Oligopeptide containing amino acid structure PQQP and preparation method and application thereof

Abstract

The present invention relates to an oligopeptide containing an amino acid structure PQQP, and a preparation method and application thereof. The oligopeptide containing a PQQP sequence of the present invention has a novel structure and is reported by the inventor for the first time. The oligopeptide can be artificially synthesized by a chemical solid phase synthesis method, or can be obtained by enzymatic hydrolysis of barley protein and separation and purification. The oligopeptide containing a PQQP sequence has a molecular weight of less than 1500 Da, can significantly promote the secretion of PYY in intestinal endocrine cells, and has the characteristics of being safe, non-toxic, non-side-effect, resistant to enzymatic hydrolysis by gastrointestinal digestive enzymes, and easy to absorb. It can be used in health foods and medicines as a functional component for improving satiety / suppressing appetite, and has very important application value.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and in particular relates to oligopeptides containing an amino acid structure PQQP and a preparation method and application thereof. Background Art

[0002] Metabolic syndrome is a group of clinical syndromes characterized by high incidence of obesity, hyperglycemia, dyslipidemia, etc., which seriously affect the health of the body. It is characterized by a combination of multiple metabolic disorders, including obesity, hyperglycemia, hypertension, dyslipidemia and hyperinsulinemia. These metabolic disorders are the pathological basis of cardiovascular and cerebrovascular diseases and diabetes. Current research believes that the common cause of metabolic syndrome is insulin resistance and hyperinsulinemia caused by obesity, especially central obesity. Therefore, reducing weight and insulin resistance is an important measure to prevent and treat metabolic syndrome.

[0003] In order to reduce body weight and insulin resistance, limiting the body's food intake is the best means. The hypothalamus and digestive tract, the body's feeding center, are key parts of the body's control of food intake regulation, and many brain-gut peptides related to food intake regulation are distributed in them. Peptide YY (PYY) is a gastrointestinal peptide hormone composed of 36 amino acids, which is mainly synthesized and secreted by endocrine L cells distributed in the ileum and colon mucosa. PYY is released by the gastrointestinal tract after a meal and is considered to be a physiological regulator of food intake and is related to long-term energy balance. Animal and clinical studies have confirmed that PYY has the function of suppressing appetite, reducing energy intake, and reducing body weight. As an appetite factor related to obesity, PYY has a certain effect on improving obesity and diabetes. Therefore, increasing the secretion of intestinal PYY is of great significance for reducing weight and insulin resistance, and then preventing and treating metabolic syndrome. The secretion of PYY is regulated by dietary factors. Therefore, the secretion of intestinal PYY can be regulated by diet to prevent and treat metabolic syndrome.

[0004] Bioactive peptides are a common dietary factor that is easily digested and absorbed by the human body and is highly safe to eat. The country attaches great importance to the development of the bioactive peptide industry. The National Development and Reform Commission and the Ministry of Industry and Information Technology clearly pointed out in the "Guiding Opinions on Promoting the Healthy Development of the Food Industry" that it is necessary to accelerate the development of functional foods, support the development of health and wellness foods such as bioactive peptides, and carry out application demonstrations. In view of the important role of PYY and the many advantages of bioactive peptides, it is of great value to find a bioactive peptide or extract that can promote the secretion of PYY. Summary of the invention

[0005] The purpose of the present invention is to provide an oligopeptide containing the amino acid structure PQQP and a preparation method and application thereof.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect, the present invention provides an oligopeptide containing an amino acid structure PQQP, which is a bioactive peptide that promotes intestinal PYY secretion, and is selected from one or more of the following seven bioactive peptides:

[0008] QPQQPF, the amino acid sequence is Gln-Pro-Gln-Gln-Pro-Phe, as shown in SEQ ID NO.1;

[0009] PQQPQPF, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Pro-Phe, as shown in SEQ ID NO.2;

[0010] LPQQPFPV, the amino acid sequence is Leu-Pro-Gln-Gln-Pro-Phe-Pro-Val, as shown in SEQ ID NO. 3;

[0011] QPFPQQPQ, the amino acid sequence is Gln-Pro-Phe-Pro-Gln-Gln-Pro-Gln, as shown in SEQ ID NO.4;

[0012] PQQPFLQPL, the amino acid sequence is Pro-Gln-Gln-Pro-Phe-Leu-Gln-Pro-Leu, as shown in SEQ ID NO.5;

[0013] QPQPYPQQP, the amino acid sequence is Gln-Pro-Gln-Pro-Tyr-Pro-Gln-Gln-Pro, as shown in SEQ ID NO.6;

[0014] PQQPQQPFFQP, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Phe-Gln-Pro, as shown in SEQ ID NO.7.

[0015] Preferably, the seven bioactive peptides are all derived from barley protein.

[0016] Barley protein contains a variety of protein subunits, among which the amino acid sequences of C-hordein, gamma prolamin and Alphaprolamin are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively. It can be found that oligopeptides containing the PQQP sequence exist in large quantities in barley protein. The oligopeptides containing the PQQP sequence can be prepared from barley protein using the above method.

[0017] The oligopeptide containing the amino acid structure PQQP provided by the present invention is derived from barley protein, and the oligopeptide containing the amino acid structure PQQP can significantly promote the secretion of intestinal peptide YY (PYY), has the characteristics of being safe, non-toxic and non-side effects, and being resistant to enzymatic hydrolysis by gastrointestinal digestive enzymes. It can be used as a functional component for improving satiety / suppressing appetite in health foods and medicines, and has very important application value.

[0018] In a second aspect, the present invention provides a polynucleotide encoding the oligopeptide containing the amino acid structure PQQP.

[0019] The third aspect of the present invention provides a method for preparing the oligopeptide containing the amino acid structure PQQP, which is artificially synthesized by genetic engineering methods, or directly obtained from barley protein by separation and purification methods, or directly prepared by chemical synthesis.

[0020] Artificially synthesizing the oligopeptide containing the amino acid structure PQQP by genetic engineering is a technical solution that can be realized by those skilled in the art. For example, it can be based on DNA recombination technology and control the sequence synthesis of the polypeptide through a suitable DNA template.

[0021] The method of directly obtaining it from barley protein by separation and purification is as follows: based on the given amino acid sequence of the oligopeptide containing the amino acid structure PQQP, conventional enzymatic hydrolysis, separation and purification methods in biological technology are used to obtain the oligopeptide containing the amino acid structure PQQP from barley.

[0022] The method for preparing by chemical synthesis is to use the traditional solid phase synthesis method to synthesize the above oligopeptide containing the amino acid structure PQQP.

[0023] In a fourth aspect, the present invention provides a method for preparing an enzymatic hydrolysis product containing the oligopeptide containing the amino acid structure PQQP, wherein barley protein is enzymatically hydrolyzed by a two-step enzymatic hydrolysis method, comprising the following steps: sequentially enzymatically hydrolyzing the barley protein with pepsin and trypsin to obtain a barley protein hydrolysis product, which is an enzymatic hydrolysis product containing the oligopeptide containing the PQQP sequence.

[0024] In one embodiment of the present invention, the method of sequentially enzymatically hydrolyzing barley protein with pepsin and trypsin comprises the following steps:

[0025] 1) Extracting barley protein from barley;

[0026] 2) enzymatically hydrolyzing the barley protein with the pepsin to obtain a first enzymatic hydrolysis product;

[0027] 3) using the trypsin to enzymatically hydrolyze the first enzymatic hydrolysis product to obtain a barley enzymatic hydrolysis product.

[0028] In one embodiment of the present invention, the method for extracting barley protein from barley comprises the following steps:

[0029] The barley is ground and defatted, then added into distilled water, the pH is adjusted to 4-6, and pre-treated with cellulase for 0.5-2h; then the pH is adjusted to 10-12 for protein extraction, after which the supernatant is centrifuged and the pH of the supernatant is adjusted to 4.4-4.6, allowed to stand and then centrifuged and washed with water; finally, the barley protein is dried.

[0030] In one embodiment of the present invention, the mass ratio of the pepsin or trypsin to the barley protein is 1:10-100.

[0031] In one embodiment of the present invention, the enzymatic hydrolysis conditions of pepsin or trypsin are 37° C. for 1-4 h.

[0032] In one embodiment of the present invention, after obtaining the barley protein hydrolysate in step 3), the following steps are further included:

[0033] The barley protein hydrolysate is separated by a Toyopearl HW-40F size exclusion chromatography column, deionized water is used as the eluent, elution and chromatography purification are performed at a certain flow rate, and different components are collected; then the effects of the different components on the secretion of PYY in enteroendocrine cells are detected, and the component with the highest activity in promoting PYY secretion is selected as the target component, that is, the enzymatic hydrolysate containing the oligopeptide containing the amino acid structure PQQP.

[0034] In a fifth aspect, the present invention provides an enzymatic hydrolysis product containing the oligopeptide containing the amino acid structure PQQP prepared based on the above preparation method.

[0035] In a sixth aspect, the present invention provides the use of the oligopeptide containing the amino acid structure PQQP, and the enzymatic hydrolysis product containing the oligopeptide containing the amino acid structure PQQP in the preparation of a product having at least one of the following functions 1) to 5):

[0036] 1) Promote PYY secretion;

[0037] 2) Slow down gastric emptying;

[0038] 3) Suppress appetite and reduce food intake;

[0039] 4) Prevent or assist in the treatment of obesity;

[0040] 5) Improve or assist in the treatment of diabetes.

[0041] The oligopeptide containing the amino acid structure PQQP and the enzymatic hydrolysis product of the oligopeptide containing the amino acid structure PQQP can significantly promote the secretion of intestinal peptide YY (PYY), and has the characteristics of being safe, having no toxic side effects, and being resistant to enzymatic hydrolysis by gastrointestinal digestive enzymes.

[0042] In a seventh aspect, the present invention provides a product comprising the oligopeptide containing the amino acid structure PQQP, or an enzymatic hydrolysis product of the oligopeptide containing the amino acid structure PQQP, wherein the product has at least one of the following functions 1) to 5):

[0043] 1) Promote PYY secretion;

[0044] 2) Slow down gastric emptying;

[0045] 3) Suppress appetite and reduce food intake;

[0046] 4) Prevent or assist in the treatment of obesity;

[0047] 5) Improve or assist in the treatment of diabetes.

[0048] The products include health foods and medicines.

[0049] Compared with the prior art, the advantages and beneficial effects of the present invention are embodied in the following aspects:

[0050] Based on a large amount of previous work by the applicant, the present invention screens out oligopeptides that can promote intestinal PYY secretion. These novel oligopeptide sequence structures involved have not been reported so far; the oligopeptides of the present invention can be prepared from food protein barley protein, and thus have the advantages of safety and no toxic side effects; the oligopeptides containing the PQQP sequence of the present invention have a molecular weight of less than 1500Da, a small molecular weight, and in addition to being able to promote intestinal PYY secretion, they are also easily absorbed by the body, thereby having good nutritional functions; the preparation method of the oligopeptides containing the PQQP sequence provided by the present invention is simple, easy to operate, convenient for industrial large-scale production, and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The liquid phase diagram and mass spectrum of the artificially synthesized oligopeptide containing the PQQP sequence;

[0052] Figure 2 The effect of seven oligopeptides on the secretion of PYY by STC-1 cells;

[0053] Figure 2 In the figure, peptides 1, 2, 3, 4, 5, 6, and 7 correspond to peptides QPQQPF, PQQPQPF, LPQQPFPV, QPFPQQPQ, PQQPFLQPL, QPQPYPQQP, and PQQPQQPFFQP, respectively;

[0054] Figure 3 The effect of different concentrations of barley protein hydrolysate on the secretion of PYY in STC-1 cells;

[0055] Figure 4 The effect of barley protein hydrolysate on PYY secretion in mice;

[0056] Figure 5 This is the spectrum of the separation of barley protein hydrolysate by HW-40F size exclusion chromatography column;

[0057] Figure 6 To investigate the effects of the isolated components on the secretion of PYY by STC-1 cells;

[0058] Figure 7 The MS / MS spectrum of the oligopeptide containing the sequence QPQQPF in the isolated component F1;

[0059] Figure 8 The MS / MS spectrum of the oligopeptide containing the sequence PQQPQPF in the isolated component F1;

[0060] Fig. 9 The MS / MS spectrum of the oligopeptide containing the LPQQPFPV sequence in the isolated component F1;

[0061] Fig.10 The MS / MS spectrum of the oligopeptide containing the sequence QPFPQQPQ in the isolated component F1;

[0062] Fig.11 The MS / MS spectrum of the oligopeptide containing the sequence PQQPFLQPL in the isolated component F1;

[0063] Fig.12 The MS / MS spectrum of the oligopeptide containing the sequence QPQPYPQQP in the isolated component F1;

[0064] Fig.13 This is the MS / MS spectrum of the oligopeptide containing the PQQPQQPFFQP sequence in the separated component F1. DETAILED DESCRIPTION

[0065] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0066] Example 1 Synthesis and Activity Evaluation of PQQP Sequence-Containing Oligopeptides

[0067] 1. Synthesis of oligopeptides containing PQQP sequence

[0068] The following 7 oligopeptides were synthesized:

[0069] QPQQPF, the amino acid sequence is Gln-Pro-Gln-Gln-Pro-Phe, as shown in SEQ ID NO.1;

[0070] PQQPQPF, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Pro-Phe, as shown in SEQ ID NO.2;

[0071] LPQQPFPV, the amino acid sequence is Leu-Pro-Gln-Gln-Pro-Phe-Pro-Val, as shown in SEQ ID NO. 3;

[0072] QPFPQQPQ, the amino acid sequence is Gln-Pro-Phe-Pro-Gln-Gln-Pro-Gln, as shown in SEQ ID NO.4;

[0073] PQQPFLQPL, the amino acid sequence is Pro-Gln-Gln-Pro-Phe-Leu-Gln-Pro-Leu, as shown in SEQ ID NO.5;

[0074] QPQPYPQQP, the amino acid sequence is Gln-Pro-Gln-Pro-Tyr-Pro-Gln-Gln-Pro, as shown in SEQ ID NO.6;

[0075] PQQPQQPFFQP, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Phe-Gln-Pro, as shown in SEQ ID NO.7.

[0076] The above 7 oligopeptides were synthesized by Zhejiang Hongtuo Technology Co., Ltd. using peptide solid phase synthesis. The purity of the synthesized peptides was verified to be greater than 95% by high performance liquid chromatography and mass spectrometry. The liquid phase diagram and mass spectrum of the representative oligopeptide PQQPQQPFFQP Figure 1 shown.

[0077] 2. Effect of PQQP-containing oligopeptides on PYY secretion in SCT-1 cells

[0078] (1) Culture of STC-1 cells

[0079] STC-1 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 100 U / mL penicillin and 0.1 mg / mL streptomycin. The cells were incubated at 37°C in a cell culture incubator containing 5% CO2 and subcultured by trypsinization when reaching 80-90% density.

[0080] (2) Determination of PYY secretion by STC-1 cells

[0081] The 7-peptide oligopeptide was prepared into a peptide solution with a molar concentration of 2 mM using Hank's buffer. STC-1 cells were cultured in a 24-well culture plate at a rate of 1.25 × 10 5 The cells were inoculated at a density of 10 cells. When the cells reached 80%-90% confluence, the cells were washed twice with Hank's buffer to remove the culture medium. The 7 oligopeptide solutions were added to the STC-1 cells and the cells were incubated in an incubator at 37°C for 2 h. After the incubation, the cells were centrifuged at 1000g for 20 min and the supernatant was taken. The PYY content was determined using a commercial PYY kit from Wuhan Cloud-Clone Technology Co., Ltd.

[0082] The effects of the seven oligopeptides on the secretion of PYY by STC-1 cells are shown in Figure 2 , it can be seen that these 7 oligopeptides can significantly promote the secretion of PYY by STC-1 cells, and the amount of PYY secretion promoted by the oligopeptide containing the PQQP sequence is 3-4 times that of the blank control group. A large number of current studies have confirmed that promoting the secretion of intestinal PYY helps to suppress appetite, reduce energy intake and body weight. PYY, as an appetite factor related to obesity, has a certain effect on improving obesity and diabetes. The oligopeptides described in this patent can significantly promote the secretion of PYY by intestinal endocrine cells STC-1, so these oligopeptides are of great significance and application value for reducing weight and insulin resistance, and then preventing and treating metabolic syndrome.

[0083] Example 2 Preparation of barley protein hydrolysate and activity evaluation

[0084] (1) Preparation of barley protein hydrolysate

[0085] Grind the barley flour and pass it through an 80 sieve, then defatted the barley flour with hexane. Soak the defatted barley flour in a beaker with distilled water at a mass ratio of 12:1, adjust the pH to 5.0 with 1mol / L HCl, and treat with cellulase at 50°C for 1h. Then adjust the pH to 11.0 with 1mol / L NaOH, centrifuge and take the supernatant after 2h of magnetic stirrer. Adjust the pH of the supernatant to its isoelectric point (pH 4.5) with 1mol / L HCl, let it stand for 1h, then centrifuge, wash the precipitate with water to neutrality, re-dissolve it with a small amount of distilled water, and freeze-dry to obtain barley protein, which is stored at 4°C for later use.

[0086] Dissolve 1g of freeze-dried protein powder in 20mL of K2HPO4-KH2PO4 phosphate buffer (0.1mol / L) containing 25mg of freshly prepared pepsin, adjust the pH of the solution to 2.0 with HCl (1mol / L), and incubate at 37°C for 2h. After the incubation, adjust the pH of the solution to 6.8 with NaOH (1mol / L), add 50mg of trypsin and continue enzymolysis for 2h. After the end, inactivate the enzyme in a boiling water bath for 8min, centrifuge and freeze-dry the supernatant to obtain barley protein hydrolysate.

[0087] (2) Activity evaluation

[0088] The barley protein hydrolysate was prepared with Hank's buffer to prepare solutions with mass concentrations of 3, 4, and 5 mg / mL, respectively. The effect of the hydrolysate on the secretion of PYY by STC-1 cells was determined by the above method. The results are shown in Figure 3 The results show that barley protein hydrolysate can significantly stimulate STC-1 cells to secrete PYY.

[0089] In addition, the effect of barley protein hydrolysate on the secretion of hormones by mouse enteroendocrine cells was evaluated at the animal level. After a one-week adaptation period, the ICR mice were randomly divided into two groups (28 mice in each group). Control group: gavage with normal saline; barley protein hydrolysate group: gavage with barley protein hydrolysate (1.0 g / kg body weight). After gavage, blood was collected by removing the eyeballs at 0, 15 min, 30 min, 60 min, 90 min, 120 min, and 150 min, respectively, and placed in a centrifuge tube containing EDTA (final concentration of 1 mg / mL) and aprotinin (final concentration of 0.6 TIU / mL). The supernatant was centrifuged and the PYY hormone content in the serum was measured by ELISA. The level of PYY in the serum of the group gavaged with normal saline was maintained at around 30 pg / mL during this period. The results of the group gavage with barley protein hydrolysate are as follows. Figure 4 As shown, it can be found that barley protein hydrolysate increases the level of PYY in mice. At 30 minutes, it has the greatest effect on the secretion of PYY by mouse enteroendocrine cells, reaching about 150 pg / mL, which is 5 times that of the oral administration of normal saline group.

[0090] Example 3 Preparation of barley protein oligopeptides that promote PYY secretion

[0091] Take Toyopearl HW-40F filler and swell and pack it in the column (the filling buffer solution is 0.1M NaCl dissolved in 50mM phosphate). The column height is 10cm and the inner diameter is 2.6cm. A water layer of 1.5-2cm should be retained on the top of the column at any time. The column can be used after balancing for about 3-4 column volumes. When the liquid level is retained at 2-3mm, start adding barley protein hydrolysate. Weigh about 20mg of barley protein hydrolysate powder and dissolve it in 2mL of distilled water. After filtering with a 0.45μm microporous filter membrane, add it to the chromatography column. The eluent is distilled water. The elution rate is 2mL / min, and the elution peak is collected. The separation spectrum of barley protein hydrolysate is shown in Figure 5 , it can be seen that the HW-40F column separates the protease hydrolysate into 4 peptide components.

[0092] The four peptide components were evaluated for activity using the above method. The results are as follows: Figure 6 As shown. It can be found that among the four peptide fractions, the F1 fraction has the best ability to stimulate STC-1 cells to secrete PYY. Therefore, the F1 fraction is a highly active oligopeptide that promotes PYY secretion.

[0093] Example 4 Identification of PQQP-containing oligopeptides from barley protein

[0094] The peptide sequences in the F1 fraction were identified by mass spectrometry. The sample was dissolved in distilled water to prepare a 1 mg / mL sample. A reverse phase column (150 μm id×150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, ) was separated, mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid / 80% acetonitrile solution, gradient elution was performed at a flow rate of 600 nL / min, separation gradient: 0-2 min, 4-8% B; 2-45 min, 8-40% B; 45-55 min, 40-60% B; 55-56 min, 60-95% B; 56-66 min, 95% B. The mass spectrometry ion source type was electrospray ionization source (ESI), positive ion scanning mode, spray voltage 2200 V, capillary temperature 270°C. Primary mass spectrometry parameter settings: scanning range 100-2000 m / z, maximum resolution 70000, automatic gain parameter 3000000. Secondary mass spectrometry parameter settings: scanning range 50-2000 m / z, maximum resolution 17500, automatic gain parameter 100000.

[0095] After mass spectrometry, the main ion peaks in the F1 component include m / z=372.688, 421.214, 463.263, 485.246, 534.301, 541.769 and 1340.680. These main molecular ion peaks were further analyzed by secondary mass spectrometry. The secondary mass spectra of these main molecular ion peaks are shown in Figure 7-Figure 13 After database matching and manual calculation, the peptides corresponding to these molecular ion peaks are:

[0096] QPQQPF, the amino acid sequence is Gln-Pro-Gln-Gln-Pro-Phe, as shown in SEQ ID NO.1;

[0097] PQQPQPF, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Pro-Phe, as shown in SEQ ID NO.2;

[0098] LPQQPFPV, the amino acid sequence is Leu-Pro-Gln-Gln-Pro-Phe-Pro-Val, as shown in SEQ ID NO. 3;

[0099] QPFPQQPQ, the amino acid sequence is Gln-Pro-Phe-Pro-Gln-Gln-Pro-Gln, as shown in SEQ ID NO.4;

[0100] PQQPFLQPL, the amino acid sequence is Pro-Gln-Gln-Pro-Phe-Leu-Gln-Pro-Leu, as shown in SEQ ID NO.5;

[0101] QPQPYPQQP, the amino acid sequence is Gln-Pro-Gln-Pro-Tyr-Pro-Gln-Gln-Pro, as shown in SEQ ID NO.6;

[0102] PQQPQQPFFQP, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Phe-Gln-Pro, as shown in SEQ ID NO.7.

[0103] Barley protein contains a variety of protein subunits, among which the amino acid sequences of C-hordein, gamma prolamin and Alphaprolamin are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively. It can be found that oligopeptides containing the PQQP sequence exist in large quantities in barley protein. The oligopeptides containing the PQQP sequence can be prepared from barley protein using the above method.

[0104] The amino acid sequence of barley C-hordein is as follows:

[0105]

[0106]

[0107] The amino acid sequence of barley gamma prolamin is as follows:

[0108]

[0109]

[0110] The amino acid sequence of barley Alpha prolamin is as follows:

[0111]

[0112]

[0113] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention. Sequence Listing <110> University of Shanghai for Science and Technology <120> Oligopeptide containing amino acid structure PQQP and preparation method and application thereof <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 6 <212> PRT <213> Barley (Hordeum vulgare) <400> 1 Gln Pro Gln Gln Pro Phe 1 5 <210> 2 <211> 7 <212> PRT <213> Barley (Hordeum vulgare) <400> 2 Pro Gln Gln Pro Gln Pro Phe 1 5 <210> 3 <211> 8 <212> PRT <213> Barley (Hordeum vulgare) <400> 3 Leu Pro Gln Gln Pro Phe Pro Val 1 5 <210> 4 <211> 8 <212> PRT <213> Barley (Hordeum vulgare) <400> 4 Gln Pro Phe Pro Gln Gln Pro Gln 1 5 <210> 5 <211> 9 <212> PRT <213> Barley (Hordeum vulgare) <400> 5 Pro Gln Gln Pro Phe Leu Gln Pro Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Barley (Hordeum vulgare) <400> 6 Gln Pro Gln Pro Tyr Pro Gln Gln Pro 1 5 <210> 7 <211> 11 <212> PRT <213> Barley (Hordeum vulgare) <400> 7 Pro Gln Gln Pro Gln Gln Pro Phe Phe Gln Pro 1 5 10 <210> 8 <211> 310 <212> PRT <213> Barley (Hordeum vulgare) <400> 8 Met Lys Thr Phe Leu Thr Phe Val Leu Leu Ala Met Val Met Ser Ile 1 5 10 15 Val Thr Thr Ala Arg Gln Leu Asn Pro Ser Ser Gln Glu Leu Gln Ser 20 25 30 Pro Gln Gln Ser Tyr Leu Gln Gln Pro Tyr Pro Gln Asn Pro Tyr Leu 35 40 45 Pro Gln Gln Pro Phe Pro Val Gln Gln Pro Phe His Thr Pro Gln Gln 50 55 60 Tyr Phe Pro Tyr Leu Pro Glu Glu Leu Ser Pro Gln Tyr Gln Ile Pro 65 70 75 80 Thr Pro Leu Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro 85 90 95 Leu Pro Arg Pro Gln Gln Pro Phe Pro Trp Gln Pro Gln Gln Pro Phe 100 105 110 Pro Gln Pro Gln Gln Pro Ile Pro Tyr Gln Pro Gln Gln Pro Phe Asn 115 120 125 Gln Gln Pro Gln Gln Ile Ile Ser Gln Gln Pro Gln Gln Pro Phe Pro 130 135 140 Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Pro Phe Pro Trp 145 150 155 160 Gln Pro Gln Gln Pro Phe Pro Gln Pro Gln Gln Pro Phe Pro Leu Gln 165 170 175 Pro Gln Gln Pro Phe Pro Trp Gln Pro Gln Gln Pro Phe Pro Gln Pro 180 185 190 Gln Gln Pro Ile Ala His Gln Pro Gln Gln Pro Phe Ser Phe Ser Gln 195 200 205 Gln Pro Gln Gln Pro Phe Pro Leu Gln Pro Gln Gln Pro Phe Pro Gln 210 215 220 Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Ile Ile Phe Gln 225 230 235 240 Gln Pro Gln Gln Ser Tyr Pro Val Gln Pro Gln Gln Pro Phe Pro Gln 245 250 255 Pro Gln Pro Val Pro Gln Gln Arg Pro Gln Gln Ala Ser Pro Leu Gln 260 265 270 Pro Gln Gln Pro Phe Pro Gln Gly Ser Glu Gln Ile Ile Pro Gln Gln 275 280 285 Pro Gln Gln Pro Phe Pro Leu Gln Pro His Gln Pro Tyr Thr Gln Gln 290 295 300 Thr Ile Trp Ser Met Val 305 310 <210> 9 <211> 274 <212> PRT <213> Barley (Hordeum vulgare) <400> 9 Met Lys Ile Leu Ile Ile Leu Ile Ile Leu Ala Met Ala Thr Thr Phe 1 5 10 15 Ala Thr Ser Glu Met Gln Val Asn Pro Ser Val Gln Val Gln Pro Thr 20 25 30 Gln Gln Gln Pro Tyr Pro Glu Ser Gln Gln Pro Phe Ile Ser Gln Ser 35 40 45 Gln Gln Gln Phe Pro Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln 50 55 60 Gln Pro Phe Pro Gln Ser Gln Gln Gln Cys Leu Gln Gln Pro Gln His 65 70 75 80 Gln Phe Pro Gln Pro Thr Gln Gln Phe Pro Gln Arg Pro Leu Leu Pro 85 90 95 Phe Thr His Pro Phe Leu Ala Phe Pro Asp Gln Leu Leu Pro Gln Pro 100 105 110 Pro His Gln Ser Phe Pro Gln Pro Pro Gln Ser Tyr Pro Gln Pro Pro 115 120 125 Leu Gln Pro Phe Pro Gln Pro Pro Gln Gln Lys Tyr Pro Glu Gln Pro 130 135 140 Gln Gln Pro Phe Pro Trp Gln Gln Pro Thr Ile Gln Leu Tyr Leu Gln 145 150 155 160 Gln Gln Leu Asn Pro Cys Lys Glu Phe Leu Leu Gln Gln Cys Arg Pro 165 170 175 Val Ser Leu Leu Ser Tyr Leu Trp Ser Lys Ile Val Gln Gln Ser Ser 180 185 190 Cys Arg Val Met Gln Gln Gln Cys Cys Leu Gln Leu Ala Gln Ile Pro 195 200 205 Glu Gln Tyr Lys Cys Thr Ala Ile Asp Ser Ile Val His Ala Ile Phe 210 215 220 Met Gln Gln Gly Gln Arg Gln Gly Val Gln Ile Val Gln Gln Gln Pro 225 230 235 240 Gln Pro Gln Gln Val Gly Gln Cys Val Leu Val Gln Gly Gln Gly Val 245 250 255 Val Gln Pro Gln Gln Leu Ala Gln Met Glu Ala Ile Arg Ser Leu Val 260 265 270 Leu Gln <210> 10 <211> 216 <212> PRT <213> Hordeum vulgare <400> 10 Met Glu Thr Phe Leu Ile Leu Ser Leu Ile Ala Ile Val Ala Thr Thr 1 5 10 15 Ala Thr Thr Ala Val Arg Val Pro Val Pro Gln Leu Gln Leu Gln Asn 20 25 30 Pro Ser Met Gln Gln Pro Gln Glu Gln Val Pro Leu Val Gln Gln Gln 35 40 45 Gln Phe Leu Gly Gln Gln Gln Thr Phe Pro Pro Gln Gln Pro Tyr Pro 50 55 60 Gln Pro Gln Pro Phe Pro Thr Gln Gln Pro Tyr Pro Gln Pro Gln Pro 65 70 75 80 Phe Pro Gln Pro Gln Pro Phe Pro Pro Gln Leu Pro Tyr Pro Gln Pro 85 90 95 Gln Pro Phe Pro Pro Gln Gln Pro Tyr Pro Gln Pro Gln Thr Gln His 100 105 110 Leu Gln Pro Gln Gln Pro Ile Ser Gln Gln Gln Path Gln Gln Gln Gln 115 120 125 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Leu Gln Gln 130 135 140 Gln Ile Leu Gln Gln Ile Leu Gln Gln Tyr Pro Leu Gly Gln Gly Ser 145 150 155 160 Phe Arg Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser Val Gln Pro 165 170 175 Gln Gln Gln Pro Gln Phe Glu Glu Ile Arg Asn Leu Ala Leu Gln Thr 180 185 190 Leu Pro Ala Met Cys Asn Ala Tyr Ile Pro Pro Tyr Cys Thr Ile Ala 195 200 205 Pro Phe Gly Ile Phe Gly Thr Asn 210 215

Claims

1. Use of an oligopeptide containing an amino acid structure PQQP in the preparation of a health food or medicine for assisting the treatment of obesity and diabetes, characterized in that: The oligopeptide containing the amino acid structure PQQP is selected from one or more of the following 7 bioactive peptides: QPQQPF, amino acid sequence is Gln-Pro-Gln-Gln-Pro-Phe; PQQPQPF, amino acid sequence is Pro-Gln-Gln-Pro-Gln-Pro-Phe; LPQQPFPV, amino acid sequence Leu-Pro-Gln-Gln-Pro-Phe-Pro-Val; QPFPQQPQ, amino acid sequence is Gln-Pro-Phe-Pro-Gln-Gln-Pro-Gln; PQQPFLQPL, amino acid sequence: Pro-Gln-Gln-Pro-Phe-Leu-Gln-Pro-Leu; QPQPYPQQP, the amino acid sequence is Gln-Pro-Gln-Pro-Tyr-Pro-Gln-Gln-Pro; PQQPQQPFFQP, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Phe-Gln-Pro.

2. A method for preparing an enzymatic hydrolysis product, characterized in that: The enzymatic hydrolysate contains the following bioactive peptides: QPQQPF, amino acid sequence is Gln-Pro-Gln-Gln-Pro-Phe; PQQPQPF, amino acid sequence is Pro-Gln-Gln-Pro-Gln-Pro-Phe; LPQQPFPV, amino acid sequence Leu-Pro-Gln-Gln-Pro-Phe-Pro-Val; QPFPQQPQ, amino acid sequence is Gln-Pro-Phe-Pro-Gln-Gln-Pro-Gln; PQQPFLQPL, amino acid sequence: Pro-Gln-Gln-Pro-Phe-Leu-Gln-Pro-Leu; QPQPYPQQP, the amino acid sequence is Gln-Pro-Gln-Pro-Tyr-Pro-Gln-Gln-Pro; PQQPQQPFFQP, the amino acid sequence is Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Phe-Gln-Pro; The preparation method of the enzymatic hydrolysis product is: using pepsin and trypsin to enzymatically hydrolyze barley protein in sequence to obtain the barley protein hydrolysis product, comprising the following steps: The barley is ground and defatted, then added into distilled water, the pH is adjusted to 4-6, and pre-treated with cellulase for 0.5-2h; then the pH is adjusted to 10-12 for protein extraction, after which the supernatant is centrifuged and the pH of the supernatant is adjusted to 4.4-4.6, the supernatant is allowed to stand and then centrifuged and washed with water; finally, the barley protein hydrolysate is obtained by drying; The mass ratio of the pepsin or trypsin to the barley protein is 1:10-100; The enzymatic hydrolysis conditions of pepsin or trypsin are 37° C. for 1-4 hours; The barley protein hydrolysate was separated by a Toyopearl HW-40F size exclusion chromatography column, with deionized water as the eluent, eluted at a certain flow rate, purified by chromatography, and different components were collected; then the effects of the different components on the secretion of PYY in enteroendocrine cells were detected, and the component with the highest activity in promoting PYY secretion was selected as the target component, that is, the enzymatic hydrolysate of the oligopeptide containing the amino acid structure PQQP.

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