Preparation method and application of food protein peptide with blood glucose-lowering activity

Abstract

The application discloses a preparation method and application of food protein peptides with blood sugar reducing activity. The food protein peptides are prepared according to the method comprising the following steps: milk protein is hydrolyzed by protease, in-vitro simulated gastrointestinal digestion and intestinal cell absorption are carried out, and milk protein enzymatic hydrolysate digestion absorption permeate with resistance to gastrointestinal digestion is obtained. The milk protein enzymatic hydrolysate digestion absorption permeate is separated, purified and sequence identified again, and the following protein peptides with blood sugar reducing activity are obtained: VSLPEWV, FLDDDL and KILDK, wherein the activity of KILDK is the strongest. The blood sugar reducing peptides prepared by the method have the characteristics of naturalness, safety and no side effects, have high blood sugar reducing activity, can be used for preventing metabolic syndrome and treating diabetes, and have good application prospect in the development of food-derived functional ingredients, functional foods and health products with blood sugar reducing function.

Description

Technical Field

[0001] This invention belongs to the field of functional foods, specifically relating to the preparation method and application of food protein peptides with hypoglycemic activity. Background Technology

[0002] With changes in diet and lifestyle, diabetes has become a common chronic disease. According to statistics from the International Diabetes Federation, as of 2019, there were 463 million people with diabetes, and the number is expected to reach 700 million by 2045.

[0003] Currently, treatments for diabetes include insulin injections and oral hypoglycemic agents (such as thiazolidinediones, biguanides, and sulfonylureas). However, these treatments are prone to adverse reactions such as hypoglycemia and liver damage, and can also lead to secondary failure effects. Therefore, developing safe, natural, and highly effective hypoglycemic peptide products derived from food has become an important part of future non-drug treatments for diabetes. For example, Chinese Patent 200910210712.5, by Li Yong et al., discloses the use of a marine collagen peptide in the preparation of drugs and foods for diabetic wound healing; Chinese Patent 201510066244.4, by Ma Hailong et al., isolates an active peptide from *Hydrilla verticillata* that can treat diabetes. However, there are currently few reports on patents regarding food protein peptides with hypoglycemic activity. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a protein peptide with hypoglycemic activity and its application.

[0005] In a first aspect, the present invention protects a method for preparing a protein peptide with hypoglycemic activity.

[0006] The method for preparing the hypoglycemic protein peptide protected by this invention includes the following steps:

[0007] 1) Dissolve milk protein in water, pretreat it, adjust the pH to 6.0-10.0, then add protease and hydrolyze to obtain milk protein hydrolysate;

[0008] 2) Dissolve the lactase hydrolysate in water, then adjust the pH to 1.5-4.0, add pepsin for the first hydrolysis; then adjust the pH to 6.0-8.5, add pancreatin for the second hydrolysis, to obtain the lactase hydrolysate digest.

[0009] 3) The lactase hydrolysate is absorbed through an intestinal monolayer, the permeate is collected, and the permeate is freeze-dried to obtain lactase hydrolysate digestion and absorption permeate, which contains the protein peptides.

[0010] In the above method, in step 1), the milk protein can be whole milk protein, casein, whey protein, lactalbumin, or lactoglobulin. Further, the milk protein is α-lactalbumin. In a specific embodiment of the present invention, the milk protein is bovine α-lactalbumin.

[0011] The ratio of milk protein to water can be 1:(2-30) (mass to volume ratio). In a specific embodiment of the present invention, the ratio of milk protein to water is 1:20 (mass to volume ratio).

[0012] The pretreatment methods include heat treatment, hydrostatic high-pressure treatment, subcritical water treatment, or ultrasonic treatment. In a specific embodiment of the present invention, the pretreatment method is heat treatment (55°C water bath) to adjust the pH to 8.5.

[0013] The protease may be any one or a mixture of proteases from microbial, plant, or animal sources. Further, the protease is an alkaline protease. In a specific embodiment of the invention, the protease is an alkaline protease derived from Bacillus licheniformis.

[0014] The ratio of milk protein to protease can be 100g:(2-20)AU. In a specific embodiment of the present invention, the ratio of milk protein to protease is 10g:1.2AU.

[0015] The hydrolysis conditions can be hydrolysis at 30–60°C for 2–10 hours. In a specific embodiment of the present invention, the hydrolysis conditions are hydrolysis at 55°C for 4 hours.

[0016] In the above method, in step 2), the ratio of the lactase hydrolysate to water can be 1:(10-50) (mass to volume ratio). In a specific embodiment of the present invention, the ratio of the lactase hydrolysate to water is 1:25.

[0017] The ratio of milk protein to pepsin can be 10g:(50000~100000)U. In a specific embodiment of the present invention, the ratio of milk protein to pepsin is 10g:75000U.

[0018] The ratio of milk protein to pancreatic enzyme can be 10g:(2000-5000)U. In a specific embodiment of the present invention, the ratio of milk protein to pepsin is 10g:3200U.

[0019] The pH adjustment to 1.5–4.0 refers to adjusting the pH to 2.0.

[0020] The pH adjustment to 6.0–8.5 refers to adjusting the pH to 7.5.

[0021] The conditions for the first hydrolysis can be hydrolysis at 30–50°C for 1–3 hours. In a specific embodiment of the present invention, the conditions for the first hydrolysis are hydrolysis at 37°C for 2 hours.

[0022] The second hydrolysis can be performed at 30–50°C for 2–4 hours. In a specific embodiment of the invention, the second hydrolysis is performed at 37°C for 3 hours.

[0023] In the above method, step 3) of the method for absorption of the intestinal monocellular layer includes the following steps: human colon adenocarcinoma cells Caco-2 are seeded in the upper chamber of a Transwell plate for in vitro culture, and a monocellular layer is formed during the in vitro culture process. The lactase digest is added to the upper chamber of the Transwell plate and incubated for 2 hours.

[0024] The freeze-drying is vacuum freeze-drying.

[0025] In the above method, step 3) further includes the following step: separating, purifying, and sequence identifying the digested and absorbed permeate of the lactase hydrolysate to obtain the protein peptide. The separation, purification, and sequence identification method refers to the method in the reference "Song, JJ, Wang, Q., Du, M., Ji, XM, & Mao, XY (2017). Identification of dipeptidylpeptidase-IV inhibitory peptides from mare whey protein hydrolysates. Journal of Dairy Science, 100(9), 6885-6894."

[0026] In the above method, the amino acid sequence of the protein peptide is sequence 1, sequence 2, or sequence 3.

[0027] The protein peptides mentioned are food-grade protein peptides.

[0028] Secondly, this invention protects a protein peptide.

[0029] The protein peptide protected by this invention is any one of the following m1)-m4):

[0030] m1) The protein peptide prepared according to the above method;

[0031] The amino acid sequence of m2 is the protein peptide shown in sequence 1;

[0032] The amino acid sequence of m3 is the protein peptide shown in sequence 2;

[0033] The amino acid sequence of m4 is the protein peptide shown in sequence 3.

[0034] Thirdly, this invention protects an enzymatic hydrolysate.

[0035] The enzymatic hydrolysate protected by this invention is the lactase hydrolysate obtained according to step 1) of the above method.

[0036] Fourthly, the present invention protects new uses of the above-mentioned protein peptides or the above-mentioned enzymatic hydrolysates.

[0037] This invention protects the use of the above-mentioned protein peptides or the above-mentioned enzymatic hydrolysates in the following X1)-X6);

[0038] X1) Lowers blood sugar;

[0039] X2) Prevention and / or treatment of diabetes;

[0040] X3) Prevention and / or treatment of metabolic syndrome.

[0041] X4) Prepare blood sugar-lowering products;

[0042] X5) Prepare products for the prevention and / or treatment of diabetes;

[0043] X6) Prepare products for the prevention and / or treatment of metabolic syndrome.

[0044] Fifthly, this invention protects a product.

[0045] The active ingredient of the product protected by this invention is the above-mentioned protein peptide or the above-mentioned enzymatic hydrolysate; the function of the product is any one of the following Y1)-Y3):

[0046] Y1) Lowers blood sugar;

[0047] Y2) Prevention and / or treatment of diabetes;

[0048] Y3) Prevention and / or treatment of metabolic syndrome.

[0049] In any of the above-described applications or products, the blood glucose reduction is manifested as increasing insulin-stimulated cellular glucose uptake or increasing insulin-stimulated cellular glucose consumption.

[0050] In any of the above-described applications or products, the diabetes is type 2 diabetes.

[0051] In any of the above-mentioned applications or products, the product may be food, health food, or medicine.

[0052] The beneficial effects of this invention are:

[0053] 1. This invention uses protease to hydrolyze milk protein and employs in vitro simulated gastrointestinal digestion and intestinal cell absorption to treat the milk protein hydrolysate, ensuring that the isolated active peptides can resist gastrointestinal digestion and absorption, thereby possessing in vivo biological activity.

[0054] 2. The digested and absorbed permeate of lactase was separated, purified, and sequenced to obtain the amino acid sequence of the hypoglycemic peptide, resulting in a product with high purity.

[0055] 3. The hypoglycemic protein peptides prepared by the present invention have the advantages of high activity and stability, high safety for consumption, no side effects, and good absorption.

[0056] This invention provides a method for preparing food protein peptides with hypoglycemic activity and their applications. The food protein peptides are prepared according to the following steps: milk protein is hydrolyzed by protease, subjected to in vitro simulated gastrointestinal digestion and intestinal cell absorption, to obtain a milk protein hydrolysate digestible permeate that resists gastrointestinal digestion; the milk protein hydrolysate digestible permeate is then separated, purified, and sequenced to obtain the following hypoglycemic protein peptides: VSLPEWV, FLDDDL, and KILDK, with KILDK exhibiting the strongest activity. The hypoglycemic peptides prepared using this method are natural, safe, and have no side effects. They possess high hypoglycemic activity and can be used to prevent metabolic syndrome and treat diabetes. They show promising application prospects in the development of food-derived functional ingredients, functional foods, and health products with hypoglycemic functions. Attached Figure Description

[0057] Figure 1 This is a secondary mass spectrum of the peptide sequence.

[0058] Figure 2 The effect of lactase hydrolysate on insulin tolerance in type 2 diabetic mice. (A) Intraperitoneal insulin tolerance test curve; (B) Area under the curve (AUC) of the intraperitoneal insulin tolerance test. Different letters represent significant differences, p < 0.05. Detailed Implementation

[0059] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0060] Example 1: Preparation method of lactase hydrolysate digestible permeate and acquisition of glycemic protein peptides

[0061] I. Preparation method of lactase hydrolysate digestion and absorption permeate

[0062] 1. Weigh 10g of milk protein (bovine milk α-lactalbumin, Davisco Foods International, USA, catalog number JE011-1-410), dissolve it in 200mL of water, adjust the pH to 8.5 in a 55℃ water bath, add 0.5g of protease (Novozymes Biotechnology, Denmark, catalog number Alcalase 2.4L, 2.4AU / g), and hydrolyze at 55℃ for 4h to obtain the milk protein hydrolysate.

[0063] 2. Dissolve 10g of the lactase hydrolysate obtained in step 1 in 250mL of water, adjust the pH to 2.0, add 0.3g of pepsin (Sigma-Aldrich, USA, catalog number P7000, 250U / mg), and hydrolyze at 37℃ for 2h. Then adjust the pH to 7.5, add 0.4g of trypsin (Sigma-Aldrich, USA, catalog number P7545, 8×USP (8U / mg)), and hydrolyze at 37℃ for 3h. Freeze-dry to obtain the lactase hydrolysate.

[0064] 3. Human colon adenocarcinoma cells Caco-2 were seeded into the upper chamber of a Transwell plate for in vitro culture. During the in vitro culture, they differentiated into a monolayer. The lactase digest obtained in step 2 was added to the upper chamber of the Transwell plate and incubated for 2 hours to simulate the intestinal absorption process. The permeate was collected and freeze-dried under vacuum to obtain the lactase digestion and absorption permeate.

[0065] II. Obtaining Glucose-Lowering Protein Peptides

[0066] The digested and absorbed permeates of the lactase obtained in step one were separated, purified, and sequenced to obtain the following hypoglycemic protein peptide sequences: VSLPEWV (sequence 1), FLDDDL (sequence 2), and KILDK (sequence 3). The methods for separation, purification, and sequence identification of the hypoglycemic protein peptides were referenced in the literature "Song, JJ, Wang, Q., Du, M., Ji, XM, & Mao, XY (2017). Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates. Journal of Dairy Science, 100(9), 6885-6894." The specific steps are as follows: the mobile phase flow rate was 1.0 mL / min, and the digested and absorbed permeates of the lactase were separated using a Sephadex G-25 gel chromatography column. The collected components were dried to obtain the hypoglycemic components of the lactase. The obtained hypoglycemic components of the lactase were separated by a chromatographic system and identified by tandem mass spectrometry to obtain the hypoglycemic active peptide sequences. The secondary mass spectra of the peptide sequences are shown in the figure. Figure 1 .

[0067] Example 2: Effect of hypoglycemic protein peptides on glucose uptake in adipocytes

[0068] I. Experimental Materials and Methods

[0069] 1. Experimental Materials

[0070] 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC), ATCC number CL-173.

[0071] 2. Experimental Methods

[0072] 3T3-L1 preadipocytes were cultured in DMEM high-glucose medium. After the cells differentiated into 3T3-L1 mature adipocytes, 4 ng / mL TNF-α (Sigma-Aldrich, USA, catalog number SRP3177) and 100 μM glycemic protein peptide were added. The cells were incubated for 4 days at 95% humidity, 5% CO2 and 37°C, with the medium changed once a day.

[0073] The experiment was divided into the following five groups:

[0074] Blank control group: 3T3-L1 mature adipocytes, without TNF-α induction and without intervention from hypoglycemic protein peptides.

[0075] TNF-α model group: 3T3-L1 mature adipocytes were used to establish an insulin resistance model by induction with TNF-α, but without intervention by hypoglycemic protein peptides.

[0076] 100 μM VSLPEWV treatment group: 3T3-L1 mature adipocytes were induced with TNF-α to establish an insulin resistance model, and simultaneously incubated with 100 μM glycemic lowering protein peptide. The glycemic lowering protein peptide was VSLPEWV obtained in step two of Example 1.

[0077] 100 μM FLDDDL treatment group: 3T3-L1 mature adipocytes were induced with TNF-α to establish an insulin resistance model, and simultaneously incubated with 100 μM glycemic protein peptide. The glycemic protein peptide was the glycemic protein peptide FLDDDL obtained in step two of Example 1.

[0078] 100 μM KILDK treatment group: 3T3-L1 mature adipocytes were used to establish an insulin resistance model by TNF-α induction, and simultaneously incubated with 100 μM glycemic lowering protein peptide. The glycemic lowering protein peptide was the KILDK glycemic lowering protein peptide obtained in step two of Example 1.

[0079] All the glycemic-lowering protein peptides added to the above-mentioned glycemic-lowering protein peptide treatment groups were artificially synthesized.

[0080] After incubation, glucose-free medium (Gibco, USA, catalog number 11966025) was added and incubated for 2 hours. Then, 800 nM insulin (Solepro Science & Technology Co., Ltd., Beijing, catalog number I8040) was added and incubated at 37°C for 30 minutes. After washing with PBS, 100 μM 2-NBDG (Life Technologies, USA, catalog number N13195) was added and incubated for 30 minutes. Finally, following the method described in the literature "Li, G., Luan, GX, He, YF, Tie, FF, Wang, ZH, Suo, YR, Ma, CJ, & Wang, H. (2018). Polyphenol stilbenes from fenugreek (trigonella foenum-graecum L.) seed improve insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. Oxidative Medicine and Cellular Longevity, 2018, 7634362", flow cytometry was used to determine the glucose uptake of adipocytes.

[0081] II. Experimental Results

[0082] The experimental results are shown in Table 1. The results indicate that treatment with 100 μM glycemic-lowering protein peptide significantly increased insulin-stimulated glucose uptake, demonstrating that the glycemic-lowering protein peptide prepared in this invention has a significant promoting effect on cellular glucose uptake. Among these, treatment with 100 μM glycemic-lowering protein peptide KILDK resulted in the most significant increase in insulin-stimulated glucose uptake.

[0083] Table 1. Effects of glucagon-lowering protein peptides on TNF-α-induced cellular glucose uptake.

[0084] Basal glucose intake Insulin-stimulated glucose uptake Blank control group <![CDATA[0.23±0.03 a ]]> <![CDATA[1.00±0.07 a ]]> TNF-α model group <![CDATA[0.17±0.02 b ]]> <![CDATA[0.26±0.04 e ]]> 100μM VSLPEWV treatment group <![CDATA[0.20±0.01 b ]]> <![CDATA[0.49±0.04 d ]]> 100μM FLDDDL treatment group <![CDATA[0.19±0.01 b ]]> <![CDATA[0.44±0.04 d ]]> 100μM KILDK treatment group <![CDATA[0.20±0.02 ab ]]> <![CDATA[0.95±0.05 c ]]>

[0085] Note: Different letters in the same column of the table represent significant differences.

[0086] Experiment Example 3: Effect of Glucose-Lowering Protein Peptides on Cellular Glucose Consumption

[0087] I. Experimental Materials and Methods

[0088] 1. Experimental Materials

[0089] 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC), ATCC number CL-173.

[0090] 2. Experimental Methods

[0091] 3T3-L1 preadipocytes were cultured in DMEM high-glucose medium. After the cells differentiated into 3T3-L1 mature adipocytes, 4 ng / mL TNF-α and 100 μM glycemic protein peptide were added and co-incubated for 4 days, with the medium changed once a day.

[0092] The experiment was divided into the following five groups:

[0093] Blank control group: 3T3-L1 mature adipocytes, without TNF-α induction and without intervention from hypoglycemic protein peptides.

[0094] TNF-α model group: 3T3-L1 mature adipocytes were used to establish an insulin resistance model by induction with TNF-α, but without intervention by hypoglycemic protein peptides.

[0095] 100 μM VSLPEWV treatment group: 3T3-L1 mature adipocytes were used to establish an insulin resistance model by TNF-α induction, and simultaneously incubated with 100 μM glycemic protein peptide. The glycemic protein peptide was VSLPEWV obtained in step two of Example 1.

[0096] 100 μM FLDDDL treatment group: 3T3-L1 mature adipocytes were used to establish an insulin resistance model by TNF-α induction, and simultaneously incubated with 100 μM glycemic protein peptide. The glycemic protein peptide was FLDDDL obtained in step two of Example 1.

[0097] 100 μM KILDK treatment group: 3T3-L1 mature adipocytes were used to establish an insulin resistance model by TNF-α induction, and simultaneously incubated with 100 μM glycemic protein peptide. The glycemic protein peptide was the KILDK glycemic protein peptide obtained in step two of Example 1.

[0098] All the glycemic-lowering protein peptides added to the above-mentioned glycemic-lowering protein peptide treatment groups were artificially synthesized.

[0099] After incubation, 800 nM insulin was added, and the mixture was incubated at 37°C for 30 min. The culture medium was collected, and the glucose concentration was determined using a glucose test kit (Nanjing Jiancheng Bioengineering Institute, catalog number A154-1-1). The glucose consumption rate was calculated according to Formula 1:

[0100] Glucose consumption rate (%) = (C 空白组 -C 实验组 ) / C 空白组 Formula 1

[0101] Among them, C 空白组 : Glucose concentration in the culture medium of the blank group (containing only culture medium and no cells); C 实验组 The glucose concentration in the culture medium of the blank control group, the TNF-α model group, and each hypoglycemic protein peptide treatment group.

[0102] II. Experimental Results

[0103] The experimental results are shown in Table 2. The results indicate that treatment with 100 μM glycemic lowering protein peptide significantly increased the glucose consumption rate stimulated by insulin, demonstrating that the glycemic lowering protein peptide prepared in this invention has a significant promoting effect on cellular glucose utilization. Among these, treatment with 100 μM glycemic lowering protein peptide KILDK resulted in the most significant increase in the glucose consumption rate stimulated by insulin.

[0104] Table 2. Effects of glucose-lowering protein peptides on TNF-α-induced cellular glucose consumption rate

[0105] Basal glucose consumption rate Insulin-stimulated glucose consumption rate Blank control group <![CDATA[27.29%±1.66% a ]]> <![CDATA[45.83%±1.46% a ]]> TNF-α model group <![CDATA[22.12%±2.03% c ]]> <![CDATA[35.63%±1.18% f ]]> 100μM VSLPEWV treatment group <![CDATA[22.67%±1.12% b ]]> <![CDATA[42.37%±0.79% cd ]]> 100μM FLDDDL treatment group <![CDATA[23.00%±0.88% b ]]> <![CDATA[41.20%±1.11% de ]]> 100μM KILDK treatment group <![CDATA[20.94%±1.09% a ]]> <![CDATA[51.94%±1.59% de ]]>

[0106] Note: Different letters in the same column of the table represent significant differences.

[0107] Experimental Example 4: Effect of lactase hydrolysate on the insulin tolerance test (IPITT) in mice.

[0108] I. Experimental Materials and Methods

[0109] 1. Experimental Materials

[0110] The lactase hydrolysate obtained in step 1 of Example 1.

[0111] 2. Experimental Methods

[0112] Thirty male C57BL / 6 mice aged 5-6 weeks were randomly divided into three groups (control group, model group, and experimental group), with 10 mice in each group. The model group and experimental group were intraperitoneally injected with sodium citrate buffer containing streptozotocin (STZ) (Sigma-Aldrich, catalog number S0130), at a dose of 30 mg / kg, for five consecutive days to establish a type 2 diabetic mouse model. The control group received an equal volume of sodium citrate buffer. Five days later, the experimental group was administered lactase hydrolysate (400 mg / kg) by gavage daily, while the control group and model group were administered an equal volume of physiological water by gavage for eight consecutive weeks. Before the experiment, mice were fasted for 6 hours but allowed free water, and basal blood glucose levels (0 min) were measured. After 8 weeks of continuous gavage administration of lactase hydrolysate, insulin tolerance tests were conducted by intraperitoneal injection of insulin (0.75 U / kgb.w.). Blood glucose levels were measured at 30 min, 60 min and 120 min after insulin injection using a Roche blood glucose meter with tail tip sampling.

[0113] II. Experimental Results

[0114] The experimental results are shown in Figure 2 The results showed that, compared with the type 2 diabetes model mice, the mice treated with lactase hydrolysate experienced a faster decrease in blood glucose levels and a lower area under the curve (AUC) after insulin injection, indicating that it improved insulin tolerance in type 2 diabetes mice and had a stable hypoglycemic effect.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> China Agricultural University <120> Preparation methods and applications of food protein peptides with hypoglycemic activity <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <400> 1 Val Ser Leu Pro Glu Trp Val 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <400> 2 Phe Leu Asp Asp Asp Leu 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Lys Ile Leu Asp Lys 1 5

Claims

1. A protein peptide, the amino acid sequence of which is shown in Sequence 3.

2. The use of the protein peptide according to claim 1 in the following (X1)-X2); X1) Prepare products that lower blood sugar; X2) Prepare products for the prevention and / or treatment of diabetes.

3. The application according to claim 2, characterized in that: The product in question is a pharmaceutical product.

4. A product, wherein the active ingredient is the protein peptide of claim 1; the function of the product is any one of the following Y1)-Y2): Y1) Lowers blood sugar; Y2) Prevention and / or treatment of diabetes.

5. The product according to claim 4, characterized in that: The product in question is a pharmaceutical product.

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