Collagen peptide with blood sugar reducing effect as well as preparation method and application thereof
By heat-treating, enzymatically hydrolyzing and purifying fish skin, collagen peptides rich in polypeptides are prepared, which solves the problem of side effects of existing drugs, achieves efficient DPP-IV inhibition and hypoglycemic effects, and is suitable for food and health products.
Patent Information
- Application Number
- CN202510672862.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-23
- Publication Date
- 2025-09-09
AI Technical Summary
Existing hypoglycemic drugs have side effects and lack natural, safe, and non-toxic food-borne DPP-IV inhibitory active substances.
The preparation method comprises the following steps: heat-treating fish skin at 95-100 DEG C for 1-2 hours, adjusting the pH to 7-8, adding protease C and neutral protease for enzymatic hydrolysis, cooling the enzymatic hydrolysis solution, centrifuging, purifying, and freeze-drying to obtain polypeptide collagen peptides rich in 4-9 amino acid residues.
The prepared collagen peptide achieved a DPP-IV inhibition rate of 69% at a final concentration of 1 mg/mL, significantly improving the fasting blood glucose and oral glucose tolerance of type 2 diabetic mice and protecting the liver. The process is simple and meets food-grade requirements.
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Figure CN120607610A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a collagen peptide with blood sugar lowering effect, a preparation method and application thereof. Background Art
[0002] In recent years, the number of people with diabetes has been increasing, and the age of onset is gradually getting younger. Over 90% of these patients have type 2 diabetes. Inhibiting dipeptidyl peptidase-IV (DPP-IV) is currently an effective treatment for type 2 diabetes. Currently, various therapeutic agents have been developed targeting different targets, primarily sulfonylureas, glinides, biguanides, thiazolidinediones, α-glucosidase inhibitors, dipeptidyl peptidase inhibitors, and sodium-glucose cotransporter-2 (SGLT-2) inhibitors. Injectable formulations include insulin and insulin analogs, and glucagon-like peptide-1 (GLP-1) receptor agonists. However, these drugs may exhibit gastrointestinal side effects, such as nausea, vomiting, indigestion, hypoglycemia, headache, and diarrhea. Therefore, developing a natural, green, safe, and non-toxic food-borne functional substance with DPP-IV inhibitory activity would be beneficial to humans.
[0003] Food-derived DPP-IV inhibitory peptides, known for their safety and lack of side effects, are widely being developed for use in blood sugar-lowering products. Existing literature reports that the structural sequences of highly active DPP-IV inhibitory peptides are primarily XP-type peptides. Therefore, protein sources high in protease inhibitors are the primary raw materials for DPP-IV production, with collagen being one of the most important raw materials for this purpose. Summary of the Invention
[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a collagen peptide with blood sugar lowering effect.
[0005] Another object of the present invention is to provide a method for preparing the aforementioned collagen peptide having blood sugar lowering effect.
[0006] Another object of the present invention is to provide a use of the above-mentioned collagen peptide having blood sugar lowering effect.
[0007] The purpose of the present invention is achieved through the following technical solutions:
[0008] A method for preparing collagen peptide with blood sugar lowering effect comprises the following steps:
[0009] (1) adding fish skin to water and heat-treating to obtain a fish skin solution;
[0010] (2) regulating the pH of the fish-skin solution, adding enzyme, carrying out enzymolysis, and after enzymolysis finishes, deactivating the enzyme to obtain an enzymolysis solution;
[0011] (3) The enzymatic hydrolysate is cooled and centrifuged, and the supernatant is collected, purified, filtered, and freeze-dried to obtain collagen peptides with hypoglycemic effect.
[0012] The heat treatment in step (1) is to soak in hot water at 95-100° C. for 1-2 hours.
[0013] The ratio of the fish skin to water in step (1) is 1 g: 1-2 mL.
[0014] The pH adjustment in step (2) is to adjust the pH to 7-8; preferably, the pH is adjusted to 7.5.
[0015] The enzyme in step (2) is at least one of Protease C, neutral protease, aminopeptidase and papain; preferably Protease C and neutral protease; more preferably an enzyme obtained by compounding Protease C and neutral protease in a mass ratio of 1:1 to 5.
[0016] The added amount of the enzyme described in step (2) is 1 to 2% of the mass of the fish skin in the raw material.
[0017] The enzymatic hydrolysis conditions in step (2) are 45-55° C. for 1-12 hours.
[0018] The centrifugation condition in step (3) is 7000-9000 rpm at 4° C. for 5-15 min.
[0019] The purification in step (3) is to add 10-20% of the total mass of activated carbon, stir and heat at 60-70°C for 40-60 minutes.
[0020] A collagen peptide with blood sugar lowering effect is prepared by the above preparation method.
[0021] The application of the above-mentioned collagen peptide with blood sugar lowering effect in the preparation of diabetes treatment drugs.
[0022] Application of the above-mentioned collagen peptide with hypoglycemic effect in the preparation of food or health care products with hypoglycemic effect.
[0023] The present invention has the following advantages and effects compared to the prior art:
[0024] (1) The preparation method provided by the present invention produces an enzymatic hydrolysate with strong DPP-IV inhibitory activity, and its DPP-IV inhibition rate reaches 69% (IC 50 value is 500μg / mL); the enzymatic hydrolysate is rich in polypeptides composed of 4 to 9 amino acid residues, accounting for as much as 45%; animal experiments have shown that the enzymatic hydrolysate can regulate the fasting blood sugar of type 2 diabetic mice, improve their oral glucose tolerance and protect the liver.
[0025] (2) The preparation method provided by the present invention has a simple process and realizes the enzymatic controlled release of the target polypeptide. The entire process can meet food grade requirements and can be applied to health products and foods related to lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 1-6 and comparative examples 1-6.
[0027] Figure 2 This is a bar graph showing the effect of the fish skin enzymatic hydrolysate obtained in Example 2 on the fasting blood glucose levels of type 2 diabetic mice.
[0028] Figure 3 This is the blood glucose-time curve obtained by oral glucose tolerance test after type 2 diabetic mice were gavaged with the fish skin hydrolysate obtained in Example 2 for 8 weeks.
[0029] Figure 4 This is a bar graph showing the effect of the fish skin enzymatic hydrolysate obtained in Example 2 on the oral glucose tolerance of type 2 diabetic mice.
[0030] Figure 5 This is the liver protection effect of the fish skin hydrolysate obtained in Example 2. DETAILED DESCRIPTION
[0031] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0032] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.
[0033] The enzymes used in the following examples include Protease C (Huapeptide Biotechnology Co., Ltd.), neutral protease (Novozymes), alkaline protease (Novozymes, trade name Alcalase 2.4L), papain (Pombo), and aminopeptidase (Tano, Japan).
[0034] Example 1 Preparation method of fish skin enzymatic hydrolysate
[0035] (1) adding fish skin to hot water at 95°C (1 g: 1.2 mL, w / v) and heat treating for 1.5 h to obtain a heat-treated fish skin solution;
[0036] (2) cooling the heat-treated fish skin solution to 40° C., adjusting the solution pH to 7.5, adding Protease C and neutral protease thereto, the added amount being 1% of the mass of the fish skin raw material, the combined mass ratio being 1:1, the enzymatic hydrolysis temperature being 50° C., and after enzymatic hydrolysis for 12 h, deactivating the enzyme in boiling water at 100° C. for 15 min to obtain an enzymatic hydrolyzate;
[0037] (3) Cool to room temperature, then centrifuge at 4°C, 8000 rpm for 10 min and collect the supernatant.
[0038] (4) 12% activated carbon was added to the supernatant, stirred and heated for 45 min (temperature was 65° C.), centrifuged and filtered to obtain a filtered enzymatic hydrolyzate.
[0039] (5) The filtered enzymatic hydrolysate is freeze-dried to obtain collagen peptides with hypoglycemic effect.
[0040] Example 2
[0041] Collagen peptide was prepared according to the method of Example 1, except that:
[0042] A protease combination with a total enzyme-substrate ratio of 0.2% is added to the fish skin solution in step (2), and then an enzymatic hydrolysis reaction is performed, with the mass ratio of Protease C to neutral protease being 1:5.
[0043] Example 3.
[0044] Collagen peptide was prepared according to the method of Example 1, except that:
[0045] A protease combination with a total enzyme-substrate ratio of 2% is added to the fish skin solution in step (2), and then an enzymatic hydrolysis reaction is performed, with the mass ratio of Protease C to neutral protease being 5:1.
[0046] Example 4.
[0047] Collagen peptide was prepared by referring to the method of Example 1, with the only difference from Example 1 being that the protease combination in step (2) was Protease C and alkaline protease.
[0048] Example 5.
[0049] Collagen peptide was prepared by referring to the method of Example 1, the only difference from Example 1 being that the protease combination in step (2) was Protease C and papain.
[0050] Example 6
[0051] Collagen peptides were prepared by referring to the method of Example 1, with the only difference from Example 1 being that the protease combination in step (2) was Protease C and aminopeptidase.
[0052] Comparative Example 1
[0053] Collagen peptides were prepared according to the method of Example 1, with the only difference from Example 1 being that the protease combination used in step (2) was papain and aminopeptidase.
[0054] Comparative Example 2
[0055] Collagen peptides were prepared by referring to the method of Example 1, with the only difference from Example 1 being that the protease combination used in step (2) was aminopeptidase and alkaline protease.
[0056] Comparative Example 3
[0057] Collagen peptides were prepared by referring to the method of Example 1, with the only difference from Example 1 being that the protease combination used in step (2) was alkaline protease and neutral protease.
[0058] Comparative Example 4
[0059] Collagen peptides were prepared according to the method of Example 1, with the only difference from Example 1 being that the protease combination used in step (2) was papain and neutral protease.
[0060] Comparative Example 5
[0061] Collagen peptides were prepared by referring to the method of Example 1, with the only difference from Example 1 being that a protease combination of aminopeptidase and neutral protease was used in step (2).
[0062] Comparative Example 6
[0063] Collagen peptides were prepared by referring to the method of Example 1, with the only difference from Example 1 being that a protease combination of neutral protease and alkaline protease was used in step (3).
[0064] Example 7 Activity Verification Experiment of Enzyme Hydrolysate
[0065] 7.1 Determination of DPP-IV Inhibition Rate
[0066] DPP-IV inhibition rate determination: Prepare a 2.5 mg / mL sample solution of fish skin hydrolysate powder in Tris-HCl buffer (pH 8.0). Add 80 μL of sample solution and 80 μL of 0.5 mM substrate (Gly-Pro-pNA) to a 96-well microtiter plate, mix, and incubate at 37°C for 10 minutes. Then, add 40 μL of 12.5 mU / mL DPP-IV reaction solution, mix thoroughly, and incubate at 37°C for 120 minutes. Measure absorbance at 405 nm every 2 minutes. Calculate the DPP-IV inhibition rate of the test sample according to the following formula.
[0067] Calculation of DPP-IV inhibition rate: Select two time points, T1 and T2, whose absorbance values change within the linear range, and calculate the slope △A / min.
[0068] Slope = (A2 - A1) / (T2 - T1);
[0069] DPP-IV inhibition rate (%) = (Slope control group - Slope sample group) * 100 / Slope control group.
[0070] The results are as follows Figure 1 As shown in Figure A. At a final concentration of 1 mg / mL, the DPP-IV inhibition rates of these fish skin hydrolysates ranged from 29% to 69%. The DPP-IV inhibitory activity of the fish skin hydrolysates obtained by different enzymatic hydrolysis techniques varied somewhat. The examples exhibited higher DPP-IV inhibition rates than the comparative examples.
[0071] 7.2 Peptide Identification and Analysis
[0072] Liquid chromatography-tandem mass spectrometry was used to study the peptidomics of the fish skin hydrolysate prepared above. The specific procedures were as follows:
[0073] (1) The lyophilized enzymatic hydrolysate was diluted with 0.1% formic acid water to a final concentration of 0.5 mg / mL (protein mass). The solution was centrifuged at 4°C, 10,000×g for 10 min, and the supernatant was collected and filtered using a 0.22 μm filter membrane. The peptide sequence was determined using an ultra-high performance liquid chromatography (UPLC) tandem quadrupole time-of-flight (Q-TOF) mass spectrometer. The mass spectrometer used an electrospray ionization (EIC) ion source and sampled in positive ion mode. The mass spectrometry acquisition range was 50-1500 m / z, and the acquisition rate was 5 Hz. The nebulizer gas pressure was 1.5 bar, the nitrogen drying gas flow rate was 8 L / min, and the nitrogen drying gas temperature was 200°C. A Waters ACQUITYUPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) was used for analysis. The injection volume was 2 μL, and the elution rate was 0.20 mL / min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 100% acetonitrile. The gradient conditions were as follows: 0-2 min, 100% A; 2-12 min, 100%-70% A; 12-14 min, 70%-10% A; 14-16 min, 10% A; 16-16.5 min, 10%-100% A; 16.5-20 min, 100% A.
[0074] (2) The obtained MS / MS spectra were analyzed using Data Analysis 4.4 (Bruker Daltonics), and the peptide content was relatively quantified by the extracted ion chromatographic peak area.
[0075] The experimental results are as follows Figure 1 As shown in B, the examples have higher relative peak areas of Gly-Pro-type peptides than the comparative examples, indicating that examples 1 to 6 contain more Gly-Pro-type peptides than comparative examples 1 to 6.
[0076] 7.3 Experimental setup for in vivo hypoglycemic efficacy evaluation
[0077] The hypoglycemic effect of the fish skin hydrolysate prepared in the above example was studied in vivo using a high-fat diet and STZ-induced type 2 diabetes mouse model. The specific operation was as follows:
[0078] (1) Experimental animals
[0079] Male C57 / 6J mice, 5 weeks old, weighing 18 ± 0.2 g, were purchased from Guangdong Medical Laboratory Animal Center.
[0080] (2) Animal breeding environment
[0081] The mice were raised at 25°C and 55% humidity with a 12-h light / dark cycle and reverse osmosis UV sterilized drinking water.
[0082] (3) Animal experimental modeling
[0083] After two weeks of adaptive feeding, the mice were divided into a normal control group and a diabetic group based on their body weight and fed a standard diet or a high-fat diet, respectively. Six weeks later, all mice were fasted for 16 hours. Mice in the diabetic group were intraperitoneally injected with 50 mg / kg of streptozotocin (STZ, dissolved in pH 4.5 citric acid solution), while mice in the normal group were injected with an equal volume of citric acid solution. Following the injection, they immediately resumed feeding and continued this process for five consecutive days. Seven days after the final injection, fasting blood glucose was monitored for three consecutive days. A blood glucose level >11.1 mM on any day was considered a successful model.
[0084] (4) In vivo hypoglycemic efficacy evaluation experiment
[0085] According to blood glucose and body weight, the mice were randomly divided into 5 groups (12 mice in each group): normal group, model group, metformin positive group (250) mg / kg, low-dose group of fish skin hydrolysate of Example 2 (300 mg / kg), and high-dose group of fish skin hydrolysate of Example 2 (600 mg / kg).
[0086] After the start of the experiment, the mice in the fish skin hydrolysate / metformin positive drug group took the corresponding dose of fish skin hydrolysate / metformin orally every day for 8 consecutive weeks; the mice in the model group and the normal group took normal saline orally for 8 consecutive weeks.
[0087] When the experiment started 2 weeks, 4 weeks, 6 weeks and 8 weeks later, after fasting for 12 hours, tail vein blood was collected, and the fasting blood glucose concentration was measured by glucometer and blood glucose test paper according to the manufacturer (Omron). In the 8th week after modeling, after fasting for 12 hours, gavage glucose solution (1.5g / kg) was administered, and the blood glucose level at 0, 30, 60, and 120min after oral glucose measurement was used to evaluate the glucose tolerance of mice. After the 8th week, mice were dissected, and blood plasma was collected, and the content of aspartate aminotransferase and alanine aminotransferase in the blood plasma was measured, and the specific assay method was with reference to the test kit instructions.
[0088] 7.4 In vivo hypoglycemic efficacy evaluation experimental results
[0089] The experimental results are as follows Figures 2 to 5 As shown by Figure 2 It can be seen that after modeling, the blood glucose level of the model group exceeded 20mmol / L, and the blood glucose level of the sample group was still higher than 20mmol / L after 2 weeks of administration, indicating that short-term intervention cannot improve the fasting blood glucose of mice. After 4 weeks of administration, the fasting blood glucose of the mice in the administration group decreased, and the fasting blood glucose of diabetic mice was significantly reduced at 6 weeks (p < 0.05). Compared with the model mice, the fasting blood glucose levels of the fish skin enzymatic hydrolysate intervention group in Example 2 decreased by 49.29% and 34.25%, respectively. After 8 weeks of gavage, the blood glucose levels of the fish skin enzymatic hydrolysate intervention group in Example 2 were still within a safe range, indicating that the collagen-derived Gly-Pro-type DPP-IV inhibitory peptide prepared in Example 2 has a hypoglycemic effect in vivo.
[0090] Depend on Figure 3 and Figure 4 It can be seen that fish skin enzymatic hydrolysate and metformin can improve oral glucose tolerance in type 2 diabetic mice. After oral administration of glucose, the blood glucose of each group of mice rose rapidly and reached a peak at 30 minutes, and then began to gradually decline. During this process, the blood glucose value of the model group was always higher than that of the other groups, indicating that the ability of diabetic mice to regulate blood glucose was weaker than that of other groups. Compared with the model group, the fish skin enzymatic hydrolysate intervention group in Example 2 was able to quickly slow down the rate of increase of blood glucose within 30 minutes, and significantly reduced AUC 0-2h , indicating that 8 weeks of intervention can effectively improve the fasting oral glucose tolerance of HFD / STZ-induced diabetic mice, significantly improve the glucose regulation ability of diabetic mice, and avoid organ damage caused by high sugar.
[0091] Depend on Figure 5 The results show that the levels of AST and ALT in the blood of diabetic mice increased compared to normal mice, indicating that the liver had been damaged to a certain extent. However, Example 2 significantly reduced the levels of ALT and ALT in the blood of diabetic mice, indicating that Example 2 has a liver-protective effect.
[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A method for preparing collagen peptide with blood sugar lowering effect, characterized in that The steps include: (1) adding fish skin to water and heat-treating to obtain a fish skin solution; (2) regulating the pH of the fish-skin solution, adding enzyme, carrying out enzymolysis, and after enzymolysis finishes, deactivating the enzyme to obtain an enzymolysis solution; (3) The enzymatic hydrolysate is cooled and centrifuged, and the supernatant is collected, purified, filtered, and freeze-dried to obtain collagen peptides with hypoglycemic effect.
2. The method for preparing the collagen peptide with hypoglycemic effect according to claim 1, characterized in that: The heat treatment in step (1) is to soak in hot water at 95-100° C. for 1-2 hours; The ratio of the fish skin to water in step (1) is 1 g: 1-2 mL.
3. The method for preparing the collagen peptide with hypoglycemic effect according to claim 1, characterized in that: The enzymes in step (2) are Protease C and neutral protease; The added amount of the enzyme described in step (2) is 1 to 2% of the mass of the fish skin in the raw material.
4. The method for preparing the collagen peptide with hypoglycemic effect according to claim 1, wherein: The pH adjustment in step (2) is to adjust the pH to 7-8; The enzymatic hydrolysis conditions in step (2) are 45-55° C. for 1-12 hours.
5. The method for preparing the collagen peptide with hypoglycemic effect according to claim 1, wherein: The centrifugation condition in step (3) is 7000-9000 rpm at 4° C. for 5-15 min; The purification in step (3) is to add 10-20% of the total mass of activated carbon, stir and heat at 60-70°C for 40-60 minutes.
6. A collagen peptide with blood sugar lowering effect, prepared by the preparation method according to any one of claims 1 to 5.
7. Use of the collagen peptide with blood sugar lowering effect according to claim 6 in the preparation of a drug for treating diabetes.
8. Use of the collagen peptide with hypoglycemic effect according to claim 6 in the preparation of food or health care products with hypoglycemic effect.