Peanut peptide for promoting cell development

Abstract

The invention belongs to the technical field of biotechnology, and discloses a peanut peptide for promoting cell development, which is characterized in that the sequence of the peanut peptide is Met Ala Ser Val Thr Gly Pro Ser Ser Ala Leu Ala Asp Ser Ile Gly, through the accurately designed amino acid sequence, the peanut peptide constructs a molecular level adaptation mechanism with a cell surface receptor, triggers a cascade reaction of a key signal channel of phosphatidylinositol-3 kinase PI3K, and can promote cell development. The activation effect not only accelerates the process of a cell cycle and increases the proportion of DNA synthetic cells in an S phase by 40% or more, but also directionally induces the mesenchymal stem cells to differentiate into functional cells such as muscle cells and nerve cells by up-regulating transcription factors MyoD and NeuroD1, in a three-dimensional cell culture model, the cell migration rate of an experimental group added with 200 [mu] g / mL peanut peptide is increased by 58% compared with a control group, and the cell migration rate of the experimental group added with 200 [mu] g / mL peanut peptide is increased by 30% or more compared with that of the control group. The branch density of the formed vascular-like network is increased by 3 times, and meanwhile, the test further proves that the peanut peptide has the function of promoting cell development and shows remarkable optimization capability on a tissue regeneration microenvironment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and particularly relates to a peanut peptide for promoting cell development. Background Art

[0002] In the field of biomedicine, the regulation of cell development is of crucial significance for tissue engineering, regenerative medicine, and disease treatment. Traditional cell development promoters mostly rely on animal-derived components or chemically synthesized substances. However, these substances have many limitations. For example, animal-derived components may cause immune rejection reactions and the risk of carrying pathogens, while chemically synthesized substances may have potential toxicity and side effects, and poor biocompatibility. Therefore, the development of a safe, efficient, and natural cell development promoter has become a research hotspot in this field.

[0003] Peanut, as a widely cultivated crop rich in high-quality protein, has abundant protein resources and a unique amino acid composition. Peanut protein contains various amino acid sequences beneficial to cell development. By converting it into peanut peptide through a specific preparation method, not only can the biological activity of peanut protein be retained, but it can also be more easily absorbed and utilized by cells. Currently, there are relatively few preparation methods for peanut peptides for promoting cell development on the market, and there are problems such as cumbersome preparation processes, low peptide segment activity, and insufficient purity. The purpose of the present invention is to provide an innovative preparation method, and by optimizing the preparation process, obtain peanut peptides with high-efficiency cell development promotion function, fill the market gap, and provide new high-quality raw materials for cell culture and biomedical research. Summary of the Invention

[0004] The purpose of the present invention is to provide a peanut peptide for promoting cell development to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A peanut peptide for promoting cell development, characterized in that: its peanut peptide sequence is Met Ala Ser Val Thr Gly Pro Ser Ser Ala Leu Ala Asp SerIle Gly.

[0006] Preferably, the peanut peptide is composed of a complex enzyme and alkaline protease. The sequence of the complex enzyme is Ile Val GlyGln Gly Pro, and the biological sequence of alkaline protease is Ile Val Lys Val Gly Asp Ile Pro Gly.

[0007] Preferably, Val-Pro-Pro is the structure of a milk-derived antihypertensive peptide, which can inhibit angiotensin-converting enzyme ACE and lower blood pressure.

[0008] Preferably, Ala-Gly-Pro proline can participate in collagen synthesis, promote fibroblast proliferation, and may have a positive effect on tissue repair and extracellular matrix formation.

[0009] Preferably, Leu-Tyr leucine is a branched-chain amino acid that can promote muscle protein synthesis; tyrosine participates in neurotransmitter synthesis and has an auxiliary effect on nerve cell development.

[0010] Preferably, Arg-Gly-Asp, the RGD sequence is a key structure for cell adhesion, can bind to integrin receptors, promote cell adhesion, migration and proliferation, and is commonly found in extracellular matrix proteins such as collagen.

[0011] Preferably, the sulfhydryl group -SH of Met-Gly-Gly methionine can scavenge free radicals and enhance antioxidant capacity.

[0012] Preferably, the specific steps of the peanut peptide method for promoting cell development are as follows: S1: Raw material pretreatment and protein extraction: Select high-quality peanuts, dry and shell them, crush the peanut kernels, and defat them by supercritical CO2 extraction to obtain low-fat peanut meal; then extract the protein by stirring with deionized water under alkaline conditions, and obtain the peanut protein extract by centrifugal separation. S2: Enzymatic hydrolysis reaction and preliminary separation: After adjusting the pH and heating the protein extract to denature, add a composite enzyme system of neutral protease and flavor protease with a mass ratio of 1:1 - 2:1 and an enzyme amount of 2% - 4%, and carry out enzymatic hydrolysis at 50 - 55 °C and pH 7.0 - 8.0 for 4 - 6 hours; after inactivating the enzyme, filter through a filter cloth for rough filtration and a microporous membrane for fine filtration in sequence to obtain the crude peanut peptide filtrate.

[0013] Preferably, the raw material pretreatment and protein extraction in S1 refer to selecting high-quality peanuts, drying and shelling them, crushing the peanut kernels, and defatting them by supercritical CO2 extraction to obtain low-fat peanut meal; then extracting the protein by stirring with deionized water under alkaline conditions, and obtaining the peanut protein extract by centrifugal separation.

[0014] Preferably, after adjusting the pH and heating the protein extract to denature, add a composite enzyme system of neutral protease and flavor protease with a mass ratio of 1:1 - 2:1 and an enzyme amount of 2% - 4%, and carry out enzymatic hydrolysis at 50 - 55 °C and pH 7.0 - 8.0 for 4 - 6 hours; after inactivating the enzyme, filter through a filter cloth for rough filtration and a microporous membrane for fine filtration in sequence to obtain the crude peanut peptide filtrate.

[0015] The beneficial effects of the present invention are as follows: 1. Through the precisely designed amino acid sequence, this peanut peptide constructs a molecular-level adaptation mechanism with cell surface receptors, triggering a cascade reaction in the phosphatidylinositol-3 kinase (PI3K) key signaling pathway. This activation effect not only accelerates the cell cycle process, increasing the proportion of DNA-synthesizing cells in the S phase by more than 40%, but also, by upregulating the transcription factors MyoD and NeuroD1, directionally induces the differentiation of mesenchymal stem cells into functional cells such as muscle cells and nerve cells. In a three-dimensional cell culture model, in the experimental group supplemented with 200 μg / mL of peanut peptide, the cell migration rate increased by 58% compared to the control group, and the density of the formed vascular-like network branches increased by 3 times. At the same time, the experiment further confirmed that this peanut peptide has the function of promoting cell development and demonstrates a significant ability to optimize the tissue regeneration microenvironment.

[0016] 2. The full-chain safety control of this invention from raw materials to processes forms the unique advantages of this peanut peptide: carefully selected non-genetically modified peanut varieties, with a protein composition matching 92% of the human essential amino acid pattern, reducing the risk of immunogenicity from the source; supercritical CO2 extraction technology completes lipid separation under mild conditions of 35 MPa and 31 °C. Compared with the traditional organic solvent method, the residue of carcinogens such as benzo[a]pyrene is reduced to below the detection limit (<0.1 μg / kg). In the membrane separation process, a polysulfone ultrafiltration membrane with a molecular weight cut-off of 5 kDa is used to effectively remove the macromolecular allergen Arah1 protein, making the allergen residue rate <0.01%. This multi-level verification from molecular compatibility to system safety enables it to seamlessly connect to application scenarios with strict safety requirements such as injection preparations and tissue engineering scaffolds.

[0017] 3. The composite enzyme system optimized by the response surface method in this invention: neutral protease: flavor protease = 3:2, achieving a degree of protein hydrolysis of 92% under the conditions of pH 7.5 and temperature 50 °C, shortening the reaction time by 40% compared with the single enzyme hydrolysis method. The integrated membrane separation system uses a cross-flow filtration mode, increasing the peptide recovery rate to 95%, while controlling the endotoxin content below 0.1 EU / mg. The gel chromatography column is equipped with an online ultraviolet detector to monitor the elution curve in real time, ensuring that the molecular weight distribution of each batch of products is concentrated in the range of 1 - 5 kDa, with a proportion >90%. HPLC detection shows that the content deviation of the main active peptide segment Leu-Tyr-Gly is controlled within ±2%. This full-process digital monitoring system enables the batch-to-batch consistency of the product to reach 98.7%, far exceeding the 85% level of the traditional process, providing a reliable guarantee for the strict quality requirements of raw materials in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the locus map of the peanut peptide that promotes cell development of this invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] As Figure 1 shown, the embodiments of the present invention provide a peanut peptide that promotes cell development, and its peanut peptide sequence is Met Ala Ser Val Thr Gly Pro Ser Ser Ala Leu Ala Asp Ser Ile Gly.

[0021] Among them, the peanut peptide is composed of a complex enzyme and alkaline protease. The sequence of the complex enzyme is Ile Val GlyGln Gly Pro, and the biological sequence of the alkaline protease is Ile Val Lys Val Gly Asp Ile Pro Gly.

[0022] Among them, Val-Pro-Pro is the structure of a milk-derived hypotensive peptide, which can inhibit angiotensin-converting enzyme ACE and reduce blood pressure.

[0023] Val-Pro-Pro is a typical active sequence of milk-derived hypotensive peptides. It consists of a tripeptide structure composed of valine Val and two prolines Pro. By binding to the zinc ion in the active center of angiotensin-converting enzyme ACE, it inhibits ACE from catalyzing the conversion of angiotensin I into vasoconstrictive II, and at the same time reduces the degradation of bradykinin. The dual mechanism dilates blood vessels and reduces blood pressure. In vitro and animal experiments have confirmed that its ACE inhibition rate reaches more than 70%, and it has the characteristics of high stability and low toxicity. It is an important natural active ingredient for the development of functional foods and antihypertensive drugs.

[0024] Among them, Ala-Gly-Pro proline can participate in collagen synthesis, promote the proliferation of fibroblasts, and may have a positive effect on tissue repair and extracellular matrix formation.

[0025] The proline in the Ala-Gly-Pro tripeptide is a characteristic amino acid of collagen, which can directly participate in the assembly of the collagen helix structure. By enhancing the expression of type I collagen gene in fibroblasts, it significantly promotes the proliferation and differentiation of fibroblasts.

[0026] Among them, Leu-Tyr leucine is a branched-chain amino acid, which can promote muscle protein synthesis; tyrosine participates in the synthesis of neurotransmitters and has an auxiliary effect on the development of nerve cells.

[0027] Leucine in Leu-Tyr, as a branched-chain amino acid, can activate the mTOR pathway to promote muscle protein synthesis and assist in the development and repair of skeletal muscle; tyrosine, as a neurotransmitter precursor, can participate in the synthesis of dopamine, etc., regulate the formation of neuronal synapses, and play an auxiliary and promoting role in the development of nerve cells.

[0028] Among them, Arg-Gly-Asp, the RGD sequence is a key structure for cell adhesion, can bind to integrin receptors, promote cell adhesion, migration and proliferation, and is commonly found in extracellular matrix proteins such as collagen.

[0029] The Arg-Gly-Asp RGD sequence, as the core structure of cell adhesion, can specifically recognize and bind to integrin receptors on the cell surface. By activating the focal adhesion kinase FAK signaling pathway, it can significantly enhance the adhesion force between cells and the extracellular matrix, and at the same time induce cytoskeletal reorganization to promote cell migration and proliferation.

[0030] Among them, the mercapto group -SH of methionine in Met-Gly-Gly can scavenge free radicals and enhance antioxidant capacity.

[0031] Methionine in Met-Gly-Gly contains a mercapto group -SH, which has strong reducibility. It can directly capture hydroxyl radicals ・OH and superoxide anions O reactive oxygen species ROS, and scavenge excessive free radicals in cells through redox reactions to reduce oxidative stress damage.

[0032] Among them, the specific steps of the peanut peptide method for promoting cell development are as follows: S1: Pretreatment of raw materials and protein extraction: Select high-quality peanuts, dry and shell them, crush the peanut kernels, and defat them by supercritical CO2 extraction to obtain low-fat peanut meal; then stir and extract the protein with deionized water under alkaline conditions, and obtain the peanut protein extract by centrifugal separation; S2: Enzymatic hydrolysis reaction and preliminary separation: After adjusting the pH and heating and denaturing the protein extract, add a composite enzyme system of neutral protease and flavor protease with a mass ratio of 1:1 to 2:1 and an enzyme amount of 2% to 4%, and carry out enzymatic hydrolysis at 50 - 55 °C and pH 7.0 - 8.0 for 4 - 6 hours; after inactivating the enzyme, filter it through a filter cloth for rough filtration and a microporous membrane for fine filtration in sequence to obtain the crude peanut peptide filtrate.

[0033] Among them, the pretreatment of raw materials and protein extraction in S1 refers to selecting high-quality peanuts, drying and shelling them, crushing the peanut kernels, and defatting them by supercritical CO2 extraction to obtain low-fat peanut meal; then stirring and extracting the protein with deionized water under alkaline conditions, and obtaining the peanut protein extract by centrifugal separation.

[0034] Among them, after adjusting the pH and heating and denaturing the protein extraction solution, a complex enzyme system of neutral protease and flavor protease is added with a mass ratio of 1:1 to 2:1 and an enzyme amount of 2% to 4%, and enzymolysis is carried out for 4 to 6 hours under the conditions of 50-55 °C and pH 7.0-8.0; after inactivating the enzyme, it is successively filtered through a filter cloth for rough filtration and a microporous filter membrane for fine filtration to obtain a crude peanut peptide filtrate. At the same time, the experiment further confirms that this peanut peptide has the function of promoting cell development and demonstrates a significant ability to optimize the tissue regeneration microenvironment.

[0035] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0036] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A peanut peptide for promoting cell development, characterized in that: Its peanut peptide biological sequence is Met Ala Ser Val Thr Gly Pro Ser Ser Ala Leu Ala Asp Ser Ile Gly.

2. The peanut peptide for promoting cell development according to claim 1, wherein: The peanut peptide is composed of a complex enzyme and alkaline protease. The sequence of the complex enzyme is Ile Val Gly Gln Gly Pro, and the biological sequence of alkaline protease is Ile Val Lys Val Gly Asp Ile Pro Gly.

3. The peanut peptide for promoting cell development according to claim 1, wherein: Val-Pro-Pro is the structure of milk-derived hypotensive peptide, which can inhibit angiotensin-converting enzyme ACE and reduce blood pressure.

4. A peanut peptide for promoting cell development according to claim 1, characterized in that: Ala-Gly-Pro proline can participate in collagen synthesis, promote fibroblast proliferation, and has a positive effect on tissue repair and extracellular matrix formation.

5. A peanut peptide for promoting cell development according to claim 1, characterized in that: Leu-Tyr leucine is a branched-chain amino acid that can promote muscle protein synthesis; tyrosine participates in neurotransmitter synthesis and has an auxiliary effect on the development of nerve cells.

6. The peanut peptide for promoting cell development according to claim 1, characterized in that: Arg-Gly-Asp, the RGD sequence is a key structure for cell adhesion, which can bind to integrin receptors, promote cell adhesion, migration and proliferation, and is commonly found in collagen extracellular matrix proteins.

7. A peanut peptide for promoting cell development according to claim 1, characterized in that: The sulfhydryl -SH of Met-Gly-Gly methionine can scavenge free radicals and enhance antioxidant capacity.

8. A preparation method of peanut peptides for promoting cell development, characterized in that: The specific steps of the preparation method of the peanut peptide for promoting cell development are as follows: S1: Raw material pretreatment and protein extraction: Select high-quality peanuts, dry and shell them, crush the peanut kernels, and defat them by supercritical CO2 extraction to obtain low-fat peanut meal; Then, stir and extract the protein with deionized water under alkaline conditions, and obtain the peanut protein extract by centrifugal separation; S2: Enzymatic hydrolysis reaction and preliminary separation: After adjusting the pH and heating the protein extract to denature, add a complex enzyme system of neutral protease and flavor protease with a mass ratio of 1:1~2:1 and an enzyme amount of 2%~4%, and carry out enzymatic hydrolysis at 50-55°C and pH 7.0-8.0 for 4-6 hours; after inactivating the enzyme, filter through a filter cloth for rough filtration and a microporous membrane for fine filtration in sequence to obtain the crude peanut peptide filtrate.

9. The preparation method of a peanut peptide for promoting cell development according to claim 8, wherein: The raw material pretreatment and protein extraction in S1 refer to selecting high-quality peanuts, drying and shelling them, crushing the peanut kernels, and defatting them by supercritical CO2 extraction to obtain low-fat peanut meal; Then, stir and extract the protein with deionized water under alkaline conditions, and obtain the peanut protein extract by centrifugal separation.

10. The preparation method of a peanut peptide for promoting cell development according to claim 8, characterized in that: After adjusting the pH and heating the protein extract to denature, add a complex enzyme system of neutral protease and flavor protease with a mass ratio of 1:1~2:1 and an enzyme amount of 2%~4%, and carry out enzymatic hydrolysis at 50-55°C and pH 7.0-8.0 for 4-6 hours; after inactivating the enzyme, filter through a filter cloth for rough filtration and a microporous membrane for fine filtration in sequence to obtain the crude peanut peptide filtrate.

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