CN110724178A - Tuna white meat ACE inhibitory peptide and preparation method thereof - Google Patents

Abstract

A tuna white meat ACE inhibitory peptide and a preparation method thereof relate to the field of preparation of ACE inhibitory peptides. The peptide segment amino acid sequence of the tuna white meat ACE inhibitory peptide is Ser-Pro or Val-Asp-Arg-Tyr-Phe, comprising the following preparation steps: 1 ) Thawing and drying tuna white meat to remove water, then defatting and drying; 2) Adding the pretreated white meat powder to distilled water, adjusting the pH value, adding protease for enzymatic hydrolysis to obtain an enzymatic hydrolysis solution; 3) Putting the enzyme The solution was subjected to enzyme-killing treatment, and the supernatant was centrifuged to obtain the enzyme-killing enzymatic solution, which was freeze-dried to obtain polypeptide powder, and the ACE inhibitory activity was measured; 4) The enzyme-killing enzymatic solution was then subjected to ultrafiltration, column chromatography, high efficiency The tuna white meat ACE inhibitory peptide is obtained by liquid chromatography purification and amino acid sequencing. The tuna white meat ACE inhibitory peptide prepared by the invention has good inhibitory activity, low preparation cost and high efficiency, and is suitable for industrial production.

Description

A kind of tuna white meat ACE inhibitory peptide and preparation method thereof

technical field

The invention relates to the field of preparation of ACE inhibitory peptides, in particular to an ACE inhibitory peptide of tuna white meat and a preparation method thereof.

Background technique

Hypertension is a systemic disease characterized by elevated arterial vaso-systolic or (and) diastolic blood pressure, leading to stroke, myocardial infarction, heart failure, dementia, liver and kidney failure, blindness and other complications. It is an important public health problem in our country and even in the world. The regulation of blood pressure in the human body is related to a variety of nervous and humoral systems, such as the sympathetic nervous system, the renin-angiotensin system (RAS), the kallikrein-kinin system (KKS) , kidney and body fluid balance system and nitric oxide-endothelin system, etc. Among them, the renin-angiotensin system and the kallikrein-kinin system play an important role in the regulation of blood pressure. The two systems of RAS and KKS interact to realize the regulation of blood pressure by the body. Among them, the RAS system is responsible for raising blood pressure, while renin converts angiotensinogen from the liver to angiotensin I, and ACE converts angiotensin I to angiotensin II, which then acts on angiotensin in tissues II receptors act to induce vasoconstriction, resulting in increased blood pressure; the KKS system is responsible for lowering blood pressure, and bradykinin can increase the production of prostaglandins and nitric oxide, resulting in vasodilation, decreased peripheral vascular resistance, and decreased blood pressure However, ACE can degrade bradykinin, so that bradykinin loses its vasodilatory effect, resulting in increased blood pressure. Therefore, ACE inhibitory peptides have the effect of lowering blood pressure. At present, the preparation methods of blood pressure lowering peptides mainly include microbial fermentation method, natural active peptide extraction method and synthetic method extraction. However, the extraction method is to directly extract natural active peptides from organisms, and the cost is relatively high. high, and the extraction efficiency is low; the microbial fermentation method is a method for preparing ACE inhibitory peptides by using the fermentation and maturation of animal products such as milk and cheese. Although this method is low in cost, the operation is complicated; It is suitable for directional synthesis in the laboratory, but not suitable for industrial production. For example, a "Isolation and Purification of Egg Yolk Polypeptide and Research on Blood-lipide-Lowering Activity" disclosed in Chinese literature, which uses egg-yolk protein as a test material, systematically studies the preparation process, separation and purification method of hydrolyzed polypeptide and its blood-lipide-lowering activity, but its The extraction cost is higher and the extraction efficiency is lower.

SUMMARY OF THE INVENTION

The present invention proposes a tuna white meat ACE inhibitory peptide and a preparation method thereof in order to overcome the problems of high cost, low efficiency, unsuitability for industrial production and low antihypertensive activity of the existing blood pressure lowering peptides.

In order to achieve the above object, the present invention adopts the following technical solutions:

A tuna white meat ACE inhibitory peptide, the peptide amino acid sequence of the tuna white meat ACE inhibitory peptide is Ser-Pro or Val-Asp-Arg-Tyr-Phe.

The present invention successfully extracts and purifies ACE inhibitory peptides with amino acid sequences of Ser-Pro and Val-Asp-Arg-Tyr-Phe from tuna white meat by enzymatic hydrolysis. Cell (HUVEC) model evaluation, has obvious hypotensive effect.

A preparation method of tuna white meat ACE inhibitory peptide, comprising the following steps:

1) Thawing and drying the white meat of tuna to remove moisture, then defatting, drying and pulverizing for subsequent use;

2) adding distilled water to the pretreated white meat powder, and after adjusting the pH value, adding protease to carry out enzymolysis to obtain an enzymolysis solution;

3) The enzymolysis solution is subjected to enzyme inactivation treatment, and then the supernatant is centrifuged to obtain the enzyme inactivation enzymolysis solution, freeze-dried to obtain polypeptide powder, and the ACE inhibitory activity is measured;

4) After ultrafiltration, column chromatography, high performance liquid chromatography purification, and amino acid sequencing are performed on the enzyme-killing enzymatic hydrolysate, the ACE inhibitory peptide of tuna white meat is prepared.

The invention uses ACE inhibitory activity as an index, extracts ACE inhibitory peptides from tuna white meat by enzymatic hydrolysis, and optimizes the enzymatic hydrolysis process with three factors (temperature, amount of enzyme added and pH), and then, after ultrafiltration, Sephadex G-25 gel layer The peptides with ACE inhibitory activity were prepared by a series of purification techniques and reversed-phase high performance liquid chromatography (RP-HPLC), and their antihypertensive activity was further evaluated by the human umbilical vein endothelial cell (HUVEC) model. hypotensive effect.

Preferably, the degreasing in step 1) is as follows: adding ethyl acetate to the tuna, immersing it for 40-60 hours, and then rotating it.

Ethyl acetate was added to the tuna for degreasing to facilitate subsequent extraction of ACE-inhibiting peptides, and rotary evaporation was used to recover ethyl acetate.

Preferably, the mass ratio of white meat powder to distilled water in step 2) is 1-3:20.

Under this ratio, the enzymatic hydrolysis 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 1-3wt%.

Preferably, the enzymatic hydrolysis temperature in step 2) is 45-65°C.

The degree of enzymatic hydrolysis had an important effect on the relative content of free amino acids in the product and the ACE inhibitory activity. When the degree of enzymatic hydrolysis is insufficient, the amino acid residues with ACE inhibitory activity cannot be exposed, and there is no ACE inhibitory activity. In the initial stage of proteolysis, the inhibitory effect of ACE is enhanced with exposure of free amino acids. However, with the progress of proteolysis, the inhibitory effect of ACE on hydrolysates reached a peak and then gradually weakened. This may be because ACE-inhibiting peptides are released at the onset of proteolysis, resulting in increased ACE-inhibitory activity of the hydrolyzate. When the hydrolysis reaches a certain level, part of the ACE inhibitory peptide is further hydrolyzed, which destroys the complete structure of the ACE inhibitory peptide and produces a weaker ACE inhibitory peptide. Therefore, the three factors in the enzymatic hydrolysis process are very important for the preparation of ACE inhibitory peptides. After a lot of experiments, the enzymatic hydrolysis process parameters within the above range have good enzymatic hydrolysis effect, and the optimal enzymatic hydrolysis process is obtained through experiments.

Preferably, the ultrafiltration step described in step 4) is to use an ultrafiltration membrane to carry out ultrafiltration and grading of the tuna white meat enzymatic hydrolysis solution at 35.1-35.6Hz and 0.5-1.2pa, and after the product components are freeze-dried The enzymatic hydrolyzate powder was obtained.

After the enzymatic hydrolyzate is subjected to ultrafiltration and classification, the ACE inhibitory activity is detected, which is used to screen the components with the best inhibitory activity in the enzymatic hydrolyzate.

Preferably, the molecular weight cut-off of the ultrafiltration membrane in step 4) is 3.5KDa.

Using an ultrafiltration membrane with a molecular weight cut-off of 3.5KDa can obtain a component with a molecular weight of less than 3.5KDa, which has a higher ACE inhibitory activity.

Preferably, the chromatography step described in step 4) is to dissolve and filter the enzymatic hydrolyzate powder, use Sephadex G-25 gel filtration chromatography, and then freeze-dry.

Filter chromatography can further separate and purify ACE inhibitory peptides.

Therefore, the present invention has the following beneficial effects: the tuna white meat ACE inhibitory peptide prepared by the present invention has good inhibitory activity, low preparation cost, high efficiency, is suitable for industrial production, and is the development of polypeptide antihypertensive drugs and the high value of processing by-products. The development has opened up new ideas, has broad application prospects, and also provides scientific references for the in-depth development and comprehensive utilization of tuna.

Description of drawings

Fig. 1 is the inhibitory rate of ACE activity of polypeptide powder under different pH in the preparation process of the present invention.

Fig. 2 is the inhibition rate of ACE activity of polypeptide powder under different enzymolysis temperatures in the preparation process of the present invention.

Fig. 3 is the inhibitory rate of ACE activity of polypeptide powder under different amounts of enzyme added in the preparation process of the present invention.

Fig. 4 is the ACE activity inhibition rate of different ultrafiltration molecular weight enzymolysate powders in the preparation process of the present invention.

Fig. 5 is the elution curve of Sephadex G-25 gel column chromatography in the preparation process of the present invention.

Fig. 6 is the ACE activity inhibition rate of different Sephadex G-25 gel column chromatography elution peaks in the preparation process of the present invention.

Fig. 7 is the spectrogram of Zorbax SB-C18 reversed-phase high performance liquid phase in the preparation process of the present invention.

Figure 8 is a standard curve of the protein of the present invention.

Figure 9 is the ET-1 standard curve of the present invention.

Figure 10 is the effect of the ACE inhibitory peptides L1 and L2 of the present invention on the proliferation activity of HUVEC.

Figure 11 is the effect of different concentrations of ACE inhibitory peptides L1 and L2 of the present invention on the NO content of human umbilical vein endothelial cells (##P<0.01, #P<0.05 vs Control group; **P<0.01, *P<0.05 vs NE Group).

Figure 12 is the effect of different concentrations of ACE inhibitory peptides L1 and L2 of the present invention on the content of ET-1 in human umbilical vein endothelial cells (##P<0.01, #P<0.05 vs Control group; **P<0.01, *P<0.05 vs NE group).

Detailed ways

The present invention will be further described below in conjunction with specific embodiments.

Embodiment 1-15: a preparation method of tuna white meat ACE inhibitory peptide, comprising the following steps:

1) Thawing and drying the white meat of tuna to remove moisture, then adding ethyl acetate to the tuna, immersing and defatting, after rotary evaporation, drying and pulverizing for subsequent use;

2) adding distilled water to the pretreated white meat powder, adjusting the pH to 8.5-10.5, adding 1-3wt% alkaline protease, and enzymatically hydrolyzing at 45-65°C for 3 hours to obtain an enzymatic hydrolysis solution;

3) The enzymatic hydrolysate was heated in a 100°C water bath for 10 min to inactivate the enzyme, then centrifuged at 4000 r for 20 min, and the supernatant was taken to obtain the enzymatic inactivation solution, which was then freeze-dried to obtain a polypeptide powder, and the ACE inhibitory activity was measured;

4) The 3.5KDa ultrafiltration membrane is used to carry out ultrafiltration and grading of the tuna white meat enzymatic hydrolysate, and the product components are freeze-dried to obtain the enzymatic hydrolysate powder; then the enzymatic hydrolysate powder and ultrapure water are made into a concentration of 50 mg/mL. The solution was centrifuged at 12000r/min for 10min at 4°C to remove insoluble impurities, filtered and chromatographed with activated Sephadex G-25, freeze-dried to prepare a solution with a concentration of 100mg/mL. Filtered with 0.45 μm microporous membrane, gradient elution was carried out with acetonitrile-water-trifluoroacetic acid (TFA) as the eluent, the flow rate was 2ml/min, purified and analyzed by high performance liquid chromatography column Zorbax SB-C18, amino acid sequencing, The ACE inhibitory peptide of tuna white meat was synthesized.

Table 1: Preparation conditions of Examples 1-15.

In the preparation process of the example, the detection method of the ACE inhibitory activity of the polypeptide powder is to use FAPGG as the substrate, and the ACE inhibitory activity is measured by the microplate reader method. The specific steps are as follows. The polypeptide hydrolyzate was added to the wells of the microplate without mixing, and then 50 μL of substrate (preheated at 37° C. for 15 minutes) was added to start the reaction. Immediately put the microplate into a microplate reader at a temperature of 37°C, and record the absorbance at a wavelength of 340 nm every 5 minutes for a total of 30 minutes. For the blank control, 40 μL of HEPES buffer was used instead of the peptide solution. The slope was calculated by plotting absorbance (ΔA 340 nm) versus time. The formula for calculating the ACE inhibition rate is as follows:

Among them, ACEI is the inhibition rate of ACE activity; ΔAc is the change of absorbance within 30 min when buffer is added; ΔAi is the change of absorbance within 30 min when inhibitor is added.

Examples 1-5 The effect of different pH on the inhibition rate of ACE activity is shown in Figure 1, the pH is 8.5, 9.0, 9.5, 10.0 and 10.5 respectively, as shown in the figure, the inhibition rate of ACE activity reaches the highest at pH 9.5 (62.45 %), indicating that at this time, the degree of enzymatic hydrolysis of tuna white meat is greater. The pH increased rapidly at 8.5-9.5, and started to decrease after 9.5. Therefore, pH 9.5 is the optimum pH for alkaline protease hydrolysis.

Examples 3, 6-9 The effects of different enzymatic hydrolysis temperatures on the inhibition rate of ACE activity are shown in Figure 2, and the enzymatic hydrolysis temperatures are 45°C, 50°C, 55°C, 60°C, and 65°C respectively; At low temperatures such as 45 °C and 50 °C, the degree of hydrolysis is small, and the ACE inhibition rate is low; as the temperature increases, the catalytic activity of the enzyme increases, the enzymatic hydrolysis speed increases, and the ACE inhibition rate increases. Until 55 °C, the product ACE The maximum inhibition rate was 68.33%; however, when the temperature was higher than 55℃, the ACE inhibition rate began to decrease, indicating that the degree of enzymatic hydrolysis of the protein decreased. This is because the protease activity is weakened 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℃.

Examples 3 and 10-13 The effects of different enzyme addition amounts on the inhibition rate of ACE activity are shown in Figure 3, and the enzyme addition amounts are 1wt%, 1.5wt%, 2wt%, 2.5wt%, and 3wt%, respectively. It can be seen from the figure that when the amount of enzyme added is less than 1.5wt%, the number of mutual binding between enzyme molecules and substrate protein molecules increases with the increase of enzyme amount, so the content of enzymatic hydrolysis products gradually increases; but , once all the substrate molecules are saturated with enzyme molecules, if the amount of enzyme continues to increase, the generated ACE inhibitory components will be excessively hydrolyzed or even inactivated, and the ACE inhibitory rate of the hydrolyzate will gradually decrease. Therefore, the optimum amount of enzyme added for the enzymatic hydrolysis of tuna white meat by alkaline protease is 1.5wt%.

Comparative Example 1: The difference from Example 11 is that the molecular weight cut-offs of the ultrafiltration membranes used are 3.5 kDa and 5 kDa, and the cut-off components are obtained with a molecular weight of 3.5-5 kDa.

Comparative Example 2: The difference from Example 11 is that the molecular weight cut-off of the ultrafiltration membrane used is 5 kDa and 10 kDa, and the cut-off component is obtained with a molecular weight of 5-10 kDa.

Comparative Example 3: The difference from Example 11 is that the molecular weight cut-off of the ultrafiltration membrane used is 10 kDa, and the components with molecular weight greater than 10 kDa are cut off.

The products after ultrafiltration in Example 11 and Comparative Examples 1-3 were marked as TLMH-I (<3.5kDa), TLMH-II (3.5-5kDa), TLMH-III (5-10kDa) and TLMH-IV (> 10kDa), after freeze-drying, a solution with a protein concentration of 1.0 mg/mL was prepared, and the ACE inhibitory activity of each component was measured. The results are shown in Figure 4. As can be seen from the figure, the ACE inhibitory activity of the component TLMH-I is the strongest, the activity of the component TLMH-II is the second, and the activity of the component TLMH-IV is the smallest. The results showed that the ACE inhibitory activity of the ultrafiltration fraction increased with decreasing molecular weight distribution range. Using ultrafiltration with a molecular weight cutoff of 3.5kDa, the enrichment of small molecular substances can be achieved by blocking macromolecular substances in the sample. The TLMH-I fraction is enriched with peptides with a molecular mass <3.5kDa, while in this fraction The possibility of being rich in ACE inhibitory peptides is greater than that of the other three components, so its ACE inhibitory activity is the highest.

The enzymatic hydrolyzate powder prepared in Example 11 was mixed with ultrapure water to form a solution with a concentration of 50 mg/mL, and centrifuged at 12000 r/min for 10 min at 4 °C to remove insoluble impurities, and activated Sephadex G-25 filtration chromatography (loading concentration is 50mg/mL, injection volume is 3ml, elution rate is 0.7ml/min), the elution results are shown in Figure 5, there are 5 elution peaks, which are respectively recorded as A1, A2, A3, A4 and A5, and then combined the solutions in the tube according to the peaks. After lyophilization, the five components were respectively prepared into 1.0 mg/mL sample solutions to determine the ACE inhibitory activity. The results are shown in Figure 6. Group A4 The antihypertensive activity was the best, and the ACE inhibition rate was up to 81.87%, which was higher than other components.

The A4 component group prepared in Example 11 was divided into a solution with a concentration of 100 mg/mL, filtered through a 0.45 μm microporous membrane, and acetonitrile-water-trifluoroacetic acid (TFA) was used as the eluent for gradient elution. The flow rate It is 2ml/min, purified and analyzed by high performance liquid chromatography column Zorbax SB-C18, and the result is shown in Figure 7. It can be seen from the figure that 6 main peaks are obtained by RP-HPLC separation, and the components of these 6 peaks are analyzed by N. End sequencing and mass spectrometry analysis, a total of 6 polypeptide sequences were determined, and their sequences were: Ser-Pro (L1, 202.21Da), Val-Asp-Arg-Tyr-Phe (L2, 698.78Da), Val-His-Gly- Val-Val (L3, 509.61 Da), Tyr-Glu (L4, 310.31 Da), Phe-Glu-Met (L5, 425.51 Da) and Phe-Trp-Arg-Val (L6, 606.73 Da), 6 peptides determined The ACE inhibition rates of the segments at different concentrations were then calculated using SPSS Statistic software to calculate the IC50 values of the 6 peptide segments, respectively: L1 (IC50=0.064mg/mL), L2 (IC50=0.284mg/mL), L3 (IC50=0.901 mg/mL), L4 (IC50=0.800 mg/mL), L5 (IC50=2.179 mg/mL), L6 (IC50=0.755 mg/mL). IC50 (half maximal inhibitory concentration) refers to the measured half inhibitory concentration of the antagonist. It can indicate that a drug or substance (inhibitor) is inhibiting some biological process or half the amount (of some substance contained in this process). Among them, the peptide whose L1 amino acid sequence is Ser-Pro has the best ACE inhibitory activity. This is because the C-terminal tripeptide structure of the ACE inhibitory peptide plays a key role in its hypotensive activity. The C-terminal amino acid of the ACE inhibitory peptide is an aromatic amino acid (including tryptophan, tyrosine, phenylalanine) or Pro ( Proline) residue short peptide has the strongest inhibitory activity, and the C-terminal tripeptide of the polypeptide contains branched chain amino acids (Ile, Leu, Val), and its ACE inhibitory activity will be stronger. In addition, peptides whose N-terminus is hydrophobic amino acid, leucine, isoleucine or basic amino acid have stronger affinity with ACE and higher inhibitory activity, except for proline; leucine, va Amino acid 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 a Pro (proline) residue, so L1 has better ACE inhibitory activity. The L2 (Val-Asp-Arg-Tyr-Phe) obtained in the present invention is a pentapeptide, but its C-terminal structural features also conform to the structure-activity relationship law of ACE-inhibiting short peptides, and its C-terminal Tyr and Phe aromatic amino acids, The N-terminal Val is a hydrophobic valine, so its activity is also very high, second only to the L2 peptide segment.

In the above experimental data, the established FAPGG-ACE in vitro evaluation model shows good ACE inhibitory activity, but its blood pressure lowering activity still needs to be confirmed by biological level experiments. Therefore, the present invention uses human umbilical vein endothelial cells. (HUVEC) model to further evaluate its antihypertensive activity.

Culture of human umbilical vein endothelial cells: HUVECs were cultured with high glucose DMEM+FBS+double antibody (penicillin-streptomycin) medium (DMEM:FBS:double antibody=9:1:0.1). The culture process is as follows: the recovered cells are placed in an incubator containing 5% CO2, cultured at 37 °C, and the medium is changed after 24 hours. When the cells grow and fuse to cover more than 85% of the bottom of the bottle, trypsinize and pass 1:2. into two bottles. HUVECs in logarithmic growth phase were used as experimental materials.

Experimental grouping and treatment: HUVEC cells in logarithmic growth phase were taken, the medium was discarded, washed twice with PBS, digested with trypsin, 2.4×105/well plate, and randomly grouped as follows:

(1) Blank control group: cells were treated without any reagents;

(2) ACE inhibitory peptide low-dose group: the final concentration of added peptide is 100 μM;

(3) Middle dose group of ACE inhibitory peptide: the final concentration of the peptide added was 200 μM;

(4) ACE inhibitory peptide high-dose group: the final concentration of added peptide is 400 μM;

(5) Captopril (Cap) group: the final concentration of Cap was 1 μM;

(6) Norepinephrine (NE) group: add NE to a final concentration of 0.5 μM

(7) Treatment group: the final concentrations of peptide and NE were 200 μM and 0.5 μM, respectively.

Cytotoxicity test (MTT method): HUVEC cells were adjusted to a cell suspension of 0.8×104 cells/well, seeded into a 96-well plate, 160 μl/well, placed in a 5% CO2 incubator, and cultured at 37°C for 24 hours After that, 20 μL of complete culture medium and 20 μL of PBS were added to blank wells, and 20 μL of complete culture medium and 20 μL of samples with final concentrations of 25 μM, 50 μM, 100 μM, 200 μM and 400 μM were added to sample wells (soluble in water with PBS, insoluble in water) (dissolved with DMSO), placed in a 5% CO2 incubator, incubated at 37 °C for 24 hours, added 20 μL MTT solution, incubated at 37 °C for 4 h, discarded the medium, added 150 μL DMSO, shaken at 37 °C for 10 min in the dark to make the reaction uniform, and the enzyme labelled The OD490nm value was measured on the instrument to determine the relative cell viability.

Determination of total protein content: In an alkaline environment, protein will reduce Cu ²⁺ to Cu ⁺ , and Cu ⁺ and BCA reagent will form a blue-violet complex, which has specific absorption at 562nm, and the absorbance at this wavelength is determined. Calculate the protein concentration of the analyte by comparing it with the standard curve. The microplate reader method operates as follows:

1. Preparation of BCA working solution. According to the quantity of standards and samples, prepare working solution according to the ratio of BCA reagent and Cu reagent 50:1, and mix well;

2. Draw a standard curve. Prepare a BSA standard solution with a final concentration of 0.5 mg/mL (take 10 μL of the BSA standard solution and dilute it to 100 μL with PBS), and add it to the 96-well plate in sequence according to the ratio in Table 2;

Table 2: BCA kit standard curve loading

After mixing, place at 37°C for 15-30min, and measure the absorbance at 562nm with a multi-function microplate reader. The protein standard curve was drawn with the protein content (g/L) as the abscissa and the absorbance value as the ordinate. The obtained protein standard curve is shown in Fig. 8, and it can be seen from the figure that the curve equation is y=0.7464x+0.1304, and R ² =0.9918, showing a good linear relationship. The cellular protein concentration of the sample can be calculated by this equation to facilitate the use of the NO kit in the later stage;

3. Sample determination. The sonicated cell samples were appropriately diluted with standard PBS solution, and 20 μL was added to a 96-well plate, and then 200 μL of BCA working solution was added. The absorbance value at 562nm was measured according to the above operation method, and the total protein content of the sample was calculated according to the standard curve.

Determination of NO content: discard the six-well plate medium, add PBS to wash the cultured cells twice, add 2 mL of PBS, and then scrape off the six-well plate with a cell scraper to make a suspension, which is sonicated in an ice bath. Make a suspension. After the pretreatment, follow the steps of the NO kit. Calculated as follows:

Determination of ET-1 content: The kit adopts double antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with endothelin 1 (ET-1) antibody, the sample, standard, and horseradish peroxidase (HRP)-labeled detection antibody were added in sequence, incubated at constant temperature and washed thoroughly. The color is developed with the substrate tetramethylbenzidine (TMB). TMB is converted into a blue substance under the catalysis of peroxidase, and converted into a final yellow substance under the action of acid. The shade of color was positively correlated with the endothelin 1 (ET-1) content in the samples. Measure the absorbance (OD value) with a microplate reader at a wavelength of 450 nm, make a standard curve, and calculate the sample concentration. The standard curve is shown in Figure 9. It can be seen from the figure that the standard concentration is used as the abscissa, and the corresponding OD value is used as the ordinate. The standard curve is drawn, and the concentration value of each sample is calculated according to the curve equation. The standard curve obtained is shown in Figure 9, and the regression equation is y=0.0029x+0.0649, R2=0.991, which has a good fitting ability.

The effect of ACE inhibitory peptides L1 and L2 on the proliferation activity of HUVECs is shown in Figure 10. It can be seen from the figure that compared with the blank control group, peptides L1 and L2 in the concentration range of 25-400 μM have no significant difference in the growth inhibition of HUVECs . This indicated that the L1 and L2ACE inhibitory peptides had no obvious toxic effect on HUVEC under this series of concentrations.

The effects of different concentrations of ACE inhibitory peptides L1 and L2 on the NO content of human umbilical vein endothelial cells are shown in Figure 11. Compared with the blank control group, the NO content in the Cap group, L1 and L2 groups all showed an upward trend, and There is a significant difference, indicating that L2 of L1 promotes the release of NO from cells similar to captopril. There was a significant difference between the NE group and the blank group, indicating that NE could significantly inhibit the release of NO from cells. The effect of L1 on promoting the release of NO from cells was concentration-dependent, while the dose of L2 had the best effect on promoting the release of NO from cells. After adding NE and medium doses of L1 or L2 at the same time, the NO content increased significantly compared with the pure NE group, indicating that a certain concentration of L1 and L2 could resist the effect of NE on NO release.

The effect of different concentrations of ACE inhibitory peptides L1 and L2 on the content of ET-1 in human umbilical vein endothelial cells is shown in Figure 12. Compared with the blank control group, the Cap group, L1 group and L2 group can significantly reduce the number of cells content of ET-1. It can be seen that L1 and Cap have similar effects, and may play a role in lowering blood pressure by inhibiting the release of ET-1 in vascular endothelial cells, and the relationship between the effect and the dose is obvious. The same as the effect of promoting the release of cellular NO, the high dose of L1 and the middle dose of L2 had the strongest inhibitory effect on the release of ET-1. Adding L1 or L2 and NE at the same time showed a significant difference compared with the NE group, indicating that a certain concentration of L1 and L2 could significantly antagonize the effect of NE on the release of ET-1.

Therefore, L1 and L2 with relatively good ACE inhibitory activities can both promote the release of NO in HUVEC cells and inhibit the production of ET-1. The combination of the two effects preliminarily shows that in the in vivo environment, these two ACE inhibitory peptides can inhibit the It exerts its obvious antihypertensive effect by affecting the function of vascular endothelial cells. It is an antihypertensive peptide with good activity and has a prospect for further research.

Claims (10)

1. The tuna white meat ACE inhibitory peptide is characterized in that the peptide segment amino acid sequence of the tuna white meat ACE inhibitory peptide is Ser-Pro or Val-Asp-Arg-Tyr-Phe.

2. A preparation method of ACE inhibitory peptide of tuna white meat is characterized by comprising 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.

3. The method for preparing ACE inhibitory peptide of tuna white meat according to claim 2, wherein the defatting in step 1) is: add ethyl acetate to tuna, immerse for 40-60h, then rotary evaporate.

4. The method for preparing ACE inhibitory peptide of tuna white meat according to claim 2, wherein the mass ratio of the white meat powder to distilled water in step 2) is 1-3: 20.

5. The method for preparing the ACE inhibitory peptide derived from tuna white meat according to claim 2, wherein the pH in step 2) is adjusted to 8.5-10.5.

6. The method for preparing ACE inhibitory peptide of tuna white meat according to claim 2, wherein the amount of enzyme added in step 2) is 1-3 wt%.

7. The method for preparing ACE inhibitory peptide of tuna white meat according to claim 2, wherein the enzymolysis temperature in step 2) is 45-65 ℃.

8. The method for preparing the ACE inhibitory peptide of the tuna white meat according to claim 2, wherein the ultrafiltration step in the step 4) is to perform ultrafiltration fractionation on the tuna white meat enzyme-deactivated enzymolysis liquid by using an ultrafiltration membrane under 35.1-35.6Hz and 0.5-1.2pa, and freeze-dry product components to obtain enzymolysis powder.

9. The method for preparing the ACE inhibitory peptide derived from tuna white meat according to claim 8, wherein the molecular weight cut-off of the ultrafiltration membrane in step 4) is 3.5 KDa.

10. The method for preparing the ACE inhibitory peptide from the white meat of tuna according to claim 2, wherein the step of chromatography in step 4) comprises dissolving and filtering the zymolyte powder, performing Sephadex G-25 gel filtration chromatography, and then freeze-drying.

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