Fish maw collagen peptide, preparation method and application
High-purity fish maw collagen peptides were prepared by a multi-enzyme combined enzymatic hydrolysis method, which solved the problems of low purity of collagen peptide extraction and low efficiency of single enzymatic hydrolysis. This method effectively improved type 2 diabetes and weight, and showed significant DPPIV inhibitory activity and liver protection effects.
Patent Information
- Application Number
- CN202512053419.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-03
AI Technical Summary
Existing collagen peptide extraction methods suffer from low purity and low efficiency due to single enzymatic hydrolysis, making it difficult to effectively improve type 2 diabetes and weight problems.
Small molecule fish maw collagen peptides with a molecular weight of 800-1200 Da were prepared by a multi-enzyme combined enzymatic hydrolysis method, including pretreatment, deep eutectic solvent extraction, separation and purification and ultrafiltration separation. High-purity fish maw collagen peptides were prepared by combining multi-enzyme combined enzymatic hydrolysis and protease inactivation treatment.
The prepared fish maw collagen peptides have significant DPPIV inhibitory activity, can effectively improve fasting blood glucose and body weight in type 2 diabetic mice, have significant liver protective effects, and the process is simple and can be used for food-grade applications.
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Figure CN121779540A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of food-derived bioactive peptide technology development and application, and in particular to a fish maw collagen peptide, its preparation method and application. Background Technology
[0002] In recent years, the incidence and mortality rates of obesity-related metabolic diseases have been rising continuously, resulting in an increasingly heavy medical burden and drawing significant national attention. Type 2 diabetes and obesity have become major global public health issues, often accompanied by pathological features such as insulin resistance and glucose and lipid metabolism disorders. According to IDF statistics, the Middle East and North Africa region has the highest prevalence rate globally at 17.6%, followed by North America and the Caribbean (15.1%). The Western Pacific region, where China is located, ranks third with a prevalence rate of 12.4%, highlighting the undeniable health challenges posed by diabetes. The number of people with diabetes worldwide is projected to continue rising. It is estimated that by 2050, the number of people with diabetes globally will increase from 588.7 million in 2024 to 852.5 million, an increase of 45%. While existing injectable and oral medications can control symptoms, long-term use can lead to side effects such as diarrhea, abdominal discomfort, hypoglycemia, weight gain, allergic reactions, heart failure, and gastrointestinal disturbances. Therefore, obtaining a safe and non-toxic dietary glucose and lipid metabolism protein regulatory peptide would be beneficial to the human body.
[0003] Collagen peptides, as natural bioactive components, are gaining increasing attention for their potential to regulate glucose and lipid metabolism. Related studies have shown that marine-derived collagen peptides exhibit good stability, small molecular weight, few side effects, and easy absorption, possessing various bioactive functions, such as angiotensin-converting enzyme (ACE) inhibition, dipeptidyl peptidase-IV (DPP-IV) inhibition, anti-inflammatory, anti-cancer, antifreeze, anti-fatigue, osteoporosis prevention, and antioxidant effects. Fish maw, with a protein content of approximately 80%, is predominantly composed of collagen, accounting for over 50% of the total protein content, making it an excellent source of collagen peptides. Summary of the Invention
[0004] The technical problem to be solved by this invention is to provide a fish maw collagen peptide that has the effects of improving weight and regulating blood sugar, thereby solving the problems of low purity and low efficiency of traditional collagen extraction and single-enzyme hydrolysis.
[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A type of fish maw collagen peptide, a small molecule collagen peptide of 800-1200 Da, has the effect of improving type 2 diabetes and assisting in weight control. Its preparation method includes the following steps: Step 1: Pre-treat the fish maw in a sodium hydroxide solution, then remove it, wash it until neutral, and cut it into small pieces; Step 2: Immerse the pretreated fish maw in a solution of deep eutectic solvent for heat treatment and extraction to obtain crude fish maw collagen stock solution; Step 3: Separate and purify the crude fish maw collagen stock solution to obtain high-purity fish maw collagen stock solution; Step 4: Add protease to the high-purity fish maw collagen stock solution for multi-enzyme combined hydrolysis. After hydrolysis, extinguish the protease to obtain fish maw collagen peptide hydrolysate. Step 5: After cooling the fish maw collagen peptide hydrolysate, centrifuge it and take the supernatant to separate it through an ultrafiltration device with a molecular cutoff of 800~1200 Da. Then freeze-dry it to obtain fish maw collagen peptide powder with the effects of regulating blood sugar and helping to control weight.
[0006] Furthermore, the fish maw collagen peptides exhibit significant inhibitory activity against DPPIV.
[0007] Furthermore, the collagen peptides are used to prepare drugs for controlling weight or improving insulin resistance or fasting blood glucose in type 2 diabetes, or to prepare functional foods.
[0008] Furthermore, the collagen peptides from the fish maw exhibited an inhibition rate of 50-100% against DPPIV; the significant difference was P<0.05.
[0009] Furthermore, after 5 weeks of administration of the aforementioned fish maw collagen peptides, the subjects' fasting blood glucose levels decreased by 58.99% or more.
[0010] Furthermore, after 5 weeks of administration of the aforementioned fish maw collagen peptides, the subjects experienced a weight loss of 29.45% or more.
[0011] Furthermore, the subjects were given oral glucose to intervene with the fish maw collagen peptides. The subjects' blood glucose levels rose rapidly and peaked at 15 minutes, then began to gradually decline.
[0012] Furthermore, intervention with the aforementioned fish maw collagen peptides for 5 weeks improved fasting blood glucose and oral glucose tolerance in diabetic subjects.
[0013] Furthermore, intervention with the aforementioned fish maw collagen peptides can achieve liver protection in the subjects.
[0014] Preferably, in step one, the pretreatment involves soaking in a 0.05-0.2 M sodium hydroxide solution at a temperature of 3-10°C for 12-48 hours.
[0015] Preferably, in step one, the washing to neutral and cutting into small pieces involves washing to neutral in ultrapure water or distilled water and cutting into pieces of 0.1–2 cm × 0.1–2 cm.
[0016] Preferably, in step two, the deep eutectic solvent is a mixed solvent of urea, citric acid, lactic acid, and water, and its volume ratio is 1-3:1-3:1-3:10-40.
[0017] Preferably, in step two, the volume ratio of fish maw to deep eutectic solvent is 1-5:5-20; the heat treatment temperature is 15-75℃, the stirring speed is 100-2000 r / min, and the extraction time is 0.5-8h.
[0018] Preferably, in step three, the crude fish maw collagen stock solution is separated and purified by a dialysis bag with a molecular weight cutoff of 3-300 kDa to obtain a first filtrate. Then, the first filtrate is separated and purified by an ultrafiltration membrane with a molecular weight cutoff of 3-300 kDa to obtain a high-purity fish maw collagen stock solution.
[0019] Preferably, in step three, the purity of the high-purity fish maw collagen stock solution reaches over 98%.
[0020] Preferably, in step five, the centrifugation conditions are: centrifugation at 5000-10000 rpm for 3-20 minutes at 4°C.
[0021] Preferably, in step four, the enzymatic hydrolysis conditions are as follows: high-purity fish maw collagen stock solution is dissolved in water to prepare fish maw collagen with a mass concentration of 0.2% to 10%, pH is 6.5 to 7.5, temperature is 45 to 55℃, and enzymatic hydrolysis is carried out for 0.5 to 24 hours under ultrasound-assisted conditions.
[0022] Preferably, in step four, the protease is two or three of papain, complex protease, flavor protease, and pepsin; wherein the weight ratio of the two proteases is 1~6:1~6, and the weight ratio of the three proteases is 1~6:1~3:1~2.
[0023] Preferably, in step four, the total amount of protease added is 4000-40000 U / g.
[0024] Preferably, in step four, the temperature for inactivating the protease is 80–100°C, and the time is 1–20 min.
[0025] This invention provides a fish maw collagen peptide as a drug for weight control, wherein the collagen peptide is the fish maw collagen peptide described in any of the above embodiments.
[0026] This invention provides a fish maw collagen peptide as a treatment to improve insulin resistance in type 2 diabetes or as a fasting blood glucose medication; the collagen peptide is the fish maw collagen peptide described in any of the above embodiments.
[0027] This invention provides a method for preparing fish maw collagen peptides, comprising the following steps: Step 1: Pre-treat the fish maw in a sodium hydroxide solution, then remove it, wash it until neutral, and cut it into small pieces; Step 2: Immerse the pretreated fish maw in a solution of deep eutectic solvent for heat treatment and extraction to obtain crude fish maw collagen stock solution; Step 3: Separate and purify the crude fish maw collagen stock solution to obtain high-purity fish maw collagen stock solution; Step 4: Add protease to the high-purity fish maw collagen stock solution for multi-enzyme combined hydrolysis. After hydrolysis, extinguish the protease to obtain fish maw collagen peptide hydrolysate. Step 5: After cooling the fish maw collagen peptide hydrolysate, centrifuge it and take the supernatant to separate it through an ultrafiltration device with a molecular cutoff of 800~1200 Da. Then freeze-dry it to obtain fish maw collagen peptide powder with the effects of regulating blood sugar and helping to control weight.
[0028] The beneficial effects of this invention are: (1) The present invention prepares fish maw collagen peptides that have the effects of improving body weight and regulating blood sugar, which can maximize the use of resources, reduce environmental pollution, and the obtained fish maw collagen has extremely high purity. Combined with multi-enzyme combined enzymatic hydrolysis, the collagen peptides produced by enzymatic hydrolysis are enzymatic hydrolysates with strong DPP IV inhibitory activity. Animal experiments have verified that the enzymatic hydrolysate can regulate the body weight and fasting blood sugar of type 2 diabetic mice, improve their oral glucose tolerance and liver function.
[0029] (2) The preparation method provided by the present invention is simple and realizes the enzymatic hydrolysis and controlled release of the target polypeptide. The entire process can meet the food grade requirements and can be applied to weight control and insulin resistance and fasting blood glucose related drugs or functional foods in type 2 diabetes. Attached Figure Description
[0030] Figure 1 It is the DPP-IV inhibition rate of the collagen hydrolysate obtained in Examples 1-6 and Comparative Examples 1-6.
[0031] Figure 2 The bar chart shows the effects of low and high doses of crude fish maw collagen from Examples 1-2, and low and high doses of fish maw collagen peptides from Example 2, on fasting blood glucose levels in obese type 2 diabetic mice.
[0032] Figure 3 The bar chart shows the effects of low and high doses of crude fish maw collagen from Examples 1-2, and low and high doses of fish maw collagen peptides from Example 2, on the fasting body weight of obese type 2 diabetic mice.
[0033] Figure 4Figures A and B show the effects of low and high doses of crude fish maw collagen in Examples 1-2, and low and high doses of fish maw collagen peptides in Example 2 on peritoneal glucose tolerance in obese type 2 diabetic mice after 5 weeks of gavage, as well as the blood glucose-time curves obtained from the peritoneal glucose tolerance test.
[0034] Figure 5 Figures A and B show the liver protection effects of low and high doses of crude fish maw collagen in Examples 1-2, and low and high doses of fish maw collagen peptides in Example 2, on obese type 2 diabetic mice after 5 weeks of gavage. Detailed Implementation
[0035] Exemplary embodiments of this application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.
[0036] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the terms "comprising," "including," "containing," and "having" are inclusive and therefore indicate the presence of the stated features, steps, operations, and ingredients, but do not exclude the presence or addition of one or more other features, steps, operations, ingredients, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated, unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.
[0037] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.
[0038] The endpoints and any values disclosed in this invention are not limited to the precise range or value, and such ranges or values should be understood to include values close to such ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0039] The present invention provides a small molecule collagen peptide derived from fish maw that has the effect of regulating blood sugar and assisting in weight control. It is obtained by extracting, purifying and enzymatically hydrolyzing collagen from fish maw, rather than directly enzymatically hydrolyzing fish maw. Its preparation method includes the following steps (1)-(5).
[0040] Step (1) involves pre-treating the fish maw, specifically by adding it to a sodium hydroxide solution, then washing it until neutral and cutting it into small pieces. The treatment involves soaking the fish maw in a 0.05–0.2 M sodium hydroxide solution at 3–10°C for 12–48 hours. Washing until neutral and cutting into small pieces can be done by washing in ultrapure water or distilled water until neutral, and then cutting into pieces of 0.1–2 cm × 0.1–2 cm.
[0041] Step (2) involves extracting collagen from crude fish maw using the eutectic solvent method. Specifically, the pretreated fish maw is immersed in the eutectic solvent and heat-treated at a predetermined temperature to obtain a crude fish maw collagen stock solution. The eutectic solvent is a mixture of urea, citric acid, lactic acid, and water, with a volume ratio of (1-3):(1-3):(1-3):(10-40). The treatment involves mixing the fish maw and the eutectic solvent at a volume ratio of (1-5):(5-20) at a predetermined temperature of 15-75°C and a rotation speed of 100-2000 r / min, and then heat-treating the mixture. The extraction time for the eutectic solvent method (heat treatment) is 0.5-8 hours to obtain the crude fish maw collagen stock solution.
[0042] Step (3): The crude fish maw collagen stock solution is separated and purified to obtain a fish maw collagen stock solution with extremely high purity. Specifically, the crude fish maw collagen stock solution is separated and purified by a dialysis bag and an ultrafiltration device. Preferably, the crude fish maw collagen stock solution is separated and purified by a dialysis bag with a molecular weight cutoff of 3 to 300 kDa to obtain a first filtrate. Then, the first filtrate is separated and purified by an ultrafiltration membrane with a molecular weight cutoff of 3 to 300 kDa to obtain a fish maw collagen stock solution with extremely high purity, up to 98% or more. After that, the fish maw collagen powder is obtained by freeze drying.
[0043] Step (4) involves multi-enzyme hydrolysis. Specifically, the above-mentioned fish maw collagen powder is dissolved in water to prepare a fish maw collagen solution. Protease is added for multi-enzyme hydrolysis. After hydrolysis, the protease is deactivated to obtain the collagen peptide hydrolysate. The hydrolysis conditions are as follows: freeze-dried fish maw collagen is mixed with water at a ratio of 1g:5-500ml to prepare a fish maw collagen solution with a mass percentage concentration of 0.2%-10%, pH 6.5-7.5, and temperature 45-55℃. Hydrolysis is carried out under ultrasound-assisted conditions for 0.5-24 hours. The protease is two or three of papain, complex protease, flavor protease, and pepsin for multi-enzyme hydrolysis. The weight ratio of two proteases can be 1-6:1-6, and the weight ratio of three proteases can be 1-6:1-3:1-2. The complex protease is a mixture of multiple proteases sold commercially, not a mixture of two proteases, and the amount of enzyme added is based on enzyme activity. The total amount of protease added is 4000–40000 U / g. The protease is inactivated at a temperature of 80–100℃ for 1–20 minutes. Papain, complex protease, flavor protease, and pepsin can all be purchased directly from the market.
[0044] Step (5): After cooling the enzymatic hydrolysate (e.g., to room temperature), centrifuge the solution and collect the supernatant for separation using an ultrafiltration device (e.g., an ultrafiltration membrane) to obtain small molecule collagen peptides (e.g., 800-1200 Da). Finally, freeze-dry the peptides to obtain collagen peptide powder with the functions of regulating blood sugar and assisting in weight control. The centrifugation conditions are 5000-10000 rpm at 4°C for 3-20 minutes. The ultrafiltration membrane has a molecular weight cutoff of 800-1200 Da.
[0045] The fish maw collagen peptide provided in this embodiment of the invention, which has the effect of regulating blood sugar and assisting in weight control, is prepared by the above-described preparation method.
[0046] The aforementioned fish maw collagen peptides, which have the effects of regulating blood sugar and assisting in weight control, can be used in the preparation of drugs or functional foods related to weight control and improvement of insulin resistance and fasting blood glucose in type 2 diabetes. The functional foods can be made by adding the aforementioned fish maw collagen peptide powder prepared in this invention to existing foods (including but not limited to conventional foods, weight control foods, foods for improving type 2 diabetes, or foods related to controlling fasting blood glucose).
[0047] Unless otherwise specified, the following implementation plan follows standard testing conditions or the testing conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.
[0048] The eutectic solvents used in the following examples include urea, lactic acid, citric acid, and water.
[0049] The enzymes used in the following examples include complex proteases, neutral proteases, papain, and pepsin.
[0050] Example 1: Preparation method of fish maw collagen (1) Soak the fish maw in a 0.1 M sodium hydroxide solution at 4℃ for 24 hours, changing the solution every 6 hours. Then wash until neutral and cut into 0.5cm×0.5cm pieces. (2) The treated fish maw and the deep eutectic solvent (the volume ratio of urea, citric acid, lactic acid and water in the deep eutectic solvent is 2:1:1:30) are heat-treated at 65°C and stirred at 1000 r / min for 5 hours to extract crude fish maw collagen solution. (3) The crude fish collagen solution was dialyzed for 24 hours using a 10 kDa dialysis bag, and then further separated and purified by an ultrafiltration device to obtain a high-purity fish collagen solution with a purity of 98.12%. After that, it was freeze-dried to obtain fish collagen powder. (4) Dissolve the above-mentioned fish maw collagen powder in water (at a weight ratio of 1:10) to prepare a 10% fish maw collagen solution. Adjust the pH of the solution to 7. Add 27000 U / (g collagen) of protease to the solution. The protease contains a complex protease, papain and flavor protease in a mass ratio of 2:6:1. Perform enzymatic hydrolysis under ultrasonic assistance at a hydrolysis temperature of 50°C. After hydrolysis for 6 hours, inactivate the enzyme at 95°C for 20 minutes to obtain fish maw collagen peptide hydrolysate. (5) Cool the fish maw collagen peptide hydrolysate to room temperature, then centrifuge at 7000 rpm for 15 min at 4℃, take the supernatant, and obtain 800~1200 Da collagen hydrolysate through an ultrafiltration device. Then freeze-dry to obtain fish maw collagen peptides that have the effect of improving body weight and regulating blood sugar.
[0051] Example 2 The method of Example 1 was used to prepare fish maw collagen peptides. The only difference from Example 1 was that the protease in step (4) was added in a ratio of 6:2:1 (w:w:w) to the complex protease, papain and flavor protease respectively.
[0052] Example 3 The method of Example 1 was used to prepare fish maw collagen peptides. The only difference from Example 1 was that 27,000 U / (g collagen) protease was added to the 10% fish maw collagen solution in step (4). The protease was a compound protease and papain added at a ratio of 1:3 (w / w) and then the enzymatic hydrolysis reaction was carried out.
[0053] Example 4 The method of Example 1 was used to prepare fish maw collagen peptides. The only difference from Example 1 was that 27,000 U / (g collagen) protease was added to the 10% fish maw collagen solution in step (4). The protease was added to the compound protease and papain in a ratio of 3:1 (w / w) and then the enzymatic hydrolysis reaction was carried out.
[0054] Example 5 The method of Example 1 was used to prepare fish maw collagen peptides. The only difference from Example 1 was that 27,000 U / (g collagen) protease was added to the 10% fish maw collagen solution in step (4). The protease was added to papain and flavor protease in a ratio of 6:1 (w / w) and then the enzymatic hydrolysis reaction was carried out.
[0055] Example 6 The method of Example 1 was used to prepare fish maw collagen peptides. The only difference from Example 1 was that 27,000 U / (g collagen) protease was added to the 10% fish maw collagen solution in step (4). The protease was added to papain and flavor protease in a ratio of 1:6 (w / w) and then the enzymatic hydrolysis reaction was carried out.
[0056] Comparative Example 1 The method of Example 1 was used to prepare fish maw collagen peptides. The only difference from Example 1 was that the protease combination used in step (4) was: complex protease, papain and pepsin in a ratio of 2:6:1 (w:w:w).
[0057] Comparative Example 2 The method of Example 2 was used to prepare fish maw collagen peptides. The only difference from Example 2 was that the protease combination used in step (4) was a complex protease, papain and pepsin, in a ratio of 6:2:1 (w:w:w).
[0058] Comparative Example 3 The method of Example 3 was used to prepare fish maw collagen peptides. The only difference from Example 3 was that the protease combination used in step (4) was flavor protease and pepsin in a ratio of 1:3.
[0059] Comparative Example 4 The method of Example 4 was used to prepare fish maw collagen peptides. The only difference from Example 4 was that the protease combination used in step (4) was a complex protease and a flavor pepsin in a ratio of 3:1.
[0060] Comparative Example 5 The method of Example 5 was used to prepare fish maw collagen peptides. The only difference from Example 5 was that the protease combination used in step (4) was pepsin and complex protease in a ratio of 6:1.
[0061] Comparative Example 6 The method of Example 6 was used to prepare fish maw collagen peptides. The only difference from Example 6 was that the protease combination used in step (4) was pepsin and complex protease in a ratio of 1:6.
[0062] Example 7 Activity Verification Experiment of Enzymatic Hydrolysate 7.1 Determination of DPPIV inhibition rate DPP-4 inhibitors are a class of drugs used to treat type 2 diabetes. They work by inhibiting the DPP-4 enzyme, which slows down the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), thereby promoting insulin secretion and inhibiting glucagon release. They are less likely to induce hypoglycemia or increase weight.
[0063] Determination of DPP-IV inhibition rate: The collagen peptides prepared in this invention were prepared into a 5 mg / mL sample solution using TrisHCl buffer (0.1 M, pH 8.0). Sample absorbance: 90 μL of the sample solution and 90 μL of 1.6 mM substrate (GlyPro-pNA) were added to an enzyme-free 1.5 mL EP tube. After mixing, the mixture was incubated at 37 °C for 10 min. Then, 180 μL of 10 U / L DPP-IV reaction solution was added, mixed thoroughly, and incubated precisely at 37 °C for 60 min. The reaction was terminated with sodium acetate (1 M, pH 4). No DPP-IV was added to the blank wells. 90 μL of TrisHCl buffer was added to both the blank and control wells; all other parameters were the same as for the sample. The DPP-IV inhibition rate of the sample was calculated using the following formula: DPP-IV inhibition rate = ×100% Among them, OD s For sample group, OD c For the control group, OD b The absorbance of the blank group at 405 nm is shown in the figure. Figure 1 As shown, the collagen peptides obtained from fish maw obtained using different enzymatic hydrolysis techniques in the above-described embodiments / comparative examples of this invention exhibit certain differences in their DPPIV inhibitory activity. Figure 1 As can be seen, the examples have a higher DPPIV inhibition rate compared to the comparative examples, with Example 2 showing a significant difference compared to the other groups (P<0.05). The protease combination in Example 2 has the best enzymatic hydrolysis effect, with a DPPIV inhibition rate close to 100%.
[0064] Figure 1-5 In the diagram, letters (e.g., b, a, bc...) are used to represent the column heights (corresponding to the vertical coordinate axes), which represent the differences between groups. The same letter indicates no significant difference between groups, while different letters indicate significant differences between groups.
[0065] 7.2 In vivo evaluation experimental setup for the effects of weight control, improvement of blood glucose and insulin resistance The effects of the fish collagen prepared in the above examples on improving obesity and insulin resistance in vivo were studied using a mouse model induced by a high-fat, high-sugar diet. The specific procedures are as follows: (1) Laboratory animals Male C57 / BL6J mice, 6 weeks old, 20±0.2 g.
[0066] (2) Animal husbandry environment They were raised at 25℃ and 55% humidity, with a light / dark cycle of 12 hours, and drinking water was sterilized by high temperature and high pressure. (3) Animal experimental modeling After two weeks of adaptive feeding, the mice were divided into a normal control group and a diabetic group according to their weight. The normal control group and the diabetic group were fed with ordinary feed and high-fat feed, respectively, for 16 weeks to simulate the natural pathogenesis of obesity-related type 2 diabetes. Fasting blood glucose was measured for three consecutive days in the 16th week. If the blood glucose level was >11.1 mM on any day and there was a significant difference between the normal control group and the diabetic group in the intraperitoneal glucose tolerance test, the model was considered to be successful. (4) Evaluation experiment on hypoglycemic efficacy in vivo The mice that were successfully modeled in (3) were randomly divided into 7 groups (8 mice in each group) according to their blood glucose and body weight: normal group, model group, metformin positive group (70 mg / kg), Example 1-2-low, Example 1-2-high, Example 2-low, and Example 2-high.
[0067] After the experiment began, mice in the crude fish maw collagen powder / fish maw collagen peptide / metformin positive drug group were orally administered the corresponding dose of crude fish maw collagen / fish maw collagen peptide / metformin daily for 5 consecutive weeks; mice in the model group and normal group were orally administered sterilized ultrapure water for 5 consecutive weeks.
[0068] At weeks 15, 16, 17, 18, 19, and 20 after the start of the experiment, mice were weighed and blood was collected from the tail vein after an 8-hour fast. Fasting blood glucose concentration was measured using a glucometer and blood glucose test strips according to the manufacturer's (Omron) instructions. At week 15 after model establishment, mice were injected intraperitoneally with a 20% glucose solution (2 g / kg) after an 8-hour fast. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after oral glucose administration using a glucometer and blood glucose test strips to evaluate glucose tolerance. After week 20, mice were dissected, plasma was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the plasma were measured. Specific measurement methods were provided in the kit instructions.
[0069] 7.3 Results of in vivo hypoglycemic efficacy evaluation experiment Experimental results are as follows Figures 2-5 As shown, by Figure 2 It was found that after modeling, the fasting blood glucose level in the model group exceeded 11.1 mmol / L, showing a significant difference from the normal group. Even after two weeks of treatment, the fasting blood glucose and body weight in the intervention groups (Examples 1-2-low / Examples 1-2-high / Examples 2-low / Examples 2-high) remained significantly different from the normal group, indicating that short-term intervention could not improve fasting blood glucose and body weight in mice. After three weeks of treatment, the fasting blood glucose and body weight of the treated mice improved, and by week 5, the fasting blood glucose of diabetic mice was significantly reduced (p < 0.05). Compared with the model group, the low-dose crude fish maw collagen intervention in Examples 1-2 did not significantly change fasting blood glucose, while the high-dose crude fish maw collagen intervention in Examples 1-2, and the low-dose and high-dose fish maw collagen peptide intervention in Example 2 reduced fasting blood glucose levels by 17.34%, 37.69%, and 58.99%, respectively. The corresponding body weight decreased by 2.92%, 12.87%, and 29.45%, respectively. Five weeks after gavage, the blood glucose levels in the high-dose intervention group of fish maw collagen peptides in Example 2 were within a safe range, and there were significant differences in weight and fasting blood glucose compared with those after intervention with undigested crude fish maw collagen. This indicates that the collagen peptides prepared in Example 2 have the effects of controlling weight, improving liver function and regulating blood glucose.
[0070] Depend on Figure 2 and Figure 3 It is known that the high doses of crude fish maw collagen in Examples 1-2, the high doses of fish maw collagen peptides in Example 2, and metformin can improve fasting blood glucose and body weight in type 2 diabetic mice, but the enzymatically hydrolyzed collagen peptides have a better effect and a more significant improvement.
[0071] Depend on Figure 4As shown in Figures A and B, the high-dose crude fish maw collagen in Examples 1-2, the high-dose fish maw collagen peptide in Example 2, and metformin can improve intraperitoneal glucose tolerance in type 2 diabetic mice. After glucose injection, the blood glucose levels of mice in the model group and the low-dose crude fish maw collagen intervention group in Examples 1-2 rose rapidly and peaked at 30 minutes. In contrast, the blood glucose levels of mice in the normal group and the high-dose crude fish maw collagen in Examples 1-2, and the low-dose and high-dose fish maw collagen peptide intervention groups in Examples 2 rose rapidly and peaked at 15 minutes, subsequently gradually declining. Throughout this process, the blood glucose levels in the model group remained higher than those in the other groups, indicating that diabetic mice have a weaker ability to regulate blood glucose than other groups. Compared with the model group, the high-dose crude fish maw collagen in Examples 1-2 and the low-dose and high-dose fish maw collagen peptide intervention groups in Example 2 could alleviate the rate of blood glucose rise more quickly and significantly reduce AUC02h. This indicates that intervention for 5 weeks can effectively improve fasting blood glucose and oral glucose tolerance in diabetic mice induced by a long-term high-fat and high-sugar diet, significantly improve the glucose regulation ability of diabetic mice, and avoid multi-organ damage caused by long-term high-sugar state.
[0072] Depend on Figure 5 The results showed that diabetic mice had increased levels of alanine aminotransferase and aspartate aminotransferase in their blood compared to normal mice, indicating some degree of liver damage. The high-dose crude fish maw collagen intervention groups in Examples 1-2, and the low-dose and high-dose fish maw collagen peptide intervention groups in Example 2, significantly reduced the levels of alanine aminotransferase and aspartate aminotransferase in the blood of diabetic mice compared to the model group. This indicates that the high-dose crude fish maw collagen intervention groups in Examples 1-2, and the low-dose and high-dose fish maw collagen peptide intervention groups in Example 2 have a liver-protective effect, and that the high-dose collagen peptides are superior to undigested collagen.
[0073] Other experimental results show that the fish maw collagen peptide prepared in this invention has significant inhibitory activity against DPPIV. The fish maw collagen peptide inhibited DPPIV by 50-100%; the significant difference was P<0.05. After 5 weeks of administration of the fish maw collagen peptide, the subjects' fasting blood glucose decreased by 58.99% or more. After 5 weeks of administration of the fish maw collagen peptide, the subjects' body weight decreased by 29.45% or more. After 5 weeks of intervention with the fish maw collagen peptide, the subjects underwent peritoneal glucose tolerance testing. The subjects' blood glucose rose rapidly and reached a peak at 15 minutes, then gradually decreased. Their peritoneal glucose tolerance trend line was similar to that of the normal group, but significantly separated from that of the model group. Intervention with the fish maw collagen peptide for 5 weeks improved fasting blood glucose and peritoneal glucose tolerance in diabetic subjects. Intervention with the fish maw collagen peptide achieved liver protection in the subjects.
[0074] As can be seen from the above embodiments, the collagen peptide and its preparation method of the present invention, which have the effects of regulating blood sugar and assisting in weight control, have the following effects: (1) The preparation method provided can maximize the use of resources, reduce environmental pollution, and the collagen obtained is extremely pure. Combined with multi-enzyme combined enzymatic hydrolysis, the collagen peptides produced by enzymatic hydrolysis have strong DPPIV inhibitory activity. Animal experiments have verified that the hydrolysate can regulate the body weight and fasting blood glucose of type 2 diabetic mice, improve their oral glucose tolerance and liver function. (2) The preparation method provided is simple and achieves the enzymatic hydrolysis and controlled release of the target peptide. The entire process can meet the food grade requirements and can be applied to weight control and insulin resistance and fasting blood glucose related drugs or functional foods in type 2 diabetes.
[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A type of fish maw collagen peptide, a small molecule collagen peptide of 800-1200 Da, which has the effect of improving type 2 diabetes and assisting in weight control, and its preparation method includes the following steps: Step 1: Pre-treat the fish maw in a sodium hydroxide solution, then remove it, wash it until neutral, and cut it into small pieces; Step 2: Immerse the pretreated fish maw in a solution of deep eutectic solvent for heat treatment and extraction to obtain crude fish maw collagen stock solution; Step 3: Separate and purify the crude fish maw collagen stock solution to obtain high-purity fish maw collagen stock solution; Step 4: Add protease to the high-purity fish maw collagen stock solution for multi-enzyme combined hydrolysis. After hydrolysis, extinguish the protease to obtain fish maw collagen peptide hydrolysate. Step 5: After cooling the fish maw collagen peptide hydrolysate, centrifuge it and take the supernatant to separate it through an ultrafiltration device with a molecular cutoff of 800~1200 Da. Then freeze-dry it to obtain fish maw collagen peptide powder with the effects of regulating blood sugar and helping to control weight.
2. The fish maw collagen peptide as described in claim 1, characterized in that: The collagen peptides from the fish maw exhibit significant inhibitory activity against DPPIV. The collagen peptides are used to prepare drugs for controlling weight or improving insulin resistance or fasting blood glucose in type 2 diabetes, or to prepare functional foods.
3. The fish maw collagen peptide as described in claim 1, characterized in that: The collagen peptides from the fish maw exhibited a 50-100% inhibition rate against DPPIV; the statistical significance was P<0.
05. After 5 weeks of administration of the aforementioned fish maw collagen peptides, the subjects' fasting blood glucose decreased by 58.99% or more. After 5 weeks of administration of the aforementioned fish maw collagen peptides, the subjects experienced a weight loss of 29.45% or more. After subjects were given oral glucose and treated with the aforementioned fish maw collagen peptides for 5 weeks, their blood glucose levels rose and peaked at 15 minutes, then began to gradually decline. Intervention with the aforementioned fish maw collagen peptides for 5 weeks improved fasting blood glucose and oral glucose tolerance in diabetic subjects. Intervention with the aforementioned fish maw collagen peptides can achieve liver protection in the subjects.
4. The fish maw collagen peptide as described in claim 1, characterized in that: In step one, the pretreatment involves soaking in a 0.05–0.2 M sodium hydroxide solution at a temperature of 3–10°C for 12–48 hours. In step one, the step of washing to neutral and cutting into small pieces involves washing in ultrapure water or distilled water until neutral and then cutting into pieces of 0.1–2 cm × 0.1–2 cm. In step two, the deep eutectic solvent is a mixed solvent of urea, citric acid, lactic acid and water, and its volume ratio is 1-3:1-3:1-3:10-40. In step two, the volume ratio of fish maw to deep eutectic solvent is 1-5:5-20; the heat treatment temperature is 15-75℃, the stirring speed is 100-2000 r / min, and the extraction time is 0.5-8h.
5. The fish maw collagen peptide as described in claim 1, characterized in that: In step three, the crude fish maw collagen stock solution is separated and purified by a dialysis bag with a molecular weight cutoff of 3-300 kDa to obtain the first filtrate. Then, the first filtrate is separated and purified by an ultrafiltration membrane with a molecular weight cutoff of 3-300 kDa to obtain high-purity fish maw collagen stock solution. In step three, the purity of the high-purity fish maw collagen stock solution reaches over 98%. In step five, the centrifugation conditions are: centrifugation at 5000-10000 rpm for 3-20 minutes at 4°C.
6. The fish maw collagen peptide as described in claim 1, characterized in that: In step four, the enzymatic hydrolysis conditions are as follows: high-purity fish maw collagen stock solution is dissolved in water to prepare fish maw collagen with a mass concentration of 0.2% to 10%, pH is 6.5 to 7.5, temperature is 45 to 55℃, and enzymatic hydrolysis is carried out for 0.5 to 24 hours under ultrasound-assisted conditions.
7. The fish maw collagen peptide according to any one of claims 1 to 6, characterized in that: In step four, the protease is two or three of papain, complex protease, flavor protease, and pepsin; wherein the weight ratio of two proteases is 1~6:1~6, and the weight ratio of three proteases is 1~6:1~3:1~2. In step four, the total amount of protease added is 4000–40000 U / g; In step four, the inactivation of the protease is carried out at a temperature of 80–100°C for 1–20 minutes.
8. A fish maw collagen peptide as a drug for controlling weight, wherein the collagen peptide is the fish maw collagen peptide according to any one of claims 1 to 7.
9. A fish maw collagen peptide for improving insulin resistance in type 2 diabetes or as a fasting blood glucose drug; wherein the fish maw collagen peptide is the fish maw collagen peptide according to any one of claims 1 to 6.
10. A method for preparing fish maw collagen peptides, comprising the following steps: Step 1: Pre-treat the fish maw in a sodium hydroxide solution, then remove it, wash it until neutral, and cut it into small pieces; Step 2: Immerse the pretreated fish maw in a solution of deep eutectic solvent for heat treatment and extraction to obtain crude fish maw collagen stock solution; Step 3: Separate and purify the crude fish maw collagen stock solution to obtain high-purity fish maw collagen stock solution; Step 4: Add protease to the high-purity fish maw collagen stock solution for multi-enzyme combined hydrolysis. After hydrolysis, extinguish the protease to obtain fish maw collagen peptide hydrolysate. Step 5: After cooling the fish maw collagen peptide hydrolysate, centrifuge it and take the supernatant to separate it through an ultrafiltration device with a molecular cutoff of 800~1200 Da. Then freeze-dry it to obtain fish maw collagen peptide powder with the effects of regulating blood sugar and helping to control weight.