CN110724178B - Tuna white meat ACE inhibitory peptide and preparation method thereof - Google Patents
Tuna white meat ACE inhibitory peptide and preparation method thereof Download PDFInfo
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
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- A61P9/12—Antihypertensives
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
An ACE inhibitory peptide of tuna white meat and a preparation method thereof relate to the field of preparation of the ACE inhibitory peptide, the peptide segment amino acid sequence of the ACE inhibitory peptide of the tuna white meat is Ser-Pro or Val-Asp-Arg-Tyr-Phe, and the preparation method comprises the following preparation steps: 1) unfreezing tuna white meat, drying to remove water, and then degreasing and drying; 2) adding distilled water into the pretreated white meat powder, adjusting the pH value, and adding protease for enzymolysis to obtain an enzymolysis solution; 3) carrying out enzyme deactivation treatment on the enzymolysis liquid, centrifuging and taking supernatant liquid to obtain enzyme deactivation enzymolysis liquid, freezing and drying to obtain polypeptide powder, and detecting ACE inhibitory activity; 4) and then carrying out ultrafiltration, column chromatography, high performance liquid chromatography purification and amino acid sequencing on the enzyme-inactivated enzymolysis liquid to obtain the ACE inhibitory peptide of the tuna white meat.
Description
Technical Field
The invention relates to the field of preparation of ACE inhibitory peptides, and particularly relates to an ACE inhibitory peptide of tuna white meat and a preparation method thereof.
Background
Hypertension is a systemic disease characterized by the rise of arterial systolic pressure or (and) diastolic pressure, and leads to various complications such as cerebral apoplexy, myocardial infarction, heart failure, dementia, liver and kidney failure, blindness and the like, and has become an important public health problem in China and even the world. The regulation of blood pressure in humans is associated with a variety of neurological and humoral systems, such as the sympathetic nervous System, the Renin-Angiotensin System (RAS), the Kallikrein-Kinin System (KKS), the renal and humoral balance systems, and the nitric oxide-endothelin System. Among them, the renin-angiotensin system and the kallikrein-kinin system play an important role in the regulation of blood pressure. The RAS system and the KKS system interact with each other to jointly realize the regulation of the body on the blood pressure. Wherein, RAS system is responsible for boosting pressure, while renin transforms angiotensinogen from liver into angiotensin I, ACE transforms angiotensin I into angiotensin II, and then acts on angiotensin II receptor in tissue to promote vasoconstriction and raise blood pressure; the KKS system is responsible for lowering blood pressure, bradykinin increases the production of prostaglandins and nitric oxide leading to vasodilation, decreased peripheral vascular resistance and hence to a decrease in blood pressure, but ACE degrades bradykinin leading to bradykinin losing vasodilation leading to an increase in blood pressure. Therefore, the ACE inhibitory peptide has the effect of reducing blood pressure, at present, the preparation method of the antihypertensive peptide mainly comprises a microbial fermentation method, a natural active peptide extraction method and a synthesis method, however, the natural active peptide is directly extracted from organisms by the extraction method, the cost is higher, and the extraction efficiency is lower; the microbial fermentation method is a method for preparing ACE inhibitory peptide by utilizing the self fermentation and maturation effects of animal products such as milk, cheese and the like, and although the method is low in cost, the operation is complex; the synthesis method has relatively high cost, is suitable for directional synthesis in a laboratory and is not suitable for industrial production. For example, a study on the separation and purification and hypolipidemic activity of yolk polypeptides disclosed in the chinese literature, which takes yolk proteins as a test material and systematically studies the preparation process, separation and purification method and hypolipidemic activity of hydrolyzed polypeptides, but the extraction cost is high and the extraction efficiency is low.
Disclosure of Invention
The invention provides an ACE inhibitory peptide of tuna white meat and a preparation method thereof, aiming at overcoming the problems that the existing antihypertensive peptide is high in cost, low in efficiency, not suitable for industrial production, low in antihypertensive activity and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the peptide fragment amino acid sequence of the tuna white meat ACE inhibitory peptide is Ser-Pro or Val-Asp-Arg-Tyr-Phe.
The ACE inhibitory peptide with the amino acid sequences of two peptide segments of Ser-Pro and Val-Asp-Arg-Tyr-Phe is successfully extracted and purified from the white tuna meat by an enzymolysis method, and has an obvious blood pressure reducing effect through evaluation of an FAPGG-ACE in-vitro evaluation model and a Human Umbilical Vein Endothelial Cell (HUVEC) model.
A preparation method of ACE inhibitory peptide of tuna white meat comprises the following steps:
1) unfreezing tuna white meat, drying to remove water, degreasing, drying and crushing for later use;
2) adding distilled water into the pretreated white meat powder, adjusting the pH value, and adding protease for enzymolysis to obtain an enzymolysis solution;
3) carrying out enzyme deactivation treatment on the enzymolysis liquid, then centrifuging to obtain supernatant liquid to obtain enzyme deactivation enzymolysis liquid, freezing and drying to obtain polypeptide powder, and detecting ACE inhibitory activity;
4) and then carrying out ultrafiltration, column chromatography, high performance liquid chromatography purification and amino acid sequencing on the enzyme-inactivated enzymolysis liquid to prepare the tuna white meat ACE inhibitory peptide.
The invention takes ACE inhibitory activity as an index, extracts the ACE inhibitory peptide of tuna white meat by an enzymolysis method, optimizes the enzymolysis process by three factors (temperature, enzyme addition amount and pH), then prepares a peptide segment with the ACE inhibitory activity by a series of purification technologies of ultrafiltration, Sephadex G-25 gel chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and further evaluates the antihypertensive activity of the peptide segment by a Human Umbilical Vein Endothelial Cell (HUVEC) model, thereby having obvious antihypertensive effect.
Preferably, the degreasing in step 1) is: add ethyl acetate to tuna, immerse for 40-60h, then rotary evaporate.
Ethyl acetate is added into tuna for degreasing, so that the subsequent extraction of ACE inhibitory peptide is facilitated, and rotary evaporation is used for recovering ethyl acetate.
Preferably, the mass ratio of the white meat powder to the distilled water in the step 2) is 1-3: 20.
In this ratio, the enzymolysis effect is better.
Preferably, the pH value is adjusted to 8.5-10.5 in step 2).
Preferably, the amount of enzyme added in step 2) is 1 to 3 wt%.
Preferably, the enzymolysis temperature in the step 2) is 45-65 ℃.
The degree of enzymatic hydrolysis has a significant influence on the relative content of free amino acids in the product and on the ACE inhibitory activity. When the degree of enzymatic hydrolysis is insufficient, amino acid residues having ACE inhibitory activity cannot be exposed, and ACE inhibitory activity is absent. In the initial phase of proteolysis, ACE inhibition is enhanced with exposure to free amino acids. However, as proteolysis proceeds, the inhibitory effect of ACE on the hydrolysate peaks and then gradually diminishes. This may be because ACE inhibiting peptides are released at the beginning of proteolysis, resulting in an increased ACE inhibiting activity of the hydrolysate. When the hydrolysis reaches a certain degree, part of ACE inhibitory peptide is further hydrolyzed, the complete structure of the ACE inhibitory peptide is destroyed, and weak ACE inhibitory peptide is generated. Therefore, the three factors in the enzymolysis process are crucial to the preparation of the ACE inhibitory peptide, and through a large number of experiments, the enzymolysis process parameters in the range have a good enzymolysis effect, and the optimal enzymolysis process is obtained through experiments.
Preferably, the ultrafiltration step in the step 4) is to perform ultrafiltration classification on the tuna white meat enzyme-killed enzymatic hydrolysate by using an ultrafiltration membrane under the conditions of 35.1-35.6Hz and 0.5-1.2pa, and freeze-dry product components to obtain the enzymatic hydrolysate powder.
After the enzymolysis liquid is subjected to ultrafiltration and classification, the ACE inhibitory activity is detected and is used for screening the component with the best inhibitory activity in the enzymolysis liquid.
Preferably, the ultrafiltration membrane in the step 4) has a molecular weight cut-off of 3.5 KDa.
The component with the molecular weight less than 3.5KDa can be obtained by using the ultrafiltration membrane with the molecular weight cutoff of 3.5KDa, and the ACE inhibitory activity of the component is higher.
Preferably, the chromatography step described in step 4) is to dissolve and filter the enzymatic hydrolysate powder, and perform Sephadex G-25 gel filtration chromatography, followed by freeze drying.
Filtration chromatography can further isolate and purify ACE inhibiting peptides.
Therefore, the invention has the following beneficial effects: the tuna white meat ACE inhibitory peptide prepared by the invention has good inhibitory activity, low preparation cost and high efficiency, is suitable for industrial production, develops a new idea for the development of polypeptide antihypertensive drugs and the high-valued development of processing byproducts, has wide application prospect, and also provides scientific reference for the deep development and comprehensive utilization of tunas.
Drawings
FIG. 1 shows ACE activity inhibition rate of polypeptide powder at different pH values in the preparation process of the present invention.
FIG. 2 shows the ACE activity inhibition ratio of polypeptide powder at different enzymolysis temperatures in the preparation process of the invention.
FIG. 3 shows the ACE activity inhibition ratio of polypeptide powder at different enzyme dosages in the preparation process of the invention.
FIG. 4 shows the ACE activity inhibition ratios of different ultrafiltrate molecular weight enzyme powders during the preparation process of the present invention.
FIG. 5 is a Sephadex G-25 gel column chromatography elution profile during the preparation of the present invention.
FIG. 6 shows ACE activity inhibition ratios of different Sephadex G-25 gel column chromatography elution peaks in the preparation process of the present invention.
FIG. 7 is a spectrum of a Zorbax SB-C18 reversed-phase high performance liquid phase during the preparation of the present invention.
FIG. 8 is a standard curve of the protein of the present invention.
FIG. 9 is a standard curve of ET-1 of the present invention.
FIG. 10 is a graph showing the effect of ACE inhibitory peptides L1 and L2 of the present invention on the proliferative activity of HUVEC.
FIG. 11 is a graph showing the effect of different concentrations of ACE inhibitory peptides L1 and L2 of the present invention on NO content in human umbilical vein endothelial cells (# # P < 0.01, # P < 0.05 vs Control; P < 0.01, # P < 0.05 vs NE).
FIG. 12 is a graph showing the effect of different concentrations of ACE inhibitory peptides L1 and L2 of the present invention on ET-1 levels in human umbilical vein endothelial cells (P # 0.01, # P < 0.05 vs Control; P # 0.01, # P < 0.05 vs NE).
Detailed Description
The invention is further described with reference to specific embodiments.
Examples 1 to 15: a preparation method of ACE inhibitory peptide of tuna white meat comprises the following steps:
1) thawing white tuna meat, drying to remove water, adding ethyl acetate into the tuna, immersing for degreasing, performing rotary evaporation, drying, and crushing for later use;
2) adding distilled water into the pretreated white meat powder, adjusting pH to 8.5-10.5, adding 1-3 wt% of alkaline protease, and performing enzymolysis at 45-65 deg.C for 3 hr to obtain enzymolysis solution;
3) heating the enzymolysis solution in a water bath at 100 ℃ for 10min for enzyme deactivation, centrifuging at 4000r for 20min, taking supernatant to obtain enzyme-deactivated enzymolysis solution, freeze-drying to obtain polypeptide powder, and measuring ACE inhibitory activity;
4) carrying out ultrafiltration classification on the tuna white meat enzymolysis liquid by using an ultrafiltration membrane of 3.5KDa, and freeze-drying product components to obtain enzymolysis powder; and then preparing the zymolyte powder and ultrapure water into a solution with the concentration of 50mg/mL, centrifuging at 12000r/min for 10min at 4 ℃, removing insoluble impurities, performing filtration chromatography by using activated Sephadex G-25, freeze-drying, preparing into a solution with the concentration of 100mg/mL, filtering by using a 0.45-micron microporous membrane, performing gradient elution by using acetonitrile-water-trifluoroacetic acid (TFA) as an eluent with the flow rate of 2mL/min, performing purification analysis by using a high performance liquid chromatography column Zorbax SB-C18, sequencing amino acids, and synthesizing to obtain the tuna white meat ACE inhibitory peptide.
Table 1: examples 1-15 preparation conditions.
In the preparation process of the embodiment, the method for detecting the ACE inhibitory activity of the polypeptide powder comprises the steps of using FAPGG as a substrate and using an enzyme-labeling method to determine the ACE inhibitory activity, and specifically comprises the following steps of adding 10 mu L of ACE solution (0.1U/mL) and 40 mu L of polypeptide hydrolysate into micropores of a microporous plate without mixing, and then adding 50 mu L of substrate (preheated at 37 ℃ for 15 minutes) to start reaction. The microplate was quickly placed in a microplate reader at 37 ℃ and the absorbance at 340nm wavelength was recorded 1 time every 5min for a total of 30 min. Blank control 40. mu.L of HEPES buffer was used instead of polypeptide solution. The slope was calculated by plotting the absorbance (. DELTA.A 340nm) against time. The formula for calculating the ACE inhibition rate is as follows:
wherein ACEI is ACE activity inhibition rate; delta Ac is the change of absorbance within 30min when buffer is added; Δ Ai is the change in absorbance over 30min upon addition of inhibitor.
Examples 1-5 the effect of different pH on the inhibition of ACE activity is shown in FIG. 1, where the pH is 8.5, 9.0, 9.5, 10.0 and 10.5, and the inhibition of ACE activity is highest at pH9.5 (62.45%), indicating that the extent of enzymolysis of tuna white meat is greater. The pH value is in a rapid rising trend at 8.5-9.5, and the pH value begins to drop after 9.5. Therefore, pH9.5 is the optimum pH for alkaline protease digestion.
The influence of different enzymolysis temperatures on the ACE activity inhibition rate in examples 3 and 6-9 is shown in FIG. 2, and the enzymolysis temperatures are 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, and 65 deg.C, respectively; as can be seen, the tuna white meat has a low degree of hydrolysis at low temperatures, such as 45 ℃ and 50 ℃, and the ACE inhibition rate is low; along with the temperature rise, the catalytic activity of the enzyme is enhanced, the enzymolysis speed is accelerated, the ACE inhibition rate is enhanced, and the ACE inhibition rate of the product reaches a maximum value of 68.33 percent until the temperature is 55 ℃; however, when the temperature is higher than 55 ℃, the ACE inhibition rate begins to decrease, which indicates that the enzymolysis degree of the protein is decreased. This is because the protease activity is reduced under high temperature conditions, resulting in a decrease in the catalytic activity of the enzyme and a decrease in the ACE inhibition rate of the product. Therefore, the optimum temperature for the action of alkaline protease on the white meat of tuna is 55 ℃.
The effect of different enzyme dosages in examples 3, 10-13 on the ACE activity inhibition rate is shown in FIG. 3, and the enzyme dosages are 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, and 3 wt%, respectively. As can be seen from the graph, when the amount of the enzyme is less than 1.5 wt%, the amount of the mutual binding between the enzyme molecules and the substrate protein molecules increases with the increase of the amount of the enzyme, and thus the content of the enzymatic hydrolysis products gradually increases; however, once all substrate molecules are saturated with enzyme molecules, if the enzyme amount is increased, the produced ACE inhibitory component is over-hydrolyzed or even inactivated, and the ACE inhibitory rate of the hydrolysate is gradually decreased. Therefore, the optimum enzyme adding amount of the alkaline protease for enzymolysis of the tuna white meat is 1.5 wt%.
Comparative example 1: the difference from example 11 is that the ultrafiltration membranes used have molecular weight cut-offs of 3.5kDa and 5kDa, which give fractions with molecular weights of 3.5-5 kDa.
Comparative example 2: the difference from example 11 is that the ultrafiltration membrane used has molecular weight cut-offs of 5kDa and 10kDa, which give a fraction with a molecular weight of 5-10 kDa.
Comparative example 3: the difference from example 11 is that the ultrafiltration membrane used has a molecular weight cut-off of 10kDa, which gives a fraction with a molecular weight of more than 10 kDa.
The ultrafiltered products of example 11 and comparative examples 1-3 were labeled TLMH-I (<3.5kDa), TLMH-II (3.5-5kDa), TLMH-III (5-10kDa) and TLMH-IV (>10kDa), respectively, and after lyophilization, solutions of 1.0mg/mL protein concentration were prepared and the ACE inhibitory activity of each fraction was determined, the results of which are shown in FIG. 4. As can be seen, the ACE inhibitory activity of the component TLMH-I is the strongest, the activity of the component TLMH-II is the second to the smallest, and the activity of the component TLMH-IV is the smallest. The results show that the ACE inhibitory activity of the ultrafiltration fraction increases with decreasing molecular weight distribution range. The ultrafiltration with the molecular weight cutoff of 3.5kDa is used, the enrichment of small molecular substances can be realized by intercepting macromolecular substances in a sample, the TLMH-I component is enriched with polypeptide with the molecular weight of less than 3.5kDa, and the component is more likely to be enriched with ACE inhibitory peptide than other three components, so the ACE inhibitory activity of the component is the highest.
The enzyme powder prepared in example 11 and ultrapure water were mixed to prepare a solution with a concentration of 50mg/mL, and centrifuged at 12000r/min at 4 ℃ for 10min to remove insoluble impurities, Sephadex G-25 activated Sephadex was used for filtration chromatography (loading concentration of 50mg/mL, injection volume of 3mL, elution rate of 0.7mL/min), the elution results are shown in FIG. 5, there are 5 elution peaks, which are respectively marked as A1, A2, A3, A4 and A5, and the solutions in the tubes were combined according to the peaks, and after lyophilization, five components were mixed to prepare sample solutions of 1.0mg/mL to measure ACE inhibitory activity, and the results are shown in FIG. 6, where the A4 component has the best pressure-reducing activity, and the ACE inhibitory rate of 81.87%, and is higher than that of the other components.
The a4 fraction prepared in example 11 was dispensed into a solution with a concentration of 100mg/mL, filtered through a 0.45 μm microporous membrane, gradient eluted with acetonitrile-water-trifluoroacetic acid (TFA) as eluent at a flow rate of 2mL/min, purified and analyzed by a high performance liquid chromatography column Zorbax SB-C18, and the results are shown in fig. 7, in which 6 main peaks were obtained by RP-HPLC separation, and the N-terminal sequencing and mass spectrometry of the components of the 6 peaks were performed to determine a total of 6 polypeptide sequences, which were: Ser-Pro (L1, 202.21Da), Val-Asp-Arg-Tyr-Phe (L2, 698.78Da), Val-His-Gly-Val-Val (L3, 509.61Da), Tyr-Glu (L4, 310.31Da), Phe-Glu-Met (L5, 425.51Da) and Phe-Trp-Arg-Val (L6, 606.73Da), determining ACE inhibition rates of 6 peptide segments at different concentrations, carrying out statistics by using SPSS statistical software, and calculating IC50 values of the 6 peptide segments, wherein the values are respectively: l1(IC 50-0.064 mg/mL), L2(IC 50-0.284 mg/mL), L3(IC 50-0.901 mg/mL), L4(IC 50-0.800 mg/mL), L5(IC 50-2.179 mg/mL), L6(IC 50-0.755 mg/mL). IC50(half maximum inhibition concentration) refers to the half inhibitory concentration of the antagonist measured. It can indicate that a drug or substance (inhibitor) is inhibiting a certain biological process or is half the amount of a certain substance (contained in this process). Among them, the peptide fragment with the amino acid sequence of L1 being Ser-Pro has the best ACE inhibitory activity. The C-terminal tripeptide structure of the ACE inhibitory peptide plays a key role in the antihypertensive activity of the ACE inhibitory peptide, the inhibitory activity of the ACE inhibitory peptide is strongest when the C-terminal amino acid of the ACE inhibitory peptide is a short peptide of an aromatic amino acid (including tryptophan, tyrosine and phenylalanine) or Pro (proline) residue, and the ACE inhibitory activity of the polypeptide is stronger when the C-terminal tripeptide of the polypeptide contains branched amino acids (Ile, Leu and Val). In addition, the peptide of which the N terminal is hydrophobic valine, leucine, isoleucine or basic amino acid has strong affinity with ACE and high inhibitory activity, except proline; leucine, valine and isoleucine are collectively referred to as branched chain amino acids. The amino acid sequence of L1 is Ser-Pro, and the amino acid at the C terminal is Pro (proline) residue, so L1 has better ACE enzyme inhibition activity. The L2(Val-Asp-Arg-Tyr-Phe) obtained in the invention is pentapeptide, but the structure characteristics of the C terminal also accord with the structure-activity relationship rule of ACE inhibition short peptide, the C terminal Tyr and Phe aromatic amino acid, and the N terminal Val is hydrophobic valine, so the activity is also high, which is next to the L2 peptide segment.
In the experimental data, the established FAPGG-ACE in vitro evaluation model shows better ACE inhibitory activity, but the blood pressure reducing activity of the model is still verified by a biological level experiment, so that the blood pressure reducing activity of the model is further evaluated by a Human Umbilical Vein Endothelial Cell (HUVEC) model.
Culturing human umbilical vein endothelial cells: HUVEC were cultured using high-glucose DMEM + FBS + diabatic (penicillin-streptomycin) medium (DMEM: FBS: diabatic-9: 1: 0.1). The culture process is as follows: putting the recovered cells into an incubator containing 5% CO2, culturing at 37 ℃, changing the liquid after 24 hours, digesting with pancreatin when the cells grow and fuse to cover more than 85% of the bottom of the bottle, and carrying out passage at a ratio of 1:2 to two bottles. HUVEC in logarithmic growth phase was taken as experimental material.
Experimental grouping and treatment: HUVEC cells in logarithmic growth phase were taken, medium was discarded, washed twice with PBS, trypsinized, plated at 2.4X 105/well and randomly grouped as follows:
(1) blank control group: treating the cells without adding any reagent;
(2) ACE inhibitory peptide low dose group: adding peptide to a final concentration of 100. mu.M;
(3) ACE inhibitory peptides medium dose groups: adding peptide to a final concentration of 200. mu.M;
(4) ACE inhibitory peptide high dose group: peptide was added to a final concentration of 400. mu.M;
(5) captopril (Cap) group: adding Cap with the final concentration of 1 mu M;
(6) noradrenaline (NE) group: NE was added to a final concentration of 0.5. mu.M
(7) Treatment groups: peptide and NE were added to final concentrations of 200. mu.M and 0.5. mu.M, respectively.
Cytotoxicity assay (MTT method): HUVEC cells are adjusted to be cell suspension of 0.8 × 104 cells/well, then inoculated to a 96-well plate, 160 μ L/well is placed in a 5% CO2 incubator, after 24 hours of culture at 37 ℃, 20 μ L of complete culture solution and 20 μ L of PBS are added to a blank well, 20 μ L of complete culture solution and 20 μ L of samples with final concentrations of 25 μ M, 50 μ M, 100 μ M, 200 μ M and 400 μ M respectively (dissolved in PBS and insoluble in DMSO), are added to a sample well, the sample well is placed in a 5% CO2 incubator, 20 μ L of LMTT solution is added after 24 hours of culture at 37 ℃, culture medium is discarded after 4 hours of culture at 37 ℃, the reaction is uniform by being shaken in dark at 37 ℃ for 10 minutes, OD490nm values are measured on a microplate reader, and the relative survival rate of the cells is measured.
Determination of total protein content: under alkaline environment, the protein converts Cu2+Reduction to Cu+，Cu+And forming a bluish purple complex with the BCA reagent, wherein the complex has specific absorption at 562nm, measuring the absorbance value at the wavelength, comparing with a standard curve, and calculating the protein concentration of the substance to be detected. The microplate reader method operates as follows:
and (1) preparing a BCA working solution. Preparing working solution according to the proportion of 50:1 of the BCA reagent and the Cu reagent according to the quantity of the standard substance and the sample, and fully and uniformly mixing;
2. and drawing a standard curve. Preparing BSA standard solution with final concentration of 0.5mg/mL (taking 10 μ LBSA standard and diluting to 100 μ L with PBS), and sequentially adding into a 96-well plate according to the proportion in Table 2;
table 2: BCA kit standard curve sample adding device
Mixing, standing at 37 deg.C for 15-30min, and measuring the light absorption value at 562nm with multifunctional microplate reader. And drawing a protein standard curve by taking the protein content (g/L) as an abscissa and the light absorption value as an ordinate. The obtained protein standard curve is shown in fig. 8, and the curve equation is that y is 0.7464x +0.1304, R20.9918, in good linear relationship. The cellular protein concentration of the sample can be calculated by the equation so as to facilitate the use of the NO kit in the later period;
3. and (4) sample determination. The cell samples after ultrasonication were diluted appropriately with standard PBS, 20. mu.L of the diluted solution was added to a 96-well plate, and then 200. mu.L of the working solution was added. The absorbance at 562nm was measured according to the above procedure, and the total protein content of the sample was calculated from the standard curve.
Determination of NO content: discarding the six-well plate culture medium, adding PBS to wash the cultured cells for two times, adding 2ml PBS, scraping the cells from the six-well plate by a cell scraper to prepare suspension, and ultrasonically crushing the suspension under the ice bath condition to prepare the suspension. After the pretreatment is finished, the operation is carried out according to the steps of the NO kit. The calculation formula is as follows:
determination of ET-1 content: the kit adopts a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). And sequentially adding a specimen, a standard substance and a detection antibody marked by Horse Radish Peroxidase (HRP) into the coated micropores previously coated with the endothelin 1(ET-1) antibody, incubating at constant temperature and thoroughly washing. Color development was performed with the substrate Tetramethylbenzidine (TMB), which was converted to a blue species catalyzed by peroxidase and to the final yellow species by the action of an acid. The shade of the color and the content of endothelin 1(ET-1) in the sample showed a positive correlation. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the concentration of the sample was calculated by drawing a standard curve. The standard curve is shown in fig. 9, in which the concentration of the standard is plotted on the abscissa and the corresponding OD value is plotted on the ordinate, and the concentration value of each sample is calculated according to the curve equation. The obtained standard curve is shown in fig. 9, the regression equation is that y is 0.0029x +0.0649, and R2 is 0.991, and the fitting capability is good.
The effect of ACE inhibitory peptides L1 and L2 on the proliferative activity of HUVECs is shown in fig. 10, where it is seen that there is no significant difference in the growth inhibition of HUVECs by the polypeptides L1 and L2 at a concentration ranging from 25 to 400 μ M, compared to the blank control group. Indicating that the L1 and L2ACE inhibitory peptide have no obvious toxic effect on HUVEC under the series of concentrations.
The influence of different concentrations of ACE inhibitory peptides L1 and L2 on NO content of human umbilical vein endothelial cells is shown in fig. 11, and it can be seen that the NO content of Cap group, L1 and L2 group all showed an upward trend and have significant difference compared with the blank control group, which indicates that L2 of L1 promotes cells to release NO similarly to captopril. Compared with the blank group, the NE group has significant difference, which indicates that the NE can significantly inhibit cells from releasing NO. The effect of L1 on promoting NO release from cells was concentration dependent, whereas the effect of L2 at doses promoting NO release from cells was the best. After the NE and the L1 or L2 with the medium dose are added simultaneously, the NO content is obviously increased compared with that of a pure NE group, which shows that the L1 and the L2 with certain concentrations can resist the effect of the NE on NO release.
The effect of different concentrations of ACE inhibiting peptides L1 and L2 on ET-1 content in human umbilical vein endothelial cells is shown in fig. 12, which shows that Cap group, L1 group and L2 group can significantly reduce the content of ET-1 in cells compared to the blank control group. Therefore, L1 has similar action with Cap, and can play a role in lowering blood pressure by inhibiting the release of ET-1 in vascular endothelial cells, and the relation between the action and the dosage is obvious. Like the effect of promoting cellular NO release, the high dose of L1 and the medium dose of L2 inhibited ET-1 release most strongly. The simultaneous addition of L1 or L2 and NE has significant difference compared with the NE group, which shows that L1 and L2 with certain concentration can significantly resist the release effect of NE promoting ET-1.
Therefore, L1 and L2 with relatively good ACE inhibitory activity can promote the release of NO in HUVEC cells and inhibit the generation of ET-1, and the combination of two effects is preliminarily demonstrated: under the in vivo environment, the two ACE inhibitory peptides can exert obvious blood pressure reducing effect through the influence on the function of vascular endothelial cells, are blood pressure reducing peptides with better activity, and have further research prospect.
Claims (1)
1. An ACE inhibitory peptide of tuna white meat is characterized in that the amino acid sequence of the inhibitory peptide is Val-Asp-Arg-Tyr-Phe.
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| CN113005166B (en) * | 2021-04-14 | 2022-08-16 | 中国海洋大学 | Cod polypeptide with xanthine oxidase inhibitory activity |
| CN113912704B (en) * | 2021-08-31 | 2023-09-26 | 北京化工大学 | Fish-source collagen hydrolysate capable of repairing alcohol-damaged human gastric mucosal cells and preparation method and application thereof |
| CN113801195B (en) * | 2021-10-15 | 2023-06-27 | 浙江海洋大学 | Tuna roe antihypertensive peptide and application thereof |
| CN114836500B (en) * | 2022-01-26 | 2024-04-30 | 青岛孺子牛生物技术有限公司 | Production and preparation method for forming functional polypeptide by utilizing enzymolysis of soybean meal |
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| Impact of processing on stability of angiotensin I-converting enzyme (ACE) inhibitory peptides obtained from tuna cooking juice;Hwang J S;《Food Research International》;20101231;第43卷(第3期);全文 * |
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