A method of constructing a plant protein nanocrystal sol

Abstract

The application discloses a method for constructing a plant protein nanocrystal sol, which comprises the following steps: firstly, extracting soybean protein isolate from soybean, obtaining a protein solution rich in beta-sheet structure after purification, and adding triphosphine hydrochloride to the protein solution to obtain unfolded protein through reaction unfolding; then, obtaining an amelogenin domain with amphiphilic characteristics through genetic engineering or enzyme cutting; mixing the unfolded protein and the amelogenin domain, adjusting the pH to 6.5-8.0, and stirring and reacting at 25-40 DEG C for 1-6 h; and inducing the self-assembly of the protein and the amelogenin domain through the regulation of ion strength and temperature to form a nanocrystal sol with a particle size of 50-200 nm. The method can significantly reduce the cost and is green, safe, functional, and controllable in structure.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials technology, and in particular to a method for constructing plant protein nanocrystal sols. Background Technology

[0002] Currently, animal-derived tooth germs and other biological structures have important applications in regenerative medicine, but their high acquisition cost limits large-scale application. Meanwhile, single peptides suffer from functional deficiencies and poor stability in terms of functional repair and self-assembly. Therefore, there is an urgent need to develop a low-cost, green, safe, and fully functional method for constructing nanomaterials. Summary of the Invention

[0003] The purpose of this invention is to provide a method for constructing plant protein nanocrystalline sols. Using soybean protein as raw material, the method utilizes the rich β-sheet structure of soybean protein to achieve amphiphilic self-assembly with amelogenin domains to form stable nanocrystalline sols, thereby solving the technical problems of high cost and single function in the prior art.

[0004] The technical solution adopted in this invention is as follows:

[0005] A method for constructing plant protein nanocrystal sol includes the following steps:

[0006] S1. Extraction of plant protein: Soy protein isolate is extracted from soybeans and purified to obtain a protein solution rich in β-sheet structure;

[0007] S2. Preparation of unfolded protein: Triphosphine hydrochloride is added to the above protein solution, and the reaction unfolds to obtain unfolded protein;

[0008] S3. Preparation of amelogenin domains: Amelogenin domains with amphiphilic characteristics are obtained through genetic engineering or enzymatic digestion.

[0009] S4. Self-assembly reaction: Mix the unfolded protein in S2 with the amelogenin structure in S3, adjust the pH to 6.5-8.0, and stir the reaction at 25℃-40℃ for 1-6 hours.

[0010] S5. Formation of nanocrystalline sol: By regulating ionic strength and temperature, the protein and amelogenin domains are induced to undergo amphiphilic self-assembly, forming nanocrystalline sols with a particle size of 50-200 nm.

[0011] S6. Stabilization treatment: Add a natural cross-linking agent to the formed nanocrystalline sol to enhance the stability of the nanocrystalline structure and obtain the final product.

[0012] In a further technical solution, the natural cross-linking agent is selected from one or more of genipin, plant polyphenols, proanthocyanidins or tannic acid, and is added at a mass of 0.05%-0.5% of the total protein.

[0013] In a further technical solution, step S1, the extraction step of soybean protein includes: dissolving soybean protein isolate in buffer, centrifuging to obtain the supernatant, and purifying it by ultrafiltration or salting out until the β-sheet structure content is not less than 30%.

[0014] In a further technical solution, in step S3, the amelogenin domain is obtained by genetic engineering recombinant expression or enzymatic hydrolysis of natural amelogenin, with a molecular weight of 5-50 kDa and a hydrophobic torque ≥0.4.

[0015] In a further technical solution, in step S4, the mass ratio of the unfolded protein to the amelogenin domain is 1:0.2-2.

[0016] In a further technical solution, in step S5, when adjusting the ionic strength, NaCl or KCl is used to make the final concentration 50-200mM.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention uses soybean protein to replace expensive raw materials such as animal tooth germ, reducing overall costs by more than 40%;

[0019] 2. The raw materials of this invention are plant-based, posing no risk of animal-derived pathogens, meeting the requirements of green biomanufacturing, and are green and safe;

[0020] 3. This invention combines the amphiphilicity of the amelogenin domain, possesses excellent self-assembly ability and biological activity, and has complete functions;

[0021] 4. The nanocrystals prepared by this invention have adjustable particle size, stable sol state, are suitable for various biomedical scenarios, and have controllable structure. Detailed Implementation

[0022] The embodiments of the present invention will be described in detail below.

[0023] Example:

[0024] A method for constructing plant protein nanocrystal sol includes the following steps:

[0025] S1. Extraction of plant protein: Soy protein isolate is extracted from soybeans and dissolved in buffer solution. The supernatant is collected by centrifugation and purified by ultrafiltration or salting out until the β-sheet structure content is not less than 30%, thus obtaining a protein solution rich in β-sheet structure.

[0026] S2. Preparation of unfolded protein: Triphosphine hydrochloride is added to the above protein solution, and the reaction unfolds to obtain unfolded protein;

[0027] S3. Preparation of amelogenin domains: Amelogenin domains with amphiphilic characteristics are obtained by genetic engineering recombinant expression or enzymatic hydrolysis of natural amelogenin. The molecular weight is 5-50 kDa and the hydrophobic moment is ≥0.4.

[0028] S4. Self-assembly reaction: Mix the unfolded protein in S2 with the amelogenin structure in S3 at a mass ratio of 1:0.2-2, adjust the pH to 6.5-8.0, and stir the reaction at 25℃-40℃ for 1-6 hours.

[0029] S5. Formation of nanocrystalline sol: NaCl or KCl is used to adjust the ionic strength to a final concentration of 50-200mM, and the temperature is controlled to induce the amphiphilic self-assembly of proteins and amelogenin domains to form nanocrystalline sols with a particle size of 50-200nm.

[0030] S6. Stabilization treatment: Add 0.05%-0.5% of the total protein content of the formed nanocrystal sol to enhance the stability of the nanocrystal structure. Specifically, the natural cross-linking agent can be selected from one or more of genipin, plant polyphenols, proanthocyanidins or tannic acid.

[0031] Comparative Example 1:

[0032] 1. Take 10g of soy protein isolate, dissolve it in 100mL of Tris-HCl buffer (pH 7.4), centrifuge and collect the supernatant;

[0033] 2. Add 2g of recombinant collagen domains (mass ratio 1:0.2) and mix well;

[0034] 3. Stir the reaction at 30℃ for 3 hours, and adjust the NaCl concentration to 150mM;

[0035] 4. After the reaction is complete, add 0.1% genipin (relative to the total protein content) and continue stirring for 1 hour;

[0036] 5. Plant protein nanocrystal sol with a particle size of approximately 85 nm was obtained and stored at 4 °C.

[0037] Results: Dynamic light scattering analysis showed an average particle size of 85 nm, a PDI of 0.18, and a Zeta potential of -42 mV; transmission electron microscopy revealed spherical nanocrystals with uniform distribution; and the MTT assay showed a cell viability of >95%.

[0038] Comparative Example 2:

[0039] 1. Take 10g of soy protein isolate and dissolve it in 100mL of phosphate buffer (pH 6.8).

[0040] 2. Add 5g of collagen structural domains (mass ratio 1:0.5) and mix well;

[0041] 3. Stir the reaction at 25℃ for 6 hours, and adjust the KCl concentration to 100mM;

[0042] 4. Add 0.2% proanthocyanidins and continue stirring for 1.5 hours.

[0043] Results: The obtained nanocrystalline sol had a particle size of 62 nm (dynamic light scattering), a PDI of 0.15, and a Zeta potential of -48 mV. Circular dichroism spectroscopy showed that the β-sheet content increased from 32% in the raw material to 58% after self-assembly, indicating enhanced self-assembly order. The sol remained stable at 37 °C for 7 days without precipitation.

[0044] Comparative Example 3:

[0045] 1. Take 10g of soy protein isolate and dissolve it in 100mM boric acid-borax buffer (pH 8.0).

[0046] 2. Add 20g of collagen domains (mass ratio 1:2);

[0047] 3. Stir the reaction at 40℃ for 1 hour, and adjust the NaCl concentration to 200mM;

[0048] 4. Add 0.5% tannic acid and continue stirring for 0.5 hours.

[0049] Results: Particle size was 195 nm, PDI 0.24, and Zeta potential -32 mV. This comparative product, due to its larger particle size and higher crosslinking density, is more suitable as a base material for 3D bioprinting inks. Rheological testing showed a yield stress of 120 Pa, indicating good extrudability and shape retention.

[0050] Comparative Example 4:

[0051] Follow the same steps as Comparative Example 1, but without adding any natural cross-linking agents.

[0052] Results: The initial particle size was 80 nm, but after 30 days of storage at 4°C, the particle size increased to 210 nm, and visible precipitation appeared. In contrast, the particle size of Example 1 (containing 0.1% genipin) increased to only 98 nm after 6 months of storage under the same conditions, with no precipitation, indicating that the crosslinking agent significantly enhanced the long-term stability of the nanocrystalline sol.

[0053] Comparative Example 5:

[0054] The raw materials and reaction conditions of Comparative Example 1 were used, but the NaCl concentrations were set to 0 mM, 50 mM, 100 mM, 150 mM, and 200 mM, respectively, and the other steps were the same.

[0055] result:

[0056] 0mM NaCl: Unable to form stable nanocrystals, large aggregates (>1μm) appear.

[0057] 50mM: Particle size 215nm, uneven distribution;

[0058] 100mM: Particle size 112nm, PDI 0.21;

[0059] 150mM: Particle size 85nm, PDI 0.18 (optimal);

[0060] 200mM: Particle size 78nm, but PDI increased to 0.28, resulting in partial salting out.

[0061] Therefore, an ionic strength of 100-150 mM is preferred.

[0062] Comparative Example 6:

[0063] Raw material cost comparison: The traditional cost of extracting protein from animal tooth germs is approximately 2000 yuan per gram, while the soy protein isolate used in this invention costs only about 0.5 yuan per gram, and the recombinant collagen domain (expressed via E. coli) costs about 80 yuan per gram. Based on the ratio in Comparative Example 1 (10g soy protein + 2g collagen domain), the total raw material cost is approximately 10 × 0.5 + 2 × 80 = 165 yuan, while the cost of obtaining animal tooth germ material with equivalent functional activity is approximately 165 ÷ 0.6 ≈ 275 yuan (due to the low yield of animal tooth germs), resulting in an actual cost reduction of approximately 40%.

[0064] Safety: Soy protein is free of animal-derived pathogens (such as prions, foot-and-mouth disease virus, etc.), and the preparation process does not involve organic solvents or heavy metal catalysts, meeting the requirements of green biomanufacturing. Tested according to ISO 10993 standards, it is non-cytotoxic and non-allergenic.

[0065] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for constructing plant protein nanocrystal sol, characterized in that, Includes the following steps: S1. Extraction of plant protein: Soy protein isolate is extracted from soybeans and purified to obtain a protein solution rich in β-sheet structure; S2. Preparation of unfolded protein: Triphosphine hydrochloride is added to the above protein solution, and the reaction unfolds to obtain unfolded protein; S3. Preparation of amelogenin domains: Amelogenin domains with amphiphilic characteristics are obtained through genetic engineering or enzymatic digestion. S4. Self-assembly reaction: Mix the unfolded protein in S2 with the amelogenin structure in S3, adjust the pH to 6.5-8.0, and stir the reaction at 25℃-40℃ for 1-6 hours. S5. Formation of nanocrystalline sol: By regulating ionic strength and temperature, amphiphilic self-assembly of proteins and amelogenin domains is induced to form nanocrystalline sols with a particle size of 50-200 nm.

2. The method for constructing plant protein nanocrystal sol according to claim 1, characterized in that, It also includes the following steps: A natural cross-linking agent is added to the formed nanocrystalline sol to enhance the stability of the nanocrystalline structure. After stabilization treatment, the final product is obtained.

3. The method for constructing plant protein nanocrystal sol according to claim 2, characterized in that, The natural cross-linking agent is selected from one or more of genipin, plant polyphenols, proanthocyanidins or tannic acid, and is added at a mass of 0.05%-0.5% of the total protein.

4. The method for constructing plant protein nanocrystal sol according to claim 1, characterized in that, In step S1, the extraction step of soybean protein includes: dissolving soybean protein isolate in buffer, centrifuging to obtain the supernatant, and purifying it by ultrafiltration or salting out until the β-sheet structure content is not less than 30%.

5. The method for constructing plant protein nanocrystal sol according to claim 1, characterized in that, In step S3, the amelogenin domain is obtained by genetic engineering recombinant expression or enzymatic hydrolysis of natural amelogenin, with a molecular weight of 5-50 kDa and a hydrophobic torque ≥0.

4.

6. The method for constructing plant protein nanocrystal sol according to claim 1, characterized in that, In step S4, the mass ratio of the unfolded protein to the amelogenin domain is 1:0.2-2.

7. The method for constructing plant protein nanocrystal sol according to claim 1, characterized in that, In step S5, when adjusting the ionic strength, NaCl or KCl is used to make the final concentration 50-200 mM.