line with the actual engineering needs.

[0046] (6) This invention is the first to combine microbial mineralization technology in the field of earth science with drug delivery systems in the field of medicine, promoting interdisciplinary integration. It not only solves the problem of limited control over mineral phase, structure and morphology in biomimetic mineralization by utilizing the regulatory characteristics of microbial metabolism, but also provides an effective new method for preparing high-load protein-mineral composite carriers, which has broad application prospects in the field of protein delivery.

[0047] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to national standards / industry standards / the contents of this disclosure; if there are no corresponding national standards / industry standards / the contents of this disclosure, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0048] Example 1 This embodiment provides a microbial mineralization preparation method for a high-load serum albumin-amorphous mineral composite carrier, the steps of which are as follows: S1, bacterial culture activation The *Pasteurella multocida* strain with preservation number ATCC 11859 was inoculated at a rate of 3% (v / v) into sterilized ATCC 1376 NH4-YE liquid medium (formulation: yeast extract 20 g / L, ammonium sulfate 10 g / L, Trisbase 15.7 g / L). It was then activated and cultured in a constant temperature shaker at 30℃ and 180 rpm for 24 h, and the optical density (OD) of the bacterial solution at a wavelength of 600 nm was measured. 600 The concentration reaches 2.5, resulting in a highly active activated microbial culture solution.

[0049] Simultaneously, the urease activity produced by the activated bacteria was detected. Urease activity was detected using a conductivity meter. The specific method was as follows: 5 mL of bacterial culture after 24 h of culture was taken and mixed with 45 mL of 1.1 mol / L urea solution. Under the condition of 28℃, the conductivity change was detected over a period of 5 min using a conductivity meter. The amount of urea hydrolyzed by urease per unit time was calculated, and the urease activity was expressed as the amount of urea hydrolyzed by urease per unit minute. The urease activity was 4.58 mmol / min / OD.

[0050] S2. Preparation of substrate solution A certain amount of human serum albumin (HSA) stock solution was mixed with a mineralization solution (containing 0.5 mol / L calcium chloride and 0.5 mol / L urea). The mixing ratio was controlled so that the final concentration of HSA in the prepared substrate solution was 2.0 mg / mL. 2+ The final concentration of urea is 20 mmol / L.

[0051] S3, co-mineralization reaction and product collection The activated bacterial solution and substrate solution were mixed at an appropriate volume ratio to achieve the desired final OD500 of the bacteria in the mixed reaction system. 600 The pH value was 2.0. The pH of the reaction system was adjusted to 8.0 using dilute sodium hydroxide solution. The mixture was then placed in a constant temperature shaker at 30°C and 170 rpm for microbial-mediated co-mineralization reaction, which lasted for 24 h.

[0052] After the reaction was completed, the entire reaction solution was centrifuged at 4℃ and 10,000 rpm for 10 min. The supernatant was collected to determine the residual protein concentration. The resulting precipitate was washed three times with ultrapure water and then freeze-dried to obtain a white HSA-amorphous mineral composite carrier powder.

[0053] By measuring the protein concentration in the supernatant before and after the reaction and calculating according to the formula, the loading of human serum albumin in the composite carrier prepared in this embodiment reached as high as 98.91%. This result is significantly higher than the 37.2% loading of the biomimetic mineralization method (chemical coprecipitation, Lei JS, Zheng Y, Meng YF, et al. Advanced Functional Materials, 2022, 32(26): 2202928.), demonstrating the outstanding effect of the microbial mineralization method of this invention in achieving efficient protein loading.

[0054] Comparative Example 1 This comparative example is modified from the one disclosed in Example 1 as follows: In step S2, HSA solution is not added; instead, an equal volume of ultrapure water is used.

[0055] Figure 1 The X-ray diffraction patterns provided in Embodiment 1 and Comparative Example 1 of the present invention are shown.

[0056] Depend on Figure 1 It is evident that the HSA-amorphous mineral composite carrier exhibits only broadened diffuse peaks, consistent with the typical characteristics of amorphous calcium carbonate (ACC). The XRD patterns of untreated HSA and bacteria also show broad peaks, representing the inherent amorphous scattering background of the organic components (proteins, bacterial cells, and extracellular polymers) in the samples. However, their peak shape, position, and intensity distribution are significantly different from the diffuse peaks of the co-mineralization products, confirming that the diffuse peaks of the co-mineralization products mainly originate from the newly formed amorphous mineral phase. The mineralization products without HSA addition consist of calcite and aragonite. This indicates that HSA, as a macromolecular inhibitor, can effectively regulate and stabilize the amorphous mineral phase, inhibiting the transformation of calcium carbonate into the crystalline phase.

[0057] Figure 2 This is a scanning electron microscope image of the mineralized product without the addition of human serum albumin, provided in Comparative Example 1 of the present invention.

[0058] Depend on Figure 2 It can be seen that the scanning electron microscope (SEM) images of the mineralized products without HSA show that the mineralized products have rhomboid and spherical morphologies, which are typical morphologies of calcite and aragonite crystals.

[0059] Figure 3 This is a scanning electron microscope image of the human serum albumin-amorphous mineral composite carrier provided in Example 1 of the present invention.

[0060] Depend on Figure 3The SEM images of the HSA-amorphous mineral composite carrier show that the product morphology is a continuous and uniform amorphous structure, with no crystalline morphology observed. This indicates that the microbial metabolism-driven mineralization reaction is relatively slow compared to chemical methods, allowing albumin to be fully and uniformly embedded in the mineral precursor. Through steric hindrance, albumin hinders the aggregation of nanoparticles, stabilizing calcium carbonate in an amorphous form and forming a uniform inorganic-organic composite structure, significantly improving the protein loading of the carrier.

[0061] Figure 4 The image shows circular dichroisms of human serum albumin before and after co-mineralization loading, as provided in Example 1 of this invention.

[0062] Depend on Figure 4 It can be seen that the co-mineralized HSA exhibits typical α-helix negative peaks at 208 nm and 220 nm, with an α-helix content of 37%, which is highly consistent with the untreated HSA (α-helix 38%). This indicates that the microbial-mediated co-mineralization process can effectively maintain the integrity of the natural secondary structure of HSA.

[0063] Example 2 This embodiment is based on the disclosure in Embodiment 1, with the following modifications: The HSA solution used in Example 1 was replaced with bovine serum albumin (BSA) at a final concentration of 2.0 mg / mL.

[0064] The protein loading of the prepared BSA-amorphous mineral composite carrier was calculated to be as high as 98.61%. This loading is significantly higher than the 37.2% loading of the carrier prepared by the biomimetic mineralization method reported in the literature (Lei JS, Zheng Y, Meng YF, et al. Advanced Functional Materials, 2022, 32(26): 2202928.), indicating that the microbial mineralization method can significantly improve the protein loading of the albumin-amorphous mineral composite carrier.

[0065] Example 3 This embodiment is based on the disclosure in Embodiment 1, with the following modifications: The HSA solution used in Example 1 was replaced with BSA at final concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL and 5 mg / mL.

[0066] The calculated protein loadings of the prepared BSA-amorphous mineral composite carriers were 96.97%, 98.41%, 98.62%, 98.66%, and 97.19% (the final concentration corresponds one-to-one with the protein loading; for example, the protein loading of the BSA-amorphous mineral composite carrier when the final concentration of the BSA solution is 0.5 mg / mL is 96.97%). This loading is significantly higher than the 37.2% loading of the carrier reported in the literature (Lei J S, Zheng Y, Meng YF, et al. Advanced Functional Materials, 2022, 32(26):2202928.), indicating that the microbial mineralization method can significantly improve the protein loading of the albumin-amorphous mineral composite carrier.

[0067] Example 4 This embodiment is based on the disclosure in Embodiment 1, with the following modifications: The HSA solution used in Example 1 was replaced with BSA at a final concentration of 3.0 mg / mL, and the final bacterial OD2.0 of the reaction system was replaced and adjusted to 1.0, 1.5, 2.5, and 3.0.

[0068] The calculated protein loadings of the prepared BSA-amorphous mineral composite carriers were 97.22%, 98.31%, 98.71%, 97.94%, and 98.23%, respectively. These loadings are significantly higher than the 37.2% loading of the biomimetic mineralization carrier reported in the literature (Lei JS, Zheng Y, Meng YF, et al. Advanced Functional Materials, 2022, 32(26): 2202928.), indicating that the microbial mineralization method can significantly improve the protein loading of the albumin-amorphous mineral composite carrier.

[0069] Example 5 This embodiment is based on the disclosure in Embodiment 1, with the following modifications: The HSA solution used in Example 1 was replaced with BSA at a final concentration of 5.0 mg / mL, and the pH of the reaction system was replaced with pH 8 and adjusted to pH 6, pH 7, pH 9 and pH 10.

[0070] The calculated protein loadings of the prepared BSA-amorphous mineral composite carriers were 95.51%, 96.22%, 96.19%, 97.21%, and 97.84%, respectively. These loadings are significantly higher than the 37.2% loading of the biomimetic mineralization carrier reported in the literature (Lei JS, Zheng Y, Meng YF, et al. Advanced Functional Materials, 2022, 32(26): 2202928.), indicating that the microbial mineralization method can significantly improve the protein loading of the albumin-amorphous mineral composite carrier.

[0071] The range descriptions used herein, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1 to 6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "including" and "contains" as used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships mentioned herein, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained through commercial purchase or prepared using existing methods.

[0072] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a high-capacity serum albumin-mineral composite carrier by microbial mineralization, characterized in that, The mineral is an amorphous mineral, and the method includes the following steps: S1. Activate the microbial liquid with mineralizing effect to obtain activated microbial liquid; S2. Mix the serum albumin solution with a mineralizing solution containing a calcium source and urea to obtain a substrate solution; S3. The activated microbial culture solution is mixed with the substrate solution, and the pH value is adjusted to 6.0-10.0 to carry out a microbial-mediated co-mineralization reaction to obtain a serum albumin-amorphous mineral composite carrier. The co-mineralization reaction includes the following parameters: reaction temperature of 28-32℃, co-mineralization time of 20-28 h, and oscillation rate of 160-180 rpm.

2. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 1, characterized in that, The bacteria in the microbial inoculum include at least one of Bacillus pasteurellii, Bacillus urealyticum, Bacillus spheroides, and Bacillus subtilis.

3. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 2, characterized in that, The preservation number of *Bacillus pasteurellus* is ATCC 11859, the preservation number of *Bacillus urealyticum* is CGMCC 1.7272, the preservation number of *Bacillus spheroidosa* is CGMCC 1.1359, and the preservation number of *Bacillus subtilis* is ATCC 6633.

4. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 1, characterized in that, In the substrate solution, the final concentration of serum albumin is 0.5–5 mg / mL, Ca 2+ The final concentration of the urea is 16–24 mmol / mL.

5. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 4, characterized in that, The serum albumin in the serum albumin solution includes at least one of human serum albumin and bovine serum albumin.

6. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 1, characterized in that, The calcium source is calcium chloride.

7. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 1, characterized in that, The volume ratio of the activated microbial culture to the substrate solution is (1-5):

1.

8. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 1, characterized in that, The final OD value of the mixture of the activated microbial inoculum and the substrate solution is 1.0 to 3.

0.

9. The microbial mineralization method for a high-load serum albumin-mineral composite carrier according to claim 1, characterized in that, The serum albumin loading in the serum albumin-mineral composite carrier is >95%.

10. The microbial mineralization method for 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.

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