Bioactive peptide as well as preparation method and application thereof

Abstract

The invention discloses a bioactive peptide as well as a preparation method and application thereof. According to the preparation method, the ginseng aqueous solution is treated in a water bath, an enzymolysis system of alkaline protease is matched, meanwhile, pediococcus pentosaceus and lactobacillus fermentum are used as mixed fermentation strains, the high-yield ginseng peptide preparation method is obtained, the heat combined enzymolysis assisted mixed fermentation process is different from the existing similar technology, a foundation is laid for high-value utilization of ginseng, and the method is suitable for industrial production. And a new thought is provided for research on preparation of functional peptides of medicinal and edible raw materials. Meanwhile, the LDALDEH (LH-7) one ginseng-derived bioactive peptide is identified by combining omics and a molecular docking technology. Cell experiments prove that the bioactive peptide can effectively play a role in neuroprotection and can be used as a neuroprotection preparation for drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and functional food technology, and particularly relates to a bioactive peptide, its preparation method and application. Background Technology

[0002] Ginseng (Panax ginseng CA Meyer) is a perennial plant and a valuable medicinal herb belonging to the Asparagaceae family. Its main components include ginsenosides, polysaccharides, amino acids, volatile oils, and polyacetylene. Historically, ginseng has been hailed as the "King of Herbs" and widely used to treat various ailments. Notably, ginseng is a natural plant rich in a variety of nutrients and bioactive compounds, such as polysaccharides, peptides, flavonoids, and minerals. These components contribute to enhancing the overall nutritional value of ginseng and may offer additional health-promoting effects. Exploring the nutritional aspects of ginseng and gaining a better understanding of its potential contributions to human health from a nutritional perspective can provide valuable insights into its overall health benefits.

[0003] The nutritional value of bioactive peptides derived from ginseng protein is noteworthy. The molecular weight of bioactive peptides lies between that of proteins and amino acids. Based on their size and structural characteristics, bioactive peptides exhibit a wide range of pharmacological functions, including antioxidant, anti-inflammatory, blood pressure-lowering, blood sugar-lowering, and sedative activities. Peptides have attracted widespread attention because they can bridge the gap between small molecule and protein drugs, thus combining the inherent advantages of both entities. Compared with dietary proteins and amino acids, food-derived bioactive peptides have significant advantages such as high safety, rapid absorption, non-toxicity, and strong biological activity.

[0004] Against this backdrop, this invention employs a water bath pretreatment of ginseng to extract its protein, followed by alkaline protease-assisted fermentation to improve the extraction efficiency of ginseng peptides. Based on this, novel ginseng peptide sequences were identified using peptidomics technology. Through molecular docking and cell experiments, a novel ginseng peptide with neuroprotective function was discovered. These results provide new insights into the high-value utilization of traditionally fermented ginseng. Summary of the Invention

[0005] In view of this, the present invention provides a bioactive peptide, its preparation method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a bioactive peptide, wherein the amino acid sequence of the bioactive peptide is: LDALDEH.

[0008] Secondly, the present invention provides a method for preparing ginseng peptides, comprising the following steps: pretreating ginseng aqueous solution with a water bath, extracting ginseng protein by acid-heat method, then hydrolyzing with alkaline protease, fermenting with mixed bacteria of Pediococcus pentosus and Lactobacillus fermentum, and obtaining ginseng peptides by centrifugation and freeze-drying, wherein the ginseng peptides contain the bioactive peptides described in claim 1.

[0009] Furthermore, the specific steps of the above-mentioned method for preparing ginseng peptides are as follows:

[0010] 1) Heat treatment of ginseng solution: Mix ginseng powder with deionized water at a ratio of 1:10, stir for 5 min to fully dissolve the protein, incubate in a 60 ℃ water bath for 30 min, cool, centrifuge at 5000 rpm for 10 min, and collect the supernatant.

[0011] 2) Extraction of ginseng protein: Adjust the pH of the supernatant obtained in step 1) to 4.0-4.5 with 1 M HCl, heat in an 80 ℃ water bath for 10 min, cool and centrifuge at 8000 rpm for 10 min, collect the precipitate and freeze dry;

[0012] 3) Enzymatic hydrolysis: Adjust the pH to 8.0-8.5 with 1 M NaOH, then add alkaline protease at a ratio of 1.0% (w / w), stir magnetically at 50℃ for 3 h, heat at 95℃ for 10 min to inactivate the enzyme, cool, and adjust the pH to 3.5-4.5 with 1 M HCl to obtain the enzymatic hydrolysate;

[0013] 4) Mixed-culture fermentation: Lactobacillus fermentum and Pediococcus pentosaceus were rapidly thawed at 37 °C and inoculated into MRS broth at a 1.0% inoculum. The cultures were activated by aerobic incubation at 37 °C for 12 h. The enzymatic hydrolysate from step 3) was sterilized at 121 °C for 20 min. 10 mL of each of the activated cultures was centrifuged at 4500 rpm for 10 min, the supernatant was discarded, and the cultures were resuspended in 10 mL of physiological saline. The OD was adjusted. 600 The optical density was adjusted to 1, and the enzyme hydrolysate was inoculated at a rate of 1.0%. Fermentation was carried out at 37 °C for 48 h, followed by centrifugation at 7000 rpm for 10 min. The supernatant was collected and freeze-dried to obtain ginseng peptides containing the bioactive peptides described in claim 1.

[0014] Furthermore, the alkaline protease hydrolysis conditions are pH 8.0-8.5, 50 °C, and hydrolysis for 3 h.

[0015] Furthermore, the microorganisms used in the mixed fermentation are Pediococcus pentosaceus and Lactobacillus fermentum, with an inoculum amount of 1.0% for both, and the fermentation conditions are 37 °C and 48 h.

[0016] Furthermore, after freeze-drying the supernatant from the mixed-culture fermentation, the structure of the active peptide with neuroprotective function was identified by combining peptidomics analysis, molecular docking technology and virtual screening technology, and the bioactive peptide described in claim 1 was obtained.

[0017] Furthermore, the peptidomics analysis was performed using an Easy-nLC 1200 nanometer liquid chromatography system coupled with an OrbitrapExploris 480 mass spectrometer to identify the structure and sequence of the most abundant active peptide.

[0018] Furthermore, the molecular docking was performed using Discovery Studio software to predict potentially neuroprotective active peptides. The docking receptors were the Keap1 crystal structure and the P38 crystal structure, with the Keap1 crystal structure PDB ID being 2FLU and the P38 crystal structure PDB ID being 1A9U.

[0019] Furthermore, the bioactive peptides obtained from screening were validated using a neural cell model. The neural cell model was constructed by inducing oxidative damage in PC12 cells using H2O2, and the validation method used was the CCK-8 assay to quantitatively detect the survival rate of neural cells.

[0020] Thirdly, the present invention provides the application of the above-mentioned bioactive peptides in the preparation of neuroprotective agents.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. This invention utilizes a combined process of water bath pretreatment, alkaline protease hydrolysis, and mixed-culture fermentation to effectively increase the peptide content in ginseng. Experimental data shows that fermentation increases the peptide content of ginseng by 1.8 times, providing a feasible solution for efficient industrial-scale preparation.

[0023] 2. The ginseng peptides screened in this invention exhibit strong binding affinity to Keap1 and P38 receptors and excellent docking performance. Cell experiments have confirmed that they can improve H2O2-induced PC12 cell damage and significantly increase cell survival rate at a concentration of 50 μM, providing novel active ingredients for the development of products related to nerve injury. Attached Figure Description

[0024] Figure 1 This is the total ion chromatogram.

[0025] Figure 2 This is a diagram showing the docking of LH-7 with the Keap1 receptor.

[0026] Figure 3 This is a diagram showing the docking of LH-7 with the P38 receptor.

[0027] Figure 4The effect of LH-7 on the survival rate of PC12 cells.

[0028] Figure 5 The effect of H2O2 on the survival rate of PC12 cells.

[0029] Figure 6 The protective effect of H2O2-induced PC12 cell damage is shown in (A) as VE and (B) as LH-7. Detailed Implementation

[0030] The present invention will be further explained and described below with reference to specific embodiments.

[0031] Example 1

[0032] The method for preparing ginseng powder by heat treatment and enzymatic hydrolysis-assisted fermentation according to the present invention is carried out according to the following steps:

[0033] I. Heat Treatment of Ginseng Solution

[0034] Mix ginseng powder with deionized water at a ratio of 1:10 and stir for 5 minutes to fully dissolve the protein. Incubate in a water bath at 60°C for 30 minutes. After the ginseng solution cools, centrifuge at 5000 rpm for 10 minutes and collect the supernatant.

[0035] II. Extraction of Ginseng Protein

[0036] The pH of the ginseng solution was adjusted to 4.0-4.5 with 1 M HCl. The solution was then heated in an 80°C water bath for 10 minutes to reduce protein solubility and induce efficient precipitation through denaturation and aggregation. After cooling, the solution was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected and lyophilized.

[0037] III. Enzymatic hydrolysis of ginseng protein solution

[0038] Adjust the pH to 8.0-8.5 with 1 M NaOH, add alkaline protease (mainly subtilisin) at 1.0% (w / w) of the substrate mass, and then place the beaker in a 50℃ magnetically stirred water bath for enzymatic hydrolysis for 3 hours. After the enzymatic hydrolysis is complete, heat the beaker in a 95℃ water bath for 10 minutes to inactivate the enzyme. After the hydrolysate cools, adjust the pH to 3.5-4.5 with 1 M HCl to meet the growth requirements of Lactobacillus fermentum and Pediococcus pentosaceus, ensuring normal proliferation and metabolism of the strains.

[0039] IV. Fermentation of Ginseng Protein Solution

[0040] 1. Strain Activation: *Lactobacillus fermentum* and *Pediococcus pentosaceus*, stored at -80℃, were rapidly thawed at 37℃. After screening multiple strain combinations and inoculation amounts, the present invention utilizes a combination of these two strains. A 1.0% (v / v) inoculation volume (i.e., 1 mL of bacterial solution) was added to 100 mL of MRS broth medium and cultured at 37℃ for 12 h to complete strain activation. This strain combination showed good compatibility with the ginseng protein hydrolysis system and significantly increased the content of small molecule peptides.

[0041] 2. Treatment of ginseng solution: The pretreated ginseng solution was sterilized in an autoclave at 121℃ for 20 min.

[0042] 3. Inoculation and Fermentation: Centrifuge 10 mL of activated probiotic solution at 4500 rpm for 10 min, discard the supernatant, and dilute the sample in 10 mL of physiological saline. Then, centrifuge each probiotic solution again at 4500 rpm for 10 min, discard the supernatant, and add 10 mL of physiological saline. Subsequently, adjust the optical density at 600 nm (OD 600 nm) for each culture to 1. Inoculate ginseng culture medium with 1.0% of this solution. Fermentate at 37°C for 48 h in an incubator. Centrifuge the sample at 7000 rpm for 10 min, then collect the supernatant and determine the peptide content.

[0043] Example 2: Determination of the content of active ingredients in ginseng solution

[0044] The peptide content of the sample was determined according to Appendix B (method for determination of peptide content) of GB / T 22492-2008 Soybean Peptide Powder.

[0045] Table 1. Ginseng peptide content before and after fermentation

[0046]

[0047] After fermentation, the peptide content increased to 1.256±0.019 g / 100g, an increase of approximately 85.0% compared to before fermentation. This improved composition is attributed to the synergistic effect of multiple processes: heat treatment in a water bath disrupts the cell walls and intracellular structure of ginseng, reducing mass transfer resistance to the dissolution of active ingredients and facilitating full contact between enzymes, microorganisms, and substrates; alkaline protease hydrolyzes large protein molecules to generate peptides; and mixed fermentation with Pediococcus pentosaceus and Lactobacillus fermentum, aided by metabolic enzymes, significantly increases peptide content through a synergistic effect.

[0048] Example 3: Structural Identification of Ginseng Peptide Sequence

[0049] Peptidomics analysis was performed using an Easy-nLC 1200 nanometer liquid chromatography system coupled with an Orbitrap Exploris 480 mass spectrometer. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Samples were separated on a reversed-phase analytical column (PepMap C18, 75 μm × 25 cm) using a linear gradient of solvent B from 5% to 50% (v / v), at a column temperature of 55 °C and a flow rate of 300 nL / min. Mass spectrometry analysis was performed on the Orbitrap instrument in data-dependent acquisition (DDA) mode, which automatically switches between full-scan MS and MS / MS scans. Full-scan MS spectra were acquired at a resolution of 12,000 in the range of 160–1500 m / z. MS / MS scans used a normalized collision energy of 27 eV, a maximum injection time of 50 ms, and an automatic gain control (AGC) target of 5 × 10⁻⁶. 5 The resolution is 17,500.

[0050] Raw LC-MS / MS data were processed in PEAKS Studio for de novo peptide sequence analysis, followed by database searching. Candidate sequences were first generated directly from the MS / MS spectra using a de novo algorithm, and then matched against a custom ginseng protein database using PEAKS DB. The total ion chromatogram of ginseng peptides is shown below. Figure 1 As shown.

[0051] Example 4 Screening of bioactive peptides

[0052] Using Discovery Studio software, 80 highly active peptides were molecularly docked with Keap1 crystal structure (PDB ID: 2FLU) and P38 crystal structure (PDB ID: 1A9U), respectively. Before docking, the 2D structure of the peptides was converted into 3D structure by minimizing energy, and neuroprotective active peptides with strong binding ability to Keap1 and P38 were screened.

[0053] Docking results are expressed as docking scores (-interaction docking energies). Higher -interaction docking energies indicate stronger interactions between the active oligopeptide and Keap1 and P38, suggesting a higher likelihood of neuroprotective function. To determine the most stable molecular docking structure with the lowest docking energy, PyMOL was used to visualize the molecular docking results. Docking energy results are shown in Tables 2 and 3.

[0054] Table 2. Docking energy of fermented ginseng peptides with Keap1 receptor

[0055]

[0056] Table 3. Docking energy of fermented ginseng peptides with P38 receptor

[0057]

[0058] Potential neuroprotective peptides were screened by evaluating their docking energy scores. The peptide with the lowest score (LH-7) was selected for visualization. Docking diagrams of LH-7 with the Keap1 receptor and with the P38 receptor are shown below. Figure 2 and Figure 3 In this study, docking models of the peptides all showed the presence of hydrogen bonding and hydrophobic interactions. Peptide LH-7 formed nine hydrogen bonds with the Keap1 active site at Val369, Val561, Leu557, Val606, Ile559, Gly564, Gln563, His516, and Thr560, with an average bond length of 2.2 Å. In addition, LH-7 also interacted with four other amino acids via hydrophobic interactions and one via electrostatic interaction.

[0059] The peptide LH-7 forms three hydrogen bonds with Met109, Asp168, and Asn114 at the P38 active site, with an average bond length of 2.2 Å. In addition, LH-7 also interacts electrostatically with five other amino acids.

[0060] The molecular docking results showed that LH-7 could effectively bind to the active sites of both proteins, thereby exhibiting neuroprotective activity.

[0061] The selected peptide LH-7 was sequenced, and its amino acid sequence was LDALDEH. LH-7 was then synthesized by Shanghai Chupeptide Biotechnology Co., Ltd. (with a purity of over 98%).

[0062] Example 5: Activity Verification of Bioactive Peptides

[0063] I. Cell Culture Methods

[0064] PC12 cells were placed in DMEM complete medium (1% penicillin / streptomycin, 10% fetal bovine serum and 89% DMEM high glucose medium) and cultured in a 37°C, 5% CO2 incubator. After a certain period of culture, cell growth and changes in the color of the culture medium were observed using an optical microscope. Cell passage was required when the cell growth area reached 80%.

[0065] II. Effects of bioactive peptide LH-7 on PC12 cell survival

[0066] PC12 cells in logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5Cells were seeded at a density of 200 μL / well in sterile 96-well plates and cultured for 24 h. After confirming good cell adhesion and growth under a microscope, the culture medium in the wells was discarded, and complete culture medium containing a series of concentrations (12.5, 25, 50, 100, 200 μM) of LH-7 was added to each well. The plates were cultured for another 24 h, and the culture medium was discarded. 10 μL of CCK−8 working solution was added to each well, and the plates were cultured in the dark for 1 h. The absorbance of the experimental wells was measured at 450 nm. Following the kit instructions, the relative cell viability of the experimental groups was calculated as follows, with the control group (no drug treatment group) having a viability of 100%:

[0067] = ×100%

[0068] The effect of LH-7 on PC12 cell survival was as follows: Figure 4 As shown.

[0069] III. Establishment of a H2O2-induced PC12 cell damage model

[0070] PC12 cells in logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / well in sterile 96-well plates and cultured for 24 h. The culture medium was discarded, and complete culture medium containing H2O2 at concentrations of 100, 200, 300, 400, and 500 μM was added to each well. After 24 h of culture, the medium was discarded, and 10 μL of CCK-8 working solution was added to each well. The plates were then incubated in the dark for 1 h. The absorbance of each well was measured at 450 nm, and cell viability was calculated using the formula. The H2O2 concentration with a lethality close to 50% was selected as the concentration for establishing the oxidative damage model.

[0071] IV. Protective effect of bioactive peptide LH-7 on H2O2-induced cell damage model

[0072] PC12 cells in logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5 Cells were inoculated at 100 μL / mL into sterile 96-well plates and cultured for 24 h. The culture medium in the wells was discarded, and complete culture medium containing a series of concentrations (12.5, 25, 50 μM) of LH-7 was added respectively. After culturing for 24 h, the culture medium was discarded, and H2O2 (400 μM) was added and cultured for 24 h. The relative cell viability was calculated according to formula (1). The following groups were formed in this experiment: the experimental group with only complete culture medium was the control group, the experimental group treated with H2O2 (400 μM) was the model group, and the experimental groups pretreated with different concentrations of LH-7 and then treated with H2O2 (400 μM) were the drug treatment groups.

[0073] This study established an H2O2-induced oxidative stress model using PC12 cells to evaluate the protective effect of bioactive peptides. Treatment with 400 μM H2O2 for 4 hours was chosen as the optimal condition for inducing oxidative stress in PC12 cells, as this point corresponds to approximately 50% cell viability. Figure 5 The effects of LH-7 (12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM) on cell viability were investigated, as shown in Figure 4. When the peptide concentration was below 50 μM, cell viability remained stable at approximately 100%; however, when the peptide concentration was above 50 μM, cell viability decreased significantly, indicating that the sample exhibited some cytotoxicity. Therefore, LH-7 concentrations below 50 μM do not exhibit significant cytotoxicity to PC12 cells; however, concentrations above 50 μM do have a certain inhibitory effect on PC12 cell growth. To ensure that subsequent experiments were not affected by cytotoxicity, LH-7 concentrations of 12.5, 25, and 50 μM were selected for further studies.

[0074] Vitamin E was selected as a positive control due to its excellent antioxidant activity. The protective effects of vitamin E and the bioactive peptide LH-7 against H2O2-damaged PC12 cells were as follows: Figure 6 As shown: at a concentration of 12.5 μM, the survival rate of VE was 57.49 ± 12.73%, and the survival rate of LH-7 was 57.87 ± 8.8%; at 25 μM, the survival rate of VE reached 67.47 ± 4.877%, and that of LH-7 increased to 61.49 ± 9.094%; at 50 μM, the survival rate of VE was 66.97 ± 9.35%, and that of LH-7 further increased to 79.99 ± 11.05%. It is evident that the protective effect of LH-7 increases with increasing concentration, and at all concentrations, the protective effect of LH-7 is superior to that of the positive control VE, demonstrating good cell protective activity.

[0075] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited. Any equivalent substitutions or changes made by those skilled in the art within the scope of the technology disclosed in the present invention should be covered within the scope of protection of the present invention.

Claims

1. A bioactive peptide, characterized in that, The amino acid sequence of the bioactive peptide is: LDALDEH.

2. A method for preparing ginseng peptides, characterized in that, The steps are as follows: ginseng aqueous solution is pretreated by water bath, ginseng protein is extracted by acid-heat method, then enzymatically hydrolyzed by alkaline protease, fermented by mixed culture of Pediococcus pentosus and Lactobacillus fermentum, and then freeze-dried by centrifugation to obtain ginseng peptides, wherein the ginseng peptides contain the bioactive peptides described in claim 1.

3. The method for preparing ginseng peptides as described in claim 2, characterized in that, The specific steps are as follows: 1) Heat treatment of ginseng solution: Mix ginseng powder with deionized water at a ratio of 1:10, stir for 5 min to fully dissolve the protein, incubate in a 60 ℃ water bath for 30 min, cool, centrifuge at 5000 rpm for 10 min, and collect the supernatant. 2) Extraction of ginseng protein: Adjust the pH of the supernatant obtained in step 1) to 4.0-4.5 with 1 M HCl, heat in an 80 ℃ water bath for 10 min, cool and centrifuge at 8000 rpm for 10 min, collect the precipitate and freeze dry; 3) Enzymatic hydrolysis: Adjust the pH to 8.0-8.5 with 1 M NaOH, then add alkaline protease at a mass fraction of 1.0%, stir magnetically at 50 ℃ for 3 h, heat at 95 ℃ for 10 min to inactivate the enzyme, cool, and adjust the pH to 3.5-4.5 with 1 M HCl to obtain the enzymatic hydrolysate; 4) Mixed-culture fermentation: Lactobacillus fermentum and Pediococcus pentosaceus were rapidly thawed at 37 °C and inoculated into MRS broth at a 1.0% inoculum. The cultures were activated by aerobic incubation at 37 °C for 12 h. The enzymatic hydrolysate from step 3) was sterilized at 121 °C for 20 min. 10 mL of each of the activated cultures was centrifuged at 4500 rpm for 10 min, the supernatant was discarded, and the cultures were resuspended in 10 mL of physiological saline. OD was adjusted. 600 The optical density was adjusted to 1, and the enzyme hydrolysate was inoculated at a rate of 1.0%. Fermentation was carried out at 37 °C for 48 h, followed by centrifugation at 7000 rpm for 10 min. The supernatant was collected and freeze-dried to obtain ginseng peptides containing the bioactive peptides described in claim 1.

4. The method for preparing ginseng peptides as described in claim 2, characterized in that, The alkaline protease hydrolysis conditions are pH 8.0-8.5, 50 ℃, and hydrolysis for 3 h.

5. The method for preparing ginseng peptides as described in claim 2, characterized in that, The mixed-culture fermentation strains were Pediococcus pentosaceus and Lactobacillus fermentum, with an inoculum amount of 1.0% for both, and the fermentation conditions were 37 ℃ and 48 h.

6. The method for preparing ginseng peptides as described in claim 2, characterized in that, After freeze-drying the supernatant from the mixed-culture fermentation, the structure of the active peptide with neuroprotective function was identified by combining peptidomics analysis, molecular docking technology and virtual screening technology, and the bioactive peptide described in claim 1 was obtained.

7. The method for preparing ginseng peptides as described in claim 6, characterized in that, The peptidomics analysis was performed using an Easy-nLC1200 nano-liquid chromatography system coupled with an Orbitrap Exploris 480 mass spectrometer to identify the structure and sequence of the most abundant bioactive peptides.

8. The method for preparing ginseng peptides as described in claim 6, characterized in that, The molecular docking was performed using Discovery Studio software to predict potentially neuroprotective active peptides. The docking receptors were the Keap1 crystal structure and the P38 crystal structure. The PDB ID of the Keap1 crystal structure was 2FLU, and the PDB ID of the P38 crystal structure was 1A9U.

9. The method for preparing ginseng peptides as described in claim 6, characterized in that, The bioactive peptides obtained from screening were validated using a neural cell model. The neural cell model was constructed by inducing oxidative damage in PC12 cells using H2O2, and the validation method used was the CCK-8 assay to quantitatively detect the survival rate of neural cells.

10. The use of the bioactive peptide as described in claim 1 in the preparation of neuroprotective agents.

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