Silk fibroin peptide solution, silk fibroin peptide powder base material as well as preparation method and application of silk fibroin peptide solution and silk fibroin peptide powder base material

Abstract

The invention provides a silk fibroin peptide solution, a silk fibroin peptide powder base material and a preparation method and application thereof, and belongs to the technical field of biology. The preparation method of the silk fibroin peptide solution comprises the following steps that silkworm cocoons and a sodium carbonate solution are mixed and boiled to remove sericin, degummed silk and a lithium bromide solution are mixed and dissolved, then impurities are removed, the fibroin solution obtained after impurity removal is subjected to enzymolysis, and the fibroin solution obtained after enzymolysis is dialyzed. The preparation method comprises the following steps: mixing a dialyzed silk fibroin peptide solution with mannitol, homogenizing and drying to obtain a silk fibroin peptide powder base material; experiments prove that the silk fibroin peptide solution and the silk fibroin peptide powder base material prepared by the invention have the effects of remarkably inhibiting XOD activity and reducing uric acid, and are of great significance in improving the utilization rate of silkworm cocoon fibroin and promoting human health.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a silk fibroin peptide solution, a silk fibroin peptide powder base material, and a preparation method and application thereof. Background Art

[0002] Silk is a natural, multifunctional protein biopolymer with a fibrous structure, primarily composed of fibroin and sericin. Extracted from silkworm cocoons, fibroin possesses numerous benefits, including radiation protection, moisturizing, antioxidant, antibacterial, and emollient properties. It has been widely used in biomedical fields such as wound healing, tissue regeneration, and drug delivery, and is attracting increasing attention as a bioactive material. Currently, the reported functions of silk fibroin peptides primarily include anti-cancer, anti-hypertensive, and cholesterol-lowering effects. Research into other functional aspects of silk fibroin peptides is crucial for enhancing the utility of silk.

[0003] The main indicator of hyperuricemia is that the uric acid level in human blood is higher than the normal value. Uric acid is an organic weak acid and the end product of purine metabolism. Xanthine oxidase (XOD) is a key enzyme in the formation of uric acid. It is mainly involved in the synthesis of uric acid, catalyzing the conversion of hypoxanthine to xanthine and then to uric acid. Therefore, inhibiting XOD activity can effectively reduce the uric acid content in the body from the source. Currently, commercial XOD inhibitory drugs are all chemically synthesized, and long-term use of such uric acid-lowering drugs can cause more serious adverse reactions. It is reported that extracts from lily, Poria cocos, Dendrobium officinale, Gardenia jasminoides, and Phellinus igniarius can exert uric acid-lowering activity by inhibiting XOD activity. The development of natural XOD activity inhibitors as candidate raw materials for uric acid-lowering drugs has become a future development trend. At present, there are no reports on the preparation of uric acid-lowering products using silkworm cocoons as raw materials. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a silk fibroin peptide solution, a silk fibroin peptide powder base material, and a preparation method and application thereof, wherein the silk fibroin peptide solution and the silk fibroin peptide powder base material have significant XOD inhibition and uric acid lowering effects.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a method for preparing a silk fibroin peptide solution, comprising the following steps: mixing silkworm cocoons with a sodium carbonate solution and boiling the mixture to remove sericin; mixing degummed silk with a lithium bromide solution and dissolving the mixture to remove impurities; enzymatically hydrolyzing the silk fibroin solution after the impurities are removed; and dialyzing the enzymatically hydrolyzed silk fibroin solution.

[0007] Preferably, the cocoons are produced from the Liangguang No. 2 silkworm species.

[0008] Preferably, the material-liquid ratio of the cocoons to the sodium carbonate solution is 1 g:20-90 mL; the boiling time is 20 min-2 h, the boiling times are 1-4 times; and the concentration of the sodium carbonate solution is 0.1-1 w / v%.

[0009] Preferably, the step of mixing and dissolving the degummed silk with the lithium bromide solution comprises: adding the lithium bromide solution to the degummed silk at a material-liquid ratio of 1 g:46-60 mL and dissolving for 4-6 hours, and then placing in a water bath at 70-85° C. for 3-6 hours.

[0010] Preferably, the enzyme comprises neutral protease and flavor protease; the total enzyme amount of the neutral protease and flavor protease is 3000-5000 U / g, and the mass ratio of the neutral protease to the flavor protease is 1:2.5-3.5.

[0011] Preferably, the mass volume ratio of the enzyme to the silk fibroin solution after impurities removal is 1 g:25-35 mL; and the enzymatic hydrolysis time is 4.5-5.5 h.

[0012] Preferably, the molecular weight cut-off used in the dialysis is 3000-4000 Da, and the dialysis time is 48-96 h.

[0013] The invention provides a silk fibroin peptide solution prepared by the preparation method.

[0014] The invention provides a silk fibroin peptide powder base material, comprising a silk fibroin peptide solution prepared by the preparation method and auxiliary materials.

[0015] The present invention provides use of the silk fibroin peptide solution obtained by the preparation method or the silk fibroin peptide powder base in preparing products for inhibiting xanthine oxidase activity and / or lowering uric acid.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses silkworm cocoons as raw materials for the first time and adopts a specific preparation method to obtain a silk fibroin peptide solution that has the effect of significantly inhibiting XOD activity and lowering uric acid. Furthermore, the silk fibroin peptide powder base prepared by mixing the silk fibroin peptide solution with auxiliary materials has a significant XOD inhibitory effect and uric acid lowering effect, which expands the use of silk fibroin in lowering uric acid and increases the added value of silk fibroin. The uric acid-lowering active ingredients prepared by the present invention are all derived from natural silkworm cocoons, will not bring too much metabolic burden to the human body, and the product preparation process is simple, the production cost is low, and it has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The inhibitory effects of different concentrations of silk fibroin peptide solutions on XOD activity. Different letters in the same group indicate differences at the P 0.05 level.

[0019] Figure 2 The inhibitory effects of different concentrations of silk fibroin peptide powder on XOD activity. Different letters in the same group indicate differences at the P 0.05 level.

[0020] Figure 3 This is the effect of silk peptide solution on the viability of HK-2 cells.

[0021] Figure 4 This is the effect of silk fibroin peptide powder base on the viability of HK-2 cells.

[0022] Figure 5 This is the effect of silk peptide solution on the uric acid content in HK-2 cells.

[0023] Figure 6 This is the effect of silk fibroin peptide powder base on the uric acid content of HK-2 cells.

[0024] Figure 7 The results show the effect of enzyme types on the inhibition of XOD activity by silk peptide solution.

[0025] Figure 8 The results show the effect of the enzyme ratio of neutral protease to flavor protease on the inhibition of XOD activity of silk peptide solution.

[0026] Figure 9 The results show the effect of enzyme dosage on the inhibition of XOD activity by silk fibroin peptide solution.

[0027] Figure 10 The results show the effect of the ratio of enzyme to silk solution on the inhibition of XOD activity of silk peptide solution.

[0028] Figure 11 The results show the effect of enzymatic hydrolysis time on the inhibition of XOD activity by silk fibroin peptide solution.

[0029] Figure 12 The results show the effect of dialysis cut-off molecular weight on the inhibition of XOD activity of silk fibroin peptide solution. DETAILED DESCRIPTION

[0030] The present invention provides a method for preparing a silk fibroin peptide solution, comprising the steps of mixing silkworm cocoons with a sodium carbonate solution and boiling to remove sericin, mixing and dissolving the degummed silk with a lithium bromide solution, and then removing impurities, enzymatically hydrolyzing the degummed silk fibroin solution, and dialyzing the hydrolyzed silk fibroin solution. The silk fibroin peptide solution prepared using this method has the effects of significantly inhibiting XOD activity and lowering uric acid.

[0031] In the present invention, the cocoons are produced from the Liangguang No. 2 silkworm eggs. The Liangguang No. 2 silkworm eggs described in the present invention are sourced from Guangdong Guangxin Seed Co., Ltd. The steps for obtaining the cocoons in the present invention include: taking the Liangguang No. 2 silkworm eggs out of the cold storage for rapid greening, hatching the silkworm eggs after 9-11 days, raising the 1st to 3rd instar larvae under the temperature and humidity conditions of 28.0-28.5°C and RH80-85%, raising the 4th to 5th instar larvae under the temperature and humidity conditions of 27-27.5°C and RH75-80%, harvesting the cocoons 5-7 days after the mature silkworms have clustered to obtain fresh cocoons. In the present invention, the silkworm eggs are preferably hatched after 10 days. In the present invention, the cocoons are preferably harvested after 6 days.

[0032] The silk cocoons of the present invention are fresh silk cocoons. As an implementation method, the fresh silk cocoons are cut open, the cocoon shells are washed with water, dried at 80-90°C, and the dried cut cocoons are cut into 1 cm 2 The water used for washing the cocoon shells is preferably ultrapure water. The temperature for drying the cocoon shells is preferably 85°C.

[0033] In the present invention, the cocoons are mixed with the sodium carbonate solution at a material-to-liquid ratio of 1 g:20-90 mL; the boiling time is 20 min-2 h, the number of boilings is 1-4 times; and the concentration of the sodium carbonate solution is 0.1-1 w / v% (w / v = g / mL). Preferably, the step of mixing the cocoons with the sodium carbonate solution and boiling to remove sericin comprises: adding the sodium carbonate solution to the cocoons at a material-to-liquid ratio of 1 g:70-90 mL and boiling for 1-2 hours; removing the silk from the cocoons for the first time, adding the sodium carbonate solution at a material-to-liquid ratio of 1 g:40-60 mL and boiling for 40 min-1.5 hours; removing the silk from the cocoons for the second time, adding the sodium carbonate solution at a material-to-liquid ratio of 1 g:20-40 mL and boiling for a third time for 20-40 min, rinsing with water to remove the sericin, and drying. The concentration of the sodium carbonate solution of the present invention is 0.1-1 w / v%. The preparation steps of the sodium carbonate solution include: accurately weighing 1-10g of anhydrous sodium carbonate, adding an appropriate amount of distilled water and stirring until completely dissolved, transferring to a 1L volumetric flask, rinsing the beaker with distilled water multiple times, adding the washing solution to the volumetric flask, adding distilled water to the scale line, and shaking well. The material-to-liquid ratio of the sodium carbonate solution added to the cocoon of the present invention is preferably 1g:75-85mL, and more preferably 1g:80mL. The first boiling time of the present invention is preferably 1.2-2.7h, and more preferably 1.5h. The material-to-liquid ratio of the sodium carbonate solution added to the silk extracted for the first time of the present invention is preferably 1g:45-55mL, and more preferably 1g:50mL. The second boiling time of the present invention is preferably 50min-1.2h, and more preferably 1h. The material-to-liquid ratio of the sodium carbonate solution added to the silk extracted for the second time of the present invention is preferably 1g:25-35mL, and more preferably 1g:30mL. The third boiling time of the present invention is preferably 25-35 minutes, more preferably 30 minutes. The present invention removes sericin to obtain degummed silk, which is then dried; the drying temperature is preferably 75-90°C, more preferably 80°C.

[0034] In the present invention, the step of mixing and dissolving the degummed silk with the lithium bromide solution comprises: adding the lithium bromide solution to the degummed silk and dissolving it for 4-6 hours according to the material-liquid ratio of the degummed silk to the lithium bromide solution of 1g:45-60mL, and then water bathing at 70-85°C for 3-6 hours. The concentration of the lithium bromide solution of the present invention is 8-10mol / L, preferably 8.5-9.5mol / L, and more preferably 9.3mol / L; the preparation of the lithium bromide solution comprises the following steps: accurately weighing 750-850g of lithium bromide (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number 108931) into a beaker, adding an appropriate amount of distilled water and stirring until completely dissolved, transferring to a 1L volumetric flask, rinsing the beaker with distilled water several times, adding the washing liquid into the volumetric flask, adding distilled water to the scale line and shaking well. The material-liquid ratio of lithium bromide solution added to the degummed silk is preferably 1 g:48-65 mL, more preferably 1 g:50 mL. The dissolution time is preferably 4.5-5.5 hours, more preferably 5 hours. The water bath temperature is preferably 75-83°C, more preferably 80°C; the water bath time is preferably 3.5-5.5 hours, more preferably 4 hours.

[0035] In the present invention, the enzyme includes a neutral protease and a flavor protease; the total enzyme amount of the neutral protease and the flavor protease is 3000-5000 U / g, preferably 3500-4500 U / g, and more preferably 4000 U / g; the mass ratio of the neutral protease to the flavor protease is 1:2.5-3.5, preferably 1:2.8-3.4, and more preferably 1:3. The mass volume ratio of the enzyme of the present invention to the silk fibroin solution after impurity removal is 1g:25-35mL, preferably 1g:28-34mL, and more preferably 1g:30mL. The enzymatic hydrolysis time of the present invention is 4.5-5.5h, preferably 4.8-5.3h, and more preferably 5h.

[0036] In the present invention, the molecular weight cut-off used for the dialysis is 3000-4000 Da, preferably 3200-3800 Da, and more preferably 3500 Da; the dialysis time is 48-96 h, preferably 60-84 h, and more preferably 72 h.

[0037] The present invention also provides a silk fibroin peptide powder base material, comprising a silk fibroin peptide solution obtained by the preparation method and an auxiliary material. In the present invention, the silk fibroin peptide solution is added to the auxiliary material at a mass ratio of 5-8% (5-8g of auxiliary material is added to 100g of silk fibroin peptide solution). The auxiliary material of the present invention comprises one or more of mannitol, xylitol or erythritol. In the process of preparing the silk fibroin peptide powder base material, the present invention performs homogenization and drying steps; the homogenization pressure is preferably 100-200MPa, more preferably 150MPa; the drying is preferably spray drying, and the drying temperature is preferably 70-80°C, more preferably 75°C.

[0038] The present invention also provides a silk fibroin peptide powder base material obtained by the preparation method.

[0039] The present invention also provides the use of the silk fibroin peptide powder base material obtained by the preparation method in preparing products for inhibiting xanthine oxidase activity and / or reducing uric acid.

[0040] In the present invention, unless otherwise specified, the raw materials or reagents used are commercially available products well known to those skilled in the art.

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Raising the silkworm Liangguang No. 2: The Liangguang No. 2 silkworm eggs (sourced from Guangdong Guangxin Seed Co., Ltd.) were taken out of the cold storage for rapid greening. After 10 days, the silkworm eggs were hatched. After the queens were collected, the 1st to 3rd instar larvae were raised under the temperature and humidity conditions of 28.0°C and RH80%, and the 4th to 5th instar larvae were raised under the temperature and humidity conditions of 27.0°C and RH75%. The cocoons were harvested 6 days after the mature silkworms were clustered to obtain fresh cocoons.

[0044] 2. Cut open the fresh silk cocoons, wash the cocoon shells with ultrapure water, and dry them at 80-90℃.

[0045] 3. Chop the dried cocoons into 1cm pieces 2For lumps of silk cocoons smaller than 0.5%, 0.5% sodium carbonate solution was added to the cocoons at a material-liquid ratio of 1 g:80 mL, and the mixture was boiled for 1.5 hours. The silk was removed, and 0.5% sodium carbonate solution was added at a material-liquid ratio of 1 g:50 mL, and the mixture was boiled for 1 hour. The silk was then removed, and 0.5% sodium carbonate solution was added at a material-liquid ratio of 1 g:30 mL, and the mixture was boiled for 30 minutes. The degummed silk was obtained by repeatedly rinsing with ultrapure water to remove residual sericin, and then drying at 80°C.

[0046] 4. According to the material-liquid ratio of degummed silk and lithium bromide solution of 1g:50mL, 9.3mol / L lithium bromide solution was added to the dried degummed silk and dissolved for 5h, then placed in a water bath at 80℃ for 4h. After the silk was fully dissolved, it was filtered and removed with a bag filter (pore size 100μm, pressure 0.2MPa), and the pressure was set to 0.2MPa.

[0047] 5. Enzymatic hydrolysis of the silk fibroin solution after impurities removal: according to the total enzyme amount of 4000U / g, the mass ratio of neutral protease and flavor protease is 1:3 to prepare a composite enzyme; according to the material-liquid ratio of composite enzyme to silk fibroin solution of 1g:30mL, the composite enzyme is added to the silk fibroin solution after impurities removal, and enzymatic hydrolysis is carried out at 50℃ and pH7.5 for 5h, and then heated to 95℃ for 10min to inactivate the enzyme.

[0048] 6. Use a dialysis bag with a pore size molecular weight cutoff of 3500Da to dialyze the enzymatically hydrolyzed silk fibroin solution in running water for 72 hours, take the solution in the dialysis bag, and obtain the purified silk fibroin peptide solution.

[0049] 7. Add 6 g of mannitol to 100 g of silk fibroin peptide solution, place the resulting mixture in a high-pressure microfluidizer and homogenize it under a pressure of 150 MPa, and spray dry it at 75°C to obtain a silk fibroin peptide powder base.

[0050] 8. Package under sterile conditions and sterilize the silk peptide powder base material by electron beam irradiation with a radiation dose of 15 kGy.

[0051] Example 2

[0052] 1. Raising the silkworm Liangguang No. 2: The Liangguang No. 2 silkworm eggs (sourced from Guangdong Guangxin Seed Co., Ltd.) were taken out of the cold storage for rapid greening. After 9 days, the silkworm eggs were hatched. After the queens were collected, the 1st to 3rd instar larvae were raised at a temperature and humidity of 28.5°C and RH85%, and the 4th to 5th instar larvae were raised at a temperature and humidity of 27.5°C and RH80%. After 5 days of mature silkworms, the cocoons were harvested to obtain fresh cocoons.

[0053] 2. Cut open the fresh silk cocoons, wash the cocoon shells with ultrapure water, and dry them at 80℃.

[0054] 3. Chop the dried cocoons into 1cm pieces 2 For lumps of silk cocoons (less than 100g), add 0.1% sodium carbonate solution to the cocoons at a material-liquid ratio of 1g:70mL, and boil continuously for 1 hour. Remove the silk, add 0.1% sodium carbonate solution at a material-liquid ratio of 1g:40mL, and boil continuously for 40 minutes. Remove the silk again, add 0.1% sodium carbonate solution at a material-liquid ratio of 1g:20mL, and boil continuously for 20 minutes. Rinse repeatedly with ultrapure water to remove residual sericin to obtain degummed silk, and dry at 75°C.

[0055] 4. According to the material-liquid ratio of degummed silk and lithium bromide solution of 1g:40mL, add 8.5mol / L lithium bromide solution to the dried degummed silk and dissolve it for 4h, then bathe it in water at 70℃ for 3h. After the silk is fully dissolved, filter and remove impurities with a bag filter (pore size 80μm, pressure 0.1MPa), and the pressure is set to 0.2MPa.

[0056] 5. Enzymatic hydrolysis of the silk fibroin solution after impurities removal: according to the total enzyme amount of 3000U / g, the mass ratio of neutral protease and flavor protease is 1:2.5 to prepare a composite enzyme; according to the material-liquid ratio of composite enzyme to silk fibroin solution of 1g:25mL, the composite enzyme is added to the silk fibroin solution after impurities removal, and enzymatic hydrolysis is carried out at 50℃ and pH7.5 for 4.5h, and then heated to 95℃ for 10min to inactivate the enzyme.

[0057] 6. Use a dialysis bag with a pore size cutoff molecular weight of 3000Da to dialyze the enzymatically hydrolyzed silk fibroin solution in running water for 48 hours, take the solution in the dialysis bag, and obtain the purified silk fibroin peptide solution.

[0058] 7. Add 5 g of mannitol to 100 g of silk fibroin peptide solution, place the resulting mixture in a high-pressure microfluidizer for homogenization at a pressure of 100 MPa, and spray dry at 70° C. to obtain a silk fibroin peptide powder base.

[0059] 8. Package under sterile conditions and sterilize the silk peptide powder base material by electron beam irradiation with a radiation dose of 15 kGy.

[0060] Example 3

[0061] 1. Raising the silkworm Liangguang No. 2: The Liangguang No. 2 silkworm eggs (sourced from Guangdong Guangxin Seed Co., Ltd.) were taken out of the cold storage for rapid greening. After 11 days, the silkworm eggs were hatched. After the queens were collected, the 1st to 3rd instar larvae were raised at a temperature and humidity of 28.0°C and RH80%, and the 4th to 5th instar larvae were raised at a temperature and humidity of 27.0°C and RH75%. After 7 days of mature silkworms, the cocoons were harvested to obtain fresh cocoons.

[0062] 2. Cut open the fresh silk cocoons, wash the cocoon shells with ultrapure water, and dry them at 90℃.

[0063] 3. Chop the dried cocoons into 1cm pieces 2 For lumps of silk cocoons (less than 100g), add 2% sodium carbonate solution to the cocoons at a material-liquid ratio of 1g:90mL of 1w / v% sodium carbonate solution and boil for 1 hour. Remove the silk, add 1% sodium carbonate solution at a material-liquid ratio of 1g:60mL and boil for 1.5 hours. Then remove the silk, add 1% sodium carbonate solution at a material-liquid ratio of 1g:40mL and boil for 40 minutes. Rinse repeatedly with ultrapure water to remove residual sericin to obtain degummed silk, which is then dried at 90°C.

[0064] 4. According to the material-liquid ratio of degummed silk and lithium bromide solution of 1g:60mL, add 9.5mol / L lithium bromide solution to the dried degummed silk and dissolve it for 6h, then bathe it in water at 85℃ for 6h. After the silk is fully dissolved, filter and remove impurities with a bag filter (pore size 120μm, pressure 0.3MPa), and the pressure is set to 0.2MPa.

[0065] 5. Enzymatic hydrolysis of the decontaminated silk fibroin solution: a composite enzyme was prepared by mixing neutral protease and flavor protease at a mass ratio of 1:3.5 with a total enzyme amount of 5000 U / g; the composite enzyme was added to the decontaminated silk fibroin solution at a material-liquid ratio of 1 g:35 mL at 50°C and pH 7.5, and enzymatic hydrolysis was performed for 5.5 h. The enzymatic inactivation was then carried out by heating to 95°C for 10 min.

[0066] 6. Use a dialysis bag with a pore size cutoff molecular weight of 4000Da to dialyze the enzymatically hydrolyzed silk fibroin solution in running water for 96 hours, take the solution in the dialysis bag, and obtain the purified silk fibroin peptide solution.

[0067] 7. Add 8 g of mannitol to 100 g of silk fibroin peptide solution, place the resulting mixture in a high-pressure microfluidizer for homogenization at a pressure of 100 MPa, and spray dry at 70°C to obtain a silk fibroin peptide powder base.

[0068] 8. Package under sterile conditions and sterilize the silk peptide powder base material by electron beam irradiation with a radiation dose of 15 kGy.

[0069] Comparative Example 1

[0070] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0071] Step 5: Enzymatically hydrolyze the silk fibroin solution after impurities removal at 55° C. and pH 7.5, with a papain to silk fibroin solution ratio of 1 g:30 mL, for 5 h, and heat to 95° C. for 10 min to inactivate the enzyme.

[0072] Comparative Example 2

[0073] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0074] Step 5: Enzyme hydrolyze the silk fibroin solution after impurity removal at 50° C. and pH 10.0, with a material-liquid ratio of alkaline protease to silk fibroin solution of 1 g:30 mL, for 5 h, and heat to 95° C. for 10 min to inactivate the enzyme.

[0075] Comparative Example 3

[0076] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0077] Step 5: Enzymatically hydrolyze the silk fibroin solution after impurity removal at 55° C. and pH 7.5 with a ratio of flavor protease to silk fibroin solution of 1 g:30 mL for 5 h, and heat to 95° C. for 10 min to inactivate the enzyme.

[0078] Comparative Example 4

[0079] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0080] Step 5: Enzymatically hydrolyze the silk fibroin solution after impurity removal at 50° C. and pH 7.5, with a neutral protease and silk fibroin solution ratio of 1 g:30 mL for 5 h, and heat to 95° C. for 10 min to inactivate the enzyme.

[0081] Comparative Example 5

[0082] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0083] The difference between step 5 and example 1 is that the mass ratio of neutral protease to flavor protease is 1:1.

[0084] Comparative Example 6

[0085] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0086] The difference between step 5 and example 1 is that the mass ratio of neutral protease to flavor protease is 1:2.

[0087] Comparative Example 7

[0088] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0089] The difference between step 5 and example 1 is that the mass ratio of neutral protease to flavor protease is 1:4.

[0090] Comparative Example 8

[0091] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0092] The difference between step 5 and example 1 is that the mass ratio of neutral protease to flavor protease is 2:1.

[0093] Comparative Example 9

[0094] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0095] The difference between step 5 and example 1 is that the mass ratio of neutral protease to flavor protease is 3:1.

[0096] Comparative Example 10

[0097] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0098] The difference between step 5 and example 1 is that the mass ratio of neutral protease to flavor protease is 4:1.

[0099] Comparative Example 11

[0100] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0101] The difference between step 5 and example 1 is that the total enzyme amount is 1000 U / g.

[0102] Comparative Example 12

[0103] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0104] The difference between step 5 and example 1 is that the total enzyme amount is 2000 U / g.

[0105] Comparative Example 13

[0106] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0107] The difference between step 5 and example 1 is that the total enzyme amount is 6000 U / g.

[0108] Comparative Example 14

[0109] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0110] The difference between step 5 and example 1 is that the material-liquid ratio of the complex enzyme to the silk fibroin solution is 1 g:10 mL.

[0111] Comparative Example 15

[0112] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0113] The difference between step 5 and example 1 is that the material-liquid ratio of the complex enzyme to the silk fibroin solution is 1 g:20 mL.

[0114] Comparative Example 16

[0115] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0116] The difference between step 5 and example 1 is that the material-liquid ratio of the complex enzyme to the silk fibroin solution is 1 g:40 mL.

[0117] Comparative Example 17

[0118] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0119] The difference between step 5 and embodiment 1 is that the enzymatic hydrolysis is carried out for 2 hours.

[0120] Comparative Example 18

[0121] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0122] The difference between step 5 and embodiment 1 is that the enzymatic hydrolysis is carried out for 3 hours.

[0123] Comparative Example 19

[0124] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0125] The difference between step 5 and embodiment 1 is that the enzymatic hydrolysis is carried out for 4 hours.

[0126] Comparative Example 20

[0127] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0128] The difference between step 5 and embodiment 1 is that the enzymolysis time is 6 hours.

[0129] Comparative Example 21

[0130] Steps 1-4 and steps 6-8 are the same as in Example 1.

[0131] The difference between step 5 and embodiment 1 is that the enzymatic hydrolysis is carried out for 7 hours.

[0132] Comparative Example 22

[0133] Steps 1-5 and steps 7-8 are the same as in Example 1.

[0134] The difference between step 6 and embodiment 1 is that the pore size molecular weight cut-off is 2000 Da.

[0135] Comparative Example 23

[0136] Steps 1-5 and steps 7-8 are the same as in Example 1.

[0137] The difference between step 6 and embodiment 1 is that the pore size molecular weight cut-off is 7000 Da.

[0138] Comparative Example 24

[0139] Steps 1-5 and steps 7-8 are the same as in Example 1.

[0140] The difference between step 6 and embodiment 1 is that the pore size molecular weight cut-off is 8000-14000 Da.

[0141] Experimental Example 1

[0142] The silk fibroin peptide solution and the silk fibroin peptide powder prepared in Example 1 were subjected to an XOD activity inhibition experiment, with allopurinol as a positive control and ultrapure water as a blank control.

[0143] The experimental steps are as follows:

[0144] (1) Preparation of sample and reagent solutions

[0145] The silk fibroin peptide solution and silk fibroin peptide powder prepared in Example 1 were diluted with ultrapure water to 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively.

[0146] Allopurinol solution: Allopurinol was dissolved in 0.4% NaOH solution and then diluted to volume with PBS to prepare allopurinol solutions of different concentrations.

[0147] Hypoxanthine solution: Accurately weigh hypoxanthine standard and dissolve it in PBS with ultrasound assistance to prepare a 0.5 mg / mL substrate solution;

[0148] XOD solution: Dilute with pre-chilled PBS to a 1 U / mL enzyme working solution.

[0149] (2) Xanthine oxidase (XOD) activity was determined using a xanthine oxidase (XOD) assay kit (colorimetric method). The kit was purchased from Nanjing Jiancheng Bioengineering Research Institute, catalog number A002-1-1.

[0150] The results of the test are as follows Figure 1 、 Figure 2 As shown in the results, both the silk fibroin peptide solution and the silk fibroin peptide powder showed significant XOD activity inhibition effects, with the inhibition rate exceeding 60% at a concentration of 0.5 mg / mL, and the inhibition rate of XOD activity at concentrations of 1.0 mg / mL and above reaching 100%.

[0151] Experimental Example 2

[0152] 1. HK-2 cells in the logarithmic growth phase were seeded in a 96-well cell culture plate at a cell seeding density of 1×10 4When the cell growth density reached 75%, silk fibroin peptide solution or silk fibroin peptide powder base of different mass concentrations were added, and the cell survival rate was detected by CCK-8 method after 24 hours.

[0153] The results are as follows Figure 3 、 Figure 4 As shown in the figure, when the concentration of silk fibroin peptide solution and silk fibroin peptide powder base was 0.1 mg / mL-5.0 mg / mL, the cell survival rate remained above 90%, indicating that the concentration within this range had no obvious toxic effect on the cells. Therefore, 1.0 mg / mL of silk fibroin peptide solution or silk fibroin peptide powder base was selected as the test concentration for evaluating the uric acid-lowering activity of HK-2 cells.

[0154] 2. Add XOD to the HK-2 cell culture medium to convert uric acid precursors into uric acid and establish a high uric acid cell model.

[0155] HK-2 cells in the logarithmic growth phase were seeded into 24-well plates. After the cells adhered and grew for 24 hours, four groups (three replicates each) were set up, namely, blank group, model group, allopurinol group (0.5 mg / mL allopurinol), and sample group (1.0 mg / mL silk fibroin peptide solution or silk fibroin peptide powder base). Complete culture medium was added to the blank group and model group, complete culture medium containing 0.5 mg / mL allopurinol was added to the allopurinol group, and complete culture medium containing 1.0 mg / mL silk fibroin peptide solution or silk fibroin peptide powder base was added to the silk fibroin peptide solution or silk fibroin peptide powder base group. After 24 hours of culture, the culture medium was removed and the cells were washed three times with PBS. Complete culture medium was added to the blank group, 2.5 mmol / L adenosine-free medium was added to the model group, 0.5 mg / mL allopurinol and 5 mmol / L adenosine-free medium was added to the allopurinol group, and 1.0 mg / mL silk fibroin solution or silk fibroin peptide powder base and 5 mmol / L adenosine-free medium was added to the silk fibroin peptide solution or silk fibroin peptide powder base group. After 30 hours of culture, XOD 0.5 U / mL was added to each group and incubated in a cell culture incubator. After 8 hours, the cell culture medium was collected and centrifuged at 3000 rpm at 4°C for 6 minutes. The supernatant was aspirated and the UA content was determined.

[0156] UA detection method: Uric acid content was detected according to the method provided in the kit instructions (the kit was purchased from Suzhou Grace Biotechnology Co., Ltd., product number G1202W). Figure 5 、 Figure 6 As shown in the results, after adding silk fibroin peptide solution or silk fibroin peptide powder base, the uric acid content in the cell culture supernatant was significantly reduced compared with the model group, showing significant uric acid-lowering activity (P < 0.05).

[0157] Experimental Example 3

[0158] The XOD activity detection method described in Experimental Example 1 was used to detect the inhibitory effect of the silk peptide solutions (0.5 mg / mL) prepared in Comparative Examples 1-24 on XOD activity. Figure 7-12 .

[0159] Figure 7 The results showed that the silk peptide solution prepared by enzymatic hydrolysis with neutral protease and flavor protease had better inhibitory effect on XOD activity.

[0160] Figure 8 The results showed that the silk peptide solution prepared with the enzyme addition mass ratio of neutral protease to flavor protease at 1:3 had the best inhibitory effect on XOD activity.

[0161] Figure 9-12 The results showed that the silk peptide solution prepared with an enzyme dosage of 4000 U / g, a material-liquid ratio of 1 g:30 mL, and an enzymatic hydrolysis time of 5 h had the best inhibitory effect on XOD activity.

[0162] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a silk fibroin peptide solution, characterized in that: The method comprises the following steps: mixing silk cocoons with sodium carbonate solution and boiling them to remove sericin, mixing degummed silk with lithium bromide solution and dissolving them to remove impurities, enzymatically hydrolyzing the silk fibroin solution after the impurities are removed, and dialyzing the enzymatically hydrolyzed silk fibroin solution.

2. The preparation method according to claim 1, wherein The silk cocoons are produced from the Liangguang No. 2 silkworm species.

3. The preparation method according to claim 1, wherein The material-liquid ratio of the cocoons to the sodium carbonate solution is 1 g: 20-90 mL; the boiling time is 20 min-2 h, and the number of boiling times is 1-4 times; and the concentration of the sodium carbonate solution is 0.1-1 w / v%.

4. The preparation method according to claim 1, wherein The step of mixing and dissolving the degummed silk with the lithium bromide solution comprises: adding the lithium bromide solution to the degummed silk at a material-liquid ratio of 1 g:46-60 mL and dissolving for 4-6 hours, and then placing in a water bath at 70-85° C. for 3-6 hours.

5. The preparation method according to claim 1, wherein The enzymes include neutral protease and flavor protease; the total enzyme amount of the neutral protease and flavor protease is 3000-5000 U / g, and the mass ratio of the neutral protease to the flavor protease is 1:2.5-3.

5.

6. The preparation method according to claim 1, wherein The mass volume ratio of the enzyme to the silk fibroin solution after impurities removal is 1 g:25-35 mL; and the enzymatic hydrolysis time is 4.5-5.5 h.

7. The preparation method according to claim 1, wherein The molecular weight cut-off used for the dialysis is 3000-4000 Da, and the dialysis time is 48-96 h.

8. The silk fibroin peptide solution prepared by the preparation method according to any one of claims 1 to 7.

9. A silk fibroin peptide powder base, characterized in that: The invention comprises a silk fibroin peptide solution prepared by the preparation method according to any one of claims 1 to 7 and auxiliary materials.

10. Use of the silk fibroin peptide solution prepared by the preparation method according to any one of claims 1 to 7 or the silk fibroin peptide powder base according to claim 9 in preparing products for inhibiting xanthine oxidase activity and / or lowering uric acid.

Images