CN114456230A - Probiotic active peptide, active peptide composition and application of probiotic active peptide and active peptide composition in preparation of products with anti-inflammatory effect - Google Patents
Probiotic active peptide, active peptide composition and application of probiotic active peptide and active peptide composition in preparation of products with anti-inflammatory effect Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to the technical field of biological medicines, and particularly discloses a probiotic active peptide, a probiotic active peptide composition and application of the probiotic active peptide and the probiotic active peptide composition in preparation of products with anti-inflammatory effects. The probiotic active peptide has a structure shown in a formula I, a formula II or a formula III. The probiotic active peptide composition comprises any two or three of probiotic active peptides with structures shown in formula I, formula II and formula III. The probiotic active peptide and the composition thereof can relieve inflammatory injury of intestinal epithelial cells caused by glucan sodium sulfate, inhibit secretion of inflammatory cytokines induced by glucan sodium sulfate and have excellent anti-inflammatory function. Therefore, the probiotic active peptide and the composition thereof have wide application prospect in preparing foods, dietary supplements or medicines with anti-inflammatory activity.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a probiotic active peptide, a probiotic active peptide composition and application of the probiotic active peptide and the probiotic active peptide composition in preparation of products with anti-inflammatory effects.
Background
Chronic colitis refers to a kind of chronic and recurrent inflammatory edema, ulcer and bleeding diseases of the intestinal tract caused by various pathogenic factors, and belongs to inflammatory bowel diseases, including ulcerative enteritis and crohn's disease. Chronic colorectal inflammation is mostly manifested as diarrhea with intestinal symptoms such as mucopurulent bloody stool, abdominal pain, abdominal distension and the like; severe chronic colorectal inflammation also presents symptoms such as toxic megacolon and colorectal carcinogenesis and intestinal massive hemorrhage. At present, drugs aiming at chronic colorectal inflammation mainly comprise aminosalicylic acid preparations, hormones, immunosuppressants, biological preparations and the like, and the treatment schemes have better prognosis for patients with mild symptoms and long-term remission.
Probiotics are a general term for active microorganisms beneficial to a host by being planted in a human body and changing the composition of flora at a certain part of the host. Studies have shown that changes in the gut microflora are closely associated with the development of chronic inflammatory related diseases in the gut and outside the gut. The probiotics can play a role in protecting patients with inflammatory bowel diseases, and analysis of results of a plurality of clinical random control experiments also shows that the probiotics have great potential and advantage in treating inflammatory bowel diseases.
By means of the practice of new methods of oral biological treatment products, bacteriophage, bacterial function editing substrates, engineering bacteria and the like, a new and wide treatment platform is provided for probiotics in treating inflammatory bowel diseases. Short chain fatty acids secreted by probiotics play an important role in the treatment of intestinal related diseases, and short chain fatty acids (such as acetic acid, propionic acid and butyric acid) produced by intestinal microbiota can directly influence cell metabolism and can regulate intestinal immune response by regulating the function of colon Treg cells. At present, there are only reports about whether the oligopeptide produced after secretion or lysis of probiotics has anti-inflammatory activity, and the action mechanism is not clear.
Disclosure of Invention
In order to overcome at least one of the technical problems of the prior art, the present invention firstly provides a probiotic active peptide.
The detailed technical scheme of the invention is as follows:
the invention firstly provides a probiotic active peptide, which has a structure shown in a formula I, a formula II or a formula III:
the inventor surprisingly found in the research that: the probiotic active peptide with the structures shown in the formula I, the formula II and the formula III has excellent anti-inflammatory effect; especially has excellent anti-enteritis effect.
Wherein the amino acid sequence of the probiotic active peptide with the structure shown in the formula I is Pro-Met-Tyr-His-Glu-Pro, which is abbreviated as LRP-1 in the following; the amino acid sequence of the probiotic active peptide with the structure shown in the formula II is Phe-Thr-Glu-Gly-Pro, and is abbreviated as LRP-2 in the following text; the amino acid sequence of the probiotic active peptide with the structure shown in the formula III is Phe-Arg-His-Ala-Leu-Ser, and is abbreviated as LRP-3 in the following.
The probiotic active peptide with the structures shown in the formula I, the formula II and the formula III can be extracted from lactobacillus rhamnosus; the synthesis can also be carried out by conventional methods for polypeptide synthesis.
The invention also provides a probiotic active peptide composition, which comprises any two or three combinations of probiotic active peptides with structures shown in formula I, formula II and formula III.
Furthermore, the research of the inventor shows that after the probiotic active peptide with the structures shown in the formula I, the formula II and the formula III is combined, the anti-inflammatory effect, especially the anti-enteritis effect is better than that of the probiotic active peptide with any one of the structures shown in the formula I, the formula II or the formula III.
Preferably, the probiotic active peptide composition comprises probiotic active peptides with structures shown in formula I, formula II and formula III;
the probiotic active peptide has the structure shown in the formula I, the formula II and the formula III, and the molar ratio of the probiotic active peptide is 1-10: 1-10.
Further preferably, the molar ratio of the probiotic active peptide with the structures shown in formula I, formula II and formula III is 1-5: 1-5.
Most preferably, the molar ratio of the probiotic active peptides having the structures shown in formula I, formula II and formula III is 1:1: 1.
The invention also provides application of the probiotic active peptide or the probiotic active peptide composition in preparation of food, dietary supplements or medicines.
Preferably, the food, dietary supplement or pharmaceutical product is a food, dietary supplement or pharmaceutical product having an anti-inflammatory effect.
Further preferably, the food, dietary supplement or medicament is a food, dietary supplement or medicament having an anti-inflammatory effect.
Most preferably, the enteritis is caused by dextran sodium sulfate.
Preferably, the enteritis is inflammatory injury of intestinal epithelial cells.
The probiotic active peptide and the probiotic active peptide composition have excellent anti-inflammatory effect, and especially have excellent anti-enteritis effect; therefore, the compound can be used as an active ingredient for preparing foods, dietary supplements or medicaments with anti-inflammatory effect, especially anti-enteritis effect.
Has the advantages that: (1) the probiotic oligopeptide or the composition thereof with anti-inflammatory activity provided by the invention has excellent anti-inflammatory activity and has wide application prospect in food, dietary supplement or medicine. (2) The probiotic active peptide can be obtained by separating and purifying from lactobacillus rhamnosus or by a synthesis method, the preparation process is simple, the operation is convenient, and the prepared probiotic oligopeptide with anti-inflammatory activity has high purity, thereby being beneficial to the application of the probiotic active peptide in food, dietary supplement or medicine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only drawings of some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows the mass spectrum of LRP-1.
FIG. 2 is a graph showing the results of high performance liquid chromatography measurement of LRP-1.
FIG. 3 is a mass spectrum of LRP-2.
FIG. 4 is a graph showing the results of high performance liquid chromatography measurement of LRP-2.
FIG. 5 is a mass spectrum of LRP-3.
FIG. 6 is a graph showing the results of high performance liquid chromatography measurement of LRP-3.
FIG. 7 is a graph showing the results of experiments in which LRP-2 and the polypeptide composition inhibited DSS-induced reduction in intestinal epithelial cell viability.
FIG. 8 is a graph showing the results of experiments in which LRP-2 and the polypeptide composition inhibited DSS-induced secretion of inflammatory factors from intestinal epithelial cells.
FIG. 9 is a graph showing the results of experiments in which LRP-2 and polypeptide compositions inhibit DSS-induced activation of the NF- κ B, NLRP3 inflammasome signaling pathway in intestinal epithelial cells.
Detailed Description
The technical solution of the present invention will be clearly and completely described with reference to the following examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of probiotic active peptides
(1) 100mg of Fmoc-Pro Wang Resin is put in a solid phase synthesis tube, N-Dimethylformamide (DMF) is added, then the mixture is kept stand for 30 minutes to fully swell the Resin, the solvent is filtered out, piperidine DMF solution is added, and the solvent is filtered out after oscillation. Dissolving Fmoc-Glu-OH, 1-hydroxybenzotriazole and O-benzotriazol-tetramethylurea hexafluorophosphate in DMF, adding N, N-diisopropylethylamine, uniformly mixing, activating in dark place, adding into resin, stirring for 2 hours under the action of nitrogen blowing at 25 ℃, carrying out suction filtration, washing with DMF and dichloromethane in sequence, and drying the solvent. And repeating the steps, sequentially adding activated Fmoc-His-OH, Fmoc-Tyr-OH, Fmoc-Met-OH and Fmoc-Pro-OH into the resin, stirring by blowing nitrogen at 25 ℃, washing the resin after complete reaction, performing rotary evaporation on the obtained filtrate to obtain a precipitate, and performing freeze drying to obtain LRP-1.
(2) 100mg of Fmoc-Pro Wang Resin is put in a solid phase synthesis tube, N-Dimethylformamide (DMF) is added, then the mixture is kept stand for 30 minutes to fully swell the Resin, the solvent is filtered out, piperidine DMF solution is added, and the solvent is filtered out after oscillation. Dissolving Fmoc-Gly-OH, 1-hydroxybenzotriazole and O-benzotriazol-tetramethylurea hexafluorophosphate in DMF, adding N, N-diisopropylethylamine, uniformly mixing, activating in dark place, adding into resin, stirring for 2 hours at 25 ℃ under the action of nitrogen blowing, carrying out suction filtration, washing with DMF and dichloromethane in sequence, and drying the solvent. And repeating the steps, sequentially adding activated Fmoc-Glu-OH, Fmoc-Thr-OH and Fmoc-Phe-OH into the resin, stirring by blowing nitrogen at 25 ℃, washing the resin after complete reaction, performing rotary evaporation on the obtained filtrate to obtain a precipitate, and performing freeze drying to obtain LRP-2.
(3) Placing 100mg Fmoc-Ser Wang Resin in a solid phase synthesis tube, adding N, N-Dimethylformamide (DMF), standing for 30 min to fully swell the Resin, filtering to remove the solvent, adding piperidine DMF solution, oscillating, and filtering to remove the solvent. Dissolving Fmoc-Leu-OH, 1-hydroxybenzotriazole and O-benzotriazol-tetramethylurea hexafluorophosphate in DMF, adding N, N-diisopropylethylamine, uniformly mixing, protecting from light, activating, adding into resin, stirring for 2 hours at 25 ℃ under the action of nitrogen blowing, carrying out suction filtration, washing with DMF and dichloromethane in sequence, and drying the solvent. And repeating the steps, sequentially adding activated Fmoc-Ala-OH, Fmoc-His-OH, Fmoc-Arg-OH and Fmoc-Phe-OH into the resin, stirring by blowing nitrogen at 25 ℃, washing the resin after complete reaction, performing rotary evaporation on the obtained filtrate to obtain a precipitate, and performing freeze drying to obtain LRP-3.
Structural analysis and molecular weight determination of probiotic active peptides: electrospray ionization mass spectrometry (HPLC-ESI-MS) was performed on a SCIEX X500R quadrupole time-of-flight (Q-TOF) mass spectrometer. The mass range was set at m/z 50-1500. Obtaining Q-TOF mass spectrum data in a positive mode, wherein the mass spectrum analysis conditions are as follows: CAD gas flow, 7L/min; the temperature of the drying gas is 550 ℃; ion spraying voltage, 5500V; and a deaggregation potential, 80V.
LRP-1 was analyzed as follows: based on mass spectrum data analysis, the following results are obtained: the ion fragment M/z 774.3311 is considered to be the fragment [ M +2H]+(ii) a The ion fragment M/z773.3295 is considered to be fragment [ M + H]+(ii) a The ion fragment m/z658.2653 is the y5 ion, m/z511.4714 is [ y4-H2O+H]+A fragment; m/z387.1665 is known as [ y4-y1-SCH3+2H]+Ions; m/z 281.0515 is considered to be [ y3-y1-CH3+H]+Ion, m/z 149.0239 is considered to be [ Met + H]+And (3) fragment. Finally, the amino acid sequence of LRP-1 can be determined to be Pro-Met-Tyr-His-Glu-Pro according to the mass spectrum data, namely the probiotic active peptide with the structure shown in the formula I.
LRP-2 was analyzed as follows: based on mass spectrum data analysis, the following results are obtained: the ion M/z551.2519 is considered to be [ M +2H]+(ii) a The ionic fragment M/z550.2474 is considered to be [ M + H]+(ii) a The ionic fragment m/z281.0514 was considered to be [ b3-H2O+H]+A fragment; m/z148.0714 is regarded as [ Phe-H ]2O+H]+Ions. Finally, the amino acid sequence of LRP-2 can be determined to be Phe-Thr-Glu-Gly-Pro, namely the probiotic active peptide with the structure shown in formula II according to the mass spectrum data.
LRP-3 was analyzed as follows: based on mass spectrum data analysis, the following results are obtained: the ion M/z730.3992 is considered to be the fragment [ M + H]+(ii) a The ion m/z 625.3592 is considered to be the y5 ion; the ion m/z495.2468 is considered to be [ y4-NH ]2+2H]+A fragment; m/z424.2099 is b4 ion; m/z287.1499 is b3 ion, m/z209.1040 is b4-b2 ion; m/z 110.0707 is considered to be [ His-COOH + H]+Ions. Finally, from the mass spectrometry data described above, it was determined that LRP-3 had an amino acid sequence of Phe-Arg-His-Ala-Leu-Ser, a probiotic active peptide having the structure shown in formula iii.
Examples of the experiments
In order to evaluate the biological activities of LRP-1, LRP-2, LRP-3 and probiotic active peptide compositions (compositions consisting of LRP-1, LRP-2, and LRP-3 in a molar ratio of 1:1:1, abbreviated as TLRP) prepared in example 1 of the present invention, the following effect experiments were performed.
Hcoepic cells at 5X 103Density of/mL was seeded in Costa 96 well plates and incubated with 1% DSS for 12 hours. The Hcoepic cells were then washed twice with cold PBS and incubated with the probiotic oligopeptide for 24 hours. Cell viability was measured by MTT assay. 5X 104Hcoepic cells/mL were incubated with 1% DSS in 6-well plates for 12 hours. Then, the Hcoepic cells were washed twice with cold PBS and incubated with LRP-1(0.10mg/mL), LRP-2(0.10mg/mL), LRP-3(0.10mg/mL), TLRP (0.10mg/mL) for 24 hours, respectively. The supernatant of the cultured cells was collected. Inflammatory epithelial cytokine content was measured using ELISA kit. Collecting cells, extracting proteins, and researching the influence of the proteins on related signal paths by a Western blotting method.
The experimental results are as follows:
the study found (as shown in FIG. 7) that LRP-1, LRP-2, LRP-3 and their compositions (TLRP) were able to significantly inhibit DSS-induced intestinal epithelial cell viability reduction. Under the same concentration condition, the inhibition effect of TLRP on DSS-induced intestinal epithelial cell viability reduction is stronger than that of LRP-1, LRP-2 and LRP-3 which are used independently. We speculate that LRP-1, LRP-2, LRP-3 in the composition (TLRP) may exert a synergistic effect, thereby reducing the damage of DSS to intestinal epithelial cells.
We further verified the anti-inflammatory activity of the probiotic active peptides and the compositions thereof with LRP-2 and TLRP, and the experimental results show that LRP-2 and TLRP can inhibit the secretion of inflammatory cytokines (IL-4, IL-6, TNF-alpha) caused by DSS exposure, and the inhibition TLRP is stronger than LRP-2 (as shown in FIG. 8).
Further studies showed that the inhibition of DSS exposure by TLRP and LRP-2 could be achieved by modulating NF- κ B and NLRP3 inflammasome (as shown in figure 9).
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A probiotic active peptide is characterized by having a structure shown as a formula I, a formula II or a formula III:
2. a probiotic active peptide composition is characterized by comprising any two or three of probiotic active peptides with structures shown as a formula I, a formula II and a formula III.
3. The probiotic bioactive peptide composition of claim 2, comprising probiotic bioactive peptides having the structures of formula i, formula ii and formula iii;
the probiotic active peptide has the structure shown in the formula I, the formula II and the formula III, and the molar ratio of the probiotic active peptide is 1-10: 1-10.
4. The probiotic active peptide composition of claim 3, wherein the molar ratio of the probiotic active peptides having the structures shown in formula I, formula II and formula III is 1-5: 1-5.
5. The probiotic bioactive peptide composition of claim 4, wherein the molar ratio of the probiotic bioactive peptides having the structures shown in formula I, formula II and formula III is 1:1: 1.
6. Use of a probiotic active peptide or probiotic active peptide composition according to any one of claims 1 to 5 in the manufacture of a food product, dietary supplement or pharmaceutical product.
7. The use according to claim 6, wherein the food, dietary supplement or pharmaceutical product is an anti-inflammatory food, dietary supplement or pharmaceutical product.
8. The use according to claim 6, wherein the food, dietary supplement or pharmaceutical product is a food, dietary supplement or pharmaceutical product having an anti-inflammatory effect.
9. The use of claim 8, wherein the enteritis is caused by dextran sodium sulfate.
10. The use of claim 8, wherein the inflammatory bowel disease is inflammatory injury of the epithelial cells of the intestinal tract.
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