A walnut hypoglycemic peptide and its preparation method and application by ultrasonic shearing-assisted enzymatic hydrolysis

Abstract

This invention relates to the field of bioactive small molecule peptide technology, specifically a walnut hypoglycemic peptide and its preparation method and application using ultrasound-assisted enzymatic hydrolysis. Addressing the bottleneck of low enzymatic hydrolysis efficiency due to the dense structure of walnut protein, a combined pretreatment of 8000-12000 r / min shearing and 300-400W ultrasound is employed. Under conditions of 4-6 min shearing, 21 min ultrasound, and an enzyme concentration of 12 kU / g, the obtained product exhibits an inhibition rate of 54.38 ± 0.77% against α-glucosidase. LC-MS / MS and molecular dynamics simulations confirm that core peptide segments (such as FFPGSP and APSKDAPMF) can stably bind to the enzyme's active site, with FFPGSP exhibiting a strong interaction with residue Glu 271. This invention improves enzymatic hydrolysis efficiency and product activity, providing a core solution for the high-value utilization of walnut resources and the development of hypoglycemic foods.

Description

Technical Field

[0001] This invention belongs to the field of bioactive small molecule peptide technology, and particularly relates to a walnut hypoglycemic peptide and its preparation method and application by ultrasonic shearing-assisted enzymatic hydrolysis. Background Technology

[0002] With the increasing aging of the global population and changes in modern lifestyles, hyperglycemia and its associated chronic metabolic diseases such as type 2 diabetes, obesity, and cardiovascular disease have become serious public health challenges. The stable maintenance of blood glucose levels depends on a complex endocrine regulatory system, in which the rate of carbohydrate breakdown and absorption is a key factor determining postprandial blood glucose stability. Studies have shown that α-glucosidase in the gut converts carbohydrates into glucose by hydrolyzing glycosidic bonds; excessive activity of α-glucosidase can lead to abnormal fluctuations in blood glucose levels, thereby inducing a vicious cycle of insulin resistance, oxidative stress, and chronic inflammation. Currently, while commonly used chemical hypoglycemic drugs are fast-acting, long-term use often results in gastrointestinal discomfort or burden on the liver and kidneys. Therefore, the search for natural, safe, and multi-target regulatory food-derived bioactive peptides has become a research hotspot in the fields of functional foods and medicine.

[0003] Walnuts are a high-quality plant protein source with a balanced amino acid composition, and their enzymatic hydrolysis can produce oligopeptides with significant biological activity. However, in the practical application of existing technologies, the preparation of highly active hypoglycemic peptides from walnut protein still faces severe technical bottlenecks. Studies have found that the spatial conformation of walnut protein is usually quite dense, with molecules highly cross-linked by strong hydrogen bonds, hydrophobic interactions, and disulfide bonds. Especially high-quality varieties, such as the Yunnan deep-striped walnut, have extremely stable protein microstructures. This compact conformation constitutes a natural physical barrier in traditional enzymatic hydrolysis processes, resulting in a severe physical shielding effect of the substrate on proteases. This makes it difficult to expose active sites buried within the molecules, leading to low enzymatic hydrolysis efficiency and uneven distribution of product activity. Existing preparation methods often employ single enzymatic hydrolysis or simple physical-assisted methods, which often only achieve limited degradation of the protein particle surface. This not only limits the degree of hydrolysis but also causes specific peptides with highly efficient inhibitory activity to be locked in the dense protein core region, preventing their release and making it difficult for the product's α-glucosidase inhibitory activity to reach the expected level. In addition, single physical modification techniques (such as pure ultrasound or single shearing treatment) often have drawbacks such as low energy utilization, uneven treatment, or difficulty in completely breaking the compact conformation.

[0004] Therefore, how to develop a composite physical modification process that can precisely disrupt the dense conformation of walnut protein (especially the dense Yunnan deep-textured walnut protein) and induce the orderly rearrangement of higher-order structures to achieve efficient release of active peptides is a core technical problem that urgently needs to be solved in the field of high-value utilization of walnut resources and development of functional foods that help lower blood sugar. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a walnut hypoglycemic peptide and its preparation method and application using ultrasonic shearing-assisted enzymatic hydrolysis, thereby solving the problems of low enzymatic hydrolysis efficiency and difficulty in releasing active peptides caused by the dense structure of walnut protein.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a walnut hypoglycemic peptide, comprising: The amino acid sequence of the walnut hypoglycemic peptide is as shown in SEQ ID NO.1, the peptide segment APSKDAPMF, the peptide segment FFPGSP, as shown in SEQ ID NO.2, or a combination of both.

[0007] Furthermore, the molecular weight of the peptide APSKDAPMF is 963.12 Da, and the molecular weight of the peptide FFPGSP is 650.73 Da.

[0008] Furthermore, the walnut hypoglycemic peptide is derived from walnut protein, which is a protein product extracted from defatted walnuts, with a protein content of ≥60%.

[0009] Secondly, the present invention also provides a method for preparing walnut hypoglycemic peptides by ultrasonic shearing-assisted enzymatic hydrolysis, comprising the following steps: Step 1: Mix the pretreated walnut protein with water at a material-to-liquid ratio of 1:25 g / mL, and perform shearing treatment to obtain a walnut protein suspension; the shearing treatment speed is 8000-12000 r / min and the time is 4-6 min. The second step is to subject the walnut protein suspension to ultrasonic treatment. The ultrasonic treatment power is 300-400W, the ultrasonic time is 21 min, and the temperature is controlled below 4℃. The third step is to add neutral protease to the ultrasonically treated suspension for enzymatic hydrolysis. The amount of enzyme added is 10,000-12,000 U / g walnut protein, the hydrolysis temperature is 50-55℃, and the hydrolysis time is 3-4 h to obtain the hydrolysate. Step 4: Centrifuge the enzymatic hydrolysate, collect the supernatant, and freeze-dry it to obtain walnut hypoglycemic peptide.

[0010] Furthermore, in step one, the walnut protein is pre-passed through a 40-60 mesh sieve.

[0011] Furthermore, in step three, the activity of the neutral protease is 50,000 U / g.

[0012] Furthermore, in step four, the centrifugation conditions are 5000-8000 r / min.

[0013] Thirdly, the present invention also provides the application of walnut hypoglycemic peptide in the preparation of products with α-glucosidase inhibitory function.

[0014] Furthermore, the product is a hypoglycemic drug, a hypoglycemic health product, or a hypoglycemic food.

[0015] Furthermore, the blood glucose-lowering drug is an anti-type 2 diabetes drug.

[0016] Compared with existing technologies, the walnut hypoglycemic peptide and its ultrasonic shearing-assisted enzymatic hydrolysis preparation method and application provided by this invention have at least the following beneficial effects: This invention employs a wet high-shear coupled ultrasonic pretreatment synergistic enzymatic hydrolysis process, which precisely disrupts the dense conformation of walnut protein, induces ordered rearrangement of higher-order structures, and achieves efficient release of active peptides. This significantly improves the enzymatic hydrolysis efficiency of walnut protein, allowing for the production of walnut hypoglycemic peptide products containing highly active hypoglycemic peptides without the need for separation and purification. The resulting product exhibits significant inhibitory effects on α-glucosidase, effectively assisting in blood sugar regulation, and possesses advantages such as natural safety, minimal side effects, and suitability for long-term consumption. Simultaneously, this invention fully utilizes walnut meal byproducts, significantly improving the comprehensive utilization rate and added value of walnut resources, and providing stable and feasible technical support for the deep processing and high-value utilization of agricultural products. Attached Figure Description

[0017] To more clearly illustrate the solution of the present invention, a brief introduction will be given to the drawings used in the description of the embodiments below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 A flowchart illustrating an ultrasonic-assisted preparation method for walnut hypoglycemic peptides provided in this embodiment of the invention; Figure 2 Molecular docking diagram of the peptide segment APSKDAPMF (SEQ ID NO.1) of a walnut hypoglycemic peptide provided in an embodiment of the present invention with α-glucosidase; Figure 3Molecular docking diagram of the peptide fragment FFPGSP (SEQ ID NO.2) of a walnut hypoglycemic peptide provided in an embodiment of the present invention with α-glucosidase; Figure 4 This is a bar chart comparing the degree of hydrolysis of walnut protein by different proteases in the embodiments of the present invention; Figure 5 This is a comparison diagram of the Zeta potentials of walnut protein under different treatment conditions in the embodiments of the present invention; Figure 6 This is a comparison diagram of the average particle size of walnut protein under different treatment conditions in the embodiments of the present invention; Figure 7 This is a comparison diagram of the surface hydrophobicity of walnut protein under different treatment conditions in the embodiments of the present invention; Figure 8 This is a comparison chart of the free sulfhydryl group content of walnut protein under different treatment conditions in the embodiments of the present invention; Figure 9 This is a comparison of the Fourier transform infrared (FTIR) spectra of walnut protein under different treatment methods in the embodiments of the present invention; Figure 10 This is a comparison of the ultraviolet absorption spectra of walnut protein under different treatment conditions in the embodiments of the present invention; Figure 11 This is a comparison of the fluorescence spectra of walnut protein under different treatment conditions in the embodiments of the present invention; Figure 12 This is a comparison of XRD patterns of walnut hypoglycemic peptides under different treatment conditions in this invention embodiment; Figure 13 This is a comparison of DSC spectra of walnut hypoglycemic peptides under different treatment conditions in the embodiments of the present invention. Detailed Implementation

[0019] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0021] Example 1

[0022] A method for preparing walnut hypoglycemic peptides by ultrasonic shearing-assisted enzymatic hydrolysis includes the following steps: 1) Raw material pretreatment: Take walnut protein (pre-pass it through a 40-60 mesh sieve to remove impurities) and set aside; 2) High-speed shear dispersion: Add distilled water to walnut protein and mix evenly at a material-to-liquid ratio of 1:25 (g / mL). Disperse the mixture using a high-shear device with a shearing speed of 8000-12000 r / min and a shearing time of 4-6 min to obtain a uniform walnut protein suspension. 3) Ultrasonic pretreatment: The walnut protein suspension was subjected to ultrasonic treatment with an ultrasonic power of 400 W and an ultrasonic time of 21 min. The temperature during the treatment process was controlled below 4℃. 4) Enzymatic hydrolysis: Adjust the temperature of the suspension to 50-55℃, add neutral protease (enzyme activity 50000 U / g), the amount of enzyme added is 12000 U / g walnut protein, and hydrolyze at a constant temperature for 3-4 h; 5) Centrifugation collection: After enzymatic hydrolysis, centrifuge at 5000-8000 r / min, discard the precipitate, and collect the supernatant; 6) Freeze-drying: The supernatant was freeze-dried to obtain walnut hypoglycemic peptide freeze-dried powder; 7) Activity and identification: The hypoglycemic activity was evaluated by α-glucosidase inhibition method, and the peptide sequences were identified by LC-MS / MS mass spectrometry. Two highly active hypoglycemic peptides (APSKDAPMF and FFPGSP) were screened and identified, corresponding to SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0023] Results: The lyophilized walnut hypoglycemic peptide powder prepared in this embodiment showed an inhibition rate of 54.38±0.77% against α-glucosidase; the product was identified as containing highly active hypoglycemic peptides APSKDAPMF and FFPGSP.

[0024] Example 2

[0025] The difference between this embodiment and Embodiment 1 is that the ultrasonic power is different in the ultrasonic pretreatment, while the other steps and parameters are the same as in Embodiment 1. The specific steps are as follows: 1) Raw material pretreatment: Take walnut protein (pre-pass it through a 40-60 mesh sieve to remove impurities) and set aside; 2) High-speed shear dispersion: Add distilled water to walnut protein and mix evenly at a material-to-liquid ratio of 1:25 (g / mL). Disperse the mixture using a high-shear device with a shearing speed of 8000-12000 r / min and a shearing time of 4-6 min to obtain a uniform walnut protein suspension. 3) Ultrasonic pretreatment: The walnut protein suspension was subjected to ultrasonic treatment with an ultrasonic power of 300 W and an ultrasonic time of 21 min. The temperature during the treatment process was controlled below 4℃. 4) Enzymatic hydrolysis: Adjust the temperature of the suspension to 50-55℃, add neutral protease (enzyme activity 50000 U / g), the amount of enzyme added is 12000 U / g walnut protein, and hydrolyze at a constant temperature for 3-4 h; 5) Centrifugation collection: After enzymatic hydrolysis, centrifuge at 5000-8000 r / min, discard the precipitate, and collect the supernatant; 6) Freeze-drying: The supernatant was freeze-dried to obtain walnut hypoglycemic peptide freeze-dried powder; 7) Activity evaluation: The hypoglycemic activity was evaluated using the α-glucosidase inhibition method. This embodiment focuses on the effect of different ultrasound powers on the hypoglycemic activity, without the need for LC-MS / MS mass spectrometry to identify peptide sequences.

[0026] Results: The lyophilized walnut hypoglycemic peptide powder prepared in this embodiment showed an inhibition rate of 44.93±1.77% against α-glucosidase. Compared with Example 1 (ultrasound power 400 W), the ultrasound pretreatment effect of walnut protein was weakened when the ultrasound power was reduced to 300 W, resulting in insufficient enzymatic hydrolysis and a significant decrease in hypoglycemic activity. This embodiment focuses on the effect of different ultrasound powers on hypoglycemic activity and does not require peptide sequence identification.

[0027] Example 3

[0028] The difference between this embodiment and Example 1 is that the amount of enzyme added in the enzymatic hydrolysis reaction is different, while the other steps and parameters are the same as in Example 1, as detailed below: 1) Raw material pretreatment: Take walnut protein (pre-pass it through a 40-60 mesh sieve to remove impurities) and set aside; 2) High-speed shear dispersion: Add distilled water to walnut protein and mix evenly at a material-to-liquid ratio of 1:25 (g / mL). Disperse the mixture using a high-shear device with a shearing speed of 8000-12000 r / min and a shearing time of 4-6 min to obtain a uniform walnut protein suspension. 3) Ultrasonic pretreatment: The walnut protein suspension was subjected to ultrasonic treatment with an ultrasonic power of 400 W and an ultrasonic time of 21 min. The temperature during the treatment process was controlled below 4℃. 4) Enzymatic hydrolysis: Adjust the temperature of the suspension to 50-55℃, add neutral protease (enzyme activity 50000 U / g), the amount of enzyme added is 10000 U / g walnut protein, and hydrolyze at a constant temperature for 3-4 h; 5) Centrifugation collection: After enzymatic hydrolysis, centrifuge at 5000-8000 r / min, discard the precipitate, and collect the supernatant; 6) Freeze-drying: The supernatant was freeze-dried to obtain walnut hypoglycemic peptide freeze-dried powder; 7) Activity evaluation: The hypoglycemic activity was evaluated using the α-glucosidase inhibition method. This example focuses on the effect of different enzyme addition amounts on hypoglycemic activity, and there is no need to identify the peptide sequence using LC-MS / MS mass spectrometry.

[0029] Results: The lyophilized walnut hypoglycemic peptide powder prepared in this embodiment showed an inhibition rate of 42.27±2.6% against α-glucosidase. Compared with Example 1 (enzyme addition of 12000 U / g), when the enzyme addition was reduced to 10000 U / g of walnut protein, the degradation of walnut protein was not thorough, and the hypoglycemic activity decreased significantly. This embodiment focuses on the effect of different enzyme addition amounts on hypoglycemic activity and does not require LC-MS / MS identification of peptide sequences.

[0030] The present invention provides a walnut hypoglycemic peptide and its ultrasonic shearing-assisted enzymatic hydrolysis preparation method and application. The molecular docking diagram of the highly active hypoglycemic peptide APSKDAPMF (SEQ ID NO.1) prepared by ultrasonic shearing enzymatic hydrolysis with α-glucosidase is shown in the figure below. Figure 2 The diagram illustrates the binding mode of this peptide to α-glucosidase. Figure 2 As can be seen, this peptide can stably bind to the active site of α-glucosidase and interact with the residues of the enzyme's active site. The molecular docking diagram of the highly active hypoglycemic peptide FFPGSP (SEQ ID NO.2) prepared by ultrasonic cleavage and enzymatic hydrolysis with α-glucosidase is shown in the figure below. Figure 3 The diagram illustrates the binding mode of this peptide to α-glucosidase. Figure 3 It is evident that this peptide can stably bind to the active site of α-glucosidase and interact with the residues of the enzyme's active site.

[0031] In this embodiment, as Figure 4 The chart shown is a bar graph comparing the degree of hydrolysis of walnut protein by different proteases. The horizontal axis represents trypsin, alkaline protease, neutral protease, and papain, and the vertical axis represents the degree of hydrolysis. This graph is used to characterize the effect of different proteases on the hydrolysis efficiency of walnut protein. Figure 4 It can be seen that the degree of hydrolysis of walnut protein in the neutral protease group is significantly higher than that in other protease groups.

[0032] In this embodiment, as Figure 5 The figure shows a comparison of Zeta potentials of walnut protein under different treatment conditions. The horizontal axis represents the control group, shearing group, ultrasonic group, ultrasonic and shearing group, enzymatic hydrolysis group, and ultrasonic-shear-enzymatic hydrolysis group, respectively. The vertical axis represents the Zeta potential value (mV). This figure is used to characterize the effect of different treatments on the colloidal stability of walnut protein hydrolysates. Figure 5 It can be seen that the absolute value of the Zeta potential of the walnut protein hydrolysis product in the ultrasonic shearing enzymatic hydrolysis group was significantly higher than that in the control group and other treatment groups.

[0033] In this embodiment, as Figure 6 The figure shows a comparison of the average particle size of walnut protein under different treatment conditions. The horizontal axis represents the control group, shearing group, ultrasonic group, ultrasonic and shearing group, enzymatic hydrolysis group, and ultrasonic shearing and enzymatic hydrolysis group, respectively. The vertical axis represents the average particle size (nm). This figure is used to characterize the effect of different treatment methods on the particle size of walnut protein hydrolysates. Figure 6 It can be seen that the average particle size of walnut protein in the ultrasonic shearing and enzymatic hydrolysis group was significantly smaller than that in the control group and other treatment groups.

[0034] In this embodiment, as Figure 7 The figure shows a comparison of the surface hydrophobicity of walnut protein under different treatment conditions. The horizontal axis represents the control group, shearing group, ultrasonic group, ultrasonic and shearing group, enzymatic hydrolysis group, and ultrasonic-shear-enzymatic hydrolysis group, respectively. The vertical axis represents the surface hydrophobicity value. This figure is used to characterize the effect of different treatment methods on the surface hydrophobicity of walnut protein hydrolysates. Figure 7 It can be seen that the surface hydrophobicity of walnut protein in the ultrasonic shearing and enzymatic hydrolysis group is significantly better than that in the control group and other treatment groups.

[0035] In this embodiment, as Figure 8 The figure shows a comparison of the free sulfhydryl content of walnut protein under different treatment conditions. The horizontal axis represents the control group, shearing group, ultrasonic group, ultrasonic and shearing group, enzymatic hydrolysis group, and ultrasonic shearing and enzymatic hydrolysis group, respectively. The vertical axis represents the free sulfhydryl content (μmol / g). This figure is used to characterize the effect of different treatments on the free sulfhydryl content of walnut protein hydrolysates. Figure 8 It can be seen that the content of free sulfhydryl groups in walnut protein in the ultrasonic shearing and enzymatic hydrolysis group was significantly higher than that in the control group and other treatment groups.

[0036] In this embodiment, as Figure 9 The image shows a comparison of Fourier transform infrared (FTIR) spectra of walnut protein under different treatments. The horizontal axis represents wavenumber in cm⁻¹, and the vertical axis represents transmittance. Curves P, S, U, S+U, E, and S+U+E correspond to the control group, shearing group, ultrasonic group, ultrasonic shearing group, enzymatic hydrolysis group, and ultrasonic shearing enzymatic hydrolysis group, respectively. Figure 9 It can be seen that in the amide I band (1600-1700 cm⁻¹), the control group has obvious characteristic absorption peaks. After different treatments, the absorption peak intensity in this band is weakened and the peak shape is blunted. The ultrasonic shearing enzymatic hydrolysis group (S+U+E) shows the most significant change, indicating that this treatment method effectively changes the secondary structure of walnut protein.

[0037] In this embodiment, as Figure 10The figure shows a comparison of the UV absorption spectra of walnut protein under different treatment conditions. The horizontal axis represents wavelength (nm), and the vertical axis represents absorbance. The curves correspond to the control group, shearing group, ultrasonic group, ultrasonic and shearing group, enzymatic hydrolysis group, and ultrasonic shearing and enzymatic hydrolysis group, respectively. This is used to characterize the effect of different treatment methods on the UV absorption characteristics of walnut protein hydrolysates. Figure 10 It is evident that the intensity of the characteristic absorption peaks of aromatic amino acids in the ultrasonic shearing and enzymatic hydrolysis group was significantly higher than that in the control group and other treatment groups.

[0038] In this embodiment, as Figure 11 The figure shows a comparison of the fluorescence spectra of walnut protein under different treatment conditions. The horizontal axis represents the emission wavelength (nm), and the vertical axis represents the fluorescence intensity. The curves correspond to the control group, shearing group, ultrasonic group, ultrasonic and shearing group, enzymatic hydrolysis group, and ultrasonic shearing and enzymatic hydrolysis group, respectively. This is used to characterize the effect of different treatment methods on the molecular conformation of walnut protein hydrolysates. Figure 11 It can be seen that the fluorescence intensity of the ultrasonic shearing and enzymatic digestion group was significantly higher than that of the control group and other treatment groups.

[0039] In this embodiment, as Figure 12 The image shows a comparison of XRD patterns of walnut hypoglycemic peptides under different treatment conditions. The horizontal axis represents the diffraction angle (2θ) in degrees; the vertical axis represents the diffraction intensity; curves P, S, U, S+U, E, and S+U+E correspond to samples prepared by different treatment methods. Figure 12 It can be seen that all samples exhibit broad diffuse diffraction peaks around 2θ = 20°, showing obvious amorphous structural characteristics. Among them, the diffraction intensity trend of the ultrasonic shearing enzymatic hydrolysis group (S+U+E) is the most gradual, and there are no obvious crystal characteristic peaks, indicating that the process basically eliminates the crystalline structure of walnut protein, forming a more uniform amorphous peptide powder.

[0040] In this embodiment, as Figure 13 The image shows a comparison of DSC spectra of walnut hypoglycemic peptides under different treatment conditions. The horizontal axis represents temperature in °C, and the vertical axis represents heat flow rate in mW / mg. Curves P, S, U, U+S, E, and S+U+E correspond to the walnut hypoglycemic peptides prepared from the control group, shearing group, ultrasonic group, ultrasonic shearing group, enzymatic hydrolysis group, and ultrasonic shearing and enzymatic hydrolysis group, respectively. Figure 13 It can be seen that within the temperature range of 30-100℃, all groups showed an endothermic peak around 38℃. Among them, the heat flow equilibrium point and baseline stability of the ultrasonic shearing enzymatic hydrolysis group (S+U+E) were significantly better than those of other groups, indicating that the ultrasonic shearing enzymatic hydrolysis process improved the thermal stability of walnut hypoglycemic peptides.

[0041] Compared with existing technologies, the walnut hypoglycemic peptide and its ultrasonic shear-assisted enzymatic hydrolysis preparation method and application described in the above embodiments employ a wet high-shear coupled ultrasonic pretreatment synergistic enzymatic hydrolysis process. This process can precisely disrupt the dense conformation, induce ordered rearrangement of higher-order structures, and achieve efficient release of active peptides, significantly improving the enzymatic hydrolysis efficiency of walnut protein. Walnut hypoglycemic peptide products containing highly active hypoglycemic peptides can be obtained without separation and purification. The obtained product has a significant inhibitory effect on α-glucosidase, effectively assisting in blood sugar regulation, and has advantages such as natural safety, few side effects, and suitability for long-term consumption. Simultaneously, this invention can fully utilize walnut meal by-products, significantly improving the comprehensive utilization rate and industrial added value of walnut resources, and providing stable and feasible technical support for the deep processing and high-value utilization of agricultural products.

[0042] Obviously, the embodiments described above are merely preferred embodiments of the present invention, and not all embodiments. The accompanying drawings illustrate preferred embodiments of the present invention, but do not limit the scope of the patent. The present invention can be implemented in many different forms; rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this invention.

Claims

1. A walnut hypoglycemic peptide, characterized in that, include: The amino acid sequence of the walnut hypoglycemic peptide is as shown in SEQ ID NO.1, the peptide segment APSKDAPMF, the peptide segment FFPGSP, as shown in SEQ ID NO.2, or a combination of both.

2. The walnut hypoglycemic peptide according to claim 1, characterized in that, The molecular weight of the peptide APSKDAPMF is 963.12 Da, and the molecular weight of the peptide FFPGSP is 650.73 Da.

3. The walnut hypoglycemic peptide according to claim 1, characterized in that, The walnut hypoglycemic peptide is derived from walnut protein, which is a protein product extracted from defatted walnuts with a protein content of ≥60%.

4. A method for preparing walnut hypoglycemic peptides using ultrasonic shearing-assisted enzymatic hydrolysis, characterized in that, Includes the following steps: Step 1: Mix the pretreated walnut protein with water at a material-to-liquid ratio of 1:25 g / mL, and perform shearing treatment to obtain a walnut protein suspension; the shearing treatment speed is 8000-12000 r / min and the time is 4-6 min. The second step is to subject the walnut protein suspension to ultrasonic treatment. The ultrasonic treatment power is 300-400 W, the ultrasonic time is 21 min, and the temperature is controlled below 4℃. The third step is to add neutral protease to the ultrasonically treated suspension for enzymatic hydrolysis. The amount of enzyme added is 10,000-12,000 U / g walnut protein, the hydrolysis temperature is 50-55℃, and the hydrolysis time is 3-4 h to obtain the hydrolysate. Step 4: Centrifuge the enzymatic hydrolysate, collect the supernatant, and freeze-dry it to obtain walnut hypoglycemic peptide.

5. The preparation method according to claim 4, characterized in that, In step one, the walnut protein is pre-passed through a 40-60 mesh sieve.

6. The preparation method according to claim 4, characterized in that, In step three, the activity of the neutral protease is 50,000 U / g.

7. The preparation method according to claim 4, characterized in that, In step four, the centrifugation conditions are 5000-8000 r / min.

8. The application of the walnut hypoglycemic peptide according to claims 1 to 3, or the walnut hypoglycemic peptide obtained by the preparation method according to claims 4 to 7, in the preparation of products with α-glucosidase inhibitory function.

9. The application according to claim 8, characterized in that, The product is a hypoglycemic drug, a hypoglycemic health product, or a hypoglycemic food.

10. The application according to claim 8, characterized in that, The blood sugar-lowering drug is an anti-type 2 diabetes drug.

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