Green cocoon sericin solution as well as preparation method and application thereof

Abstract

The invention provides a green cocoon sericin solution as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method of the green cocoon sericin solution comprises the following steps: mixing green cocoons with water, boiling, collecting a degumming solution, mixing the degumming solution with enzyme, carrying out enzymolysis, carrying out enzyme deactivation, and concentrating; the enzyme is papain and flavourzyme. The green cocoon sericin oral liquid is obtained by mixing a green cocoon sericin solution with auxiliary materials. Experiments prove that the green cocoon sericin solution and the green cocoon sericin oral liquid have a remarkable XOD inhibition effect, and are of great significance in improving the utilization rate of natural colored sericin and promoting human health.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a green cocoon sericin solution, a preparation method thereof, and an application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder, which is divided into two types: primary and secondary. Purine metabolism disorder leads to an increase in the content of uric acid in the body. When uric acid cannot be metabolized and excreted in time, it will accumulate in the body. Excessive accumulation will form hyperuricemia, and severe cases will further cause complications such as gout in the body. Uric acid is the final product of purine catabolism, and xanthine oxidase (XOD) is the key enzyme in this metabolic process and an important target for anti-hyperuricemic drugs.

[0003] At present, the anti-hyperuricemic drugs clinically used mainly include XOD inhibitors allopurinol, febuxostat, and uricosuric drugs probenecid, benzbromarone, etc. The varieties of drugs are few, and there are adverse reactions to varying degrees. For example, allopurinol can cause bone marrow suppression, leukopenia, and may produce adverse reactions in the gastrointestinal and nervous systems; febuxostat is prone to cause abnormal liver function; probenecid may cause gastrointestinal symptoms such as nausea and vomiting; benzbromarone may cause liver function disorders and hepatitis. Therefore, the research and development of more natural anti-hyperuricemic bioactive substances have positive significance for the treatment of hyperuricemia.

[0004] Natural cocoons are mainly divided into three categories according to color: white cocoons, yellow-red cocoons, and green cocoons. The degumming solution of natural colored cocoons contains not only sericin protein, but also components such as carotenoid substances and flavonoid compounds, and has a variety of biological activities. The sericin of natural cocoons has long been discarded as waste during the raw silk processing process, resulting in serious waste of resources. There are only some research reports on its antioxidant and skin care effects, and no research on its anti-hyperuricemic effect has been reported. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a green cocoon sericin solution, a preparation method thereof, and an application thereof. The green cocoon sericin solution and the green cocoon sericin oral liquid prepared by this method have the function of significantly inhibiting the activity of XOD, and thus have the effect of reducing uric acid.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method of a green cocoon sericin solution, including the following steps: mixing cocoons with water and boiling to collect the degumming solution, mixing the degumming solution with an enzyme for enzymatic hydrolysis, inactivating the enzyme, and concentrating; the enzyme is papain and flavor protease; the cocoon is a green cocoon.

[0008] Preferably, the green cocoon is produced by the green cocoon silkworm species 301.

[0009] Preferably, the mass-volume ratio of the cocoon to water is 1 g: 20-60 mL.

[0010] Preferably, the boiling time is 1-3 h, and the number of boiling times is 1-3 times.

[0011] Preferably, the total amount of the enzyme is 3000-5000 U / g.

[0012] Preferably, the mass ratio of papain to flavor protease is 1: 1.5-2.5.

[0013] Preferably, the mass-volume ratio of the enzyme to the degumming solution is 1 g: 6-14 mL.

[0014] The present invention also provides a green cocoon sericin solution prepared by the preparation method.

[0015] The present invention provides a green cocoon sericin oral liquid, comprising the green cocoon sericin solution prepared by the preparation method and excipients.

[0016] The present invention provides the use of the green cocoon sericin solution prepared by the preparation method or the green cocoon sericin oral liquid in the preparation of products for inhibiting xanthine oxidase activity and / or reducing uric acid.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention for the first time uses green cocoons as raw materials, and the green cocoon sericin solution obtained by the specific preparation method of the present invention has a significant XOD inhibitory effect. Further, the green cocoon sericin oral liquid prepared by mixing the green cocoon sericin solution with excipients still has a significant XOD inhibitory effect, expands the use of sericin in reducing uric acid, and improves the added value of sericin. The uric acid-reducing active ingredients of the present invention all come from natural green 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. Description of the Drawings

[0019] Figure 1 Shows the inhibitory effect of green cocoon sericin solutions with different concentrations on XOD activity. Among them, different letters in the same group indicate that there are differences in P at the 0.05 level.

[0020] Figure 2 Shows the inhibitory effect of green cocoon sericin oral liquids with different concentrations on XOD activity. Among them, different letters in the same group indicate that there are differences in P at the 0.05 level.

[0021] Figure 3 Shows the influence results of enzyme types on the inhibitory effect of green cocoon sericin solutions on XOD activity.

[0022] Figure 4Results of the effect of the enzyme addition ratio of papain to flavor protease on the inhibitory activity of green cocoon sericin solution against XOD.

[0023] Figure 5 Results of the effect of the enzyme dosage on the inhibitory activity of green cocoon sericin solution against XOD.

[0024] Figure 6 Results of the effect of the material-liquid ratio of the enzyme to the degummed solution after impurity removal on the inhibitory activity of green cocoon sericin solution against XOD.

[0025] Figure 7 Results of the effect of the enzymolysis time on the inhibitory activity of green cocoon sericin solution against XOD. Detailed implementation manners

[0026] The present invention provides a method for preparing a green cocoon sericin solution, which includes the following steps: mixing cocoons with water and boiling to collect the degummed solution, mixing the degummed solution with an enzyme for enzymolysis, inactivating the enzyme, and concentrating; the enzyme is papain and flavor protease; the cocoons are green cocoons. The green cocoons in the present invention are produced by the green cocoon silkworm breed 301. The silkworm breed 301 in the present invention is a natural green cocoon silkworm and has been included in the National Silkworm Genetic Resource Variety List (2024 Edition) and is recorded in "Chinese Silkworm Variety Annals" (published by the Agricultural Publishing House in 1987). The papain and flavor protease in the present invention can be obtained through commercially available channels well-known to those skilled in the art without special instructions.

[0027] In the present invention, the natural green cocoon silkworm breed 301 is taken out of the cold storage for embryo activation. After 8 - 10 days, the silkworm eggs hatch. After collecting the newly hatched larvae, the 1st - 3rd instar larvae are reared under the temperature and humidity conditions of 28.0 - 28.5°C and RH80 - 85%, and the 4th - 5th instar larvae are reared under the temperature and humidity conditions of 27 - 27.5°C and RH75 - 80%. The cocoons are harvested 5 - 8 days after the mature silkworms mount the cocoon frame to obtain natural green cocoons. The present invention preferably hatches the silkworm eggs after 9 days. The present invention preferably harvests the cocoons after 6 days.

[0028] As an implementable manner, the present invention cuts open fresh natural cocoons, washes the cocoon shells with water, dries them at 40 - 60°C, and cuts the dried cut cocoons into blocks within 1 cm 2 for subsequent experiments. The water used by the present invention to wash the cocoon shells is preferably ultrapure water. The temperature for drying the cocoon shells by the present invention is preferably 50°C.

[0029] In the present invention, the mass-volume ratio of the cocoon to water is 1 g: 20 - 60 mL, the boiling time is 1 - 3 h, and the number of boiling times is 1 - 3 times. As an implementable embodiment, the step of mixing and boiling the cocoon with water in the present invention includes: in the cocoon shell, adding water at a material-liquid ratio of 1 g: 40 - 60 mL for the first boiling for 1 - 3 h, and collecting the first degumming solution; then, adding water at a material-liquid ratio of 1 g: 20 - 40 mL to the remaining cocoon shell after collecting the degumming solution for the second boiling for 1 - 2 h, and collecting the second degumming solution; combining the two degumming solutions. When boiling for the first time in the present invention, the mass-volume ratio of the cocoon shell to water is preferably 1 g: 45 - 55 mL, more preferably 1 g: 50 mL; the boiling time for the first time in the present invention is preferably 2 h. When boiling for the second time in the present invention, the mass-volume ratio of the cocoon shell to water is preferably 1 g: 25 - 35 mL, more preferably 1 g: 30 mL; the boiling time for the second time in the present invention is preferably 1.5 h. The present invention filters the combined degumming solution to remove impurities in the degumming solution. The present invention uses a bag filter for filtration and impurity removal. The set pore size of the bag filter is 80 - 120 μm, preferably 100 μm, and the pressure is set to 0.1 - 0.5 MPa, preferably 0.2 MPa.

[0030] In the present invention, the total amount of the enzyme is 3000 - 5000 U / g, preferably 3500 - 4500 U / g, more preferably 4000 U / g. The mass ratio of papain to flavor protease in the present invention is 1: 1.5 - 2.5, preferably 1: 1.8 - 2.3, more preferably 1: 2. As an implementable mode, when the total amount of the enzyme in the present invention is 4000 U / g and the mass ratio of papain to flavor protease is 1: 2, the amount of papain is 1333.3 U / g and the amount of flavor protease is 2666.7 U / g.

[0031] In the present invention, the mass-volume ratio of the enzyme to the degumming solution is 1 g: 6 - 14 mL, preferably 1 g: 8 - 12 mL, more preferably 1 g: 10 mL. The enzymolysis time in the present invention is 130 - 170 min, preferably 140 - 160 min, more preferably 150 min; the enzymolysis temperature is 50 - 60 °C, preferably 52 - 58 °C, more preferably 55 °C; the pH of the enzymolysis is 7.0 - 8.0, preferably 7.3 - 7.9, more preferably 7.5.

[0032] In the present invention, the temperature for inactivating the enzyme is 85 - 99 °C, preferably 90 - 98 °C, more preferably 95 °C; the time for inactivating the enzyme is 5 - 15 min, preferably 8 - 13 min, more preferably 10 min; the vacuum concentration is up to a soluble solid content of 8 - 18%, preferably 10 - 15%, more preferably 13%.

[0033] The present invention also provides a green cocoon sericin solution prepared by the said preparation method.

[0034] The present invention also provides a green cocoon sericin oral liquid, which comprises the green cocoon sericin solution prepared by the said preparation method and excipients. In the present invention, the green cocoon sericin solution is added with excipients according to a ratio of 0.005 - 0.01 w / v% (0.005 - 0.01 g of excipients are added to 100 mL of the green cocoon sericin solution). The excipients of the present invention include one or more of xylitol, maltitol, sorbitol, mannitol or erythritol.

[0035] The present invention also provides the application of the green cocoon sericin solution prepared by the said preparation method or the said green cocoon sericin oral liquid in the preparation of products for reducing uric acid.

[0036] In the present invention, unless otherwise specified, all components or reagents are commercially available products well-known to those skilled in the art.

[0037] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Example 1

[0039] 1. Rearing the natural green cocoon silkworm variety 301: Take out the natural green cocoon silkworm eggs 301 from the cold storage for hibernation awakening. After 9 days, the silkworm eggs hatch. After collecting the newly hatched silkworms, rear the 1st - 3rd instar larvae under the temperature and humidity conditions of 28.0°C and RH80%, and rear the 4th - 5th instar larvae under the temperature and humidity conditions of 27°C and RH75%. After 6 days when the mature silkworms climb onto the cocooning frame, harvest the cocoons to obtain natural green cocoons.

[0040] 2. Cut open the fresh natural green cocoons, wash the cocoon shells with ultrapure water, and dry them at 50°C.

[0041] 3. Cut the dried green cocoon shells into pieces within 1 cm 2 in size, add ultrapure water at a material - liquid ratio of 1 g:50 mL and continuously boil for 2 h, and collect the first degumming solution. Then add the green cocoons at a material - liquid ratio of 1 g:30 mL of ultrapure water and continuously boil for 1.5 h, and collect the second degumming solution. Combine the two degumming solutions and filter them with a bag - type filter (pore size 100 μm, pressure 0.2 MPa) to remove impurities in the degumming solution, and obtain the degumming solution after impurity removal.

[0042] 4. Mix papain and flavor protease at a total enzyme amount of 4000 U / g and a mass ratio of 1:2 to obtain a composite enzyme. According to the material-liquid ratio of 1 g of the composite enzyme to 10 mL of the degummed solution after impurity removal, add the composite enzyme to the degummed solution after impurity removal, and carry out enzymatic hydrolysis at 55 °C and pH 7.5 for 150 min. Heat to 95 °C and keep for 10 min to inactivate the enzyme, and then vacuum concentrate to a soluble solid content of 13 w / v% (13 g of green cocoon sericin in 100 mL of concentrated solution) to obtain a green cocoon sericin solution.

[0043] 5. Add a mixture of xylitol and maltitol with a mass ratio of 1:1 to the green cocoon sericin solution at a ratio of 0.008 w / v% (add 0.008 g of the xylitol and maltitol mixture to 100 mL of the concentrated green cocoon sericin solution), homogenize, and centrifuge to remove impurities to obtain a green cocoon sericin oral liquid.

[0044] 6. Carry out ultra-high pressure sterilization treatment on the green cocoon sericin oral liquid at a pressure of 400 MPa for 20 min (temperature 24 °C), and fill it into sterile oral liquid bottles (specification 10 mL).

[0045] Example 2

[0046] 1. Raise the natural green cocoon silkworm variety 301: Take out the natural green cocoon silkworm eggs 301 from the cold storage for hatching. After 8 days, the silkworm eggs hatch. After collecting the newly hatched silkworms, raise the 1st - 3rd instar larvae at a temperature of 28.5 °C and a relative humidity of 85%, and raise the 4th - 5th instar larvae at a temperature of 27.5 °C and a relative humidity of 80%. Harvest the cocoons 5 days after the mature silkworms climb onto the cocooning frame to obtain natural green cocoons.

[0047] 2. Cut open the fresh natural green cocoons, wash the cocoon shells with ultrapure water, and dry at 40 °C.

[0048] 3. Cut the dried green cocoon shells into pieces within 1 cm 2 in size, add ultrapure water at a material-liquid ratio of 1 g:40 mL and boil continuously for 1 h, and collect the first degummed solution. Then add the green cocoons at a material-liquid ratio of 1 g:20 mL of ultrapure water and boil continuously for 1 h, and collect the second degummed solution. Combine the two degummed solutions and filter them with a bag filter (pore size 80 μm, pressure 0.1 MPa) to remove impurities in the degummed solution to obtain a degummed solution after impurity removal.

[0049] 4. Mix papain and flavor protease at a total enzyme amount of 3500 U / g and a mass ratio of 1:1.5 to obtain a composite enzyme. According to the material-liquid ratio of 1 g of the composite enzyme to 6 mL of the degummed solution after impurity removal, add the composite enzyme to the degummed solution after impurity removal, and carry out enzymatic hydrolysis at 50 °C and pH 7.0 for 130 min. Heat to 90 °C and keep for 8 min to inactivate the enzyme, and then vacuum concentrate to a soluble solid content of 8 w / v% to obtain a green cocoon sericin solution.

[0050] 5. Add the green cocoon sericin solution at a ratio of 0.005 w / v% to a mixture of xylitol and maltitol with a mass ratio of 1:0.5, homogenize, and centrifuge to remove impurities to obtain the green cocoon sericin oral liquid.

[0051] 6. Ultra-high pressure sterilize the green cocoon sericin oral liquid at a pressure of 400 MPa for 20 min (temperature 24 °C), and fill it into sterile oral liquid bottles (specification 10 mL).

[0052] Example 3

[0053] 1. Raise the natural green cocoon silkworm variety 301: Take out the natural green cocoon silkworm eggs 301 from the cold storage for hibernation awakening. After 10 days, the silkworm eggs hatch. After collecting the newly hatched silkworms, raise the 1st - 3rd instar larvae under the temperature and humidity conditions of 28 °C and RH 80%, and raise the 4th - 5th instar larvae under the temperature and humidity conditions of 27 °C and RH 75%. Harvest the cocoons 8 days after the mature silkworms climb onto the cocooning frame to obtain natural green cocoons.

[0054] 2. Cut open the fresh natural green cocoons, wash the cocoon shells with ultrapure water, and dry them at 60 °C.

[0055] 3. Cut the dried green cocoon shells into pieces within 1 cm 2 in size, add ultrapure water at a material - liquid ratio of 1 g:60 mL and boil continuously for 3 h, and collect the first degumming solution. Then add the green cocoons to ultrapure water at a material - liquid ratio of 1 g:40 mL and boil continuously for 2 h, and collect the second degumming solution. Combine the two degumming solutions and filter them with a bag - type filter (pore size 120 μm, pressure 0.3 MPa) to remove impurities in the degumming solution to obtain the degumming solution after impurity removal.

[0056] 4. Mix papain and flavor protease at a total enzyme amount of 4500 U / g with a mass ratio of 1:2.5 to obtain a composite enzyme; according to the material - liquid ratio of the composite enzyme to the degumming solution after impurity removal of 1 g:14 mL, add the composite enzyme to the degumming solution after impurity removal, and carry out enzymatic hydrolysis at 50 °C and pH 7.5 for 170 min, heat to 98 °C, and keep heating for 13 min to inactivate the enzyme, and vacuum - concentrate to a soluble solid content of 18 w / v% to obtain the green cocoon sericin solution.

[0057] 5. Add the green cocoon sericin solution at a ratio of 0.01 w / v% to a mixture of xylitol and maltitol with a mass ratio of 1:1.5, homogenize, and centrifuge to remove impurities to obtain the green cocoon sericin oral liquid.

[0058] 6. Ultra - high pressure sterilize the green cocoon sericin oral liquid at a pressure of 400 MPa for 20 min (temperature 24 °C), and fill it into sterile oral liquid bottles (specification 10 mL).

[0059] Comparative Example 1

[0060] The difference from Example 1 is that the natural green-cocoon silkworm variety 301 is replaced with the white-cocoon silkworm variety "Liangguang No. 2" (from Guangdong Guangxin Seed Industry Co., Ltd.), and the silkworm eggs hatch after 10 days during the feeding process.

[0061] Comparative Example 2

[0062] The difference from Example 1 is that the natural green-cocoon silkworm variety 301 is replaced with the natural yellow-cocoon silkworm variety "Yuecan Yellow Cocoon No. 1" (from Sericulture and Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences), and the silkworm eggs hatch after 10 days during the feeding process.

[0063] Comparative Example 3

[0064] Steps 1-3 and 5-6 are the same as those in Example 1.

[0065] Step 4: Under the conditions of 55°C and pH 7.5, add papain to the green-cocoon sericin solution at a material-liquid ratio of 1 g:10 mL, enzymatically hydrolyze for 150 min, heat to 95°C, keep for 10 min to inactivate the enzyme, and vacuum concentrate to a soluble solid content of 13%.

[0066] Comparative Example 4

[0067] Steps 1-3 and 5-6 are the same as those in Example 1.

[0068] Step 4: Under the conditions of 50°C and pH 10.0, add alkaline protease to the green-cocoon sericin solution at a material-liquid ratio of 1 g:10 mL, enzymatically hydrolyze for 150 min, heat to 95°C, keep for 10 min to inactivate the enzyme, and vacuum concentrate to a soluble solid content of 13%.

[0069] Comparative Example 5

[0070] Steps 1-3 and 5-6 are the same as those in Example 1.

[0071] Step 4: Under the conditions of 55°C and pH 7.5, add flavor protease to the green-cocoon sericin solution at a material-liquid ratio of 1 g:10 mL, enzymatically hydrolyze for 150 min, heat to 95°C, keep for 10 min to inactivate the enzyme, and vacuum concentrate to a soluble solid content of 13%.

[0072] Comparative Example 6

[0073] Steps 1-3 and 5-6 are the same as those in Example 1.

[0074] Step 4: Under the conditions of 50°C and pH 7.5, add neutral protease to the green-cocoon sericin solution at a material-liquid ratio of 1 g:10 mL, enzymatically hydrolyze for 150 min, heat to 95°C, keep for 10 min to inactivate the enzyme, and vacuum concentrate to a soluble solid content of 13%.

[0075] Comparative Example 7

[0076] Steps 1-3 and 5-6 are the same as those in Example 1.

[0077] The difference in Step 4 is that the mass ratio of papain to flavorzyme is 1:1.

[0078] Comparative Example 8

[0079] Steps 1-3 and 5-6 are the same as those in Example 1.

[0080] The difference in Step 4 is that the mass ratio of papain to flavorzyme is 1:3.

[0081] Comparative Example 9

[0082] Steps 1-3 and 5-6 are the same as those in Example 1.

[0083] The difference in Step 4 is that the mass ratio of papain to flavorzyme is 2:1.

[0084] Comparative Example 10

[0085] Steps 1-3 and 5-6 are the same as those in Example 1.

[0086] The difference in Step 4 is that the mass ratio of papain to flavorzyme is 3:1.

[0087] Comparative Example 11

[0088] Steps 1-3 and 5-6 are the same as those in Example 1.

[0089] The difference in Step 4 is that the total enzyme amount is 500 U / g.

[0090] Comparative Example 12

[0091] Steps 1-3 and 5-6 are the same as those in Example 1.

[0092] The difference in Step 4 is that the total enzyme amount is 1000 U / g.

[0093] Comparative Example 13

[0094] Steps 1-3 and 5-6 are the same as those in Example 1.

[0095] The difference in Step 4 is that the total enzyme amount is 2000 U / g.

[0096] Comparative Example 14

[0097] Steps 1-3 and 5-6 are the same as those in Example 1.

[0098] The difference in Step 4 is that the total enzyme amount is 6000 U / g.

[0099] Comparative Example 15

[0100] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0101] The difference in Step 4 is that the ratio of the complex enzyme to the degummed solution after impurity removal is 1 g:2 mL.

[0102] Comparative Example 16

[0103] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0104] The difference in Step 4 is that the ratio of the complex enzyme to the degummed solution after impurity removal is 1 g:5 mL.

[0105] Comparative Example 17

[0106] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0107] The difference in Step 4 is that the ratio of the complex enzyme to the degummed solution after impurity removal is 1 g:15 mL.

[0108] Comparative Example 18

[0109] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0110] The difference in Step 4 is that the enzymatic hydrolysis is carried out for 90 min.

[0111] Comparative Example 19

[0112] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0113] The difference in Step 4 is that the enzymatic hydrolysis is carried out for 120 min.

[0114] Comparative Example 20

[0115] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0116] The difference in Step 4 is that the enzymatic hydrolysis is carried out for 180 min.

[0117] Comparative Example 21

[0118] Steps 1-3 and Steps 5-6 are the same as those in Example 1.

[0119] The difference in Step 4 is that the enzymatic hydrolysis is carried out for 210 min.

[0120] Experimental Example 1

[0121] An inhibitory effect experiment on the XOD activity was conducted on the green cocoon sericin solution and green cocoon sericin oral liquid prepared in Example 1, or the white cocoon sericin solution and white cocoon sericin oral liquid prepared in Comparative Example 1, or the yellow cocoon sericin solution and yellow cocoon sericin oral liquid prepared in Comparative Example 2. Allopurinol was used as the positive control, and ultrapure water was used as the blank control.

[0122] The experimental steps are as follows:

[0123] (1) Preparation of sample and reagent solutions

[0124] The green cocoon sericin solution and green cocoon sericin oral liquid prepared in Example 1, or the white cocoon sericin solution and white cocoon sericin oral liquid prepared in Comparative Example 1, or the yellow cocoon sericin solution and yellow cocoon sericin oral liquid prepared in Comparative Example 2 were respectively diluted with ultrapure water to 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, and 40 mg / mL.

[0125] Allopurinol solution: Allopurinol was dissolved with 0.4% NaOH solution and then made up to volume with PBS to prepare allopurinol solutions with different concentrations (1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL).

[0126] Hypoxanthine solution: The hypoxanthine standard was accurately weighed, added with PBS and dissolved with ultrasonic assistance to prepare a substrate solution of 0.5 mg / mL.

[0127] Xanthine oxidase (XOD) solution: It was diluted with pre-cooled PBS into an enzyme working solution of 1 U / mL.

[0128] (2) The XOD activity was determined using a xanthine oxidase (XOD) test kit (colorimetric method), which is a product from Nanjing Jiancheng Bioengineering Institute of Test Kits, with the product number A002-1-1. The determination results are as Figure 1 and Figure 2 shown. Both the green cocoon sericin solution and the green cocoon sericin oral liquid showed significant inhibitory effects on XOD activity. The inhibition rate increased with the increase in concentration. The inhibition rate was 53.2% at a concentration of 10 mg / mL, and the inhibition rate of XOD activity reached 100% at a concentration of 20 mg / mL and above. In the concentration range of 1 mg / mL - 40 mg / mL, the green cocoon sericin solution was significantly higher than the white cocoon sericin solution and the yellow cocoon sericin solution, and the green cocoon sericin oral liquid was significantly higher than the white cocoon sericin oral liquid and the yellow cocoon sericin oral liquid.

[0129] Experimental Example 2

[0130] Using the XOD activity detection method described in Experimental Example 1, the inhibitory effect of the green cocoon sericin solution (at a concentration of 10 mg / mL) prepared in Comparative Examples 3 - 21 on XOD activity was detected. The results are as Figures 3 - 7 .

[0131] Figure 3 The results show that the green cocoon sericin solution prepared by the combined enzymatic hydrolysis of papain and flavor protease has a better inhibitory effect on XOD activity.

[0132] Figure 4 The results show that the green cocoon sericin solution prepared when the enzyme addition ratio of papain to flavor protease is 1 g:2 mL has the best inhibitory effect on XOD activity.

[0133] Figures 5 - 7 The results show that the green cocoon sericin solution prepared with an enzyme dosage of 4000 U / g, a material-liquid ratio of 1 g:10 mL, and an enzymatic hydrolysis time of 150 min has the best inhibitory effect on XOD activity.

[0134] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a green cocoon sericin solution, characterized in that it comprises the following steps: mixing cocoons with water, boiling to collect the degumming solution, mixing the degumming solution with an enzyme for enzymatic hydrolysis, inactivating the enzyme, and concentrating; the enzyme is papain and flavorzyme; the cocoons are green cocoons.

2. The preparation method according to claim 1, characterized in that, The green cocoons are produced by the green cocoon silkworm breed 301.

3. The preparation method according to claim 1, characterized in that, The mass-volume ratio of the cocoons to water is 1 g: 20-60 mL.

4. The preparation method according to claim 1, characterized in that, The boiling time is 1-3 h, and the number of boiling times is 1-3 times.

5. The preparation method according to claim 1, characterized in that, The total amount of the enzyme is 3000-5000 U / g, and the mass ratio of papain to flavorzyme is 1: 1.5-2.

5.

6. The preparation method according to claim 1, characterized in that, The mass-volume ratio of the enzyme to the degumming solution is 1 g: 6-14 mL.

7. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis time is 130-170 min, the enzymatic hydrolysis temperature is 50-60 °C, and the pH of the enzymatic hydrolysis is 7.0-8.

0.

8. A green cocoon sericin solution prepared by the preparation method according to any one of claims 1-7.

9. A green cocoon sericin oral liquid, characterized in that, It includes a green cocoon sericin solution prepared by the preparation method according to any one of claims 1-7 and excipients.

10. Use of the green cocoon sericin solution prepared by the preparation method according to any one of claims 1-7 or the green cocoon sericin oral liquid according to claim 9 in the preparation of a product for inhibiting xanthine oxidase activity and / or reducing uric acid.

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