Basa fish swimming bladder polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a Basa fish swimming bladder polypeptide as well as a preparation method and application thereof. The Basa fish swimming bladder polypeptide contains an amino acid sequence as shown in SEQ ID NO: 1. The Basa fish swimming bladder polypeptide is extracted from swimming bladders of Basa fish, and in animal experiments, compared with diabetic mice not taking the Basa fish swimming bladder polypeptide, the diabetic mice taking the Basa fish swimming bladder polypeptide shows good sugar tolerance, and the organism has good regulation ability on glucose. The preparation method provided by the invention comprises the following steps: carrying out enzymolysis on homogenate of Basa fish swim bladder by adopting an alkaline protease and animal protease A3 double-enzymolysis mode, and then carrying out ultrafiltration on enzymatic hydrolysate to obtain the Basa fish swim bladder polypeptide. Compared with other dual-enzymolysis matching, the dual-enzymolysis matching of the alkaline protease and the animal protease A3 is adopted, so that the swim bladder of Basa fish can be better subjected to enzymolysis, and the Basa fish swim bladder polypeptide with a better blood glucose reducing effect is obtained.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a basa fish swim bladder polypeptide, a preparation method and uses thereof. Background Art

[0002] Diabetes is a common clinical metabolic syndrome characterized by continuously elevated blood glucose levels. If not managed promptly and effectively, hyperglycemia will cause various physical dysfunctions and damages, seriously threatening the physical health of patients, and has become a global public health challenge that urgently needs to be solved.

[0003] As a by-product in fish processing, the swim bladder is an aquatic resource with high protein, low fat and high nutritional value. It is regarded as a good product for nourishing the body and supplementing qi and blood due to its rich nutritional components. Chemically, the swim bladder is mainly composed of collagen and some other bioactive substances. The collagen in the swim bladder, due to its unique molecular structure and biological activity, can moisturize the skin, making the skin delicate and smooth, and has the effect of beauty care. In addition to collagen, the swim bladder may also contain some peptide substances with special physiological functions, which may have special effects on the human body and need further exploration and research. Summary of the Invention

[0004] The purpose of the present invention is to provide a basa fish swim bladder polypeptide with hypoglycemic function and a preparation method thereof.

[0005] To achieve the above purpose, in the first aspect of the present invention, a basa fish swim bladder polypeptide is proposed. The basa fish swim bladder polypeptide contains the amino acid sequence shown in SEQ ID NO: 1.

[0006] In the second aspect of the present invention, a preparation method of a basa fish swim bladder polypeptide is proposed, which is used to prepare the basa fish swim bladder polypeptide proposed in the first aspect of the present invention, and includes:

[0007] S1. Pretreat the basa fish swim bladder to obtain a homogenate, add pure water to the obtained homogenate, and heat and stir to obtain a mixed solution;

[0008] S2. Adjust the pH of the mixed solution obtained in step S1 to alkaline, then add alkaline protease for the first enzymatic hydrolysis. After the first enzymatic hydrolysis ends, add animal protease A3 for the second enzymatic hydrolysis under alkaline conditions. After the second enzymatic hydrolysis ends, an enzymatic hydrolysate is obtained;

[0009] S3. Inactivate the enzyme, cool, and centrifuge the enzymatic hydrolysate obtained in step S2 to take the supernatant. Ultrafilter and purify the supernatant to obtain the basa fish swim bladder polypeptide.

[0010] Further, the pretreatment in step S1 is to remove the grease on the basa fish swim bladder, and then wash, boil, and crush the basa fish swim bladder polypeptide after removing the grease.

[0011] Further, the ratio of the homogenate to pure water in step S1 is 1:30 - 1:40 g / mL, and the mixture is stirred at 85 - 95 °C for 1 - 2 h to obtain a mixed solution.

[0012] Further, the dosage of alkaline protease in step S2 is 1.0% - 2.0% of the dry weight of the homogenate. The first enzymatic hydrolysis is carried out at 50 - 60 °C and pH 8 - 9 for 3 - 4 h.

[0013] Further, adding animal protease A3 in step S2 for the second enzymatic hydrolysis under alkaline conditions means adding 0.5% - 1.0% of animal protease A3 based on the dry weight of the homogenate, and carrying out the enzymatic hydrolysis at 40 - 45 °C and pH 7 - 8 for 2 - 3 h.

[0014] Further, the ultrafiltration in step S3 is to filter the enzymatic hydrolysate with a 2 kDa ultrafiltration membrane.

[0015] The Pangasius bocourti swim bladder polypeptide proposed in the first aspect of the present invention or the Pangasius bocourti swim bladder polypeptide obtained by the preparation method proposed in the second aspect of the present invention has the effect of lowering blood sugar.

[0016] The present invention extracts a Pangasius bocourti swim bladder polypeptide from the swim bladder of Pangasius bocourti. In animal experiments, compared with diabetic mice that did not ingest the Pangasius bocourti swim bladder polypeptide, diabetic mice that ingested the Pangasius bocourti swim bladder polypeptide of the present invention showed good glucose tolerance and good regulatory ability of the body to glucose.

[0017] The present invention proposes a method for preparing the Pangasius bocourti swim bladder polypeptide described in the present invention. The homogenate of the Pangasius bocourti swim bladder is enzymatically hydrolyzed by a double-enzymatic hydrolysis method using alkaline protease and animal protease A3, and then the enzymatic hydrolysate is ultrafiltered to obtain the Pangasius bocourti swim bladder polypeptide. Compared with other double-enzymatic hydrolysis combinations, the double-enzymatic hydrolysis combination of alkaline protease and animal protease A3 in the present invention can better enzymatically hydrolyze the swim bladder of Pangasius bocourti to obtain the Pangasius bocourti swim bladder polypeptide with a better blood sugar-lowering effect in the present invention. Description of the Drawings

[0018] Figure 1 It is a graph of the experimental results of the inhibitory degree of each component after ultrafiltration on α-glucosidase in Example 1 of the present invention;

[0019] Figure 2 It is a graph of the experimental results of the inhibitory degree of each component in gel chromatography on α-glucosidase in Example 1 of the present invention;

[0020] Figure 3 It is a graph of the experimental results of the inhibitory degree of each fraction in reverse-phase liquid chromatography on α-glucosidase in Example 1 of the present invention;

[0021] Figure 4 This is the mass spectrometry diagram in Example 1 of the present invention;

[0022] Figure 5 This is the experimental result diagram of double enzymatic hydrolysis combination in Example 2 of the present invention;

[0023] Figure 6 This is the glucose tolerance curve diagram of mice in Example 3 of the present invention;

[0024] Figure 7 This is the area diagram under the glucose tolerance curve in Example 3 of the present invention. Detailed implementation manners

[0025] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions from beginning to end. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention. For those not specified in the embodiments in terms of specific technologies or conditions, the technologies or conditions described in the literature in the art or according to the product specifications are followed. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. In the following embodiments, unless otherwise clearly stated, "%" all refers to weight percentage.

[0026] 1. In the present invention, the direct drying method is used to measure the moisture content of the homogenate. Except for the direct drying method, other methods for measuring the moisture content can be applied to the measurement of the moisture content of the homogenate in the present invention.

[0027] 2. In the following text, MW is the abbreviation of Molecular Weight, which means molecular weight.

[0028] 3. In the present invention, the components after ultrafiltration are purified by gel chromatography and reverse-phase liquid chromatography respectively. The purification method of the present invention is not limited to gel chromatography and reverse-phase liquid chromatography. Other purification methods that can further subdivide the components with a molecular weight of 1 kDa and below can be applied to the present invention.

[0029] 4. In the present invention, the normal feed for feeding the control group of mice is the experimental mouse and rat compound granular feed purchased from Beijing Huafukang Biotechnology Co., Ltd., and the high-fat feed is the irradiated and sterilized experimental animal customized purified feed purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0030] The preparation method of the Pangasius catfish swim bladder polypeptide in the present invention is as follows:

[0031] S1. Pretreat the Pangasius catfish swim bladder to obtain a homogenate, add pure water to the obtained homogenate, and heat and stir to obtain a mixed solution;

[0032] S2. Adjust the pH of the mixture obtained in step S1 to alkaline, then add alkaline protease for the first enzymatic hydrolysis. After the first enzymatic hydrolysis is completed, add animal protease A3 and carry out the second enzymatic hydrolysis under alkaline conditions. After the second enzymatic hydrolysis is completed, an enzymatic hydrolysate is obtained.

[0033] S3. Inactivate the enzyme, cool, and centrifuge the enzymatic hydrolysate obtained in step S2 to obtain the supernatant. Ultrafilter and purify the supernatant to obtain the Pangasius catfish swim bladder polypeptide.

[0034] Example 1: Preparation of Pangasius catfish swim bladder polypeptide

[0035] After thawing the Pangasius catfish swim bladder, remove the fat with surgical scissors, wash, drain, boil it in boiling water, and wait for it to cool. Then break the Pangasius catfish swim bladder to obtain a homogenate, and measure the moisture content of the homogenate by the direct drying method. Weigh 30 g of the homogenate, add pure water according to the solid-liquid ratio of 1:35 g / mL, stir and boil at 90 °C for 1 h to obtain a mixture. Immediately adjust the temperature of the mixture to 55 °C and the pH to 8, add alkaline protease according to 1% of the dry weight of the homogenate, and carry out enzymatic hydrolysis for 3 h. During the enzymatic hydrolysis process, adjust the pH with 1 M sodium hydroxide to maintain the pH at 8. After the enzymatic hydrolysis with alkaline protease is completed, adjust the pH to 7 and the temperature to 40 °C, add animal protease A3 according to 0.5% of the dry weight of the swim bladder, and carry out enzymatic hydrolysis for 2 h. During the enzymatic hydrolysis process, adjust the pH with 1 M sodium hydroxide to maintain the pH at 7. After the enzymatic hydrolysis is completed, an enzymatic hydrolysate is obtained. Raise the temperature of the enzymatic hydrolysate to 90 °C to inactivate the enzyme for 10 min. After the enzyme inactivation is completed, wait for it to cool, place the enzymatic hydrolysate in a centrifuge tube, centrifuge at 9000 rpm and 25 °C for 10 min, and collect the supernatant.

[0036] Perform fractional separation on the supernatant obtained above. Use ultrafiltration membranes with molecular weights of 1 kDa, 2 kDa, and 5 kDa to perform fractional separation on the obtained supernatant, and divide the obtained components into three groups: P1 (MW < 1 kDa), P2 (1 kDa < MW < 2 kDa), and P3 (2 kDa < MW < 5 kDa), and measure the inhibitory degree of each component on the activity of α-glucosidase. The results are as Figure 1 shown. It can be seen from Figure 1 that P1 has the highest inhibitory degree on the activity of α-glucosidase, and further purify P1 by gel chromatography.

[0037] Prepare a solution of P1 with a concentration of 10 mg / mL, separate it by Sephadex G-25 column chromatography, elute with ultrapure water at a flow rate of 1 mL / min, collect each chromatographic peak in the chromatogram at 220 nm, and divide the P1 component into five components: P1-1, P1-2, P1-3, P1-4, and P1-5 according to different molecular weights, and test the inhibitory degree of the above five components on the activity of α-glucosidase. The results are as Figure 2As shown in the figure. Among them, P1-2 has the highest inhibitory effect on α-glucosidase activity, and the P1-2 component is further purified.

[0038] The P1-2 component was further purified using a reversed-phase liquid chromatography column. After freeze-drying P1-2, it was formulated into a solution with a mass concentration of 20 mg / mL and filtered through a 0.45 μm membrane. The chromatographic column was CST C18, the injection volume was 100 μL, and the flow rate was set at 1 mL / min. The elution conditions were that mobile phase A was ultrapure water + 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile. After collecting the samples of different elution peak fractions, rotary evaporation and concentration were carried out, and then freeze-drying was performed to obtain five groups of fractions, namely P1-2-1, P1-2-2, P1-2-3, P1-2-4, and P1-2-5, and the inhibitory activity of each fraction against α-glucosidase was measured. The experimental results are as Figure 3 shown. Among them, the P-1-2-3 fraction has the highest inhibitory effect on α-glucosidase activity. The P-1-2-3 fraction was subjected to mass spectrometry analysis and identification. The mass spectrometry results are as Figure 4 shown. The P-1-2-3 fraction is the Pangasius hypophthalmus swim bladder polypeptide proposed in the present invention, and the amino acid sequence of the obtained Pangasius hypophthalmus swim bladder polypeptide is as shown in SEQ ID NO: 1.

[0039] The Pangasius hypophthalmus swim bladder polypeptide contains the amino acid sequence shown in SEQ ID NO: 1: PVGPPGPR; SEQ ID NO: 1.

[0040] Example 2: Verification of the effect of double enzymolysis

[0041] (1) Group 1: Double enzymolysis using alkaline protease and animal protease A3

[0042] After thawing the Pangasius hypophthalmus swim bladder, the fat was removed with surgical scissors, washed, drained, boiled in boiling water, and after cooling, the Pangasius hypophthalmus swim bladder was broken to obtain a homogenate. The moisture content of the homogenate was measured by the direct drying method. Weigh 30 g of the homogenate, add pure water according to a solid-to-liquid ratio of 1:35 g / mL, and stir and boil at 90 °C for 1 h to obtain a mixed solution. Immediately, the temperature of the mixed solution was adjusted to 55 °C and the pH was adjusted to 8. According to 1% of the dry weight of the homogenate, alkaline protease was added and enzymolyzed for 3 h. During the enzymolysis process, 1 M sodium hydroxide was used to adjust the pH to maintain it at 8. After the alkaline protease enzymolysis was completed, the pH was adjusted to 7 and the temperature was adjusted to 40 °C. According to 0.5% of the dry weight of the swim bladder, animal protease A3 was added and enzymolyzed for 2 h. During the enzymolysis process, 1 M sodium hydroxide was used to adjust the pH to maintain it at 7. After the enzymolysis was completed, an enzymolysis solution was obtained. The temperature of the enzymolysis solution was raised to 90 °C to inactivate the enzyme for 10 min. After the enzyme inactivation was completed and cooled, the enzymolysis solution was placed in a centrifuge tube and centrifuged at 9000 rpm and 25 °C for 10 min to collect the supernatant A.

[0043] (2) Group 2: Double enzymolysis with alkaline protease and neutral protease

[0044] After thawing the Pangasius catfish swim bladder, remove the fat with surgical scissors, wash, drain, boil in boiling water, wait for cooling, break the Pangasius catfish swim bladder to obtain homogenate, and measure the moisture content of the homogenate by direct drying method. Weigh 30 g of the homogenate, add pure water according to the solid-liquid ratio of 1:35 g / mL, stir and boil at 90 °C for 1 h to obtain a mixed solution. Immediately adjust the temperature of the mixed solution to 55 °C and the pH to 8, add alkaline protease according to 1% of the dry weight of the homogenate, enzymolyze for 3 h, and adjust the pH with 1 M sodium hydroxide during the enzymolysis process to keep the pH at 8. After the enzymolysis with alkaline protease is completed, adjust the pH to 7 and the temperature to 45 °C, add neutral protease according to 0.5% of the dry weight of the swim bladder, enzymolyze for 2 h, adjust the pH with 1 M sodium hydroxide during the enzymolysis process to keep the pH at 7, and obtain an enzymolysis solution after the enzymolysis is completed. Raise the temperature of the enzymolysis solution to 90 °C to inactivate the enzyme for 10 min. After the enzyme inactivation is completed and cooled, place the enzymolysis solution in a centrifuge tube, centrifuge at 9000 rpm and 25 °C for 10 min, and collect the supernatant B.

[0045] (3) Group 3: Double enzymolysis with alkaline protease and trypsin

[0046] After thawing the Pangasius catfish swim bladder, remove the fat with surgical scissors, wash, drain, boil in boiling water, wait for cooling, break the Pangasius catfish swim bladder to obtain homogenate, and measure the moisture content of the homogenate by direct drying method. Weigh 30 g of the homogenate, add pure water according to the solid-liquid ratio of 1:35 g / mL, stir and boil at 90 °C for 1 h to obtain a mixed solution. Immediately adjust the temperature of the mixed solution to 55 °C and the pH to 8, add alkaline protease according to 1% of the dry weight of the swim bladder, enzymolyze for 3 h, and adjust the pH with 1 M sodium hydroxide during the enzymolysis process to keep the pH at 8. After the enzymolysis with alkaline protease is completed, adjust the pH to 7 and the temperature to 45 °C, add trypsin according to 0.5% of the dry weight of the swim bladder, enzymolyze for 2 h, adjust the pH with 1 M sodium hydroxide during the enzymolysis process to keep the pH at 7, and obtain an enzymolysis solution after the enzymolysis is completed. Raise the temperature of the enzymolysis solution to 90 °C to inactivate the enzyme for 10 min. After the enzyme inactivation is completed and cooled, place the enzymolysis solution in a centrifuge tube, centrifuge at 9000 rpm and 25 °C for 10 min, and collect the supernatant C.

[0047] Add α-glucosidase to the supernatants collected from the above three groups of double-enzymolysis combinations, and observe the inhibition of the supernatants obtained from different double-enzymolysis combinations on the activity of α-glucosidase. The experimental steps are as follows:

[0048] Control tube: Add 50 μL of α-glucosidase solution and 25 μL of PBS solution to a centrifuge tube;

[0049] Blank tube: Add 75 μL of PBS solution to a centrifuge tube;

[0050] Sample tubes: Add 50 μL of α-glucosidase solution and 25 μL of sample solution (the sample solutions are supernatant A, supernatant B, supernatant C, and the chemical inhibitor Acarbose, which are respectively prepared as sample tube 1, sample tube 2, sample tube 3, and sample tube 4) into centrifuge tubes;

[0051] Sample control tubes: Add 50 μL of PBS solution and 25 μL of sample solution (the sample solutions are supernatant A, supernatant B, supernatant C, and the chemical inhibitor Acarbose, which are respectively prepared as sample control tube 1, sample control tube 2, sample control tube 3, and sample control tube 4) into centrifuge tubes.

[0052] Incubate the above tubes in a water bath at 37 °C for 10 minutes. Subsequently, add 25 μL of PNPG to each tube and react for another 15 minutes at 37 °C. Finally, add 100 μL of Na2CO3 solution to terminate the reaction. Measure the absorbance of the reaction mixture at a wavelength of 405 nm using a microplate reader.

[0053] The calculation formula for inhibitory activity is as follows: α-glucosidase (%) = 1 - (ODc - ODd) / (ODa - ODb) × 100

[0054] ODa: Absorbance of the control tube; ODb: Absorbance of the blank tube; ODc: Absorbance of the sample tube (absorbances of sample tube 1, sample tube 2, and sample tube 3 respectively); ODd: Absorbance of the sample control tube. (Absorbances of sample control tube 1, sample control tube 2, and sample control tube 3 respectively)

[0055] The results are as Figure 5 shown. The chemical inhibitor Acarbose of α-glucosidase is used as a positive control to evaluate the inhibitory effects of Group 1, Group 2, and Group 3 on α-glucosidase. The inhibition rates are as Figure 1 shown. The inhibition rates of Acarbose, Group 1, Group 2, and Group 3 are Acarbose: 57.67 ± 0.25%, Group 1: 39.85 ± 0.16%, Group 2: 3.33 ± 0.23%, Group 3: 12.61 ± 0.28% respectively. It can be seen from Figure 1 this that the inhibitory rate of the Pangasius hypophthalmus swim bladder polypeptide obtained by enzymatic hydrolysis with alkaline protease and animal protease A3 on α-glucosidase is much higher than the double-enzymatic hydrolysis effects of other groups.

[0056] Example 3: Verification of the hypoglycemic effect of Pangasius hypophthalmus swim bladder polypeptide in vivo

[0057] Fifty 6-8-week-old male C57 mice were randomly divided into five groups of 10 mice each, namely the control group (NC), the model group (Model), the P-1-2-3 (L) group, the P-1-2-3 (M) group, and the P-1-2-3 (H) group. The control group was fed a normal diet for three months, while the model group (Mode1), the P-1-2-3 (L) group, the P-1-2-3 (M) group, and the P-1-2-3 (H) group were fed a high-fat diet for three months to induce a T2DM model and obtain diabetic mice. The administration of the above five groups of mice was as follows:

[0058] (1) Control group (NC): Each mouse was intragastrically administered 10 mL / kg.bw of ultrapure water daily;

[0059] (2) Model group (Model): Each mouse was intragastrically administered 10 mL / kg.bw of ultrapure water daily;

[0060] (3) P-1-2-3 (L) group: The sample of SEQ ID NO: 1 was formulated into a final concentration of 10 mg / mL, and each mouse was intragastrically administered 20 mg / kg.bw daily;

[0061] (4) P-1-2-3 (M) group: The sample of SEQ ID NO: 1 was formulated into a final concentration of 10 mg / mL, and each mouse was intragastrically administered 30 mg / kg.bw daily;

[0062] (5) P-1-2-3 (H) group: The sample of SEQ ID NO: 1 was formulated into a final concentration of 10 mg / mL, and each mouse was intragastrically administered 50 mg / kg.bw daily.

[0063] Gavage was continuously performed for 4 weeks. Every day at 9 am, the status of the mice was observed on time, water and feed were provided, and then gavage was carried out. The body weight of the mice was recorded every 3 days.

[0064] After the last weighing of the body weight, the mice were fasted for 4 h, and then an oral glucose tolerance test was performed on the mice. The mice's tails were cut to collect blood, and a blood glucose meter was used to detect the fasting blood glucose value, which was recorded as the blood glucose value at 0 min. Subsequently, a 20% glucose solution was intragastrically administered, and then the fasting blood glucose values were detected and recorded at 30 and 120 min respectively. The experimental results are as Figure 6 and Figure 7 shown.

[0065] The oral glucose tolerance test is a key evaluation index for diabetes, mainly detecting the glucose tolerance degree of diabetic mice and the ability to handle elevated blood glucose through an oral glucose tolerance test. As Figure 6 and Figure 7It can be seen that after intragastric administration of glucose, the blood glucose level of the model group was significantly higher than that of the control group and the PVGPPGPR group (i.e., mice in groups P-1-2-3(L), P-1-2-3(M), and P-1-2-3(H)). There may be a certain time delay in the hypoglycemic effect of basa swim bladder polypeptide (PVGPPGPR). Although the blood glucose level in group P-1-2-3(H) increased rapidly in the early stage, judging from the overall trend of the line graph, after 30 minutes, the blood glucose level of mice in group P-1-2-3(H) decreased significantly faster than that of other groups and finally could be maintained at a lower blood glucose level. Therefore, it can improve the blood glucose regulation ability of mice.

[0066] Compared with the model group, PVGPPGPR significantly reduced the total incremental area (AUC) under the glucose tolerance curve. The total incremental area (AUC) under the glucose tolerance curve reflects the overall exposure level of blood glucose in mice during the entire experiment. In the glucose tolerance test, the larger the area under the glucose tolerance curve, the greater the degree and duration of blood glucose elevation in mice during the test, indicating that there may be certain defects in their blood glucose regulation mechanism.

[0067] The above results indicate that PVGPPGPR can significantly improve the glucose tolerance of diabetic mice and increase the body's ability to regulate glucose.

[0068] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. A basa fish swim bladder polypeptide, characterized in that, The basa swim bladder polypeptide contains the amino acid sequence shown in SEQ ID NO:

1.

2. A preparation method of basa fish swim bladder polypeptide, characterized in that, The method for preparing the basa swim bladder polypeptide described in claim 1 includes: S1. Pretreat the basa swim bladder to obtain a homogenate, add pure water to the obtained homogenate, and heat and stir to obtain a mixture; S2. Adjust the pH of the mixture obtained in step S1 to alkaline, then add alkaline protease for the first enzymatic hydrolysis. After the first enzymatic hydrolysis ends, add animal protease A3 and carry out the second enzymatic hydrolysis under alkaline conditions. After the second enzymatic hydrolysis ends, obtain an enzymatic hydrolysate; S3. Inactivate the enzyme, cool, and centrifuge the enzymatic hydrolysate obtained in step S2 to take the supernatant. Ultrafilter and purify the supernatant to obtain the basa swim bladder polypeptide.

3. The preparation method of the basa fish swim bladder polypeptide according to claim 2, characterized in that, The pretreatment described in step S1 is to remove the grease on the basa swim bladder, and then wash, boil, and crush the basa swim bladder polypeptide after removing the grease.

4. The preparation method of basa swim bladder polypeptide according to claim 2, characterized in that, The material-liquid ratio of the homogenate to the pure water in step S1 is 1:30 - 1:40 g / mL, and the mixture is stirred at 85 - 95 °C for 1 - 2 h to obtain the mixture.

5. The preparation method of basa fish swim bladder polypeptide according to claim 2, wherein The dosage of the alkaline protease in step S2 is 1.0% - 2.0% of the dry weight of the homogenate. The first enzymatic hydrolysis is carried out at 50 - 60 °C and pH 8 - 9 for 3 - 4 h.

6. The preparation method of basa swim bladder polypeptide according to claim 2, wherein The addition of animal protease A3 for the second enzymatic hydrolysis under alkaline conditions in step S2 is to add 0.5% - 1.0% of the dry weight of the homogenate of animal protease A3, and carry out the enzymatic hydrolysis at 40 - 45 °C and pH 7 - 8 for 2 - 3 h.

7. The preparation method of basa swim bladder polypeptide according to claim 2, characterized in that, The ultrafiltration in step S3 is to filter the enzymatic hydrolysate with a 2 kDa ultrafiltration membrane.

8. The basa swim bladder polypeptide described in claim 1 or the basa swim bladder polypeptide obtained by the preparation method described in any one of claims 2 - 7 has the effect of lowering blood sugar.

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