A bioactive peptide with weight loss efficacy, a preparation thereof and applications thereof

Abstract

The present invention discloses a bioactive peptide, a preparation and an application thereof with a weight loss effect, belonging to the technical field of bioactive peptides. The amino acid sequences of the bioactive peptide with a weight loss effect of the present invention are at least one of QLGIK and NTDKQVT. The present invention isolates and purifies two bioactive peptides with a weight loss effect from defatted Antarctic krill protein, and preliminarily identifies their components and sequences by liquid chromatography-mass spectrometry technology. Chemical synthesis is carried out according to the identified peptide sequences, and the weight loss effects of the synthesized single peptide and compound peptide are detected. These bioactive peptides have good inhibitory activities against pancreatic lipase and α-amylase, and thus have good application prospects in the preparation of products with a weight loss effect, laying a foundation for the high-value utilization of Antarctic krill protein and promoting the research and development and application of functional active peptides.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and particularly relates to a bioactive peptide with weight loss efficacy, a preparation thereof, and applications thereof. Background Art

[0002] The fat component in food is mainly triglyceride. After the food is ingested by the body, the fat insoluble in water is first emulsified into chylomicrons in the stomach so as to be hydrolyzed by water-soluble lipase in the duodenum. Then the lipase hydrolyzes to generate fatty acids and monoglycerides, which become soluble mixed micelles under the action of bile acid micelles, and are then absorbed in the gastrointestinal tract, and the body re-synthesizes fat. If too much high-fat food is ingested, it will eventually cause the accumulation of body fat and form obesity. In order to effectively prevent, control, and treat obesity and reduce the adverse effects caused by obesity on the body, many technical methods have been studied and tried. Among them, the methods for controlling and treating obesity include physical and chemical methods. The physical method refers to reducing fat by controlling diet, exercise, and liposuction surgery; the chemical method mainly achieves the purpose of reducing fat through the chemical action of some drugs and health foods. Among these technologies, taking weight loss drugs is an effective and convenient method for preventing or assisting in the treatment of weight loss diseases.

[0003] Currently, functional food ingredients with lipid-lowering and weight loss effects that have been discovered in research include substances such as alkaloids, saponins, L-carnitine, enzymes, and phenolphthalein. The weight loss drugs composed of these active substances often achieve the purpose of controlling weight or losing weight by affecting gastrointestinal reactions and regulating the nervous system, reducing appetite or inhibiting the absorption of glucose, protein, and lipids in the gastrointestinal tract. However, their side effects are often proportional to the dosage of the drug, that is, the greater the dosage, the greater the side effects, which can cause insomnia, fatigue, diarrhea, vomiting, elevated blood pressure, etc., and even tachycardia, arrhythmia, and heart failure. The side effects and safety hazards of weight loss drugs have become the key factors restricting their application in the food industry.

[0004] The molecular weight of bioactive peptides is generally less than 6000 Da, containing at least 2 amino acids, having good functionality and special activity, and can regulate special physiological functions and life activities of the body. They play key roles in a variety of biological processes, including hormone regulation, immune response, and cell signal transduction. Natural bioactive peptides with weight loss efficacy isolated from natural substances, including functional peptides with the functions of inhibiting pancreatic lipase and α-amylase activities and active peptides that reduce fat content by regulating appetite hormones and affecting the proliferation and differentiation of preadipocytes, are a kind of safe and side-effect-free functional ingredient, and have great potential to replace traditional weight loss drugs. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a bioactive peptide with weight loss efficacy, a preparation thereof and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A bioactive peptide with weight loss efficacy, the amino acid sequence of the bioactive peptide with weight loss efficacy is at least one of QLGIK and NTDKQVT.

[0008] The application of the above-mentioned bioactive peptide with weight loss efficacy in the preparation of a product with weight loss efficacy.

[0009] On the basis of the above solution, the product is a food, a drug or a health product.

[0010] On the basis of the above solution, the product further contains excipients acceptable in food, pharmacy or health products.

[0011] A bioactive peptide preparation with weight loss efficacy, the active ingredient of the preparation is a bioactive peptide shown by at least one of QLGIK and NTDKQVT.

[0012] On the basis of the above solution, the concentration of the active ingredient in the bioactive peptide preparation with weight loss efficacy is 0.5 mg / mL.

[0013] On the basis of the above solution, the weight loss efficacy is to have pancreatic lipase and α-amylase inhibitory activities.

[0014] Advantages of the technical solution of the present invention

[0015] The present invention uses defatted Antarctic krill protein as a raw material, and separates bioactive peptides with weight loss efficacy through enzymatic hydrolysis, ultrafiltration, gel chromatography and liquid chromatography separation techniques, and preliminarily identifies their components and sequences by liquid chromatography-mass spectrometry technology. And according to the identified peptide sequences, chemical synthesis is carried out to obtain two bioactive peptides QLGIK and NTDKQVT with weight loss efficacy. After detection, both peptide segments and their compound peptide segments have good pancreatic lipase and α-amylase inhibitory activities, and have good application prospects in the preparation of products with weight loss efficacy, laying a foundation for the high-value utilization of Antarctic krill protein and promoting the research and application of functional active peptides.

[0016] Traditional weight loss drugs, such as orlistat, Qsymia, phenolphthalein, etc., may cause endocrine disorders, nutritional imbalances, and increased nerve excitability in the body. Long-term use will affect the normal secretion function of the gastrointestinal tract and pose potential health risks. The bioactive peptide isolated in this invention has a good weight loss effect. The raw material is the natural and non-toxic Antarctic krill protein, which is green and safe. No organic reagents are introduced during the preparation process, and there are no side effects. The operation is simple, and the requirements for equipment are relatively low. It has the potential to be used as a new weight loss product in the field of health food. Description of the Drawings

[0017] Figure 1 Effect of different proteases on the hydrolysis degree of Antarctic krill protein (wherein, the data marked with different lowercase letters are significantly different, P < 0.05);

[0018] Figure 2 Effect of different proteases on the pancreatic lipase inhibitory activity of Antarctic krill enzymolysis products (wherein, the data marked with different lowercase letters are significantly different, P < 0.05);

[0019] Figure 3 Inhibitory activity of components with different molecular weights after ultrafiltration on pancreatic lipase (wherein, the data marked with different lowercase letters are significantly different, P < 0.05);

[0020] Figure 4 Inhibitory activity of components with different molecular weights after ultrafiltration on α-amylase (wherein, the data marked with different lowercase letters are significantly different, P < 0.05);

[0021] Figure 5 Separation and purification of components by gel filtration chromatography;

[0022] Figure 6 Inhibitory activity of each component after gel filtration chromatography on pancreatic lipase (wherein, the data marked with different lowercase letters are significantly different, P < 0.05);

[0023] Figure 7 Inhibitory activity of each component after gel filtration chromatography on α-amylase (wherein, the data marked with different lowercase letters are significantly different, P < 0.05);

[0024] Figure 8 Secondary mass spectrometry diagram of Antarctic krill peptide segment QLGIK;

[0025] Figure 9 Secondary mass spectrometry diagram of Antarctic krill peptide segment NTDKQVT;

[0026] Figure 10 Primary mass spectrometry diagram of biosynthetic peptide segment QLGIK;

[0027] Figure 11First mass spectrometry of biosynthetic peptide NTDKQVT;

[0028] Figure 12 Inhibitory activities of various biosynthetic peptides against pancreatic lipase (where the data marked with different lowercase letters are significantly different, P < 0.05);

[0029] Figure 13 Inhibitory activities of various peptides after biosynthesis against α-amylase (where the data marked with different lowercase letters are significantly different, P < 0.05). Specific implementation manners

[0030] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way.

[0031] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The test materials, reagents, drugs, etc. used in the following examples can be obtained through general channels unless otherwise specified.

[0032] In the following examples,

[0033] 1. Method for determining the degree of hydrolysis of Antarctic krill protein:

[0034] Dissolve 80 mg of OPA in 2 mL of absolute ethanol, 200 μL of β-mercaptoethanol, 5 mL of 10% SDS (w / v), and 92.8 mL of 0.1 mol / L sodium tetraborate, and prepare a 100 mL OPA reagent solution. Take 40 μL of the Antarctic krill enzymatic hydrolysate and mix it with 4 mL of the OPA reagent, incubate at room temperature for 2 min, and measure the absorbance at a wavelength of 340 nm. Place the Antarctic krill protein in 6 mol / L HCl and react at 115 °C for 24 h. Use the serine standard curve to determine the number of free amino groups, which is used as the number of free amino acids produced by the complete hydrolysis of Antarctic krill protein.

[0035] The degree of hydrolysis of Antarctic krill protein is calculated according to the following formula:

[0036]

[0037] In the formula: (NH2) t represents the number of free amino acids in the enzymatic hydrolysate at time t; (NH2) t0 represents the number of free amino acids without enzymatic hydrolysis; (NH2) TIndicates the number of free amino acids after complete hydrolysis.

[0038] 2. Method for determining pancreatic lipase inhibitory activity:

[0039] Using p-nitrophenyl butyrate (0.005 mol / L) as the substrate, take the enzymolysis product of Euphausia superba and pancreatic lipase (1.25 mg / mL) in PBS (0.01 mol / L, pH 7.4), place them in a 96-well plate and adjust the volume of the reaction mixture to 150 μL with PBS, incubate at 37 °C for 30 min, select a multifunctional microplate reader, and measure the absorbance at a wavelength of 405 nm. The composition of each component of the reaction system is shown in Table 1.

[0040] Table 1 Composition of each system for determining pancreatic lipase inhibitory activity

[0041]

[0042] Result calculation: The inhibitory effect of the enzymolysis product of Euphausia superba on lipase activity is calculated according to the following formula:

[0043]

[0044] Where, I: Inhibition rate of the enzymolysis product of Euphausia superba on pancreatic lipase; A: Blank tube; a: Blank control tube; B: Inhibition tube; b: Background control tube; " / " indicates no sample addition.

[0045] 3. Method for determining α-amylase inhibitory activity

[0046] Using soluble starch as the substrate and 3,5-dinitrosalicylic acid (DNS) as the color reagent, the α-amylase activity is determined by spectrophotometry. Add 0.25 mL of 4 units / mL α-amylase and 0.25 mL of the enzymolysis product of Euphausia superba to 0.5 mL of 0.2 mol / L PBS at pH 6.9 respectively, incubate at 37 °C for 10 min, then add 0.5 mL of 1% soluble starch solution, react accurately for 5 min, finally add 1 mL of DNS reagent, boil for 10 min, quickly cool with running water, dilute five times, and measure the absorbance at a wavelength of 540 nm. The composition of each component of the reaction system is shown in Table 2.

[0047] Table 2 Composition of each system for determining α-amylase activity

[0048]

[0049] Result calculation: The inhibitory effect of the enzymolysis product of Euphausia superba on α-amylase activity is calculated according to the following formula:

[0050]

[0051] Wherein, I: the inhibition rate of the enzymolysis product of Antarctic krill on α-amylase; A: blank tube; a: blank control tube; B: inhibition tube; b: background control tube; " / " indicates no sample addition.

[0052] The Antarctic krill used in the examples was purchased from Qingdao Antarctic Wellcome Biotechnology Co., Ltd. (Qingdao, China).

[0053] Example 1

[0054] A bioactive peptide with weight loss efficacy, and the amino acid sequence of the bioactive peptide is shown as SEQ ID NO:1 or SEQ ID NO:2.

[0055] SEQ ID NO:1: QLGIK;

[0056] SEQ ID NO:2: NTDKQVT.

[0057] Example 2

[0058] A method for extracting a bioactive peptide with weight loss efficacy from Antarctic krill, the steps are as follows:

[0059] (1) Weigh 100 g of washed Antarctic krill meat without head and shell, place it in a beaker, and homogenize it with deionized water (4 °C) with a liquid-to-solid ratio of 3.17 mL / g. Subsequently, adjust the pH to 11.38 with 2 mol / L sodium hydroxide, let it stand for 0.5 h, and then centrifuge at 10000 g for 10 min at 4 °C to obtain the supernatant. The whole extraction process is repeated 3 times. Finally, adjust the pH of the collected supernatant to 4.5 with 2 mol / L phosphoric acid, let it stand for 1.0 h, centrifuge at 10000 g for 10 min at 4 °C, collect the precipitate and freeze-dry it at -80 °C for storage, which is Antarctic krill protein.

[0060] (2) Weigh 5 g of the Antarctic krill protein extracted in step (1), add acetone solution according to the ratio of 1:4 (w:v), stir with a constant temperature magnetic stirrer for 3 h and then let it stand for 0.5 h until the liquid is clear. The upper red liquid is Antarctic krill oil. After pouring it out, continue to add acetone, stir for 2 h and then let it stand for 0.5 h. Repeat the above operation until the upper liquid is colorless. Recover the defatted Antarctic krill protein in the fume hood.

[0061] (3)Weigh the defatted Antarctic krill protein prepared in step (2) and dissolve it in deionized water at a ratio of 2% (w / v). Add alkaline protease to the solution. The addition amount of alkaline protease is 3000 U of alkaline protease per gram of Antarctic krill protein. After stirring evenly at low speed, adjust the pH value to 9.5 and the temperature to 55 °C, and place it in a constant temperature incubator for enzymatic reaction for 4 h. After enzymatic hydrolysis, heat the reaction solution in boiling water for 15 min to inactivate the protease, terminate the reaction, and quickly cool it to 4.0 °C with ice water. Centrifuge at 12000 g for 15 min and retain the supernatant (referred to as AKPH), and freeze-dry it to obtain the enzymatic hydrolysis product of Antarctic krill protease.

[0062] Effects of Different Proteases on the Degree of Hydrolysis and Pancreatic Lipase Inhibitory Activity of Enzymatic Hydrolysis Products of Antarctic Krill

[0063] Use acid protease, neutral protease, trypsin, pepsin, and papain to carry out enzymatic hydrolysis of Antarctic krill protein respectively. The temperature and pH of enzymatic hydrolysis are carried out at the optimal temperature and pH of each protease (Table 3), and the remaining conditions are the same as those of alkaline protease. After enzymatic hydrolysis is completed, measure the effects of different proteases on the degree of hydrolysis and pancreatic lipase inhibitory activity of the enzymatic hydrolysis products of Antarctic krill. The results are as Figure 1 、 Figure 2 shown.

[0064] Table 3 Optimal Temperature and Optimal pH of Different Proteases

[0065]

[0066] The degree of protein hydrolysis is one of the important indicators to evaluate the biological activity of enzymatic hydrolysis products. Different types of proteases have different degrees of hydrolysis of proteins at different times. A good hydrolysis effect can release more biologically active groups. As Figure 1 can be seen, the degree of hydrolysis of alkaline protease on Antarctic krill protein is 38.64% ± 1.15, which is significantly higher than that of the enzymatic hydrolysis products of the other 5 proteases (P < 0.05). This is because under the condition of sufficient substrate, alkaline protease has more cleavage sites, good heat resistance and hydrolysis performance, and the protein is more easily hydrolyzed into small molecular peptides.

[0067] Some dietary fats in the human body can be hydrolyzed by pancreatic lipase. When the activity of pancreatic lipase is inhibited by small molecular peptides, dietary fats cannot be decomposed into short-chain fatty acids, resulting in the body's inability to absorb fats. Therefore, the inhibitory activity of Antarctic krill enzymatic hydrolysis products on pancreatic lipase is selected as the main index to evaluate its lipid-lowering and weight-loss activity. As Figure 2It can be seen that, compared with the other five proteases, the enzymatic hydrolysate obtained by alkaline protease has a stronger inhibitory effect on pancreatic lipase, which is 34.20% ± 1.28. Alkaline protease is a serine protease, and the active center of such proteases contains serine residues. Using alkaline protease for digestion is more conducive to improving the lipid-lowering and weight-loss activity. Therefore, alkaline protease is selected as the best hydrolysis enzyme.

[0068] The amino acid composition of the enzymatic hydrolysate of Antarctic krill protease was analyzed using an amino acid analyzer. The steps are as follows:

[0069] The freeze-dried sample was treated with 6 mol / L HCl at 110 °C for 24 h under a nitrogen atmosphere. The digested mixture was transferred to a centrifuge tube and dried under reduced pressure at 50 °C. The dried sample was dissolved in distilled water twice. Finally, the dried sample was dissolved in sodium citrate buffer (pH 2.2) and filtered through a 0.22 µm filter before being tested on the machine. The results are shown in Table 4.

[0070] Table 4 Amino acid composition and content of the enzymatic hydrolysate of alkaline protease

[0071]

[0072] The efficacy of bioactive peptides is closely related to the amino acid composition and content and their arrangement order on the peptide chain. As can be seen from Table 4, the content of hydrophobic amino acids such as valine, leucine, phenylalanine, and isoleucine in the enzymatic hydrolysate of alkaline protease is relatively rich, with contents of 3.05 g / 100 g, 3.03 g / 100 g, 2.57 g / 100 g, and 2.16 g / 100 g, respectively. These amino acids help to improve the biological activity of small peptides and have potential lipid-lowering and weight-loss activities, which are consistent with the structure and functional characteristics of weight-loss peptides. At the same time, the content of essential amino acids such as lysine, tryptophan, methionine, and threonine in the enzymatic hydrolysate of Antarctic krill is also very rich, with high nutritional value.

[0073] (4) The enzymatic hydrolysate of Antarctic krill alkaline protease was fractionated into different molecular weights using 10 KDa and 3 KDa ultrafiltration centrifugal tubes. The ultrafiltration parameters were set to 4000 × g for 25 min until all the liquid passed through the membrane. The fractions >10 KDa, 3 - 10 KDa, and <3 KDa of the peptide segments were collected. All fractions were freeze-dried and stored in a sealed bottle at -20 °C, and the inhibitory activities of pancreatic lipase and α-amylase of different molecular weight fractions were measured to analyze their weight-loss efficacy.

[0074] Ultrafiltration centrifugal tubes can efficiently and quickly concentrate small molecules such as peptides, nucleic acids, antigens, and antibodies, and have advantages including high flow rate, high throughput, protein adsorption characteristics with high concentration multiples, and high recovery rates. The soluble protein hydrolysate produced by alkaline protease was ultrafiltered using an ultrafiltration centrifugal tube and divided into three parts: MW > 10KDa, MW between 3 and 10KDa, and MW < 3KDa. The separation and enrichment of small molecule peptides were achieved through ultrafiltration separation means. From Figure 3 it can be seen that the inhibitory rate (56.99% ± 2.17) of the fraction with MW < 3kDa on pancreatic lipase was significantly higher (P < 0.05) than that of the two fractions with MW > 10KDa and MW between 3 and 10KDa (30.88% ± 2.91, 47.37% ± 4.93), and it could better inhibit the activity of pancreatic lipase. This may be because macromolecules are transformed into small molecule active fragments, the fraction with a smaller molecular weight has a higher content of short peptides, and short peptides are more likely to exhibit better biological activity.

[0075] α - Amylase can decompose starch, and amylase inhibitors reduce carbohydrate absorption by hindering the decomposition of starch into glucose in the small intestine. Therefore, the inhibitory activity of Antarctic krill enzymolysis products on α - amylase was selected as another index to evaluate its lipid - lowering and weight - reducing activities. From Figure 4 it can be seen that the lipid - lowering and weight - reducing activity of the fraction with MW < 3kDa was the most obvious, its inhibitory activity on α - amylase was 19.03% ± 0.76, and there were more short peptides with a smaller molecular weight retained in the protease hydrolysate. All in all, the fraction with MW < 3kDa could significantly (P < 0.05) inhibit the activities of pancreatic lipase and α - amylase, and had the potential of lipid - lowering and weight - reducing functional characteristics, so it was selected for further separation and purification.

[0076] (5)The fraction with ultrafiltered MW < 3kDa was further separated and purified using a Sephadex G - 25 gel filtration column (2.0 * 40 cm). First, it was equilibrated with ultrapure water, and then eluted with ultrapure water at a flow rate of 0.3 mL / min. Each part of the eluate was monitored at 280 nm, and the collected peaks were obtained.

[0077] The enzymolysis fraction with MW < 3KDa was further separated and purified on a Sephadex G - 25 gel filtration column. The elution results are as Figure 5 shown. The distilled water elution fraction showed a total of three peaks, named F1, F2, and F3 respectively, and the eluted fractions were collected according to the elution time. The lower the molecular weight, the longer the retention time of the separated peptide. Therefore, it can be inferred that F1 has the largest molecular weight, F2 was the second to be separated out in the chromatographic column, with a molecular weight in the middle, and F3 has the smallest molecular weight.

[0078] The weight - reducing effects of the above three different elution fractions were evaluated. The results are as Figure 6 andFigure 7 as shown

[0079] It can be seen from Figure 6 that the F2 fraction obtained by separation on a Sephadex G-25 gel filtration column had the highest pancreatic lipase inhibitory activity, which was 64.11 ± 0.36%, 1.87 times that of the enzymatic hydrolysis product before separation, and there were significant differences between it and other peaks (P < 0.05). The F1 fraction and the F3 fraction were 36.10 ± 0.48% and 52.14 ± 2.05% respectively.

[0080] It can be seen from Figure 7 that the α-amylase inhibitory activity of the F2 fraction obtained by separation on a Sephadex G-25 gel filtration column was significantly higher than that of the enzymatic hydrolysis product before separation and the F1 and F3 fractions (P < 0.05), reaching 33.51% ± 0.57. This is mainly attributed to the fact that in natural protein molecules, their hydrophobic groups are wrapped in the folded structure regions inside the protein molecules, and when the protein is hydrolyzed, the internal hydrophobic groups will be exposed, which will in turn increase the surface hydrophobicity. As the separation and purification progress continuously, these exposed groups will be further concentrated into smaller peptide segments.

[0081] (6) Select the F2 fraction with the best weight loss effect after gel filtration chromatography for LC-MS / MS analysis. After centrifugation and drying, the digested polypeptide sample was redissolved in Nano-LC mobile phase A (0.1% formic acid / water), bottled and loaded for online LC-MS / MS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3μm, 100Å) for rinsing and desalting. The liquid phase was an EasynLC 1200 nanoliter liquid phase system (ThermoFisher, USA). After desalting and retention on the pre-column, the sample was separated by the analytical column. The specifications of the analytical column were a C18 reversed-phase chromatographic column (Acclaim PepMap RSLC, 75μm×25cm C18-2μm 100Å). The gradient used in the experiment was that mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30 minutes. The mass spectrometry was performed using a ThermoFisher QExactive system (ThermoFisher, USA) combined with a nano spray Nano Flex ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the heating temperature of the ion transfer tube was 275°C. The mass spectrometry scanning mode was in the data-dependent acquisition working mode (DDA, Data Dependent Analysis). The resolution of the first-level mass spectrometry scan was 70000, the scanning range was 350 - 2000 m / z, and the maximum injection time was 100 ms. In each DDA cycle, at most 20 secondary spectra with charges from 2+ to 5+ were collected, and the maximum injection time of the secondary mass spectrometry ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 25 seconds.

[0082] Table 5 Amino acid sequences of Antarctic krill weight loss peptides identified by LC-MS / MS

[0083]

[0084] The peptide mixture F2 was analyzed using an LC-MS / MS liquid chromatography-mass spectrometry high-resolution mass spectrometry platform to detect the mass-to-charge ratio (m / z) of the analyte and compare it with the theoretical value, so as to accurately identify the target analyte, which plays a key role in the identification of the amino acid sequence of polypeptides. The information of the two peptide segments identified in the sub-fraction F2 is shown in Table 5. The amino acid sequences were identified as Gln-Leu-Gly-Ile-Lys (QLGIK) and Asn-Thr-Asp-Lys-Gln-Val-Thr (NTDKQVT), and the number of amino acids was 5 and 7 respectively.

[0085] The secondary mass spectrometry diagrams of Antarctic krill weight loss peptide segments QLGIK and NTDKQVT are as Figure 8 、 Figure 9As shown, clear mass spectra were obtained from the peptide ions, and some b- and y-ions derived from the peptide ions were also shown.

[0086] Two peptide segments QLGIK and NTDKQVT (purity > 95%) obtained by LC-MS / MS analysis were synthesized by the biosynthetic method. Their primary mass spectra are as Figure 10 , Figure 11 shown. The measured exact molecular weights were 558.4 and 805.6 respectively, and they were compared with the previous theoretical values. The molecular weights were basically the same, verifying the accuracy of the synthesized peptides.

[0087] Example 3

[0088] A bioactive peptide preparation with weight loss efficacy, and its active ingredient is at least one of the bioactive peptides with the amino acid sequences shown as QLGIK and NTDKQVT.

[0089] Accurately weigh 2 mg of QLGIK (QK) and NTDKQVT (NT) respectively, dissolve them in 4 mL of deionized water, and dissolve them mutually to prepare the composite peptide segment QLGIK + NTDKQVT (QK + NT). The concentration of each peptide segment is 0.5 mg / mL. Its lipid-lowering and weight loss activities were further verified by measuring the inhibitory activities of pancreatic lipase and α-amylase. The results are as Figure 12 , Figure 13 shown. The three peptide segments QK, NT, and QK + NT all showed high lipid-lowering and weight loss activities. The inhibitory activities of pancreatic lipase were 74.08% ± 1.40, 65.52% ± 1.98, and 69.78% ± 0.73 respectively, and the inhibitory activities of α-amylase were 43.47% ± 1.25, 36.83% ± 0.74, and 40.18% ± 0.72 respectively. Among them, the inhibitory activities of pancreatic lipase and α-amylase of QK were significantly higher than those of NT and QK + NT (P < 0.05). This may be because the molecular weights of the two peptide segments are different, and the molecular weight is an important factor affecting the biological activity of polypeptides. At the same concentration, the smaller the molecular weight of the peptide segment, the stronger the overall biological activity. QK has the lowest molecular weight, giving it the strongest lipid-lowering and weight loss activity.

[0090] The above results show that both the two peptide segments QLGIK and NTDKQVT have appropriate amino acid numbers, structures, and peptide segment lengths, meeting the characteristics of natural weight loss peptides, which is beneficial to the development and utilization of bioactive peptides and weight loss products, and has great potential as a functional ingredient in the fields of health food and cosmetic medicine.

[0091] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in any other form. Any person skilled in the relevant art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A bioactive peptide with weight loss effect, characterized in that: The amino acid sequence of the bioactive peptide with weight loss effect is any one of QLGIK and NTDKQVT.

2. Use of the bioactive peptide with weight loss effect as claimed in claim 1 in the preparation of a product with weight loss effect.

3. The use of the bioactive peptide with weight loss effect according to claim 2 in the preparation of a product with weight loss effect, characterized in that: The product is food, medicine or health product.

4. The use of the bioactive peptide with weight loss effect according to claim 3 in preparing a product with weight loss effect, characterized in that: The product further comprises excipients acceptable to food, medicine or health care products.

5. A bioactive peptide preparation having weight loss effect, characterized in that: The active ingredient of the preparation is at least one of the bioactive peptides shown in QLGIK and NTDKQVT.

6. The bioactive peptide preparation with weight loss efficacy according to claim 5, characterized in that: The concentration of the active ingredient is 0.5 mg / mL.

7. The bioactive peptide preparation with weight loss effect according to claim 5 or 6, characterized in that: The weight loss effect is that it has the activity of inhibiting pancreatic lipase and α-amylase.

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