A mixed sweet potato peptide composition for inhibiting alpha-glucosidase activity

Abstract

The present invention relates to the field of biotechnology, and in particular to a mixed sweet potato peptide composition for inhibiting alpha-glucosidase activity. The amino acid sequence of the composition is shown in SEQ ID NO.1-2. The present invention provides a mixed sweet potato peptide composition for inhibiting alpha-glucosidase activity, comprising two sweet potato protein peptides that have good inhibition of alpha-glucosidase activity and can delay starch digestion in vivo, and inhibition kinetics show that it is a mixed inhibitor.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a mixed sweet potato peptide composition for inhibiting alpha-glucosidase activity. Background Art

[0002] Diabetes is a widespread metabolic disorder. Long-term hyperglycemia can lead to chronic damage and dysfunction of various tissues, and cause complications such as retinal damage and chronic renal failure. Therefore, controlling blood sugar, especially postprandial blood sugar, is an effective treatment for diabetes, and delaying carbohydrate metabolism is the main measure to control postprandial blood sugar. α-Glucosidase is a key enzyme involved in carbohydrate metabolism, mainly located in the brush border cells of the small intestinal mucosa. The mechanism of α-glucosidase inhibitors to control postprandial blood sugar is mainly to inhibit various α-glucosidases in the small intestine, slow down the rate of carbohydrate decomposition into glucose, thereby slowing down the absorption of glucose and reducing postprandial hyperglycemia symptoms. Although synthetic α-glucosidase inhibitors are already on the market (such as acarbose, etc.), they have certain toxic side effects, so there is a broad prospect for discovering more efficient and safe α-glucosidase inhibitors from natural products.

[0003] Sweet potato (Dioscorea esculenta (Lour.) Burkill) is now the fifth largest food crop in my country and is widely planted throughout China, ranking first in the world in terms of total output and planting area. Sweet potato is mainly used for starch processing, and its processing byproducts contain relatively rich nutrients such as protein, dietary fiber, and trace elements, with the characteristics of high output and high water content. It is estimated that the amount of wastewater generated by the production of sweet potato starch reaches 20 million to 40 million tons each year. Direct disposal will lead to serious environmental pollution and waste of resources.

[0004] At present, there are literature reports on the antioxidant and hypotensive activities of sweet potato protein peptides, but there are no reports on the preparation of active peptides that inhibit α-glucosidase from sweet potato protein. From the perspective of enzyme inhibition kinetics, enzyme inhibitors are divided into competitive inhibitors, anti-competitive inhibitors, non-competitive inhibitors, mixed inhibitors, etc. The application of sweet potato peptides as enzyme preparations for α-glucosidase has not been reported. Summary of the invention

[0005] The technical problem to be solved by the present invention is that currently there are literature reports on the antioxidant and blood pressure lowering activities of sweet potato protein polypeptides, but there is no report on the preparation of active peptides for inhibiting α-glucosidase from sweet potato protein.

[0006] To solve the above problems, the present invention provides a mixed sweet potato peptide composition for inhibiting α-glucosidase activity, comprising two sweet potato protein peptides that have good inhibition of α-glucosidase activity and can delay starch digestion in vivo. Inhibition kinetics show that the composition is a mixed inhibitor.

[0007] To achieve the above object, the present invention is achieved by the following technical means: a mixed sweet potato peptide composition for inhibiting α-glucosidase activity, the amino acid sequence of which is shown in SEQ ID NO.1-2.

[0008] SEQ ID NO.1:

[0009] Amino acid sequence Trp-Glu-Ser-Gly-Pro-Trp, abbreviated as WESGPW.

[0010] SEQ ID NO.2:

[0011] Amino acid sequence Gly-Thr-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Lys-Glu, abbreviation GTPPPPPPKE.

[0012] Furthermore, the above sweet potato peptide composition is artificially synthesized.

[0013] The application of the above-mentioned sweet potato peptide composition in the preparation of products for inhibiting the activity of α-glucosidase. The two sweet potato peptides have the characteristics of competitive and non-competitive inhibition, and can not only compete with the substrate for the corresponding α-glucosidase catalytic active site, but also interact with the inactive site. Since the number of enzyme catalytic sites is certain, when the sweet potato peptides preferentially bind to the active site of the enzyme, the corresponding substrate cannot bind to the active site on the enzyme, reducing the binding rate between the enzyme and the oligosaccharide; at the same time, they bind to the inactive site of the enzyme to form an oligopeptide-enzyme-substrate ternary complex, so that the glycosidic bond of the oligosaccharide cannot be cleaved, and a large amount of oligosaccharides cannot be catalyzed by the enzyme into monosaccharides that can be absorbed by the human body, thereby avoiding postprandial hyperglycemia.

[0014] The beneficial effects of the present invention are:

[0015] (1) Two new peptides were obtained from sweet potato peptides. The inhibition kinetics showed that they were mixed-type inhibitors of α-glucosidase, non-toxic and non-sensitizing. They not only had high α-glucosidase inhibitory activity in vitro, but also showed α-glucosidase inhibitory activity in vivo, providing new raw materials for products that inhibit α-glucosidase activity.

[0016] (2) Both novel peptides are derived from sweet potato protein, opening up a new direction for the research of sweet potato protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the chemical structural formula of the polypeptide shown in SEQ ID NO.1.

[0018] Figure 2 It is the chemical structural formula of the polypeptide shown in SEQ ID NO.2.

[0019] Figure 3 It is the secondary mass spectrum of the polypeptide shown in SEQ ID NO.1.

[0020] Figure 4 It is the secondary mass spectrum of the polypeptide shown in SEQ ID NO.2.

[0021] Figure 5 This is the docking result of P1 (WESGPW) and P2 (GTPPPPPPKE) with α-glucosidase.

[0022] Figure 6 The inhibitory activity of P1(A) and P2(B) on α-glucosidase.

[0023] Figure 7 It is the blood glucose level within 3 hours after the meal of P1 and P2. Among them, Figure A represents the effect of P1 and P2 on the blood glucose level of normal mice after intragastric administration of starch; Figure B represents the effect of P1 and P2 on the blood glucose level of normal mice after intragastric administration of maltose; Figure C is the area under the curve of blood glucose after intragastric administration of maltose; Figure D is the area under the curve of blood glucose after intragastric administration of starch.

[0024] Figure 8 This is the enzymatic reaction rate diagram of WESGPW and GTPPPPPPKE to α-glucosidase.

[0025] Fig. 9 It is a Lineweaver-Burk diagram. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0028] Embodiment 1:

[0029] A mixed sweet potato peptide composition for inhibiting alpha-glucosidase activity includes an amino acid sequence as shown in SEQ ID NO.1-2.

[0030] SEQ ID NO.1:

[0031] WESGWP.

[0032] SEQ ID NO.2:

[0033] GTPPPPPPPE.

[0034] Its chemical structure and secondary mass spectrum are as follows Figure 1-4 As shown in the figure, the two sweet potato peptides have the characteristics of competitive and non-competitive inhibition. They can not only compete with the substrate for the corresponding α-glucosidase catalytic active site, but also interact with the inactive site. Since the number of enzyme catalytic sites is fixed, when the sweet potato peptides preferentially bind to the active site of the enzyme, the corresponding substrate cannot bind to the active site on the enzyme, reducing the binding rate between the enzyme and the oligosaccharide; at the same time, they bind to the inactive site of the enzyme to form an oligopeptide-enzyme-substrate ternary complex, so that the glycosidic bond of the oligosaccharide cannot be cleaved, and a large number of oligosaccharides cannot be catalyzed by the enzyme into monosaccharides that can be absorbed by the human body, thereby avoiding postprandial hyperglycemia.

[0035] Embodiment 2:

[0036] The use of the sweet potato peptide composition described in Example 1 in the preparation of a product for inhibiting α-glucosidase activity.

[0037] Embodiment 3:

[0038] The method for preparing the above sweet potato peptide composition comprises the following steps:

[0039] (1) The sweet potatoes are washed and peeled, and 4 to 5 times the volume of water is added to crush and extract. The extract is allowed to stand for 6 to 8 hours and then filtered through gauze to remove the sweet potato fiber and large granular starch to obtain a supernatant. The supernatant is adjusted to a pH value of 4, allowed to stand and precipitate, and then centrifuged to obtain a precipitate. The precipitate is freeze-dried to obtain the sweet potato crude protein.

[0040] (2) Weigh an appropriate amount of sweet potato crude protein, add distilled water at a ratio of 1:20 (w / v), heat at 90°C for 10 min, and then high-pressure treat at 200 MPa for 6 min. Then adjust the pH to 10 with 1 mol / L NaOH, add 4% (w / w) alkaline protease, and hydrolyze in a 55°C constant temperature water bath for 3 h. After the enzymatic hydrolysis is completed, centrifuge at 4°C, 10,000 r / min for 15 min to obtain the supernatant to obtain the sweet potato enzymatic hydrolysate (SPP).

[0041] (3) The enzymatic hydrolyzate obtained in Example 1 was filtered through a microporous filter membrane (pore size 0.22 μm), and then treated in an ultrafiltration system with an ultrafiltration membrane having a molecular weight Mw cutoff value of 3 to obtain sweet potato peptide SPP-1 with Mw < 3 kDa.

[0042] (4) The sweet potato peptide is identified through separation, purification and sequence identification.

[0043] LC-MS / MS identification of sweet potato protein α-glucosidase inhibitory peptide sequence. After desalting, the peptide sample was centrifuged and dried, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and bottled for online LCMS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3μm, ), and then 20 μl volume was used for desalting. The sample was desalted and retained on the pre-column and then chromatographically processed on a C18 reverse phase column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm ) separation, the gradient used in the experiment was 5% to 38% of mobile phase B (80% acetonitrile, 0.1% formic acid) in 60 min. The mass spectrometer was operated with a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a nanospray Nano Flex ion source (ThermoFisher, USA), the spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 320 °C.

[0044] Effect verification:

[0045] 1. Perform molecular docking of the peptide with α-glucosidase (PDB ID: 2QMJ). Select the best docking conformation based on the binding energy. After the docking result file pdbqt is converted into a pdb file, it is imported into the Pymol software and the online website PLIP (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) for result visualization analysis. Use ChemDraw to draw the chemical structure, and then perform molecular docking of P1-P2 with α-glucosidase (such as Figure 5As shown). The binding energy required for the docking of peptides with α-glucosidase is shown in Table 1. The binding energy of the ligand and the receptor is used to evaluate the degree of binding between the two. The smaller the binding energy, the easier it is for the two to bind and the stronger the interaction. The binding energy of P1 and P2 with α-glucosidase is -9kcal / mol. Therefore, it is speculated that P1 and P2 have strong α-glucosidase inhibitory activity. The interaction force generated with α-glucosidase is shown in Table 2. P1 forms hydrogen bonds with the amino acid residues Asp203, Thr205, Ser448, Arg526, and Asp542 of α-glucosidase, and forms hydrophobic interaction forces with Tyr299, Trp406, Trp441, Asp542, Phe575, and Ala576. P2 forms hydrogen bonds with the amino acid residues Thr205, Asn207, Asn209, Arg526, Asp542, Gln603, and Tyr605 of α-glucosidase, and forms hydrophobic interactions with Thr205, Asn207, Tyr299, Trp406, Phe450, Leu473, Lys480, Gln603, and Tyr605. The binding of active peptides to macromolecules mainly depends on a variety of intermolecular forces such as hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic interactions, and hydrogen bonding plays an important role in stabilizing the docking complex. In addition, Asp 542 and Arg 526 are considered to be essential amino acid residues for the binding of the active pocket of α-glucosidase to the substrate. Therefore, P1 and P2 can occupy the key binding sites of α-glucosidase through hydrogen bonding, and bind to α-glucosidase under the influence of hydrophobic interaction, thereby inhibiting α-glucosidase in the body and delaying the rise of blood sugar.

[0046] Table 1: Binding energy required for peptide docking with α-glucosidase

[0047]

[0048] Table 2: Interaction forces with α-glucosidase

[0049]

[0050] 2. The peptides in Example 1 were artificially synthesized, and the purity of the synthesized polypeptides was >95%. After synthesis, the ability of each peptide to inhibit α-glucosidase was measured. Take 200 μL of sample and add 200 μL of α-glucosidase (0.2U / mL). Mix the above solution and incubate at 37°C for 5 minutes. Then add 200 μL of 2.5mM p-nitrobenzene-α-glucoside solution (pNPG) and 0.1M phosphate buffer (pH 6.8) to 2mL and incubate at 37°C for 20 minutes. Finally, add 200 μL of 0.4M sodium carbonate to stop the reaction and measure the absorbance at 405nm. The calculation formula for α-glucosidase inhibition rate is as follows:

[0051] α-Glucosidase inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100%

[0052] Where: A1—absorbance value of sample group,

[0053] A2—absorbance value of sample background,

[0054] A3—absorbance value without sample,

[0055] A4—Absorbance value of blank group without sample.

[0056] like Figure 6 As shown, the IC of P1 and P2 50 The values ​​were 4.137±0.640mg / mL and 3.239±0.37mg / mL, respectively. This indicated that P1 and P2 showed strong α-glucosidase inhibitory activity in in vitro experiments, and the activity of P2 was stronger than that of P1.

[0057] 3. After mice were gavaged with P1, P2 and starch or maltose, blood glucose levels within 3 hours after the meal were analyzed. Figure 7 AB results showed that the postprandial blood glucose of mice increased first and reached the maximum value within 0.5h. The blood glucose concentration decreased over time and returned to the initial level within 3h. After gavage with starch, the three pure peptides could inhibit the increase of postprandial blood glucose content; compared with the control group, the acarbose, P1, and P2 groups significantly reduced the area under the blood glucose curve (p<0.05), which was consistent with the results of in vitro inhibition of α-glucosidase activity. Compared with the results of the starch test, the effects of the two peptides on the postprandial blood glucose levels of mice induced by maltose showed the same trend. Therefore, P1 and P2 have obvious hypoglycemic effects in vivo, and it is speculated that these two peptides may have an inhibitory effect on α-glucosidase in vivo.

[0058] 4. Plot the enzymatic reaction rates of WESGPW and GTPPPPPPPKE on α-glucosidase at different peptide concentrations, e.g. Figure 8As shown, all straight lines pass through the origin and show a good linear relationship. Moreover, the slope of the straight line decreases with the continuous increase of peptide, indicating that the activity of α-glucosidase is not completely inhibited, but its catalytic rate gradually decreases. It is reported that irreversible inhibitors can form a stable enzyme-inhibitor complex with the enzyme through covalent interaction, completely inactivating the enzyme. Therefore, the experimental results show that WESGPW and GTPPPPPPKE are reversible α-glucosidase inhibitors.

[0059] The Lineweaver-Burk plot was used to study the inhibition type and inhibition kinetic parameters of the inhibitor. The maximum initial digestion rate (Vmax) calculated according to the intercept (1 / Vmax) of the Lineweaver-Burk equation line has hardly changed (Table 3). At the same time, the inhibition constant (Ki) calculated according to the slope of the equation (Km / vmax) increases with the increase in the concentration of QAGPDPW (Table 3). The fitting curves of WESGPW and GTPPPPPPPKE both intersect in the second quadrant, which is consistent with the mixed inhibition type. It can be seen from Table 3 that the maximum reaction rate values ​​of WESGPW and GTPPPPPPPKE at different concentrations decrease with increasing concentration. The concentration of the inhibitor corresponding to the Ki value of 50% of the enzyme binding to the inhibitor reflects the inhibitory strength of the inhibitor. The smaller the Ki value, the stronger the inhibition ability. Through calculation, the inhibition is ranked as GTPPPPPPPKE>WESGPW, which is consistent with the above IC 50 The conclusions are consistent.

[0060] Table 3

[0061]

[0062] Toxicity prediction was performed using ToxinPred ( https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php ). Allergenicity prediction was performed using AllerTOP v2.0 ( http: / / www.ddg-pharmfac.net / AllerTOP / ). It has been proven that the above sweet potato peptides are non-toxic and non-allergenic.

[0063] Finally, it should be noted that although the above embodiments describe the specific implementation of the present invention, they are not intended to limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. All modifications or equivalent replacements should be included in the protection scope of the present invention.

Claims

1. A mixed sweet potato peptide composition for inhibiting α-glucosidase activity, comprising a polypeptide whose amino acid sequence is shown in SEQ ID NO.1-2; the sweet potato peptide composition is artificially synthesized.

2. Use of the sweet potato peptide composition according to claim 1 in the preparation of a product for inhibiting α-glucosidase activity.

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