Application of millet prolamine K3ZAN2 in product preparation

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of millet prolamine K3ZAN2 to preparation of a product, the product is used for inhibiting the activity of alpha-glucosidase, and the amino acid sequence of the protein is shown as SEQ ID NO: 1. The millet prolamin K3ZAN2 is derived from a natural crop millet, so that the protein has relatively high biological safety and biocompatibility, can be used as an active component for efficiently inhibiting the activity of alpha-glucosidase to control the blood sugar level, and further can play an important role in management of metabolic diseases such as diabetes and the like, and has a good application prospect. The method can also be applied to development of food, medicines, health care products, kits or alpha-glucosidase inhibitors and other related products, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of millet prolamin K3ZAN2 in the preparation of products. Background Art

[0002] Diabetes, as a globally prevalent metabolic disease, is characterized by elevated blood glucose levels, which in turn lead to disorders in the metabolism of body fat, carbohydrates, and proteins. By inhibiting the activity of α-glucosidase, the digestion rate of starch can be delayed and the postprandial blood glucose level can be regulated. Therefore, inhibiting the activity of α-glucosidase has gradually become an important target for the treatment of metabolic diseases such as diabetes.

[0003] Currently, α-glucosidase inhibitor drugs have been widely used in the treatment of diabetes, such as acarbose, voglibose, etc. These drugs bind to α-glucosidase to prevent its decomposition of carbohydrates, thereby reducing the postprandial blood glucose peak, reducing blood glucose fluctuations, and effectively controlling the blood glucose level of diabetic patients. However, although these chemically synthesized drugs have good inhibitory effects, they may cause side effects such as gastrointestinal discomfort and allergic reactions, and their therapeutic effects are limited in some patients.

[0004] Foxtail millet (Setaria italica), as a cereal with various health benefits, is rich in nutritional components such as dietary fiber, polyphenolic compounds, and minerals. The millet obtained after processing foxtail millet has similar nutritional components to foxtail millet and is more easily digested and absorbed by the human body. Relevant research shows that using it as a dietary supplement helps to alleviate metabolic diseases such as diabetes and obesity. Specifically, the glycemic index (GI) of millet is relatively low, and the blood glucose rises slowly after consumption, which helps to maintain the stability of the body's blood glucose level.

[0005] Therefore, screening out the active components in millet that inhibit the activity of α-glucosidase is of great significance for developing new α-glucosidase inhibitor products with good inhibitory effects on α-glucosidase activity, high biocompatibility, and low toxicity and side effects. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, the present invention provides the application of millet prolamin K3ZAN2 in the preparation of products. The millet prolamin K3ZAN2 of the present invention is derived from the natural crop millet. Therefore, this protein has high biosafety and biocompatibility. It can not only control blood glucose levels as an active component that efficiently inhibits the activity of α-glucosidase, and thus play an important role in the management of metabolic diseases such as diabetes, but also be applied to the development of related products such as food, medicine, health products, test kits, or α-glucosidase inhibitors, with broad application prospects.

[0007] This invention is made based on the inventor's discovery and recognition of the following problems:

[0008] In the process of researching the bioactive components with α-glucosidase inhibitory activity in millet, the millet protein was first separated to obtain a millet prolamin mixture with a significant blood sugar-lowering effect. Then, through a large number of experiments and molecular simulation work, the monomer protein K3ZAN2 with α-glucosidase inhibitory activity was identified and screened from millet prolamin for the first time. The amino acid sequence of this protein is: methionine-lysine-isoleucine-phenylalanine-isoleucine-valine-leucine-alanine-leucine-leucine-threonine-leucine-alanine-alanine-serine-serine-alanine-serine-alanine-glutamine-glutamine-proline-glycine-tyrosine-tyrosine-glycine-serine-leucine-histidine-proline-cysteine-glycine-glutamic acid-phenylalanine-leucine-arginine-glutamine-arginine-cysteine-proline-alanine-valine-threonine-glycine-tryptophan-proline-tyrosine-alanine-tryptophan-serine-arginine-threonine-tryptophan-glutamine-proline-serine-serine-cysteine-methionine-alanine-methionine-histidine-glutamine-glutamine-cysteine-cysteine-glutamine-glutamine-leucine-arginine-glutamine-valine-glutamic acid-proline-leucine-histidine-arginine-cysteine-glutamine-glutamic acid-valine-cysteine-serine-leucine-valine-glutamic acid-threonine-threonine-valine-glutamine-glutamine-methionine-methionine-leucine-glutamine-glycine-serine-glutamic acid-tyrosine-tyrosine-glutamic acid-leucine-glutamine-histidine-alanine-alanine-leucine-glycine-alanine-lysine-asparagine-leucine-proline-alanine-methionine-cysteine-glycine-isoleucine-serine-leucine-proline-serine-tyrosine-cysteine-threonine-threonine-proline-cysteine-alanine-isoleucine-alanine-glycine-glycine-glycine-alanine-cysteine-cysteine (MKIFIVLALLTLAASSASAQQPGYYGSLHPCGEFLRQRCPAVTGWPYAWSRTWQPSSCMAMHQQCCQQLRQVEPLHRCQEVCSLVETTVQQMMLQGSEYYELQHAALGAKNLPAMCGISLPSYCTTPCAIAGGGACC, SEQ ID NO: 1); Then, the inventor further verified the interaction mechanism between this protein and α-glucosidase through molecular docking and molecular dynamics simulation techniques, and confirmed that it has a significant effect of inhibiting α-glucosidase activity, thus providing new ideas and new choices for the development of new α-glucosidase inhibitor products based on natural proteins.

[0009] In the first aspect of the present invention, there is provided an application of a protein in the preparation of a product. According to an embodiment of the present invention, the product is used to inhibit α-glucosidase activity, and the amino acid sequence of the protein is as shown in SEQ ID NO: 1. According to the application of the embodiment of the present invention, the prolamin K3ZAN2 is derived from the natural crop millet, and this protein has high biosafety and biocompatibility. Using this protein as an active ingredient for inhibiting α-glucosidase activity lays a foundation for the development of related products with high efficiency in inhibiting α-glucosidase activity, and further broadens the application scope of this protein.

[0010] According to an embodiment of the present invention, the above application may further have the following additional technical features:

[0011] According to an embodiment of the present invention, the product includes: food, medicine, health product, kit or α-glucosidase inhibitor.

[0012] In the second aspect of the present invention, there is provided a product. According to an embodiment of the present invention, the product is used to inhibit α-glucosidase activity; the product contains a protein with an amino acid sequence as shown in SEQ ID NO: 1. The product according to the embodiment of the present invention contains prolamin K3ZAN2, enabling the product to efficiently inhibit α-glucosidase activity and further broadening the application scope of this protein.

[0013] According to an embodiment of the present invention, the above product may further have the following additional technical features:

[0014] According to an embodiment of the present invention, the product includes: food, medicine, health product, kit or α-glucosidase inhibitor.

[0015] In the third aspect of the present invention, there is provided an application of a protein in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: controlling blood glucose level; preventing, alleviating and / or treating diabetes; wherein, the amino acid sequence of the protein is as shown in SEQ ID NO: 1. According to the application of the embodiment of the present invention, the prolamin K3ZAN2 is derived from the natural crop millet, and this protein has high biosafety and biocompatibility and can efficiently inhibit α-glucosidase activity. Applying it to the preparation of drugs can prevent, alleviate and / or treat diabetes, control blood glucose level, etc., and further broadens the application scope of this protein.

[0016] In the fourth aspect of the present invention, a pharmaceutical composition is proposed. According to an embodiment of the present invention, the pharmaceutical composition contains a protein with an amino acid sequence as shown in SEQ ID NO: 1. The pharmaceutical composition according to the embodiment of the present invention, by adding a protein with an amino acid sequence as shown in SEQ ID NO: 1, namely millet prolamin K3ZAN2, uses this protein as a pharmaceutically active ingredient for efficiently inhibiting the activity of α-glucosidase, and then exerts effects such as preventing, alleviating and / or treating diabetes and controlling blood glucose levels of the pharmaceutical composition.

[0017] According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may further have the following additional technical features:

[0018] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0019] In the fifth aspect of the present invention, an application of a protein in the preparation of hypoglycemic foods or health products is proposed. According to an embodiment of the present invention, the amino acid sequence of the protein is as shown in SEQ ID NO: 1. According to the application of the embodiment of the present invention, millet prolamin K3ZAN2 is derived from the natural crop millet. This protein has high biosafety and biocompatibility, and can efficiently inhibit the activity of α-glucosidase. Applying it to the preparation of hypoglycemic foods or health products can effectively control blood glucose levels, further broadening the application scope of this protein.

[0020] In the sixth aspect of the present invention, a hypoglycemic food or health product is proposed. According to an embodiment of the present invention, the hypoglycemic food or health product contains a protein with an amino acid sequence as shown in SEQ ID NO: 1. The hypoglycemic food or health product according to the embodiment of the present invention, by adding a protein with an amino acid sequence as shown in SEQ ID NO: 1, namely millet prolamin K3ZAN2, can efficiently inhibit the activity of α-glucosidase. The addition of this protein enables the hypoglycemic food or health product to have effects such as controlling blood glucose levels.

[0021] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0023] Figure 1 It is a test result graph of the α-glucosidase inhibition rate of millet protein in Example 2 of the present invention. Among them, the superscripts a, b, and ab in the figure are significance markers, and the presence of different letters indicates significance between groups;

[0024] Figure 2 It is the secondary mass spectrometry result diagram of millet prolamin in Example 3 of the present invention;

[0025] Figure 3 It is the protein coverage result diagram of millet prolamin K3ZAN2 in Example 3 of the present invention;

[0026] Figure 4 It is the structure simulation diagram of millet prolamin K3ZAN2 in Example 4 of the present invention;

[0027] Figure 5 It is the molecular docking schematic diagram of millet prolamin K3ZAN2 and α-glucosidase in Example 4 of the present invention. Among them, A is the 3D docking result between millet prolamin K3ZAN2 and α-glucosidase, B and C are the interaction display diagrams of amino acid hydrogen bonds, and D is the 2D interaction schematic diagram of millet prolamin K3ZAN2 and α-glucosidase: the green dotted line represents hydrogen bonds and electrostatic forces, while hydrophobic interactions are shown as pink and red arcs, and the numbers on each dotted line represent the force distance

[0028] Figure 6 It is the molecular dynamics simulation analysis result diagram of millet prolamin K3ZAN2 and α-glucosidase in Example 4 of the present invention. Among them, A is the root mean square deviation (RMSD) analysis of the complex of millet prolamin K3ZAN2 and α-glucosidase, B is the (RMSF) analysis of the complex of millet prolamin K3ZAN2 and α-glucosidase, C is the radius of gyration (Rg) of the complex of millet prolamin K3ZAN2 and α-glucosidase, D is the solvent accessible surface area analysis (SASA), E is the hydrogen bond number analysis of the complex, F-I is the principal component analysis (PCA) of the complex of millet prolamin K3ZAN2 and α-glucosidase, J is the dynamic correlation matrix analysis result (DCCM), and K is the free energy landscape analysis result (FEL) diagram. Detailed implementation manners

[0029] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0031] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0032] As used herein, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0033] As used herein, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where such events or conditions occur, and the cases where such events or conditions do not occur.

[0034] As used herein, the term "PPI mechanism" refers to the Protein-Protein Interaction (PPI) mechanism, that is, a molecular mechanism in which proteins form specific bindings through non-covalent bonds (such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals forces, etc.) to achieve biological functions.

[0035] Use of a protein in the preparation of a product

[0036] The present invention provides a use of a protein in the preparation of a product. According to an embodiment of the present invention, the product is used to inhibit α-glucosidase activity, and the amino acid sequence of the protein is as shown in SEQ ID NO: 1. According to the application of the embodiment of the present invention, the prolamin K3ZAN2 is derived from the natural crop millet, and this protein has high biosafety and biocompatibility. Using this protein as an active ingredient for inhibiting α-glucosidase activity lays a foundation for the development of related products with high-efficiency α-glucosidase activity inhibition function, and further broadens the application scope of this protein.

[0037] According to an embodiment of the present invention, the product includes: food, medicine, health care product, kit or α-glucosidase inhibitor. Thus, the prolamin K3ZAN2 is derived from the natural crop millet, and this protein has high biosafety and biocompatibility and can be applied to the development of various different types of products with α-glucosidase activity inhibition function, mainly including food, medicine, health care product, kit or α-glucosidase inhibitor, etc.

[0038] Product

[0039] The present invention provides a product. According to an embodiment of the present invention, the product is used to inhibit the activity of α-glucosidase; the product contains a protein with an amino acid sequence as shown in SEQ ID NO: 1. The product according to the embodiment of the present invention contains prolamin K3ZAN2, enabling the product to efficiently inhibit the activity of α-glucosidase and further broadening the application scope of this protein.

[0040] According to an embodiment of the present invention, the product includes: food, medicine, health product, kit or α-glucosidase inhibitor. Thus, prolamin K3ZAN2 can be added to food, medicine, health product, kit or α-glucosidase inhibitor to efficiently inhibit the activity of α-glucosidase, further broadening the application scope of this protein.

[0041] Use of a protein in the preparation of a drug

[0042] The present invention provides a use of a protein in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: controlling blood glucose level; preventing, alleviating and / or treating diabetes; wherein, the amino acid sequence of the protein is as shown in SEQ ID NO: 1. According to the application of the embodiment of the present invention, prolamin K3ZAN2 is derived from the natural crop millet. This protein has high biosafety and biocompatibility and can efficiently inhibit the activity of α-glucosidase. Applying it to the preparation of a drug can prevent, alleviate and / or treat diabetes, control blood glucose level, etc., further broadening the application scope of this protein.

[0043] Pharmaceutical composition

[0044] The present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition contains a protein with an amino acid sequence as shown in SEQ ID NO: 1. According to the pharmaceutical composition of the embodiment of the present invention, by adding a protein with an amino acid sequence as shown in SEQ ID NO: 1, namely prolamin K3ZAN2, and using this protein as a drug active ingredient for efficiently inhibiting the activity of α-glucosidase, the pharmaceutical composition can thus exert effects such as preventing, alleviating and / or treating diabetes, controlling blood glucose level, etc.

[0045] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Exemplarily, it is well known in the art that the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, which may include but is not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.; pharmaceutically acceptable excipients are well known in the art and include but are not limited to starch, lactose or dextrin; examples of diluents may include but are not limited to calcium carbonate, sodium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0046] Use of a protein in the preparation of a hypoglycemic food or health product

[0047] The present invention provides a use of a protein in the preparation of a hypoglycemic food or health product. According to an embodiment of the present invention, the amino acid sequence of the protein is as shown in SEQ ID NO: 1. According to the application of the embodiment of the present invention, the millet prolamin K3ZAN2 is derived from the natural crop millet. This protein has high biosafety and biocompatibility, and can efficiently inhibit the activity of α-glucosidase. Applying it to the preparation of a hypoglycemic food or health product can effectively control blood sugar levels, further broadening the application scope of this protein.

[0048] Hypoglycemic food or health product

[0049] The present invention provides a hypoglycemic food or health product. According to an embodiment of the present invention, the hypoglycemic food or health product contains a protein with an amino acid sequence as shown in SEQ ID NO: 1. According to the hypoglycemic food or health product of the embodiment of the present invention, by adding a protein with an amino acid sequence as shown in SEQ ID NO: 1, namely millet prolamin K3ZAN2, it can efficiently inhibit the activity of α-glucosidase, and the addition of this protein enables the hypoglycemic food or health product to have effects such as controlling blood sugar levels.

[0050] The following will explain the solution of the present invention in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0051] Example 1: Extraction and separation of millet protein

[0052] First, defat sufficient millet flour with n - hexane to obtain defatted millet flour, and then gradually separate millet protein on the basis of the traditional Osboren method. The specific steps are as follows:

[0053] 1. Extraction of millet albumin

[0054] Mix the defatted millet flour with pure water at a ratio of 0.17 (g / mL), and extract it in a 45 °C water bath for 4 h with stirring during this period; then centrifuge at 8000 rpm for 20 min and collect supernatant I and precipitate I respectively. Adjust the pH of supernatant I to 4.1 (isoelectric point to precipitate albumin); collect the precipitate, wash the precipitate 5 times with a small amount of pure water, and centrifuge at 8000 rpm for 20 min to remove the supernatant after each washing. Finally, freeze - dry the precipitate to obtain a millet albumin sample.

[0055] 2. Extraction of millet globulin

[0056] Mix precipitate I with 0.5 M NaCl solution at a ratio of 0.17 (g / mL), and extract it in a 45 °C water bath for 4 h with stirring during this period; then centrifuge at 8000 rpm for 20 min and collect supernatant II and precipitate II respectively. Adjust the pH of supernatant II to 4.3 (isoelectric point to precipitate albumin); collect the precipitate, wash the precipitate 5 times with a small amount of 0.5 M NaCl solution, and centrifuge at 8000 rpm for 20 min to remove the supernatant after each washing. Finally, freeze - dry the precipitate to obtain a millet globulin sample.

[0057] 3. Extraction of millet prolamin

[0058] Mix precipitate II with 70% ethanol solution at a ratio of 0.17 (g / mL), and extract it in a 45 °C water bath for 4 h with stirring during this period; then centrifuge at 8000 rpm for 20 min and collect supernatant III and precipitate III respectively. Place supernatant III in a dialysis bag and dialyze it with distilled water for 48 h. After dialysis is completed, freeze - dry the dialysis solution to obtain a millet prolamin sample.

[0059] 4. Extraction of millet glutelin

[0060] Mix precipitate III with 0.1 M NaOH solution at a ratio of 0.17 (g / mL), and extract it in a 45 °C water bath for 4 h with stirring during this period; then centrifuge at 8000 rpm for 20 min and collect supernatant IV and precipitate IV respectively. Adjust the pH of supernatant IV to 5.3 (isoelectric point to precipitate millet glutelin); collect the precipitate, wash the precipitate 5 times with a small amount of 0.1 M NaOH solution, and centrifuge at 8000 rpm for 20 min to remove the supernatant after each washing. Finally, freeze - dry the precipitate to obtain a millet glutelin sample.

[0061] 5. Determination of Protein Content

[0062] The protein contents of the albumin sample, globulin sample, prolamin sample, and glutelin sample of millet obtained in Steps 1 - 5 were determined by the Kjeldahl method.

[0063] The results showed that the purities of the albumin sample, globulin sample, prolamin sample, and glutelin sample of millet were 51.53 ± 2.23%, 84.39 ± 1.18%, 87.89 ± 0.45%, and 73.50 ± 0.51% respectively.

[0064] Example 2: α - Glucosidase Inhibition Test

[0065] The inventors conducted an α - glucosidase inhibition test, and the specific steps were as follows:

[0066] Albumin sample group, globulin sample group, prolamin sample group, and glutelin sample group of millet: 20 μL of α - glucosidase solution (1.0 mg mL -1 , dissolved in 0.05 M PBS, pH = 6.9), 20 μL of each protein sample solution (the protein samples obtained in Example 1 were adjusted to 4 mg mL according to the purity) -1 , dissolved in 0.05 M PBS, pH = 6.9), and 50 μL of PBS (0.05 M, pH = 6.9) were added to a 96 - well plate and incubated at 37 °C for 10 min.

[0067] Albumin sample blank group, globulin sample blank group, prolamin sample blank group, and glutelin sample blank group of millet: 20 μL of each protein sample solution (the protein samples obtained in Example 1 were adjusted to 4 mg mL according to the purity) -1 , dissolved in 0.05 M PBS, pH = 6.9), and 50 μL of PBS (0.05 M, pH = 6.9) were added to a 96 - well plate and incubated at 37 °C for 10 min.

[0068] Control group: 20 μL of α - glucosidase solution (1.0 mg mL -1 , dissolved in 0.05 M PBS, pH = 6.9), 20 μL of 0.05 M phosphate buffer (pH = 6.8), and 50 μL of PBS (0.05 M, pH = 6.9) were added to a 96 - well plate and incubated at 37 °C for 10 min.

[0069] Control blank group: 90 μL of PBS (0.05 M, pH = 6.9) was added to a 96 - well plate and incubated at 37 °C for 10 min.

[0070] After the incubation was completed, 20 μL of PNPG solution (1.0 mM) was added to each well of the above groups to initiate the hydrolysis reaction. After incubation at 37 °C for 20 min, 20 μL of 0.2 M Na2CO3 was added to terminate the hydrolysis reaction. Then, the absorbance of the sample solution in each well was recorded at 405 nm using a microplate reader (ThermoFisher Scientific, Waltham, MA, USA). All sample solutions were measured three times, and the α-glucosidase inhibition rates of millet albumin, millet globulin, millet prolamin, and millet glutelin were calculated using Equation 1.

[0071]

[0072] In Equation 1: ODA is the absorbance value of the protein sample group; ODa is the absorbance value of the blank group of the protein sample; ODB is the absorbance value of the control group; ODb is the absorbance value of the control blank group.

[0073] The test results of the α-glucosidase inhibition rate of millet protein are shown in Figure 1 .

[0074] The results showed that the α-glucosidase inhibition rates of millet albumin, millet globulin, and millet glutelin were 39.23 ± 6.69%, 25.13 ± 7.54% (P < 0.5), and 33.64 ± 5.63% (P < 0.5), respectively. Compared with the former three, the α-glucosidase inhibition rate of millet prolamin was as high as 49.69 ± 8.57%, indicating that millet prolamin has a better α-glucosidase inhibition effect.

[0075] Example 3: Screening and obtaining millet prolamin K3ZAN2

[0076] 1. Shotgun proteomics analysis of millet prolamin

[0077] The inventors selected millet prolamin with a better α-glucosidase inhibition effect for shotgun proteomics analysis. The specific steps are as follows:

[0078] After enzymatically digesting the prolamin sample obtained in Example 1 with trypsin solution, mass spectrometry analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) based on EASY-nanoLC1200, operating in data-dependent acquisition mode (DDA) to achieve automatic switching between MS and MS / MS data. The mass spectrometry parameters were set as follows: the first-stage mass spectrometry scan range was 350 - 1500 m / z, the resolution was 60,000, the normalized AGC target value was 300%, and the maximum injection time was 25 ms; the second-stage mass spectrometry resolution was 15,000, the normalized AGC target value was 50%, the maximum injection time was 22 ms, the collision energy was 30%, and the dynamic exclusion time was 30 s.

[0079] Subsequently, the obtained mass spectrometry data was analyzed using PEAKS Studio version 10.6 software, and protein identification was performed using the Uniprot-Setaria italica database (version 2024, 35701 entries) (https: / / www.uniprot.org / uniprotkb?query=Setaria+italica). The parameter settings included trypsin digestion, a precursor ion mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.02 Da, a maximum of 2 missed cleavage sites, a fixed modification of Carbamidomethylation (+57.02 Da) for Cys, variable modifications of Oxidation (+15.99 Da) for Met, Deamidation (+0.98 Da) for Asn / Gln, and Acetylation (+42.01 Da) for the protein N-terminus; the protein identification criterion was 1% FDR, and each protein contained at least 1 specific peptide.

[0080] The secondary mass spectrometry results of prolamin are shown in Figure 2 .

[0081] The results showed that: the secondary mass spectrometry (MS / MS) analysis successfully resolved the peptide sequences of prolamin. Obvious b-ion (blue) and y-ion (red) signals were visible in the spectra, indicating that the peptide was cleaved at specific sites; the main y-ion peaks (such as y9, y14, y21, etc.) and b-ion peaks (such as b5, b8, b9, etc.) were all in good agreement with the theoretical values, with an error within ±0.02 Da, verifying the accuracy of the peptide; thus, it was confirmed that the peptide was QEVCSLVETTVQQMMLQGSEYYELQHAALGAK, which, as a characteristic peptide of the K3ZAN2 protein, further supported its identification as being derived from prolamin.

[0082] 2、Alphfold Structure Modeling

[0083] The inventors downloaded the amino acid sequences of 5 proteins in FASTA format from the Uniprot database (https: / / www.uniprot.org / ) and constructed 3D models of these proteins through the official AlphaFold 3 (https: / / alphafoldserver.com / ); and based on multiple key mass spectrometry parameters and the Alphfold prediction template scores, mainly including protein sequence coverage (Coverage%), mass spectrometry score (-10lgP), number of specific peptides (#Unique), protein expression abundance (AreaX), total number of matching spectra corresponding to the protein (#Spec), and Alphfold pTM score, etc., finally screened out the core millet prolamins I-V.

[0084] The results of shotgun proteomics and structure screening of millet prolamins are shown in Table 1, and the protein coverage results of millet prolamin K3ZAN2 are shown in Figure 3 。

[0085] Table 1 Results of Shotgun Proteomics and Structure Screening of Millet Prolamins

[0086]

[0087] In Table 1, Accession is the unique accession number of each protein in the UniProt database; -10lgP is the mass spectrometry search score, and a high score (such as >50 or 100) indicates high confidence; Coverage(%) is the protein sequence coverage in the sample; Area is the protein expression abundance in the sample; #Peptides is the number of peptides identified and matched in the sample; #Unique is the number of specific peptides contained in the identified protein; #Spec is the total number of matching spectra corresponding to the identified protein; Avg.Mass is the average molecular weight; pTM is the template modeling score.

[0088] Amino acid sequence of protein K3ZAN2:

[0089] MKIFIVLALLTLAASSASAQQPGYYGSLHPCGEFLRQRCPAVTGWPYAWSRTWQPSSCMAMHQQCCQQLRQVEPLHRCQEVCSLVETTVQQMMLQGSEYYELQHAALGAKNLPAMCGISLPSYCTTPCAIAGGGACC (SEQ ID NO: 1)

[0090] Amino acid sequence of protein K3YKH0:

[0091] MPSPSALGKRDGGDGILAALSRSQLAAHGREAATMAKKLLRSTGKAAWIAGTTFLVLVVPLIIEMDREQQLNDLELQQQTLLGGPPAPIK (SEQ ID NO: 2)

[0092] Amino acid sequence of protein K4AG30:

[0093] MASRIALVVLLALSAVATTTATCPQNFPLMMGIGMMDPCMQSCMMQQPFTMVSSFSPIMGMGSMVPCMQSCMMQRAFTIGGSPLLAMLMQQSPLAMLQQQCCMQSMMQGMMSPQCRCGAMCQMMHLQQIRIAMQLPFMWNTAAMMMQPTYWRQPFPSCAC (SEQ ID NO: 3)

[0094] Amino acid sequence of protein K3YX48:

[0095] MALRALYNEIRGMKVREVPAYLKPRLTWENVKKSADQAVDRYIEKYIETSKPDPLFHVCFGGMAFSYLVALPWERAHLAHLEEMERTGGKHH (SEQ ID NO: 4)

[0096] Amino acid sequence of protein K4AGP2:

[0097] MAGRLTAAGSRILGGGGARAAAAALRHRAGMGLPVGRHIVPDKPLPTNDELLWDN GTPFPEPCIDRLAPHIGKYEALAWLCGGLGFFATLGVAAAVNDKASKIPYTPKVYPYDNL RVELGDRP (SEQ ID NO: 5)

[0098] Results showed that shotgun proteomics analysis identified five kinds of prolamins in millet, among which prolamin K3ZAN2 had the highest confidence level (-10lgP = 323), a coverage of 66%, 50 peptide segments (#Peptides), and 49 specific peptide segments (#Unique), indicating its high abundance and high reliability in the prolamin sample of millet; the pTM score predicted by AlphaFold structure showed that the pTM of prolamin K3ZAN2 was 0.61, indicating that the overall structure prediction was relatively reliable, while the pTM scores of prolamins K4AG30, K3YX48, and K4AGP2 were all low (<0.4), indicating that there was a large uncertainty in the prediction of their overall topological structures.

[0099] The above results indicated that prolamin K3ZAN2 was the prolamin with the highest confidence level, the largest coverage, and the strongest reliability of the predicted structure in shotgun proteomics analysis, and it was speculated that it might play an important role in function.

[0100] Example 4: Molecular docking and molecular dynamics simulation of prolamin K3ZAN2 in millet

[0101] Based on the mass spectrometry results and the prediction credibility of Alphfold, the inventors used the prolamin K3ZAN2 with the highest credibility as the ligand and α-glucosidase as the receptor to further determine the application potential of prolamin in reducing blood sugar through molecular docking and molecular dynamics simulation. The specific steps are as follows:

[0102] 1. Structure analysis of prolamin K3ZAN2

[0103] The structural simulation of prolamin K3ZAN2 is shown in Figure 4 .

[0104] The results showed that the structure of millet prolamin K3ZAN2 is characterized by a dominant α-helix architecture and consists of 137 amino acids, with glutamine (GLN) having the highest proportion, reaching 12.15%; it contains a total of six key α-helical domains. The N-terminal region (residues 1-25) exhibits a highly dynamic disordered conformation and forms a unique "rhinoceros horn"-shaped protrusion at the top. This protrusion structure is connected to the main structure through an ultra-short α-helix composed of GLY / A:26 to LEU / A:28. The subsequent conformational conversion region contains two characteristic structural elements: a short disordered segment (residues 28-29) is connected to the second α-helix (HIS / A:29-ARG / A:36), and then transitions to the third α-helix region (TYR / A:47-TRP / A:53) through a loop structure (residues 37-46). Its C-terminal domain contains three extended α-helices (SER / A:56-GLN / A:71, ARG / A:77-LEU / A:94, TYR / A:99-MET / A:115); these helices are stabilized by each other through a hydrophobic core and a hydrogen bond network, ultimately forming a triangular pyramid-like "fortress"-shaped folding unit of millet prolamin K3ZAN2.

[0105] 2. Molecular Docking

[0106] Retrieve the three-dimensional structure of α-glucosidase (PDB ID: 3A4A) from the RCSB database (https: / / www.rcsb.org / ), and then import the protein receptor (α-glucosidase) and ligand (millet prolamin K3ZAN2) into HADDOCK for molecular docking. Before molecular docking, remove water molecules and add hydrogen atoms; analyze the binding force between millet prolamin K3ZAN2 and α-glucosidase through PDBePISA, and use LigPlus software to analyze the interaction between the active pocket residues and the combined conformation ligand. Then use PyMOL software to visualize the binding of the ligand and the receptor.

[0107] The schematic diagram of the molecular docking of millet prolamin K3ZAN2 and α-glucosidase is shown in Figure 5 .

[0108] The results showed that the molecular binding energy of millet prolamin K3ZAN2 and α-glucosidase is -12.8 kcal / mol, indicating that millet prolamin K3ZAN2 and α-glucosidase have good affinity; specifically, the Thr11 OG1 and O atoms of millet prolamin form distances of and with the active site residues Thr285 OG1 and OG on Ser282 of α-glucosidase, respectively, forming two hydrogen bonds ( Figure 5-B); In addition, Ser18O forms a hydrogen bond with the sub-site OG on Ser311 with a certain length; the sub-site NE2 on Gln 64 forms a hydrogen bond with Phe 321O with a certain length; in addition, OE1 and CD on the Gln20 amino acid residue form hydrogen bonds with Asn317 and Leu 318O with lengths of and respectively; finally, Gly 26O forms a hydrogen bond with NZ on the positively charged Lys432 amino acid residue with a length of ; ( Figure 5 -C); A total of 7 hydrogen bonds are formed, indicating that the prolamin K3ZAN2 from millet forms a relatively stable complex with α-glucosidase, thereby inhibiting the activity of α-glucosidase; on the other hand, strong hydrophobic interactions are observed between the hydrophobic amino acid residues Gly23, Leu9, Ala17, Met59 of prolamin K3ZAN2 from millet and the amino acid residues Leu318, Phe321, Thr285 of α-glucosidase Figure 5 -D), promoting the mutual formation of a stable binding between prolamin K3ZAN2 from millet and α-glucosidase and enhancing the binding stability.

[0109] 3. Molecular dynamics simulation

[0110] (1) System preparation

[0111] First, the α-glucosidase and prolamin K3ZAN2 from millet are constructed into a complex using the Gromacs 2023 software. During the simulation, the classical AMBER14SB_parmbsc1 force field is selected and applied to the complex of α-glucosidase and prolamin K3ZAN2 from millet, and the pdb2gmx module in Gromacs is used to generate a topology file for the complex; then the TIP3P water model is used to solvate the system to ensure that the distance from the protein atoms to the water tank boundary is at least 1 nm to simulate the solvent effect in a real biological environment; finally, by adding Na + or Cl - ions to neutralize the charge of the system, making the whole system electrically neutral and avoiding the influence of charge imbalance on the simulation results.

[0112] (2) Energy minimization

[0113] Then, the steepest descent algorithm is used for energy minimization to remove the bad contacts and high-energy conformations in the system. The convergence criterion is set to 0.01 kcal / mol, and the maximum number of steps allowed is 50000. The positions of all atoms are optimized to obtain a stable initial conformation.

[0114] (3) System equilibrium

[0115] Then, the entire system is heated up in the NVT ensemble, and harmonic constraints with a force constant of are added to the protein backbone to maintain the structural stability of the protein during the heating process, so that the entire system is heated up to 310 K within a cycle of 100 ps to simulate the dynamic behavior of biomolecules at physiological temperature; after the heating is completed, 100 ps of equilibration is carried out in the NPT ensemble, the time step is set to 2 fs, the Langevin algorithm is used to control the temperature to remain at 310 K, and the Berendsen algorithm is used to control the pressure to remain at 1 atm to simulate the thermodynamic properties of biomolecules under physiological conditions.

[0116] (4) Molecular dynamics simulation and related analysis

[0117] Molecular dynamics simulation is carried out using the Gromacs-2023.4 GPU version. The total number of steps (nsteps) is set to 50000000, and one trajectory conformation is saved every 10 ps, and a total of 100 ns of simulation is carried out to fully sample the dynamic behavior of the system; finally, the complex is annealed from 310 K to 300 K for 102 ns to simulate the temperature change process of biomolecules under actual biochemical experimental conditions; then molecular dynamics simulation analysis is carried out, including root mean square deviation (RMSD), further calculation of root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) based on the simulation trajectory from 60 to 100 ns; and based on the Bio3D toolkit and the last 30 ns MD trajectory, principal component analysis (PCA) is described, and the dynamic cross-correlation matrix (DCCM) is used to study the characteristic changes of the correlated motions between residues; by calculating the Gibbs free energy of each protein molecule and plotting it as a free energy landscape (FEL), and the gmx_MMPBSA method is used to determine the binding free energy (ΔG) between prolamin K3ZAN2 and α-glucosidase.

[0118] The results of molecular dynamics simulation analysis of prolamin K3ZAN2 and α-glucosidase are shown in Figure 6 , and the results of the binding energy determination of prolamin K3ZAN2 protein and α-glucosidase are shown in Table 2.

[0119] Table 2 Results of the binding energy determination of prolamin K3ZAN2 protein and α-glucosidase

[0120] Energy Component Average SD(Prop.) SD SEM(Prop.) SEM BOND 2294.28 41.55 41.55 1.31 1.31 ANGLE 5884.81 60.43 60.43 1.91 1.91 DIHED 9320.79 36.78 36.78 1.16 1.16 VDWAALS -6202.9 36.5 36.5 1.15 1.15 EEL -52031.32 164.47 164.47 5.2 5.2 1-4VDW 2676.11 22.01 22.01 0.7 0.7 1-4EEL 32579.48 64.77 64.77 2.05 2.05 EGB -10257.92 133.05 133.05 4.21 4.21 ESURF 199.97 2.86 2.86 0.09 0.09 GGAS -5478.76 199.48 170.56 6.31 5.39 GSOLV -10057.95 133.08 131.63 4.21 4.16 TOTAL -15536.71 239.8 83.68 7.58 2.64

[0121] In Table 2, Energy Component refers to the energy components, including bond energy (BOND), angle energy (ANGLE), dihedral angle energy (DIHED), van der Waals interaction (VDWAALS), electrostatic interaction (EEL), 1-4 van der Waals interaction (1-4VDW), 1-4 electrostatic interaction (1-4EEL), generalized Born solvation energy (EGB), surface tension term (ESURF), gas-phase total energy (GGAS), solvation total energy (GSOLV), and total energy (TOTAL). Average is the average value of this energy term, which is the average energy of multiple frame data (such as multiple time points in molecular dynamics simulation). SD(Prop.) is the propagation standard deviation of this energy term, indicating the fluctuation of the energy value. SD is the standard deviation, used to measure the dispersion degree of the data. SEM(Prop.) is the propagation standard error of the mean, that is, the mean standard error calculated by the propagation method, measuring the reliability of the mean. SEM is the standard error of the mean, indicating the degree of uncertainty of the average value. SEM = SD / √n (n is the number of samples).

[0122] The results show that the binding energy between millet prolamin K3ZAN2 and α-glucosidase is relatively low, indicating that a stable complex is formed between the two. Among them, the electrostatic interaction contributes most significantly to the binding energy, while the van der Waals force and solvation effect also affect the binding stability to a certain extent. These results suggest that millet prolamin K3ZAN2 binds to α-glucosidase through electrostatic attraction and hydrophobic interaction, thereby affecting its activity.

[0123] (1) Regarding the stability of the complex

[0124] The results of RMSD (root mean square deviation) analysis show that the complex formed by α-glucosidase and millet prolamin K3ZAN2 exhibits large fluctuations in the initial stage of the simulation (0 - 40 ns), indicating that the system has not reached a stable state. As the simulation time extends, the RMSD value tends to be stable during 40 - 100 ns and finally stabilizes at about 0.2 nm, indicating that the complex has formed a relatively stable binding state ( Figure 6 -A); Further, the results of RMSF (root mean square fluctuation) analysis show that the RMSF values of the residues Thr 11 (0.2790 nm), Ser 18 (0.2430 nm), Gln 20 (0.2152 nm), Gly 26 (0.1851 nm), and Gln 64 (0.1364 nm) of millet prolamin K3ZAN2 are all higher than the overall average value (0.1183 nm) of the entire system, indicating that these residues have higher flexibility during the binding process. Figure 6-B); Meanwhile, the root mean square radius of gyration (Rg) analysis results show that the entire system fluctuates greatly within 0 - 10 ns, but the amplitude gradually decreases after 10 - 40 ns and stabilizes at around 1.9 nm after 40 ns ( Figure 6 -C), indicating that the flexible region of zein K3ZAN2 has undergone dynamic adaptive adjustment during the complex formation process.

[0125] (2) Regarding the solvent accessibility of the complex structure

[0126] The solvent accessible surface area (SASA) analysis results show that in the initial stage of the simulation (0 - 45 ns), the SASA value fluctuates within a large range (290 - 320 nm 2 ), presumably related to the dynamic rearrangement of protein interactions; but after 45 ns, the SASA value tends to be stable, with an average value maintained at 310 nm 2 , and the fluctuation amplitude is small ( Figure 6 -D), indicating that zein K3ZAN2 maintains a relatively stable conformation after the complex formation.

[0127] (3) Regarding hydrogen bond interactions and binding free energy

[0128] The number of hydrogen bonds between zein K3ZAN2 and α - glucosidase shows significant dynamic changes ( Figure 6 -E). In the initial stage of the simulation (0 - 25 ns), the number of hydrogen bonds gradually increases during the protein interaction process (10 - 20). As the simulation time extends, the number of hydrogen bonds tends to be stable after 25 ns and finally maintains between 15 - 20. The formation of these hydrogen bonds may presumably inhibit the enzyme catalytic activity by hindering the substrate from entering the enzyme active site or changing the local conformation of the enzyme active site; further, the principal component analysis (PCA) and dynamic cross - correlation matrix (DCCM) analysis results show that there is a significant dynamic synergistic effect between Thr 11, Ser 18, Gln 20, Gly 26 and Gln 64 of zein K3ZAN2 and Ser 282, Thr 285, Ser 311, Phe 321 and Lys 432 of α - glucosidase ( Figure 6 -F - L); further, the calculated binding free energy of zein K3ZAN2 and α - glucosidase shows that the total binding free energy of the complex is - 15536.71 kJ / mol, indicating that its binding process is spontaneous and stable. The main contribution to its free energy comes from electrostatic interactions (EEL, - 52031.32 kJ / mol) and van der Waals interactions (VDWAALS, - 6202.9 kJ / mol). Among them, the electrostatic interaction is mainly dominated by hydrogen bonds, further verifying the key role of hydrogen bonds in the inhibition mechanism.Figure 6 -E)

[0129] The above results indicate that the flexible region of millet prolamin K3ZAN2 has dynamic adaptability during the binding process with α-glucosidase. These key flexible residues (Thr 11, Ser 18, Gln 20, Gly 26, Gln 64) form stable complexes with the active site of α-glucosidase through hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces. This protein-enzyme interaction leads to conformational changes in the active site of the enzyme or directly hinders the entry of the substrate, thereby inhibiting the catalytic activity of α-glucosidase. Accordingly, millet prolamin K3ZAN2 can inhibit the catalytic activity of α-glucosidase through the PPI mechanism and can be used as an α-glucosidase inhibitor in the development of related products such as blood glucose level control.

[0130] Example 5: In vitro recombinant expression of millet prolamin K3ZAN2 and verification of α-glucosidase inhibitory ability

[0131] The inventors carried out in vitro recombinant expression of the screened millet prolamin K3ZAN2 and verified its α-glucosidase inhibitory ability in vitro. The specific steps are as follows:

[0132] (1) Recombinant expression

[0133] First, the target gene was cloned into the pET30a vector after codon optimization, and a His tag was fused at the C-terminus to facilitate subsequent purification. After the recombinant plasmid was verified by sequencing, it was transformed into an Escherichia coli expression host to induce protein expression. After protein expression, the bacterial cells were collected by centrifugation and lysed by sonication to obtain the soluble and inclusion body protein fractions. The soluble protein was purified by Ni 2 + affinity chromatography and eluted with different concentrations of imidazole gradients. If the protein mainly exists in the form of inclusion bodies, a combination of denaturing purification and gradient renaturation strategies was used to improve the renaturation efficiency and optimize the protein activity of millet prolamin K3ZAN2. Thus, finally, highly purified recombinant protein K3ZAN2 was obtained to support subsequent functional verification.

[0134] (2) Verification of α-glucosidase inhibitory ability

[0135] Referring to the α-glucosidase inhibition test method in Example 2, the in vitro function of highly purified recombinant protein K3ZAN2 was verified.

[0136] The results showed that the expression of millet prolamin K3ZAN2 using an Escherichia coli vector, and the obtained highly purified recombinant protein K3ZAN2 was verified by the α-glucosidase inhibition test to have a high ability to inhibit the activity of α-glucosidase.

[0137] Therefore, the millet prolamin K3ZAN2 can be used as an α-glucosidase inhibitor in the development of related products such as blood glucose level control.

[0138] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0139] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a protein in the preparation of a product, characterized in that, The product is used to inhibit the activity of α-glucosidase, and the amino acid sequence of the protein is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein The product includes: Food, medicine, health care product, kit or α-glucosidase inhibitor.

3. A product, characterized in that, The product is used to inhibit the activity of α-glucosidase; The product contains a protein with an amino acid sequence shown in SEQ ID NO:

1.

4. The product according to claim 3, wherein The product includes: Food, medicine, health care product, kit or α-glucosidase inhibitor.

5. Use of a protein in the preparation of a drug, characterized in that, The drug has at least one of the following uses: Controlling blood glucose levels; Preventing, alleviating and / or treating diabetes; Wherein, the amino acid sequence of the protein is shown in SEQ ID NO:

1.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a protein with an amino acid sequence shown in SEQ ID NO:

1.

7. The pharmaceutical composition according to claim 6, wherein The pharmaceutical composition further includes pharmaceutically acceptable excipients.

8. Use of a protein in the preparation of hypoglycemic foods or health products, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

1.

9. A hypoglycemic food or health product, characterized in that, The hypoglycemic food or health care product contains a protein with an amino acid sequence shown in SEQ ID NO: 1.

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