Animal feather keratin extraction method

Abstract

The invention discloses an animal feather keratin extraction method which comprises the following steps: (1) mixing pretreated animal feathers with a tea saponin solution, and stirring to obtain degreased feathers; mixing the degreased feathers with an ethanol solution, and performing ultrasonic treatment to obtain loose feathers; (2) preparing liquid drops from the water phase and the oil phase through a micro-fluidic chip process, standing and curing the collected liquid drops, demulsifying and cleaning to obtain sodium alginate-CaCl2 microspheres; (3) mixing the loose feathers, water, the sodium alginate-CaCl2 microspheres and keratinase, then adjusting the pH value, and carrying out enzymolysis; finally, taking supernate; and (4) adjusting the pH value of the supernate, precipitating, dialyzing the precipitated mixed solution, and freeze-drying to obtain the animal feather keratin. According to the method disclosed by the invention, the slow release of calcium ions is realized by adding the sodium alginate-CaCl2 microspheres, so that the enzyme utilization rate is improved.

Description

Technical Field

[0001] This invention belongs to the field of keratin extraction technology, and more specifically, relates to a method for extracting keratin from animal feathers. Background Technology

[0002] Feathers are a significant solid waste product of poultry farming and a largely untapped biological resource. Keratin, the core component of feathers, accounts for 80% to 90% of their dry weight and is a uniquely structured and stable natural fibrous protein. The keratin macromolecule contains numerous disulfide bonds, hydrogen bonds, and other cross-linked structures, giving it excellent mechanical strength. Feather keratin is rich in various amino acids, with an extremely high content of cysteine, which is essential for forming disulfide bonds and maintaining the stable spatial structure of keratin. It is also rich in smaller amino acids such as glycine and serine. This amino acid composition gives keratin good biocompatibility, moisturizing properties, and cell affinity, enabling its application in human-related fields. Keratin can be extracted from waste feathers using biological or chemical methods and is widely used in medical tissue engineering, drug delivery systems, cosmetic moisturizers, and environmentally friendly biodegradable materials, achieving the recycling and utilization of waste.

[0003] Animal feather keratin can be extracted using physical, chemical, and biological methods. The extraction process primarily involves the breaking of hydrogen bonds and salt bonds, and the reconstruction of disulfide bonds. Physical methods are the earliest processing methods, mainly using high-temperature, high-pressure steam treatment. Physical methods can hydrolyze some disulfide bonds, softening and partially degrading the feathers, and are commonly used in the production of feed-grade keratin powder. However, this process is energy-intensive, the high temperatures easily damage amino acids, and the extracted product has a wide molecular weight distribution, resulting in lower functionality and value.

[0004] Chemical methods are currently the most widely used approach in research, and their core principle is to break disulfide bonds using chemical reagents. Chemical methods are further divided into acid-base hydrolysis, oxidation, and reduction methods. Acid-base hydrolysis uses strong acids or bases at high temperatures to hydrolyze peptide and disulfide bonds, yielding short keratin peptides or amino acid mixtures; however, this method completely destroys the higher-order structure of proteins. Oxidation methods use oxidants such as formic acid and hydrogen peroxide to oxidize disulfide bonds to sulfonic acid groups, yielding soluble keratin. The extracted product has good stability, but the oxidation process may damage sensitive amino acids such as tryptophan. Reduction methods use reducing agents such as mercaptoethanol, sulfite, and sodium borohydride to reduce disulfide bonds (-SS-) to thiol groups (-SH), thereby dissolving keratin. This method has a high extraction rate, but subsequent removal of the reagents and reformation of disulfide bonds are required for material preparation.

[0005] Biological methods utilize specific microorganisms or their secreted keratinase to specifically degrade feathers under mild conditions. Enzymatic hydrolysis is characterized by mild conditions, high specificity, low energy consumption, and environmental friendliness, and it better preserves the natural structure and activity of keratin, making it a current research hotspot. The catalytic performance of keratinase depends on its precise spatial conformation; keratinase molecules contain 2-3 specific Ca2+ groups. 2+ Combined with pocket, Ca 2+ Enzyme activity and stability can be enhanced by locking the enzyme's three-dimensional structure through the formation of polydentate coordination bonds with 3-4 oxygen atoms within the pocket. Current techniques typically involve directly adding CaCl2 to improve keratinase activity, but the initial addition of Ca... 2+ Excessive concentration can cause conformational distortion of enzyme molecules, resulting in a sharp drop in enzyme activity of 30%-40%; in the later stages, Ca... 2+ Furthermore, adsorption or precipitation can reduce the concentration to below 1 mM, thereby losing its stabilizing effect and resulting in enzyme utilization of less than 50%. Therefore, developing a precise sustained-release calcium ion extraction method combined with efficient enzymatic hydrolysis has become a core requirement for breaking through the high-value utilization of feather keratin. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting keratin from animal feathers. This method achieves slow release of calcium ions by adding sodium alginate-CaCl2 microspheres, thereby improving enzyme utilization.

[0007] To achieve the above objectives, the present invention provides a method for extracting keratin from animal feathers, the extraction method comprising: (1) The pretreated animal feathers and tea saponin solution are mixed and stirred to obtain defatted feathers; then the defatted feathers are mixed with ethanol solution and ultrasonically treated to obtain loose feathers; (2) Droplets were prepared by microfluidic chip process of aqueous phase and oil phase. After the collected droplets were allowed to stand and solidify, they were demulsified and washed to obtain sodium alginate-CaCl2 microspheres. The aqueous phase is prepared by the following steps: mixing sodium alginate solution and Ca-EDTA solution evenly, and filtering with a filter membrane to obtain the aqueous phase; the oil phase is prepared by the following steps: mixing electronic fluorinated liquid and fluorinated surfactant evenly to obtain the oil phase; (3) Mix the loose feathers, water, sodium alginate-CaCl2 microspheres and keratinase, then adjust the pH and perform enzymatic hydrolysis; finally, take the supernatant. (4) Adjust the pH of the supernatant, precipitate, and then dialyze and freeze-dry the precipitate mixture to obtain the animal feather keratin.

[0008] According to the present invention, preferably, the pretreatment of the animal feathers includes: removing stems and impurities from the animal feathers, rinsing and trimming them; The animal feathers are at least one of chicken feathers, duck feathers, and goose feathers; The term "shortening" refers to cutting the material into short segments of 1-3 cm.

[0009] In this invention, preferably, the feather shafts and dust are removed by hand, the feathers are rinsed 2-3 times with deionized water, cut into 1-3 cm short segments, and drained.

[0010] According to the present invention, preferably, in step (1), the tea saponin solution is an aqueous solution of tea saponin with a mass concentration of 0.5-2%; The ratio of animal feathers to tea saponin solution was 1:(18-22) g / mL; The stirring process is carried out at a temperature of 45-55℃ for 25-35 minutes.

[0011] In this invention, the tea saponin used is tea saponin extracted from camellia oil cake residue, with a purity ≥90%; the stirring speed is preferably 50-80 rpm.

[0012] In this invention, after degreasing treatment with tea saponin solution, the feather sebum removal rate is ≥85% and the keratin denaturation rate is ≤3%.

[0013] According to the present invention, preferably, in step (1), the ethanol solution is an aqueous ethanol solution with a mass concentration of 8-15%; The ratio of animal feathers to ethanol solution was 1:(18-22) g / mL; The ultrasonic treatment is intermittent, consisting of 25-35 seconds of operation followed by a 25-35 second pause; the total duration of the intermittent treatment is 10-20 minutes. The frequency of the ultrasonic treatment is 15-25kHz, and the power is 140-155W; The system temperature was maintained at 45-50 ℃ during the ultrasound process.

[0014] In this invention, the total time of the intermittent processing is the sum of the working time and the pause time; the resulting loose feather surface will form 50-100 nm micropores.

[0015] In this invention, the ratio of animal feathers to tea saponin solution and the ratio of animal feathers to ethanol solution, as well as the weight of animal feathers, refer to the weight of animal feathers before pretreatment.

[0016] According to the present invention, preferably, in step (2), the working parameters for preparing droplets by microfluidic chip technology include: aqueous phase flow rate of 6-10 μL / min and oil phase flow rate of 13-17 μL / min; The time for the droplets to stand and solidify is 0.5-1 hour; The sodium alginate-CaCl2 microspheres have a particle size of 1-2 μm.

[0017] In this invention, preferably, the droplets are solidified in an acetic acid oil phase; the preparation method of the acetic acid oil phase includes: mixing glacial acetic acid and the oil phase evenly to obtain the acetic acid oil phase, wherein the volume percentage of glacial acetic acid is 1-2% based on the total volume of the acetic acid oil phase.

[0018] In this invention, in step (2), the microspheres are washed three times by centrifugation with PBS buffer (pH=7.4); in the sodium alginate-CaCl2 microspheres, Ca²⁺ + Loading capacity ≥ 0.5 mmol / g microspheres.

[0019] According to the present invention, preferably, in step (2), the sodium alginate solution is an aqueous solution of sodium alginate with a mass concentration of 1-2%; The Ca-EDTA solution is prepared by a method comprising the following steps: mixing an aqueous solution of CaCl2 and an aqueous solution of EDTA, adjusting the pH to 7-7.5, and adding water to make up to a final volume to obtain the Ca-EDTA solution; wherein the concentration of Ca-EDTA in the Ca-EDTA solution is 200-250 mmol / L; The volume ratio of the sodium alginate solution to the Ca-EDTA solution is (0.5-1.5):1; Filter using a 0.2-0.3μm filter membrane; The content of fluorinated surfactants is 1.5-2.5 wt% based on the total weight of the oil phase. The electronic fluorinated liquid is a fluorocarbon oil.

[0020] In this invention, the crosslinking mechanism of sodium alginate and CaCl2 in the preparation of sodium alginate-CaCl2 microspheres is as follows: Figure 2 As shown.

[0021] According to the present invention, preferably, in step (3), the ratio of the loose feathers to water is 1:(18-22) g / mL; The amount of keratinase added was 0.1-0.3 wt% based on the total weight of the loose feathers, water, sodium alginate-CaCl2 microspheres, and keratinase. The amount of sodium alginate-CaCl2 microspheres added is such that when the Ca²⁺ in the sodium alginate-CaCl2 microspheres is... + When 100% released, the Ca²⁺ in the enzymatic hydrolysis solution... + The concentration ranges from 1.2 to 5.0 mmol / L; Adjust the pH to 9.0-9.5; The enzymatic hydrolysis is performed at a temperature of 50-60℃ for 4-6 hours. Centrifuge at 8000-10000 rpm and collect the supernatant.

[0022] In this invention, in an alkaline environment, the carboxyl groups on the sodium alginate molecular chain completely dissociate into -COO. - The negative charge density is significantly increased. Excess -COO - The electrostatic repulsion between them will weaken the interaction with Ca² + Coordination binding leads to partial Ca² + The microspheres detach from their cross-linking sites and enter the solution, thereby enhancing enzyme activity. During enzymatic hydrolysis, the microspheres release Ca²⁺ in stages. + Release rate: 25%-35% at 2 hours, 50%-55% at 4 hours, and 95%-100% at 6 hours.

[0023] According to the present invention, preferably, in step (3), the activity of the keratinase is ≥5000 KerU / g, and the source is Bacillus subtilis; the stirring speed during the enzymatic hydrolysis is 50-80 rpm.

[0024] According to the present invention, preferably, in step (4), the pH of the supernatant is adjusted to 5.5-6.5; Dialysis is performed using dialysis bags for 24-36 hours. The freeze-drying process involves pre-freezing followed by vacuum freeze-drying.

[0025] The technical solution of the present invention has the following beneficial effects: (1) The sodium alginate-CaCl2 microspheres of the present invention release Ca in stages 2+ This avoids initial high-concentration enzyme denaturation and subsequent low-concentration enzyme inactivation, thus improving enzyme utilization. 2+ Low residue rate.

[0026] (2) In this invention, the keratin extraction rate is over 87%; the molecular weight of the product is concentrated in 5-12 kDa, which meets the standards for cosmetic and medical grade.

[0027] (3) In this invention, tea saponin is used to replace NaOH. When the pH is neutral, the disulfide bond will be broken and the α-helix retention rate is >85%. The COD value of the degreasing waste liquid is low and it can be directly used for irrigation. Both environmental protection costs and raw material costs are reduced.

[0028] (4) In this invention, low-frequency ultrasound assists in ethanol penetration, which significantly shortens the time for feathers to loosen and improves the pretreatment efficiency; intermittent ultrasound avoids ethanol from damaging the structure, and the fiber integrity is preserved by more than 90%.

[0029] (5) In this invention, tea saponin and sodium alginate are industrial-grade raw materials, and the cost is only 1 / 5 to 1 / 3 of that of L-cysteine; the preparation of microfluidic microspheres can be scaled up in pilot-scale (≥10 g per hour), and the ultrasonic, centrifugal and other equipment are conventional industrial equipment and do not require special customization.

[0030] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0031] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the invention.

[0032] Figure 1 A flowchart of a keratin extraction method according to an embodiment of the present invention is shown.

[0033] Figure 2 A diagram illustrating the crosslinking mechanism of sodium alginate and CaCl2 according to the present invention is shown. Detailed Implementation

[0034] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0035] The present invention is further illustrated by the following examples: In the following embodiments: the fluorosurfactant used is DuPont's Capstone FS-81; the fluorocarbon oil (electronic fluorinated liquid) used is 3M's HFE-7500; The keratinase used had an activity ≥5000 KerU / g and was derived from Bacillus subtilis.

[0036] In the following examples: Ca²⁺ in sodium alginate-CaCl₂ microspheres + The loading amount is the ratio of the molar amount of calcium ions in the microspheres to the dry weight of the microspheres. The mass of calcium ions in the microspheres is measured by ultraviolet spectrophotometer. Keratin extraction rate was calculated based on nitrogen content and measured using the Kjeldahl method. α-helix retention rate: The α-helix retention rate was determined by Fourier transform infrared spectroscopy; Keratin molecular weight: Keratin molecular weight was determined using SDS-PAGE.

[0037] Example 1

[0038] like Figure 1 As shown in the figure, this embodiment provides a keratin extraction method, as detailed below: Take 100 g of fresh chicken feathers, remove stems and impurities, rinse 3 times, and cut into 2 cm short segments; prepare 2000 mL of 1% tea saponin aqueous solution (20 g tea saponin + 1980 mL deionized water), add the feathers, and stir at 50℃ for 30 min; rinse twice and drain; add 2000 mL of 10% ethanol aqueous solution, and treat intermittently with ultrasound at 20 kHz for 15 min (power 150 W, intermittent treatment is 30 s working and 30 s pausing, and the system temperature is maintained at 48℃ during ultrasound), to obtain loose feathers with an α-helix retention rate of 92%.

[0039] Preparation of sodium alginate-CaCl2 microspheres: (1) Mix 1.2 mL of 2 M CaCl2 aqueous solution with 4.8 mL of 0.5 M EDTA aqueous solution, adjust the pH to 7.2 with 2 M NaOH aqueous solution, and make up to 10 mL with deionized water to obtain Ca-EDTA solution (the concentration of Ca-EDTA in Ca-EDTA solution is 240 mmol / L); mix 2% sodium alginate aqueous solution and Ca-EDTA solution at a volume ratio of 1:1, and use 0.22 (1) Filter the aqueous phase through a μm filter membrane; (2) Add a fluorinated surfactant to the fluorocarbon oil and mix evenly to obtain the oil phase; wherein, based on the total weight of the oil phase, the content of the fluorinated surfactant is 2wt%; (3) Load the aqueous phase and oil phase into syringes and install them into the injection pump, and connect them to the microfluidic chip; set the flow rate of the aqueous phase to 8μL / min and the flow rate of the oil phase to 15μL / min, and form droplets in the chip; observe the droplet morphology under a real-time microscope, and start collecting after stabilization; collect the droplets into a centrifuge tube containing 15mL of acetic acid oil phase (the preparation method of acetic acid oil phase is: mix 1ml of glacial acetic acid and 99mL of oil phase by ultrasonication to obtain acetic acid oil phase), and let stand for 30min to solidify; add 5mL of demulsifier (ethanol: n-hexane = 1:1, v / v) to the centrifuge tube and shake for 5min to demulsify; remove the upper oil phase, and centrifuge and wash 3 times with PBS buffer (pH=7.4) (centrifugation rate 3000 rpm, 5 min). (min / batch) to obtain sodium alginate-CaCl2 microspheres (particle size 1.5 μm, Ca²⁺). + Loading capacity: 0.55 mmol / g.

[0040] Enzymatic hydrolysis: 100g of loose feathers were added to 2000mL of deionized water, followed by 3.63g of sodium alginate-CaCl2 microspheres (Ca²⁺). + When 100% released, the total Ca² in the enzymatic hydrolysis solution +Add 0.1 g of keratinase (concentration 2 mM), adjust the pH to 9.0 with 1 M NaOH aqueous solution, and incubate at 55℃ for 5 h (stirring speed 60 rpm during incubation); centrifuge at 10000 rpm for 20 min, collect the supernatant, adjust the pH to 6.0 with 1 M HCl aqueous solution (to reach the isoelectric point), and precipitate for 30 min; place the precipitate mixture into a 3 kDa molecular weight cutoff dialysis bag, and dialyze with deionized water at 4℃ for 30 h, changing the dialysate 3-4 times during the process to remove residual Ca²⁺. + Tea saponins and small molecule peptides were extracted; pre-frozen at -50℃ for 2 h and freeze-dried under vacuum for 14 h to obtain keratin. The keratin extraction rate was 87.6% and the molecular weight of keratin was 7 kDa.

[0041] Example 2

[0042] like Figure 1 As shown in the figure, this embodiment provides a keratin extraction method, as detailed below: Take 100 g of fresh chicken feathers, remove stems and impurities, rinse 3 times, and cut into 2 cm short segments; prepare 2000 mL of 1% tea saponin aqueous solution (20 g tea saponin + 1980 mL deionized water), add the feathers, and stir at 50 ℃ for 30 min; rinse twice and drain; add 2000 mL of 10% ethanol aqueous solution, and treat intermittently with ultrasound at 20 kHz for 15 min (power 150 W, intermittent treatment is 30 s working and 30 s pausing, and the system temperature is maintained at 48 ℃ during ultrasound), to obtain loose feathers with an α-helix retention rate of 92%.

[0043] Preparation of sodium alginate-CaCl2 microspheres: (1) Mix 1.2 mL of 2 M CaCl2 aqueous solution with 4.8 mL of 0.5 M EDTA aqueous solution, adjust the pH to 7.2 with 2 M NaOH aqueous solution, and make up to 10 mL with deionized water to obtain Ca-EDTA solution (the concentration of Ca-EDTA in Ca-EDTA solution is 240 mmol / L); mix 2% sodium alginate aqueous solution and Ca-EDTA solution at a volume ratio of 1:1, and use 0.22 (1) Filter the aqueous phase through a μm filter membrane; (2) Add a fluorinated surfactant to the fluorocarbon oil and mix evenly to obtain the oil phase; wherein, based on the total weight of the oil phase, the content of the fluorinated surfactant is 2wt%; (3) Load the aqueous phase and oil phase into syringes and install them into the injection pump, and connect them to the microfluidic chip; set the flow rate of the aqueous phase to 8μL / min and the flow rate of the oil phase to 15μL / min, and form droplets in the chip; observe the droplet morphology under a real-time microscope, and start collecting after stabilization; collect the droplets into a centrifuge tube containing 15mL of acetic acid oil phase (the preparation method of acetic acid oil phase is: mix 1ml of glacial acetic acid and 99mL of oil phase by ultrasonication to obtain acetic acid oil phase), and let stand for 30min to solidify; add 5mL of demulsifier (ethanol: n-hexane = 1:1, v / v) to the centrifuge tube and shake for 5min to demulsify; remove the upper oil phase, and centrifuge and wash 3 times with PBS buffer (pH=7.4) (centrifugation rate 3000 rpm, 5 min). (min / batch) to obtain sodium alginate-CaCl2 microspheres (particle size 1.5 μm, Ca²⁺). + Loading capacity: 0.55 mmol / g.

[0044] Enzymatic hydrolysis: 100g of loose feathers were added to 2000mL of deionized water, followed by 5.43g of sodium alginate-CaCl2 microspheres (Ca²⁺). + When 100% released, the total Ca² in the enzymatic hydrolysis solution + Add 0.2 g of keratinase (concentration 3 mM), adjust the pH to 9.5 with 1 M NaOH aqueous solution, and incubate at 55℃ for 5 h (stirring speed 60 rpm during incubation); centrifuge at 10000 rpm for 20 min, collect the supernatant, adjust the pH to 6.0 with 1 M HCl aqueous solution (to reach the isoelectric point), and precipitate for 30 min; place the precipitate mixture into a 3 kDa molecular weight cutoff dialysis bag, and dialyze with deionized water at 4℃ for 30 h, changing the dialysate 3-4 times during the process to remove residual Ca²⁺. + Tea saponins and small molecule peptides were extracted; pre-frozen at -50℃ for 2 h and freeze-dried under vacuum for 14 h to obtain keratin. The keratin extraction rate was 89.3% and the molecular weight of keratin was 8 kDa.

[0045] Example 3

[0046] like Figure 1As shown in the figure, this embodiment provides a keratin extraction method, as detailed below: Take 100 g of fresh chicken feathers, remove stems and impurities, rinse 3 times, and cut into 2 cm short segments; prepare 2000 mL of 1% tea saponin aqueous solution (20 g tea saponin + 1980 mL deionized water), add the feathers, and stir at 50 ℃ for 30 min; rinse twice and drain; add 2000 mL of 10% ethanol aqueous solution, and treat intermittently with ultrasound at 20 kHz for 15 min (power 150 W, intermittent treatment is 30 s working and 30 s pausing, and the system temperature is maintained at 48 ℃ during ultrasound), to obtain loose feathers with an α-helix retention rate of 92%.

[0047] Preparation of sodium alginate-CaCl2 microspheres: (1) Mix 1.2 mL of 2 M CaCl2 aqueous solution with 4.8 mL of 0.5 M EDTA aqueous solution, adjust the pH to 7.2 with 2 M NaOH aqueous solution, and make up to 10 mL with deionized water to obtain Ca-EDTA solution (the concentration of Ca-EDTA in Ca-EDTA solution is 240 mmol / L); mix 2% sodium alginate aqueous solution and Ca-EDTA solution at a volume ratio of 1:1, and use 0.22 (1) Filter the aqueous phase through a μm filter membrane; (2) Add a fluorinated surfactant to the fluorocarbon oil and mix evenly to obtain the oil phase; wherein, based on the total weight of the oil phase, the content of the fluorinated surfactant is 2wt%; (3) Load the aqueous phase and oil phase into syringes and install them into the injection pump, and connect them to the microfluidic chip; set the flow rate of the aqueous phase to 8μL / min and the flow rate of the oil phase to 15μL / min, and form droplets in the chip; observe the droplet morphology under a real-time microscope, and start collecting after stabilization; collect the droplets into a centrifuge tube containing 15mL of acetic acid oil phase (the preparation method of acetic acid oil phase is: mix 1ml of glacial acetic acid and 99mL of oil phase by ultrasonication to obtain acetic acid oil phase), and let stand for 30min to solidify; add 5mL of demulsifier (ethanol: n-hexane = 1:1, v / v) to the centrifuge tube and shake for 5min to demulsify; remove the upper oil phase, and centrifuge and wash 3 times with PBS buffer (pH=7.4) (centrifugation rate 3000 rpm, 5 min). (min / batch) to obtain sodium alginate-CaCl2 microspheres (particle size 1.5 μm, Ca²⁺). + Loading capacity: 0.55 mmol / g.

[0048] Enzymatic hydrolysis: 100g of loose feathers were added to 2000mL of deionized water, followed by 7.24g of sodium alginate-CaCl2 microspheres (Ca²⁺). + When 100% released, the total Ca² in the enzymatic hydrolysis solution +Add 0.1 g of keratinase (concentration 4 mM), adjust the pH to 9.5 with 1 M NaOH aqueous solution, and incubate at 50℃ for 5 h (stirring speed 60 rpm during incubation); centrifuge at 10000 rpm for 20 min, collect the supernatant, adjust the pH to 6.0 with 1 M HCl aqueous solution (to reach the isoelectric point), and precipitate for 30 min; place the precipitate mixture into a 3 kDa molecular weight cutoff dialysis bag, and dialyze with deionized water at 4℃ for 30 h, changing the dialysate 3-4 times during the process to remove residual Ca²⁺. + Tea saponins and small molecule peptides were extracted; pre-frozen at -50℃ for 2 h and freeze-dried under vacuum for 14 h to obtain keratin. The keratin extraction rate was 87.9% and the molecular weight of keratin was 8 kDa.

[0049] Example 4

[0050] like Figure 1 As shown in the figure, this embodiment provides a keratin extraction method, as detailed below: Take 100 g of fresh chicken feathers, remove stems and impurities, rinse 3 times, and cut into 2 cm short segments; prepare 2000 mL of 1% tea saponin aqueous solution (20 g tea saponin + 1980 mL deionized water), add the feathers, and stir at 50 ℃ for 30 min; rinse twice and drain; add 2000 mL of 10% ethanol aqueous solution, and treat intermittently with ultrasound at 20 kHz for 15 min (power 150 W, intermittent treatment is 30 s working and 30 s pausing, and the system temperature is maintained at 48 ℃ during ultrasound), to obtain loose feathers with an α-helix retention rate of 92%.

[0051] Preparation of sodium alginate-CaCl2 microspheres: (1) Mix 1.2 mL of 2 M CaCl2 aqueous solution with 4.8 mL of 0.5 M EDTA aqueous solution, adjust the pH to 7.2 with 2 M NaOH aqueous solution, and make up to 10 mL with deionized water to obtain Ca-EDTA solution (the concentration of Ca-EDTA in Ca-EDTA solution is 240 mmol / L); mix 2% sodium alginate aqueous solution and Ca-EDTA solution at a volume ratio of 1:1, and use 0.22 (1) Filter the aqueous phase through a μm filter membrane; (2) Add a fluorinated surfactant to the fluorocarbon oil and mix evenly to obtain the oil phase; wherein, based on the total weight of the oil phase, the content of the fluorinated surfactant is 2wt%; (3) Load the aqueous phase and oil phase into syringes and install them into the injection pump, and connect them to the microfluidic chip; set the flow rate of the aqueous phase to 8μL / min and the flow rate of the oil phase to 15μL / min, and form droplets in the chip; observe the droplet morphology under a real-time microscope, and start collecting after stabilization; collect the droplets into a centrifuge tube containing 15mL of acetic acid oil phase (the preparation method of acetic acid oil phase is: mix 1ml of glacial acetic acid and 99mL of oil phase by ultrasonication to obtain acetic acid oil phase), and let stand for 30min to solidify; add 5mL of demulsifier (ethanol: n-hexane = 1:1, v / v) to the centrifuge tube and shake for 5min to demulsify; remove the upper oil phase, and centrifuge and wash 3 times with PBS buffer (pH=7.4) (centrifugation rate 3000 rpm, 5 min). (min / batch) to obtain sodium alginate-CaCl2 microspheres (particle size 1.5 μm, Ca²⁺). + Loading capacity: 0.55 mmol / g.

[0052] Enzymatic hydrolysis: 100g of loose feathers were added to 2000mL of deionized water, followed by 9.05g of sodium alginate-CaCl2 microspheres (Ca²⁺). + When 100% released, the total Ca² in the enzymatic hydrolysis solution + Add 0.2 g of keratinase (5 mM concentration), adjust the pH to 9.5 with 1 M NaOH aqueous solution, and incubate at 50 °C for 5 h (stirring speed at 60 rpm during incubation). Centrifuge at 10,000 rpm for 20 min, collect the supernatant, adjust the pH to 6.0 with 1 M HCl aqueous solution (to reach the isoelectric point), and precipitate for 30 min. Place the precipitate mixture into a 3 kDa molecular weight cutoff dialysis bag and dialyze with deionized water at 4 °C for 30 h, changing the dialysate 3-4 times during this period to remove residual Ca²⁺. + Tea saponins and small molecule peptides were extracted; pre-frozen at -50℃ for 2 h and freeze-dried under vacuum for 14 h to obtain keratin. The keratin extraction rate was 90.1% and the molecular weight of keratin was 7 kDa.

[0053] Example 5

[0054] like Figure 1As shown in the figure, this embodiment provides a keratin extraction method, as detailed below: Take 100 g of fresh chicken feathers, remove stems and impurities, rinse 3 times, and cut into 2 cm short segments; prepare 2000 mL of 1% tea saponin aqueous solution (20 g tea saponin + 1980 mL deionized water), add the feathers, and stir at 50 ℃ for 30 min; rinse twice and drain; add 2000 mL of 10% ethanol aqueous solution, and treat intermittently with ultrasound at 20 kHz for 15 min (power 150 W, intermittent treatment is 30 s working and 30 s pausing, and the system temperature is maintained at 48 ℃ during ultrasound), to obtain loose feathers with an α-helix retention rate of 92%.

[0055] Preparation of sodium alginate-CaCl2 microspheres: (1) Mix 1.2 mL of 2 M CaCl2 aqueous solution with 4.8 mL of 0.5 M EDTA aqueous solution, adjust the pH to 7.2 with 2 M NaOH aqueous solution, and make up to 10 mL with deionized water to obtain Ca-EDTA solution (the concentration of Ca-EDTA in Ca-EDTA solution is 240 mmol / L); mix 2% sodium alginate aqueous solution and Ca-EDTA solution at a volume ratio of 1:1, and use 0.22 (1) Filter the aqueous phase through a μm filter membrane; (2) Add a fluorinated surfactant to the fluorocarbon oil and mix evenly to obtain the oil phase; wherein, based on the total weight of the oil phase, the content of the fluorinated surfactant is 2wt%; (3) Load the aqueous phase and oil phase into syringes and install them into the injection pump, and connect them to the microfluidic chip; set the flow rate of the aqueous phase to 8μL / min and the flow rate of the oil phase to 15μL / min, and form droplets in the chip; observe the droplet morphology under a real-time microscope, and start collecting after stabilization; collect the droplets into a centrifuge tube containing 15mL of acetic acid oil phase (the preparation method of acetic acid oil phase is: mix 1ml of glacial acetic acid and 99mL of oil phase by ultrasonication to obtain acetic acid oil phase), and let stand for 30min to solidify; add 5mL of demulsifier (ethanol: n-hexane = 1:1, v / v) to the centrifuge tube and shake for 5min to demulsify; remove the upper oil phase, and centrifuge and wash 3 times with PBS buffer (pH=7.4) (centrifugation rate 3000 rpm, 5 min). (min / batch) to obtain sodium alginate-CaCl2 microspheres (particle size 1.5 μm, Ca²⁺). + Loading capacity: 0.55 mmol / g.

[0056] Enzymatic hydrolysis: 100g of loose feathers were added to 2000mL of deionized water, followed by 9.05g of sodium alginate-CaCl2 microspheres (Ca²⁺). + When 100% released, the total Ca² in the enzymatic hydrolysis solution +Add 0.1 g of keratinase (5 mM concentration), adjust the pH to 9.5 with 1 M NaOH aqueous solution, and incubate at 55 ℃ for 6 h (stirring speed at 60 rpm during incubation). Centrifuge at 10000 rpm for 20 min, collect the supernatant, adjust the pH to 6.0 with 1 M HCl aqueous solution (to reach the isoelectric point), and precipitate for 30 min. Place the precipitate mixture into a 3 kDa molecular weight cutoff dialysis bag and dialyze with deionized water at 4 ℃ for 30 h, changing the dialysate 3-4 times during the process to remove residual Ca²⁺. + Tea saponins and small molecule peptides were extracted; pre-frozen at -50℃ for 2 h and freeze-dried under vacuum for 14 h to obtain keratin. The keratin extraction rate was 89.7% and the molecular weight of keratin was 7 kDa.

[0057] Example 6

[0058] like Figure 1 As shown in the figure, this embodiment provides a keratin extraction method, as detailed below: Take 100 g of fresh chicken feathers, remove stems and impurities, rinse 3 times, and cut into 2 cm short segments; prepare 2000 mL of 1% tea saponin aqueous solution (20 g tea saponin + 1980 mL deionized water), add the feathers, and stir at 50 ℃ for 30 min; rinse twice and drain; add 2000 mL of 10% ethanol aqueous solution, and treat intermittently with ultrasound at 20 kHz for 15 min (power 150 W, intermittent treatment is 30 s working and 30 s pausing, and the system temperature is maintained at 48 ℃ during ultrasound), to obtain loose feathers with an α-helix retention rate of 92%.

[0059] Preparation of sodium alginate-CaCl2 microspheres: (1) Mix 1.2 mL of 2 M CaCl2 aqueous solution with 4.8 mL of 0.5 M EDTA aqueous solution, adjust the pH to 7.2 with 2 M NaOH aqueous solution, and make up to 10 mL with deionized water to obtain Ca-EDTA solution (the concentration of Ca-EDTA in Ca-EDTA solution is 240 mmol / L); mix 2% sodium alginate aqueous solution and Ca-EDTA solution at a volume ratio of 1:1, and use 0.22 (1) Filter the aqueous phase through a μm filter membrane; (2) Add a fluorinated surfactant to the fluorocarbon oil and mix evenly to obtain the oil phase; wherein, based on the total weight of the oil phase, the content of the fluorinated surfactant is 2wt%; (3) Load the aqueous phase and oil phase into syringes and install them into the injection pump, and connect them to the microfluidic chip; set the flow rate of the aqueous phase to 8μL / min and the flow rate of the oil phase to 15μL / min, and form droplets in the chip; observe the droplet morphology under a real-time microscope, and start collecting after stabilization; collect the droplets into a centrifuge tube containing 15mL of acetic acid oil phase (the preparation method of acetic acid oil phase is: mix 1ml of glacial acetic acid and 99mL of oil phase by ultrasonication to obtain acetic acid oil phase), and let stand for 30min to solidify; add 5mL of demulsifier (ethanol: n-hexane = 1:1, v / v) to the centrifuge tube and shake for 5min to demulsify; remove the upper oil phase, and centrifuge and wash 3 times with PBS buffer (pH=7.4) (centrifugation rate 3000 rpm, 5 min). (min / batch) to obtain sodium alginate-CaCl2 microspheres (particle size 1.5 μm, Ca²⁺). + Loading capacity: 0.55 mmol / g.

[0060] Enzymatic hydrolysis: 100g of loose feathers were added to 2000mL of deionized water, followed by 9.05g of sodium alginate-CaCl2 microspheres (Ca²⁺). + When 100% released, the total Ca² in the enzymatic hydrolysis solution + Add 0.1 g of keratinase (5 mM concentration), adjust the pH to 9.0 with 1 M NaOH aqueous solution, and incubate at 50 °C for 6 h (stirring speed at 60 rpm during incubation). Centrifuge at 10000 rpm for 20 min, collect the supernatant, adjust the pH to 6.0 with 1 M HCl aqueous solution (to reach the isoelectric point), and precipitate for 30 min. Place the precipitate mixture into a 3 kDa molecular weight cutoff dialysis bag and dialyze with deionized water at 4 °C for 30 h, changing the dialysate 3-4 times during the process to remove residual Ca²⁺. + Tea saponins and small molecule peptides were extracted; pre-frozen at -50℃ for 2 h, and then freeze-dried under vacuum for 14 h to obtain keratin. The keratin extraction rate was 90.6%, and the molecular weight of keratin was 7 kDa.

[0061] Test case

[0062] The release rate of sodium alginate-CaCl2 microspheres prepared in Example 1 was tested. The specific test methods and results are as follows.

[0063] Ca was plotted using ultraviolet spectrophotometry. 2+ The standard curve was prepared as follows: A 1000 μg / mL calcium ion standard stock solution was prepared using solid calcium carbonate. Accurately measured volumes of 0, 0.5, 1.0, 2.0, 3.0, and 4.0 mL of the stock solution were placed in separate 50 mL volumetric flasks. 5 mL of acetate-sodium acetate buffer (pH 5.0) and 2 mL of azoarsine III colorimetric reagent were added to each flask. The solution was then diluted to the mark with ultrapure water, shaken well, and allowed to stand for 15 min to obtain standard solutions with concentrations of 0, 10, 20, 40, 60, and 80 μg / mL. After preheating the UV-Vis spectrophotometer for 30 min, the wavelength was adjusted to 650 nm. Using a 0 μg / mL blank colorimetric solution as a reference, the absorbance of each concentration of standard solution was measured sequentially, and the readings were recorded (average value was used for parallel samples). The standard curve was obtained by plotting calcium ion concentration on the x-axis and average absorbance (A) on the y-axis: y = 0.0311x + 0.0008R. 2 =0.9998. 0.5 g of calcium alginate microspheres were released into 500 mL of PBS buffer (pH 6.8), and stirred at 25 °C. Every 1 hour, 2 mL of the release solution was drawn using a 5 mL syringe and filtered through a 0.22 μm hydrophilic membrane. Immediately, 2 mL of PBS buffer at the same temperature and pH was added to the conical flask, and stirring continued. 1.0 mL of the filtered release solution was placed in a 10 mL stoppered colorimetric tube. Following the standard curve colorimetric reaction procedure, 0.5 mL of masking agent (triethanolamine), 1.5 mL of buffer (acetic acid-sodium acetate buffer, pH 5.0), and 0.6 mL of colorimetric reagent (azoarsine III colorimetric reagent) were added, and the volume was adjusted to 10 mL. The solution was then allowed to stand in the dark for 15 min. Using a blank PBS buffer (pH 6.8) solution after the same colorimetric treatment as a control, the absorbance was measured at λmax. Based on Ca... 2+ Standard curve, calculate calcium ion concentration (C t ) and release amount (released molar amount n) t After the release reaction is complete, calculate the total calcium content (total molar amount n) in the microspheres. 总 According to calcium ion release rate (%) = n t / n 总 The calcium ion release rate was calculated, and the release rate was 26% at 2h, 53% at 4h, and 98% at 6h.

[0064] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for extracting keratin from animal feathers, characterized in that, The extraction method includes: (1) The pretreated animal feathers and tea saponin solution are mixed and stirred to obtain defatted feathers; then the defatted feathers are mixed with ethanol solution and ultrasonically treated to obtain loose feathers; (2) Droplets were prepared by microfluidic chip process of aqueous phase and oil phase. After the collected droplets were allowed to stand and solidify, they were demulsified and washed to obtain sodium alginate-CaCl2 microspheres. The aqueous phase is prepared by the following steps: mixing sodium alginate solution and Ca-EDTA solution evenly, and filtering with a filter membrane to obtain the aqueous phase; the oil phase is prepared by the following steps: mixing electronic fluorinated liquid and fluorinated surfactant evenly to obtain the oil phase; (3) Mix the loose feathers, water, sodium alginate-CaCl2 microspheres and keratinase, then adjust the pH and perform enzymatic hydrolysis; finally, take the supernatant. (4) Adjust the pH of the supernatant, precipitate, and then dialyze and freeze-dry the precipitate mixture to obtain the animal feather keratin.

2. The method for extracting keratin from animal feathers according to claim 1, wherein, The pretreatment of the animal feathers includes: removing stems and impurities, rinsing, and trimming the animal feathers; The animal feathers are at least one of chicken feathers, duck feathers, and goose feathers; The term "shortening" refers to cutting the material into short segments of 1-3 cm.

3. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (1), the tea saponin solution is an aqueous solution of tea saponin with a mass concentration of 0.5-2%; The ratio of animal feathers to tea saponin solution was 1:(18-22) g / mL; The stirring process is carried out at a temperature of 45-55℃ for 25-35 minutes.

4. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (1), the ethanol solution is an aqueous ethanol solution with a mass concentration of 8-15%; The ratio of animal feathers to ethanol solution was 1:(18-22) g / mL; The ultrasonic treatment is intermittent, consisting of 25-35 seconds of operation followed by a 25-35 second pause; the total duration of the intermittent treatment is 10-20 minutes. The ultrasonic treatment frequency is 15-25kHz, and the power is 140-155W; The system temperature was maintained at 45-50 ℃ during the ultrasound process.

5. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (2), the working parameters for preparing droplets using microfluidic chip technology include: aqueous phase flow rate of 6-10 μL / min and oil phase flow rate of 13-17 μL / min; The time for the droplets to stand and solidify is 0.5-1 hour; The sodium alginate-CaCl2 microspheres have a particle size of 1-2 μm.

6. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (2), the sodium alginate solution is an aqueous solution of sodium alginate with a mass concentration of 1-2%; The Ca-EDTA solution is prepared by a method comprising the following steps: mixing CaCl2 aqueous solution and EDTA aqueous solution, adjusting the pH to 7-7.5, and adding water to make up to a final volume to obtain the Ca-EDTA solution; The concentration of Ca-EDTA in the Ca-EDTA solution is 200-250 mmol / L; The volume ratio of the sodium alginate solution to the Ca-EDTA solution is (0.5-1.5):1; Filter using a 0.2-0.3μm filter membrane; The content of fluorinated surfactants is 1.5-2.5 wt% based on the total weight of the oil phase. The electronic fluorinated liquid is a fluorocarbon oil.

7. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (3), the ratio of the loose feathers to water is 1:(18-22) g / mL; The amount of keratinase added is 0.1-0.3 wt% based on the weight of the loose feathers. The amount of sodium alginate-CaCl2 microspheres added is such that when the Ca²⁺ in the sodium alginate-CaCl2 microspheres is... + When 100% released, the Ca²⁺ in the enzymatic hydrolysis solution... + The concentration ranges from 1.2 to 5.0 mmol / L; Adjust the pH to 9.0-9.5; The enzymatic hydrolysis is performed at a temperature of 50-60℃ for 4-6 hours. Centrifuge at 8000-10000 rpm and collect the supernatant.

8. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (3), the keratinase activity is ≥5000 KerU / g and the source is Bacillus subtilis; the stirring speed during enzymatic hydrolysis is 50-80 rpm.

9. The method for extracting keratin from animal feathers according to claim 1, wherein, In step (4), the pH of the supernatant is adjusted to 5.5-6.5; Dialysis is performed using dialysis bags for 24-36 hours. The freeze-drying process involves pre-freezing followed by vacuum freeze-drying.

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