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CN-122011111-A - Bioactive peptide and preparation method and application thereof

CN122011111ACN 122011111 ACN122011111 ACN 122011111ACN-122011111-A

Abstract

The invention discloses a bioactive peptide, a preparation method and application thereof. The invention adopts water bath to treat ginseng aqueous solution, matches with an enzymolysis system of alkaline protease, and simultaneously adopts pediococcus pentosaceus and lactobacillus fermentum as mixed bacteria fermentation strains to obtain the preparation method of the high-yield ginseng peptide. Meanwhile, by combining histology and molecular docking technology, LDALDEH (LH-7) 1 ginseng-derived bioactive peptides are obtained through identification. 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.

Inventors

  • YANG ZIQING
  • YANG KUN
  • YANG JING

Assignees

  • 通化吉通药业有限公司

Dates

Publication Date
20260512
Application Date
20260415

Claims (10)

  1. 1. A bioactive peptide is characterized in that the amino acid sequence of the bioactive peptide is LDALDEH.
  2. 2. A preparation method of ginseng peptide is characterized by comprising the steps of pretreating ginseng aqueous solution by water bath, extracting ginseng protein by an acid heating method, carrying out enzymolysis by alkaline protease, fermenting by mixing Pediococcus pentosaceus and Lactobacillus fermentum, centrifuging and freeze-drying to obtain the ginseng peptide, wherein the ginseng peptide comprises the bioactive peptide of claim 1.
  3. 3. The method for preparing ginseng peptide according to claim 2, comprising the specific steps of: 1) Heat treatment of Ginseng radix solution, mixing Ginseng radix powder with deionized water at a ratio of 1:10, stirring for 5 min to dissolve protein completely, cooling in water bath at 60deg.C 30: 30min, centrifuging at 5000: 5000 rpm for 10: 10min, and collecting supernatant; 2) Extracting ginseng protein, namely adjusting the pH of the supernatant obtained in the step 1) to 4.0-4.5,80 ℃ by using 1M HCl, heating in a water bath to 10 min, cooling, centrifuging to 10 min at 8000 rpm, collecting precipitate and freeze-drying; 3) Enzymolysis, namely regulating the pH value to 8.0-8.5 by using 1M NaOH, adding alkaline protease according to the mass fraction of 1.0%, magnetically stirring and hydrolyzing for 3 hours at 50 ℃, heating for 10 minutes at 95 ℃ to inactivate enzymes, and regulating the pH value to 3.5-4.5 by using 1M HCl after cooling to obtain enzymolysis liquid; 4) Mixing bacteria, namely quickly thawing lactobacillus fermentum and pediococcus pentosaceus at 37 ℃, inoculating the lactobacillus fermentum and pediococcus pentosaceus to MRS broth with an inoculum size of 1.0%, aerobically incubating the broth at 37 ℃ for 12h to activate strains, sterilizing the enzymolysis liquid in the step 3) at 121 ℃ for 20min, taking 10mL of each of the two activated strain bacterial liquids, centrifuging at 4500 rpm for 10min, discarding the supernatant, re-suspending the supernatant by 10mL normal saline, adjusting the optical density of OD 600 to 1, inoculating the enzymolysis liquid with an inoculum size of 1.0%, fermenting at 37 ℃ for 48 h, centrifuging at 7000 rpm for 10min, collecting the supernatant and freeze-drying to obtain the ginseng peptide containing the bioactive peptide according to claim 1.
  4. 4. The method for preparing ginseng peptide according to claim 2, wherein the alkaline protease is enzymatically hydrolyzed at ph8.0-8.5, 50 ℃ and 3 h.
  5. 5. The method for preparing ginseng peptide according to claim 2, wherein the mixed bacteria are Pediococcus pentosaceus and Lactobacillus fermentum, the inoculation amount is 1.0%, the fermentation condition is 37 ℃ and the fermentation is 48 h.
  6. 6. The method for preparing ginseng peptide according to claim 2, wherein the bioactive peptide according to claim 1 is obtained by combining a peptide group analysis, a molecular docking technique and a virtual screening technique after the supernatant obtained by mixed fermentation is freeze-dried, and identifying the structure of the bioactive peptide having a neuroprotective function.
  7. 7. The method for preparing ginseng peptide according to claim 6, wherein the peptide group analysis uses Easy-nLC 1200 nm liquid chromatography system in combination with Orbitrap Exploris 480,480 mass spectrometer to identify the structure and sequence of the active peptide with the highest peptide content.
  8. 8. The method of claim 6, wherein the molecular docking uses Discovery Studio software to predict the potential neuroprotective active peptide, and the docking acceptors are Keap1 crystal structure and P38 crystal structure, keap1 crystal structure PDB ID:2FLU, P38 crystal structure PDB ID:1A9U.
  9. 9. The method of claim 6, wherein the bioactive peptides obtained by screening are subjected to activity verification by using a nerve cell model, the nerve cell model is constructed by using H 2 O 2 to induce PC12 cell oxidative damage, and the verification method is used for quantitatively detecting the survival rate of nerve cells by using a CCK-8 method.
  10. 10. Use of a bioactive peptide according to claim 1 in the preparation of a neuroprotective formulation.

Description

Bioactive peptide and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicines and functional foods, and particularly relates to a bioactive peptide, a preparation method and application thereof. Background Ginseng (Panax ginseng c.a. Meyer) is a perennial plant, and is also a valuable herb belonging to the agavaceae family. The main components of the composition comprise ginsenoside, polysaccharide, amino acid, volatile oil and polyacetylene. Historically, ginseng was known as the "king of herbal medicine" and is widely used for the treatment of various diseases. Notably, ginseng is a natural plant that is rich in various nutrients and bioactive compounds such as polysaccharides, peptides, flavonoids, and minerals. These ingredients help to increase the overall nutritional content of the ginseng and may have additional health promoting effects. Exploring the nutritional aspects of ginseng and better understanding its potential contribution to human health from a nutritional perspective may provide valuable insight into its overall health benefits. The nutritional value of bioactive peptides derived from ginseng proteins is of concern. The molecular weight of the bioactive peptide is between that of the protein and the amino acid. Depending on their size and structural characteristics, bioactive peptides exhibit a wide range of pharmacological functions including antioxidant, anti-inflammatory, antihypertensive, hypoglycemic and sedative activities. Peptides are receiving great attention because of their ability to bridge the gap between small molecules and protein drugs, thus fusing the inherent advantages of both entities. Compared with dietary proteins and amino acids, the bioactive peptide from food sources has the remarkable characteristics of high safety, quick absorption, no toxicity, strong bioactivity and the like. Based on the background, the ginseng is pretreated by water bath, protein is extracted, and then the ginseng is subjected to enzymolysis auxiliary fermentation treatment by alkaline protease, so that the extraction efficiency of ginseng peptide is improved. Based on the peptide group technique, the new ginseng peptide sequence is identified, and a new ginseng peptide is found to have the nerve protection function through molecular docking and cell experiment. These results provide a new idea for the research on the high-value utilization of traditional fermented ginseng. Disclosure of Invention In view of the above, the invention provides a bioactive peptide, a preparation method and application thereof. In order to achieve the above purpose, the present invention adopts the following technical scheme: In a first aspect, the invention provides a bioactive peptide, which has the amino acid sequence LDALDEH. In a second aspect, the invention provides a preparation method of ginseng peptide, which comprises the steps of pretreating ginseng aqueous solution by water bath, extracting ginseng protein by an acid heating method, carrying out enzymolysis by alkaline protease and fermentation by pediococcus pentosaceus-lactobacillus fermentum mixed bacteria, and carrying out centrifugal freeze-drying to obtain the ginseng peptide, wherein the ginseng peptide comprises the bioactive peptide of claim 1. Further, the preparation method of the ginseng peptide comprises the following specific steps: 1) Heat treatment of Ginseng radix solution, mixing Ginseng radix powder with deionized water at a ratio of 1:10, stirring for 5 min to dissolve protein completely, cooling in water bath at 60deg.C 30: 30min, centrifuging at 5000: 5000 rpm for 10: 10min, and collecting supernatant; 2) Extracting ginseng protein, namely adjusting the pH of the supernatant obtained in the step 1) to 4.0-4.5,80 ℃ by using 1M HCl, heating in a water bath to 10 min, cooling, centrifuging to 10 min at 8000 rpm, collecting precipitate and freeze-drying; 3) Enzymolysis, namely adjusting the pH value to 8.0-8.5 by using 1M NaOH, adding alkaline protease according to the proportion of 1.0% (w/w), magnetically stirring and hydrolyzing for 3 hours at 50 ℃, heating for 10 minutes at 95 ℃ to inactivate the enzyme, and adjusting the pH value to 3.5-4.5 by using 1M HCl after cooling to obtain enzymolysis liquid; 4) Mixing bacteria, namely quickly thawing lactobacillus fermentum and pediococcus pentosaceus at 37 ℃, inoculating the lactobacillus fermentum and pediococcus pentosaceus to MRS broth with an inoculum size of 1.0%, aerobically incubating the broth at 37 ℃ for 12h to activate strains, sterilizing the enzymolysis liquid in the step 3) at 121 ℃ for 20min, taking 10mL of each of the two activated strain bacterial liquids, centrifuging at 4500 rpm for 10min, discarding the supernatant, re-suspending the supernatant by 10mL normal saline, adjusting the optical density of OD 600 to 1, inoculating the enzymolysis liquid with an inoculum size of 1.0%, fermenting at 37 ℃