CN115197295A - Antioxidant peptide, incubated eggshell membrane hydrolysate containing antioxidant peptide and application of incubated eggshell membrane hydrolysate - Google Patents
Antioxidant peptide, incubated eggshell membrane hydrolysate containing antioxidant peptide and application of incubated eggshell membrane hydrolysate Download PDFInfo
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- CN115197295A CN115197295A CN202111667462.5A CN202111667462A CN115197295A CN 115197295 A CN115197295 A CN 115197295A CN 202111667462 A CN202111667462 A CN 202111667462A CN 115197295 A CN115197295 A CN 115197295A
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Images
Classifications
-
- 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
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
Abstract
The invention discloses an antioxidant peptide, an incubated eggshell membrane hydrolysate containing the antioxidant peptide and application thereof. The amino acid sequence of the antioxidant peptide is any one of the following: LWNPR; MDGWPR; DGGLPPM. The antioxidant peptide MDGWPR has high ORAC value, ABTS and DPPH free radical scavenging activity; the antioxidant peptide LWNPR shows high ORAC value and ABTS free radical scavenging activity and cellular antioxidation; the antioxidant peptide DGGLPPM showed high ORAC value and DPPH free radical scavenging activity. The antioxidant peptides of the invention have the potential of targeted combination with antioxidant protein Keap 1. The hatching eggshell membrane hydrolysate contains one or more than one antioxidant peptide, can promote proliferation of chondrocytes, protect chondrocytes and macrophages from oxidative stress damage, can effectively regulate intracellular antioxidant signal pathways, and can be applied to foods, medicines, health products and beauty products.
Description
Technical Field
The invention relates to the technical field of biological small molecule active peptides, in particular to antioxidant peptides, an incubation eggshell membrane hydrolysate containing the antioxidant peptides and application thereof.
Background
Free radicals have high activity and extremely strong oxidation reaction capability, and can attack macromolecules in vivo through oxidation reaction to further cause the damage of cell structures and functions, so that the damage of organism tissues and organs is caused, the imbalance of continuous oxidative stress and organism defense is caused, and the diseases such as osteoarthritis, heart disease, diabetes, cancer and the like are caused. In addition, food spoilage is caused primarily by lipid oxidation and secondary lipid oxidation. Many synthetic antioxidants, such as Butylhydroxyanisole (BHA) and Butylhydroxytoluene (BHT), although having high antioxidant activity, are limited in their use because they may induce side effects such as DNA damage and body toxicity. The antioxidant extracted from natural resources, such as antioxidant peptide, can be used in food and health product with high safety.
The Keapl-Nrf2 signal pathway is the most important endogenous antioxidant signal pathway in the cell. When the cells are exposed to oxidative stress, the Nrf2 is released from the Keap1-Nrf2 complex and enters the cell nucleus to promote the expression of antioxidant genes. Natural ingredient derived antioxidants can alleviate H by activating the Keap1-Nrf2 pathway 2 O 2 Induced oxidative stress, inflammation and cartilage degradation. Modulation of the Keap1-Nrf2 signaling pathway is expected to be a viable approach to the treatment of osteoarthritis.
The eggshell is an important component of hatchery waste and is composed of a calcium carbonate shell, membranes, embryonic derived proteins and active peptides, and other contaminants (including microorganisms) that coat the eggshell. The hatching eggshell membrane is reported to contain a large amount of bioactive proteins, peptides, enzymes and a plurality of microbial protein factors, and the utilization rate of the current hatching eggshells is low, most of the hatching eggshells are discarded, and only a small part of the hatching eggshells are added into feed and fertilizer, so that the great waste of biological resources and the environmental pollution are caused.
At present, polypeptide preparation of fresh egg shell membranes has been reported, for example, chinese patent CN 108323764B reports that fresh egg membranes can prepare protein hydrolysate with bone joint protection effect, but the structure and sequence of active polypeptide which plays a specific role are not provided. However, no report is made at home and abroad about the application of hatching eggshell membranes in the preparation of bioactive peptides, and the development of hatching eggshell membrane antioxidant peptides is beneficial to the high-valued utilization of hatching eggshell membrane waste resources, and a brand new source is provided for preparing antioxidant peptides.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides antioxidant peptide, hatching eggshell membrane hydrolysate containing the antioxidant peptide and application thereof. The invention hydrolyzes the hatching eggshell membrane by proper proteolytic enzyme to obtain hatching eggshell membrane hydrolysate, separates and purifies the hydrolysate by ultrafiltration, sephadex chromatography and reversed-phase high performance liquid chromatography, measures the chemical and cell antioxidant activity of each component, performs peptide group identification by LC-MS/MS, screens active peptide sequences by combining with bioinformatics means, synthesizes in a solid phase, and verifies the activity, thereby completing the invention.
In order to achieve the purpose, the invention adopts the following technical scheme:
an antioxidant peptide, which is characterized in that the amino acid sequence is any one of the following sequences:
LWNPR(Leu-Trp-Asn-Pro-Arg)；
MDGWPR(Met-Asp-Gly-Trp-Pro-Arg)；
DGGLPPM(Asp-Gly-Gly-Leu-Pro-Pro-Met)。
a hatching eggshell membrane hydrolysate comprising one or more of the following antioxidant peptides;
an antioxidant peptide with the amino acid sequence of LWNPR (Leu-Trp-Asn-Pro-Arg);
antioxidant peptide with amino acid sequence of MDGWPR (Met-Asp-Gly-Trp-Pro-Arg);
the amino acid sequence of the antioxidant peptide is DGGLPPM (Asp-Gly-Gly-Leu-Pro-Pro-Met).
A food comprises the above antioxidant peptide or incubated eggshell membrane hydrolysate.
An antioxidant medicine contains the above antioxidant peptide or hydrolysate of incubated eggshell membrane.
A health product contains the above antioxidant peptide or hydrolysate of eggshell membrane.
Wherein the usage amount of the hydrolysate of incubated eggshell membrane in food, antioxidant medicine or health product is 4-40mg/kg.
An antioxidant cosmetic contains the above antioxidant peptide or hydrolysate of incubated eggshell membrane.
Preferably, the addition amount of said hatching eggshell membrane hydrolysate is 0.1-10wt%.
Preferably, the cosmetic product comprises a facial mask, a cream and an emulsion.
Use of the above antioxidant peptide or incubated eggshell membrane hydrolysate in the manufacture of an antioxidant.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method prepares the antioxidant peptide by using the hatching eggshell membrane for the first time, reasonably utilizes waste resources of a hatching field, avoids the pollution to the environment, and has the advantages of novel raw materials, environmental protection and sustainable development;
(2) The molecular weight of the antioxidant peptide provided by the invention is below 1000Da and is easy to absorb.
(3) The antioxidant peptide has high antioxidant activity, wherein the ORAC values of the antioxidant peptides MDGWPR, LWNPR and DGGLPPM are respectively 4.59, 4.46 and 2.46 times of GSH. The antioxidant peptide and the hydrolysate containing the antioxidant peptide can protect inflammatory cells (RAW 264.7) and a chondrocyte line (SW 1353) from being damaged by oxidative stress, and can promote the proliferation of the chondrocyte line (SW 1353).
(4) The method relates to bioinformatics screening and in-vitro and intracellular antioxidant activity verification, and researches and verifies the antioxidant activity of antioxidant peptide and incubated eggshell membrane hydrolysate containing the antioxidant peptide from multiple angles.
(5) The novel antioxidant peptide disclosed by the invention can effectively regulate and control a Keap1-Nrf2 signal path of an organism antioxidant path besides in-vitro antioxidant activity, and has the potential of targeted combination with Keap 1.
Drawings
FIG. 1 is the ORAC values of different molecular weight ultrafiltration components of incubated eggshell membrane hydrolysate (different letters indicate significant difference P < 0.05) in example 2 of the present invention;
FIG. 2 is the result of the antioxidant capacity measurement of the preferred hatching eggshell membrane antioxidant peptide (different letters in the same index indicate that there is a significant difference P < 0.05);
FIG. 3 is a mass spectrum of incubated eggshell membrane antioxidant peptide LWNPR;
FIG. 4 is a mass spectrum of incubated eggshell membrane antioxidant peptide MDGWPR;
FIG. 5 is a mass spectrum of DGGLPPM;
FIG. 6 shows the effect of antioxidant peptides LWNPR on the expression of Keap1-Nrf2 signaling pathway-related proteins in cells;
FIG. 7 shows the molecular docking results of antioxidant peptide LWNPR and key protein Keap1 of cell antioxidant.
Detailed Description
The present invention is further described with reference to the following examples, which should not be construed as limiting the scope of the invention as claimed.
The antioxidant peptide and the hatching eggshell membrane hydrolysate containing the same have the characteristics of novel raw materials, low cost, environmental friendliness and higher activity of the antioxidant peptide. Finally, 3 polypeptides with high antioxidant activity were isolated and identified from the incubated eggshell membrane hydrolysate. The amino acid sequences of the antioxidant peptides are respectively as follows: LWNPR, MDGWPR and DGGLPPM are searched and compared with BIOPEP of the existing active peptide database, and are not reported, and are novel antioxidant peptides, and through chemical antioxidant activity verification, the peptides related to the invention show high free radical scavenging activity, can promote the proliferation of a chondrocyte line (SW 1353), and can obviously improve H 2 O 2 Induced cell viability of chondrocytes and immune cells.
The specific polypeptide sequence and activity assay results are as follows:
(1) The antioxidant peptides MDGWPR, LWNPR and DGGLPPM show high ORAC values, the determination results are all superior to the results of positive control GSH, and meanwhile, the antioxidant peptides MDGWPR and LWNPR show high ABTS free radical scavenging activity and the determination results are remarkably higher than the results of GSH. The antioxidant peptides LWNPR, DGGLPPM, MDGWPR show high DPPH free radical scavenging activity, wherein the result of MDGWPR is better than that of GSH.
(2) Preferably, the hatching eggshell membrane antioxidant peptide LWNPR can increase H 2 O 2 Induced expression of RAW264.7 cells Nrf2 and HO-1 activates a Keap1-Nrf2 signal channel to exert antioxidant activity.
(3) The molecular docking result shows that hatching eggshell membrane antioxidant peptides MDGWPR, LWNPR and DGGLPPM can occupy the binding site of Nrf2 in the Keap1 Kelch structural domain, inhibit the ubiquitination degradation of Nrf2 and activate the Keap1-Nrf2 signal pathway.
(4) The hatching eggshell membrane hydrolysate containing the antioxidant peptide sequence can obviously improve H 2 O 2 The cell survival rate of the induced SW1353 cell oxidative damage model is higher when the concentration is 0.75mg/mL compared with that of the model group,the survival rate of SW1353 cells can be improved by 67.58 percent.
(5) 0.75mg/mL of incubated eggshell membrane hydrolysate containing the antioxidant peptide sequence of the invention can promote the proliferation of SW1353 cells, and compared with a blank group, the cell proliferation index reaches 3.
(6) The incubated eggshell membrane hydrolysate containing the antioxidant peptide sequence can obviously improve H 2 O 2 Cell viability in the induced RAW264.7 cell oxidative damage model at a concentration of 0.75mg/mL, in combination with H 2 O 2 Compared with a model group, the survival rate of the RAW264.7 cells can be improved by 55.81%.
It should be noted that, as a typical example, the three antioxidant peptides of the present invention can be obtained by separation and purification from the hydrolysate of incubated eggshell membrane, or can be prepared by other methods known in the art, such as chemical synthesis and genetic engineering.
The antioxidant peptide of the present invention may be used as an antioxidant after being separated from a hatching eggshell membrane hydrolysate containing the antioxidant peptide, or may be used as an antioxidant in a form contained in a hatching eggshell membrane hydrolysate.
The antioxidant peptide of the present invention is used for determining the ORAC value by the method described in Habinshuti I, mu T H, zhang M.ultrasonic microwave-assisted enzymatic production and chromatography of antioxidant peptides from sweet potato protein [ J ]. Ultrason Sonochem,2020, 69.
ABTS, determination of the clearance of DPPH free radicals, the determination of the clearance of DPPH free radicals is described in reference to (dying Wang, mengting Ma, zhipen Yu, shuang-kui Du.preparation and identification of the antioxidant peptides from chemically linked proteins [ J ]. Food Chemistry,2021,352 129399.) and (Qiaozhi Zhang, xiaohong Tong, yang Li, huangan Wang, zhongjiang Wang, baokun Qi, xian Sui, lianzhou J.purification and characterization of the antioxidant peptides from Alca-hydrolybe antibody (glucamine L.) and reaction 20136. Nutritional sample J.12. Additive J..
The present invention will be described more specifically with reference to examples.
Example 1
< preparation of hydrolysate of incubated eggshell Membrane >
a. Obtaining of hatching eggshell membrane:
is prepared from the waste of incubated eggshell in culture plant by tearing off the membrane of incubated eggshell, washing with pure water, and oven drying.
b. Preparation of hatching eggshell membrane hydrolysate:
incubating eggshell membrane with pure water at a ratio of 1: 10-1: 60 (w/v), adjusting the pH value of the solution to 8.0-10.0, adding metalloprotease and serine protease according to the enzyme adding amount of 1-5% (w/w), and reacting for 8-10 h at 50-60 ℃. After the reaction is finished, the beaker filled with the hydrolysate is placed in hot water at the temperature of 80-100 ℃ for 10-30 min to inactivate the protease, then the beaker is placed in room temperature or cold water, and after the temperature is cooled to the room temperature, the pH value of the hydrolysate is adjusted to 7.0-7.6 by using hydrochloric acid solution. Centrifuging the hydrolysate at 8000-12000 r/min for 10-30 min, and storing the supernatant at-20 ℃ for later use, wherein the supernatant is the hydrolysate of the incubated eggshell membrane.
Example 2
< separation by Ultrafiltration >
1) Ultrafiltration of the crude polypeptide: thawing the prepared incubated eggshell membrane hydrolysate at 4 ℃, then taking the supernatant, filtering the supernatant through a 30kDa filter membrane, and intercepting polypeptides with the molecular weight of more than 30kDa in the supernatant to obtain a first ultrafiltrate; performing second ultrafiltration with ultrafiltration membrane with molecular weight cutoff of 10kDa to obtain second ultrafiltrate; and performing third ultrafiltration on the second ultrafiltrate by using an ultrafiltration membrane with the molecular weight cutoff of 3kDa to obtain a third ultrafiltrate, finally obtaining four ultrafiltration components with different molecular weights, wherein the four ultrafiltration components are respectively more than 30kDa, 30-10 kDa, 10-3 kDa and less than 3kDa, and freeze-drying the ultrafiltration components with different molecular weights for subsequent activity determination.
2) Measuring the Oxidative Radical Absorption Capacity (ORAC) of the different molecular weight fractions obtained in 1) respectively, and selecting the fraction with the largest ORAC value for amino acid sequence identification. ORAC values of the different molecular weight ultrafiltration fractions of incubated eggshell membrane hydrolysate are shown in FIG. 1, with the ultrafiltration fraction < 3kDa showing the highest ORAC value and being significantly higher than the ORAC values of the other ultrafiltration fractions.
Example 3
< Mass Spectrometry identification of incubated eggshell membrane antioxidant peptides >
The amino acid sequence of the < 3kDa ultrafiltration fraction was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS was coupled with a Thermo LTQ linear ion trap mass spectrometer (Thermo Fisher, san Jose, CA, USA) using an eks ignent nano LC instrument (eks Technologies, dublin, CA, USA), an LC Packins PepMap 300C 18 column (75 μm × 150mm pore size
particle size 5 μm; dionex, sunnyvale, CA, USA). Eluent A contained 2% ACN, 0.1% formic acid, eluent B contained 80% ACN, 0.1% formic acid, and incubated eggshell membrane hydrolysate having a molecular weight of less than 3kDa was separated at a flow rate of 300 nL/min. The elution conditions were: 0 to 85min,5 percent of B; 85-95min, 5-50 percent of B;95 to 125min,50 to 95 percent of B. The eluted peptide sequence was evaluated using MS/MS data with a spray voltage of 2.2kV, a m/z scan range of 400-2000, a data acquisition time of 110min, a capillary temperature of 200 ℃ and a normalized collision energy of 35%. Finally, MS/MS data were analyzed using Mascot (version 2.1.0, matrix sciences, london, UK).
Example 4
< screening of antioxidant peptides and verification of antioxidant Activity Using bioinformatics >
According to the results of mass spectrometric identification in example 3, combined with bioinformatics, by PeptideRanker website (C.) (http:// distilldeep.ucd.ie/PeptideRankerV) predicting the possibility that the antioxidant peptide has activity, the maximum value is 1, and the larger the result is, the higher the possibility that the polypeptide has activity is shown. Simultaneously combines with ProtParam tool website (https:// web.expasy.org/protparam/) Predicting the molecular weight, hydrophilicity, hydrophobic amino acid content, isoelectric point of the polypeptide, preferably the molecular weight, hydrophilicity, hydrophobic amino acid content, isoelectric point and activity of the polypeptide identified in incubated eggshell membrane hydrolysate asAs shown in Table 1 below, polypeptides with a predicted activity greater than 0.80 are preferred.
TABLE 1 molecular weight, hydrophilic, hydrophobic amino acid content, isoelectric point of polypeptides identified in incubated eggshell membrane hydrolysates
Note: negative values of GRAVY indicate hydrophilicity, with smaller values being more hydrophilic.
Preferably, the antioxidant peptides contained in the incubated eggshell membrane hydrolysate were obtained by solid phase synthesis and the antioxidant index thereof was measured, the results of which are listed in table 2 below.
TABLE 2 antioxidant peptides contained in incubated eggshell membrane antioxidant peptide hydrolysate and antioxidant activity thereof
Note: different letters represent significant differences (P < 0.05), tables 4, 5.
From the results of table 2 and fig. 2, it can be found that the antioxidant peptides MDGWPR, LWNPR, DGGLPPM exhibit high ORAC values, and compared with the positive control GSH, the results of the above three antioxidant peptides are all higher than the positive control GSH; the antioxidant peptides MDGWPR and LWNPR show high ABTS values, and compared with positive control GSH, the results of the two antioxidant peptides are higher than that of the positive control GSH; the antioxidant peptides MDGWPR show high DPPH value, the result is slightly higher than the result of positive control GSH, and the DPPH values of the antioxidant peptides LWNPR and DGGLPPM are similar to the result of the positive control GSH, so that the antioxidant peptides MDGWPR and DGGLPPM show good antioxidant activity. The results in the table show that the antioxidant peptide MDGWPR simultaneously shows higher ORAC value, ABTS value and DPPH value, and the results are all better than the results of the positive control GSH. The antioxidant peptides LWNPR showed both high ORAC and ABTS values, and it was found that these peptides were excellent in antioxidant activity.
The invention provides mass spectrograms of the above 3 antioxidant peptides:
wherein, fig. 3 is a mass spectrum of hatching eggshell membrane antioxidant peptide LWNPR;
FIG. 4 is a mass spectrum of incubated eggshell membrane antioxidant peptide MDGWPR;
FIG. 5 is a mass spectrum of DGGLPPM.
The invention provides an antioxidant peptide which can be used for food and medicine and shows high antioxidant activity, and a hatching eggshell membrane hydrolysate containing the antioxidant peptide, has the potential of being used as an antioxidant and added into food and medicine, and provides a theoretical basis for the antioxidant.
Example 5
<Antioxidant peptide pair H 2 O 2 Protective effects of induced SW1353 cells>
Preferably, the pair of incubated eggshell membrane hydrolysates comprising the antioxidant peptide sequence of the invention described above is H 2 O 2 The protective effect of the model of oxidative damage induced by SW1353 cells can be determined as follows:
culture of sw1353 cells: SW1353 cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin) at 37 ℃ with 5% CO 2 A saturated humidity incubator. Cell monolayer culture and subculture by digestion with 0.25% trypsin solution.
2. Study of hatching eggshell membrane hydrolysate on the proliferative effect of SW1353 cells: SW1353 cells at 4X 10 5 Inoculating the cells per mL in a 6-well plate, culturing for 24h, adding incubated eggshell membrane hydrolysate (dissolved in cell culture solution, filtered through a 0.22 μm filter membrane to remove impurities and bacteria) to the final concentration of 0.75mg/mL, and culturing for 24h to determine the cell survival rate.
SW1353 cell oxidative damage model (H) 2 O 2 Model group) establishment: SW1353 cells at 4X 10 5 The cells were seeded in 6-well plates at a concentration of 200. Mu.M/mL and cultured for 48H 2 O 2 Solution (in DMEM) and incubated for 1h.
Determination of SW1353 cell viability: SW1353 cells at 4X 10 5 Inoculating into 6-well plate at cell concentration per mL, culturing for 24 hr, adding hydrolysate of incubated eggshell membrane (dissolved in cell culture solution, filtered with 0.22 μ M filter membrane to remove impurities and bacteria), culturing for 24 hr, and adding H at final concentration of 200 μ M into each well 2 O 2 And (5) culturing the solution for 1h. Removing the culture medium, adding 100 μ L of CCK-8 solution (10%) per well, standing at 37 deg.C, 5% 2 After the culture is continued for 1h in the saturated humidity incubator, the light absorption value A of each hole is measured by an enzyme-labeling instrument 450nm 。
5. Cell viability was calculated according to the following formula:
wherein A represents A in the experimental group 450nm ；A 0 A representing blank group 450nm ；A 1 A representing normal group 450nm ；
The results are shown in tables 3 and 4 below:
TABLE 3 results of incubated eggshell membrane hydrolysate on the proliferation of SW1353 cells
| Group of | Blank group | Polypeptide treatment group (0.75 mg/mL polypeptide pretreatment) |
| SW1353 cell survival rate% | 99.95±3.76 | 299.57±5.64 *** |
Note: "x" indicates P <0.001, with very significant differences.
TABLE 4 hatching eggshell membrane hydrolysate pairs H 2 O 2 Results of protective Effect of induced SW1353 cells
As can be seen from Table 3, 0.75mg/mL pretreatment of incubated eggshell membrane hydrolysate promoted the proliferation of SW1353 cells, and the cell survival rate was increased 2.00-fold compared to the blank group. From table 4, it can be found that the survival rate of SW1353 cells after pretreatment of 0.75mg/mL hatching eggshell membrane hydrolysate is improved by 67.58% compared with that of the model group, which indicates that the hatching eggshell membrane hydrolysate containing the antioxidant peptide sequence of the present invention can significantly improve H 2 O 2 Cell viability in the model of oxidative damage to induced SW1353 cells.
Example 6
<Antioxidant peptide pair H 2 O 2 Protection of induced RAW264.7 cells>
The hatching eggshell membrane hydrolysate containing the antioxidant peptide sequence can obviously improve H 2 O 2 The specific operation method for inducing the cell survival rate of the RAW264.7 cell oxidative damage model is the same as that in example 5.
The results are shown in Table 5 below, comparing H to the blank 2 O 2 The cell survival rate of the model group was significantly decreased. 0.75mg/mL incubated eggshell membrane hydrolysate, after pretreatment, was reacted with H 2 O 2 Compared with a model group, the survival rate of the RAW264.7 cells can be improved by 55.81 percent.
TABLE 5 incubated eggshell membrane hydrolysate pairs H 2 O 2 Results of protective Effect of induced RAW264.7 cells
Example 7
< Effect of the preferred antioxidant peptide LWNPR on intracellular antioxidant pathways based on western blot study >
RAW264.7 cells (5.1X 10) 5 Individual cells/well) were seeded into 6-well plates and cultured for 24h. Preferred antioxidant peptides LWNPR (500 and 1000. Mu.M) are pretreated 24h, 175. Mu.M H 2 O 2 After 1h of treatment, RAW264.7 cells were harvested and lysed with RIPA buffer containing PMSF and phosphatase inhibitor. After analysis of the total protein content (BCA kit), the proteins were separated on a 12% SDS-PAGE gel (40. Mu.g) and transferred onto a PVDF membrane. 5mL of blocking solution was added for 1h, the blocking solution was removed, primary antibody diluted with blocking solution was added, and incubation was performed slowly overnight at 4 ℃. Washing with TBST for 3 times, adding TBST diluted secondary antibody, incubating at 24 deg.C for 1h, washing with TBST for 3 times, adding ECL liquid to the washed PVDF membrane, allowing chemical reaction for 1-2 min, placing in dark environment, developing and fixing, and scanning to obtain western blot result.
As shown in fig. 6: in comparison with blank group, H 2 O 2 The expression of the Nrf2 and HO-1 proteins of the group is reduced, and the expression of the Keap1 is increased; after pretreatment of antioxidant peptides LWNPR at 500 and 1000 mu M, the expression of Nrf2 and HO-1 is increased, and the expression of Keap1 is reduced, which shows that the antioxidant peptides for hatching eggshell membranes provided by the invention can activate the expression of Keap1-Nrf2 signal pathway related proteins, thereby protecting inflammatory cells (RAW 264.7) from H 2 O 2 Damage by induced oxidative stress.
Example 8
< Effect of optimizing antioxidant peptide LWNPR on intracellular antioxidant pathways based on molecular docking study >
Molecular docking is carried out through a CDOCKER program of Discovery Studio (DS) 2018 client software, hatching eggshell membrane antioxidant peptide is used as a ligand, and Keap1 (PDB ID:2 FLU) is used as a receptor. Docking results are expressed by a-CDOCKER interaction energy (-CIE) score, higher scores indicate more compact binding, and specific values of preferred hatching eggshell membrane antioxidant peptides are shown in Table 6.
TABLE 6 molecular docking results of incubated eggshell membrane antioxidant peptides with Keap1
| Peptide sequence | –CIE |
| DKLPGF | 51.0025 |
| ASNTDFFLR | 76.8399 |
| LWNPR | 63.0303 |
| MDGWPR | 57.7207 |
| DGGLPPM | 56.0518 |
All the hatching eggshell membrane antioxidant peptides related by the invention can be successfully docked with the active site of Keap 1. The LWNPR is used as a preferred hatching eggshell membrane antioxidant peptide to research the binding mechanism of the hatching eggshell membrane antioxidant peptide and Keap 1. The results are shown in FIG. 7, in which LWNPR forms 6 conventional hydrogen bonds with Ser363, arg380, asn382 and Arg415 of Keap1, 1 carbon hydrogen bond with Tyr572, and 3 pi-alkyl bonds with His436, tyr572 and Phe 577. All of the amino acid residues described above are key residues of the Keap1-Kelch domain in the Keap l-Nrf2 interaction binding site. The hatching egg shell membrane antioxidant peptide can occupy the binding site of Nrf2 in the Keap1-Kelch structural domain, so that the interaction of the Keap1-Nrf2 is damaged, the ubiquitination degradation of the Nrf2 is inhibited, the Keap1-Nrf2 signal pathway is activated, and the potential of targeting antioxidant key protein Keap1 is realized.
The antioxidant peptide of the present invention and the incubated eggshell membrane hydrolysate containing the same can be used alone as an antioxidant, but can synergistically enhance antioxidant activity when used in combination with 1 or more other substances having antioxidant activity or extracts containing these substances.
The antioxidant peptide and the incubated eggshell membrane hydrolysate containing the antioxidant peptide can be used as medicines, preferably antioxidant foods, medicines, health products and beauty products.
In the production of beverages, nutritional supplements and tablets for preventing food autoxidation or achieving physiological antioxidant effects by oral ingestion, the antioxidant peptide of the present invention and incubated eggshell membrane hydrolysate containing the same may be added thereto as an active ingredient to exert the above-mentioned effects.
The above embodiments describe the technical solutions provided by the present invention in detail, and the specific examples are applied herein to explain the principles and embodiments of the present invention, and the descriptions of the above embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.
Claims (9)
1. An antioxidant peptide, characterized in that the amino acid sequence of the antioxidant peptide is any one of the following sequences:
LWNPR(Leu-Trp-Asn-Pro-Arg)；
MDGWPR(Met-Asp-Gly-Trp-Pro-Arg)；
DGGLPPM(Asp-Gly-Gly-Leu-Pro-Pro-Met)。
2. a hatching egg shell membrane hydrolysate comprising one or more of the following antioxidant peptides:
an antioxidant peptide with the amino acid sequence of LWNPR (Leu-Trp-Asn-Pro-Arg);
antioxidant peptide with amino acid sequence of MDGWPR (Met-Asp-Gly-Trp-Pro-Arg);
the amino acid sequence of the antioxidant peptide is DGGLPPM (Asp-Gly-Gly-Leu-Pro-Pro-Met).
3. A food product comprising the antioxidant peptide of claim 1 or the incubated eggshell membrane hydrolysate of claim 2.
4. An antioxidant pharmaceutical product comprising the antioxidant peptide according to claim 1 or the incubated eggshell membrane hydrolysate according to claim 2.
5. A health product comprising the antioxidant peptide according to claim 1 or the incubated eggshell membrane hydrolysate according to claim 2.
6. An antioxidant cosmetic preparation comprising the antioxidant peptide according to claim 1 or the incubated eggshell membrane hydrolysate according to claim 2.
7. The antioxidant cosmetic article as claimed in claim 6, wherein the addition amount of said incubated eggshell membrane hydrolysate is 0.1-10wt%.
8. The antioxidant cosmetic product of claim 6, wherein the cosmetic product comprises a mask, a cream, or a lotion.
9. Use of the antioxidant peptide of claim 1 or the incubated eggshell membrane hydrolysate of claim 2 in the manufacture of an antioxidant.
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