CN117964691A - Bird's nest peptide II with skin elasticity protecting and anti-inflammatory effects and application thereof - Google Patents
Bird's nest peptide II with skin elasticity protecting and anti-inflammatory effects and application thereof Download PDFInfo
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- CN117964691A CN117964691A CN202410156762.4A CN202410156762A CN117964691A CN 117964691 A CN117964691 A CN 117964691A CN 202410156762 A CN202410156762 A CN 202410156762A CN 117964691 A CN117964691 A CN 117964691A
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- bird
- nest
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- nest peptide
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a bird's nest peptide II with the effects of protecting skin elasticity and resisting inflammation and application thereof. The amino acid sequence of the bird's nest peptide II is DPFYGGEYLK. The bird's nest peptide II has high elastase inhibition rate, can inhibit the degradation of elastin in skin and delay skin aging. The bird's nest peptide II also has the effect of improving inflammation, and has important significance for improving the economic value and the utilization value of bird's nest.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to a bird's nest peptide II with skin elasticity protecting and anti-inflammatory effects and application thereof.
Background
The nidus Collocaliae is nidus Collocaliae of Porifera of Uighur family and multiple species of Porifera, which is formed by mixing and coagulating saliva and fluff. The "Ben Cao gang mu Shi Yi" carries: the bird's nest is Gan Danping in flavor, can nourish lung yin greatly, resolve phlegm and relieve cough, tonify and clear, and is a holy medicine for conditioning consumptive disease. For all diseases, it is indicated for those who cannot clear descending due to lung deficiency. "Bencao Renewness" carry: "bird's nest is effective in invigorating primordial qi, moistening lung, nourishing yin, treating cough, hemoptysis, hematemesis, and activating fire to source, moisturizing intestine and stimulating appetite". Edible bird's nest contains several nutrients including protein, saccharide, lipid, vitamins, amino acids and several important inorganic elements, and its main functional components are sialic acid and epidermal growth factor, and has the functions of resisting oxidation, resisting senility, raising immunity, etc. Therefore, nidus Collocaliae has been used as a health food for nourishing, caring skin and preserving health.
Elastin is an insoluble, macromolecular fibrous protein in the extracellular matrix that, together with microfibrils, constitutes elastic fibers in connective tissue. The elastin accounts for 2-4% of the total protein content of the skin, has high extensibility and toughness, can endow the skin with stronger elasticity and recoil force, and is responsible for restoring the deformed skin to a normal form. Skin aging is a major concern in today's society and is mainly characterized by thinning of the epidermis, flattening of the junction of the dermis and overall degradation of the extracellular matrix (ECM) of the dermis. Elastic fibers play an important role in skin aging, and if elastin is degraded and destroyed, it can cause hypoelasticity, sagging, and various wrinkles. High expression of elastase enzymes can cause enzymatic hydrolysis of elastin and damage of elastin. Therefore, elastase inhibition is one of the indexes for examining the anti-skin aging ability of the product.
Although the content of protein in the bird's nest is high, the content of glycoprotein digestion-resistant components in the bird's nest is high, and digestion products comprise insoluble substances and proteins with high molecular weight, so that the glycoprotein digestion-resistant components cannot be effectively absorbed and utilized. And the solubility of total sugar in the bird's nest polypeptide is lower than 40%, and the mass fraction of undissolved sialic acid reaches about 56%. Caco-2 cell simulated absorption also showed limited absorption of bird's nest digestion products. The existing preparation methods of bird's nest peptides are studied very much, for example: patent CN110468176a discloses a preparation method for obtaining small-molecule bird's nest peptide by utilizing bromelain enzymolysis, patent CN115807049a discloses a preparation method for high-antioxidant-activity bird's nest peptide, and patent CN115261433a discloses a processing method for high-activity anti-fatigue peptide. However, the bird's nest peptide prepared by the method has large molecular weight, high peptide content, low sugar content and low sialic acid content. There is currently little research on the inhibition of elastase activity and anti-inflammatory effects of cubilose peptides.
Disclosure of Invention
In order to solve the technical problems, the invention fully utilizes the efficacy of the polypeptide in the bird's nest, promotes the digestion and absorption of the bird's nest in human body, improves the health level of people and the application value of the bird's nest, and provides the following technical scheme:
In a first aspect, the invention provides a bird's nest peptide II with skin elasticity protection and anti-inflammatory effects, and the amino acid sequence of the bird's nest peptide II is DPFYGGEYLK (SEQ ID NO: 2).
In a second aspect, the present invention provides a polypeptide mixture comprising the bird's nest peptide ii according to the first aspect.
Preferably, the ratio of glycopeptides in the polypeptide mixture is 0.2 to 0.8, for example: 0.20, 0.40, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80.
Preferably, the sialic acid content of the polypeptide mixture is 9 to 18wt%, for example: 9.0wt%, 11.0wt%, 13.0 wt%, 15.0wt%, 16.25wt%, 17.50wt% and 18.0wt%.
Preferably, the content of polypeptides with relative molecular mass of 4000-6000 Da in the polypeptide mixture is more than or equal to 60%, for example: 60%, 60.25%, 65.38%, 68.31%, 70%, 75%, 80%.
Preferably, the content of sugar chains with a relative molecular mass of 3000-4000 Da in the polypeptide mixture is greater than or equal to 55%, for example: 55%, 56.28%, 58.30%, 60.25%, 63.15%, 66.37%, 70.25%.
Preferably, the content of peptide chains with a relative molecular mass of 1000-2000 Da in the polypeptide mixture is greater than or equal to 65%, for example: 65%, 66.35%, 68.25%, 70.24%, 70.26%, 72.35%, 75.16%.
In a third aspect, the present invention provides a method for preparing a mixture of polypeptides according to the second aspect, comprising the steps of:
(1) Foaming and crushing the bird's nest to obtain bird's nest pulp;
(2) Adding protease into the bird's nest pulp, carrying out enzymolysis under 100-300 MPa of hydrostatic pressure, inactivating enzyme, and centrifuging to obtain supernatant;
(3) And (3) carrying out alcohol precipitation, centrifugation, rotary evaporation and drying on the supernatant to obtain the polypeptide mixture.
Preferably, the foaming in the step (1) is to add the bird's nest into water and then thoroughly mix the mixture uniformly.
Further, the mass of the water is 20-50 times of the dry weight of the bird's nest, for example: 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times.
Preferably, the edible bird nest after foaming is crushed by a colloid mill, and the tooth grinding gap of the colloid mill is 80-120 mu m. Thereby enabling the particles in the bird's nest slurry to reach the micron level.
Preferably, the grinding time of the colloid mill is 20-40 min, for example: 20min, 25min, 30min, 35min, 40min.
Preferably, the protease in step (2) is selected from any one or a combination of more of flavourzyme, papain, alkaline protease.
Further, when the protease is flavourzyme, adjusting the pH of the bird's nest slurry to 7.0-8.0, adding the flavourzyme according to the enzyme-to-base ratio of 5000-7000U/mg (enzyme activity/dry weight of bird's nest), mixing, vacuum packaging in a polyethylene bag, immediately placing in a high hydrostatic pressure (HPP) device, and treating for 10-14 h under the conditions of 45-55 ℃ and 100-300 MPa of hydrostatic pressure.
Further, when the protease is papain, adjusting the pH of the bird's nest slurry to 5.5-7.5, adding papain with an enzyme-to-base ratio of 5000-7000U/mg (enzyme activity/dry weight of bird's nest), mixing, vacuum packaging in a polyethylene bag, immediately placing in a high hydrostatic pressure (HPP) device, and treating for 10-14 h at 37-60 ℃ under a hydrostatic pressure of 100-300 MPa.
Further, when the protease is alkaline protease, adjusting the pH of the bird's nest slurry to 9.5-10.5, adding the alkaline protease with the enzyme-to-enzyme ratio of 5000-7000U/mg (enzyme activity/dry weight of bird's nest), mixing, vacuum packaging in a polyethylene bag, immediately placing in a high hydrostatic pressure (HPP) device, and treating for 10-15 h at 35-50 ℃ under the hydrostatic pressure of 100-300 MPa.
Preferably, the enzyme deactivation temperature in step (2) is 90 to 100 ℃, for example: 90 ℃, 92 ℃, 95 ℃, 97 ℃, 100 ℃. The enzyme deactivation time is 10 to 30 minutes, for example: 10min, 15min, 20min, 25min, 30min.
Preferably, the centrifugal rotational speed in step (2) is 5000 to 10000r/min, for example: 5000r/min, 6000r/min, 7000r/min, 8000r/min, 9000r/min and 10000r/min.
Preferably, the centrifugation time in step (2) is 15 to 30min, for example: 15min, 20min, 25min, 30min.
Preferably, the process of alcohol precipitation, centrifugation and spin-steaming in step (3) is repeated at least 1 time.
Preferably, in the alcohol precipitation in the step (3), the volume fraction of the absolute ethanol is 40-60% of the total system, for example: 40%, 45%, 50%, 55%, 60%.
Preferably, the alcohol precipitation time in step (3) is 10 to 30min, for example: 10min, 15min, 20min, 25min, 30min.
Preferably, the centrifugal speed in step (3) is 3000 to 5000r/min, for example: 3000r/min, 3500r/min, 4000r/min, 4500r/min, 5000r/min.
Preferably, the centrifugation time in step (3) is 15 to 30min, for example: 10min, 15min, 20min, 25min, 30min.
Preferably, the spin-steaming speed in step (3) is 100-200 r/min, for example: 100r/min, 120r/min, 150r/min, 180r/min, 200r/min.
Preferably, the spin-steaming temperature in step (3) is 30 to 50 ℃, for example: 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃.
Preferably, the drying in step (3) is freeze-drying or low-temperature drying, and more preferably freeze-drying.
Further, the freeze drying is to quick freeze the concentrated solution after spin steaming, and then freeze-dry.
Further, the freeze-drying temperature is-90 to-60 ℃, for example: -90 ℃, -85 ℃, -80 ℃, -75 ℃, -70 ℃, -65 ℃, -60 ℃.
Further, the quick freezing time is 10-12 hours, for example: 10h, 10.5h, 11h, 11.5h, 12h.
Further, the lyophilization time is 12 to 24 hours, for example: 12h, 14h, 16h, 18h, 20h, 22h, 24h.
In a fourth aspect, the present invention provides a composition comprising the bird's nest peptide ii of the first aspect or the polypeptide mixture of the second aspect.
Preferably, the composition includes food, health products, cosmetics and medicines.
Preferably, the composition comprises auxiliary materials which are allowed to be added in foods, health care products, cosmetics or medicines.
In a fifth aspect, the present invention provides the use of a cubilose peptide ii according to the first aspect or a polypeptide mixture according to the second aspect for the preparation of a product having elastase inhibition.
Preferably, the product is a food, a health product, a cosmetic or a pharmaceutical product.
Preferably, the product comprises auxiliary materials which are allowed to be added in foods, health products, cosmetics or medicines.
Further, the administration dose of the bird's nest peptide II is 10mg/kg body weight/day to 30mg/kg body weight/day, for example: 10mg/kg body weight/day, 15mg/kg body weight/day, 20mg/kg body weight/day, 25mg/kg body weight/day, 30mg/kg body weight/day.
In a sixth aspect, the present invention provides the use of the bird's nest peptide ii of the first aspect or the polypeptide mixture of the second aspect for the preparation of an anti-inflammatory product.
Preferably, the anti-inflammatory product is a food, a health product, a cosmetic or a pharmaceutical product.
Preferably, the anti-inflammatory product comprises auxiliary materials which are allowed to be added in foods, health products, cosmetics or medicines.
Further, the administration dose of the bird's nest peptide II is 10mg/kg body weight/day to 30mg/kg body weight/day, for example: 10mg/kg body weight/day, 15mg/kg body weight/day, 20mg/kg body weight/day, 25mg/kg body weight/day, 30mg/kg body weight/day.
The invention has the beneficial effects that:
The bird's nest peptide II has double effects of inhibiting elastase and resisting inflammation, has the elastase inhibition rate of 80.75%, and can inhibit degradation of elastin in skin and delay skin aging. The polypeptide mixture containing the bird's nest peptide has wide application prospect in the fields of cosmetics, foods, medicines and the like, promotes the further development of bird's nest products, and improves the economic value and the utilization value of bird's nest.
Drawings
FIG. 1 is a schematic flow chart of separation, identification and functional study of bird's nest peptide in the invention.
Detailed Description
The technical scheme of the present invention will be clearly and completely described below with reference to the embodiments and the accompanying drawings. The described embodiments are only some, but not all, embodiments of the invention. Other embodiments, which are apparent to those of ordinary skill in the art based on the embodiments of the invention, are within the scope of the invention without making any inventive effort.
Unless otherwise indicated, all methods and reagents used in the examples of the present invention were conventional in the art.
The main reagents used in the examples are as follows:
Flavourzyme (S10153, shanghai-derived leaf biotechnology Co., ltd.), papain (S10011, shanghai-derived leaf biotechnology Co., ltd.), alkaline protease (S10154, shanghai-derived leaf biotechnology Co., ltd.).
Fmoc-Tyr (tBu) -WANG RESIN (substitution 0.4mmol/g, degree of crosslinking 1%, particle size 100-200 mesh), fmoc-Lys (Boc) -WANG RESIN (substitution 0.4mmol/g, degree of crosslinking 1%, particle size 100-200mesh)、Fmoc-Asp-OH、Fmoc-Ala-OH、Fmoc-Arg-OH、Fmoc-Phe-OH、Fmoc-Glu-OH、Fmoc-Leu-OH、Fmoc-Pro-OH、Fmoc-Val-OH、Fmoc-Tyr-OH、Fmoc-Gly-OH、Fmoc-Thr-OH、Fmoc-Ser-OH、Fmoc-Cys-OH were all purchased from Nanjing peptide industry Biotechnology Co., ltd.).
EXAMPLE 1 preparation of polypeptide mixtures
1.1 Preparation and use of polypeptide mixtures
(1) Foaming the bird's nest: the bird's nest (dry weight) is fully soaked in 20 times of water, the water temperature is 25 ℃, and the time is 2 hours.
(2) Crushing by a colloid mill: setting a tooth grinding gap of a colloid mill to be 100 mu m, and carrying out colloid mill treatment on the feed liquid obtained in the step (1) for 30min.
(3) High hydrostatic pressure assists enzymolysis: and (3) regulating the pH value of the feed liquid obtained in the step (2) to 7.5 by utilizing HCL and NaOH, adding flavourzyme (the enzyme adding amount is 7000U/mg), mixing, vacuum packaging in a polyethylene bag, immediately placing in HPP equipment, and treating for 12 hours under the conditions of 50 ℃ and hydrostatic pressure of 200 MPa.
(4) Enzyme deactivation and centrifugation: inactivating enzyme of the enzymolysis liquid obtained in the step (3) for 20min at 95 ℃, cooling to room temperature, centrifuging for 20min at 5000r/min by a centrifuge, and taking supernatant.
(5) Secondary alcohol precipitation and rotary evaporation concentration: adding absolute ethyl alcohol into the supernatant obtained in the step (4) to enable the volume fraction of the ethyl alcohol in a final system to reach 60%, and stirring and uniformly mixing. Standing for more than 10min, centrifuging at 3500r/min for 20min to separate precipitate and supernatant, transferring all supernatant to rotary steaming bottle, and rotary evaporating at 100deg.C in water bath at 100r/min to remove ethanol until the volume in bottle is basically unchanged. Pouring out the concentrated solution, adding a small amount of water for washing, recovering by a rotary steaming bottle, and measuring the total volume of the concentrated solution. And (3) adding absolute ethyl alcohol again according to the measured volume of the concentrated solution to ensure that the volume fraction of the ethyl alcohol in a final system reaches 60%, and stirring and uniformly mixing. Standing for more than 10min, centrifuging at 3500r/min for 20min, and separating precipitate and supernatant. The supernatant was again transferred to a rotary evaporator for rotary evaporation to remove ethanol.
(6) And (3) separation and preparation: and (3) subpackaging the concentrated solution obtained in the step (5) into glass plates, carrying out quick freezing at-80 ℃ for 12 hours on each plate by about 25-30 mL, and then freeze-drying in a freeze dryer for 24 hours to obtain a powdery polypeptide mixture.
(7) Sialic acid content determination: adding 1% phosphoric acid solution into the enzymolysis product powder, hydrolyzing in boiling water bath, and centrifuging to obtain supernatant. Adding phthalic diammine hydrochloride solution, mixing with supernatant in equal volume, derivatizing in a light-proof water bath at 80deg.C for 40min, cooling, filtering with 0.45mm filter membrane, and measuring sialic acid content by high performance liquid chromatography to obtain enzymolysis product with sialic acid content of 16.25wt%.
(8) Determination of glycopeptide ratio: measuring total sugar content by phenol-sulfuric acid method, weighing enzymolysis product powder, adding 1mL of 5% phenol and 5mL of concentrated sulfuric acid, mixing uniformly by vortex oscillation, standing at room temperature for 20min, measuring absorbance at 490nm, and measuring total sugar content of enzymolysis product to be 35.46%. And (3) measuring the total peptide content by adopting a biuret method, preparing an enzymolysis sample into a proper concentration, adding biuret (the sample to be detected is a biuret reagent=3:2, v/v), uniformly mixing on a vortex mixer, taking supernatant, measuring absorbance at 540nm, and measuring that the total peptide content of an enzymolysis product is 47.28%, and the glycopeptide ratio of the enzymolysis product is 0.75.
(9) Determination of polypeptide molecular weight distribution: the molecular weight distribution of the polypeptide mixture is determined by a high performance liquid chromatograph, then the glycosylase PNCaseF enzyme specificity is adopted to break off the glycopeptide bond, so that the sugar chains in the polypeptide are completely separated, then the component of the nidus Collocaliae polysaccharide is collected by a Sephadex G-100 sephadex column, the molecular weight of the sugar chains in the polypeptide mixture is determined, and the molecular weight distribution of the peptide chains in the polypeptide mixture is determined by the high performance liquid chromatograph, and the result is shown in a table 1.
TABLE 1 relative molecular weight distribution of polypeptide mixtures
As can be seen from Table 1, the relative molecular weight distribution of the polypeptide mixture is mainly concentrated at 4000-6000 Da, the percentage of peptides with molecular weight reaches 68.31%, the relative molecular weight distribution of sugar chains is mainly concentrated at 3000-4000 Da, the percentage of sugar chains with molecular weight reaches 66.37%, the relative molecular weight distribution of peptide chains is mainly concentrated at 1000-2000 Da, and the percentage of peptide chains with molecular weight reaches 70.26%.
Example 2 amino acid sequence analysis of polypeptide mixtures
And (3) sequencing and identifying the peptide chain amino acid sequence in the polypeptide mixture by adopting an ultra-high performance liquid chromatography ionization tandem mass spectrometer (UPLC-ESI-MS/MS). Specific chromatographic conditions: ACCLAIM PEPMAP C 18 column (75 μm. Times.25 cm), mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile solution containing 0.1% formic acid; the loading was 5.0. Mu.L and the elution flow rate was 300.0nL/min. The mass spectrum positive charge spray voltage was 2.0kV.
And then, carrying out database comparison by a Mascot Server on-line system to select 5 peptide fragments with high matching degree and relative strength of more than 10 9 for De novo analysis, wherein the result is shown in Table 2.
TABLE 2 peptide chain amino acid sequences in polypeptide mixtures
EXAMPLE 3 Synthesis of bird's nest peptide
3.1 Synthesis of nidus Collocaliae peptide I (SEQ ID NO: 1)
By adopting a solid phase synthesis process, synthesizing bird's nest peptide (Asp-Ala-Arg-Phe-Glu-Asp-Leu-Pro-Val-Tyr, SEQ ID NO: 1) according to the direction from the C end to the N end, wherein the specific process is as follows:
4.0g Fmoc-Tyr (tBu) -WANG RESIN with a substitution of 0.4mmol/g was weighed, added to the solid phase reaction column, washed twice with 20mL DMF, the solvent removed and swollen with 60mL DMF for 30min. Washing with DMF twice, adding piperidine-DMF mixed solution (volume ratio 1:3) and stirring for 20min, and monitoring the reaction completion by ninhydrin color development. Washing with DMF and DCM 5 times each, dissolving 2.17g (6.40 mmol) Fmoc-Val-OH and 1.04g (7.60 mmol) HOBt in DMF, adding 1.20mL (7.6 mmol) DIC under ice bath condition, adding into the solid phase reaction column after removing solvent after stirring for 8min in dark, adding 0.08g (0.64 mmol) DMAP, stirring under nitrogen protection for 3h, and monitoring the reaction completion by ninhydrin chromogenic method. The solvent was removed and washed 5 times with DMF to give Fmoc-Val-Tyr-WANG RESIN. According to the coupling method, corresponding Fmoc protected amino acid is added in sequence to the peptide, and the peptide chain is extended by condensation coupling in sequence. After the end of the last coupling reaction, the resin was washed 4 times with DCM, DMF, meOH portions each.
The resin was blown dry with nitrogen and transferred to a round bottom flask, and was cut 3 times with 209mL of acetic acid (HOAc) -Trifluoroethanol (TFE) -DCM mixed solution (volume ratio 1:3:6), with reaction times of 30min, 15min, and 5min in this order. Suction filtration, concentration of the filtrate to one fourth of the original volume, adding the concentrated solution into 10 times of diethyl ether for precipitation, and standing in a refrigerator overnight. Suction filtering, washing the filter cake with a small amount of diethyl ether for 6 times, and vacuum drying to obtain the fully protected crude product of the bird's nest peptide, wherein the yield is 90.60%.
Analysis and identification of synthetic bird's nest peptides according to the method of example 2: by adopting a method of sequencing and identification,
Dissolving the crude product of the cubilose peptide in a proper amount of DMSO, and separating and purifying by using a high performance liquid chromatograph, wherein a chromatographic column is a C18 reversed phase column, and eluent is obtained: solution A was an aqueous solution of 0.1% TFA, solution B was an aqueous solution of acetonitrile containing 0.1% TFA, and the detection wavelength was 220nm. And freeze-drying the purified liquid to obtain a finished product of the bird's nest peptide, wherein the purity of the finished product is 99.2%.
3.2 Synthesis of nidus Collocaliae peptide II-V
4 Peptide chains with the amino acid sequences shown in SEQ ID NO. 2-5 are synthesized according to the method of 3.1.
Example 4 determination of elastase inhibition Rate of bird's nest peptide
An elastase inhibition rate reaction system was prepared according to table 3, and the elastase inhibition rate was calculated according to formula (1). The specific process is as follows:
2mL of elastase PPE solution with concentration of 2mg/mL prepared by using a borate solution preheated at 37 ℃ is added with 2mL of the flavourzyme enzymolysis product solution with different concentrations, fully vortex and mix uniformly, and shake for 20min at 37 ℃ in a 400r/min shaking table. Immediately thereafter, 5mL of phosphate buffer (pH 6.0) at 0.5mol/L was added, and after vortexing, the mixture was centrifuged at 5000r/min for 15min, the supernatant was aspirated and its absorbance at 495nm was determined. The elastase inhibition rate of the bird's nest peptide was calculated and the results are shown in table 4.
TABLE 3 elastase inhibition ratio reaction System composition and volume
TABLE 4 elastase inhibition of bird's nest peptides
Note that bird's nest peptides I-V respectively represent sequences 1 (SEQ ID NO: 1) to 5 (SEQ ID NO: 5)
As can be seen from Table 4, the elastase inhibition rates of the bird's nest peptides I-III are significantly higher than that of the bird's nest peptides IV-V and the unhydrolyzed bird's nest, both being higher than 70%. Compared with unhydrolyzed nidus Collocaliae, the inhibition rates of elastase of the nidus Collocaliae peptides I-III are respectively increased by 48.21%, 32.36% and 21.79%, which indicates that the nidus Collocaliae peptide I, the nidus Collocaliae peptide II and the nidus Collocaliae peptide III in the nidus Collocaliae polypeptide mixture prepared by adopting the method of combining high hydrostatic pressure assisted enzymolysis and secondary alcohol precipitation have obvious inhibition effects on elastase. The bird's nest peptides I-V and unhydrolyzed bird's nest can delay the degradation of skin elastin and the decline of skin elasticity, wherein the bird's nest peptides I-III have stronger effect and are more suitable for developing products such as beauty, health care or medicines related to skin aging resistance.
Example 5 improvement of blood inflammation in mice by bird's nest peptide
5.1 Animal model establishment: 32C 57BL/6J male mice at 8 weeks of age were selected. The blank group, the model group, the bird's nest group and the bird's nest peptide group are divided into 8 groups according to the administration design. All mice were given a1 week adaptation period, and 0.2mL of 0.9% saline was given daily gavaged for the control and model groups at week 2, and the equivalent volume of nidus Collocaliae was gavaged for the nidus Collocaliae group, and the equivalent volume of nidus Collocaliae peptide (200 mg/kg/d) was gavaged for the nidus Collocaliae peptide group for 7 consecutive weeks. At the last 1 week of feeding, the control group was intraperitoneally injected with 0.2mL of 0.9% physiological saline and the other groups were intraperitoneally injected with an equal volume of LPS solution (2 mg/kg) for 1 week continuously, fasted for 12 hours, and the mice were sacrificed.
5.2 Blood index determination: blood cell counts including inflammatory cells such as leukocytes, neutrophils, lymphocytes, etc. were performed using fresh 50 μl whole blood with a fully automatic blood cell analyzer. And taking a proper amount of fresh blood, standing at room temperature for 30min, centrifuging at 4 ℃ for 15min at 3000r/min, taking supernatant to prepare serum, measuring the levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the serum by using a commercial Elisa kit, measuring the absorbance of a 96-well plate at 490nm according to the instruction of the kit, and quantifying the inflammatory factors according to a standard curve. The effect of bird's nest peptide on mouse hemogram index is shown in Table 5, and the effect of bird's nest peptide on mouse blood inflammatory factor is shown in Table 6.
TABLE 5 influence of bird's nest peptides on mouse hemogram index
The group I to the group V of bird's nest peptides respectively represent a group of sequences 1 (SEQ ID NO: 1) to a group of sequences 5 (SEQ ID NO: 5)
As can be seen from table 5, the model group showed significant differences in white blood cell, neutrophil, monocyte, lymphocyte content (p < 0.05) compared to the normal group, indicating successful modeling. After the mice are subjected to dry prognosis, each intervention group (bird's nest group and bird's nest peptide group) has obvious callback effect on the contents of leucocytes, neutrophils, monocytes and lymphocytes in the blood of the mice compared with the model group. The differences between the bird's nest peptides I-III group and the normal group are not obvious. However, compared with the bird's nest group, the mouse hemogram index taking the bird's nest peptide has more obvious callback effect, and the bird's nest peptides I-III have better effect.
TABLE 6 influence of bird's nest peptide on blood inflammatory factor of mice
The group I to the group V of bird's nest peptides respectively represent a group of sequences 1 (SEQ ID NO: 1) to a group of sequences 5 (SEQ ID NO: 5)
As can be seen from table 6, the model group has significant differences (p < 0.05) between the three inflammatory factors (TNF- α, IL-1β, IL-6) compared with the normal group, which indicates that the modeling was successful, and each intervention group (nidus Collocaliae group, nidus Collocaliae peptide group) has obvious callback effect on TNF- α, IL-1β, IL-6 content in blood of mice compared with the model group after the mice are subjected to dry prognosis. The differences between the bird's nest peptides I-III group and the normal group are not obvious. However, compared with the bird's nest group, the mice taking bird's nest peptides I-III have more obvious callback effect on inflammatory factors in blood. The bird's nest peptide prepared by the invention has obvious effect of improving the blood inflammation of mice, and the bird's nest peptides I-III have better effect.
Example 6 improvement of brain inflammation in mice with bird's nest peptide
6.1 Animal model establishment: the method for constructing the animal model was as in example 5.1.
6.2 Brain inflammatory factor determination: after obtaining the cerebral cortex of the mice, the cerebral cortex is homogenized, the levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum are measured by using a commercial Elisa kit, the operation steps are guided according to the description of the kit, the absorbance of a 96-well plate is measured at 490nm, and the inflammatory factors are quantified according to a standard curve. The results are shown in Table 7.
TABLE 7 Effect of bird's nest peptides on mouse brain inflammatory factors
The group I to the group V of bird's nest peptides respectively represent a group of sequences 1 (SEQ ID NO: 1) to a group of sequences 5 (SEQ ID NO: 5)
As can be seen from table 7, the three inflammatory factors (TNF- α, IL-1β, IL-6) all showed significant differences (p < 0.05) compared to the normal group, indicating that the modeling was successful, and the intervention group (nidus Collocaliae group, nidus Collocaliae peptide group) had significant callback effects on TNF- α, IL-1β, IL-6 content in the cerebral cortex of the mice compared to the model group after the mice were subjected to dry prognosis. The differences between the bird's nest peptides I-III group and the normal group are not obvious. However, compared with the bird's nest group, the mice taking bird's nest peptides I-III have more obvious callback effect on inflammatory factors in cerebral cortex. The bird's nest peptide prepared by the invention has obvious effect of improving brain inflammation of mice, and the bird's nest peptides I-III have better effect.
Example 7 improvement of liver inflammation in mice with bird's nest peptide
7.1 Animal model establishment: the method for constructing the animal model was as in example 5.1.
7.2 Liver inflammatory factor assay: after the liver of the mice is obtained, the liver is homogenized, the levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum are measured by using a commercial Elisa kit, the operation steps are guided according to the description of the kit, the absorbance of a 96-well plate is measured at 490nm, and the inflammatory factors are quantified according to a standard curve. The results are shown in Table 8.
TABLE 8 influence of bird's nest peptides on mouse liver inflammatory factors
The group I to the group V of bird's nest peptides respectively represent a group of sequences 1 (SEQ ID NO: 1) to a group of sequences 5 (SEQ ID NO: 5)
As can be seen from table 8, the three inflammatory factors (TNF- α, IL-1β, IL-6) all showed significant differences (p < 0.05) compared to the normal group, indicating successful modeling, and the intervention groups (nidus Collocaliae group, nidus Collocaliae peptide group) had significant callback effects on TNF- α, IL-1β, IL-6 content in the liver of mice compared to the model group after the mice were subjected to dry prognosis. The differences between the bird's nest peptides I-III group and the normal group are not obvious. However, compared with the bird's nest group, the mice taking bird's nest peptides I-III have more obvious callback effect on inflammatory factors in liver. The bird's nest peptide prepared by the invention has obvious effect of improving liver inflammation of mice, and the bird's nest peptides I-III have better effect.
Example 8 improvement of bird's nest peptide on pulmonary inflammation in mice
8.1 Animal model establishment: the method for constructing the animal model was the same as that of example 5.1.
8.2 Pulmonary inflammatory factor assay: after the lung of the mice is obtained, the lung is homogenized, the levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum are measured by using a commercial Elisa kit, the operation steps are guided according to the description of the kit, the absorbance of a 96-well plate is measured at 490nm, and the inflammatory factors are quantified according to a standard curve. The results are shown in Table 9.
TABLE 9 Effect of bird's nest peptides on mouse pulmonary inflammatory factors
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The group I to the group V of bird's nest peptides respectively represent a group of sequences 1 (SEQ ID NO: 1) to a group of sequences 5 (SEQ ID NO: 5)
As can be seen from table 9, the three inflammatory factors (TNF- α, IL-1β, IL-6) all showed significant differences (p < 0.05) compared to the normal group, indicating that the modeling was successful, and the intervention groups (nidus Collocaliae group, nidus Collocaliae peptide group) had significant callback effects on TNF- α, IL-1β, IL-6 content in the lung tissue of the mice compared to the model group after the mice were subjected to dry prognosis. The differences between the bird's nest peptides I-III group and the normal group are not obvious. However, compared with the bird's nest group, the mice taking the bird's nest peptide have more obvious callback effect on inflammatory factors in lung tissues. The bird's nest peptide prepared by the invention has obvious improvement effect on lung inflammation, and the bird's nest peptides I-III have better effect.
The experiment shows that the bird nest peptide prepared by the method of combining high hydrostatic pressure assisted enzymolysis with secondary alcohol precipitation has obvious effect of improving inflammation in blood, brain, liver and lung of mice, and the improvement effect is higher than that of bird nest groups taken, and the bird nest peptide can be applied to production of foods, health care products and medicines by decomposing bird nest into polypeptides with smaller molecular weight to exert the anti-inflammatory effect.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the content of the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (8)
1. The bird's nest peptide II with the effects of protecting skin elasticity and resisting inflammation is characterized in that: the amino acid sequence of the bird's nest peptide II is DPFYGGEYLK.
2. A polypeptide mixture characterized by: the polypeptide mixture comprises the bird's nest peptide II of claim 1.
3. A composition characterized by: the composition comprises the bird's nest peptide II as claimed in claim 1 or the polypeptide mixture as claimed in claim 2.
4. A composition according to claim 3, wherein: the composition also comprises auxiliary materials which are allowed to be added in foods, health care products, cosmetics or medicines.
5. Use of the cubilose peptide of claim 1 or the polypeptide mixture of any one of claims 2-3 for the preparation of a product having elastase inhibitory activity.
6. Use of the cubilose peptide of claim 1 or the polypeptide mixture of any one of claims 2-3 for the preparation of an anti-inflammatory product.
7. Use according to claim 5 or 6, characterized in that: the administration dosage of the bird's nest peptide II is 10mg/kg body weight/day-30 mg/kg body weight/day.
8. Use according to claim 5 or 6, characterized in that: the product is food, health product, cosmetic or medicine.
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| CN114848791A (en) * | 2022-04-20 | 2022-08-05 | 厦门市燕之屋丝浓食品有限公司 | Application of small molecular cubilose peptide for preventing and improving skin inflammation |
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| CN114848791A (en) * | 2022-04-20 | 2022-08-05 | 厦门市燕之屋丝浓食品有限公司 | Application of small molecular cubilose peptide for preventing and improving skin inflammation |
| CN116120420A (en) * | 2022-05-16 | 2023-05-16 | 魏珂 | Bird's nest polypeptide composition, preparation method thereof and application thereof in anti-aging and whitening |
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