Method for extracting cashmere and wool keratin from disulfhydryl compound

Abstract

The invention relates to a method for extracting cashmere and wool keratin by using a disulfydryl reducing agent. The method comprises the following steps: (1) pretreating cashmere and wool fibers; preparing a disulfydryl reducing agent solution, then adding urea and lauryl sodium sulfate, and adjusting the pH value to obtain an extraction solution; adding pretreated cashmere and wool fibers, introducing nitrogen for protection, heating and stirring to obtain a cashmere and wool keratin extracting solution; and (2) centrifuging the cashmere and wool keratin extracting solution to obtain a supernatant, dialyzing the supernatant, taking out a protein solution in a dialysis bag after dialysis, adjusting the pH value of the solution to an isoelectric point, separating out keratin in the solution, centrifuging to obtain a precipitate, and freeze-drying the precipitate to obtain the cashmere and wool keratin. The keratin powder prepared by the preparation method disclosed by the invention is large in density, high in strength and uniform in pores, meanwhile, the relative molecular weight is relatively high and is 15-25KDa, the price of the extract is low, and the keratin powder has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biological resource extraction and utilization, and particularly relates to a method for extracting cashmere wool keratin by using a dithiol compound. Background Art

[0002] Cashmere wool is a precious animal fiber that contains a large amount of keratin. Cashmere wool macromolecules are mainly bonded by molecular attraction, salt bonds, hydrogen bonds and disulfide bonds. Compared with other structural proteins, cashmere wool is a biomaterial with a higher keratin content. Due to its inherent biological properties and excellent biocompatibility, keratin-based biomaterials have been widely studied in the past few decades. Unlike other natural polymers such as starch, collagen, and chitosan, the complex three-dimensional structure of keratin requires strong reducing and alkaline conditions for dissolution and extraction. The most commonly used methods for keratin extraction are oxidation, reduction, steam explosion, microbial method, microwave irradiation and the use of ionic liquids. The reduction method is used to extract the intact keratin molecular structure. Urea and sodium dodecyl sulfate are added in an alkaline environment to break the intramolecular hydrogen bonds and increase the solubility rate.

[0003] Protein-based biomaterials are the materials of choice for tissue engineering because they are similar to the natural extracellular matrix and can be made into a variety of shapes. Both plant proteins (soy protein, corn protein) and animal proteins (keratin, silk, collagen) have been made into fibers, films, hydrogels, and nanoparticles for medical applications.

[0004] Traditional keratin extraction methods usually involve strong acid and alkali treatment, enzymatic decomposition or the action of other chemicals, which are often accompanied by environmental pollution risks and high costs. In addition, the quality of the obtained keratin is uneven, which limits its wide application in high-end cosmetics, medicine and textile industries. Therefore, finding more environmentally friendly and cost-effective extraction methods has become the focus of current research. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for extracting cashmere and wool keratin using a dithiol compound. The keratin powder prepared by the method has high density, high strength, uniform pores, and a high relative molecular weight of 15-25KDa. The extract is cheap and has good market application prospects.

[0006] The present invention provides a method for extracting cashmere wool keratin using a dithiol compound, comprising the following steps:

[0007] (1) pretreating cashmere wool fibers; preparing a dithiol compound solution, then adding urea and sodium dodecyl sulfate, adjusting the pH, and obtaining an extraction solution; adding the pretreated cashmere wool fibers, passing nitrogen protection, heating and stirring, and obtaining a cashmere wool keratin extract;

[0008] (2) The cashmere wool keratin extract is centrifuged to obtain a supernatant, the supernatant is dialyzed, and after the dialysis is completed, the pH is adjusted to the isoelectric point, the precipitate is centrifuged to obtain a precipitate, and the precipitate is freeze-dried to obtain cashmere wool keratin.

[0009] Preferably, the pretreatment in step (1) comprises washing the cashmere wool fibers with anhydrous ethanol at 30-40°C and drying at 50-60°C.

[0010] Preferably, the dithiol compound in step (1) comprises at least one of diethylmercaptan and diethylene glycol.

[0011] Preferably, the bath ratio of the cashmere wool fiber to the extraction solution in step (1) is 1:20.

[0012] Preferably, in step (1), the amount of the bis-thiol compound used is 0.5-1 mol / L, the amount of dodecyl sulfuric acid used is 0.04 mol / L-0.06 mol / L, the amount of urea used is 4-8 mol / L, and the pH is adjusted to 8.5-10.

[0013] Preferably, the heating and stirring conditions in step (1) are 80-90° C. and the time is 4-6 h.

[0014] Preferably, the dialysis treatment in step (2) uses deionized water, the dialysis time is 72-96 hours, and the water is changed every 6 hours.

[0015] Preferably, the isoelectric point pH in step (2) is 4.6.

[0016] Preferably, the centrifugal rate in step (2) is 10000-12000 r / min, and the centrifugal time is 3-5 min.

[0017] Preferably, the freeze-drying treatment time in step (2) is 48-72 hours, and the freeze-drying temperature is -70--60°C.

[0018] Beneficial Effects

[0019] (1) The present invention uses diethyl mercaptan or diethylene glycol as a reducing agent to extract keratin, which not only enhances the extraction efficiency but also retains the activity of the protein. Diethyl mercaptan and diethylene glycol are easier to handle and have no negative impact on the environment.

[0020] (2) The dithiol functional groups of diethylmercaptan and diethylene glycol used in the present invention can specifically break the disulfide bonds in the keratin molecules. This targeted destruction helps to more efficiently and thoroughly unravel the protein network, thereby improving the extraction efficiency of keratin.

[0021] (3) The extraction method of the present invention is not limited to cashmere wool, but is also applicable to keratin from various sources, including but not limited to human hair, wool and other animal hair materials, which broadens the selection range of raw materials and improves resource utilization.

[0022] (4) The extraction method of the present invention improves the extraction rate, reduces the production cost, simplifies the processing flow, and is expected to accelerate the commercialization process of keratin, especially in the cosmetics, pharmaceutical, textile and other industries, creating more possibilities for the development of high value-added products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the effect of the amount of diethylmercaptan on the solubility rate of cashmere wool and the extraction rate of keratin.

[0024] Figure 2 This paper shows the effect of diethylene glycol dosage on cashmere wool dissolution rate and keratin extraction rate.

[0025] Figure 3 This is the effect of urea on the dissolution rate of cashmere wool and the extraction rate of keratin.

[0026] Figure 4 The relative molecular weight of keratin extracted by different reducing agents.

[0027] Figure 5 SEM images of keratin extracted by different reducing agents.

[0028] Figure 6 Infrared images of keratin extracted with different reducing agents.

[0029] Figure 7 This is the test curve of thiol content. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0031] Example 1

[0032] (1) Cut the cashmere wool fibers into pieces, add anhydrous ethanol and perform ultrasonic cleaning at 30°C to remove lanolin and dust, and dry them at 60°C for later use.

[0033] (2) Prepare diethylmercaptan solutions with concentrations of 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L, respectively, add 6 mol / L urea and 0.02 mol / L sodium dodecyl sulfate (SDS), adjust the pH to 8.5 with NaOH solution, and obtain an extraction solution.

[0034] (3) Add 1 g of pretreated cashmere wool fiber to the extraction solution (bath ratio is 1:20), introduce nitrogen protection, heat and stir at 85° C. for 6 h to obtain a cashmere wool keratin extract.

[0035] (4) The cashmere wool keratin extract is centrifuged to obtain a supernatant, and the supernatant is dialyzed using deionized water for 72 hours, with the water changed every 6 hours. After the dialysis is completed, the protein solution in the dialysis bag is taken out, the pH of the solution is adjusted to the isoelectric point, keratin is precipitated in the solution, and a precipitate is obtained by centrifugation at a centrifugal speed of 10000 r / min and a centrifugal time of 4 minutes. The precipitate is freeze-dried for 48 hours at a freeze-drying temperature of -70°C to obtain cashmere wool keratin.

[0036] Depend on Figure 1 It can be seen that diethylmercaptan has a strong reducing property and can effectively break the stable disulfide bonds (SS) in proteins, which is one of the key stabilizing forces in the molecular structure of keratin. When the concentration of diethylmercaptan reaches 1 mol / L, its large amount of active sulfhydryl groups (-SH) can fully react with the disulfide bonds on the cysteine ​​residues in keratin, reducing them to single sulfhydryl groups, thereby destroying the spatial conformation of keratin. This process makes the molecular structure of keratin loose and easy to dissolve, thereby improving the extraction efficiency.

[0037] Example 2

[0038] (1) Cut the cashmere wool fibers into pieces, add anhydrous ethanol and perform ultrasonic cleaning at 30°C to remove lanolin and dust, and dry them at 60°C for later use.

[0039] (2) Prepare diethylene glycol solutions with concentrations of 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L, respectively, add 6 mol / L urea and 0.02 mol / L sodium dodecyl sulfate (SDS), and adjust the pH to 8.5 with NaOH solution.

[0040] (3) Add 1 g of pretreated cashmere wool fiber to the extraction solution (bath ratio is 1:20), introduce nitrogen protection, heat and stir at 85° C. for 6 h to obtain a cashmere wool keratin extract.

[0041] (4) The cashmere wool keratin extract is centrifuged to obtain a supernatant, and the supernatant is dialyzed using deionized water for 72 hours, with the water changed every 6 hours. After the dialysis is completed, the protein solution in the dialysis bag is taken out, the pH of the solution is adjusted to the isoelectric point, keratin is precipitated in the solution, and a precipitate is obtained by centrifugation at a centrifugal speed of 10000 r / min and a centrifugal time of 4 minutes. The precipitate is freeze-dried for 48 hours at a freeze-drying temperature of -70°C to obtain cashmere wool keratin.

[0042] Depend on Figure 2 It can be seen that when the concentration of diethyl mercaptan is 0.6 mol / L, the extraction rate and dissolution rate of keratin begin to increase significantly. The main function of the reducing agent is to destroy the tertiary structure of cashmere and wool fibers, open the disulfide bonds in the peptide bond segments, and then dissolve the cashmere and wool fibers to obtain small molecule proteins. When the concentration of the reducing agent diethyl ester is 1.0 mol / L, the dissolution rate and extraction rate of keratin are the largest. At this concentration, diethyl ester can efficiently break the disulfide bonds (SS) in keratin, which are an important part of maintaining the complex three-dimensional structure of keratin. The concentration of 1.0 mol / L provides sufficient reducing agent molecules to widely contact and reduce all disulfide bonds in the keratin structure, thereby making the keratin structure more flexible and easy to extract.

[0043] Example 3

[0044] (1) Cut the cashmere wool fibers into pieces, add anhydrous ethanol and perform ultrasonic cleaning at 30°C to remove lanolin and dust, and dry them at 60°C for later use.

[0045] (2) Prepare a 0.6 mol / L diethylene glycol solution, add 0 mol / L, 2 mol / L, 4 mol / L, 6 mol / L, 8 mol / L urea and 0.02 mol / L sodium dodecyl sulfate (SDS), and adjust the pH to 8.5 with NaOH solution.

[0046] (3) Add 1 g of pretreated cashmere wool fiber to the extraction solution (bath ratio is 1:20), introduce nitrogen protection, heat and stir at 85° C. for 6 h to obtain a cashmere wool keratin extract.

[0047] (4) The cashmere wool keratin extract is centrifuged to obtain a supernatant, and the supernatant is dialyzed using deionized water for 72 hours, with the water changed every 6 hours. After the dialysis is completed, the protein solution in the dialysis bag is taken out, the pH of the solution is adjusted to the isoelectric point, keratin is precipitated in the solution, and a precipitate is obtained by centrifugation at a centrifugal speed of 10000 r / min and a centrifugal time of 4 minutes. The precipitate is freeze-dried for 48 hours at a freeze-drying temperature of -70°C to obtain cashmere wool keratin.

[0048] Depend on Figure 3 It can be seen that at a urea concentration of 8 mol / L, urea molecules begin to penetrate into the gaps between keratin molecules, competitively interact with polar groups in the protein, and gradually weaken the secondary and tertiary structures of keratin, especially by destroying hydrogen bonds. This process relaxes the helical and folded structures of keratin, enhances the affinity of keratin to the solvent, and improves the extraction efficiency.

[0049] Example 4

[0050] (1) Cut the cashmere wool fibers into pieces, add anhydrous ethanol and perform ultrasonic cleaning at 30°C to remove lanolin and dust, and dry them at 60°C for later use.

[0051] (2) Prepare 0.8 mol / L L-cysteine, 0.8 mol / L dithiothreitol, 0.6 mol / L diethylene glycol, and 0.6 mol / diethylmercaptan, respectively. The remaining conditions are 0.02 mol / L SDS and 8 mol / L urea.

[0052] (3) Add 1 g of pretreated cashmere wool fiber to the extraction solution (bath ratio is 1:20), introduce nitrogen protection, heat and stir at 85° C. for 6 h to obtain a cashmere wool keratin extract.

[0053] (4) The cashmere wool keratin extract is centrifuged to obtain a supernatant, and the supernatant is dialyzed using deionized water for 72 hours, with the water changed every 6 hours. After the dialysis is completed, the protein solution in the dialysis bag is taken out, the pH of the solution is adjusted to the isoelectric point, keratin is precipitated in the solution, and a precipitate is obtained by centrifugation at a centrifugal speed of 10000 r / min and a centrifugal time of 4 minutes. The precipitate is freeze-dried for 48 hours at a freeze-drying temperature of -70°C to obtain cashmere wool keratin.

[0054] The molecular weight of cashmere wool keratin was tested by SDS-polyacrylamide gel electrophoresis. The separation gel was 15%, the concentration gel was 5%, the sample volume was 10 μL, the voltage was 90 V, the indicator protein range was 10 000-170 000 g / mol, and Coomassie Brilliant Blue G250 was used for staining.

[0055] Ellman's reagent (DTNB) was used to test the thiol content in the extracted keratin. The thiol group reacts with DTNB to produce a color reaction, generating a yellow compound with a maximum absorption peak at a wavelength of 412nm. 0.1g of keratin sample, 10μLL of DTNB, react for 1h in the dark, and measure the absorbance at 412nm.

[0056] Depend on Figure 4It can be seen that the relative molecular mass of keratin extracted by L-cysteine ​​and dithiothreitol is 10-15kDa, and the relative molecular mass of keratin extracted by diethyl mercaptan and diethylene glycol is 10-25kDa, and the effect is better than the former, which proves that diethyl mercaptan and diethylene glycol have low reducing properties, the internal peptide chain macromolecules of cashmere and wool fibers are not destroyed, and the macromolecular keratin is retained.

[0057] Figure 5 SEM images of keratin extracted with different reducing agents.

[0058] Depend on Figure 6 It can be seen that keratin mainly consists of long-chain peptide bonds, and the characteristic peaks are amide bonds (-CONH-), specifically amide A, amide I, amide II and amide III. L-cysteine ​​and dithiothreitol destroy a large number of primary structures (hydrogen bonds) of cashmere fibers during the extraction of keratin, and there is an obvious characteristic peak on amide A. The characteristic peaks of diethylmercaptan and diethylene glycol near amide A are not obvious, and the peptide chain is well retained, so the relative molecular weight of the obtained keratin is relatively large.

[0059] Figure 7 In order to use glutathione as a standard reagent for testing thiol groups, a standard curve is established, and the thiol content in keratin is calculated according to the standard curve. The obtained standard curve can be used to calculate the thiol content in different keratins.

[0060] As shown in Table 1, the keratin protein extracted by using diethylene glycol as a reducing agent has a higher content of thiol groups.

[0061] Table 1

[0062]

[0063] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or variations within the scope of the claims, which does not affect the essence of the present invention.

Claims

1. A method for extracting cashmere wool keratin using a dithiol compound, comprising the following steps: (1) Pre-treating cashmere wool fibers; A dithiol compound solution is prepared, and then urea and sodium dodecyl sulfate are added, and the pH is adjusted to obtain an extraction solution; Add the pretreated cashmere wool fibers, introduce nitrogen protection, heat and stir to obtain cashmere wool keratin extract; (2) The cashmere wool keratin extract is centrifuged to obtain a supernatant, the supernatant is dialyzed, and after the dialysis is completed, the pH is adjusted to the isoelectric point, the precipitate is centrifuged to obtain a precipitate, and the precipitate is freeze-dried to obtain cashmere wool keratin.

2. The method according to claim 1, characterized in that: The pretreatment in step (1) comprises washing the cashmere wool fibers with anhydrous ethanol at 30-40°C and drying at 50-60°C.

3. The method according to claim 1, characterized in that: The bis-mercapto compound in step (1) includes at least one of diethyl mercaptan and diethylene glycol.

4. The method according to claim 1, characterized in that: The bath ratio of the cashmere wool fiber to the extraction solution in the step (1) is 1:

20.

5. The method according to claim 1, characterized in that: In the step (1), the amount of the bis-mercapto compound used is 0.5-1 mol / L, the amount of dodecyl sulfuric acid used is 0.04 mol / L-0.06 mol / L, the amount of urea used is 4-8 mol / L, and the pH is adjusted to 8.5-10.

6. The method according to claim 1, characterized in that: The heating and stirring conditions in step (1) are 80-90° C. and the time is 4-6 h.

7. The method according to claim 1, characterized in that: The dialysis treatment in step (2) uses deionized water, the dialysis time is 72-96 hours, and the water is changed every 6 hours.

8. The method according to claim 1, characterized in that: The isoelectric point pH in the step (2) is 4.

6.

9. The method according to claim 1, characterized in that: The centrifugal speed in step (2) is 10000-12000 r / min, and the centrifugal time is 3-5 min.

10. The method according to claim 1, characterized in that: The freeze-drying treatment time in step (2) is 48-72 hours, and the freeze-drying temperature is -70--60°C.

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