Corn peptide with neuroprotective effect as well as preparation method and application thereof

Abstract

The invention provides a corn peptide with a neuroprotective effect as well as a preparation method and application thereof. Wherein the functional peptide fragment comprises a peptide fragment GL, a peptide fragment FA and a peptide fragment FQ, and the functional peptide fragment has good biological safety; the nerve cell injury caused by A beta1-42 can be effectively inhibited, and the survival rate of injured cells is improved; the mitochondrial membrane potential intensity of nerve cells under A beta1-42 injury can be improved, so that the mitochondrial membrane potential dysfunction is improved; the cell membrane damage of nerve cells under Abeta1-42 damage can be inhibited, so that the release of lactic dehydrogenase is reduced; the active oxygen content and malondialdehyde content of nerve cells damaged by A beta1-42 can be reduced, and the superoxide dismutase content and glutathione peroxidase can be improved, so that the oxidation-antioxidation effect of the body is promoted to be maintained in a balanced state, the negative effects of oxidative stress and the like are avoided, and the nerve protection effect is further achieved.

Description

Technical Field

[0001] The present invention relates to biotechnology, in particular to a corn peptide with neuroprotective effect and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disorder of the central nervous system that primarily occurs in the elderly or pre-elderly. Key features of the disease include progressive cognitive impairment and behavioral impairment. AD is the most common form of dementia, potentially accounting for 60-70% of cases. AD is the most common type of dementia in the elderly, and the risk of developing the disease increases with age. Symptoms of AD include memory impairment, aphasia, apraxia, agnosia, and impaired visual-spatial abilities. In addition, patients experience impairment in abstract thinking and calculation, often accompanied by changes in personality and behavior.

[0003] Alzheimer's disease is closely associated with abnormal protein deposits in the brain (particularly beta-amyloid plaques, also known as Aβ plaques) and an imbalance of metal ions (such as copper and zinc). These abnormalities impair neuronal function, leading to a range of symptoms such as memory loss, cognitive decline, and behavioral abnormalities. Currently, Alzheimer's disease treatment is challenging, relying primarily on a combination of medications, non-drug therapies, and lifestyle adjustments to alleviate symptoms and slow disease progression. However, existing neuroprotective products have shown limited efficacy in clinical trials and have failed to significantly alter the disease course. Some neuroprotective products may also have serious side effects and be toxic to cells or the body. Furthermore, the complexity of the nervous system makes it difficult to elucidate the mechanisms of action of neuroprotective products, hindering their further development and targeted use. Therefore, there is an urgent need to develop products that are side-effect-free and can clearly provide neuroprotective benefits to overcome these shortcomings. Summary of the Invention

[0004] The present invention provides a functional peptide segment with neuroprotective effect, corn peptide, which has good biosafety and can effectively inhibit Aβ 1-42 It can reduce the damage to nerve cells caused by the drug, improve the survival rate of damaged cells, increase the mitochondrial membrane potential strength of nerve cells, inhibit the destruction of nerve cell membranes, reduce the active oxygen content and malondialdehyde content of nerve cells, and increase the superoxide dismutase content and glutathione peroxidase, thereby playing a neuroprotective role.

[0005] The present invention provides a method for preparing the corn peptide, which is simple and fast to operate and convenient for large-scale production.

[0006] The present invention provides a use of the above-mentioned functional peptide segment, and / or the above-mentioned corn peptide, and / or the corn peptide prepared by the above-mentioned preparation method in the preparation of related products with neuroprotective effects.

[0007] The present invention provides a functional peptide segment with neuroprotective effect, wherein the functional peptide segment includes peptide segment GL, peptide segment FA and peptide segment FQ.

[0008] The present invention provides a corn peptide with neuroprotective effect, wherein the corn peptide includes the above-mentioned functional peptide segment.

[0009] The corn peptide as described above, wherein, based on the mass of the corn peptide, the content of peptide segment GL is ≥30 μg / g, the content of peptide segment FA is ≥35 μg / g, and the content of peptide segment FQ is ≥35 μg / g.

[0010] The corn peptide as described above, wherein the moisture content of the corn peptide is 1%-10%; and / or,

[0011] Ash content of corn peptides ≤ 5%; and / or,

[0012] The protein content of corn peptides is ≥85%; and / or,

[0013] Acid-soluble protein content in corn peptides ≥ 80%; and / or,

[0014] The mass content of peptides with a molecular weight of less than 5000 Da in corn peptides is 100%; and / or,

[0015] The mass content of peptides with a molecular weight of less than 1000 Da in corn peptides is ≥ 95%; and / or,

[0016] The mass content of peptides with a molecular weight of 189-1000 Da in corn peptides is ≥ 75%; and / or,

[0017] The non-essential amino acid content of corn peptides is 40%-60%; and / or,

[0018] The essential amino acid content of corn peptides is 15%-35%; and / or,

[0019] The hydrophobic amino acid content of corn peptides is 25-45%; and / or,

[0020] The aromatic amino acid content in corn peptides is 1%-10%.

[0021] The corn peptide as described above, wherein the corn peptide is obtained by enzymatic hydrolysis of corn protein raw material;

[0022] The enzymatic hydrolysis treatment includes a first enzymatic hydrolysis using alkaline protease and a second enzymatic hydrolysis using neutral protease.

[0023] The present invention provides a method for preparing the above-mentioned corn peptide, which comprises the following steps:

[0024] The corn protein raw material is subjected to enzymatic hydrolysis to obtain corn peptides;

[0025] The enzymatic hydrolysis treatment includes a first enzymatic hydrolysis using alkaline protease and a second enzymatic hydrolysis using neutral protease.

[0026] The preparation method as described above, wherein the enzyme activity of the alkaline protease is 580,000-650,000 DU / g; and / or,

[0027] Based on each gram of zein, the amount of alkaline protease added is 0.8-1.0 wt%; and / or,

[0028] The enzyme activity of the neutral protease is 380,000-420,000 U / g; and / or,

[0029] Based on each gram of zein, the addition amount of neutral protease is 1.0-1.2wt%.

[0030] The preparation method as described above, wherein the conditions of the first enzymatic hydrolysis include: time of 2-4 h, pH value of 8.0-9.0, temperature of 50-60° C.; and / or,

[0031] The conditions of the second enzymatic hydrolysis include: a time of 1-3 hours, a pH value of 6.5-7.5, and a temperature of 50-60° C.; and / or,

[0032] The zein raw material is obtained by mixing zein and water in a mass to volume ratio of 1:(8-12).

[0033] The preparation method as described above, wherein the enzymatic hydrolysis treatment further comprises separation and purification of the enzymatic hydrolyzate;

[0034] Separation and purification processes include centrifugation, filtration, ultrafiltration, and spray drying.

[0035] The present invention provides a use of the above-mentioned functional peptide segment, and / or the above-mentioned corn peptide, and / or the corn peptide prepared by the above-mentioned preparation method in the preparation of related products with neuroprotective effects.

[0036] The present invention provides a functional peptide segment with neuroprotective effect, wherein the functional peptide segment includes peptide segment GL, peptide segment FA and peptide segment FQ. The functional peptide segment does not produce toxicity to nerve cells and has good biosafety; it can effectively inhibit β-amyloid peptide (1-42) (Aβ 1-42 ) caused by nerve cell damage, improve the survival rate of damaged cells, and thus play a neuroprotective role. At the same time, the above functional peptides can increase the 1-42The mitochondrial membrane potential strength of damaged nerve cells can be improved, thereby improving mitochondrial membrane potential dysfunction and avoiding affecting the biological activity in cells; it can inhibit Aβ 1-42 The cell membrane of damaged nerve cells is destroyed, thereby reducing the release of lactate dehydrogenase; it can also reduce Aβ 1-42 It can reduce the active oxygen content and malondialdehyde content of damaged nerve cells, and increase the content of superoxide dismutase and glutathione peroxidase, thereby promoting the body's oxidation-antioxidation function to maintain a balanced state, avoiding negative effects such as oxidative stress, DNA damage, and lipid peroxidation, and thus playing a neuroprotective role. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the gel chromatogram of the corn peptide in Example 3;

[0038] Figure 2 This is a graph showing the cell survival rate under the intervention of corn peptide in Example 6;

[0039] Figure 3 This is the result graph of cell survival rate under the intervention of functional peptide segments in Example 6;

[0040] Figure 4 Aβ in Example 6 1-42 Graph showing cell survival results under intervention;

[0041] Figure 5 For example 6 corn peptide, Aβ 1-42 Graph showing cell survival results under intervention;

[0042] Figure 6 For the functional peptide segment and Aβ in Example 6 1-42 Graph showing cell survival results under intervention;

[0043] Figure 7 This is the result of the mitochondrial membrane potential intensity under the intervention of corn peptide in Example 6;

[0044] Figure 8 This is a graph showing the results of lactate dehydrogenase content determination under the intervention of corn peptide in Example 6;

[0045] Figure 9 This is a graph showing the results of measuring the relative level of reactive oxygen species under the intervention of corn peptide in Example 6;

[0046] Figure 10 This is a graph showing the results of measuring malondialdehyde content under the intervention of corn peptide in Example 6;

[0047] Figure 11 This is a graph showing the results of superoxide dismutase content determination under the intervention of corn peptide in Example 6;

[0048] Figure 12This is a graph showing the results of glutathione peroxidase content determination under the intervention of corn peptide in Example 6;

[0049] Figure 13 This is the result diagram of mitochondrial membrane potential intensity under the intervention of functional peptides in Example 6;

[0050] Figure 14 This is a graph showing the results of lactate dehydrogenase content determination under the intervention of functional peptide segments in Example 6;

[0051] Figure 15 This is a graph showing the results of measuring the relative level of reactive oxygen species under the intervention of the functional peptide segment in Example 6;

[0052] Figure 16 This is a graph showing the results of measuring malondialdehyde content under the intervention of the functional peptide segment in Example 6;

[0053] Figure 17 This is a graph showing the results of superoxide dismutase content determination under the intervention of functional peptides in Example 6;

[0054] Figure 18 This is a graph showing the results of glutathione peroxidase content determination under the intervention of the functional peptide segment in Example 6;

[0055] Figure 19 This is the protein band diagram of the protein immunoblotting experiment under the intervention of corn peptide in Example 6;

[0056] Figure 20 This is the result diagram of the relative expression of PKA under the intervention of corn peptide in Example 6;

[0057] Figure 21 This is the result diagram of the relative expression of CREB under the intervention of corn peptide in Example 6;

[0058] Figure 22 This is the result diagram of the relative expression of BDNF under the intervention of corn peptide in Example 6;

[0059] Figure 23 This is the protein band diagram of the protein immunoblotting experiment under the intervention of the functional peptide segment in Example 6;

[0060] Figure 24 This is the result diagram of the relative expression of PKA under the intervention of functional peptide segments in Example 6;

[0061] Figure 25 This is the result diagram of the relative expression of CREB under the intervention of the functional peptide in Example 6;

[0062] Figure 26 This is the result diagram of the relative expression of BDNF under the intervention of functional peptides in Example 6. DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific embodiments listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0064] In a first aspect, the present invention provides a functional peptide with neuroprotective effects, comprising peptide segments GL, FA, and FQ. Peptide segment GL is a dipeptide segment composed of glycine and leucine (Gly-Leu, GL), peptide segment FA is a dipeptide segment composed of phenylalanine and alanine (Phe-Ala, FA), and peptide segment FQ is a dipeptide segment composed of phenylalanine and glutamine (Phe-Gln, FQ).

[0065] After experimental verification, the above functional peptides will not cause toxicity to nerve cells at all experimental concentrations and have good biosafety; they can effectively inhibit β-amyloid peptide (1-42) (Aβ 1-42 ) caused by the decrease in the survival rate of nerve cells, thereby playing a neuroprotective role. At the same time, the above functional peptides can increase the Aβ 1-42 The effect of the damaged mitochondrial membrane potential on the nerve cells can improve the mitochondrial membrane potential dysfunction; it can inhibit Aβ 1-42 The cell membrane of damaged nerve cells is destroyed, thereby reducing the release of lactate dehydrogenase; it can also reduce Aβ 1-42 It reduces the levels of reactive oxygen species and malondialdehyde in damaged nerve cells, and increases the levels of superoxide dismutase and glutathione peroxidase, thereby promoting the body's oxidation-antioxidation balance, avoiding negative effects such as oxidative stress, DNA damage, and lipid peroxidation, and thus playing a neuroprotective role. Experimental verification has shown that peptides GL, FA, and FQ can play a neuroprotective role when used alone.

[0066] In addition, experiments have shown that functional peptides GL, FA, and FQ can inhibit Aβ by activating the PKA-CREB-BNDF signaling pathway in nerve cells. 1-42 Induce adverse reactions caused by nerve cells, thereby reducing damage to nerve cells and providing protection for nerve cells.

[0067] The second aspect of the present invention provides a corn peptide with neuroprotective effect, comprising the above-mentioned functional peptide segment.

[0068] Corn peptides are hydrolyzed products of corn protein. They are small-molecule polypeptides obtained through targeted enzymatic cleavage of corn protein and specific small peptide isolation techniques. Corn peptides possess the inherent advantages of corn protein while offering many new benefits, such as direct absorption, strong solubility, excellent stability, and high safety, thus offering promising development prospects.

[0069] Experimental verification shows that corn peptides can effectively inhibit Aβ 1-42 It can also improve the survival rate of damaged cells and play a neuroprotective role; it can also increase the Aβ 1-42 The mitochondrial membrane potential strength of damaged nerve cells can be improved, thereby improving mitochondrial membrane potential dysfunction and avoiding affecting the biological activity in cells; it can also inhibit Aβ 1-42 The damaged nerve cell membrane is destroyed, thereby reducing the release of lactate dehydrogenase; at the same time, it can reduce Aβ 1-42 It reduces the active oxygen content and malondialdehyde content of damaged nerve cells, and increases the content of superoxide dismutase and glutathione peroxidase, thereby promoting the body's oxidation-antioxidation function to maintain a balanced state, avoiding negative effects such as oxidative stress, DNA damage, and lipid peroxidation; and ultimately plays a neuroprotective role.

[0070] Experiments have also found that corn peptides can also inhibit Aβ by activating the PKA-CREB-BNDF signaling pathway in nerve cells. 1-42 Induce adverse reactions caused by nerve cells, thereby reducing damage to nerve cells and providing protection for nerve cells.

[0071] In the above technical solution, based on the mass of corn peptides, the content of peptide segment GL is ≥30 μg / g, the content of peptide segment FA is ≥35 μg / g, and the content of peptide segment FQ is ≥35 μg / g.

[0072] The present invention can promote corn peptide to inhibit Aβ by regulating the functional peptide segments and their content in the corn peptide composition to meet the above range. 1-42 Caused by nerve cell damage, improve the survival rate of damaged cells; increase Aβ 1-42 The mitochondrial membrane potential strength of damaged nerve cells inhibits Aβ 1-42 Damage to the cell membrane of damaged nerve cells; reduce Aβ 1-42 It reduces the active oxygen content and malondialdehyde content of damaged nerve cells, and increases the content of superoxide dismutase and glutathione peroxidase, thereby promoting the body's oxidation-antioxidation function to maintain a balanced state; it plays a neuroprotective role through multiple mechanisms.

[0073] In the above technical solution, the moisture content of corn peptide is 1%-10%, indicating that the moisture content of corn peptide is appropriate, which can inhibit the growth of fungi to a certain extent and play a role in the preservation of corn peptide; the ash content of corn peptide is ≤5%, indicating that the corn peptide has fewer impurities and higher quality; the protein content of corn peptide is ≥85%, indicating that the corn peptide has rich protein nutrition; the acid-soluble protein content of corn peptide is ≥80%, indicating that the corn peptide contains many low-molecular-weight substances, which is beneficial to human absorption; the mass content of peptides with a molecular weight of <5000 Da in corn peptide is 100%, the mass content of peptides with a molecular weight of <1000 Da is ≥95%, and the molecular weight is 189-1000 The mass content of Da peptide is ≥75%, indicating that the molecular weight of corn peptide is small, and it has the potential for high absorption and utilization rate, high blood-brain barrier penetration rate, and high digestion and transport speed in the gastrointestinal tract; the content of non-essential amino acids in corn peptide is 40%-60%, and the content of essential amino acids is 15%-35%, indicating that the amino acid composition and content of corn peptide can better meet the normal physiological metabolism and daily activities of the human body; the content of hydrophobic amino acids in corn peptide is 25-45%, indicating that the corn peptide is rich in hydrophobic amino acids, which helps to enhance the solubility of corn peptide in lipids, and then remove free radicals in lipids to exert antioxidant effects; the content of aromatic amino acids in corn peptide is 1%-10%, and aromatic amino acids can provide protons to electron-deficient free radicals to maintain the stability of the substance, indicating that the corn peptide has antioxidant potential. It can be understood that the neuroprotective effect of corn peptide can be enhanced by meeting at least one of the above schemes.

[0074] In the above technical solution, corn peptides are obtained by enzymatically hydrolyzing corn protein raw materials; wherein the enzymatic hydrolysis includes a first enzymatic hydrolysis using alkaline protease and a second enzymatic hydrolysis using neutral protease.

[0075] Zein is a byproduct of corn kernels after starch production in the food industry or purification in the brewing industry. Its protein is rich in nutrients and has a unique flavor and color, making it suitable for use as feed. However, it has many drawbacks, such as poor water solubility, strong hydrophobicity, and difficulty in digestion, which limit its application. By preparing zein into corn peptides, the large zein molecules can be converted into small zein molecules that are absorbable, have a well-defined characteristic structure, and possess biological activity, thereby enhancing their biological activity. This also gives zein higher added value and a wider range of applications, thus increasing its economic value.

[0076] The present invention is not limited to a specific type of corn protein raw material, as long as the protein content (dry basis) of the corn protein is not less than 85%. It is understood that the corn protein of the present invention can be in a solid form or a liquid form.

[0077] The present invention uses alkaline protease for the first enzymatic hydrolysis and neutral protease for the second enzymatic hydrolysis. Alkaline protease refers to an enzyme that can hydrolyze protein peptide bonds under alkaline conditions, with an optimal pH range of 9-11. Neutral protease is an endonuclease that can be used in various protein hydrolysis processes. Under certain temperatures and pH values, it can hydrolyze large molecular proteins into products such as amino acids. By regulating the enzymatic hydrolysis of corn protein, the present invention can ensure that the final corn peptides meet the predetermined functional peptide segment composition.

[0078] The third aspect of the present invention provides a method for preparing the above-mentioned corn peptide, comprising the following steps:

[0079] The corn protein raw material is subjected to enzymatic hydrolysis to obtain corn peptides;

[0080] The enzymatic hydrolysis treatment includes a first enzymatic hydrolysis using alkaline protease and a second enzymatic hydrolysis using neutral protease.

[0081] Extensive research has been conducted to determine how to ensure that enzymatic hydrolysis products of corn protein contain the desired peptides GL, FA, and FQ. The choice of enzyme preparation and the order in which they are used have a significant impact on the results. During this research, it was discovered that enzymatic hydrolysis of corn protein with both alkaline and neutral proteases can yield corn peptides containing peptides GL, FA, and FQ. This preparation method is simple and rapid, making it amenable to large-scale production.

[0082] In the above technical solution, regulating the enzymatic activity and addition amount of alkaline protease or the enzymatic activity and addition amount of neutral protease also helps to promote the enzymatic reaction, thereby promoting the release of functional peptides. Specifically, the enzymatic activity of alkaline protease is 580,000-650,000 DU / g; based on each gram of corn protein, the addition amount of alkaline protease is 0.8-1.0wt%; the enzymatic activity of neutral protease is 380,000-420,000 U / g; and / or, based on each gram of corn protein, the addition amount of neutral protease is 1.0-1.2wt%. It is understandable that if at least one of the enzymatic activity of alkaline protease, the addition amount of alkaline protease, the enzymatic activity of neutral protease, and the addition amount of neutral protease meets the above range, the release of functional peptides can be promoted, thereby enhancing the neuroprotective effect of corn peptides.

[0083] In the above technical solution, regulating the temperature, time, and pH of the enzymatic hydrolysis treatment helps promote the enzymatic hydrolysis reaction. Specifically, the conditions for the first enzymatic hydrolysis include: time of 2-4 hours, pH of 8.0-9.0, and temperature of 50-60°C; the conditions for the second enzymatic hydrolysis include: time of 1-3 hours, pH of 6.5-7.5, and temperature of 50-60°C.

[0084] In the actual operation process, the pH value can be first adjusted to the optimal enzymatic hydrolysis pH environment of alkaline protease, that is, the pH value is 8.0-9.0, to obtain an enzymatic hydrolysis solution that is conducive to the first enzymatic hydrolysis treatment of alkaline protease, wherein the pH value can be adjusted using sodium hydroxide NaOH or hydrochloric acid HCl. Subsequently, at the appropriate enzymatic hydrolysis temperature of alkaline protease, that is, 50-60°C, alkaline protease is added to the enzymatic hydrolysis solution and enzymolysis is carried out for 2-4 hours, so that the alkaline protease fully acts on the protein in the enzymatic hydrolysis solution and releases more functional peptides; then, the pH value is adjusted to the optimal enzymatic hydrolysis pH environment of neutral protease, that is, the pH value is 6.5-7.5, to obtain a first enzymatic hydrolysis solution that is conducive to the second enzymatic hydrolysis treatment of neutral protease, and neutral protease is added to the first enzymatic hydrolysis solution and enzymolysis is carried out for 1-3 hours, so that the neutral protease fully acts on the protein or polypeptide in the first enzymatic hydrolysis solution and releases more functional peptides.

[0085] To better enrich functional peptides, zein can be first mixed with water to obtain a zein solution, which can then be used as the zein raw material for enzymatic hydrolysis. Specifically, zein and water can be mixed in a mass-to-volume ratio of 1:(8-12), i.e., 8-12 mL of water per 1 g of zein. Zein solution exhibits a certain degree of fluidity and a larger surface area than zein powder, facilitating subsequent enzymatic hydrolysis. Adding too little water results in poor fluidity, which is detrimental to the enzyme's action and can reduce enzymatic hydrolysis efficiency. Adding too much water results in an excessively large reaction volume during enzymatic hydrolysis, increasing the load on subsequent processing (such as separation and purification) and the associated processing costs.

[0086] It is understandable that if at least one of the enzymatic hydrolysis temperature, enzymatic hydrolysis time, enzymatic hydrolysis pH value, and the method of obtaining the corn protein raw material meets the above range, the release of functional peptide segments can be promoted, thereby enhancing the neuroprotective effect of corn peptides.

[0087] In addition, after the enzymatic hydrolysis treatment is completed, an enzyme inactivation treatment can be performed to deactivate the catalytic activity of the enzyme preparation. The present invention does not limit the enzyme inactivation method, and conventional enzyme inactivation methods in the art can be used for enzyme inactivation, such as heating to 95°C and maintaining for 15 minutes.

[0088] After the enzyme is inactivated, centrifugation can be performed to collect the supernatant for subsequent operations. Specifically, the centrifugation speed can be 2500-3500 rmp / min and the centrifugation time can be 10-20 min.

[0089] In the above technical solution, the enzymatic hydrolysis treatment also includes separation and purification treatment of the enzymatic hydrolyzate; wherein the separation and purification treatment includes centrifugation treatment, filtration treatment, ultrafiltration treatment, and spray drying treatment.

[0090] In detail, after obtaining the enzymatic hydrolysate, the enzymatic hydrolysate can be first allowed to stand and cool at room temperature (25°C); then, it can be centrifuged at 5000-7000 r / min for 5-15 min to obtain a supernatant; the supernatant is then filtered with a ceramic filter membrane with a diameter of 150-250 nm to remove large particle impurities in the supernatant; the filtered liquid is then ultrafiltered with a 1000 Da ultrafiltration membrane to further remove substances with a molecular weight greater than 1000 Da, thereby obtaining a corn peptide solution with a molecular weight mainly below 1000 Da; finally, the corn peptide solution is spray dried by spray drying, and the spray drying conditions are controlled by adjusting the feed temperature (20-30°C), feed rate (10-20 mL / min), inlet air temperature (130-140°C) and inlet air pressure (15-25 kPa). The spray drying time is 5-15 min, and the powdered corn peptide is obtained for better storage and use.

[0091] The fourth aspect of the present invention provides the use of the above-mentioned functional peptide segment, the above-mentioned corn peptide, and the corn peptide prepared by the above-mentioned preparation method in the preparation of related products with neuroprotective effects.

[0092] It is understood that the aforementioned related products include, but are not limited to, foods, health supplements, and pharmaceuticals. Extensive research data demonstrates that the functional peptides of the present invention, including peptides GL, FA, and FQ, exhibit significant neuroprotective effects. These peptides can be used not only in conventional foods and health supplements, such as neuroprotective foods and health supplements, but also in the preparation of neuroprotective pharmaceuticals. This broadens the application scope of corn peptides and provides new raw materials for neuroprotective products.

[0093] The technical solutions of this application are further explained below with reference to specific examples. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. All reagents used, unless otherwise specified, were commercially available or publicly available.

[0094] Example 1: Preparation of corn peptide

[0095] Corn gluten meal was used as the raw material. Distilled water was added at a mass-to-volume ratio of 1:10, and the mixture was homogenized at 25,000 × g for 10 minutes in a homogenizer. The pH of the homogenate was adjusted to 8.5 with NaOH, and the temperature was adjusted to 55°C. 0.9 wt% alkaline protease (based on the weight of the corn gluten meal) was added to the homogenate, and enzymatic hydrolysis was carried out for 3 hours. After the enzymatic hydrolysis, the pH was adjusted to 7.0, and 1.1 wt% neutral protease (based on the weight of the corn gluten meal) was added. Enzymatic hydrolysis was continued for another 2 hours to obtain an enzymatic hydrolyzate. The enzymatic activity of the alkaline protease was 620,000 DU / g, and that of the neutral protease was 400,000 U / g. After the enzymatic hydrolysis, the mixture was heated to 90°C and held for 15 minutes to inactivate the enzymes.

[0096] The enzymatic hydrolysate after enzyme inactivation was allowed to stand and cool at room temperature (25°C); then, it was centrifuged at 6000 r / min for 10 minutes to obtain a supernatant; the supernatant was filtered with a ceramic filter membrane with a diameter of 200 nm to remove large particle impurities in the supernatant; the filtered liquid was then ultrafiltered with a 1000 Da ultrafiltration membrane to further remove substances with a molecular weight greater than 1000 Da, thereby obtaining a corn peptide solution with a molecular weight mainly below 1000 Da; finally, the corn peptide solution was dried by spray drying. The drying conditions were controlled by adjusting the feed temperature (25°C), feed rate (14 mL / min), inlet air temperature (135°C) and inlet air pressure (20 kPa). The spray drying time was 10 minutes to obtain corn peptide powder.

[0097] Example 2: Determination of corn peptide composition

[0098] With reference to the methods of national standards GB 5009.3-2016, GB 5009.4-2016, GB 5009.5-2016, and GB 22729-2008, the moisture content, ash content, protein content, and acid-soluble protein content of the corn peptide powder in Example 1 were respectively tested.

[0099] Testing revealed that the corn peptide had a moisture content of 5.68%, an ash content of 2.59%, a protein content of 89.39%, and an acid-soluble protein content of 84.00%. These results indicate that the corn peptide has an appropriate moisture content, which can inhibit fungal growth to a certain extent, thus contributing to its shelf life. The corn peptide also has a low ash content and excellent quality. Furthermore, the corn peptide has a high protein content, providing rich protein nutrition. Furthermore, acid-soluble proteins are proteins that dissolve in acidic environments and have a low molecular weight. Hydrolyzates with a relative molecular weight of less than 1000 Da account for as much as 98.66%. Therefore, the high acid-soluble protein content of the corn peptide indicates that it contains a high concentration of low-molecular-weight substances, which facilitates its absorption by the human body.

[0100] Example 3: Determination of molecular weight distribution of corn peptides

[0101] The molecular weight distribution of the corn peptide powder in Example 1 was determined using an LC-20A high performance liquid chromatography (HPLC) system, and the chromatographic data were processed using gel permeation chromatography (GPC) software. -3 g / mL solution, and filter it with a filter membrane to remove possible impurities or particulate matter. The filtered standard solution is then sampled for detection, and a relative molecular weight calibration curve is drawn based on the molecular weight analysis results of the standard solution. Subsequently, the corn peptide powder in Example 1 is prepared into a 2 mg / mL sample solution with ultrapure water, and filtered with a 0.2 μm polytetrafluoroethylene filter membrane to ensure that the sample solution is free of impurities, and then sampled for detection. The HPLC detection conditions are shown in Table 1, the molecular weight distribution of corn peptides is shown in Table 2, and the gel chromatogram of corn peptides is shown in Table 2. Figure 1 As shown. When corn peptide enters the HPLC column, the column is filled with a stationary phase. Through the flow of the mobile phase, different components are separated according to their molecular weight and the different retention times in the stationary phase. Figure 1 The larger the peak area, the higher the content of the component.

[0102] Molecular weight is closely related to the body's absorption and utilization rate, blood-brain barrier penetration rate, and antioxidant function. For example, small-molecule peptides are digested and transported faster in the gastrointestinal tract than large-molecule peptides or proteins. Peptides with a molecular weight of less than 3000 Da generally have stronger antioxidant biological activity. Testing has shown that the molecular weight of corn peptides is relatively small, with all molecular weights being 5000 Da and below; molecular weights below 1000 Da account for as high as 98.6625%; and molecular weights are mainly concentrated in the 189-1000 Da range, accounting for 77.4402%. These results indicate that corn peptides have the potential for high absorption and utilization rate, high blood-brain barrier penetration rate, high antioxidant function, and high digestion and transit rates in the gastrointestinal tract.

[0103] Table 1

[0104]

[0105] Table 2

[0106]

[0107] Example 4: Determination of amino acid composition of corn peptides

[0108] Referring to the method of national standard GB 5009.124-2016, the amino acid composition and content of the corn peptide powder in Example 1 were detected using an 835-50 fully automatic amino acid analyzer. The test results are shown in Table 3, where hydrolyzed amino acids refer to the decomposition of proteins into single amino acids by hydrolysis; free amino acids refer to single amino acid molecules that are not combined with other amino acids or compounds.

[0109] After testing, it was found that corn peptide is composed of 17 amino acids, including 8 essential amino acids, 8 non-essential amino acids and 1 semi-essential amino acid. The content of non-essential amino acids is 52.091% and the content of essential amino acids is 27.40%. This shows that the amino acid composition and content of corn peptide can meet the needs of normal physiological metabolism and daily activities of the human body. Some of the amino acids can undergo chemical changes and modifications to become essential amino acids under the normal physiological metabolism of the body, such as tyrosine can be converted into phenylalanine.

[0110] The hydrophobic amino acid content in corn peptides is 37.356%. Hydrophobic amino acid residues generally carry hydrophobic groups. Therefore, the presence of hydrophobic amino acids in corn peptides helps enhance their solubility in lipids, thereby scavenging free radicals in lipids and exerting an antioxidant effect.

[0111] The content of aromatic amino acids in corn peptides is 5.266%. Aromatic amino acids can donate protons to electron-deficient free radicals to maintain the stability of substances. Therefore, the presence and content of aromatic amino acids in corn peptides are very critical.

[0112] Furthermore, the type and content of amino acids are closely linked to the biological activities of corn peptides, such as their regulatory effects on the nervous system. Table 3 shows that corn peptides contain high levels of six amino acids: glutamic acid (Glu), leucine (Leu), alanine (Ala), aspartic acid (Asp), proline (Pro), and serine (Ser), with glutamic acid being the highest. Studies have shown that glutamic acid is one of the most important amino acids in protein composition. It participates in metabolism as a carbon and nitrogen nutrient and is one of the key amino acids that ensures the normal functioning of various circulations in the body. It is also a crucial neurotransmitter in the central nervous system, an excitatory neurotransmitter that plays a crucial role in the proper functioning of mitochondria. Glutamic acid can be converted into glutamine, which promotes protein synthesis and maintains acid-base balance. Glutamic acid and aspartic acid are flavor-producing amino acids and are important umami amino acids, indicating that corn peptides are nutritious and functional plant extracts with a savory taste. Furthermore, leucine is one of the amino acids reported to have high antioxidant activity. The above results show that the amino acid composition and content distribution of corn peptides not only help to enhance its flavor, but also help to exert its biological functional activity, especially the regulation of the nervous system, maintaining the stable operation of the digestive system, enhancing memory function, and improving antioxidant activity.

[0113] Table 3

[0114]

[0115] Example 5: Structural Identification and Peptide Quantification of Corn Peptides

[0116] In this example, high-peak intensity mass-to-charge ratio information is collected through precursor ion (Q3) scanning to obtain peptide precursor ion information. Peaks of different heights represent different mass-to-charge ratios, and the mass-to-charge ratio at the higher peak is selected for secondary scanning; the precursor ion at the high-peak intensity is fragmented through product ion (PIS) scanning to obtain fragment ions of the corresponding peptide, so as to obtain product ion information of the corresponding peptide; the a and y ions that appear after further dissociation by secondary mass spectrometry are related to the amide bond in the polypeptide chain structure, and the peptide bond breakage has a certain regularity. The ion fragments that appear after the amide bond breakage at the N-terminus are marked as b, and the ion fragments that appear after the amide bond breakage at the C-terminus are marked as y. These fragment ions will be further dehydrated and deaminated, and amino acids have corresponding characteristic ions after mass spectrometry collision breakage. Therefore, the precursor ion and product ion information of the peptide segment can be obtained. The specific steps are as follows:

[0117] Purified water was added to the corn peptide powder obtained in Example 1 to prepare a 2 mg / mL corn peptide solution. The solution was centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm nylon filter membrane to obtain the test sample. The sample was subjected to Q3 scanning, PIS scanning, multiple reaction monitoring (MRM) optimization, and peptide sequence quantification on an LCMS-8060 instrument to obtain the structural peptides (see Table 4 for details) and peptide content (see Table 5 for details).

[0118] Chromatographic conditions included: mobile phase A: pure water (containing 0.1% (v / v) formic acid); mobile phase B: 80% acetonitrile (containing 0.1% (v / v) formic acid); flow rate: 10 µL / min; elution time: 65 min; analytical column: 75 µm × 150 mm; C18 trap column: 300 A, 5 µm. Elution conditions included: first gradient elution time: 0-1 min, mobile phase A 2%; second gradient elution time: 1-23 min, mobile phase A 2%-40%; third gradient elution time: 23-25 min, mobile phase A 40%-80%; fourth gradient elution time: 25-26 min, mobile phase A 80%; fifth gradient elution time: 26-30 min, mobile phase A 80%-2%. Scanning modes included: Full MS, dd-MS2; resolution: Full MS 35000, dd-MS2 17500; scan range: 350-1800 m / z; scanning ion: positive ion mode; collision energy: 29% normalized collision energy. In the resulting secondary mass spectra, peptides are assigned corresponding ordinates (ion degree) and abscissas (ion mass ratio (m / z)). The m / z of each peptide was used as the precursor ion for analysis. In positive ion mode, collision energies were set at -35 V and -25 V, respectively. The fragment ion information corresponding to the m / z of the major chromatographic peaks is shown in Table 6. The liquid chromatography conditions are shown in Table 7, the gradient elution program sequence is shown in Table 8, and the mass spectrometry conditions are shown in Table 9. Voltage optimization for the standards was performed using the automatic optimization function of the HPLC-MS / MS system. The resulting MRM optimization parameters are shown in Table 10.

[0119] Table 4

[0120]

[0121] Table 5

[0122]

[0123] Table 6

[0124]

[0125] Table 7

[0126]

[0127] Table 8

[0128]

[0129] Table 9

[0130]

[0131] Table 10

[0132]

[0133] As shown in Table 4, the structural peptides in corn peptides are all small, short peptides. Molecular weight is closely related to human absorption and utilization, blood-brain barrier penetration, and antioxidant activity. For example, small peptides are digested and transported more rapidly in the gastrointestinal tract than larger peptides or proteins. Therefore, the corn peptides of the present invention have the potential for high absorption and utilization, high blood-brain barrier penetration, high antioxidant activity, and rapid digestion and transit in the gastrointestinal tract.

[0134] The structural peptides in Table 4 were subjected to bioactivity scoring and ADMET prediction methods (both computer-based operations). The bioactivity index was used as the evaluation criterion, and finally 11 active peptides were screened and the contents of the 11 active peptides were obtained, as shown in Table 5.

[0135] Finally, the 11 active peptides in Table 5 were subjected to molecular docking experiments. According to the experimental results of molecular docking, the three best docking functional peptides were selected for functional experimental verification. The functional peptides were GL, FA and FQ.

[0136] Example 6: Functional experiments of functional peptides in corn peptides

[0137] (1) Cell culture method

[0138] Human neuroblastoma cells (SHSY5Y) were inoculated into T25 culture flasks in DMEM / F12 containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin solution at 5% CO. 2Culture the cells in a 37°C incubator and observe their growth. Replace the culture medium daily or every other day depending on cell growth. When the cells reach approximately 80% of the flask, aspirate the culture medium and wash away any residual culture medium and cellular waste with 1-2 mL of phosphate-buffered saline (PBS). Add 1 mL of 0.05% trypsin and let it rest for 30 seconds. Observe under a microscope to see if the cells shrink, become round, and detach. Add 4 mL of fresh culture medium to dislodge the cells and evenly distribute the cell suspension. Centrifuge at 1000 rpm / min for 3 minutes, add 3 mL of fresh culture medium, and evenly distribute the suspension. Subculture the cells at a 1:3 ratio. Subsequent experiments were performed when the cells reached the logarithmic phase.

[0139] (2) Cytotoxicity analysis

[0140] Cytotoxicity analysis is a key method for evaluating the safety of the substance to be tested for organisms. After SHSY5Y cells are treated with different concentrations of corn peptides or functional peptides for 24 hours, the CCK8 method is used to detect the cell survival rate, which can quickly and sensitively reflect the cell proliferation and cytotoxicity. The CCK8 method determines whether corn peptides or functional peptides affect the growth of SHSY5Y cells by detecting the cell survival rate after intervention with different concentrations of corn peptides or functional peptides, and it is necessary to determine whether corn peptides or functional peptides affect the growth of SHSY5Y cells without adding β-amyloid peptide (1-42) (abbreviated as Aβ 1-42 ) under the condition of corn peptide on the survival rate of SHSY5Y cells, in order to eliminate the interference in the experiment, and finally select the corn peptide or functional peptide concentration most suitable for the growth of SHSY5Y cells for subsequent experiments. The specific operation is as follows:

[0141] The cells in the logarithmic growth phase in (1) were cultured at a rate of 5×10 4Cells were plated into 96-well plates per well. When the cells reached approximately 70% of the well, the old culture medium was aspirated and washed with PBS. Fresh culture medium was then added to serve as the blank control group (or NC group). Alternatively, culture medium containing functional peptides (GL, FA, FQ) was added to the wells to achieve final concentrations of functional peptides of 300 μg / mL, 250 μg / mL, 200 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL, respectively, serving as the functional peptide intervention group. Alternatively, culture medium containing corn peptides was added to the wells to achieve final concentrations of corn peptides of 12 mg / mL, 10 mg / mL, 8 mg / mL, 6 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0.25 mg / mL, respectively. In addition, wells containing no cells but only the same volume of fresh culture medium served as blank groups. Six replicate wells were set up for each group. After 24 h of culture, the old culture medium was removed and washed with PBS. 100 μL of new culture medium and 10 μL of CCK8 reagent were added to each well. The 96-well plate was placed in an incubator and cultured for 1-4 h. The absorbance was measured at 450 nm. The sample concentration that was non-toxic to the cells was selected for subsequent experiments. The results are shown in the figure. Figure 2 and Figure 3 As shown. Among them, the calculation method of cell viability is as follows:

[0142]

[0143] Among them, A sample Indicates the absorbance value of corn peptide or functional peptide intervention group, A blank Indicates the absorbance intervention value of the blank group, A control It represents the absorbance value of the blank control group.

[0144] according to Figure 2As shown, no concentration of corn peptides induced toxicity to SHSY5Y cell growth or decreased cell survival after 24 hours of treatment. Compared with the NC group, corn peptides at concentrations of 0.25 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, and 8 mg / mL showed significant differences (P>0.05). Furthermore, no statistical differences were observed between any two corn peptides at concentrations of 8 mg / mL, 6 mg / mL, and 4 mg / mL (P>0.05). Cell survival rates at these three concentrations were 101.3±1.185%, 101.2±0.6297%, and 100.8±0.1640%, respectively. Corn peptides neither promoted nor inhibited SHSY5Y cell proliferation. Therefore, a concentration of 8 mg / mL was selected as the high-dose group (COP-H) of corn peptide, a concentration of 6 mg / mL was selected as the medium-dose group (COP-M) of corn peptide, and a concentration of 4 mg / mL was selected as the low-dose group (COP-L) of corn peptide for subsequent experiments.

[0145] according to Figure 3 It can be seen that after 24 hours of intervention with functional peptides at various concentrations, there was no significant difference compared with the NC group (P>0.05). For the functional peptide GL, when its final concentration was 200 μg / mL, 150 μg / mL, and 100 μg / mL, the cell viability was 101.2±1.4890%, 100.8±0.7312%, and 102.5±2.0050%, respectively; for the functional peptide FA, when its final concentration was 200 μg / mL, 150 μg / mL, and 100 μg / mL, the cell viability was 99.9±1.3660%, 100.1±2.3330%, and 100.7±3340%, respectively; for the functional peptide FQ, when its final concentration was 200 μg / mL, 150 μg / mL, and 100 When the concentrations of GL, FA, and FQ were 200 μg / mL, 150 μg / mL, and 100 μg / mL, respectively, the cell viability was 98.84±0.8009%, 101.8±0.7407%, and 100.7±2.0700%, indicating that the above concentrations had no effect on cell growth. Therefore, for GL, FA, and FQ, the concentrations of 200 μg / mL, 150 μg / mL, and 100 μg / mL were selected for subsequent experiments.

[0146] (3) Cell damage assay

[0147] Aβ 1-42 It is an important indicator of Alzheimer's disease. It will be deposited in the brain and cause oxidative stress, which in turn will aggravate Aβ 1-42 The accumulation of Aβ 1-42The accumulation of Aβ induces cytotoxicity, and the toxic cells will also trigger a signal cascade reaction of apoptotic proteins. In order to simulate the symptoms of neuronal cell damage in Alzheimer's disease, we constructed Aβ 1-42 Injury model, testing different concentrations of Aβ 1-42 Effects of intervention on SHSY5Y cell viability 24 h after intervention:

[0148] Aβ was placed in a centrifuge tube. 1-42 The powder was dissolved to 1 mM with hexafluoroisopropanol (HFIP), incubated at room temperature for 60 min to fully dissolve it, and the tube was opened and left in a fume hood overnight to form a peptide film. The peptide film was dissolved to 5 mM with dimethyl sulfoxide (DMSO), and the culture medium was added until the DMSO concentration in the system was <1% (v / v). The cells in the logarithmic growth phase of (1) were cultured at a rate of 5×10 4 / well of a 96-well plate, and when the cells grew to about 70% in the well, the old culture medium was aspirated and washed with PBS, and then new culture medium was added to set up the blank control group (or NC group); alternatively, Aβ-containing 1-42 culture medium to make Aβ 1-42 The final concentrations of Aβ were 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, and 0.5 μM, respectively. 1-42 Intervention group. Six replicate wells were set up in each group. After 24 h of culture, the old culture medium was removed and washed with PBS. 100 μL of new culture medium and 10 μL of CCK8 reagent were added to each well. The 96-well plate was placed in an incubator and cultured for 1-4 h. The absorbance was measured at 450 nm. The results are shown as follows: Figure 4 As shown in Figure 2, the calculation method of cell viability is the same as (2). The concentration of Aβ1-42 that has a half-inhibitory effect on cells (i.e., the concentration of Aβ1-42 when cell viability is close to 50%) is selected for subsequent experiments.

[0149] After testing, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, and 0.5 μM Aβ 1-42 After intervention, the cell viabilities were 31.22±1.55%, 35.94±1.47%, 41.45±2.41%, 48.71±1.33%, 58.06±1.33%, and 65.26±4.01%, respectively. Figure 4 It can be seen that compared with the NC group, the cell survival rates of the other groups decreased, and there were extremely significant statistical differences (P<0.0001). Therefore, 2 μM Aβ was selected to produce a half-inhibitory effect on the cells. 1-42 To intervene in cells and thus construct Aβ 1-42 injury model, and the Aβ 1-42The injury model was set as the model group (MC).

[0150] Collect the cells in the logarithmic growth phase (1) from the culture flask and count them. Adjust the cell concentration in the cell suspension to a volume of 100 μL per well and a cell density of 5×10 4 / well into the 96-well plate, placed in the incubator for 24 hours. When the cells grow to about 70% in the well, aspirate the old culture medium and wash with PBS, then add new culture medium or Aβ-containing 1-42 The culture medium makes Aβ 1-42 The final concentration was 2 μM. After culturing for 24 hours, the old culture medium was removed and washed with PBS. New culture medium or 100 μL of culture medium containing corn peptide was added to each well to make the final concentration of corn peptide in the well 8 mg / mL, 6 mg / mL, and 4 mg / mL, respectively. After culturing for 24 hours, the old culture medium was removed and washed with PBS. 100 μL of new culture medium and 10 μL of CCK8 reagent were added to each well. The 96-well plate was placed in an incubator and cultured for 1-4 hours. The absorbance was measured at 450 nm. The results are shown as follows: Figure 5 The calculation method of cell viability is the same as (2). 1-42 Inducing SHSY5Y cell damage has a protective effect.

[0151] After testing, no Aβ was used 1-42 The survival rate of cells treated with corn peptide (NC group) was 99.39±1.94%, and 2 μM Aβ 1-42 The survival rate of cells without corn peptide intervention (MC group) was 43.71±5.35%, and the survival rate of cells without corn peptide intervention (MC group) was 43.71±5.35%. 1-42 The survival rate of cells treated with 8 mg / mL corn peptide (COP-H group) was 72.25±2.15%, and the survival rate of cells treated with 2 μM Aβ 1-42 The survival rate of cells treated with 6 mg / mL corn peptide (COP-M group) was 68.81 ± 4.02%, and the survival rate of cells treated with 2 μM Aβ 1-42 The survival rate of cells intervened with 4 mg / mL corn peptide (COP-L group) was 65.93±3.35%. Figure 5 The results showed that Aβ 1-42The injury model was successfully constructed, and the cell survival rate was improved after intervention with corn peptides. Compared with the MC group, the cell survival rates of the COP-H and COP-M groups increased by approximately 28% and 25%, respectively, with extremely significant statistical differences (P<0.0001). The cell survival rate of the COP-L group increased by approximately 22%, with significant statistical differences (P<0.001). With the increase in corn peptide concentration, the cell survival rate also increased significantly, indicating that corn peptides can inhibit Aβ in a concentration-dependent manner. 1-42 The survival rate of SHSY5Y cells decreases under injury, thereby protecting nerve cells.

[0152] Collect the cells in the logarithmic growth phase (1) from the culture flask and count them. Adjust the cell concentration in the cell suspension to a volume of 100 μL per well and a cell density of 5×10 4 / well into the 96-well plate, placed in the incubator for 24 hours. When the cells grew to about 70% in the well, aspirate the old culture medium and wash with PBS, then add the Aβ 1-42 The culture medium makes Aβ 1-42 The final concentration was 2 μM. After culturing for 24 hours, the old culture medium was removed and washed with PBS. 100 μL of culture medium containing different functional peptides (GL, FA, FQ) was added to each well so that the final concentrations of the functional peptides in the wells were 200 μg / mL, 150 μg / mL, and 100 μg / mL, respectively. After culturing for 24 hours, the old culture medium was removed and washed with PBS. 100 μL of new culture medium and 10 μL of CCK8 reagent were added to each well. The 96-well plate was placed in an incubator and cultured for 1-4 hours. The absorbance was measured at 450 nm. The results are shown as follows: Figure 6 As shown in the figure, the calculation method of cell viability is the same as (2). In addition, the setting is not used. β1-42 The cells treated with functional peptides were NC group, and 2 μM Aβ was used. 1-42 The cells without functional peptide intervention were the MC group. 1-42 Inducing SHSY5Y cell damage has a protective effect.

[0153] according to Figure 6It can be seen that compared with the MC group, the cell survival rate increased after intervention of SHSY5Y cells with different concentrations of functional peptides, and there was a significant statistical difference (P<0.001). For the functional peptide GL, when its final concentrations were 200 μg / mL, 150 μg / mL, and 100 μg / mL, the cell viability was 82.70±2.425%, 76.49±3.276%, and 69.52±3.480%, respectively. For the functional peptide FA, when its final concentrations were 200 μg / mL, 150 μg / mL, and 100 μg / mL, the cell viability was 77.11%±2.719%, 70.50±2.352%, and 56.15±5.437%, respectively. For the functional peptide FQ, when its final concentrations were 200 μg / mL, 150 μg / mL, and 100 μg / mL, the cell viability was 77.07±2.043%, 67.93±2.819%, and 63.64±4.399%, respectively. Among them, the cell survival rate of GL with a final concentration of 200 μg / mL increased by 33% compared with the MC group, and it had the best effect on improving cell damage. The above results show that the functional peptides GL, FA, and FQ can inhibit Aβ in a concentration-dependent manner. 1-42 The decreased survival rate of SHSY5Y cells under injury may play a protective role on nerve cells through this pathway.

[0154] (4) Determination of indicators in damaged cells under the intervention of corn peptides

[0155] The cells in the logarithmic growth phase of (1) were cultured at a rate of 5×10 4 / well into a 96-well plate, placed in an incubator for 24 hours. When the cells grow to about 70% in the well, aspirate the old culture medium and wash with PBS, then add new culture medium or Aβ-containing 1-42 The culture medium makes Aβ 1-42 The final concentration was 2 μM. After culturing for 24 hours, the old culture medium was removed and washed with PBS. New culture medium or 100 μL of culture medium containing corn peptide was added to each well to make the final concentration of corn peptide in the well 8 mg / mL, 6 mg / mL, and 4 mg / mL, respectively. After culturing for 24 hours, the old culture medium was removed and washed with PBS. The above cells were collected and the corresponding indicators were measured according to the instructions of the mitochondrial membrane potential kit, lactate dehydrogenase kit, reactive oxygen species kit, malondialdehyde kit, superoxide dismutase kit, and glutathione peroxidase kit. The results are shown in the figure. Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 、 Figure 11 and Figure 12 shown.

[0156] Figure 7 The figure shows the result of mitochondrial membrane potential intensity. The normal physiological metabolic activities of nerve cells are closely related to the normal functioning of mitochondria. When a large number of free radicals attack cells, ROS will accumulate in the cells to cause oxidative stress reactions. ROS further attack mitochondria, causing their functions to be impaired. The interaction between oxidative stress and abnormal mitochondrial function will form a vicious circle, thereby aggravating mitochondrial dysfunction, aggravating the damage to nerve cells, and causing Alzheimer's disease to become more serious. In this assay, the JC-1 staining reagent in the mitochondrial membrane potential kit showed potential-dependent accumulation in the mitochondria. It existed as a monomer at low concentrations and produced green fluorescence at about 529 nm. At higher concentrations, J-aggregates were formed, and the fluorescence signal shifted to red (about 590 nm). Therefore, Figure 7 The red / green fluorescence ratio can represent the mitochondrial membrane potential, and a decrease in the red / green fluorescence ratio indicates a decrease in mitochondrial membrane potential. Figure 7 The results showed that without the use of Aβ 1-42 The mitochondrial membrane potential of cells treated with corn peptide (NC group) was the highest; 2 μM Aβ 1-42 The mitochondrial membrane potential of cells not treated with corn peptide (MC group) decreased sharply; compared with the MC group, the mitochondrial membrane potential of cells treated with 2 μM Aβ decreased significantly. 1-42 and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42 The mitochondrial membrane potential of cells treated with 4 mg / mL corn peptide (COP-L group) increased. Compared with the MC group, the COP-H group showed an extremely significant statistical difference (P<0.0001), and the COP-M and COP-L groups showed significant statistical differences (P<0.001). Figure 7 The results showed that corn peptide can improve Aβ 1-42 The degree of mitochondrial damage in treated SHSY5Y cells.

[0157] Figure 8 This figure shows the results of lactate dehydrogenase (LDH) content measurement. Under normal conditions, LDH is an enzyme present in the cell cytoplasm. When cells are exposed to stimuli such as oxidative stress, the cell membrane ruptures and LDH is released outside the cell. Therefore, measuring the LDH content in the cell culture supernatant can indicate the degree of cell damage; higher LDH levels indicate greater cell damage. Figure 8 The results showed that without the use of Aβ 1-42 The LDH content of cells treated with corn peptide (NC group) was the lowest; 2 μM Aβ 1-42The LDH content of cells not treated with corn peptide (MC group) increased sharply; compared with the MC group, the LDH content of cells treated with 2 μM Aβ increased significantly. 1-42 and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42 The LDH content of cells treated with 4 mg / mL corn peptide (COP-L group) decreased. Compared with the MC group, the COP-H group, COP-M group, and COP-L group all showed extremely significant statistical differences (P<0.0001), while there was no statistical difference between the COP-H group and the NC group (P>0.05, P=0.0873). Figure 8 The results showed that different concentrations of corn peptides could reduce Aβ 1-42 COP-H inhibits Aβ release in SHSY5Y cells 1-42 The cell damage caused by toxicity is the strongest.

[0158] Figure 9 The figure shows the relative level of reactive oxygen species (ROS). Under normal circumstances, ROS, as a natural by-product of oxygen metabolism, is at a low level in the body. As a "redox messenger", it participates in intracellular signal transmission and regulation, and plays an important role in the cell cycle, gene expression, and maintenance of the body's internal environment homeostasis. However, when the body is stimulated, the level of ROS will increase sharply, exceeding the body's own clearance and processing capacity, causing an imbalance in the body's oxidation-antioxidation effects and oxidative stress, leading to DNA damage, lipid peroxidation, changes in protein structure and function, cell membrane damage, and ultimately the death of the body's cells. It may also cause various diseases (such as Alzheimer's disease). In this assay, the reactive oxygen species kit uses the fluorescent probe DCFH-DA to detect Aβ 1-42 The ROS induction in SHSY5Y cells was detected. Figure 9 The results showed that without the use of Aβ 1-42 The relative level of ROS in cells treated with corn peptide (NC group) was the lowest; 2 μM Aβ 1-42 The relative level of ROS in cells without corn peptide intervention (MC group) increased dramatically; compared with the MC group, the level of ROS in cells treated with 2 μM Aβ 1-42 and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42The relative levels of ROS in cells treated with 4 mg / mL corn peptide (COP-L group) decreased, showing a concentration-dependent gradient within a certain concentration range. Compared with the MC group, the COP-H and NC groups showed extremely significant statistical differences (P<0.0001), the COP-M and COP-L groups showed significant statistical differences (P<0.001), and there was no statistical difference between the COP-H and NC groups (P>0.05). Figure 9 The results showed that corn peptides could reduce the excessive production of ROS in SHSY5Y cells and inhibit Aβ 1-42 Induction of oxidative stress in SHSY5Y cells.

[0159] Figure 10 This figure shows the results of malondialdehyde (MDA) content measurement. Oxygen free radicals produced during lipid peroxidation can damage cells and induce apoptosis. Their cytotoxicity can also affect the activity of various enzymes, causing them to inactivate. MDA is a marker of the degree of cellular lipid peroxidation damage; higher MDA levels indicate a greater degree of cellular lipid peroxidation damage. Figure 10 The results showed that without the use of Aβ 1-42 The MDA content of cells treated with corn peptide (NC group) was the lowest; 2 μM Aβ 1-42 The MDA content of cells without corn peptide intervention (MC group) increased sharply, indicating that the model was successful; compared with the MC group, the MDA content of cells without corn peptide intervention (MC group) increased sharply, indicating that the model was successful; compared with the MC group, the MDA content of cells without corn peptide intervention (MC group) increased sharply, indicating that the model was successfully established ... 1-42 and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42 The MDA content of cells treated with 4 mg / mL corn peptide (COP-L group) decreased. Compared with the MC group, the COP-H group and the COP-M group showed significant statistical differences (P<0.001), and the COP-L group showed a statistical difference (P<0.005). Compared with the NC group, there was no statistical difference in the COP-H group, the COP-M group, and the COP-L group (P>0.05). Figure 10 The results showed that corn peptides could reduce lipid peroxidation caused by intracellular oxidative stress.

[0160] Figure 11This chart shows the results of superoxide dismutase (SOD) content measurement. SOD is an antioxidant enzyme commonly found in natural organisms that can directly scavenge metabolic free radicals and superoxide anion radicals. It can reduce cell damage and scavenge ROS to maintain the oxidation-antioxidation balance, eliminating harmful substances produced by the body's metabolism. The level of SOD reflects the body's ability to scavenge oxygen free radicals; higher SOD levels indicate a stronger ability to scavenge oxygen free radicals. Figure 11 The results showed that without the use of Aβ 1-42 The SOD content of cells treated with corn peptide (NC group) was the highest; 2 μM Aβ 1-42 The SOD content of cells not treated with corn peptide (MC group) decreased sharply; compared with the MC group, the SOD content of cells treated with 2 μM Aβ decreased significantly. 1-42 and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42 The SOD content of cells treated with 4 mg / mL corn peptide (COP-L group) and 10 mg / mL corn peptide (COP-L group) increased, and there were extremely significant statistical differences (P<0.0001). Figure 11 The results showed that corn peptides can increase the content of cellular SOD and enhance the ability of intracellular antioxidant enzymes to resist intracellular oxidative stress, thereby improving Aβ 1-42 Induced cell damage.

[0161] Figure 12 This figure shows the results of glutathione peroxidase (GSH-Px) content measurement. GSH-Px is an antioxidant enzyme that is ubiquitous and closely linked to various antioxidant reactions in living organisms. It can specifically catalyze the reduction of H2O2 by glutathione (GSH) to generate oxidized glutathione (GSSG), thereby protecting the body's biomembranes from damage caused by ROS and maintaining the normal physiological functions of related cells. It also has protective effects on the liver and the body's immunity. Figure 12 The results showed that without the use of Aβ 1-42 The GSH-Px content of cells treated with corn peptide (NC group) was the highest; 2 μM Aβ 1-42 The GSH-Px content of cells not treated with corn peptide (MC group) decreased sharply; compared with the MC group, the GSH-Px content of cells treated with 2 μM Aβ 1-42 and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42The GSH-Px content of cells intervened with 4 mg / mL corn peptide (COP-L group) increased, and the COP-H group had an extremely significant statistical difference (P<0.0001), the COP-M group had a statistical difference (P<0.05), and the COP-L group had a significant statistical difference (P<0.001). Figure 12 The results showed that corn peptides can increase the content of cellular GSH-Px, enhance the ability of intracellular antioxidant enzymes to resist intracellular oxidative stress, and thus improve Aβ 1-42 Induced cell damage.

[0162] (5) Determination of indicators in damaged cells under the intervention of functional peptides

[0163] The cells in the logarithmic growth phase of (1) were cultured at a rate of 5×10 4 / well into the 96-well plate, placed in the incubator for 24 hours. When the cells grew to about 70% in the well, aspirate the old culture medium and wash with PBS, then add 1-42 The culture medium makes Aβ 1-42 The final concentration was 2 μM. After culturing for 24 hours, the old culture medium was removed and washed with PBS. Culture medium containing different functional peptides (GL, FA, FQ) was added to each well so that the final concentrations of the functional peptides in the wells were 200 μg / mL, 150 μg / mL, and 100 μg / mL, respectively. After culturing for 24 hours, the old culture medium was removed and washed with PBS. The above cells were collected and the corresponding indicators were measured according to the instructions of the mitochondrial membrane potential kit, lactate dehydrogenase kit, reactive oxygen species kit, malondialdehyde kit, superoxide dismutase kit, and glutathione peroxidase kit. In addition, no Aβ was used. 1-42 The cells treated with functional peptides were NC group, and 2 μM Aβ was used. 1-42 The cells that were not intervened by functional peptides were the MC group. Figure 13 、 Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 and Figure 18 shown.

[0164] Figure 13 This is the result diagram of mitochondrial membrane potential intensity. Figure 13The results showed that the mitochondrial membrane potential intensity was the highest in the NC group, while it decreased sharply in the MC group. Compared with the MC group, the mitochondrial membrane potential intensity increased after intervention with different concentrations of functional peptides, with extremely significant statistical differences (P<0.0001). There was no statistical difference between the three concentrations of each functional peptide (P>0.05). The mitochondrial membrane potential intensity of functional peptides GL and FA increased with increasing intervention concentration, showing a concentration-dependent increase. Figure 13 The results showed that functional peptides GL, FA, and FQ could improve Aβ 1-42 The extent of mitochondrial damage after treatment of nerve cells.

[0165] Figure 14 This is the result of lactate dehydrogenase (LDH) content determination. Figure 14 The results showed that LDH levels were lowest in the NC group, while those in the MC group increased dramatically. Compared with the MC group, LDH levels decreased to varying degrees after intervention with different concentrations of the functional peptides. The FA peptide concentrations of 150 μg / mL and 100 μg / mL showed statistically significant differences (P < 0.001), while the other groups showed extremely significant differences (P < 0.0001). Furthermore, GL improved LDH levels in a concentration-dependent manner. Figure 14 The results showed that functional peptides GL, FA, and FQ could reduce Aβ 1-42 Processing LDH release from neurons, thereby inhibiting Aβ 1-42 Toxic neuronal damage.

[0166] Figure 15 The graph shows the results of the relative level determination of reactive oxygen species (ROS). Figure 15 The results showed that the relative ROS level was lowest in the NC group, while it increased dramatically in the MC group, with a statistically significant difference compared to the NC group (P<0.0001). Compared to the MC group, ROS levels decreased after intervention with different concentrations of the functional peptides, with statistically significant differences (P<0.0001). Furthermore, the improvement in ROS levels by GL and FQ was concentration-dependent, indicating that the effect of the peptides is directly related to their concentration, and changes in concentration affect their ability to reduce ROS production and inhibit oxidative stress. Figure 15 The results showed that functional peptides GL, FA, and FQ can reduce the excessive production of ROS in nerve cells and inhibit Aβ 1-42 Induces oxidative stress in nerve cells, showing potential neuroprotective effects.

[0167] Figure 16 This is the result of malondialdehyde (MDA) content determination. Figure 16The results showed that MDA levels were lowest in the NC group, while those in the MC group increased dramatically. Compared with the MC group, intervention with different concentrations of functional peptides resulted in varying degrees of decreases in MDA levels, with statistically significant differences (P < 0.0001). Furthermore, no statistical differences were found between the three concentrations of each functional peptide (P > 0.05). GL and FA showed the greatest improvement, with no statistical difference compared with the NC group (P > 0.05). Furthermore, FQ showed a concentration-dependent effect on MDA levels. Figure 16 The results showed that functional peptides GL, FA, and FQ could reduce lipid peroxidation caused by oxidative stress in nerve cells.

[0168] Figure 17 This is the result of superoxide dismutase (SOD) content determination. Figure 17 The results showed that the SOD content was highest in the NC group, while the SOD content in the MC group decreased sharply. Compared with the MC group, SOD content increased significantly after intervention with different concentrations of functional peptides, and the differences were extremely significant (P<0.0001). At the same time, there was no statistical difference between the three concentrations of the functional peptide GL (P>0.05). In addition, the functional peptides GL, FA, and FQ showed a concentration-dependent effect on the increase in SOD content. Figure 17 The results showed that functional peptides GL, FA, and FQ can increase the content of cellular SOD and enhance the ability of intracellular antioxidant enzymes to resist intracellular oxidative stress, thereby improving Aβ 1-42 Induced neuronal damage.

[0169] Figure 18 This is the result of glutathione peroxidase (GSH-Px) content determination. Figure 18 Results showed that GSH-Px levels were highest in the NC group, while those in the MC group decreased dramatically. Compared with the MC group, GSH-Px levels increased after intervention with different concentrations of functional peptides. While 100 μg / mL FQ increased GSH-Px levels, the difference was not statistically significant (P>0.05). 100 μg / mL FA showed a significant difference (P<0.01), while the remaining groups showed highly significant differences (P<0.0001). Furthermore, the three concentrations of the functional peptide GL showed no statistical differences between any two groups (P>0.05), and none of the three concentrations of the functional peptide GL showed any statistical difference compared with the NC group (P>0.05). Furthermore, 200 μg / mL FA and FQ also showed no statistical difference compared with the NC group (P>0.05). GL, FA, and FQ at 200 μg / mL had the strongest effect on increasing GSH-Px levels. Figure 18The results showed that functional peptides GL, FA, and FQ can increase the content of cellular GSH-Px, enhance the ability of intracellular antioxidant enzymes to resist intracellular oxidative stress, and thus improve Aβ 1-42 Induced neuronal damage.

[0170] All of the above intracellular indicators are related to oxidative stress. Oxidative stress is caused by an imbalance between ROS production and degradation, leading to ROS accumulation. Excessive ROS can lead to lipid peroxidation, affecting cell membrane fluidity and permeability, thereby inhibiting neuronal signaling and affecting neurotransmitter transmission, ultimately causing damage to the structure and function of brain and neuronal cells. Certain enzymes and non-enzymatic antioxidants (such as SOD and GSH-Px) can scavenge peroxides, thereby preventing neuronal damage. These experimental results indicate that the functional peptides GL, FA, and FQ have a potent inhibitory effect on oxidative stress. They participate in the expression of multiple substances involved in the oxidative stress process, inhibiting and scavenging free radicals, and ultimately promoting a stable balance in the body. Therefore, the functional peptides GL, FA, and FQ are expected to improve memory and other cognitive impairments caused by Alzheimer's disease by inhibiting oxidative stress.

[0171] (6) Corn peptide inhibits Aβ 1-42 Damage mechanism research

[0172] When neurons communicate with each other, protein kinase A (PKA) acts on the downstream cyclic adenosine monophosphate response element binding protein (CREB), prompting CREB phosphorylation. The activated CREB then acts on the downstream brain-derived neurotrophic factor (BDNF), prompting increased BDNF expression, thereby promoting and balancing the expression and differentiation of neurons. In addition, the PKA-mediated signaling pathway plays a vital role in the formation of neural protrusions and signal transduction, and can regulate nerves. Studies have confirmed that the PKA-CREB-BDNF signaling pathway has a certain protective effect on neurons in a variety of brain injury diseases. Therefore, in this experiment, protein immunoblotting technology was used to study the effect of corn peptide on the inhibition of Aβ 1-42 Whether the damage mechanism is related to the above-mentioned cell signaling pathways and whether it can have a protective effect on neurons.

[0173] The cells in the logarithmic growth phase of (1) were cultured at a rate of 5×10 4 / well into the 96-well plate, when the cells grow to about 70% in the well, aspirate the old culture medium and wash with PBS, then add new culture medium or Aβ-containing 1-42 The culture medium makes Aβ 1-42The final concentration was 2 μM. After 24 hours of culture, the old culture medium was removed and washed with PBS. Fresh culture medium or 100 μL of culture medium containing corn peptide was added to each well to achieve final corn peptide concentrations of 8 mg / mL, 6 mg / mL, and 4 mg / mL, respectively. After 24 hours of culture, the old culture medium was removed and washed with PBS. Cell lysis buffer containing protease inhibitors, phosphatase inhibitors, and phenylmethylsulfonyl fluoride was added to each well. Lysis was carried out on ice for 30 minutes. The cells were centrifuged at 4°C and 12,000 rpm for 15 minutes, and the supernatant was collected. The protein concentration of the supernatant in each well was determined using a protein quantification kit (BCA assay). The supernatant was mixed with loading buffer at a volume ratio of 1:4 and diluted to the same concentration in each well. The protein was then boiled in a 100°C water bath for 5 minutes to denature the protein. Equal amounts of protein were loaded for western blotting, and the procedures were as follows: electrophoresis (80 V, 30 min; 120 V, 60 min), transfer (200 mA, 60 min), blocking for 2 h, incubation with primary antibody (PKA and primary antibody diluent were prepared at a volume ratio of 1:1000; or, CREB and primary antibody diluent were prepared at a volume ratio of 1:1000; or, BDNF and primary antibody diluent were prepared at a volume ratio of 1:1000) overnight (4°C), washing with TBST (10 min / 3 times), incubation with secondary antibody (mouse antibody and secondary antibody diluent were prepared at a volume ratio of 1:10000) for 2 h, washing with TBST (10 min / 3 times), development with chemiluminescent substrate (ECL) and photography, processing the bands with ImageJ software and calculating the grayscale value. The results are shown in Figure 2. Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 shown.

[0174] Figure 19 This is the protein band diagram of the protein immunoblotting experiment. Figure 20 The results of the relative expression of PKA are shown in Figure 2. Figure 21 The relative expression results of CREB are shown in Figure 2. Figure 22 The results of the relative expression of BDNF are shown in Figure 2. Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 The results showed that without the use of Aβ 1-42 The relative expression levels of PKA, CREB and BDNF in cells treated with corn peptide (NC group) were the highest; 2 μM Aβ 1-42 The relative expression of PKA, CREB and BDNF in cells without corn peptide intervention (MC group) decreased sharply. 1-42and 8 mg / mL corn peptide-intervention cells (COP-H group), 2 μM Aβ 1-42 and 6 mg / mL corn peptide-intervention cells (COP-M group), 2 μM Aβ 1-42 The relative expression of CREB increased in the COP-H, COP-M, and COP-L groups compared with the MC group; there was no statistical difference between the COP-H and COP-M groups compared with the NC group (P>0.05). The relative expression of BDNF also increased in the COP-H, COP-M, and COP-L groups compared with the MC group; there was no statistical difference between the COP-H and COP-M groups compared with the NC group (P>0.05). The above results indicate that different concentrations of corn peptides can effectively improve the expression of PKA, CREB, and BDNF proteins in cells, effectively upregulate the expression of these three proteins, and thus inhibit Aβ. 1-42 Inducing adverse reactions caused by SHSY5Y cells, thereby reducing damage to nerve cells and providing protection for nerve cells.

[0175] (7) Functional peptides inhibit Aβ 1-42 Damage mechanism research

[0176] The cells in the logarithmic growth phase of (1) were cultured at a rate of 5×10 4 / well into the 96-well plate, when the cells grew to about 70% in the well, the old culture medium was aspirated and washed with PBS, and then the Aβ-containing 1-42 The culture medium makes Aβ 1-42The final concentration was 2 μM. After 24 h of culture, the old culture medium was removed and the cells were washed with PBS. Culture medium containing different functional peptides (GL, FA, and FQ) was added to each well to achieve final concentrations of 200 μg / mL, 150 μg / mL, and 100 μg / mL, respectively. After 24 h of culture, the old culture medium was removed and the cells were washed with PBS. The cells were digested and collected from each well. The cells were centrifuged at 1000 rpm for 5 min. Cell lysis buffer containing protease inhibitors and phosphatase inhibitors was added (the volume ratio of lysis buffer, protease inhibitors, and phosphatase inhibitors was 100:1:1). The cells were lysed on ice for 30 min and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected. The protein concentration of the supernatant in each well was determined using a protein quantification kit (BCA assay). The supernatant was mixed with loading buffer at a volume ratio of 1:4 and diluted to the same concentration in each well. The proteins were then boiled in a 100°C water bath for 5 min to denature the proteins. 20 μg of protein was loaded for SDS-PAGE electrophoresis. Electrophoresis was initially performed at a constant voltage of 80 V for 30 min. Once the protein sample entered the lower PAGE gel, the electrophoresis was switched to 120 V for 60 min, and the electrophoresis was stopped when the protein sample reached the bottom of the PAGE gel. A sponge gasket, filter paper, gel strip, PVDF membrane, filter paper, and sponge gasket were placed in the transfer apparatus, in order from the negative electrode (black bottom) to the positive electrode. The PAGE gel was transferred at a constant current of 200 mA for 60 min. The PAGE gel was blocked with 5% skim milk powder for 2 h and washed three times with TBST for 10 min. The gel was then incubated overnight at 4°C with the primary antibody. The primary antibody was prepared by mixing PKA with the primary antibody diluent at a volume ratio of 1:1000, CREB with the primary antibody diluent at a volume ratio of 1:1000, or BDNF with the primary antibody diluent at a volume ratio of 1:1000. The cells were washed again with TBST for 3 times within 10 minutes and incubated with secondary antibody for 2 hours. The secondary antibody was prepared by mixing mouse antibody and secondary antibody diluent at a volume ratio of 1:10000. The cells were washed again with TBST for 3 times within 10 minutes and then developed with chemiluminescent substrate (ECL) and photographed. The bands were processed with ImageJ software and the grayscale values were calculated. The results are shown in Figure 2. Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 shown.

[0177] Figure 23 This is the protein band diagram of the protein immunoblotting experiment. Figure 24 The results of the relative expression of PKA are shown in Figure 2. Figure 25 The relative expression results of CREB are shown in Figure 2. Figure 26 The results of the relative expression of BDNF are shown in Figure 2. Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 Results showed that the relative expression levels of PKA, CREB, and BDNF were highest in the NC group, while those in the MC group decreased significantly. Compared with the MC group, the relative expression levels of PKA in the NC group were statistically significantly different (P<0.0001), GL (P<0.01), and FQ (P<0.05). There was no statistical difference between the NC group and the GL group (P>0.05). Compared with the MC group, the relative expression levels of CREB in the GL group were statistically significantly different (P<0.0001), while those in the FA and FQ groups were statistically significantly different (P<0.01). There was no statistical difference between the NC group and the GL group (P>0.05). Compared with the MC group, the relative expression levels of BDNF in the GL and FQ groups were statistically different (P<0.05), with a statistically significant difference in the FA group (P<0.0001). The above results show that the functional peptides GL, FA, and FQ act on Aβ 1-42 After damaged nerve cells, it can effectively improve the expression of three proteins, PKA, CREB, and BDNF, and effectively upregulate the expression of these three proteins, with GL having the most significant upregulation effect. Functional peptides GL, FA, and FQ can inhibit Aβ by activating the memory proteins PKA, CREB, and BNDF in the PKA-CREB-BNDF signaling pathway in nerve cells. 1-42 Induce adverse reactions caused by nerve cells, thereby reducing damage to nerve cells and providing protection for nerve cells.

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A functional peptide segment with neuroprotective effect, characterized in that: The functional peptide segments include peptide segment GL, peptide segment FA and peptide segment FQ.

2. A corn peptide with neuroprotective effect, characterized in that: The corn peptide comprises the functional peptide segment according to claim 1.

3. The corn peptide according to claim 2, characterized in that Based on the mass of the corn peptide, the content of the peptide segment GL is ≥30 μg / g, the content of the peptide segment FA is ≥35 μg / g, and the content of the peptide segment FQ is ≥35 μg / g.

4. The corn peptide according to claim 2 or 3, characterized in that The moisture content of the corn peptide is 1%-10%; and / or, Ash content of corn peptides ≤ 5%; and / or, The protein content of corn peptides is ≥85%; and / or, Acid-soluble protein content in corn peptides ≥ 80%; and / or, The mass content of peptides with a molecular weight of less than 5000 Da in corn peptides is 100%; and / or, The mass content of peptides with a molecular weight of less than 1000 Da in corn peptides is ≥ 95%; and / or, The mass content of peptides with a molecular weight of 189-1000 Da in corn peptides is ≥ 75%; and / or, The non-essential amino acid content of corn peptides is 40%-60%; and / or, The essential amino acid content of corn peptides is 15%-35%; and / or, The hydrophobic amino acid content of corn peptides is 25-45%; and / or, The aromatic amino acid content in corn peptides is 1%-10%.

5. The corn peptide according to any one of claims 2 to 4, characterized in that The corn peptide is obtained by enzymatically hydrolyzing corn protein raw materials; The enzymatic hydrolysis treatment includes using alkaline protease to perform a first enzymatic hydrolysis, and then using neutral protease to perform a second enzymatic hydrolysis.

6. The method for preparing the corn peptide according to any one of claims 2 to 5, characterized in that: The steps include: performing enzymatic hydrolysis on a corn protein raw material to obtain the corn peptide; The enzymatic hydrolysis treatment includes using alkaline protease to perform a first enzymatic hydrolysis, and then using neutral protease to perform a second enzymatic hydrolysis.

7. The preparation method according to claim 6, characterized in that The alkaline protease has an enzyme activity of 580,000-650,000 DU / g; and / or, Based on each gram of the zein, the amount of the alkaline protease added is 0.8-1.0 wt%; and / or, The enzymatic activity of the neutral protease is 380,000-420,000 U / g; and / or, Based on each gram of the zein, the neutral protease is added in an amount of 1.0-1.2 wt %.

8. The preparation method according to claim 6 or 7, characterized in that The conditions of the first enzymatic hydrolysis include: a time of 2-4 h, a pH value of 8.0-9.0, and a temperature of 50-60° C.; and / or, The conditions of the second enzymatic hydrolysis include: a time of 1-3 hours, a pH value of 6.5-7.5, and a temperature of 50-60° C.; and / or, The corn protein raw material is obtained by mixing corn protein and water in a mass volume ratio of 1: (8-12).

9. The preparation method according to any one of claims 6 to 8, characterized in that After the enzymatic hydrolysis treatment, the enzymatic hydrolyzate is further subjected to separation and purification treatment; The separation and purification process includes centrifugation, filtration, ultrafiltration and spray drying.

10. Use of the functional peptide segment according to claim 1, and / or the corn peptide according to any one of claims 2 to 5, and / or the corn peptide prepared by the preparation method according to any one of claims 6 to 9 in the preparation of related products with neuroprotective effects.

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