Preparation method of highly active tartary buckwheat albumin antihypertensive peptide

Abstract

This invention discloses a method for preparing highly active tartary buckwheat albumin antihypertensive peptides, belonging to the food and pharmaceutical fields. The invention involves extracting tartary buckwheat albumin from tartary buckwheat, enzymatically hydrolyzing it using two proteases essential for human digestion, and optimizing the pepsin-trypsin hydrolysis process to obtain tartary buckwheat albumin peptides. The inhibition rate of ACE activity was detected in vitro. The tartary buckwheat albumin peptides were then separated into peptides of different molecular weights by gel filtration chromatography, and their in vitro ACE activity inhibition rates were measured separately. Finally, the amino acid sequence of the peptide segment exhibiting the highest inhibition rate against ACE activity was identified using nano-HPLC-MS / MS.

Description

Technical Field

[0001] This invention relates to a method for preparing highly active tartary buckwheat albumin antihypertensive peptides, belonging to the fields of food and pharmaceuticals. Background Technology

[0002] Hypertension is a common chronic cardiovascular disease. Long-term hypertension can easily lead to stroke, coronary heart disease, and other diseases, and most hypertensive patients need lifelong medication. Angiotensin-converting enzyme (ACE) has two main functions: 1. catalyzing the conversion of angiotensin I to angiotensin II; 2. inactivating bradykinin. Because of these two functions, ACE has become an ideal target for treating hypertension, heart failure, type 2 diabetes, and diabetic nephropathy. Currently, drug treatment for hypertension is relatively mature. Drugs such as perindopril and captopril can inhibit ACE, but taking these drugs can easily cause a series of side effects, such as drug resistance, cough, and angioedema. Therefore, safe and non-toxic food-derived animal and plant proteins have come into focus. As early as 1979, Oshima et al. obtained antihypertensive peptides by hydrolyzing gelatin with bacterial collagenase. Increasing research has shown that animal and plant proteins can be used to prepare effective antihypertensive active peptides, providing new ideas for the treatment of hypertension and a theoretical basis for regulating blood pressure through diet therapy and other methods.

[0003] Studies have shown that hydrophobic amino acids and proline in bioactive peptides play a crucial role in their ACE inhibitory activity. Koyama et al. first discovered the buckwheat inhibitory peptide Ser-Thr-Hyp, which has a hypotensive effect on spontaneously hypertensive rats; Li et al., in their study of buckwheat protein hydrolysis, found that whole buckwheat that had not undergone protein extraction, after being hydrolyzed with pepsin, significantly reduced the ACE IC50. 50 The protein content was 0.36 mg / ml, while whole buckwheat that had not undergone protein extraction, after hydrolysis with pepsin, chymotrypsin, and trypsin, had an IC50 of 0.36 mg / ml. 50 The protein concentration was reduced to 0.14 mg / ml, indicating that the addition of chymotrypsin and trypsin after pepsin treatment significantly improved the ACE inhibition rate of buckwheat protein. However, although the enzymatic hydrolysate obtained from the direct hydrolysis of whole buckwheat has an inhibitory effect on ACE, it cannot completely rule out the influence of other components in buckwheat on the ACE inhibitory ability.

[0004] Tartary buckwheat, also known as Tatar buckwheat, is a type of buckwheat with high nutritional and medicinal value. Pharmacological studies have shown that tartary buckwheat has hypoglycemic, hypotensive, and lipid-lowering effects, as well as antioxidant properties. The regulatory effects of tartary buckwheat protein on lipid metabolism and its ability to improve glucose metabolism have been confirmed. Tartary buckwheat protein contains 17 amino acids, including 8 essential amino acids, making it a complete protein. Currently, the antihypertensive peptides derived from tartary buckwheat have a low ACE inhibition rate. Summary of the Invention

[0005] [Technical Issues]

[0006] The technical problem to be solved by this invention is that the existing antihypertensive peptides derived from tartary buckwheat have a low inhibition rate on ACE.

[0007] [Technical Solution]

[0008] This invention provides a method for preparing protein peptides derived from tartary buckwheat that have blood pressure-lowering functions, comprising the following steps:

[0009] (1) Tartary buckwheat albumin was extracted from tartary buckwheat using the Osborne method;

[0010] (2) Take 0.1-0.9g of the tartary buckwheat albumin obtained in step (1), add it to phosphate buffer, adjust the pH of the system to 2-3, add pepsin (2500U / mg) accounting for 3-6% of the tartary buckwheat albumin protein, and enzymatically hydrolyze at 37℃ for 2h. Then adjust the pH of the system to 7-8, add trypsin (1500U / mg) accounting for 3-6% of the tartary buckwheat albumin protein, and enzymatically hydrolyze at 37℃ for 1h to obtain the enzymatic hydrolysate.

[0011] (3) The enzyme hydrolysate was filtered through a microfiltration membrane to obtain a filtrate. Then, the peptides of different molecular weights in the enzyme hydrolysate filtrate were separated by gel filtration chromatography to screen out the tartary buckwheat protein active peptide with the best ACE inhibition effect.

[0012] (4) The amino acid sequence of the tartary buckwheat protein active peptide with the best inhibitory effect was identified by nano-HPLC-MS / MS.

[0013] In some embodiments of the present invention, step (1) includes the following steps:

[0014] ① Grinding: Grind the buckwheat in a grinder and pass it through a 60-80 mesh sieve for later use;

[0015] ② Defatting: Add petroleum ether to buckwheat flour and stir for 6-10 hours, changing the petroleum ether several times during the process, until the supernatant is clear and air-dry for later use;

[0016] ③ Extraction of buckwheat albumin: Weigh 300g of defatted buckwheat powder, add 10 times the volume of water, stir and extract at 40℃ for 2h, then centrifuge at 5000r / min for 15min. The supernatant obtained by centrifugation is the albumin extract. Adjust the pH of the albumin extract to the isoelectric point of albumin, precipitate for 1h, centrifuge and collect the precipitate. Freeze-dry the precipitate to obtain buckwheat albumin.

[0017] In some embodiments of the present invention, the pepsin used in step (2) may be porcine pepsin.

[0018] In some embodiments of the present invention, the trypsin used in step (2) may be porcine trypsin.

[0019] In some embodiments of the present invention, step (3) involves filtering the enzymatic hydrolysate through a 0.22 μm microfiltration membrane, passing the filtrate through a Superdux peptide 10 / 300GL gel column with water as the mobile phase and controlling the flow rate at 0.4 mL / min. The filtrate is collected at 280 nm using an AKTAavant system according to the peaks, yielding three peaks: F1, F2, and F3. After diluting the three peak components to the same concentration, their inhibition rates against ACE are measured. The results show that component F3 has the highest inhibition rate against ACE.

[0020] In some embodiments of the present invention, step (4) uses nano-HPLC-MS / MS to identify the amino acid sequence of the tartary buckwheat albumin active peptide with the best inhibitory effect, and a total of ten tartary buckwheat albumin antihypertensive active peptides are obtained.

[0021] This invention provides an active peptide with blood pressure lowering function, the amino acid sequences of which are FLR, LPRL, LFGK, TLFR, IPRL, ​​VVLK and SFFK.

[0022] The active peptide can be used to prepare antihypertensive products. Excipients may be added to the product to serve as excipients, carriers, improve stability, solubilize, aid solubility, and / or provide sustained or controlled release.

[0023] [Beneficial Effects]

[0024] This invention extracts tartary buckwheat albumin from tartary buckwheat, performs enzymatic hydrolysis using two proteases essential for human digestion, and optimizes the pepsin-trypsin hydrolysis process to obtain tartary buckwheat albumin peptides. The inhibitory rate on ACE activity is then detected in vitro. The tartary buckwheat albumin peptides are further separated into peptides of different molecular weights by gel filtration chromatography, and their in vitro ACE activity inhibition rates are measured separately. Finally, the amino acid sequence of the peptide with the highest ACE activity inhibition rate is identified using nano-HPLC-MS / MS. This invention yields 10 highly active tartary buckwheat albumin peptides with antihypertensive properties, whose amino acid sequences are IFR, LRF, FLR, LPRL, FLK, LFGK, TLFR, IPRL, ​​VVLK, and SFFK. Among these, FLR, LPRL, LFGK, TLFR, IPRL, ​​VVLK, and SFFK are peptides with ACE inhibitory activity discovered for the first time in this invention.

[0025] The highly active tartary buckwheat albumin antihypertensive peptide prepared by this invention can be encapsulated to enhance its stability after entering the human body; or it can be made into an oral liquid product to increase the added value of tartary buckwheat. The polypeptide sequence contained in the tartary buckwheat albumin antihypertensive peptide prepared by the process of this invention is clearly defined and unique to this invention, providing a theoretical basis for the use of highly active tartary buckwheat albumin antihypertensive peptide as food or medicine.

[0026] The tartary buckwheat protein peptides prepared by the method of this invention have an IC50 value without separation and purification. 50 The concentration was as low as 0.255 mg / mL; after separation by gel filtration chromatography, the inhibition rate of ACE was significantly improved, reaching 71.83% at 0.1 mg / mL. The antihypertensive peptide isolated from buckwheat albumin in this invention has a better inhibitory effect on ACE activity.

[0027] This invention utilizes two proteases for continuous hydrolysis. Compared to hydrolysis methods such as alkaline proteases, the hydrolysis is gentler, causes less damage to buckwheat protein, and is simpler. Compared to other processes that use pepsin and trypsin for hydrolysis, this invention has a shorter hydrolysis time, consumes less protease, and produces buckwheat protein peptides with smaller molecular weights, making them easier for the human body to absorb. Attached Figure Description

[0028] Figure 1 The preparation process of buckwheat albumin antihypertensive peptides.

[0029] Figure 2 The IC50 of the enzymatic hydrolysate for ACE under optimal enzymatic hydrolysis conditions of buckwheat albumin 50 .

[0030] Figure 3 The peak spectrum is obtained by gel filtration chromatography of the enzymatic hydrolysate of tartary buckwheat albumin after hydrolysis by pepsin-trypsin.

[0031] Figure 4 This study compares the inhibition rates of different components collected by gel filtration chromatography from the enzymatic hydrolysate of tartary buckwheat albumin after hydrolysis by pepsin-trypsin. Detailed Implementation

[0032] The moisture and protein content of buckwheat albumin were determined using the methods specified in GB 5009.3-2016 and GB 5009.5-2016, respectively.

[0033] The ACE inhibition rate was determined as follows: In the experimental group, 100 μL of the simulated substrate FAPGG (furanoacryl tripeptide) was added, followed by 50 μL of tartary buckwheat albumin hydrolysate for a 18-fold dilution, and finally 50 μL of ACE (60 mU / mL). In the blank group, 50 μL of Tris-HCl buffer was used instead of the tartary buckwheat albumin hydrolysate. The reaction was carried out at 37℃ for 30 min. The inhibition effect was characterized by the decrease in absorbance at 345 nm after ACE digestion of the simulated substrate in both the blank and experimental groups. The ACE inhibition rate was calculated using the following formula:

[0034]

[0035] In the formula, A1 is the decrease in absorbance of the blank group; A2 is the decrease in absorbance of the experimental group.

[0036] Peptide content determination: The peptide content in the tartary buckwheat protein hydrolysate before gel filtration chromatography was determined using the trichloroacetic acid method. Approximately 5g of hydrolysate was weighed, and the volume was adjusted to 25mL with trichloroacetic acid. The mixture was mixed, allowed to precipitate, and centrifuged for 20min. The supernatant was retained and filtered through double-layered filter paper. 10mL of the filtrate was added to a digestion tube, along with 3g of potassium sulfate, 0.2g of copper sulfate pentahydrate (blue powder), and 10mL of concentrated sulfuric acid. Digestion was performed in a graphite digester. After digestion, the peptide content in the hydrolysate was determined using the Kjeldahl method.

[0037] Determination of the degree of hydrolysis: The degree of hydrolysis of buckwheat protein was determined by formaldehyde titration. Take 5 mL of hydrolysate, add 60 mL of decarbonated distilled water, stir well, and adjust the pH to 8.20. Prepare a neutral formaldehyde solution using 50 mL of formaldehyde solution and 3 mL of 0.5% (m / v) phenolphthalein solution, and adjust the pH to 8.20. Add 20 mL of the neutral formaldehyde solution and titrate with 0.1 mol / mL standard NaOH solution to pH 9.20. Record the volume of NaOH solution consumed as V1. Replace the hydrolysate sample with an equal volume of distilled water to perform a blank experiment, and record the volume of NaOH consumed as V0. Calculate the amino nitrogen content in the hydrolysate. Calculate the degree of protein hydrolysis (DH).

[0038]

[0039]

[0040] In the formula, V1 is the volume of NaOH consumed in the titration of the experimental group samples, in milliliters (mL);

[0041] V0 represents the volume of NaOH consumed in the titration of the blank sample, in milliliters (mL).

[0042] N is the concentration of the standard NaOH solution used in the experiment, in moles per liter (mol / L);

[0043] V represents the volume of the sample taken, in milliliters (mL).

[0044] Gel filtration chromatography was used to separate the proteolytic products of tartary buckwheat extract: The proteolytic solution of tartary buckwheat extract was filtered through a 0.22 μm microfiltration membrane. The filtered proteolytic solution was then passed through a Superdux peptide 10 / 300GL gel column with water as the mobile phase and a flow rate controlled at 0.4 mL / min. Peaks were collected at 280 nm using an AKTA avant system. The antihypertensive active peptide fraction was collected as three peaks: F1, F2, and F3. The three peak fractions were diluted to the same concentration, and their inhibition rate against ACE was measured.

[0045] Peptide sequence determination: The antihypertensive peptide with the best inhibitory effect obtained after gel filtration chromatography was analyzed by nano-HPLC-MS / MS. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with tandem EASY-nanoLC 1200. A total of 3 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated by a gradient of 60 min, with the column flow rate controlled at 300 nL / min, column temperature at 40 °C, and electrospray voltage at 2 kV. The gradient started at 2% B phase, increased non-linearly to 35% at 47 min, increased to 100% within 1 min, and maintained for 12 min. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: Scan range (m / z): 200-1800; Resolution: 70000; AGC target: 3e6; Maximum injection time: 50ms; (2) HCD-MS / MS: Resolution: 17500; AGC target: 1e5; Maximum injection time: 45ms; Collision energy: 28; Dynamic exclusion time: 30s.

[0046] The pepsin used in the following examples has an activity of 2500 U / mg and is derived from pigs.

[0047] The trypsin used in the following examples has an activity of 1500 U / mg and is derived from pigs.

[0048] The following are examples and comparative examples for preparing tartary buckwheat antihypertensive active peptides. The results of the index determination are shown in Table 1 below.

[0049] Example 1: Preparation of enzymatic hydrolysate of tartary buckwheat albumin

[0050] (1) Albumin was extracted from buckwheat.

[0051] Grinding: Grind the buckwheat in a grinder and pass it through a 60-80 mesh sieve for later use;

[0052] Defatting: Add petroleum ether to buckwheat flour and stir for 6-10 hours, changing the petroleum ether several times during the process, until the supernatant is clear. Air dry for later use.

[0053] Extraction of buckwheat albumin: Weigh 300g of defatted buckwheat powder, add 10 times the volume of water, stir and extract at 40℃ for 2h, centrifuge at 5000r / min for 15min, the supernatant is the albumin extract, adjust the pH to the isoelectric point of albumin, precipitate for 1h, centrifuge and collect the precipitate, freeze dry to obtain buckwheat albumin.

[0054] (2) Enzymatic hydrolysis

[0055] A 1.6% concentration of tartary buckwheat protein suspension was prepared using phosphate buffer. The pH of the suspension was adjusted to 2 using 1M hydrochloric acid solution. Pepsin (4% of the tartary buckwheat protein mass) was added for 2 hours of enzymatic hydrolysis. The pH was then adjusted to 7 using 1M sodium hydroxide solution. Trypsin (5.5% of the tartary buckwheat protein mass) was added for 2 hours of enzymatic hydrolysis, followed by boiling to inactivate the enzymes. After cooling and centrifugation, the tartary buckwheat protein hydrolysate was obtained. The ACE inhibition rate, degree of hydrolysis, and peptide content were measured. The results are shown in Table 1.

[0056] Example 2: Preparation of buckwheat protein hydrolysate and isolation of ACE inhibitory peptides

[0057] (1) Extracting albumin from buckwheat: The specific steps are the same as step (1) in Example 1 above.

[0058] (2) Enzymatic hydrolysis

[0059] A 2% concentration of tartary buckwheat protein suspension was prepared using phosphate buffer. The pH of the suspension was adjusted to 2 using 1M hydrochloric acid solution. Pepsin (4% of the tartary buckwheat protein mass) was added and hydrolyzed for 2 hours. The pH was then adjusted to 7 using 1M sodium hydroxide solution. Trypsin (5% of the tartary buckwheat protein mass) was added and hydrolyzed for 1 hour. The enzyme was then inactivated by boiling. After cooling, the tartary buckwheat protein hydrolysate was obtained by centrifugation. The ACE inhibition rate, degree of hydrolysis, and peptide content were measured. The results are shown in Table 1.

[0060] (3) The protease hydrolysate from tartary buckwheat was filtered through a 0.22 μm microfiltration membrane. The filtered hydrolysate was then passed through a Superdux peptide 10 / 300GL gel column with water as the mobile phase at a flow rate of 0.4 mL / min. Peaks were collected at 280 nm using an AKTA avant system. Three peaks, F1, F2, and F3, were collected from the antihypertensive active peptide fraction. The three peak fractions were diluted to the same concentration, and the inhibition rate against ACE was measured. The results are as follows: Figure 4 As shown, the F3 component exhibited the highest ACE inhibition rate.

[0061] (4) Determination of peptide sequence

[0062] The antihypertensive peptide with the best inhibitory effect obtained after gel filtration chromatography was analyzed by nano-HPLC-MS / MS. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC 1200 in series. A total of 3 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated by a gradient over 60 min, with the column flow rate controlled at 300 nL / min, the column temperature at 40 °C, and the electrospray voltage at 2 kV. The gradient started at 2% B phase, increased non-linearly to 35% at 47 min, increased to 100% within 1 min, and maintained for 12 min. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: Scan range (m / z): 200-1800; Resolution: 70000; AGC target: 3e6; Maximum injection time: 50ms; (2) HCD-MS / MS: Resolution: 17500; AGC target: 1e5; Maximum injection time: 45ms; Collision energy: 28; Dynamic exclusion time: 30s.

[0063] Comparative Example 1

[0064] (1) Extracting albumin from buckwheat: The specific steps are the same as step (1) in Example 1 above.

[0065] (2) Enzymatic hydrolysis

[0066] A 2% buckwheat protein suspension was prepared using phosphate buffer. The pH of the suspension was adjusted to 2 using 1M hydrochloric acid solution without adding pepsin. After reacting for 2 hours, the pH of the suspension was adjusted to 7 using 1M sodium hydroxide solution without adding trypsin. After enzymatic hydrolysis for 1 hour, the supernatant was obtained by boiling and centrifugation. The inhibition rate, degree of hydrolysis, and peptide content of ACE were then determined.

[0067] Comparative Example 2

[0068] (1) Extracting albumin from buckwheat: The specific steps are the same as step (1) in Example 1 above.

[0069] (2) Enzymatic hydrolysis

[0070] A 2% tartary buckwheat albumin suspension was prepared using phosphate buffer. The pH of the suspension was adjusted to 2 using 1M hydrochloric acid solution. 4% pepsin was added, and after reacting for 2 hours, the enzyme was inactivated by boiling and centrifugation was performed to obtain the supernatant. The inhibition rate, degree of hydrolysis, and peptide content of ACE were then measured.

[0071] Comparative Example 3

[0072] (1) Extracting albumin from buckwheat: The specific steps are the same as step (1) in Example 1 above.

[0073] (2) Enzymatic hydrolysis

[0074] A 2% concentration of tartary buckwheat albumin suspension was prepared using phosphate buffer. The pH of the suspension was adjusted to 7 using 1M sodium hydroxide solution. Trypsin with a protein content of 5% was added and hydrolyzed for 1 hour, followed by boiling to inactivate the enzyme. After cooling, the tartary buckwheat albumin hydrolysate was obtained by centrifugation. The ACE inhibition rate, degree of hydrolysis, and peptide content were then measured.

[0075] Comparative Example 4

[0076] (1) Extracting albumin from buckwheat: The specific steps are the same as step (1) in Example 1 above.

[0077] (2) Enzymatic hydrolysis

[0078] A 2% concentration of tartary buckwheat albumin suspension was prepared using phosphate buffer. The pH of the suspension was adjusted to 2 using 1M hydrochloric acid solution. 4% pepsin was added, and the reaction was carried out for 2 hours. Then, the pH of the suspension was adjusted to 7 using 1M sodium hydroxide solution. Trypsin with a protein content of 6% was added and hydrolyzed for 2 hours. The enzyme was then inactivated by boiling. After cooling, the tartary buckwheat albumin hydrolysate was obtained by centrifugation. The ACE inhibition rate, degree of hydrolysis, and peptide content were measured.

[0079] Comparative Example 5

[0080] (1) Extracting albumin from buckwheat: The specific steps are the same as step (1) in Example 1 above.

[0081] (2) Enzymatic hydrolysis

[0082] A 2% tartary buckwheat albumin suspension was prepared using phosphate buffer. The enzymatic hydrolysis temperature was 50℃, the pH of the hydrolysate was 9.0, and alkaline protease was added at a concentration of 20 kU / g (based on substrate). The hydrolysis was carried out for 2 hours, and the inhibition rate, degree of hydrolysis, and peptide content of ACE were measured.

[0083] Table 1. Detection results of each enzyme hydrolysate

[0084]

[0085] As can be seen from Comparative Examples 1, 2, and 3, no bioactive peptides can be produced without the addition of any enzymes. However, when only pepsin or trypsin is added, although some bioactive peptides are produced, fewer peptides have an inhibitory effect on ACE, the inhibition rate is low, and the degree of hydrolysis and peptide content are also low. This may be related to the fact that pepsin and trypsin alone hydrolyze tartary buckwheat protein at fewer cleavage sites.

[0086] As can be seen from Examples 1 and 2 and Comparative Example 4, in the preparation of antihypertensive active peptides, under the same enzyme action, different enzymatic hydrolysis conditions resulted in significant differences in the ACE inhibitory effect, peptide content, and degree of hydrolysis. During the enzymatic hydrolysis reaction, the peptide content gradually increased with increasing trypsin dosage, substrate concentration, and hydrolysis time. The inhibition rate reached its maximum when the substrate concentration was 2%, the trypsin dosage was 5% of the protein content, and hydrolysis lasted for 1 hour. This indicates that the peptide obtained under these hydrolysis conditions had a good inhibitory effect on ACE. Adding more trypsin may destroy the already generated effective inhibitory peptides, increase the viscosity of the reaction system, and hinder the reaction. While the content and degree of hydrolysis increased with prolonged hydrolysis time, the peptides effective against ACE were over-hydrolyzed, leading to a gradual decrease in the ACE inhibition rate. Therefore, only by adding the optimal amount of enzyme and hydrolyzing for the optimal time under the optimal substrate concentration conditions can the peptide with the best inhibition rate be obtained. Under optimal conditions, the proportion of bioactive peptides with a content of less than 3000 Da in the enzymatic hydrolysate reached 90%, of which more than 66% were peptides with a content of less than 1000 Da. This indicates that under these conditions, the tartary buckwheat protein was hydrolyzed more thoroughly. Moreover, studies have shown that peptides with better ACE inhibition effects are mostly short peptides, which can also explain why the antihypertensive active peptides obtained in this invention have a better inhibitory effect.

[0087] As can be seen from Comparative Example 5, although alkaline protease hydrolysis of tartary buckwheat protein can also yield bioactive peptides with ACE inhibitory effects, the alkaline protease hydrolysis is more intense compared to that of the present invention. The continuous pepsin-trypsin hydrolysis process used in the present invention provides a better evaluation of the in vitro ACE inhibitory activity of tartary buckwheat protein. This method can better reflect the changes in the spatial structure, amino acid sequence, and molecular weight of tartary buckwheat protein peptides after digestion by gastrointestinal digestive enzymes in the human body.

[0088] Depend on Figure 4 As can be seen, the enzymatic hydrolysate prepared under the optimal enzymatic hydrolysis conditions in Example 2 was separated into three components by gel filtration chromatography. Among them, the inhibition rate of ACE by component F3 was 71.83% when the peptide concentration was 0.1 mg / mL, which was a significant improvement compared to the inhibition rate before separation. After gel column filtration, the active ingredients were effectively enriched, and it can be concluded that it contains highly active tartary buckwheat protein peptides, which can be identified.

[0089] Ten bioactive peptides were identified from the F3 fraction in step (4) of Example 2 using nano-HPLC-MS / MS analysis. Their bioactivity was predicted using the Peptide Ranker method and ranked according to their scores. All ten buckwheat protein peptides had bioactivity scores greater than 0.5, indicating a high probability of bioactivity. Studies have reported that peptides with good ACE inhibition effects are often highly hydrophobic. Therefore, the average hydrophobicity was calculated using a peptide property calculator. The average hydrophobicity of the peptides obtained in this invention is high, suggesting that the ten buckwheat protein peptides obtained in this invention may have high ACE inhibitory activity. In addition, studies have found that the presence of hydrophobic amino acids Tyr, Phe, Trp, Lys, and Pro at the C-terminus of peptides, as well as the presence of Arg, can significantly improve the ACE inhibition rate. Among the seven previously unreported tartary buckwheat protein peptides discovered in this invention, FLR, LFGK, TLFR, VVLK, and SFFK all contain arginine or hydrophobic amino acids at their C-terminus, thus suggesting that they have high ACE inhibitory activity. Furthermore, LPRL and IPRL may also significantly enhance the ACE inhibitory activity of peptides due to the presence of Leu at their C-terminus. The above inferences were verified by detecting their ACE inhibition rate.

[0090] The efficacy of the predicted potential ACE-inhibiting peptides was validated by solid-phase synthesis at Sangon Biotech Co., Ltd. (Shanghai, China). The peptide purity was verified as 99% by HPLC, and the molecular weight of the synthesized peptide was confirmed using LC-MS / MS before its in vitro ACE-inhibiting activity was determined.

[0091] Table 2

[0092]

[0093]

[0094] The preparation process of this invention is simple, time-saving, requires a small amount of enzyme, and is low in cost. The tartary buckwheat albumin peptides obtained by this invention have a high in vitro ACE inhibition rate, the obtained polypeptides are short, and many polypeptides are the first to be isolated and identified by this invention. Therefore, this invention has significant application prospects in terms of process, mechanism of action, acceptance, and number of consumers. Microencapsulation technology can be used to make microcapsules with the tartary buckwheat ACE-inhibiting peptides obtained by this invention as the core material. These microcapsules can withstand high temperature and high acid / alkali environments, and are slowly released after entering the human body, exhibiting strong stability. Furthermore, it can be made into tartary buckwheat polypeptide oral liquid products, increasing its commercial value and enhancing the added value of tartary buckwheat.

[0095] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An active peptide with blood pressure lowering function, characterized in that, The amino acid sequences are FLR, TLFR, IPRL, ​​VVLK, and SFFK.

2. The application of the active peptide with blood pressure lowering function as described in claim 1 in the preparation of blood pressure lowering products.

3. A blood pressure-lowering product containing active peptides, characterized in that, It contains at least one peptide selected from FLR, TLFR, IPRL, ​​VVLK, and SFFK.

4. The product according to claim 3, characterized in that, It also contains auxiliary ingredients.

5. The product according to claim 4, characterized in that, The excipients are used for shaping, acting as carriers, improving stability, solubilizing, aiding solubility, and / or controlled release.

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