A method for preparing silk fibroin peptide by separating crystalline region and non-crystalline region of tussah silk fibroin

Abstract

The application discloses a method for preparing silk fibroin peptides by separating crystalline regions and non-crystalline regions of tussah silk fibroin. First, the tussah silk fibroin is reacted with 85% phosphoric acid at room temperature for 6-8 hours to loosen the crystalline regions, then water is added to adjust the mass concentration of the phosphoric acid to 60-65%, and the reaction is carried out at a temperature of 50-60 DEG C for 4-6 hours, so that the crystalline regions and the non-crystalline regions exist in solid and liquid respectively, then the solid and liquid are separated, and the solid and the liquid are hydrolyzed, desalted and freeze-dried respectively, so that the tussah silk fibroin peptides with a molecular weight of less than 1000 Da are obtained. The specific amino acid component of the crystalline region tussah silk fibroin peptide is significantly increased, and the crystalline region tussah silk fibroin peptide has the effect of promoting skin cell proliferation and repairing. The non-crystalline region tussah silk fibroin peptide has a significant whitening performance.

Description

Technical Field

[0001] This invention belongs to the field of high-value utilization technology of tussah silk, and particularly relates to a method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin. Background Technology

[0002] Tussah silkworm ( Antherae pernyi Silk includes fibroin and sericin, the outer layer of fibroin. Tussah silk fibroin, which is degummed tussah silk, is a high-molecular-weight heavy-chain protein. Tussah silk fibroin peptides, obtained by hydrolyzing tussah silk fibroin, not only have no toxic side effects on cells and the body, but also possess multiple skincare benefits such as moisturizing, sun protection, antibacterial properties, antioxidant effects, and whitening. Meanwhile, tussah silk fibroin includes crystalline and amorphous regions. The crystalline region exhibits an α-helix and β-sheet molecular conformation formed by polyalanine, while the amorphous region exhibits a random coil conformation. Due to the complex structure of tussah silk fibroin, existing methods for preparing tussah silk fibroin peptides all involve first completely dissolving the tussah silk into a molten silk solution, followed by subsequent preparations.

[0003] Chinese patent application No. 02102964.4 discloses a method for "preparing silk peptides and silk amino acids by acid hydrolysis of silk". The specific technical solution is to first melt the silk, that is, to mix the silk with 80-85% phosphoric acid at a ratio of 1:2-8, and then heat it to 50-120°C. Under stirring, the silk is completely dissolved into a melt, and then silk peptides and silk amino acids are prepared. The prepared silk peptides have a wide molecular weight distribution and their functions are not concentrated.

[0004] Chinese patent number ZL201810289358.9 discloses a "method for preparing tussah silk fibroin peptides by step-temperature controlled hydrolysis." The specific technical solution involves first mixing degummed tussah silk with 85% ammonium phosphate at a mass ratio of 1:9-10, reacting at 10-25℃ for 3-5 days to obtain a molten silk solution, and then preparing tussah silk fibroin peptides through step-temperature controlled hydrolysis. This method can produce various silk fibroin peptide products with concentrated molecular weight distribution and strong functionality (moisturizing, hypoglycemic, and hypolipidemic).

[0005] To date, there are no reports on separating the crystalline and non-crystalline regions of tussah silk and then preparing two different silk fibroin peptides with different functions. Summary of the Invention

[0006] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin.

[0007] The technical solution of this invention is: a method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin, which is carried out in sequence according to the following steps: Step 1. Immerse tussah silk fibroin in a phosphoric acid solution with a mass concentration of 85% and react at 20-30℃ for 6-8 hours. Then add water to adjust the phosphoric acid mass concentration to 60-65% and react at 50-60℃ for 4-6 hours. After that, perform solid-liquid separation to obtain solid and liquid respectively. Step 2. Immerse the obtained solid in 85% phosphoric acid and dissolve at 20-30℃ for 6 hours. Then, heat to 100℃ and hydrolyze at a constant temperature for 4-8 hours. After desalting, concentrate and dry into powder to obtain crystalline tussah silk fibroin peptide. Step 3. Heat the obtained liquid to 90℃ for 4-8 hours to hydrolyze, desalt, concentrate and dry into powder to obtain the non-crystalline region of tussah silk fibroin peptide.

[0008] The mass ratio of tussah silk fibroin to an 85% phosphoric acid solution in step 1 is 1:7-9.

[0009] The mass ratio of the solid in step 2 to the 85% phosphoric acid solution is 1:9.

[0010] This invention first reacts tussah silk fibroin with 85% phosphoric acid at room temperature for 6-8 hours to loosen the amorphous region. Then, water is added to adjust the phosphoric acid concentration to 60-65%, and the reaction is carried out at 50-60℃ for 4-6 hours, so that the crystalline and amorphous regions exist in the solid and liquid respectively. The solid and liquid are then separated, and the solid and liquid are hydrolyzed, desalted, and freeze-dried respectively to obtain tussah silk fibroin peptides with a molecular weight of less than 1000 Da in both crystalline and amorphous regions. The crystalline region of the tussah silk fibroin peptides has a significantly increased alanine content, which promotes skin cell proliferation and thus repairs the skin. The amorphous region of the tussah silk fibroin peptides has significant whitening properties. Attached Figure Description

[0011] Figure 1 This is a schematic diagram comparing the effects of different treatment groups on skin cell proliferation and repair in embodiments of the present invention.

[0012] Figure 2 A schematic diagram comparing the whitening effects of different tussah silk fibroin peptides. Detailed Implementation

[0013] The present invention discloses a method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin, which is carried out in sequence according to the following steps: Step 1. Immerse tussah silk fibroin in an 85% phosphoric acid solution with a mass ratio of 1:8. React at room temperature (20-30℃) for 6 hours. Then add water to adjust the phosphoric acid concentration to 60% and react at 55℃ for 5 hours. After that, perform solid-liquid separation to obtain solid and liquid respectively. Step 2. Immerse the obtained solid in 85% phosphoric acid solution at a mass ratio of 1:9. Dissolve at room temperature (20-30℃) for 6 hours, then heat to 100℃ and hydrolyze at a constant temperature for 6 hours. After desalting, concentrate and dry into powder to obtain crystalline tussah silk fibroin peptide. Step 3. Heat the obtained liquid to 90℃ for 6 hours to hydrolyze, desalt, concentrate and dry into powder to obtain the non-crystalline region of tussah silk fibroin peptide.

[0014] Comparative Example: Preparation of Tussah silk fibroin peptides With a phosphoric acid concentration of 85%, tussah silk fibroin was added at a mass ratio of 1:8. The mixture was reacted at around 25°C for 6 hours at room temperature, then heated to 100°C for 6 hours to hydrolyze. After desalting, the mixture was concentrated and dried into powder to obtain whole tussah silk fibroin peptide.

[0015] Experiment 1: Detection of Amino Acid Components in Tussah Silk Peptides The crystalline tussah silk fibroin peptides, the amorphous tussah silk fibroin peptides obtained in Example 1, and the whole tussah silk fibroin peptides obtained in the comparative example were used as samples.

[0016] Weigh a suitable amount of well-mixed sample, add 10 mL of 1:1 hydrochloric acid solution to the hydrolysis tube, mix well, and place the hydrolysis tube in an electric heating oven at 110℃±1℃ for 22 h for hydrolysis. Remove and cool to room temperature. Open the hydrolysis tube and filter the hydrolysate into a 25 mL volumetric flask. Rinse the hydrolysis tube several times with a small amount of water, transferring the washings to the same 25 mL volumetric flask. Finally, dilute to the mark with water and mix well. Accurately pipette 0.5 mL of the filtrate into a 15 mL test tube, dry it with nitrogen, and dilute to 10 mL with 0.02 mol / L hydrochloric acid solution. Shake well, filter through a 0.22 μm microporous membrane, and then perform analysis. Chromatographic column: sulfonic acid cation exchange resin; flow rate: separation column 0.4 ml / min; derivatization column 0.35 ml / min; injection volume: 500 μL; wavelength: 570 nm and 440 nm; column temperature: separation column 57℃; derivatization column 135℃.

[0017] The content of each amino acid in the sample:

[0018] X—The content of each amino acid in the sample, in g / 100g; C—The concentration of each amino acid in the sample, calculated based on the concentration of the standard, in mg / L; C0—The concentration values ​​of each amino acid in the blank control, calculated based on the concentration of the standard, in mg / L; V—Volume at constant volume, in mL; N—Dilution factor; m — Sample weight, in grams.

[0019] The results of the comparison of amino acid composition of each sample are shown in Table 1. According to the calculation of amino acid content, compared with the comparative example, the alanine content of crystalline tussah silk fibroin peptide increased by 48.86%, and compared with non-crystalline tussah silk fibroin peptide, the alanine content increased by 31.81 g / 100g.

[0020] Table 1

[0021] Experiment 2: The cell proliferation and repair effects of tussah silk fibroin peptides Mouse embryonic fibroblasts (Balb / c3T3) were removed from liquid nitrogen at -196°C, rapidly thawed in a 37°C water bath, centrifuged at low speed (1000 r / min, 5 min), and the culture medium in the cryovials was removed. DMEM culture medium (pH 7.2-7.4) containing 10% fetal bovine serum was added, and the cells were cultured in a 37°C, 5% CO2 incubator. When the cells showed good growth, they were passaged. Cells in the logarithmic growth phase were collected, the culture medium was discarded, and the cells were digested with 0.25% trypsin. The enzyme solution was discarded, and 5 mL of DMEM culture medium containing 10% fetal bovine serum was added and the cells were mixed by pipetting. The cell suspension was counted using a hemocytometer. Cells were then counted at a rate of 5 × 10⁶ cells / mL. 4 Cells were seeded into 96-well plates at a seeding density of cells / well, with 200 μL of cell suspension added to each well. After cell attachment, samples of crystalline and non-crystalline tussah silk fibroin peptides and whole tussah silk fibroin peptides were added at concentrations of 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively, with corresponding blank control groups. The cells were cultured in a CO2 incubator at 37°C and 5% CO2 concentration for 48 h, and the OD value of cell growth changes was measured.

[0022] The results are as follows Figure 1 As shown, compared with other groups, the crystalline region of tussah silk fibroin peptides significantly promoted cell proliferation at 48 h, with a significantly better effect than the control group.

[0023] Experiment 3: Determination of the whitening properties of tussah silk fibroin peptides The skin-whitening performance was evaluated by measuring the absorbance of the dopaquinone produced in the reaction and calculating the tyrosinase inhibition rate. Test reagents, crystalline zone, non-crystalline zone tussah silk fibroin peptide, and comparative whole tussah silk fibroin peptide sample solutions were prepared using PBS phosphate buffer (pH 6.8). The test reagents and sample solutions were added sequentially to the ELISA plate. After adding 200 U / mL of tyrosinase solution, the plate was incubated at 37 °C for 10 min, followed by the addition of L-DOPA substrate and a 10 min reaction. The absorbance at 475 nm was then measured. The tyrosinase inhibition rate was calculated using the following formula.

[0024]

[0025] In the formula: A is 100 μL PBS solution + 50 μL tyrosinase + 50 μL substrate; B is 150 μL PBS solution + 50 μL tyrosinase; C is 50 μL PBS solution + 50 μL sample + 50 μL tyrosinase + 50 μL substrate; D is 100 μL PBS solution + 50 μL sample + 50 μL tyrosinase.

[0026] The results are as follows Figure 2 As shown, compared with other groups, the tyrosinase inhibition rate of the non-crystalline region of tussah silk fibroin peptide was significantly improved, indicating that it has excellent whitening properties.

Claims

1. A method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin, characterized in that... Follow these steps in sequence: Step 1. Immerse tussah silk fibroin in a phosphoric acid solution with a mass concentration of 85% and react at 20-30℃ for 6-8 hours. Then add water to adjust the phosphoric acid mass concentration to 60-65% and react at 50-60℃ for 4-6 hours. After that, perform solid-liquid separation to obtain solid and liquid respectively. Step 2. Immerse the obtained solid in 85% phosphoric acid and dissolve at 20-30℃ for 6 hours. Then, heat to 100℃ and hydrolyze at a constant temperature for 4-8 hours. After desalting, concentrate and dry into powder to obtain crystalline tussah silk fibroin peptide. Step 3. Heat the obtained liquid to 90℃ for 4-8 hours to hydrolyze, desalt, concentrate and dry into powder to obtain the non-crystalline region of tussah silk fibroin peptide.

2. The method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin according to claim 1, characterized in that: The mass ratio of tussah silk fibroin to an 85% phosphoric acid solution in step 1 is 1:7-9.

3. The method for preparing silk fibroin peptides by separating the crystalline and non-crystalline regions of tussah silk fibroin according to claim 2, characterized in that: The mass ratio of the solid in step 2 to the 85% phosphoric acid solution is 1:9.

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