High-molecular-weight keratin based on reversible redox catalysis as well as preparation method and application of high-molecular-weight keratin

Abstract

The invention discloses high-molecular-weight keratin based on reversible oxidation-reduction catalysis and a preparation method and application thereof, and belongs to the technical field of biomass high-molecular material processing.The pH of a first extracting solution or a second extracting solution is adjusted to 7-10, then crushed keratin raw materials are added, the first extracting solution is a hydrogen bond breaking agent aqueous solution, and the second extracting solution is a hydrogen bond breaking agent aqueous solution; adding a thiol catalyst and a reducing agent to form a second extracting solution to obtain a first mixed system or a second mixed system; the first mixed system is subjected to a stirring reaction at the constant reduction potential of-0.5 V to-1.5 V at the temperature of 20-50 DEG C, the second mixed system is subjected to a stirring reaction at the temperature of 20-50 DEG C, undissolved residues and impurities are removed, and then a product is separated and dried, so that the soluble keratin with high molecular weight and a complete secondary structure can be efficiently extracted from the waste keratin; and the application requirements of high-performance 3D printing bio-ink and the like are met.

Description

Technical Field

[0001] This invention belongs to the field of biomass polymer material processing technology, specifically a high molecular weight keratin based on reversible redox catalysis, its preparation method, and its application. Background Technology

[0002] Keratin is one of the most abundant natural proteins on Earth, widely found in biomass waste such as wool, feathers, hair, and hooves. Its unique amino acid sequence and α-helix / β-sheet secondary structure endow it with excellent biocompatibility, biodegradability, and mechanical properties, making it a promising candidate for applications in biomedicine, tissue engineering, and cosmetics. However, the extremely stable and insoluble structure formed by numerous hydrogen bonds, hydrophobic interactions, and high-density disulfide bonds between natural keratin molecular chains limits its high-value utilization.

[0003] To achieve the regeneration and utilization of keratin, it must first be dissolved. Existing technologies mainly include the following methods: Traditional chemical methods: such as hydrolysis or reduction using strong acids, strong bases, sulfides (such as sodium hydrosulfide) or sulfites. These methods are subject to harsh conditions (high temperature, extreme pH), which severely damage the peptide backbone and secondary structure of keratin, resulting in severe degradation of the product, leading to low molecular weight (e.g., diffuse distribution in the range of 5-30 kDa), poor purity, and dark color. In addition, the process generates a large amount of toxic and harmful sulfur-containing wastewater, causing serious environmental pollution. Ionic liquid (ILs) / eutectic solvent (DES) method: These new solvents can effectively destroy the hydrogen bond network to dissolve keratin under relatively mild conditions. However, their disadvantages are: (1) high cost, which limits large-scale industrial application; (2) high viscosity, which leads to difficulties in mass transfer and complicated separation and recovery processes after dissolution from keratin; (3) some ionic liquids have potential biotoxicity, which does not conform to the concept of green chemistry. Enzymatic / biological methods: using microbial enzyme systems such as keratinase to degrade keratin. This method is the mildest and most environmentally friendly. However, its mechanism of action is degradation rather than dissolution, that is, it destroys the overall structure by breaking peptide bonds. The molecular weight of the product is too low, so the product obtained is mainly a mixture of short peptides and amino acids, which completely loses the high molecular weight and natural secondary structure required as a structural material, and cannot be used to prepare high-performance materials (such as 3D printing inks, fibers or films).

[0004] Therefore, current keratin dissolution technologies cannot simultaneously achieve mild reaction conditions, product performance, and cost / environmental friendliness. They cannot efficiently extract high-molecular-weight (>40 kDa) soluble keratin with intact secondary structures from waste keratin under mild (room temperature, neutral pH), green (water-based, non-toxic), and low-cost conditions. This greatly hinders the widespread application of keratin as a high-performance biomaterial, especially in the field of 3D printing, which has stringent requirements for the molecular weight and structure of raw materials. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a high molecular weight keratin based on reversible redox catalysis, its preparation method, and its application. This solves the problem of not being able to efficiently extract high molecular weight (>40 kDa) soluble keratin with intact secondary structure from waste keratin under mild (room temperature, neutral pH), green (water-based, non-toxic), and low-cost conditions. It enables the efficient extraction of high molecular weight soluble keratin with intact secondary structure under green, low-cost, and mild conditions to meet the needs of applications such as high-performance 3D printing bio-inks.

[0006] This invention is achieved through the following technical solution: A method for preparing high molecular weight keratin based on reversible redox catalysis includes the following steps: Step 1: Adjust the pH of the first or second extract to 7.0-10.0, then add defatted and crushed keratin raw material. The first extract is an aqueous solution of hydrogen bond disruptor. Add thiol catalyst and reducing agent to the first extract to form the second extract, thus obtaining the first or second mixed system. Step 2: The first mixture is stirred and reacted under anaerobic conditions at 20-50°C and a constant reduction potential of -0.5 V to -1.5 V to obtain the first reaction solution; The second mixture was stirred and reacted under anaerobic conditions at 20-50°C to obtain the second reaction solution; Step 3: Remove undissolved residues and impurities from the first or second reaction solution, then separate the product and dry it to obtain high molecular weight keratin.

[0007] A further improvement of the present invention is that: The defatted and crushed keratin raw material described in step 1 is obtained through the following process: The keratin raw material is stirred in a nonionic surfactant aqueous solution at 30-50°C for 1-2 hours, then kept at constant weight at 60-80°C, and finally processed into short fibers or fine powder with a length of 0.5-5 mm to obtain the defatted pulverized keratin raw material.

[0008] The hydrogen bond disruptor mentioned in step 1 is one or more of arginine, arginine hydrochloride, guanidine salt, modified sugar alcohol, betaine and urea, and the reducing agent is L-ascorbic acid or tris(2-carboxyethyl)phosphine.

[0009] The concentration of the hydrogen bond disruptor in the first extract is 2-4 M, the concentration of the thiol catalyst in the second extract is 0.01-0.2 M, and the concentration of the reducing agent is 0.2-1.5 M.

[0010] In step 1, the ratio of defatted crushed keratin raw material to the first or second extract is 1g: (10-50)mL.

[0011] Step 2: The stirring reaction of both the first and second mixing systems is carried out for 1-8 hours.

[0012] Step 3: Centrifuge or filter the first or second reaction solution to obtain a supernatant or filtrate. Then separate the product from the supernatant or filtrate and dry it to obtain high molecular weight keratin.

[0013] Step 3: Dialyze the supernatant or filtrate in a 3.5-14 kDa dialysis bag for 48-72 hours, changing the deionized water every 6-8 hours to obtain a keratin solution. Dry the keratin solution as follows to obtain high molecular weight keratin: Pre-freeze at -85 to -75°C for 12 to 18 hours or cool from room temperature to -55 to -45°C at a rate of 1 to 2°C / min and hold for 4 to 8 hours. Then, sublimate and dry the resulting solid at a pressure below 10 Pa for 24 to 48 hours, and then raise it to room temperature at a rate of 0.1 to 0.3°C / min and hold for 6 to 12 hours.

[0014] A high molecular weight keratin obtained by the preparation method of high molecular weight keratin based on reversible redox catalysis as described in any one of the above.

[0015] The above-mentioned applications of high molecular weight keratin in 3D printing.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses a method for preparing high molecular weight keratin based on reversible redox catalysis. Natural keratin has a dense structure, with numerous disulfide bonds (-SS-) encapsulated by a hydrogen bond network and hydrophobic interactions, making them difficult for reducing agents to access. The defatted, pulverized keratin raw material is directly added to an aqueous solution of a hydrogen bond disruptor for a stirred reaction at a constant reduction potential. Alternatively, a thiol catalyst and a reducing agent are added to the hydrogen bond disruptor aqueous solution for a direct stirred reaction. The hydrogen bond disruptor possesses biocompatibility and swelling-exposure properties, allowing it to penetrate the keratin fiber interior. Like a molecular crowbar, it preferentially disrupts and replaces the hydrogen bond network between keratin peptide chains. This causes gentle swelling and unfolding of the keratin, loosening its three-dimensional structure and fully exposing the previously deeply embedded disulfide bonds, creating conditions for subsequent reduction reactions. After the disulfide bonds are fully exposed, a stirred catalytic reaction at a constant reduction potential or a direct stirred catalytic reaction with added thiol catalysts and a reducing agent can efficiently and gently cleave them precisely. For electrochemical catalysis: the working electrode, acting as the cathode, serves as a clean electron source. At a set constant reduction potential, it directly transfers electrons to the exposed disulfide bonds (-SS-), reducing them to two independent thiol groups (-SH + HS-). This process achieves precise cleavage of the cross-linking points, dissociating the insoluble keratin network into soluble high-molecular-weight single chains without introducing any chemical reducing agent impurities, making it a truly atom-economical reaction. Direct stirring catalysis forms a catalytic cycle, with the reducing agent acting as the final electron provider. However, its efficiency in directly reducing keratin disulfide bonds is relatively low. Thiol catalysts are first reduced to their active form by the reducing agent, and then rapidly cleave the keratin disulfide bonds through a highly efficient thiol-disulfide bond exchange reaction, becoming oxidized themselves. Subsequently, the oxidized catalyst is immediately regenerated by an excess of the main reducing agent and re-participates in the next cleavage cycle. This catalytic mechanism greatly improves reduction efficiency and significantly reduces the amount of strong reducing agent required. This invention solves the problems of existing technologies that either severely damage the main chain under harsh conditions or fail to sever crosslinks under mild conditions by employing a two-step synergistic strategy of swelling and exposure followed by precise shearing. This breakthrough enables the preparation of high-molecular-weight, structurally intact soluble keratin under green and mild conditions. It constructs a mild water-based dissolution system with the synergistic effect of hydrogen bond disruptors and reversible redox catalysis. The reaction is carried out in an aqueous solution within a mild temperature range (20-50°C) and near-neutral pH (7-10), avoiding the acid-base hydrolysis of keratin peptide chains in traditional chemical methods. The wool keratin obtained by this method, as analyzed by SDS-PAGE electrophoresis, has a molecular weight mainly distributed between 60-75 kDa (corresponding to the α-keratin subunit), consistent with the molecular weight of natural keratin subunits; while the molecular weight of products obtained by the traditional sodium sulfite method is dispersed between 5-30 kDa.Circular dichroism (CD) analysis revealed that the product of this invention retained approximately 25-35% of the α-helical structure, while the α-helical structure content of products from the traditional strong base method was less than 5%. The entire system uses water as a solvent, and the hydrogen bond disruptor is biocompatible, non-toxic, and inexpensive. The electrochemical method consumes only electrical energy and introduces no additional chemical pollutants, making it a truly atom-economical reaction. The use of a catalytic system significantly reduces the amount of reducing agent required, lowering costs and simplifying subsequent processing. The synergistic effect of the hydrogen bond disruptor and redox catalysis (the hydrogen bond disruptor causes keratin structure swelling, and the reversible redox catalysis efficiently contacts and breaks internal disulfide bonds) results in a dissolution efficiency far exceeding that of a single component. The reversibility of the electrochemical method provides unprecedented convenience for process control (start / stop) and product application (in-situ gelation).

[0017] This invention relates to a high-molecular-weight, structurally intact soluble keratin, whose aqueous solution can be formulated into a high-concentration (>20% w / v) fluid with excellent shear-thinning behavior, making it an ideal precursor for 3D printing bio-inks. The printed scaffold can be rapidly cured after mild oxidation (such as air, low-concentration H2O2, or electrochemical oxidation) to form a three-dimensional structure with good mechanical strength and cell compatibility. Attached Figure Description

[0018] Figure 1 This is a comparison of the SDS-PAGE electrophoresis patterns of soluble keratin extracted by the methods of Example 1 and Comparative Example 3 of the present invention.

[0019] Figure 2 Comparison of circular dichroism (CD) spectra of soluble keratin extracted by different methods of this invention.

[0020] Figure 3 This is a schematic diagram of the electrochemical catalytic dissolution process in Example 1 of the present invention.

[0021] Figure 4 The rheological properties curve of the keratin 3D printing ink prepared by the method of the present invention is shown.

[0022] Figure 5 The white flocculent keratin powder was extracted according to Example 1.

[0023] Figure 6 The yellow flocculent keratin powder was extracted according to Example 2.

[0024] Figure 7 The yellowish-brown keratin powder was extracted according to Comparative Example 3.

[0025] Figure 8 Macroscopic photograph of a tissue engineering scaffold 3D printed using the keratin ink of this invention.

[0026] Figure 9This is an SDS-PAGE electrophoresis pattern of soluble keratin extracted by the method in Example 2 of this invention.

[0027] In the figure: 31- Nickel foam; 32- Platinum sheet; 33- Ag / AgCl electrode. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications should be considered within the scope of protection of this invention.

[0029] This invention discloses a method for preparing high molecular weight keratin based on reversible redox catalysis. This method can extract high molecular weight, structurally intact, soluble keratin from waste keratin resources (such as waste wool, feathers, and human hair). The prepared soluble keratin can be used in high-end materials fields such as bio-3D printing inks. The specific preparation steps are as follows: Step 1: Pretreatment of keratin raw materials 1.1 Cleaning and degreasing (removing surface lipids): Immerse the keratin material (e.g., wool, feathers, or human hair) in an aqueous solution containing 0.1-1.0% (w / v) of a nonionic surfactant (such as Tween-20 or Triton X-100) at 30-50°C and wash with agitation for 1-2 hours. Then rinse repeatedly with plenty of deionized water until the wash solution is neutral and foam-free.

[0030] 1.2 Drying: The cleaned raw materials are dried in a forced-air drying oven at 60-80°C until constant weight.

[0031] 1.3 Mechanical processing: The dried raw materials are processed into short fibers or fine powder with a length of 0.5-5 mm by a crusher or shearer to increase their specific surface area for later use.

[0032] Step 2: Preparation of the water-based mild dissolution system in the reaction vessel 2.1 Addition of hydrogen bond disruptors: Dissolving component A (hydrogen bond disruptor) in deionized water to a final concentration of 2.0 M - 4.0 M is a key parameter for achieving mild and efficient dissolution in this invention. Its mechanism of action involves first disrupting the internal hydrogen bond network of keratin, causing the fiber structure to swell and exposing deeply embedded disulfide bonds. When the concentration is below 2.0 M, the driving force for hydrogen bond disruption is insufficient, resulting in inadequate swelling of the keratin fibers and a large number of disulfide bonds remaining trapped inside the fibers. This leads to inefficient subsequent reduction reactions, requiring longer reaction times or higher temperatures, and significantly reducing the final keratin solubility (typically below 50%), failing to meet the requirements of high-value applications for efficient raw material supply. When the concentration is above 4.0 M, although the solubility improves slightly, the process operability deteriorates drastically: the solution viscosity increases exponentially, leading to difficulties in stirring, uneven mass transfer, high energy consumption, and even the formation of clumps and gels, making subsequent separation and purification steps difficult. Excess component A needs to be removed in subsequent dialysis steps. High concentrations undoubtedly prolong dialysis time and increase pure water consumption, leading to a disproportionate increase in production costs. When the concentration increases from 4.0 M to 5.0 M, the keratin solubility improves by less than 5%, but the aforementioned process and cost issues are amplified dramatically, resulting in a decrease in overall techno-economic benefits. A concentration between 2.0 M and 4.0 M provides sufficient hydrogen bond breaking capacity to ensure sufficient keratin swelling and efficient disulfide bond exposure, thereby achieving a solubility exceeding 80% under mild conditions. Simultaneously, the system has moderate viscosity, good fluidity, and is easy to handle and process. Furthermore, purification costs are controllable, making the overall process optimally economical and feasible.

[0033] Component A is selected from one or more of arginine, arginine hydrochloride, guanidine salt, modified sugar alcohol (such as sorbitol), betaine and urea.

[0034] 2.2 Construction of a reversible redox mechanism: Option B1 (electrochemical catalysis): No additional chemical reagents are required. The solution of component A is used as the electrolyte, and the working electrode (WE, nickel foam), the reference electrode (RE, Ag / AgCl), and the counter electrode (CE, platinum sheet) are set in the reactor.

[0035] Option B2 (chemical catalysis): Add a thiol catalyst (0.01 M - 0.2 M, such as L-glutathione) and a mild primary reducing agent (0.2 M - 1.5 M, such as L-ascorbic acid or tris(2-carboxyethyl)phosphine) to the solution. The abbreviation for tris(2-carboxyethyl)phosphine is TCEP.

[0036] 2.3 pH adjustment: Use a 1 M NaOH or HCl solution to adjust the pH of the entire dissolution system to the range of 7.0-10.0, preferably pH 8.5-9.5.

[0037] Step 3: Catalytic dissolution of keratin 3.1 Feeding: Add the keratin raw material processed in step one to the dissolution system in step two according to a solid-liquid ratio of 1g: (10-50)mL.

[0038] 3.2 Reaction condition control: Temperature: The temperature of the reaction system is controlled at 20-50°C, preferably 30-40°C.

[0039] Atmosphere: To prevent the mercapto groups from being prematurely oxidized by oxygen in the air, the reaction is carried out under an inert atmosphere such as nitrogen or argon.

[0040] Stirring: Continuous mechanical or magnetic stirring is carried out throughout the reaction process to ensure uniform mass transfer.

[0041] Reaction time: The reaction lasts for 1-8 hours. During this time, most or all of the keratin fibers can be observed to dissolve, and the solution becomes clear or translucent.

[0042] 3.3 Redox Catalysis: If scheme B1 is adopted, a constant reduction potential of -0.5 V to -1.5 V (vs. Ag / AgCl) is applied by a potentiostat until the reaction is completed.

[0043] If scheme B2 is adopted, then it is only necessary to maintain the set reaction conditions.

[0044] Step 4: Purification and Recovery of Products 4.1 Solid-liquid separation: After the reaction is complete, the crude product solution is centrifuged at 6000-10000 rpm for 10-20 minutes, or filtered through a 0.45μm filter membrane to remove undissolved residues and impurities.

[0045] 4.2 Dialysis: Take the supernatant or filtrate and place it in a dialysis bag with a molecular weight cutoff of 3.5 kDa to 14 kDa (preferably 8-14 kDa). Dialyze the bag in a large volume of deionized water for 48-72 hours, changing the deionized water every 6-8 hours. This step aims to thoroughly remove small molecule impurities such as hydrogen bond breakers, salt ions, and residual reducing agents from the system.

[0046] 4.3 Freeze-drying: Dispense the purified keratin solution (concentration 10-20 mg / mL) after dialysis into freeze-drying vials or trays, ensuring the liquid layer thickness does not exceed 1.5 cm to facilitate heat and mass transfer. Then, proceed with freeze-drying as follows: 1. Pre-freezing stage: Pre-freeze the container containing the keratin solution in an ultra-low temperature freezer at -80°C for 12-18 hours, or directly on the shelf of a freeze dryer, setting the program to slowly cool from room temperature to -55 to -45°C at a rate of 1-2°C / min, and hold at this temperature for 4-8 hours. Ensuring that the solution freezes completely and uniformly into a solid is crucial for obtaining a product with good morphology.

[0047] 2. First drying stage: Start the vacuum system of the freeze dryer. Once the vacuum level reaches below 10 Pa, begin programmed temperature rise on the shelves. Set the shelf temperature to -20°C to -10°C, and perform sublimation drying on the solid under these conditions. This stage needs to last 24 to 48 hours to ensure complete sublimation of the ice crystals in the sample. This can be determined by monitoring the sample temperature and shelf temperature to ensure they are consistent.

[0048] 3. Secondary drying stage: After the first drying cycle, the shelf temperature is slowly increased to 22-25°C (room temperature, constant vacuum) at a rate of 0.1-0.3°C / min, and drying continues at this temperature and high vacuum for 6 to 12 hours. This step aims to remove residual bound water from the sample, ensuring the product is sufficiently dry for long-term stable storage.

[0049] 4. Final product: After the drying process is complete, turn off the vacuum pump and backfill with dry nitrogen or argon gas to atmospheric pressure, then remove the sample. The final product is a white or pale yellow, fluffy, fibrous solid powder, which is high-purity, high-molecular-weight soluble keratin (also known as S-thiolated keratin). Seal the product and store it in the dark at -20°C or lower.

[0050] Methods for determining key performance indicators Solubility (%): The solubility was determined by gravimetric method. The residual solid after the reaction was filtered, washed, dried, and weighed (M_residual). Solubility (%) = [(M_initial - M_residual) / M_initial] × 100%.

[0051] Molecular weight distribution (kDa): Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used. Keratin samples were treated with reducing loading buffer before electrophoresis and stained with Coomassie Brilliant Blue R-250. The major molecular weight distribution of the samples was determined by comparison with standard proteins of known molecular weight.

[0052] α-helix content (%): Circular dichroism (CD) chromatography was used. Keratin samples were prepared as 0.1 mg / mL aqueous solutions and scanned within the wavelength range of 190–260 nm. The mean residue molar ellipticity [θ] at 222 nm was measured. 222 And it is calculated using the following generally accepted formula: α-helix content (%) = {(-[θ]} 222 + 3000) / 39000} × 100%.

[0053] Example 1: Mild dissolution of Merino wool using an electrochemical catalytic system (preferred embodiment of the invention) This embodiment aims to illustrate the process of preparing high molecular weight soluble keratin from Merino wool using an electrochemical catalytic system.

[0054] 1. Raw material pretreatment: Take 10.0 g of clean Merino wool and cut it into short fibers of 1-3 mm. Place it in 200 mL of 40°C warm water containing 0.5% (w / v) Triton X-100 and degrease it with magnetic stirring for 2 hours. Rinse repeatedly with plenty of deionized water until neutral, then dry in a 60°C oven to constant weight. Shear it using a high-speed rotary shearing machine, with the target fiber length set at 3 mm. Collect the short fibers with a length within the range of 3 mm (±0.5 mm) through sieve sorting for subsequent experiments to obtain pretreated wool.

[0055] 2. Preparation of the dissolution system and setup of the apparatus: Weigh 126.4 g of L-Arginine Hydrochloride (MW ≈ 210.66 g / mol) solid powder into a beaker, add 150 mL of deionized water, and stir magnetically at room temperature until completely dissolved. After complete dissolution, transfer the solution to a 200 mL volumetric flask, rinse the beaker 2-3 times with deionized water, and transfer the washings to the volumetric flask as well. Finally, dilute to the mark with deionized water and mix well. This yields a 200 mL L-Arginine Hydrochloride solution with a final concentration of 3.0 M. Add this solution to a 250 mL jacketed glass electrolytic cell (see [link to relevant documentation]). Figure 3This solution was used as the electrolyte. In the electrolytic cell, a large-area nickel foam (labeled 31) was used as the working electrode (WE), a platinum sheet (labeled 32) as the counter electrode (CE), and an Ag / AgCl electrode (labeled 33) as the reference electrode (RE). The pH of the electrolyte was precisely adjusted to 9.0 using 1 M NaOH solution.

[0056] 3. Catalytic dissolution: Add 5.0 g of pretreated wool fiber to the above system and purge with nitrogen for 15 minutes to remove oxygen, then maintain the nitrogen atmosphere. Start a magnetic stirrer and connect a potentiostat to apply a constant reduction potential of -1.2 V (vs. Ag / AgCl). Maintain the reaction temperature at 35°C using a jacketed water bath. After 4 hours of reaction, it was observed that most of the wool fiber had dissolved, forming a homogeneous, slightly viscous, pale yellow, transparent solution.

[0057] 4. Purification and Recovery: The reaction solution was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected. The supernatant was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against a large volume of deionized water for 72 hours, with the deionized water changed every 6 hours. Finally, the dialyzed solution was freeze-dried as follows to obtain 4.3 g of... Figure 5 The white, fluffy, flocculent solid powder shown is high molecular weight soluble keratin: The dialyzed solution was dispensed into freeze-drying vials with a liquid layer thickness of 1.5 cm, and then freeze-dried according to the following steps: 1. Pre-freezing stage: Place the container containing the solution directly on the shelf of the freeze dryer, set the program to slowly cool from room temperature to -50°C at a rate of 1°C / min, and maintain this temperature for 6 hours; 2. First drying stage: Start the vacuum system of the freeze dryer. Once the vacuum level reaches 8 Pa, begin programmed temperature increase on the shelves. Set the shelf temperature to -20°C to -10°C, and perform sublimation drying on the solid under these conditions. This stage needs to last for 36 hours to ensure complete sublimation of the ice crystals in the sample. This can be determined by monitoring the sample temperature and shelf temperature to ensure they are consistent.

[0058] 3. Secondary drying stage: After the first drying cycle, the shelf temperature was slowly increased to room temperature at a rate of 0.2°C / min, and then dried for another 8 hours at this temperature and under a high vacuum of 8 Pa.

[0059] 4. Final product: After the drying process is complete, turn off the vacuum pump and backfill with dry nitrogen to atmospheric pressure, then remove the sample.

[0060] 5. Performance Characterization: Solubility: The calculated solubility is (5.0 g - (5.0 g - 4.3 g)) / 5.0 g = 86%.

[0061] Molecular weight analysis: A portion of the sample was analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 1 As shown in lane 1, the product exhibits a concentrated and dense main band within the 60-75 kDa molecular weight range, consistent with the molecular weight of the natural keratin subunit, indicating that the product has a high molecular weight and a concentrated distribution.

[0062] Secondary structure analysis: A portion of the sample was analyzed by circular dichroism (CD) chromatography, and the results are as follows: Figure 2 The solid curve is shown. The spectrum exhibits two typical α-helical negative characteristic peaks at 222 nm and 208 nm. Calculations indicate that the α-helical structure content is approximately 31%, suggesting that the product retains the natural secondary structure well.

[0063] Example 2: A chemical catalytic system gently dissolves chicken feathers. This embodiment aims to illustrate the universality of the present invention for keratin (β-keratin) from different sources.

[0064] 1. Raw material pretreatment: Take 10.0 g of waste chicken feathers, cut them into small pieces, and then degrease, wash, and dry them according to the method in Example 1.

[0065] 2. Preparation of the dissolving system: In a 250 mL three-necked flask, add 135 mL of deionized water and dissolve 54.6 g of sorbitol (final concentration 3 M), 3.07 g of L-glutathione (GSH, final concentration 0.1 M), and 26.4 g of L-ascorbic acid (final concentration 0.75 M). Adjust the pH to 8.5 with 1 M NaOH.

[0066] 3. Catalytic dissolution: Add 5.0 g of pretreated chicken feather powder to the above system, and react with magnetic stirring at 45°C for 6 hours under nitrogen protection.

[0067] 4. Purification and Recovery: The same centrifugation, dialysis (MWCO 3.5 kDa), and freeze-drying procedures as in Example 1 were followed to finally obtain 3.9 g of [the product / material]. Figure 6 The pale yellow flocculent solid shown.

[0068] 5. Performance Characterization: The solubility rate is 78%. For example... Figure 9 SDS-PAGE analysis showed that the main band was distributed in the range of 10-15 kDa, which is consistent with the molecular weight of the subunits of avian β-keratin.

[0069] Comparative Example 1: No redox system (only hydrogen bond breakers) This comparative example aims to demonstrate the necessity of redox systems.

[0070] The operation steps are the same as those in Example 1, with the only difference being: In step 3, the potentiostat remained connected but no reduction potential was applied (i.e., open circuit). After 4 hours, only slight swelling of the wool fibers was observed, the solution remained mostly clear, and a large amount of fiber remained undissolved. After filtration, washing, and drying, the undissolved wool residue weighed 4.86 g. The calculated solubility was less than 3%. This result indicates that hydrogen bond disruptors alone cannot effectively dissolve keratin under mild conditions.

[0071] Comparative Example 2: No hydrogen bond disruptor (only redox system) This comparative example aims to demonstrate the necessity of hydrogen bond disruptors.

[0072] The operation steps are the same as those in Example 1, with the only difference being: In step 2, arginine hydrochloride was not added; instead, a 0.1 M Tris-HCl buffer solution (pH 9.0) was used as the electrolyte. After 4 hours, the solution became slightly turbid, and most of the wool fibers remained intact. After filtration, washing, and drying, the undissolved wool residue weighed 4.28 g. The calculated solubility was less than 15%. This result indicates that without a hydrogen bond disruptor to cause keratin structure swelling, even with a redox system, it is impossible to efficiently access and break the internal disulfide bonds.

[0073] Comparative Examples 1 and 2 fully demonstrate that the synergistic effect between hydrogen bond disruptors and reversible redox catalysts is the key to achieving efficient and mild dissolution, which is the core innovation of this invention.

[0074] Comparative Example 3: Traditional Sodium Sulfite Method This comparative example employs a typical method found in the prior art.

[0075] 1. Dissolve: Take 5.0 g of the same pretreated wool as in Example 1 and add it to 200 mL of 0.5 M sodium sulfite solution, adjusting the pH to 9.0. Heat the mixture to 95°C and stir vigorously for 4 hours.

[0076] 2. Purification and Recovery: The same centrifugation, dialysis, and freeze-drying procedures as in Example 1 were followed to finally obtain 4.5 g of [the product / method / etc.]. Figure 7 The image shows a yellowish-brown hard powder.

[0077] 3. Performance Characterization: Solubility: Approximately 90%.

[0078] Molecular weight analysis: SDS-PAGE electrophoresis results are as follows: Figure 1 As shown in lane 2, there are no clear bands in the figure; instead, there is a diffuse tail extending from 30 kDa down to the low molecular weight region, indicating that the product has undergone severe degradation.

[0079] Secondary structure analysis: CD spectrum as shown Figure 2 As shown by the dashed curve, the spectrum exhibits a single strong negative peak near 200 nm, a typical characteristic of random coil structures. No α-helix characteristic peaks are observed at 222 nm and 208 nm. Calculations show that the α-helix content is less than 5%, indicating that its native structure has been almost completely destroyed.

[0080] It should be noted that lipids account for approximately 1-2% of waste keratin, primarily existing on the surface and in the spaces between keratin fibers through physical adsorption or encapsulation. These lipids exert a certain hydrophobic shielding effect, slightly hindering the penetration of hydrogen bond disruptors and reducing agents. However, the core advantage of this invention lies in the fact that high concentrations of hydrogen bond disruptors (such as arginine) possess inherent surface activity and solubilizing capabilities, enabling them to penetrate or bypass most of the lipid layer and act within the keratin. Simultaneously, mechanical stirring and gentle heating further promote mass transfer. Therefore, even without defatting, the synergistic dissolution system can effectively cleave the hydrogen bonds and disulfide bonds of keratin, achieving dissolution.

[0081] After degreasing: the solubility rate can reach over 85%, the reaction time is short, the resulting keratin solution is clear, the product is highly pure, and the color is white.

[0082] Without degreasing: The solubility can still reach a level with industrial application value (65-75%), but a longer reaction time is required (e.g., 5 hours), and the solution will contain a small amount of emulsified lipid impurities, and the product color will be yellowish.

[0083] For high-value applications, defatting is essential. It enables the preparation of high-purity products such as medical-grade hydrogels and cosmetic additives. Degreasing pretreatment eliminates lipid impurities at the source, greatly simplifying subsequent purification processes, reducing costs, and ensuring the biocompatibility and stability of the final product. From this perspective, defatting improves both product purity and process economy.

[0084] Example 3: Preparation and Application of High Molecular Weight Keratin 3D Printing Ink This embodiment illustrates the application of high molecular weight keratin prepared by the method of the present invention in the field of 3D printing.

[0085] 13D printing ink preparation: Take 25 g of the white flocculent keratin powder obtained in Example 1 and dissolve it in 100 mL of phosphate buffered saline (PBS, pH 7.4) to prepare a 25% (w / v, 25 g / 100 mL) high-concentration keratin solution. This solution is a homogeneous, high-viscosity fluid at room temperature and can be used as a 3D printing ink.

[0086] 2. Rheological performance testing: A portion of the ink was subjected to rheological analysis, and the results are as follows: Figure 4 As shown, this ink exhibits excellent shear-thinning behavior, meaning it has high viscosity at low shear rates, which is beneficial for maintaining the shape after printing; at high shear rates (such as when passing through the print head), the viscosity decreases rapidly, ensuring a smooth printing process.

[0087] 33D Printing and Curing: Using an extrusion 3D bioprinter, the above ink was printed into a porous mesh scaffold of 10 mm × 10 mm × 2 mm at room temperature. After printing, several drops of 0.03% H2O2 solution were added to the scaffold, or it was placed in air to oxidize for 12 hours, which cured the scaffold by reforming the disulfide bond cross-linking network.

[0088] 4. Final Product: After curing, a white hydrogel scaffold with regular morphology, precise porous structure, and certain mechanical strength was obtained, as shown in the macroscopic photograph. Figure 8 As shown, this scaffold demonstrates the enormous application potential of the product of this invention as a high-performance biomaterial.

[0089] The results of the above embodiments and comparative examples clearly demonstrate that the method and system provided by the present invention successfully solve the problem that the prior art cannot simultaneously achieve mild conditions, high product performance, and green low cost, and have significant progress and practicality.

[0090] Summary table of key data for examples and comparative examples

[0091] Table Analysis: Examples 1 and 2 demonstrate the effectiveness and versatility of the method of the present invention, which can obtain high solubility and high quality products under extremely mild conditions.

[0092] The results of Comparative Examples 1 and 2 present a stark contrast, as each removed a core component from the system, resulting in almost no dissolution. This irrefutably demonstrates the synergistic effect between the hydrogen bond disruptor and the redox system, which is the core inventive point of this invention.

[0093] Comparative Example 3 represents the level of the prior art. Although the solubility is high, it comes at the cost of harsh reaction conditions and devastating damage to product performance (low molecular weight, loss of structure, poor color), highlighting the significant advantages of the present invention in terms of product performance and process greening.

Claims

1. A method for preparing high molecular weight keratin based on reversible redox catalysis, characterized in that, Includes the following steps: S1, adjust the pH of the first or second extract to 7.0-10.0, then add the crushed keratin raw material. The first extract is an aqueous solution of hydrogen bond disruptor. Add thiol catalyst and reducing agent to the first extract to form the second extract, and obtain the first or second mixed system. S2, the first mixture is stirred and reacted under anaerobic conditions at 20-50°C and a constant reduction potential of -0.5 V to -1.5 V to obtain the first reaction solution; The second mixture was stirred and reacted under anaerobic conditions at 20-50°C to obtain the second reaction solution; S3, remove undissolved residues and impurities from the first or second reaction solution, then separate the product and dry it to obtain high molecular weight keratin.

2. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 1, characterized in that, The pulverized keratin raw material mentioned in S1 is defatted and pulverized keratin raw material, specifically obtained through the following process: The keratin raw material is stirred in a nonionic surfactant aqueous solution at 30-50°C for 1-2 hours, then kept at constant weight at 60-80°C, and finally processed into short fibers or fine powder with a length of 0.5-5 mm to obtain the defatted and pulverized keratin raw material.

3. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 1, characterized in that, The hydrogen bond disruptor described in S1 is one or more of arginine, arginine hydrochloride, guanidine salt, modified sugar alcohol, betaine, and urea, and the reducing agent is L-ascorbic acid or tris(2-carboxyethyl)phosphine.

4. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 3, characterized in that, The concentration of the hydrogen bond disruptor in the first extract is 2-4 M, the concentration of the thiol catalyst in the second extract is 0.01-0.2 M, and the concentration of the reducing agent is 0.2-1.5 M.

5. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 4, characterized in that, The ratio of pulverized keratin raw material to the first or second extract in S1 is 1g: (10-50)mL.

6. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 1, characterized in that, S2 The stirring reaction of the first and second mixing systems was carried out for 1-8 hours.

7. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 1, characterized in that, S3 centrifuges or filters the first or second reaction solution to obtain a supernatant or filtrate. Then, the product of the supernatant or filtrate is separated and dried to obtain high molecular weight keratin.

8. The method for preparing high molecular weight keratin based on reversible redox catalysis according to claim 7, characterized in that, S3. Dialyze the supernatant or filtrate in a 3.5-14 kDa dialysis bag for 48-72 hours, changing the deionized water every 6-8 hours to obtain a keratin solution. Dry the keratin solution as follows to obtain high molecular weight keratin: Pre-freeze at -85 to -75°C for 12 to 18 hours or cool from room temperature to -55 to -45°C at a rate of 1 to 2°C / min and hold for 4 to 8 hours. Then, sublimate and dry the resulting solid at a pressure below 10 Pa for 24 to 48 hours, and then raise it to room temperature at a rate of 0.1 to 0.3°C / min and hold for 6 to 12 hours.

9. A high molecular weight keratin obtained by the preparation method of high molecular weight keratin based on reversible redox catalysis as described in any one of claims 1 to 8.

10. The application of high molecular weight keratin as described in claim 9 in 3D printing.

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