A microbially-derived active peptide, and methods of making and using the same

Abstract

The application discloses a microorganism-derived active peptide and a preparation method and application thereof. The active peptide has small molecular weight, high activity, simple preparation process, and can be used for health care and treatment of diseases related to aging, and has wide application prospects in the fields of food, health care products, medicine and biology.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically a microbial bioactive peptide, its preparation method, and its application. Background Technology

[0002] Bioactive peptides are beneficial molecular polymers that lie between amino acids and proteins. They can be composed of two amino acids or dozens of amino acids linked by peptide bonds, and are formed from the 20 natural amino acids in proteins in different compositions and arrangements. Bioactive peptides are multifunctional compounds derived from proteins, possessing various metabolic and physiological regulatory functions, playing a crucial role in growth, development, and metabolism. Bioactive peptides are among the most important bioactive substances in the human body, and their secretion in organisms contributes to the human life cycle from infancy to old age. The secretion of bioactive peptides varies greatly across different age groups. Sufficient secretion during youth and middle age makes the body less susceptible to disease; secretion significantly decreases in middle age, leading to a sub-healthy state; and severely insufficient secretion in old age causes peptide imbalance, resulting in aging symptoms and inducing disease, ultimately leading to death. The use of bioactive peptides has the potential to break this life cycle, slowing down aging and restoring youth. Compared to biological macromolecules and free amino acids, bioactive peptides are digested quickly, absorbed well, and have high biological value and nutritional value. Currently, research on bioactive peptides is developing rapidly, and scientists worldwide are paying close attention to them. Some bioactive peptides have already been industrially produced as functional foods, while others are being used as drugs. The research and development of bioactive peptides, as an emerging field of biotechnology internationally, has enormous market potential.

[0003] Stem cells are a type of seed cell with the potential for self-replication and multi-directional differentiation. Scientists use stem cell therapy to transplant their own or another person's stem cells into a patient's body. Under specific stimulation, the transplanted stem cells differentiate into the desired functional cells, replacing diseased cells and restoring damaged tissue to normal. Stem cell therapy is a leading, groundbreaking, and disruptive biotechnology, representing the third type of medical treatment after drug therapy and surgery, and signifying a new medical revolution.

[0004] The human body possesses sufficient stem cells to maintain normal bodily functions; however, the differentiation capacity of stem cells decreases significantly with age. Therefore, finding safe, effective, and easily operable substances that can induce stem cell differentiation is of great significance both from the perspective of human health development and from the perspective of disease research and treatment. Summary of the Invention

[0005] This invention addresses the current status and future prospects by isolating and purifying an active peptide that can induce stem cell differentiation from microbial secretions. The active peptide has a small molecular weight, high activity, and a simple preparation process. It can be used for the health care and treatment of aging-related diseases and has broad application prospects in the fields of food, health products, and pharmaceuticals.

[0006] The technical solution adopted to achieve the above objectives is:

[0007] A bioactive peptide derived from microorganisms, wherein the amino acid sequence of the bioactive peptide has the general formula XPY; the amino acid sequence of P is shown in Seq-1; X is absent or is Lys-Tyr; and Y is absent or is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0008] Furthermore, the amino acid sequence of the active peptide is as shown in Seq-1, Seq-2, Seq-3, or Seq-4.

[0009] Furthermore, the active peptide can promote stem cell differentiation.

[0010] Furthermore, the amino acid sequence of the active peptide also includes any adjustment or modification to the Seq-1, Seq-2, Seq-3, or Seq-4 active peptide sequence as the core, and the application of the adjusted or modified sequence still has the function of promoting stem cell differentiation.

[0011] The source of the microorganisms includes any microorganism.

[0012] Furthermore, the source of the microorganism may be one or more of the following: live strains, fire-extinguished strains, dried strains, strain metabolites, strain lysates, or strain post-biotics.

[0013] Furthermore, the active peptide is extracted and purified from the microorganism, and then a specific sequence is artificially synthesized. Alternatively, the active peptide can be obtained by gene expression in the host cell.

[0014] Furthermore, the preparation methods of the active peptides, in addition to the artificial synthesis of active peptides, include, but are not limited to, direct extraction by separation and extraction by hydrolysis with proteases.

[0015] The present invention also provides a nucleic acid molecule that can encode the active peptide or is reverse complementary to the nucleotide sequence encoding the active peptide.

[0016] The present invention also provides a recombinant vector containing the nucleotide sequence of the nucleic acid molecule.

[0017] The present invention also provides a host cell whose genome is doped with the aforementioned nucleic acid molecules.

[0018] The active peptides, nucleic acid molecules, recombinant vectors, or host cells described in this invention can be used to prepare functional foods / drugs / cosmetics / health products that induce stem cell differentiation.

[0019] Furthermore, the dosage form of the article includes pharmaceutically acceptable carriers, including but not limited to one or more of fillers, wetting agents, disintegrants, binders, or lubricants.

[0020] Furthermore, the state of the article includes one or more of the following: emulsion, oil, gel, paste, powder, talc, lotion, custard, foam, cream, gel, ointment, suspension, aerosol, vapor, cryo-liquid, spray, or mist.

[0021] The active peptides, nucleic acid molecules, recombinant vectors, or host cells described in this invention are used in the preparation of microbial preparations / stem cell therapy products / fermented products / beauty and health products / functional products / pharmaceuticals.

[0022] Furthermore, microbial preparations include any other microorganisms as well as acceptable excipients or adjuvants.

[0023] Furthermore, the excipients include one or more of the following: diluent, preservative, buffer, disintegrant, antioxidant, suspending agent, colorant, and excipient.

[0024] Furthermore, the fermented product is prepared by culturing fermenting bacteria and collecting the fermentation product; the fermentation product includes any one of the active peptides or compositions mentioned above.

[0025] Furthermore, the fermented products include one or more of fermented milk, milk beverages, fermented fruit and vegetable beverages, synbiotic preparations, or foods containing prebiotics.

[0026] The above applications are applied to mammals, including but not limited to humans.

[0027] The advantages and positive effects of this invention are as follows:

[0028] The active peptides described in this invention are derived from the gastrointestinal microorganisms of mammals such as humans and mice. Compared to traditional stem cell therapies, they are simpler to operate, have no ethical issues, and have no toxic side effects, thus having a positive significance for promoting the development and industrial production of stem cell therapy products. Cell experiments show that this strain or its secretions can significantly induce stem cell differentiation. Animal experiments show that this strain or its secretions can significantly enhance wound healing ability. It can be used to prepare functional formulations, foods, and drugs that induce stem cell differentiation, and has broad application prospects. Attached Figure Description

[0029] Figure 1This is a high-performance liquid chromatogram of the active peptide in a preferred embodiment of the present invention;

[0030] Figure 2 This is a mass spectrometry image of the active peptide in a preferred embodiment of the present invention;

[0031] Figure 3 The effect of the active peptides in the preferred embodiment of the present invention on intestinal stem cells;

[0032] Figure 4 The effect of the active peptide in the preferred embodiment of the present invention on pancreatic stem cells;

[0033] Figure 5 The effect of the active peptides in the preferred embodiment of the present invention on adipose-derived mesenchymal stem cells;

[0034] Figure 6 The effect of the active peptide in the preferred embodiment of the present invention on the wound healing ability of mice. Detailed Implementation

[0035] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0036] This invention discloses a microbial-derived bioactive peptide, its preparation method, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the specific embodiments:

[0038] Example 1

[0039] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide has the general formula XPY; wherein the amino acid sequence of P is shown in Seq-1, wherein X is Lys-Tyr, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0040] Example 2

[0041] The method for preparing a microbial-derived small molecule bioactive peptide as described in Example 1 includes the following three steps.

[0042] S1. Isolation and Purification of Active Peptides: Microbial culture broth was obtained and air-dried under natural conditions. The air-dried powder was dissolved in 50% methanol-water to prepare a 0.1 g / mL solution. Separation was performed using silica gel column chromatography with a mobile phase of 50% methanol, a flow rate of 0.5 mL / min, and absorbance measured at 208 nm using the eluent. The desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase of 50% methanol, flow rate of 0.5 mL / min, detection wavelength of 208 nm. Effective chromatographic peaks were collected and the peptides were air-dried to obtain the active peptides. Figure 1 The image shows the high-performance liquid chromatogram of the active peptide.

[0043] S2, Purity and Structure Determination:

[0044] The collected active peptides were detected as a single peak by liquid chromatography, and their specific structures were determined by high performance liquid chromatography-mass spectrometry. The amino acid sequence was determined to be XPY; the amino acid sequence of P is shown in SEQ-1, X is Lys-Tyr, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu. Figure 2 This is a mass spectrometry diagram for identifying the active peptide.

[0045] S3, Artificially Synthetic Active Peptides:

[0046] The amino acid sequence determined by high performance liquid chromatography-mass spectrometry was synthesized by Suzhou Pharmaceutical Technology Co., Ltd. using a peptide solid-phase synthesis method. The general formula of the amino acid sequence is XPY. The amino acid sequence of P is shown in SEQ-1, X is Lys-Tyr, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0047] Example 3

[0048] The effect of the active peptide on stem cell differentiation capacity: Intestinal stem cells were extracted and cultured under conditions that maintained cell stemness (preventing differentiation). After cell stabilization, the intestinal stem cells were randomly divided into a control group and an experimental group. The experimental group was added to the bacterial culture medium at a ratio of 10 μg / mL and cultured in a 37℃ incubator for 48 h.

[0049] Experimental data show that stem cells in the group treated with the added active peptide underwent significant differentiation, while stem cells in the control group maintained their stemness without any change. This indicates that the active peptide can significantly enhance the differentiation capacity of stem cells. Figure 3 The diagram shows the effect of the strain on intestinal stem cells.

[0050] The same procedure was used to measure pancreatic stem cells and mesenchymal stem cells, such as... Figure 4, 5 As shown.

[0051] Example 4

[0052] The effect of the active peptide on the wound healing ability of mice: As described in Example 3, the bacterial culture medium was obtained. A skin scratch model was constructed in 24-week-old aged mice. Six aged mice of different sexes and batches had circular wounds with a diameter of 1 cm made on each side and were randomly divided into a control group and an experimental group. The experimental group was treated with the active peptide (1 mg / mL 5 μL) daily, while the control group was treated with sterile water 5 μL daily for 5 consecutive days.

[0053] Experimental data showed that, compared with the control group, the experimental group mice treated with the aforementioned active peptides exhibited significantly enhanced wound healing ability. Figure 6 The graph shows the effect of the active peptide on the wound healing ability of mice.

[0054] Example 5

[0055] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide has the general formula XPY; wherein the amino acid sequence of P is shown in Seq-2, wherein X is Lys-Tyr, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0056] Example 6

[0057] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula XPY; wherein the amino acid sequence of P is shown in Seq-3, wherein X is Lys-Tyr, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0058] Example 7

[0059] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula XPY; wherein the amino acid sequence of P is shown in Seq-4, wherein X is Lys-Tyr, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0060] Example 8

[0061] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula PY; wherein the amino acid sequence of P is shown in Seq-1, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0062] Example 9

[0063] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula XP; wherein the amino acid sequence of P is shown in Seq-1, and X is Lys-Tyr.

[0064] Example 10

[0065] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula PY; wherein the amino acid sequence of P is shown in Seq-2, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0066] Example 11

[0067] A bioactive peptide derived from microorganisms can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula XP; the amino acid sequence of P is shown in Seq-2, and X is Lys-Tyr.

[0068] Example 12

[0069] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula PY; wherein the amino acid sequence of P is shown in Seq-3, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0070] Example 13

[0071] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula XP; wherein the amino acid sequence of P is shown in Seq-3, and X is Lys-Tyr.

[0072] Example 14

[0073] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula PY; wherein the amino acid sequence of P is shown in Seq-4, and Y is Asp-Ala-Asp-Tyr-Ser-Ser-Leu.

[0074] Example 15

[0075] A microbial bioactive peptide that can induce stem cell differentiation, wherein the amino acid sequence of the bioactive peptide is of the general formula XP; wherein the amino acid sequence of P is shown in Seq-4, and X is Lys-Tyr.

[0076] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A bioactive peptide derived from microorganisms, characterized in that, The amino acid sequence of the active peptide is KYGVAPMVPIVY.

2. A method for preparing the microbial-derived bioactive peptide according to claim 1, characterized in that, Includes the following steps: (1) Cultivate peptide-producing microorganisms and collect fermentation supernatant or cell lysate; (2) The fermentation supernatant or cell lysate is separated and purified to obtain the active peptide.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule can encode the active peptide of claim 1.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, The host cell contains the recombinant vector of claim 4, or its genome is integrated with the nucleic acid molecule of claim 3.

6. The use of the active peptide of claim 1 or the nucleic acid molecule of claim 3 in the preparation of cell culture reagents for inducing stem cell differentiation.

7. The use of the active peptide of claim 1 or the nucleic acid molecule of claim 3 in the preparation of a medicament for promoting tissue repair or wound healing.

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