Vinasse polypeptide with alpha-glucosidase inhibitory activity and preparation method thereof

Abstract

The invention relates to a small cellar sauced vinasse hydrolysis peptide with alpha-glucosidase inhibitory activity and a preparation method thereof. Five small cellar Maotai-flavor vinasse hydrolytic peptides with alpha-glucosidase inhibitory activity are prepared through vinasse protein preparation, enzymolysis, separation and purification and identification, and the sequences of the five small cellar Maotai-flavor vinasse hydrolytic peptides are AAPFYAL, LAYPQ, IRLPFNQI, INPALPW and ATPVFL respectively. The molecular docking analysis result shows that the interaction between the vinasse polypeptide and the amino acid residues of the alpha-glucosidase active pocket is mainly based on hydrogen bonds, electrostatic binding hydrophobic interaction and pi-pi interaction. Pharmacophore construction and matching analysis show that the vinasse polypeptide has a common characteristic structure composed of a positive ion center and three hydrogen bond donors and plays a key role in enzyme inhibition. The small cellar sauced vinasse polypeptide with alpha-glucosidase inhibitory activity prepared by the invention can be used for developing health-care foods and medicines for reducing blood sugar.

Description

Technical Field

[0001] The present invention belongs to the technical field of the development of food-derived bioactive peptides, and particularly relates to a distiller's grains polypeptide with α-glucosidase inhibitory activity and a preparation method thereof. Background Art

[0002] α-Glucosidase (EC 3.2.1.20) is an important target for regulating blood glucose, which is widely distributed on the outer epidermal folds of the human small intestine. It mainly increases blood glucose concentration by hydrolyzing polysaccharides such as water-soluble starch and glycogen to release glucose. By inhibiting the activity of α-glucosidase, blood glucose concentration can be controlled. Therefore, α-glucosidase inhibitors have become an important approach for the treatment of type II diabetes. In recent years, food-derived polypeptides have been proven to be effective inhibitors of α-glucosidase, so they have attracted much attention in the development of hypoglycemic products.

[0003] Currently, a variety of α-glucosidase inhibitory peptides have been isolated from food raw materials. The IC 50 values of α-glucosidase inhibitory peptides LDLQR, AGGFR, and LDNFR isolated from wheat germ protein are 8.59 ± 0.25 mM, 8.66 ± 0.33 mM, and 9.21 ± 0.22 mM (Journal of Agricultural and Food Chemistry 69.50 (2021): 15231 - 15239.); the inhibitory rate of α-glucosidase inhibitory peptide LPLLR derived from walnut kernel protein reached 50.12% at a concentration of 2000 μM, and it can increase glycogen synthesis and glucose uptake, as well as reduce gluconeogenesis by activating the IRS-1 / PI3K / Akt and AMPK signaling pathways (Journal of Functional Foods 69 (2020): 103944.). The IC 50 values of LSMSFPPF, VPKIPPP, and MPGPPSD isolated from ginkgo seeds are 454.33 ± 32.45 μM, 1446.81 ± 66.98 μM, and 943.82 ± 73.10 μM respectively (Food Chemistry 404 (2023): 134481.). The above studies indicate that plant seeds and fruits, etc. are important sources of food-derived α-glucosidase inhibitory peptides.

[0004] Distillers' grains are the residues after brewing raw materials such as sorghum and wheat, and are the main by-products of the liquor industry. As an important nutrient component in distillers' grains, the polypeptide molecules produced by the enzymatic hydrolysis of protein have strong antibacterial, antioxidant and hypoglycemic potential. Small cellar fermentation is a special liquor brewing process, especially the small cellar Maotai-flavor process, whose raw material composition and fermentation conditions are quite different from those of traditional liquor brewing processes. However, the activity of inhibiting α-glucosidase by the hydrolyzed peptides of small cellar Maotai-flavor distillers' grains is not yet clear, which restricts the processing and utilization of small cellar Maotai-flavor distillers' grains and their application in the development of products such as hypoglycemic products. Summary of the Invention

[0005] The purpose of the present invention is to provide a distillers' grains polypeptide with α-glucosidase inhibitory activity and its preparation method.

[0006] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a distillers' grains polypeptide with α-glucosidase inhibitory activity, which is composed of any one of the following amino acid residues: AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, ATPVFL.

[0007] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a preparation method of a distillers' grains polypeptide with α-glucosidase inhibitory activity, which includes the following steps:

[0008] Step 1: Preparation of distillers' grains protein

[0009] Dry the distillers' grains, then crush them to obtain distillers' grains powder, and then extract the distillers' grains protein by the alcohol-alkali method;

[0010] Step 2: Enzymatic hydrolysis of distillers' grains protein

[0011] Use alkaline protease to enzymatically hydrolyze the distillers' grains protein, and centrifuge the obtained enzymatic hydrolysate to take the supernatant to obtain a crude extract of distillers' grains polypeptide;

[0012] Step 3: Separation and purification of distillers' grains polypeptide

[0013] Use ultrafiltration and liquid chromatography to separate and purify the crude extract of the distillers' grains polypeptide, and based on the difference in molecular weight, separate the fraction with a molecular weight < 3 kDa;

[0014] Perform freeze-drying treatment on the fraction with a molecular weight < 3 kDa, then dissolve it in water to prepare a solution, and then separate it using a preparative reversed-phase high-performance liquid chromatograph; the mobile phase is a mixed system of solvent A and solvent B; solvent A is acetonitrile containing 0.1% formic acid by mass fraction, and solvent B is water containing 0.1% formic acid by mass fraction;

[0015] Separation is carried out step by step using a preparative reversed-phase high-performance liquid chromatograph, and the mobile phase is a mixed system of solvent A and solvent B; the elution gradient in the first stage is: 0 - 45 min, 5% - 50% solvent A; 45 - 50 min, 50% - 100% solvent A; 50 - 55 min, 100% - 5% solvent A; 55 - 60 min, 5% solvent A remains unchanged; the elution gradient in the second stage is: 0 - 30 min, 5% - 15% A; 30 - 35 min, 15% - 50% A; 35 - 40 min, 50% - 5% A; 40 - 45 min, 5% A remains unchanged.

[0016] The preferred technical solution is: in step 1, the distiller's grains powder with a particle size less than or equal to 60 mesh is subjected to protein extraction using the alcohol-alkali method.

[0017] The preferred technical solution is: using a 0.5 mol / L sodium hydroxide solution and ethanol mixed in a volume ratio of 2 - 5:1 as the extractant, with a solid-liquid ratio of 1:30 - 80, stirring at 20 - 28 °C for 1 - 3 h, then centrifuging at a speed of 5000 - 8000 r / min for 10 - 20 min, taking the supernatant, then adjusting the pH value of the supernatant to 4.0, and stirring at 20 - 28 °C for 1 - 3 h; centrifuging again at a speed of 5000 - 8000 r / min for 10 - 20 min, and finally taking out the precipitate; washing the precipitate with distilled water until neutral, freeze-drying it and storing it refrigerated to obtain distiller's grains protein.

[0018] The preferred technical solution is: in step 2, dissolve the distiller's grains protein in a PBS solution with a pH value of 7.4, then adjust the pH value to 8.5, and at the same time add an alkaline protease solution to make the enzyme activity reach 5000 - 20000 U / g, and carry out oscillatory enzymatic hydrolysis of the sample at 52 - 57 °C with the pH value maintained at 8.5 for 3 - 5 h to obtain distiller's grains hydrolysate peptides. After the enzymatic hydrolysis is completed, carry out enzyme inactivation treatment, centrifuge at a speed of 7000 - 9000 r / min for 5 - 15 min to obtain a hydrolysate peptide solution; freeze-dry to form a freeze-dried powder.

[0019] The preferred technical solution is: in step 3, use an ultrafiltration tube equipped with a 3 kDa ultrafiltration membrane and a 10 kDa ultrafiltration membrane to divide the crude extract of distiller's grains polypeptides into three components of >3 kDa, 3 - 10 kDa, and <10 kDa based on the difference in molecular weight.

[0020] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are:

[0021] 1. The present invention discovers new peptide segments from distiller's grains, and the peptide segment sequences with the highest α-glucosidase inhibitory activity are heptapeptide AAPFYAL, pentapeptide LAYPQ, octapeptide IRLPFNQI, heptapeptide INPALPW, and hexapeptide ATPVFL, and their IC 50 are 363.77 ± 11.47, 550 ± 13.96, 637.26 ± 25.53, 756.08 ± 19.02, and 780.77 ± 11.61 μM, respectively.

[0022] 2. Through molecular docking analysis, the results of the present invention show that the main interactions between AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, and ATPVFL and the amino acid residues of α-glucosidase are hydrogen bonds, electrostatic interactions, and hydrophobic interactions. However, there are similarities and differences in the specific interaction sites.

[0023] 3. The α-glucosidase inhibitory peptides of the present invention can be applied to the development of health foods and drugs for the purpose of lowering blood sugar. Description of the Drawings

[0024] Figure 1 Liquid chromatogram of distiller's grains polypeptides with Mw < 3 kDa.

[0025] Figure 2 Inhibitory rate of each distiller's grains polypeptide component on α-glucosidase after the first liquid chromatography purification (2 mg / mL); different letters indicate significant differences in the inhibitory rate of each distiller's grains polypeptide component on α-glucosidase (p < 0.05).

[0026] Figure 3 Liquid chromatogram of the distiller's grains polypeptide in fraction F2.

[0027] Figure 4 Inhibitory rate of each distiller's grains polypeptide component on α-glucosidase after the second liquid chromatography purification (2 mg / mL); different letters indicate significant differences in the inhibitory rate of each distiller's grains polypeptide component on α-glucosidase (p < 0.05).

[0028] Figure 5 Secondary mass spectrum of distiller's grains polypeptides.

[0029] Figure 6 Schematic diagram of the molecular structure of distiller's grains polypeptides.

[0030] Figure 7 Schematic diagram of molecular docking of the interaction between distiller's grains polypeptides and α-glucosidase.

[0031] Figure 8 Pharmacophore heat map.

[0032] Figure 9Pharmacophore molecular superposition diagrams of AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, and ATPVFL. Detailed implementation manners

[0033] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this embodiment.

[0034] Please refer to Figures 1 - 9 It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in the art to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they do not have any technical substance. Any modification of the structure, change in the proportional relationship, or adjustment of the size. The following embodiments are provided to better understand the present invention, rather than to limit the present invention.

[0035] Unless otherwise specified, the experimental materials used in the following examples were all obtained from regular biochemical reagent stores. Small cellar sauce-flavored distiller's grains: Purchased from Anhui Xuanjiu Group Co., Ltd. Ethanol, sodium hydroxide, hydrochloric acid, and acarbose were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd. α-Amylase, alkaline protease, 4-nitrophenyl-β-D-glucopyranoside (abbreviated as pNPG) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. α-Glucosidase was from Shanghai Yuanye Bio-Technology Co., Ltd. The synthesized polypeptides (AAPFYAL, ATPVFL, LAYPQ, IRLPFNQI, INPALPW, and SSSSAVVG) were from Hefei Guotai Co., Ltd.

[0036] The preparation of PBS (pH = 7.4) is as follows: Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 mL of distilled water, adjust the pH to 7.4, and then add water to make up the volume to 1 L. If different pH values need to be adjusted, just add an appropriate amount of hydrochloric acid or sodium hydroxide solution on this basis.

[0037] Example 1: Preparation of distiller's grains polypeptide with α-glucosidase inhibitory activity

[0038] (1) Preparation of distiller's grains protein: Accurately weigh 5.0 g of the powder of sauce-flavored distiller's grains sample, use a 0.5 mol / L sodium hydroxide solution and ethanol mixed at a ratio of 3:1 (mL / mL) as the extractant, and the solid-liquid ratio is 1:40. Stir and extract the distiller's grains protein for 1.5 h, take the supernatant after centrifugation, then adjust the pH to 4.0, stir for 1.5 h, separate and wash the precipitate until it is neutral, and freeze-dry to obtain the distiller's grains protein extract for standby.

[0039] (2) Protease hydrolysis of distillers' grains: Weigh a certain amount of distillers' grains protein according to a substrate concentration of 1.5% to prepare a solution with a corresponding concentration. Add alkaline protease under the conditions of pH 8.5 and a temperature of 55°C. The enzyme addition amount is fixed at 10,000 U / g of crude protein. During hydrolysis, 0.05 mol / L NaOH standard solution is added drop by drop to keep the pH constant. After 2.5 hours of enzymatic hydrolysis, enzyme inactivation treatment is carried out, and the supernatant is taken by centrifugation.

[0040] (3) Isolation and purification of distillers' grains polypeptides: Use an ultrafiltration tube equipped with two ultrafiltration membranes (3 kDa and 10 kDa) to fractionate the hydrolyzed peptide solution obtained in the previous step. Based on the difference in molecular weight (MW), the separated components are divided into three components: >3 kDa, 3 - 10 kDa, and <10 kDa. These three components are collected, concentrated, and freeze-dried respectively, and then each is formulated into a 2 mg / mL solution to analyze their inhibitory activities against α-glucosidase.

[0041] It was found from the results in Table 1 that compared with the distillers' grains polypeptides with molecular weights of 3 - 10 kDa and greater than 10 kDa, the peptides with a molecular weight less than 3 kDa showed a significant enhancement in inhibitory effect, and the inhibition rate increased to 47.78%. This is because the peptides with larger molecular weights are hindered in the spatial binding to the active site of the enzyme, resulting in their inhibitory activity being lower than that of small peptides, which is consistent with the previous research conclusions.

[0042] Table 1: Hypoglycemic activities of ultrafiltered peptide segments

[0043] Component α - Glucosidase Inhibition Rate / % <3 kDa <![CDATA[47.78±1.89 a > 3kDa - 10kDa <![CDATA[33.33±1.53 b > >10kDa <![CDATA[25.56±0.99 c >

[0044] Example 2: Further purification of distillers' grains polypeptides

[0045] The fraction with Mw < 3 kDa after ultrafiltration is freeze-dried and formulated into a 50 mg / ml solution, and separated using a preparative reversed-phase high-performance liquid chromatograph (RP-HPLC). This step contains two stages, and the mobile phases are acetonitrile (A) with a mass fraction of 0.1% formic acid and an aqueous solution (B) of 0.1% formic acid.

[0046] The first-stage liquid chromatography conditions: detection wavelength 220 nm, room temperature, flow rate 10 mL / min. Elution program: 0 - 45 min, 5% - 50% A; 45 - 50 min, 50% - 100% A; 50 - 55 min, 100% - 5% A; 55 - 60 min, 5% A remains unchanged.

[0047] Compare the hypoglycemic activities of different components of distillers' grains active peptides separated by the first-stage liquid chromatography, and select the component with the highest activity for the next-stage separation.

[0048] Second-stage liquid chromatography conditions: detection wavelength 220 nm, room temperature, flow rate 8 mL / min. Elution program: 0 - 30 min, 5% - 15% A; 30 - 35 min, 15% - 50% A; 35 - 40 min, 50% - 5% A; 40 - 45 min, 5% A unchanged.

[0049] The distillers' grains polypeptide separation in the first stage of the above process was collected in the manner of Figure 1 According to the elution time, this fraction was divided into twelve sub-fractions (F1 - F12), and their inhibitory rates against α-glucosidase were tested.

[0050] From Figure 2 The results showed that the inhibitory rates among the fractions were different. Among them, fraction F2, that is, the fraction with an elution time of 7.5 - 10 min, showed the highest inhibitory rate against α-glucosidase, reaching 68.22 ± 1.89%.

[0051] Similarly, the distillers' grains polypeptide separation in the second stage of the above process was collected in the manner of Figure 3 According to the elution time, this fraction was divided into five sub-fractions (F2-1 - F2-5), and their inhibitory rates against α-glucosidase were tested.

[0052] From Figure 4 The results showed that the inhibitory rates among the fractions were different. Among them, fraction F2-2, that is, the fraction with an elution time of 8.3 - 9.8 min, showed the highest inhibitory rate against α-glucosidase, reaching 77.33 ± 1.15%.

[0053] Nano LC-MS / MS sequencing was performed on the most active F2-2 distillers' grains polypeptide: An appropriate amount of the sample was desalted by a C 18 desalting column. The sample was analyzed by LC-MS / MS equipped with an online nano-spray ion source. The whole system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with EASY-nanoLC1200. A total of 2 μL of the sample was loaded (analysis column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated with a 60-min gradient. The column flow rate was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 4% of phase B and increased to 50% in a non-linear gradient in 53.6 minutes, and increased to 95% in 40 seconds and maintained for 5.6 minutes.

[0054] The mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions. The mass spectrometry parameters are set as follows: (1) MS: scan range (m / z): 100 - 1500; resolution: 120,000; Normalized AGC target: 200%; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 50,000; Normalized AGC target: 200%; maximum injection time: 25 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s.

[0055] The tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The databases were uniprot_Sorghum_bicolor (version 2024, 34001 entries); uniprot_Triticum_aestivum (version 2024, 105062 entries) sequences, with none set. The database search parameters were as follows: fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm. The protein cutoff value was at least 1 unique peptide; the peptide cutoff value was -10lgP ≥ 20.

[0056] Six distiller's grains polypeptides with the strongest α-glucosidase inhibitory activity were isolated by the above steps. The amino acid sequences, molecular weights, charges, and hydrophobicities of each component are shown in Table 2. The mass spectra are as Figure 5 shown.

[0057] Table 2: Sequences of distiller's grains polypeptide segments with the best α-glucosidase inhibitory activity

[0058]

[0059]

[0060] Note: The amino acid abbreviations in the sequence are: A - alanine (Ala); P - proline (Pro); F - phenylalanine (Phe); Y - tyrosine (Tyr); L - leucine (Leu); Q - glutamine (Gln); I - isoleucine (Ile); R - arginine (Arg); N - asparagine (Asn); W - tryptophan (Trp); T - threonine (Thr); V - valine (Val); S - serine (Ser); G - glycine (Gly).

[0061] Example 3: Verification experiment

[0062] According to the results obtained from mass spectrometry analysis, the inhibitory activities of AAPFYAL (SEQ ID NO.1), LAYPQ (SEQ ID NO.2), IRLPFNQI (SEQ ID NO.3), INPALPW (SEQ ID NO.4), ATPVFL (SEQ ID NO.5), and SSSSAVVG (SEQ ID NO.6) against α-glucosidase were compared.

[0063] As shown in Table 3, the IC 50 of AAPFYAL (SEQ ID NO.1), LAYPQ (SEQ ID NO.2), IRLPFNQI (SEQ ID NO.3), INPALPW (SEQ ID NO.4), and ATPVFL (SEQ ID NO.5) was between 300 and 900 μM, among which AAPFYAL showed the highest IC 50 of 730.2 ± 71.54 μM. At the same time, we found that the IC 50 of SSSSAVVG (SEQ ID NO.6) exceeded 1000 μM, and it can be judged that it has no contribution to the inhibitory effect on α-glucosidase. These results confirmed the existence of polypeptides with hypoglycemic function in the F2-2 fraction, clearly demonstrating that in vitro digestion effectively released peptides with potential α-glucosidase inhibitory ability from the protein in XiaoJiao Jiangxiang distiller's grains.

[0064] Table 3: The half-inhibitory rate concentration (IC 50 )

[0065] Serial Number Sequence <![CDATA[IC 50 (μM)]]> 1 AAPFYAL 363.77±11.47 2 LAYPQ 550±13.69 3 IRLPFNQI 637.26±25.53 4 INPALPW 756.08±19.02 5 ATPVFL 780.77±11.61 6 SSSSAVVG 5068.08±13.69

[0066] Example 4: Analysis and Docking of Distiller's Grains Polypeptides with α-Glucosidase

[0067] Using the molecular docking method, different distiller's grains polypeptides were further screened to clarify the binding conformation of distiller's grains polypeptides with α-glucosidase. The three-dimensional structure diagram of α-glucosidase (PDB code: 2QMJ) was downloaded from the Protein Data Bank. In this method, HPEPDOCK (http: / / huanglab.phys.hust.edu.cn / hpepdock / ) was used for docking, and the docking site was around acarbose, and other parameters were set to default values. The result with the best score on the website was selected as the binding mode of distiller's grains polypeptide-enzyme, and the distiller's grains polypeptide-enzyme conformation was visualized and processed by PyMOL software. Figure 6 Schematic diagrams of the molecular structures of AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, and ATPVFL distiller's grains polypeptides.

[0068] By Figure 7The results showed that these four polypeptides all had complex interaction points with the deep pocket of the α-glucosidase model (MGAM). In terms of electrostatic interactions and hydrogen bonds, the tyrosine of AAPFYAL (SEQ ID NO.1), the leucine of LAYPQ (SEQ ID NO.2), the arginine of IRLPFNQI (SEQ ID NO.3), the isoleucine of INPALPW (SEQ ID NO.4), and the alanine of ATPVFL (SEQ ID NO.5) could interact with the deep pocket of MGAM in a complex manner, and all could form favorable electrostatic interactions with the D443 residue of MGAM. In addition, the leucine of LAYPQ (SEQ ID NO.2), the asparagine of INPALPW (SEQ ID NO.4), and the threonine of ATPVFL (SEQ ID NO.5) could also form hydrogen bonds with the D542 residue, while the arginine of IRLPFNQI (SEQ ID NO.3), the isoleucine of INPALPW (SEQ ID NO.4), and the alanine of ATPVFL (SEQ ID NO.5) could also form hydrogen bonds with D327. These interaction patterns were similar to those of the FDA-approved drug acarbose reported in the relevant literature.

[0069] In terms of hydrophobic and π-π interactions, the phenylalanine of AAPFYAL (SEQ ID NO.1), the leucine of LAYPQ (SEQ ID NO.2), the proline of IRLPFNQI (SEQ ID NO.3), the proline of INPALPW (SEQ ID NO.4), and the proline of ATPVFL (SEQ ID NO.5) can all form such strong interactions with F575. In addition, the interaction sites of AAPFYAL (SEQ ID NO.1) and LAYPQ (SEQ ID NO.2) with the interior of the MGAM active pocket are similar in terms of hydrophobic and π-π interactions. The tyrosine of AAPFYAL (SEQ ID NO.1) and the leucine of LAYPQ (SEQ ID NO.2) form strong interactions with the internal residues F575, Y299, and W406 of the MGAM pocket. In contrast, IRLPFNQI (SEQ ID NO.3) shows a slightly different binding mode, with its arginine and phenylalanine forming strong hydrophobic interactions with the residues M444 and L577 near the pocket, respectively. And F450 is the common binding site of AAPFYAL (SEQ ID NO.1), LAYPQ (SEQ ID NO.2), and IRLPFNQI (SEQ ID NO.3), corresponding to the leucine, proline, and isoleucine on the three polypeptides, respectively. The interaction of ATPVFL (SEQ ID NO.5) with MGAM is between the above two interaction modes. Its threonine, phenylalanine, and alanine form strong hydrophobic interactions with M444, L577, and W406, Y299, respectively, and its alanine also forms a strong hydrophobic interaction with the residue I328. The hydrophobic binding site of INPALPW (SEQ ID NO.4) is the most different from the above four peptides, and its main binding sites are tryptophan and alanine, which are completely different from other peptides.

[0070] Meanwhile, we found that these AAPFYAL (SEQ ID NO.1), LAYPQ (SEQ ID NO.2), IRLPFNQI (SEQ ID NO.3), and INPALPW (SEQ ID NO.4) can form strong interaction forces with certain residues outside the pocket. For example, the glutamine of LAYPQ (SEQ ID NO.2) can form a hydrogen bond with D474, the isoleucine at the C-terminus of IRLPFNQI (SEQ ID NO.3) can form hydrogen bonds with N209 and N207, and the tryptophan at the C-terminus of INPALPW (SEQ ID NO.4) can form a hydrogen bond with T544. The interaction between AAPFYAL (SEQ ID NO.1) and MGAM is the most active, where the alanine at its N-terminus can form an electrostatic interaction with D549 and hydrogen bonds with P206 and T544. This situation where both ends interact with the enzyme simultaneously may be the reason why these inhibitory peptides can maintain high activity.

[0071] Therefore, AAPFYAL (SEQ ID NO.1), LAYPQ (SEQ ID NO.2), IRLPFNQI (SEQ ID NO.3), INPALPW (SEQ ID NO.4), and ATPVFL (SEQ ID NO.5) have the potential to competitively interact with the substrate within the active site of MGAM, potentially effectively hindering MGAM activity. The specific secondary bond action sites on the polypeptide and the enzyme are shown in Table 4.

[0072] Table 4: Interaction sites and secondary bond types between polypeptides and proteins

[0073]

[0074] Example 5: Construction of the pharmacophore model of α-glucosidase inhibitory peptides

[0075] Ten α-glucosidase inhibitory peptides were selected as the training set. The data set of the pharmacophore model training set is shown in Table 5. In addition, the peptides in the training set are similar in length compared to the finally obtained peptides (AAPFYAL, LAYPQ, IRLPFNQI, and ATPVFL), and the inhibitory activity ranges of these three peptides are within the inhibitory activity range of the training set. The structure and energy optimization of these peptides were completed using Discovery Studio v 2019 under the condition of pH 7.40. Finally, the common features of the peptides in the training set were identified through the HipHop algorithm, generating ten pharmacophore models. Subsequently, these four peptides were matched with these models. Figure 8 A heat map showing the matching degree between the pharmacophore model and the peptides is presented. Red indicates a higher matching degree, blue indicates the opposite, and Figure 9The superposition effect of four peptides with the highest-scoring pharmacophore is shown.

[0076] Table 5. Pharmacophore model training set

[0077]

[0078]

[0079] The results of the training set are shown in Table 6. Ten hypothesis experiments were conducted, and the "Rank" score ranged from 134.71 to 136.90. The "Features" of these models were "PAAA", namely one positive ion center, three hydrogen bond donor features, and the maximum fitting value was 4. In addition, the values of Direct Hit and Partial Hit were "1111111111" and "0000000000" respectively. These results indicate that these four pharmacophore features are highly consistent with each peptide segment in the training set. Although all these hypotheses consist of one positive ion center and three AA features, the spatial positions of these features are different.

[0080] Table 6. Pharmacophore features of the training set

[0081] Serial Number Features Rank Direct Hit Partial Hit Max Fit 1 PAAA 145.010 1111111111 0000000000 4 2 PAAA 144.988 1111111111 0000000000 4 3 PAAA 144.949 1111111111 0000000000 4 4 PAAA 144.949 1111111111 0000000000 4 5 PAAA 144.609 1111111111 0000000000 4 6 PAAA 143.148 1111111111 0000000000 4 7 PAAA 142.985 1111111111 0000000000 4 8 PAAA 142.981 1111111111 0000000000 4 9 PAAA 142.478 1111111111 0000000000 4 10 PAAA 141.918 1111111111 0000000000 4

[0082] It can be seen from Figure 8 that the four peptides match well with Hypo 1. As Figure 9 it can be seen, these four peptides (AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, and ATPVFL) have one positive ion center and three hydrogen bond donor features. This indicates that these four peptides may have good α-glucosidase inhibitory activity similar to that of the training set. The pharmacophore features and molecular docking results further indicate that hydrogen bonds play an important role in α-glucosidase inhibition.

[0083] Example 6: A distiller's grains polypeptide with α-glucosidase inhibitory activity and its preparation method

[0084] A distiller's grains polypeptide with α-glucosidase inhibitory activity, consisting of the following amino acid residues: AAPFYAL.

[0085] A distiller's grains polypeptide with α-glucosidase inhibitory activity, consisting of the following amino acid residues: LAYPQ.

[0086] A distiller's grains polypeptide with α-glucosidase inhibitory activity, consisting of the following amino acid residues: IRLPFNQI.

[0087] A distiller's grains polypeptide with α-glucosidase inhibitory activity, consisting of the following amino acid residues: INPALPW.

[0088] A distiller's grains polypeptide with α-glucosidase inhibitory activity, which is composed of the following amino acid residues: ATPVFL.

[0089] A preparation method of a distiller's grains polypeptide with α-glucosidase inhibitory activity, comprising the following steps:

[0090] Step 1: Preparation of distiller's grains protein

[0091] Dry and crush the Jiangxiang-flavor distiller's grains to obtain distiller's grains powder, and then extract the distiller's grains protein by the alcohol-alkali method.

[0092] Step 2: Enzymatic hydrolysis of distiller's grains

[0093] Use alkaline protease to enzymatically hydrolyze the distiller's grains protein, and centrifuge the obtained enzymatic hydrolysate to take the supernatant to obtain a crude extract of distiller's grains polypeptide; claim 7 has been described

[0094] Step 3: Separation and purification of distiller's grains polypeptide

[0095] Separate and purify the crude extract of the distiller's grains polypeptide by ultrafiltration and liquid chromatography, and separate the fraction with a molecular weight < 3 kDa based on the difference in molecular weight;

[0096] Perform freeze-drying on the fraction with a molecular weight < 3 kDa, then prepare it into a sample with water, and then separate it using a preparative reversed-phase high-performance liquid chromatograph; the mobile phase is a mixed system of solvent A and solvent B; solvent A is acetonitrile containing 0.1% formic acid, and solvent B is water containing 0.1% formic acid; the first-stage elution gradient is: 0 - 45 min, 5% - 50% A; 45 - 50 min, 50% - 100% A; 50 - 55 min, 100% - 5% A; 55 - 60 min, 5% A remains unchanged; collect the eluate from 7.5 - 10 min; the second-stage elution gradient is: 0 - 30 min, 5% - 15% A; 30 - 35 min, 15% - 50% A; 35 - 40 min, 50% - 5% A; 40 - 45 min, 5% A remains unchanged; collect the eluate from 8.3 - 9.8 min.

[0097] The preferred technical solution is: in step 1, mix the distiller's grains powder with a particle size less than or equal to 100 with n-hexane at a volume ratio of 1:1.5 - 5, stir for 0.5 - 2 h, then take the filter residue, dry it to obtain defatted distiller's grains powder; mix the defatted distiller's grains powder with ethanol at a volume ratio of 1:5 - 15, perform ultrasonic dephenolization for 0.5 - 2 h and then centrifuge, take out the precipitate to obtain defatted and dephenolized distiller's grains powder; mix the defatted and dephenolized distiller's grains powder with distilled water at a volume ratio of 1:5 - 15 to obtain a suspension, and use the alkali solution acid precipitation method for protein extraction.

[0098] The preferred technical solution is as follows: A 0.5 mol / L sodium hydroxide solution and ethanol mixed at a ratio of 3:1 (mL / mL) are used as the extractant, the solid-liquid ratio is 1:40, and the mixture is stirred at 20 - 28 °C for 1.5 h. Then, it is centrifuged at a speed of 5000 - 8000 r / min for 10 - 20 min. The supernatant is taken, and the pH value of the supernatant is adjusted to 4.0, and it is stirred at 20 - 28 °C for 1.5 h; it is centrifuged again at a speed of 5000 - 8000 r / min for 10 - 20 min, and finally the precipitate is taken out; the precipitate is washed with distilled water until neutral, freeze-dried and then stored refrigerated to obtain distillers' grains protein.

[0099] The preferred technical solution is as follows: In step 2, the distillers' grains protein is dissolved in a PBS solution with a pH value of 7.4, and the pH value of the solution is adjusted to 8.5. At the same time, an alkaline protease solution is added to make the enzyme activity reach 10000 U / g. The sample is subjected to oscillating enzymatic hydrolysis at 55 °C with the pH value maintained at 8.5 for 2.5 h to obtain distillers' grains hydrolysate peptides. After the enzymatic hydrolysis is completed, enzyme inactivation treatment is carried out, and it is centrifuged at a speed of 7000 - 9000 r / min for 5 - 15 min to obtain a hydrolysate peptide solution; it is freeze-dried to form a freeze-dried powder.

[0100] The preferred technical solution is as follows: In step 3, an ultrafiltration cup equipped with a 3 kDa ultrafiltration membrane and a 10 kDa ultrafiltration membrane is used. Based on the difference in molecular weight, the components separated from the crude extract of distillers' grains polypeptides are divided into three components: >3 kDa, 3 - 10 kDa, and <10 kDa.

[0101] A composition with α-glucosidase inhibitory activity, characterized in that it contains polypeptides AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, and ATPVFL. It also includes a stabilizer, an emulsifier, and a flavoring agent.

[0102] The above are only preferred embodiments for explaining the present invention, and are not intended to limit the present invention in any form. Therefore, any modification or change to the present invention made under the same inventive spirit should still be included within the scope intended to be protected by the present invention.

Claims

1. A distiller's grains polypeptide with α-glucosidase inhibitory activity, characterized in that: Composed of any one of the following amino acid residues: AAPFYAL, LAYPQ, IRLPFNQI, INPALPW, ATPVFL.

2. A preparation method of distiller's grains polypeptide with α-glucosidase inhibitory activity as described in claim 1, characterized in that: Including the following steps: Step 1: Distiller's grains protein preparation Dry the distiller's grains, then crush to obtain distiller's grains powder, and then extract the distiller's grains protein by the alcohol-alkali method; Step 2: Distiller's grains protein hydrolysis Hydrolyze the distiller's grains protein with alkaline protease, centrifuge the obtained hydrolysate to take the supernatant, and obtain a crude extract of distiller's grains polypeptide; Step 3: Separation and purification of distiller's grains polypeptide Separate and purify the crude extract of the distiller's grains polypeptide by ultrafiltration and liquid chromatography, and based on the difference in molecular weight, separate the fraction with a molecular weight < 3 kDa; Perform freeze-drying treatment on the fraction with a molecular weight < 3 kDa, then dissolve it in water to form a solution, and then separate it using a preparative reversed-phase high-performance liquid chromatograph; the mobile phase is a mixed system of solvent A and solvent B; solvent A is acetonitrile containing 0.1% formic acid by mass fraction, and solvent B is water containing 0.1% formic acid by mass fraction; Use the preparative reversed-phase high-performance liquid chromatograph to perform separation step by step, and the mobile phase is a mixed system of solvent A and solvent B; the elution gradient in the first stage is: 0 - 45 min, 5% - 50% solvent A; 45 - 50 min, 50% - 100% solvent A; 50 - 55 min, 100% - 5% solvent A; 55 - 60 min, 5% solvent A remains unchanged; the elution gradient in the second stage is: 0 - 30 min, 5% - 15% A; 30 - 35 min, 15% - 50% A; 35 - 40 min, 50% - 5% A; 40 - 45 min, 5% A remains unchanged.

3. The preparation method of distiller's grains polypeptide with α-glucosidase inhibitory activity according to claim 2, characterized in that: In step 1, use the alcohol-alkali method to extract protein from distiller's grains powder with a particle size less than or equal to 60 mesh.

4. The preparation method of distiller's grains polypeptide with α-glucosidase inhibitory activity according to claim 3, characterized in that: Use a 0.5 mol / L sodium hydroxide solution and ethanol mixed at a volume ratio of 2 - 5:1 as the extractant, with a solid-liquid ratio of 1:30 - 80, stir at 20 - 28 °C for 1 - 3 h, then centrifuge at a speed of 5000 - 8000 r / min for 10 - 20 min, take the supernatant, then adjust the pH value of the supernatant to 4.0, and stir at 20 - 28 °C for 1 - 3 h; centrifuge again at a speed of 5000 - 8000 r / min for 10 - 20 min, and finally take out the precipitate; wash the precipitate with distilled water until neutral, perform freeze-drying and then store it refrigerated to obtain distiller's grains protein.

5. The preparation method of distiller's grains polypeptide with α-glucosidase inhibitory activity according to claim 2, characterized in that: In step 2, dissolve the distiller's grains protein in a PBS solution with a pH value of 7.4, then adjust the pH value to 8.5, and at the same time add alkaline protease solution to make the enzyme activity reach 5000 - 20000 U / g, and perform oscillatory hydrolysis of the sample at 52 - 57 °C with the pH value maintained at 8.5 for 3 - 5 h to obtain distiller's grains hydrolysate peptides. After the hydrolysis is completed, perform enzyme inactivation treatment, centrifuge at a speed of 7000 - 9000 r / min for 5 - 15 min to obtain a hydrolysate peptide solution; perform freeze-drying to form a lyophilized powder.

6. The preparation method of the distiller's grains polypeptide with α-glucosidase inhibitory activity according to claim 2, wherein: In Step 3, an ultrafiltration tube equipped with a 3 kDa ultrafiltration membrane and a 10 kDa ultrafiltration membrane was used to divide the components separated from the crude extract of distillers' grains polypeptides into three components, namely >3 kDa, 3-10 kDa, and <10 kDa, based on the difference in molecular weight.

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