Egg white-sourced protein-based biological hydrogel as well as preparation method and application thereof

Abstract

The invention provides an egg white-sourced protein-based biological hydrogel as well as a preparation method and application thereof. The method comprises the following steps: separating and purifying ovomucoid in egg white by adopting an ethanol fractional precipitation method, and accurately controlling the final concentration of ethanol in a system in two ethanol precipitation processes, so that the final concentration a of ethanol in the system in the primary ethanol precipitation process and the final concentration b of ethanol in the system in the secondary ethanol precipitation process meet the condition that a is greater than or equal to 40 vol% and less than or equal to 62%; other miscellaneous proteins, salts and other small molecular impurities in the egg white can be removed, and the ovomucoid is efficiently separated and purified from the egg white; the modified ovomucoid obtained by covalently modifying the ovomucoid separated and purified by the method by using a modifier has the rheological characteristics of rapid crosslinking, shear thinning and the like, and can form the protein-based biological hydrogel under the action of a photoinitiator. The rapid prototyping and mechanical properties of the hydrogel are met, and the hydrogel has a wide application prospect in preparation of 3D biological printing materials, 4D biological printing materials, artificial organs, artificial tissues and dressings.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomaterials, and particularly relates to a protein-based biohydrogel from egg white as well as a preparation method and application thereof. BACKGROUND

[0002] Due to the three-dimensional porous structure, high water content and adjustable mechanical and biological properties, hydrogels have become a key matrix material in the fields of tissue engineering, regenerative medicine and bioprinting. Natural proteins as the building blocks of hydrogels can endow the material with excellent biocompatibility, degradability and inherent cell recognition sites and signal activity, and thus are considered as an ideal choice to replace traditional synthetic polymers.

[0003] Egg white, as the most abundant protein resource in poultry eggs, accounts for about 60% of the total egg mass. It is a highly complex natural protein system mainly including more than ten single proteins such as ovalbumin, ovotransferrin, ovomucoid, lysozyme, etc. These single proteins can form a gel network under physiological concentration through heat, salt or pH induction, showing a natural "self-gel" potential. In addition to the above main proteins, there are also glycoproteins, protease inhibitors and various growth factors, and the molecular weight, isoelectric point, glycosylation degree, thermal / chemical stability and biological activity of each component are significantly different.

[0004] The existing technology generally takes "whole egg white" as the object of research, ignoring the specific advantages of different single proteins in gel network formation, mechanical regulation, degradation kinetics and cell interaction, which may lead to the following problems: (1) functional redundancy and performance compromise: whole egg white-derived hydrogels often dilute key active sites due to the presence of a large number of non-target proteins, making it difficult to achieve precise functional design for specific tissue regeneration needs; (2) poor process controllability: the composition fluctuation of different batches of egg white makes it difficult to standardize quality parameters (such as degree of substitution, crosslinking density, and light initiator concentration), affecting batch consistency; (3) lagging mechanism research: the protein-protein interaction in whole egg white system is complex, and the independent contribution of single protein in gel formation, cell adhesion, growth factor presentation and immune regulation cannot be accurately analyzed, which further limits the rational design of high-performance and multifunctional hydrogels.

[0005] So far, there is no report on the systematic separation of single protein components in egg white and the directional construction of single protein hydrogel based on its molecular characteristics (isoelectric point, glycosylation site, sulfhydryl content, thermal denaturation temperature, enzyme cutting site, etc.) at home and abroad. If each major protein in egg white can be separated and purified, the differences in gelation behavior, mechanical properties, degradation mode and biological activity are analyzed, and then single protein components are selected or combined according to different tissue engineering application scenarios, it is expected to break through the performance bottleneck of the existing "whole egg white" hydrogel, and promote the egg white derived protein-based biological hydrogel to the direction of precision medicine and personalized regenerative medicine. SUMMARY

[0006] In order to solve the problem of performance decline of the prepared protein hydrogel due to the fluctuation of components between batches of whole egg white and the complexity of protein types in the process of preparing protein hydrogel by using "whole egg white" in the prior art, the present application provides an egg white derived protein-based biological hydrogel and a preparation method and application thereof.

[0007] According to a first aspect of the present application, a preparation method of an egg white derived protein-based biological hydrogel is provided, comprising the following steps: S1. Adding ethanol to egg white to make the final concentration of ethanol in the system a, and standing to obtain a first mixed solution; S2. Centrifuging the first mixed solution to obtain a first supernatant and a precipitate; S3. Resuspending the precipitate with ethanol to obtain a resuspension, and centrifuging the resuspension to obtain a second supernatant; S4. Mixing the first supernatant and the second supernatant, and adding ethanol to make the final concentration of ethanol in the system b, and standing to obtain a second mixed solution; Wherein a and b represent volume fraction, a and b satisfy 40 vol%≤a<b≤62%; S5. Separating and purifying ovalbumin from the second mixed solution, modifying the ovalbumin with a modifier to obtain modified ovalbumin, and making the modified ovalbumin form a protein-based biological hydrogel under the action of a photoinitiator; The modifier includes at least one of methacrylic anhydride, methacrylic ester, methacrylic amide, acrylic ester, styrene, hydroxyethyl methacrylate, lactic acid methacrylate, methacrylic acid modified caprolactone, norbornene, maleimide, vinyl sulfone, tetrazine, tyramine, catechol, azide-alkyne, imine, hydrazone compound and disulfide.

[0008] Among the above modifiers, methacrylate lactate is a monomer formed by the esterification reaction of lactic acid and methacrylic acid, methacrylate-modified caprolactone is a monomer formed by the esterification reaction of caprolactone and methacrylic acid, and azide compound-alkyne is a compound formed by the cycloaddition reaction of azide compound and alkyne.

[0009] To address the issue of uncontrollable performance caused by the complexity of egg white protein systems and the unclear function of individual proteins, this invention provides a method for preparing protein-based biohydrogels. Using egg white as the raw material, a stepwise ethanol precipitation method (primary and secondary ethanol precipitation) is employed to separate and purify ovomucoid in the egg white. During this process, the final ethanol concentration in the system is precisely controlled during both precipitation stages, ensuring that the final ethanol concentration 'a' in the primary precipitation stage and the final ethanol concentration 'b' in the secondary precipitation stage satisfy a0 = 40%. The relationship vol%≤a<b≤62% has several advantages. First, it can remove other impurities in egg white, such as ovalbumin and ovotransferrin, as well as small molecule impurities like salts, and efficiently separate and purify a single protein, ovomucoid (OVC), from egg white. The OVC obtained through this method is then covalently modified with a modifier, and the modified OVC forms a protein-based biohydrogel under the action of a photoinitiator. Second, egg white is a widely available and inexpensive raw material, and the above process can be scaled up to industrial production using conventional centrifugation and dialysis equipment. Third, the separation and extraction of OVC primarily uses an ethanol / water system, avoiding the use of highly toxic chemical reagents (such as urea and guanidine) or complex enzymatic hydrolysis processes, making the process greener, safer, and more environmentally friendly. Fourth, the separation and purification method minimizes damage to the protein's natural structure and biological activity during the extraction of OVC from egg white, resulting in a more refined product after covalent modification with the modifier. Ovomucin possesses rapid cross-linking and shear-thinning rheological properties. Under the action of a photoinitiator, it can form a protein-based biohydrogel, satisfying the requirements for rapid hydrogel molding and mechanical properties. Furthermore, polymers with different degrees of substitution (i.e., modified ovomucin) can be obtained by adjusting the amount of modifier. The relative standard deviation of mechanical test results for the same batch is <5%, significantly improving batch-to-batch variation. Fourth, the prepolymer obtained by covalently modifying the isolated and purified ovomucin and dissolving it in a buffer solution exhibits shear-thinning properties. It can gel quickly under photoinitiation, making it suitable for various applications requiring rapid molding and on-demand control of mechanical properties. Fifth, the protein-based biohydrogel is derived from a single natural protein component (i.e., ovomucin in egg white), possessing excellent biocompatibility, biodegradability, controllable mechanical properties, and a stable three-dimensional network structure, providing a safe, economical, and sustainable novel natural protein hydrogel platform for tissue engineering and regenerative medicine.

[0010] The ovomucoid obtained by the method provided by this invention, after being modified by a modifier, can be solidified and shaped under the action of a photoinitiator to obtain a protein-based biohydrogel derived from egg white. The photoinitiator can be a biocompatible ultraviolet photoinitiator or a biocompatible visible photoinitiator. The biocompatible ultraviolet photoinitiator includes 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone and / or ethyl 2,4,6-trimethylbenzoylphenylphosphonate (TPO), and the biocompatible visible photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) and / or 2959.

[0011] Preferably, in step S1, before adding ethanol to the egg white, the following homogenization process is performed on the egg white: the egg white is homogenized at 13~17℃ and 10000~12000 r / min for 25~35 s, stopped for 4~6 s, and this is considered one homogenization process. The egg white is repeatedly homogenized, and the total homogenization time of the egg white is 2~4 min.

[0012] Ovalbumin in natural egg white does not exist as a single molecule, but rather forms an insoluble complex gel network with proteins such as lysozyme through electrostatic interactions. Before performing a single ethanol precipitation treatment on egg white, homogenization is performed, with the rate and time of this homogenization process carefully controlled. Under high shear forces of 10,000–12,000 r / min, the natural complex gel structure of egg white can be effectively physically broken down, dissociating ovomucoid from the gel network and dispersing it in solution as more uniform monomers or smaller polymers. This lays a solid foundation for efficient and sufficient contact and precipitation of the target ovomucoid with ethanol, improving the final yield of ovomucoid and reducing batch-to-batch variations. This enhances the stability and repeatability of the overall performance of the protein-based biohydrogel prepared using ovomucoid. Furthermore, during the homogenization process, controlling the temperature between 13–17°C and employing short, intermittent cycles (25–35 s working, 4–6 s stopping) is crucial. The s) mode can minimize the heat generated by mechanical shearing and friction during homogenization, and avoid protein denaturation and inactivation due to local overheating, thus preventing protein damage. This allows the egg white to achieve physical fragmentation and full homogenization while preserving the natural spatial conformation and biological activity of oval mucin and other useful components as much as possible, thus retaining the key molecular structural basis for the subsequent preparation of functional hydrogels with ideal rheological properties and rapid cross-linking ability.

[0013] Preferably, in S1, a satisfies 40 vol% ≤ a ≤ 45 vol%.

[0014] Preferably, in S2, b satisfies 60 vol% ≤ b ≤ 62 vol%.

[0015] This method utilizes the differences in solubility of different proteins in egg white in an ethanol-water system to control the final ethanol concentrations a and b in the two ethanol precipitation processes to the aforementioned range. In the first ethanol precipitation process, the final ethanol concentration a is controlled between 40% and 45% (v / v) to selectively precipitate and remove most of the major impurity proteins while maximizing the retention of ovomucoid in the supernatant, achieving preliminary separation and enrichment of ovomucoid. In the second ethanol precipitation process, the final ethanol concentration b is controlled between 60% and 62% (v / v) to efficiently separate and purify the ovomucoid enriched in the first ethanol precipitation process. Thus, the two ethanol precipitation processes, through a gradient of ethanol concentration from low to high, achieve a precise purification process that selectively removes impurity proteins and efficiently precipitates the target protein from a complex mixture, improving the purity of the final ovomucoid and ensuring that the prepared biohydrogel possesses excellent, batch-to-batch consistent gelation and mechanical properties.

[0016] Preferably, in S5, the separation and purification of oval mucin from the second mixture includes the following steps: centrifuging the second mixture at 4°C and 3300~3500 g for 15~25 min, collecting the supernatant, reacting the supernatant in a water bath at 64~66°C for 15~25 min, cooling down, and obtaining oval mucin.

[0017] Preferably, after reacting the supernatant in a water bath at 64-66°C for 15-25 min and then cooling it down, the following steps are also included: dialyzing the obtained reaction solution to retain molecules with a molecular weight below 10 kDa until the ethanol concentration in the reaction solution is <0.01 vol%, obtaining a dialysate; centrifuging the dialysate at 4°C and 3300-3500 g for 15-25 min; collecting the supernatant and filtering it using a 0.22 μm filter membrane to obtain a filtrate; and freeze-drying the filtrate to obtain ovomucoid.

[0018] Preferably, in step S5, modifying ovomucin with a modifier to obtain modified ovomucin includes the following steps: dissolving ovomucin in a buffer solution to prepare an ovomucin solution, adding the modifier dropwise to the ovomucin solution and reacting at 24-26°C in the dark for 12-16 h to obtain modified ovomucin.

[0019] Preferably, the buffer solution is prepared from sodium carbonate solution and sodium bicarbonate solution, and the pH value of the buffer solution is 8.4~8.6. Preferably, the concentration of the sodium carbonate solution is 40-45 g / L, and the concentration of the sodium bicarbonate solution is 30-35 g / L.

[0020] Preferably, the solid-liquid ratio of ovomucoid to buffer is 0.5~2 g: 15~25 mL.

[0021] Preferably, the mass of ovomucoid in the ovomucoid solution is m (mg), and the volume of the modifier is V (μL). m and V satisfy the condition that m:V = 1:(5~20).

[0022] According to a second aspect of the present invention, an egg white-derived protein-based biohydrogel is provided, which is prepared by the above-described method for preparing an egg white-derived protein-based biohydrogel.

[0023] According to a third aspect of the present invention, the application of the above-mentioned protein-based biohydrogel derived from egg white in the preparation of 3D bioprinting materials, 4D bioprinting materials, artificial organs, artificial tissues, and dressings is provided.

[0024] The ovomucin obtained by the method provided in this invention, after covalent modification with a modifier, exhibits rapid cross-linking characteristics and rheological properties such as shear thinning. The modified ovomucin obtained after covalent modification with the modifier can form a protein-based biohydrogel under the action of a photoinitiator, satisfying the requirements of rapid prototyping and mechanical properties of hydrogels. It has broad application prospects in the preparation of 3D bioprinting materials (such as 3D bioprinted vascular analogs), 4D bioprinting materials, artificial organs, artificial tissues (such as skin tissue engineering scaffolds), and dressings. Attached Figure Description

[0025] Figure 1 A schematic diagram of OVCMA hydrogel formed by photocrosslinking of OVCMA powder prepared in step 1 in LAP solution.

[0026] Figure 2 The image shows the electrophoretic results of egg white (EW) and three independently extracted ovomucoid proteins (OVC-1, OVC-2, OVC-3) using SDS-PAGE technology for Test Example 2.

[0027] Figure 3 Example 3 is a test result of the protein concentration of ovomucoid (OVC) obtained during the preparation of protein-based biohydrogels derived from egg white using the preparation method provided in Example 1.

[0028] Figure 4 Example 4 is a test result of the degree of substitution of the protein-based biohydrogel derived from egg white prepared three times independently (1st, 2nd, 3rd) using the preparation method provided in Example 1.

[0029] Figure 5The image shows the results of a scan to detect the shear rate change of the protein-based biohydrogel derived from egg white prepared by the method provided in Example 1 for Test Example 5. Detailed Implementation

[0030] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0031] Example 1 A method for preparing an egg white-derived protein-based biohydrogel includes the following steps: S1. Select fresh brown-shelled eggs within 24 hours post-laying with a Haugh unit ≥75. Disinfect the egg surface with 75% alcohol spray and store at 4℃ for no more than 3 hours. Manually break the shells in a clean bench using a stainless steel egg white and yolk separator sterilized at 121℃ for 30 minutes to ensure no visible yolk residue remains in the egg white. Accurately measure the egg white volume V0 using a graduated cylinder and add an equal volume of deionized water to obtain a 2V0 volume of diluted egg white solution. Then, place the diluted egg white solution in a high-speed disperser and homogenize at 11000 r / min for 3 minutes, pausing for 5 seconds every 30 seconds. During homogenization, use an ice bath to control the system temperature at 15℃±2℃. Using a peristaltic pump, add pre-cooled anhydrous ethanol (-20℃) to the homogenized egg white solution at a rate of 5 mL / min until the final ethanol concentration in the system is a (a=43). vol%, assuming the current volume is 2V0, the volume of anhydrous ethanol to be added is V1 = [0.43 × (2V0 + V1) → V1 = 1.51V0), and mechanical stirring is performed at a speed of 200 r / min while adding the ethanol to obtain the first mixture; S2. The first mixture was centrifuged at high speed using a floor centrifuge at 4°C, 3400×g for 20 min. After centrifugation, the first supernatant and precipitate were obtained. S3. Take the first supernatant and store it at 4℃ for later use. Add 43% ethanol (2 times the original precipitate volume, recorded volume V2) to resuspend the precipitate and obtain the resuspended liquid. Centrifuge the resuspended liquid at high speed using a floor centrifuge at 4℃, 3400×g, for 20 min. After centrifugation, obtain the second supernatant. S4. Combine the first and second supernatants (total volume denoted as V3) and use a peristaltic pump to add pre-cooled anhydrous ethanol (pre-cooled to -20℃) at a constant rate of 5 mL / min until the final ethanol concentration in the system is b (b = 61 vol%). After completion, transfer to a 2 L beaker, seal with aluminum foil, and let stand at 4℃ in the dark for 12-14 h to obtain the second mixture. Calculate the volume of ethanol to be added to achieve a final ethanol concentration of 61 vol%, V4 = (0.61 × (V3 + V4)). (0.43×V3)→V4=0.85V3; S5. Centrifuge the second mixture that has been left to stand overnight at 4°C and 3400×g for 20 min to obtain the third supernatant. Place the third supernatant in a water bath preheated to 65°C for heat denaturation, then keep it at a constant temperature in a 250 mL glass beaker for 20 min (±0.5°C), and immediately place it in an ice-water bath for 5 min to cool it to below 10°C to obtain the protein solution. Then, dialyze the protein solution using a 10 kDa MWCO dialysis tube at 4°C with electromagnetic stirring for 4 h, with 1 L of deionized water for every 10 mL of protein solution. Change the water twice, dialyzing for 2 h each time, until the ethanol concentration of the dialysate is <0.01%. Centrifuge the dialysate at 4°C and 3400g for 20 min, collect the supernatant, and filter it through a 0.22 μm filter membrane to obtain the filtrate. Aliquot the filtrate into 50 mL lyophilization bottles and pre-freeze at -80°C for 4 h (thickness ≤1 cm). Place the pre-frozen sample in a Labconco FreeZone. A 6 L freeze dryer is set to a cold trap temperature of [temperature value missing]. 50℃, vacuum degree ≤0.05 mbar, freeze-drying procedure is as follows: 40℃, 6 h → The mixture was lyophilized at 20℃ for 4 h, then 0℃, then 4 h, then 20℃ for 6 h to obtain a fluffy white powder, which is ovomucoid (OVC) powder. 4.24 g of Na₂CO₃ (purity ≥99.5%) and 3.36 g of NaHCO₃ (purity ≥99.7%) were weighed, dissolved, and diluted to 100 mL of deionized water. The solutions were filtered through a 0.22 μm filter membrane to obtain sodium carbonate and sodium bicarbonate solutions. Sodium carbonate solution was added dropwise to the sodium bicarbonate solution and stirred at 25℃ until homogeneous. The pH of the solution was monitored with a pH meter until it reached 8.50 ± 0.02, at which point the addition of sodium carbonate solution was stopped, yielding a buffer solution, which was prepared and used immediately. The OVC powder was accurately weighed (with an error of ±1 mg) according to the ratio of 20 mL buffer solution: 1 g OVC powder and added to the buffer solution. The mixture was magnetically stirred at 25℃ and 200 r / min for 30 min, and sonicated at 40 kHz if necessary. Remove air bubbles with min to obtain a 5% (w / v) ovalbumin solution; add methacrylic anhydride (MA, 94% purity) to... Store at 20°C protected from light. Before use, allow to return to room temperature. In a light-protected fume hood, add methacrylic anhydride dropwise to the ovomucoid solution at a ratio of 1:10 (μL MA / mg OVC) using a micro-injection pump at a rate of 1 mL / min, while simultaneously stirring gently at 100 r / min using a top-mounted stirrer. Incubate the resulting liquid at 25°C protected from light for 12–16 h. Maintain a slight positive pressure of nitrogen at the top of the reactor to prevent oxidation of the methacrylic anhydride. After the reaction, dilute with two volumes of 4°C deionized water to terminate residual anhydride activity, yielding the reaction solution. Dialyze the reaction solution using a 10 kDa MWCO dialysis tube at 4°C and 100 r / min for 72 h, using 1 L of purified water for every 10 mL of reaction solution. Change the solution every 8 h. The dialysis is considered complete when the conductivity of the dialysate is ≤2 μS / cm. Aliquot the resulting dialysate into 50 mL lyophilized bottles and dry. Pre-freeze at 80℃ for 4 h, then place the pre-frozen sample in a Labconco FreeZone 6 L freeze dryer, setting the cold trap temperature to [temperature value missing]. 50℃, vacuum degree ≤0.05 mbar, freeze-drying procedure is as follows: 40℃, 6 h → White, spongy ovomucoid-methacrylic anhydride (OVCMA) powder was obtained by reflux at 20℃ for 4 h, followed by 0℃, 4 h, and then reflux at 20℃ for 6 h. The OVCMA powder was dissolved in a 0.5% LAP solution at a certain concentration. After complete dissolution, the solution was placed under 405 nm blue light and irradiated with 60 mW power for 30 s. After the above photocrosslinking, OVCMA hydrogel (i.e., the protein-based biohydrogel of this embodiment) was obtained. The OVCMA hydrogel was then freeze-dried according to the above freeze-drying operation and procedure to obtain OVCMA hydrogel powder. A schematic diagram of the OVCMA hydrogel formed after photocrosslinking of the OVCMA powder in LAP solution is shown below. Figure 1 As shown.

[0032] Example 2 This embodiment provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S1, during the homogenization process of the diluted egg white solution, the rotation speed is 13000 r / min and an ice bath is used to control the system temperature at 20℃±2℃.

[0033] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0034] Example 3 This embodiment provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S1, during the homogenization process of the diluted egg white solution, the rotation speed is 9000 r / min and an ice bath is used to control the system temperature at 10℃±2℃.

[0035] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0036] Example 4 This embodiment provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S1, a peristaltic pump is used to uniformly add anhydrous ethanol pre-cooled at -20℃ to the homogenized egg white dilution solution, and the addition rate of anhydrous ethanol is controlled at 7 mL / min.

[0037] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0038] Comparative Example 1 This comparative example provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S1, anhydrous ethanol is added during the preparation of the first mixture until the final ethanol concentration in the system is a = 25 vol.

[0039] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0040] Comparative Example 2 This comparative example provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S4, the first mixture and the second mixture are combined and anhydrous ethanol is added until the final ethanol concentration in the system is b = 70 vol.

[0041] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0042] Comparative Example 3 This comparative example provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S1, anhydrous ethanol is added during the preparation of the first mixture until the final ethanol concentration in the system is a = 61 vol.

[0043] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0044] Comparative Example 4 This comparative example provides a method for preparing a protein-based biohydrogel derived from egg white. Compared with Example 1, the difference in composition is that in step S4, the first mixture and the second mixture are combined and anhydrous ethanol is added until the final ethanol concentration in the system is b = 43 vol.

[0045] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0046] Comparative Example 5 This comparative example provides a method for preparing a protein-based biohydrogel derived from egg white. The difference in composition compared to Example 1 is that the oval mucin powder is prepared through the following steps: Fresh brown-shelled eggs (within 24 hours post-laying, Haugh units ≥ 75) are selected. The egg surface is disinfected with 75% alcohol spray and stored at 4°C for no more than 3 hours. The eggs are then manually cracked in a clean bench. A stainless steel egg white and yolk separator sterilized at 121°C for 30 minutes is used to ensure no visible yolk residue remains in the egg white. The volume V0 of the egg white is accurately measured using a graduated cylinder, and an equal volume of deionized water is added to obtain a 2V0 volume of diluted egg white solution. The diluted egg white solution is then placed in a high-speed disperser and homogenized at 11000 r / min for 3 minutes, with a 5-second pause every 30 seconds. During homogenization, an ice bath is used to control the system temperature at 15°C ± 2°C. A peristaltic pump is used to inject 5% of the homogenized egg white solution into the diluted solution at a rate of 5%. Add pre-cooled anhydrous ethanol at a rate of mL / min until the final ethanol concentration in the system is 25 vol%. Obtain a mixed solution and adjust the pH of the mixed solution to 4.5~5. After magnetic stirring for 15~30 min, centrifuge. The supernatant obtained is ultrafiltered and freeze-dried to obtain oval mucin powder.

[0047] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0048] Test Example 1 This test aims to calculate the OVC yield by measuring the mass of ovomucoid (OVC) powder obtained during the preparation of protein-based biohydrogels derived from egg white using the methods provided in Examples 1-4 and Comparative Examples 1-5. The results are shown in Table 1. The OVC yield (w / w) is calculated based on the OVC protein in the egg white, and the formula is as follows: OVC yield (%) = mass of OVC powder / (egg mass × 0.066 × 0.11), where egg mass × 0.066 × 0.11 is the theoretical mass of OVC contained in the egg used.

[0049] Table 1 OVC Yield

[0050] From the OVC yield results in Table 1, we can see that: The methods for preparing protein-based biohydrogels derived from egg white provided in Comparative Examples 1 and 3 adjusted the final ethanol concentration (a) of the system to 25 vol% and 61 vol%, respectively, during the first alcohol precipitation process. The methods for preparing protein-based biohydrogels derived from egg white provided in Comparative Examples 2 and 4 adjusted the final ethanol concentration (b) of the system to 70 vol% and 43 vol%, respectively, during the second alcohol precipitation process. The method for preparing protein-based biohydrogels derived from egg white provided in Comparative Example 5 only used a single alcohol precipitation to separate and purify ovomucoid (OVC), and the final ethanol concentration of the system was adjusted to 25 vol% during the first alcohol precipitation process. Compared with Comparative Examples 1-5, the methods for preparing protein-based biohydrogels derived from egg white provided in Examples 1-4 adjusted the final ethanol concentration (a) of the system to 43 vol% during the first alcohol precipitation process and the final ethanol concentration (b) of the system to 61 vol% during the second alcohol precipitation process. The OVC yields of Examples 1-4 were all higher than those of Comparative Examples 1-3. The lower OVC yield of Comparative Example 1 was due to the lower ethanol concentration (25 vol%) during the first alcohol precipitation process. The high ethanol concentration (70 vol%) in Comparative Example 2 has no effect on most proteins in egg white, resulting in excessive and large precipitates during the second ethanol precipitation. This leads to excessive supernatant entrainment during centrifugation, causing some loss of OVC and thus reducing the OVC yield. The OVC yield in Comparative Example 2 was only 14% because the high ethanol concentration (70 vol%) during the second ethanol precipitation caused most of the OVC to precipitate, leaving only a small portion in the supernatant, significantly reducing the yield. Comparative Example 3 used an excessively high ethanol concentration (61 vol%) during the first ethanol precipitation, causing OVC to precipitate during the first precipitation. Excessive precipitation also entrained OVC from the supernatant, further reducing the OVC yield. Although the methods for preparing protein-based biohydrogels derived from egg white provided in Comparative Examples 4 and 5 showed significantly higher OVC yields during the separation and purification of OVC compared to Examples 1 and 4, Comparative Example 4 had a lower ethanol concentration (only 43 vol%) during the second ethanol precipitation. (vol%) Some more hydrophilic proteins, such as ovotransferrin, remain in the supernatant, resulting in high-quality protein powder and excessively high yield. In this case, the protein powder contains too many impurity proteins, resulting in low purity of OVC. In contrast, in the purification of ovomucin, only one alcohol precipitation was used, and the final ethanol concentration of the system was 25 vol, with a pH of 4.5-5. In addition to OVC, the proteins remaining in the supernatant were ovotransferrin, lysozyme, and other proteins with high electrical points. OVC was also affected to some extent, with a small portion of OVC precipitating, resulting in low purity of OVC.

[0051] Compared to Example 1, Example 2 used a rotation speed higher than 12000 r / min and a system temperature higher than 17°C during the homogenization process of the egg white dilution, which to some extent damaged the protein structure, resulting in a significant decrease in the yield of OVC. Example 3 used a rotation speed lower than 10000 r / min and a system temperature lower than 10°C during the homogenization process of the egg white dilution, which led to incomplete homogenization, resulting in an unstable and decreased yield of OVC, as well as reduced purity and more impurity proteins. In Example 4, the anhydrous ethanol was added at an excessively high rate (7 mL / min) during the process of uniformly adding anhydrous ethanol to the homogenized egg white dilution using a peristaltic pump. The excessively rapid addition of anhydrous ethanol led to excessively high local ethanol concentrations in the system, resulting in increased OVC loss and a decrease in the yield of OVC.

[0052] The above results demonstrate that the method for preparing protein-based biohydrogels derived from egg white provided in Example 1 takes into account the activity, yield, and purity of OVCs, and is efficient, simple, and easy to scale up for production.

[0053] Test Example 2 This test aims to study the purity and molecular weight distribution of egg white (EW) and ovomucoid (OVC) extracted during the three independent preparations of protein-based biohydrogels derived from egg white using the preparation method provided in Example 1. The study verifies the purity and molecular weight distribution of egg white (EW) and the three independently extracted ovomucoid (OVC). Using SDS-PAGE technology and protein molecular weight standards as a reference, the differences in purity and molecular weight distribution between whole egg white protein and the three independently extracted OVCs are visually demonstrated, thus proving the reproducibility and high purity characteristics of the extraction process described in this invention. The specific experimental steps are as follows: 1. Test sample (1) Egg white (EW): The diluted egg white solution obtained in step S1 of Example 1; (2) Ovulminin (OVC): Following the method for preparing protein-based biohydrogels derived from egg white provided in Example 1, three complete OVC extractions were performed independently on different batches of eggs. The extracted OVCs were denoted as OVC-1, OVC-2, and OVC-3, respectively.

[0054] 2. Sample Preparation (1) EW sample: Take 50 μL of the egg white dilution obtained in step S1 of Example 1, add 50 μL of 2× loading buffer (denaturing / reducing Tricine protein loading buffer, purchased from Shanghai Yamei Biomedical Technology Co., Ltd.), vortex mix, and then incubate in a water bath at 95℃ for 5 min. Then, centrifuge the resulting mixture at 10000×g for 30 s to obtain the EW sample for later use. (2) OVC samples: Weigh 2.0 mg of each of OVC-1, OVC-2 and OVC-3 lyophilized powders and dissolve them in 1.0 mL of deionized water to prepare OVC-1 solution, OVC-2 solution and OVC-3 solution with a concentration of 2 mg / mL. Take 20 μL of OVC-1 solution, OVC-2 solution and OVC-3 solution respectively and mix them with 20 μL of 2× loading buffer. After mixing, incubate in a water bath at 95℃ for 5 min. Centrifuge the resulting mixture briefly at 10000×g for 30 s to obtain OVC-1 sample, OVC-2 sample and OVC-3 sample for later use. 3. Sample loading (1) Remove commercial Super-PAGE TM Precast gel (Bis-Tris, 4-12%, purchased from Shanghai Yamei Biomedical Technology Co., Ltd.), remove the bottom transparent seal, vertically insert the precast gel into the electrophoresis tank, tighten the side plates, slowly pour 1×Tris-Glycine-SDS buffer into the inner tank until the liquid level just covers the upper edge of the short plate, add buffer to the outer tank to the mark, let stand for 1 min to check for leaks; smoothly pull out the comb with both hands, immediately rinse each lane with buffer to remove fragments of gel and air bubbles; slowly add the prestained protein marker (purchased from Shanghai Yamei Biomedical Technology Co., Ltd.) and the samples of each experimental group (EW sample, OVC-1 sample, OVC-2 sample, OVC-3 sample) to the wall with a thin pipette tip, keeping the volume of each well consistent and not overflowing, according to the sample addition order shown in Table 2, add 10 μL of experimental group sample to each well; Table 2 Sample loading order

[0055] (2) Electrophoresis: Cover the tank and confirm that the red and black electrodes are correctly matched. Run the stacking gel at a constant voltage of 80 V (about 20 min), and then run it at a constant voltage of 120 V until the bromophenol blue front is 1 cm away from the bottom of the gel (about 90 min). Throughout the process, the electrophoresis tank is placed in an ice water bath to ensure that the gel temperature is <15℃. (3) Staining: Remove the gel and place it in Coomassie Brilliant Blue Rapid Staining Solution (purchased from Shanghai Yamei Biomedical Technology Co., Ltd.). Shake gently at room temperature for 1 hour. After the staining is complete, transfer it to PBS buffer and wash twice for 10 minutes each time. (4) Imaging and molecular weight calculation Images were taken using the white light transmission mode of an imaging system. Pre-stained protein bands were used as standards, and the apparent molecular weight of the target protein was estimated based on the migration of the sample bands. The SDS-PAGE electrophoresis results are as follows: Figure 2 As shown.

[0056] Depend on Figure 2 It can be seen that the EW sample showed multiple distinct bands at 76 kDa (ovotransferrin), 45 kDa (ovalbumin), 28 kDa (ovomucoid main band), and 14 kDa (lysozyme), with significant background diffusion; while the OVC-1, OVC-2, and OVC-3 samples all showed only a single band at 28 kDa, with a clear band and clean background, without any extraneous bands at 45 kDa and 14 kDa. The migration distances of the OVC bands extracted independently in the three extractions were consistent, which is consistent with the theoretical molecular weight of oval mucin reported in the literature.

[0057] The above SDS-PAGE electrophoresis results demonstrate that the method for preparing protein-based biohydrogels derived from egg white provided by this invention can effectively separate ovomucoid (OVC) with a molecular weight of 28 kDa from other components in egg white (including ovotransferrin, ovalbumin, and lysozyme). The method exhibits good reproducibility across three independent batches, with single bands, no contaminating proteins, and a purity ≥95%. The OVC prepared in this way can be directly used for subsequent methacrylic anhydride reaction (OVCMA preparation), which helps to ensure the specificity and uniformity of the covalent modification reaction.

[0058] Test Example 3 This test case aims to detect the protein concentration of ovomucoid (OVC) obtained during three independent (1st, 2nd, 3rd) preparations of protein-based biohydrogels derived from egg white using the preparation method provided in Example 1. The specific experimental procedures are as follows: Approximately 2 mg (accurate to 0.1 mg) of OVC powder obtained after freeze-drying in step S5 of the preparation of protein-based biohydrogels derived from egg white, prepared three independently (1st, 2nd, and 3rd) using the method provided in Example 1, was placed in a clean, dry 10 mL colorimetric tube. 1.0 mL of pre-warmed deionized water at 25°C was added, and the tube was gently inverted 5 times, then allowed to stand for 5 min to allow the protein to fully dissolve. The tube was then vortexed at maximum speed for 3 s, followed by centrifugation at 4°C and 13000×g for 2 min to remove insoluble particles. 100 μL of the supernatant was collected and 9.9 mL of deionized water was added. The mixture was inverted and mixed thoroughly to obtain a 100-fold diluted test solution. 3 mL of this test solution was placed in a 1 cm optical path quartz cuvette, and 3 mL of room-temperature equilibrated Bradford working solution (Coomassie Brilliant Blue G-250) was quickly added. 0.01% (w / v), dissolved in a mixture of 5% ethanol and 8.5% phosphoric acid, was immediately and gently stirred with a disposable plastic rod. The mixture was then incubated at 25°C in the dark for 10 min. The sample was zeroed using a blank cuvette (3 mL deionized water + 3 mL working solution). The absorbance was read at 595 nm using a spectrophotometer. The measured value was then substituted into the 0–25 μg / mL BSA standard curve (R0). 2 Calculate the diluted concentration C1 (≥0.995), multiply by 100 and divide by 1000 to obtain the actual OVC concentration (mg / mL). For each batch, the blank, standard, and 15 μg / mL quality control were simultaneously measured three times each. Experimental results are as follows: Figure 3 As shown.

[0059] Depend on Figure 3 It can be seen that during the preparation of protein-based biohydrogels derived from egg white in three independent (1st, 2nd, 3rd) processes using the preparation method provided in Example 1, the protein concentration differences of the three batches of ovomucoid (OVC) were within an acceptable range, proving that the preparation method provided in Example 1 can stably produce ovomucoid and has good repeatability and batch-to-batch consistency.

[0060] Test Example 4 This test example aims to detect the degree of substitution (i.e., the ratio of the amino concentration difference between unmodified OVC and the corresponding OVCMA to the amino concentration of unmodified OVC) of protein-based biohydrogels derived from egg white prepared three independently (1st, 2nd, 3rd) using the preparation method provided in Example 1. The specific experimental procedures are as follows: In the preparation of OVCMA powder, methacrylic anhydride was added dropwise to the ovomucoid solution at three gradients (1:5, 1:10, and 1:20 μL MA / mg OVC, i.e., MA:OVC = 0.2, 0.1, and 0.05 μL / mg) using a micro-injection pump to obtain three types of OVCMA powder to be tested. 2.00 mg (accurate to 0.01 mg) of the OVCMA powder to be tested was dissolved in 10.00 mL of 0.1 mol / L sodium bicarbonate buffer solution (filtered through a 0.22 μm filter and preheated to 25°C). After gently shaking 10 times and standing for 5 min, sonication at 40 kHz for 30 s was performed if necessary to aid dissolution. Simultaneously, an unmodified OVC solution of the same concentration was prepared as a control. 0.5 mL of each sample solution was transferred to a 1 cm optical path quartz cuvette, and 0.5 mL of 0.1% (w / v) OVC solution was added. In a 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution, gently stir and react at 37°C in the dark for 2 h. Then, add 0.5 mL of 10% (v / v) sodium dodecyl sulfate (SDS) solution and 0.25 mL of 1M hydrochloric acid (HCl) solution and vortex thoroughly to stop the reaction. Use a blank of 0.5 mL PBS buffer + 0.5 mL 0.1% TNBS + 0.5 mL 10% SDS + 0.25 mL 1M HCl. Read the absorbance A at 335 nm. 335 Each sample was tested in triplicate. The amino concentration in the corresponding sample could be obtained by using a standard curve. The degree of substitution is the ratio of the difference in amino concentration between unmodified OVC and the corresponding OVCMA to the amino concentration of unmodified OVC. The absorbance of L-alanine at 64, 32, 16, 8, 0.8, and 0 μg / mL was measured simultaneously for each batch to form a standard curve. If A... 335 For results >1.5, dilution and retesting are performed to ensure reliability. The degree of substitution test results for the protein-based biohydrogel derived from egg white prepared using the method provided in Example 1 are as follows: Figure 4 As shown.

[0061] Depend on Figure 4 It can be seen that the degree of substitution of OVCMA can be controlled by adding different amounts of methacrylic anhydride, and the amount of methacrylic anhydride added is the main factor in adjusting the degree of substitution.

[0062] Test Example 5 This test case aims to scan and detect the shear rate change of the protein-based biohydrogel derived from egg white prepared by the method provided in Example 1. The specific experimental procedures are as follows: 0.1 g, 0.2 g, and 0.4 g of the OVCMA powder obtained in Example 1 were added to 5 mL of PBS buffer (pH 7.4) and magnetically stirred for 30 min at room temperature until completely dissolved to prepare OVCMA prepolymer solutions with target concentrations of 20%, 40%, and 80% (w / v). These solutions were then sterilized using a 0.22 μm filter membrane and set aside for later use. The sterilized OVCMA prepolymer solutions of different concentrations were placed on an Anton Paar MCR 302 rheometer in a 25°C constant temperature water bath, using a 25 mm parallel plate-PP25 clamp with a 1 mm gap. The shear rate γ used in the shear rate scan program was 0.01 s⁻¹. -1 Increased to 1000 s -1 The shear rate change of the protein-based biohydrogel derived from egg white prepared in Example 1 was scanned and tested according to the above test method. The curve of the apparent viscosity as a function of shear rate of the protein-based biohydrogel derived from egg white prepared in Example 1 is shown below. Figure 5 As shown.

[0063] Depend on Figure 5 It can be seen that the viscosity of OVCMA hydrogel decreases significantly with increasing shear rate, proving that OVCMA hydrogel has shear-thinning properties, which can be applied to bioprinting.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A method for preparing a protein-based biohydrogel derived from egg white, characterized in that, Includes the following steps: S1. Add ethanol to the egg white to make the final concentration of ethanol in the system a, and obtain the first mixture; S2. Centrifuge the first mixture to obtain a first supernatant and a precipitate; S3. The precipitate is resuspended in ethanol to obtain a resuspended solution, and the resuspended solution is centrifuged to obtain a second supernatant. S4. Mix the first supernatant and the second supernatant, add ethanol to make the final concentration of ethanol in the system b, let stand, and obtain the second mixture. Wherein, 'a' and 'b' both represent volume fractions, and 'a' and 'b' satisfy 40 vol% ≤ a < b ≤ 62%; S5. Oval mucin is separated and purified from the second mixture, and modified with a modifier to obtain modified ovum mucin, which is then used to form the protein-based biohydrogel under the action of a photoinitiator. The modifier includes at least one of the following: methacrylic anhydride, methacrylate, methacrylamide, acrylate, styrene, hydroxyethyl methacrylate, lactate methacrylate, methacrylic acid-modified caprolactone, norbornene, maleimide, vinyl sulfone, tetraazine, tyramine, catechol, azide-alkyne, imine, hydrazone, and disulfide.

2. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 1, characterized in that, In step S1, before adding ethanol to the egg white, the following homogenization process is performed on the egg white: the egg white is homogenized at 13-17°C and 10000-12000 r / min for 25-35 s, stopped for 4-6 s, and this is considered one homogenization process. The egg white is repeatedly homogenized, and the total homogenization time of the egg white is 2-4 min.

3. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 1, characterized in that: In S1, 'a' satisfies 40 vol% ≤ a ≤ 45 vol% In S2, b satisfies 60 vol% ≤ b ≤ 62 vol%.

4. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 1, characterized in that, In step S5, the separation and purification of ovomucin from the second mixture includes the following steps: centrifuging the second mixture at 4°C and 3300-3500 g for 15-25 min, collecting the supernatant, reacting the supernatant in a water bath at 64-66°C for 15-25 min, cooling down, and obtaining the ovomucin.

5. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 4, characterized in that, After reacting the supernatant in a water bath at 64-66°C for 15-25 min and then cooling it down, the following steps are also included: dialyzing the obtained reaction solution to retain molecules with a molecular weight below 10 kDa until the ethanol concentration in the reaction solution is <0.01 vol%, obtaining a dialysate; centrifuging the dialysate at 4°C and 3300-3500 g for 15-25 min; collecting the supernatant and filtering it using a 0.22 μm filter membrane to obtain a filtrate; and freeze-drying the filtrate to obtain the ovomucoid.

6. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 1, characterized in that, In step S5, modifying the oval mucin with the modifier to obtain the modified oval mucin includes the following steps: dissolving the oval mucin in a buffer solution to prepare an oval mucin solution, adding the modifier dropwise to the oval mucin solution and reacting at 24-26°C in the dark for 12-16 h to obtain the modified oval mucin.

7. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 6, characterized in that: The buffer solution is prepared from sodium carbonate and sodium bicarbonate solutions, and the pH value of the buffer solution is 8.4-8.

6. And / or, The concentration of the sodium carbonate solution is 40-45 g / L, and the concentration of the sodium bicarbonate solution is 30-35 g / L.

8. The method for preparing the protein-based biohydrogel derived from egg white as described in claim 6, characterized in that: The solid-liquid ratio of the ovomucoid to the buffer solution is 0.5~2 g: 15~25 mL. And / or, Let m be the mass of the oval mucin in the oval mucin solution, in mg, and V be the volume of the modifier, in μL. Let m and V satisfy the condition m:V = 1:(5~20).

9. A protein-based biohydrogel derived from egg white, characterized in that: The protein-based biohydrogel is prepared by the method for preparing protein-based biohydrogel derived from egg white as described in any one of claims 1 to 8.

10. The application of the protein-based biohydrogel derived from egg white as described in claim 9 in the preparation of 3D bioprinting materials, 4D bioprinting materials, artificial organs, artificial tissues, and dressings.

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