A yeast peptide, and a preparation method and application thereof

Abstract

The present application provides a kind of yeast peptide and its preparation method and application.The yeast peptide provided by the present application contains the following sequence peptide segment: phenylalanine-valine-alanine-leucine-proline, valine-leucine-isoleucine-leucine-lysine and alanine-glycine-leucine-threonine-leucine.The present application provides angiotensin converting enzyme (ACE) inhibitory activity and in vivo blood pressure lowering activity.

Description

Technical Field

[0001] This invention belongs to the field of food and pharmaceutical technology, specifically relating to the preparation of a yeast peptide that can lower blood pressure, and the specific blood pressure lowering effect of the peptide. Background Technology

[0002] Hypertension (systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg) is a common cardiovascular disease. According to the WHO, over 270 million people in China suffer from hypertension, but only 13.8% of patients have it effectively controlled. Hypertension easily leads to stroke, kidney failure, blindness, myocardial infarction, and cerebral infarction. Therefore, the prevention and treatment of hypertension are of great significance to the health and development of society. The regulation of blood pressure in the human body is carried out through a complex mechanism involving multiple interconnected metabolic pathways. The most important regulatory mechanisms are the renin-angiotensin system and the kininase-kinin system. Angiotensin-converting enzyme (ACE) plays a crucial role in this process. ACE can convert the inactive form of angiotensin I (Ang I) into angiotensin II (Ang II), which has vasoconstrictive effects, and inactivate the vasodilatory peptide bradykinin. Therefore, ACE inhibitors are commonly used drugs for the treatment of hypertension in clinical practice.

[0003] Existing synthetic ACE inhibitors, such as lisinopril, can cause side effects like allergies, dizziness, and liver toxicity, and are unsuitable for the prevention of hypertension or the treatment of mild cases. Natural ACE-inhibiting peptides, however, have attracted research attention due to their wide availability and low toxicity. Recent studies have found that ACE-inhibiting peptides not only lower blood pressure by inhibiting ACE activity but also by alleviating oxidative stress and regulating vascular cortical function. Furthermore, numerous studies have demonstrated the crucial role of gut microbiota in maintaining host metabolic balance. Alterations in the host's gut microbiota significantly impact the development and management of diseases such as hypertension, diabetes, obesity, and hyperuricemia. Unabsorbed dietary protein hydrolysates regulate gut microbiota composition, thereby affecting host metabolism. Studies have shown that dietary protein hydrolysates can promote blood pressure regulation by modulating gut microbiota composition. Therefore, dietary protein hydrolysates with ACE-inhibiting activity, in vivo antioxidant activity, and gut microbiota-regulating effects hold great potential for the prevention and treatment of hypertension. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a method for preparing and applying yeast peptides.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] A yeast peptide characterized by containing the following sequence peptide segments: (phenylalanine-valine-alanine-leucine-proline)FVALP, (valine-leucine-isoleucine-leucine-lysine)VLILK, (alanine-glycine-leucine-threonine-leucine)AGLTL.

[0007] This invention provides a yeast peptide, which contains the following peptides:

[0008] First peptide: phenylalanine-valine-alanine-leucine-proline

[0009] Second peptide: valine-leucine-isoleucine-leucine-lysine

[0010] And, the third peptide: alanine-glycine-leucine-threonine-leucine.

[0011] Preferably, the mass ratio of the first peptide, the second peptide, and the third peptide in the yeast peptide is 0.3-1:0.3-1:0.3-1;

[0012] Preferably, the mass ratio of the first peptide, the second peptide, and the third peptide in the yeast peptide is 0.3-0.5: 0.3-0.9: 0.5-1.

[0013] The present invention also provides a method for preparing the yeast peptides, the method comprising the following steps: digesting yeast milk after nucleic acid removal using a first enzyme, and separating the light phase, which is the yeast peptide.

[0014] Alternatively, the yeast milk after nucleic acid removal can be enzymatically hydrolyzed using a second enzyme to break down the cell wall, and the heavy phase can be separated by enzymatic hydrolysis using a third enzyme. The resulting light phase is yeast peptides.

[0015] Preferably, the pH of the yeast milk after nucleic acid removal is adjusted to 8-9, and then enzymatic hydrolysis is performed using the first enzyme;

[0016] Alternatively, adjust the pH of the yeast milk after nucleic acid removal to 5-6, and then use a second enzyme for enzymatic hydrolysis.

[0017] Preferably, the preparation method further includes purifying the yeast peptide using membrane separation.

[0018] Preferably, the preparation method further includes obtaining powdered yeast peptides by spray drying or freeze drying.

[0019] Preferably, the mass ratio of the first enzyme to the dry weight of the yeast milk after nucleic acid removal is 0.1% to 0.5%, more preferably 0.2% to 0.3%, based on dry matter mass.

[0020] The denucleic acid-free yeast milk in this invention can be yeast milk that has undergone fermentation and denucleated, or it can be a mixture of denucleated yeast powder and water, with a mass concentration of 8-15%.

[0021] Preferably, the first enzyme is a mixture of thermophilic protease and papain;

[0022] Preferably, the first enzyme is a mixture of thermophilic protease and papain in a mass ratio of 1:1;

[0023] Alternatively, preferably, the first enzyme is a neutral protease Protin SD-NY 10 or a neutral protease Protease A 2SD.

[0024] Preferably, the mass ratio of the second enzyme to the yeast milk after nucleic acid removal is 0.1% to 1% based on dry matter mass, and more preferably 0.3% to 0.5%.

[0025] Preferably, the second enzyme is β-glucanase, mannanase, or cellulase.

[0026] Preferably, the mass ratio of the third enzyme to the yeast milk after nucleic acid removal is 0.1% to 0.5%, more preferably 0.2% to 0.3%;

[0027] Preferably, the third enzyme is a mixture of thermophilic protease and papain, or a complex protease (Protin SD-NY), or a neutral protease (Protease A 2SD).

[0028] Preferably, the third enzyme is a mixture of thermophilic protease and papain in a mass ratio of 1-2:1-2.

[0029] The present invention also provides a peptide having the following sequence: phenylalanine-valine-alanine-leucine-proline.

[0030] The present invention also provides a peptide having the following sequence: valine-leucine-isoleucine-leucine-lysine.

[0031] The present invention also provides a peptide having the following sequence: alanine-glycine-leucine-threonine-leucine.

[0032] The present invention also provides the application of the yeast peptide described herein or the yeast peptide prepared by the method described herein, or the peptide described herein, in the preparation of products such as angiotensin-converting enzyme inhibitors, antioxidants, antihypertensive agents, or intestinal microbiome modulators.

[0033] Preferably, the product is a pharmaceutical, food, or health product.

[0034] The present invention also provides a product for inhibiting angiotensin-converting enzyme activity, wherein the product contains the yeast peptide described above or the yeast peptide prepared by the preparation method described above or the peptide described above.

[0035] Preferably, the content of the yeast peptide in the product or the yeast peptide prepared by the preparation method is 65-85 wt%.

[0036] In this invention, the yeast peptide obtained by the above method exhibits significant inhibitory activity against ACE in vitro, IC50. 50 It is 0.082 mg / mL.

[0037] To verify the in vivo blood pressure-lowering effect of yeast peptides, an acute gavage experiment and a four-week gavage experiment were conducted in essential hypertensive rats (SHR).

[0038] Studies have shown that the yeast peptides obtained using the above-mentioned technical methods have a significant in vivo blood pressure lowering effect, which is achieved through the following pathways: inhibiting the activity of ACE in SHR and reducing serum Ang II levels; improving oxidative stress in SHR, increasing the expression levels of ACE2 and angiotensin II receptor type 2 (AT2R), and decreasing the expression level of angiotensin II receptor type 1 (AT1R); increasing the level of intestinal short-chain fatty acids and improving intestinal microbial dysbiosis caused by hypertension.

[0039] This invention provides a method for preparing a yeast peptide with antihypertensive effects, which is obtained by hydrolyzing edible yeast with a complex protease. The hydrolysate has good potential and application prospects as a health product and drug for the prevention and treatment of hypertension. Attached Figure Description

[0040] Figure 1 IC50 of yeast peptides against ACE 50 ;

[0041] Figure 2 The figure shows the effect of yeast peptides on the expression level of ACE2 protein in SHR kidneys; Note: * indicates a significant difference (p<0.05), ** indicates a significant difference (p<0.01), *** indicates a significant difference (p<0.001);

[0042] Figure 3 The image shows the effect of yeast peptides on the expression level of AT1R protein in SHR kidneys; Note: * indicates a significant difference (p<0.05), ** indicates a significant difference (p<0.01), *** indicates a significant difference (p<0.001).

[0043] Figure 4The image shows the effect of yeast peptides on the expression level of AT2R protein in SHR kidneys; Note: * indicates a significant difference (p<0.05), ** indicates a significant difference (p<0.01), and *** indicates a significant difference (p<0.001).

[0044] Figure 5 The figure shows the effect of yeast peptides on the α-diversity index of SHR gut microbiota, where Figure 5 Figure A shows the effect of yeast peptides on the SHR gut microbiota Sobs index. Figure 5 Figure B shows the effect of yeast peptides on the ACE index of SHR gut microbiota. Figure 5 Figure C shows the effect of yeast peptides on the Shannon index of gut microbiota in SHR. Figure 5 D shows the effect of yeast peptides on the Simpson index of gut microbiota in SHR.

[0045] Figure 6 The effect of yeast peptides on the Firmicutes to Bacteroidetes ratio (F / B) in the gut of SHR is shown. Detailed Implementation

[0046] This invention provides a yeast peptide containing the following peptides: a first peptide: phenylalanine-valine-alanine-leucine-proline, a second peptide: valine-leucine-isoleucine-leucine-lysine, and a third peptide: alanine-glycine-leucine-threonine-leucine.

[0047] In one specific embodiment of the present invention, the method for preparing the yeast peptide includes the following steps:

[0048] Step 1: Extract nucleic acids from edible yeast milk, and collect the yeast milk after nucleic acid removal, or add water to edible denucleated yeast powder to obtain yeast milk;

[0049] Step 2: Directional enzymatic hydrolysis of proteins in yeast milk after nucleic acid removal;

[0050] Step 3: Centrifuge. The light phase is the yeast peptide, which can also be further purified by membrane separation.

[0051] Step 4: For easy preservation, the above light phase or membrane separation permeate can be further spray-dried or freeze-dried to obtain powdered yeast peptides.

[0052] The denucleated yeast milk used in this invention can be any conventional food-grade denucleated yeast milk or yeast milk obtained by mixing yeast powder with water after denucleating by any conventional method, or it can be yeast milk obtained by mixing any commercially available conventional food-grade denucleated yeast powder with water.

[0053] In one specific embodiment of the present invention, the method for preparing the yeast peptide includes the following steps:

[0054] Step 1: Extract nucleic acids from edible yeast milk and collect the yeast milk after nucleic acid removal;

[0055] Step 2: Directional enzymatic hydrolysis of the cell wall in the denucleated yeast milk, followed by centrifugation;

[0056] Step 3: The centrifuged recombinant phase from Step 2 is digested with protease;

[0057] Step 4: Centrifuge in step 3. The light phase is the yeast peptide, which can also be further purified by membrane separation.

[0058] Step 5: For easy preservation, the above light phase or membrane separation permeate can be further spray-dried or freeze-dried to obtain powdered yeast peptides.

[0059] In one embodiment of the present invention, the first enzyme may be a neutral protease known in the art. Specifically, for example, commercially available Protin SD-NY 10 (sold by Amano Amano Enzyme Trading (Shanghai) Co., Ltd.) may be used in the present invention.

[0060] In one embodiment of the present invention, the first enzyme may be a neutral protease known in the art. Specifically, for example, commercially available Protease A 2SD (sold by Amano Amano Enzyme Trading (Shanghai) Co., Ltd.) may be used in the present invention.

[0061] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] Table 1 Experimental Materials / Instruments and Manufacturers

[0063]

[0064]

[0065]

[0066] Laboratory animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0067] Example 1: Preparation of yeast peptides

[0068] Take 1 kg of denucleotide-free yeast powder, add purified water to prepare a homogeneous solution with a mass fraction of approximately 10%, adjust the pH to 8.5, heat to 75℃ and stir for 2 hours, then centrifuge to collect the heavy phase. Disperse the heavy phase with an appropriate amount of water, adjust the pH to 8.2, add 3 g of a mixture of Thermoase C and Papain (1:1), and hydrolyze for 12 hours. Centrifuge to collect the supernatant, which is the yeast peptide. Freeze-dry into powder.

[0069] Example 2: Preparation of yeast peptides

[0070] Take 1 kg of denucleotide-free yeast powder, add purified water to prepare a homogeneous solution with a mass fraction of approximately 10%, adjust the pH to 7.5, heat to 90℃ and incubate for 2 hours, centrifuge and collect the heavy phase; after dispersing the heavy phase with an appropriate amount of water, adjust the pH to 5.5, add 4 g of β-glucanase, and enzymatically hydrolyze for 12 hours, then centrifuge and collect the heavy phase; after dispersing the heavy phase with an appropriate amount of water, adjust the pH to 8.0, add 2 g of complex protease (Protin SD-NY 10), and enzymatically hydrolyze for 10 hours. Centrifuge and collect the supernatant, which is the yeast peptide. Spray dry into powder.

[0071] Example 3: Identification of yeast polypeptides

[0072] Two micrograms of the samples prepared in Examples 1 and 2 were weighed respectively, and chromatographic gradient separation was performed for 60 min. Detection was then performed using a Thermo Orbitrap-Lumos ultra-high resolution mass spectrometer. Specific instrument parameters are as follows: High-performance liquid chromatography (Easyn LC1200); Column: C18, 3 μm, 100A, 75 μm × 15 cm; Sample loading: 2 μg. Mobile phase: A: 0.1% Formic acid in water; B: 0.1% Formic acid in 80% Acetonitrile / H2O. The chromatographic gradient is shown in Table 2 below.

[0073] Table 2

[0074]

[0075]

[0076] The mass spectrometry acquisition parameters are as follows: Orbitrap-Lumos mass spectrometer, spray voltage: 2.0 kV, capillary temperature: 320℃, S-lens RF Level: 40, collision energy: 35% NCE, isolation window: 1.2 Da, resolution settings: Level 1 120,000@m / z 200, AGC Target 4e5, maximum ion implantation time for Level 1 50 ms, precursor ion scan range: m / z 300-1500; resolution settings: Level 2 15,000@m / z 200, AGC Target 5e4, maximum ion implantation time for Level 1 22 ms, daughter ion scan range: start from m / z 110.

[0077] Data Analysis: Mass spectrometry data of the samples were analyzed using PEAKS Studio software. Peaks searches were performed using the yeast species database (uniport-saccharomyces+cerevisiae.fasta). Specific search parameters were set as follows: trypsin partial digestion mode, primary precursor ion window of 10 ppm, secondary ion window of 0.05 Da, missed cleavage setting of 2, and modifications: fixed Carbamidomethylation on C, variable Oxidation on M, and variable Deamidation on NQ.

[0078] The peptide identification results obtained in Example 1 are shown in Table 3 below. The peptide identification results obtained in Example 2 are shown in Table 4 below.

[0079] Table 3

[0080]

[0081] Table 4

[0082]

[0083]

[0084] As shown in Tables 3 and 4, the following peptide sequences FVALP, AGLTL, and VLILK were prepared in Examples 1 and 2.

[0085] Example 4: Method for identifying the ACE inhibitory activity of peptides

[0086] The identified peptides were retrieved online from the BIOPEP Active Peptide Database, and ACE (angiotensin-converting enzyme, a target for lowering blood pressure) inhibitory peptides already included were analyzed. Peptides from the peptide library were molecularly docked with ACE (using Autodock 4.2 docking software), and the binding free energy ΔG (in kJ / mol) obtained from the docking was used for screening. A lower ΔG suggests the peptide may have higher inhibitory activity against the target.

[0087] Among the identified peptides, (phenylalanine-valine-alanine-leucine-proline) FVALPΔG is -13.4 KJ / mol, (valine-leucine-isoleucine-leucine-lysine) VLILKΔG is -13.22 KJ / mol, and (alanine-glycine-leucine-threonine-leucine) AGLTLΔG is -12.73 KJ / mol, which are peptides with relatively low free energy and high content.

[0088] These three peptides were synthesized in vitro, and their bioactivity was then verified. The specific methods are as follows:

[0089] Three chemically synthesized peptides were prepared into different concentrations using deionized water. Rabbit-derived ACE was prepared into a 310 mU solution using 0.1 M borate buffer at pH 8.3. The substrate hippurylhistylleucine (HHL) was prepared into a 5 mM solution using borate buffer. The specific assay method was as follows: 10 μL of ACE solution and 25 μL of sample solution (yeast hydrolysate of different concentrations) were added to a 1.5 mL EP tube and incubated at 37 °C for 10 min. Then, 50 μL of HHL solution was added, and the reaction was incubated at 37 °C for 1 h. After the reaction, 0.5 mL of 0.5 M HCl solution was added. The reaction solution was filtered through a 0.22 μm aqueous membrane and the product hippuric acid was determined by high-performance liquid chromatography (HPLC). The specific high-performance liquid chromatography (HPLC) conditions were as follows: 25% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid) as the mobile phase, flow rate of 0.8 mL / min, detection wavelength of 228 nm, column temperature of 30 °C, and sample loading volume of 10 μL. The ACE inhibition rate (X) was calculated using the following formula:

[0090] X = (Control A – A1) / Control A × 100%

[0091] In the formula: X is the ACE inhibition rate (%), A control is the peak area of ​​hippuric acid in the blank control, and A1 is the peak area of ​​hippuric acid in the experimental group.

[0092] IC 50 Calculation of IC 50 It is the concentration of the inhibitor that inhibits 50% of ACE activity, obtained by measuring the ACE inhibition rate corresponding to different concentrations of the sample.

[0093] The test results are shown in Table 5. As shown in Table 5, FVALP, VLILK, and AGLTL in this invention all exhibit good ACE inhibitory activity, with IC50 values ​​reaching the μM level. The inhibition rate was highest, especially at a concentration of 0.15 mg / mL.

[0094] Table 5. In vitro ACE inhibitory activity of the synthesized peptides

[0095]

[0096] Example 5: Determination of the in vitro ACE inhibitory activity of yeast peptides

[0097] The lyophilized yeast peptides prepared in Example 2 were prepared into different concentrations using deionized water. Rabbit-derived ACE was prepared into a 310 mU solution using 0.1 M borate buffer at pH 8.3. The substrate hippurylhistylleucine (HHL) was prepared into a 5 mM solution using borate buffer. The specific assay method was as follows: 10 μL of ACE solution and 25 μL of sample solution (yeast hydrolysate of different concentrations) were added to a 1.5 mL EP tube and incubated at 37 °C for 10 min. Then, 50 μL of HHL solution was added, and the reaction was incubated at 37 °C for 1 h. After the reaction, 0.5 mL of HCl solution (0.5 M) was added. The reaction solution was filtered through a 0.22 μm aqueous membrane and the product hippuric acid was determined by high-performance liquid chromatography (HPLC). The specific high-performance liquid chromatography (HPLC) conditions were as follows: 25% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid) as the mobile phase, flow rate of 0.8 mL / min, detection wavelength of 228 nm, column temperature of 30 °C, and sample loading volume of 10 μL. The ACE inhibition rate (X) was calculated using the following formula:

[0098] X = (Control A – A1) / Control A × 100%

[0099] In the formula: X is the ACE inhibition rate (%), A control is the peak area of ​​hippuric acid in the blank control, and A1 is the peak area of ​​hippuric acid in the experimental group.

[0100] IC 50 Calculation of IC 50 It is the concentration of the inhibitor that inhibits 50% of ACE activity, obtained by measuring the ACE inhibition rate corresponding to different concentrations of the sample.

[0101] Experimental results are as follows Figure 1 As shown, from Figure 1 It can be seen that the inhibitory activity of ACE increases with increasing peptide concentration. The ACE inhibition rate is approximately 63% at 0.15 mg / mL, and the increase in inhibition rate is not significant with further increases in concentration (the ACE inhibition rate is approximately 73% at 0.295 mg / mL).

[0102] Example 6: Determination of the in vivo blood pressure-lowering effect of yeast polypeptides

[0103] I. Effects of a single dose on essential hypertensive rats

[0104] Experimental material information:

[0105] Twenty-four 9-week-old clean-grade male essential hypertensive rats (SHR) were randomly divided into four groups: a model group, a positive control group (Lisinopril), a low-dose yeast peptide group (YP-200) prepared in Example 2, and a high-dose yeast peptide group (YP-400) prepared in Example 2. Six 9-week-old clean-grade male Wistar rats were used as the normal control group. All experimental animals had free access to food and water, and the environment was maintained at 24±2℃ with 12 hours of light per day. The animals were allowed acclimatization for 7 days after transport, during which blood pressure was measured using the tail artery pressure method. All rats were preheated at 37℃ for 15 minutes before blood pressure measurement. Initial blood pressure was recorded for each rat group before the experiment, and the drug administration details are as follows:

[0106] 1) Normal control group: The same volume of physiological saline was administered by gavage daily during the experimental period;

[0107] 2) Model group: The same volume of physiological saline was administered by gavage daily during the experimental period;

[0108] 3) Positive control group (Lisinopril): Lisinopril (Zhejiang Huahai Pharmaceutical Co., Ltd.) administered by gavage at a daily dose of 2.5 mg / kg BW during the experimental period;

[0109] 4) Low-dose yeast peptide group (YP-200): The daily oral dose of yeast peptide during the experimental period was 200 mg / kg BW.

[0110] 5) High-dose yeast peptide group (YP-400): The daily oral dose of yeast peptide was 400 mg / kg BW during the experimental period.

[0111] All the above reagents were dissolved in physiological saline. The corresponding samples were administered to each group via gavage. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured at 0, 1, 2, 4, 6, and 8 hours after gavage using the tail artery manometry method. Measurements were taken five times, and the average values ​​were calculated. The results are shown in Tables 6 and 7.

[0112] Table 6. Effect of a single dose of yeast peptide on SHR systolic blood pressure (mmHg)

[0113]

[0114] Note: For the same group of systolic pressures relative to 0h, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0115] Table 7. Effect of a single dose of yeast peptide on diastolic blood pressure in SHR (mmHg)

[0116]

[0117] Note: For diastolic blood pressure relative to 0h in the same group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0118] As shown in Tables 6 and 7, the acute experimental results after a single dose indicate that oral administration of 200 and 400 mg / kg BW of yeast peptide significantly reduced systolic and diastolic blood pressure in SHR rats. In the YP-200 group, systolic and diastolic blood pressure decreased by 12 mmHg after 4 hours of administration, while in the YP-400 group, they decreased by 17 mmHg and 16 mmHg, respectively. This demonstrates that higher doses of yeast peptide have a stronger acute antihypertensive effect.

[0119] II. Effects of continuous drug administration on essential hypertensive rats

[0120] After completing the acute experiment with a single dose, each group continued to be administered the same sample and dose by gavage daily for 4 weeks. On days 7, 14, 21, and 28, the systolic and diastolic blood pressure of each group of rats was measured 24 hours after the previous administration. The measurements were taken 5 times, and the average values ​​were taken as shown in Tables 8 and 9.

[0121] Table 8. Effect of continuous administration of yeast peptide on systolic blood pressure in SHR (mmHg)

[0122]

[0123] Note: For systolic blood pressure relative to week 0 within the same group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; for systolic blood pressure in different groups relative to the model group at the same time point, ... # This indicates that p < 0.05. ## This indicates that p < 0.01. ### This indicates that p < 0.001.

[0124] Table 9. Effects of continuous administration of yeast peptides on diastolic blood pressure in SHR (mmHg)

[0125]

[0126] Note: For diastolic blood pressure relative to week 0 within the same group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; for diastolic blood pressure in different groups relative to the model group at the same time point, ... # This indicates that p < 0.05. ## This indicates that p < 0.01. ### This indicates that p < 0.001.

[0127] The results in Tables 8 and 9 show that the continuous dosing experiment demonstrated that after four consecutive weeks of administration of yeast peptides at doses of 200 and 400 mg / kg BW, both systolic and diastolic blood pressure were significantly reduced compared to the model group. Specifically, in the high-dose group, systolic and diastolic blood pressure at week 4 showed a significant decrease compared to week 0, with reductions of 11 mmHg and 10 mmHg, respectively. In the low-dose group, both systolic and diastolic blood pressure at week 0 showed some decrease compared to week 0, but this was not statistically significant. This study indicates that a 10 mmHg reduction in blood pressure can significantly reduce the risk of cardiovascular disease.

[0128] In conclusion, both single-dose and continuous-dose experiments have demonstrated that yeast peptides have the potential to be used as antihypertensive drugs or functional health foods.

[0129] Example 7: Effects of yeast polypeptides on the SHR renin-angiotensin system

[0130] After the experiment, rats in the model group, positive control, YP-200 group and YP-400 group in Example 6 were fasted for 12 hours and blood was collected. Serum was obtained by centrifugation. Then, the rats were euthanized and dissected in accordance with the regulations for the management of experimental animals. The kidneys, colons and livers of the rats were taken and stored at -80℃.

[0131] I. Effects of yeast peptides on ACE activity and Ang II content in SHR (Synthetic Angiotensin II)

[0132] In the fourth week, serum ACE activity was measured: according to the definition of enzyme activity, HHL was used as a substrate to perform an enzyme reaction to measure the ACE activity of each group of rat serum. The results are shown in Table 8.

[0133] Determination of serum Ang II levels in the fourth week: The serum Ang II levels of rats in each group were measured using an ELISA kit, and the results are shown in Table 10.

[0134] Table 10 Effects of yeast peptides on ACE activity and Ang II content in SHR in vivo

[0135]

[0136] Note: Serum ACE activity relative to the model group. # This indicates that p < 0.05. ## This indicates that p < 0.01.### * indicates p < 0.001; relative to the serum Ang II content in the model group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0137] Table 10 shows that the serum ACE activity and Ang II levels in SHR, after continuous administration of 200 mg / kg and 400 mg / kg yeast peptides for 4 weeks, significantly decreased serum ACE activity compared to the model control, and further significantly decreased serum Ang II levels, with the high-dose group showing a more significant effect. This indicates that yeast peptides can lower blood pressure by inhibiting ACE activity in SHR and reducing Ang II levels.

[0138] II. Effects of yeast peptides on the expression of key proteins in the renin-angiotensin system (RAS)

[0139] Determination of expression levels of key proteins in the kidney: A suitable amount of kidney sample was added to 9 times its mass of RIPA and disrupted using a tissue homogenizer. The homogenate was then centrifuged at 12,000 rpm, and the supernatant was collected as the total protein extract. Using β-actin as an internal control protein, the relative protein levels of angiotensin-converting enzyme 2 (ACE2), angiotensin type 1 receptor (AT1R), and angiotensin type 2 receptor (AT2R) in the kidney tissue homogenate were determined by Western blotting. The experimental results are as follows: Figures 2-4 As shown in the electrophoresis results, yeast peptides increased the expression levels of ACE2 and angiotensin II receptor 2 (AT2R) (ACE2 / Actin ratios in the model group, lisinopril, yeast peptide-200, and yeast peptide-400 groups were 0.75, 0.711, 0.83, and 0.99, respectively; AT2R / Actin ratios were 0.69, 0.56, 0.70, and 0.94, respectively), and decreased the expression level of angiotensin II receptor 1 (AT1R) (AT1R / Actin ratios in the model group, lisinopril, yeast peptide-200, and yeast peptide-400 groups were 0.975, 0.925, 0.735, and 0.755, respectively). Figure 2 The figure shows the effect of yeast peptides on the expression level of ACE2 protein in SHR kidneys; Figure 3 The figure shows the effect of yeast peptides on the expression level of AT1R protein in SHR kidneys; Figure 4 The figure shows the effect of yeast peptides on the expression level of AT2R protein in SHR kidneys; * indicates a significant difference (p<0.05), ** indicates a significant difference (p<0.01), and *** indicates a significant difference (p<0.001).

[0140] ACE2 can degrade Ang II, thereby lowering blood pressure. AT1R is a receptor that transmits vasoconstriction signals, while AT2R is a receptor that transmits vasodilation signals. Experimental results showed that after SHR received 200 mg / kg and 400 mg / kg of yeast peptides for four consecutive weeks, the expression levels of ACE2 and AT2R increased, while the expression level of AT1R decreased, with the high-dose group showing a more significant effect. These results indicate that yeast peptides lower blood pressure by regulating the expression of key proteins in the body's RAS system, and the higher the dose, the more significant the effect.

[0141] Example 8: Effects of yeast peptides on SHR oxidative stress levels

[0142] Determination of oxidative stress levels: Following the requirements and procedures of the malondialdehyde (MDA) assay kit, glutathione (GSH) assay kit, and superoxide dismutase (SOD) activity assay kit, the activities of MDA, GSH, and SOD in the serum of the model group, positive control, YP-200 group, and YP-400 rats in Example 7 were measured. The oxidative stress levels of each group of rats were comprehensively judged, and the results are shown in Table 11.

[0143] Table 11 Effects of yeast peptides on oxidative stress levels in SHR patients

[0144]

[0145] Note: Relative to the model group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0146] The results in Table 11 show that after four consecutive weeks of administration of yeast peptides at doses of 200 mg / kg and 400 mg / kg, SHR showed a significant reduction in serum MDA levels and an increase in GSH levels and SOD activity. MDA is a product of lipid oxidation, GSH is an endogenous antioxidant, and SOD is an important endogenous antioxidant enzyme. Oxidative stress can induce elevated blood pressure, and yeast peptides can significantly reduce oxidative stress in SHR, thereby contributing to a reduction in blood pressure.

[0147] Example 9: Effects of yeast peptides on short-chain fatty acid levels in the SHR gut

[0148] Determination of intestinal short-chain fatty acid levels: During the last 2-3 days of the experiment, feces from rats in each group of Example 6 were collected, and the intestinal short-chain fatty acid levels were characterized by measuring the short-chain fatty acids in fresh feces. The specific method was as follows: 1) Weigh 0.1g of fecal sample into a 2mL EP tube, add 1mL of deionized water, and vortex repeatedly to disperse the sample; 2) Centrifuge the vortexed sample for 10min, take out 0.8mL of supernatant, add 0.1mL of concentrated hydrochloric acid, and mix for 5min; 3) Add 5mL of diethyl ether, mix repeatedly and extract for 20min, and centrifuge for 10min to separate the organic and aqueous phases; 4) Transfer the upper organic phase to a new 10mL EP tube, add 0.5mL of 1M NaOH solution, mix and extract for 20min; 5) After extraction, centrifuge, take out the lower aqueous phase, add 0.1mL of concentrated hydrochloric acid, filter through a 0.22μm filter membrane, and use for high performance liquid chromatography determination. The mobile phase used in high-performance liquid chromatography (HPLC) was acetonitrile and 0.025% phosphoric acid solution in a ratio of 5:95; the flow rate was 1 mL / min, and the wavelength was 210 nm. Standards of acetic acid, propionic acid, and butyric acid at different concentrations were determined by HPLC to obtain standard curves. The concentrations of the three short-chain fatty acids in the feces of different groups of rats were calculated using the standard curves, and the results are shown in Table 12.

[0149] Table 12 Effects of yeast peptides on short-chain fatty acids in SHR gut

[0150]

[0151] Note: Relative to the model group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0152] The results in Table 12 show that after four consecutive weeks of administration of yeast peptides at doses of 200 mg / kg and 400 mg / kg, the levels of the three main short-chain fatty acids in the intestines of SHR (Self-Rich Patients) were increased to some extent. Short-chain fatty acids play a positive role in regulating blood pressure, and yeast peptides can increase the level of short-chain fatty acids in the intestines of SHR, thereby contributing to lowering blood pressure.

[0153] Example 10: Effects of yeast peptides on the gut microbiota of SHR

[0154] Gut microbial diversity analysis: After collecting colon contents from rats in each group, microbial genomes were extracted. DNA from the V3-V4 region of the bacterial 16S rRNA gene was then amplified, purified, and quantified. The purified samples were sequenced. Low-quality reads were filtered after sequencing, then assembled and filtered again to obtain valid data for OTU clustering. After obtaining the OTUs, species annotation, α-diversity analysis, principal coordinate analysis (PCoA), and species composition analysis were performed sequentially. The analysis results are as follows: Figures 5-6 As shown. Among them. Figure 5 Figure A shows the effect of yeast peptides on the SHR gut microbiota Sobs index. Figure 5 Figure B shows the effect of yeast peptides on the ACE index of SHR gut microbiota. Figure 5 Figure C shows the effect of yeast peptides on the Shannon index of gut microbiota in SHR. Figure 5 D shows the effect of yeast peptides on the Simpson index of gut microbiota in SHR. Figure 6 The effect of yeast peptides on the Firmicutes to Bacteroidetes ratio (F / B) in the gut of SHR is shown.

[0155] Alpha-diversity analysis of the gut microbiota in rats of each group revealed that the Sobs (Richness), ACE index, Shannon index, and Simpson index in the model group were significantly lower than those in the normal group, while gavage administration of yeast peptides significantly improved all four indicators of gut microbiota α-diversity in SHR rats. Numerous studies have shown that gut microbiota α-diversity is significantly reduced in hypertensive patients and hypertensive animal models, and gavage administration of 400 mg / kg yeast peptides can significantly improve the α-diversity of gut microbiota in SHR rats. PCoA results showed that the microbiota structure of the YP-200 and YP-400 groups of rats differed significantly from that of the model group. Phylogenetic stacking diagrams showed that gavage administration of yeast peptides could regulate the microbiota of SHR rats, causing it to develop towards the composition of the normal rat microbiota. Figure 6 It can be seen that the ratio of Firmicutes to Bacteroidetes (F / B) in the YP-400 group was significantly lower than that in the model group. Literature indicates that the F / B value is positively correlated with hypertension, and yeast peptides can regulate blood pressure by modulating the abundance of different bacterial communities.

[0156] Example 11: A blood pressure-lowering functional food

[0157] Ten parts by weight of the yeast peptide prepared in Example 2 were dissolved in 71.5 parts by weight of water along with 5 parts by weight of coconut milk powder, 5 parts by weight of red grape concentrate, and 8 parts by weight of erythritol. After complete dissolution, 0.5 parts by weight of edible flavoring powder was added to prepare 1000g of a food product with blood pressure-lowering function. After shaking well, the product was UHT sterilized (135℃, 5-8s) and bottled into small glass bottles, each containing 30ml.

[0158] Example 12: A blood pressure lowering drug

[0159] 20 parts by weight of the yeast peptide prepared in Example 2 were mixed with 20 parts by weight of Ginkgo biloba extract and 59.5 parts by weight of sorbitol until homogeneous. Then, 0.5 parts by weight of magnesium stearate were added and mixed until homogeneous to prepare 1000g of a drug with antihypertensive function. The above materials were compressed into tablets using a direct compression method, with each tablet weighing 1g. It is recommended to take 1-2 tablets daily.

[0160] Example 13: A blood pressure lowering health food

[0161] Ten parts by weight of the yeast peptide prepared in Example 2 were mixed with 20 parts by weight of kudzu root extract and 69 parts by weight of resistant dextrin until homogeneous. Then, 1 part by weight of silica was added and mixed until homogeneous to prepare 1000g of a health food product with blood pressure lowering function. The product was then filled into hard capsules with a content of 0.5g using a capsule machine. The dosage is 2-3 capsules twice daily.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A yeast peptide composition, characterized in that, The yeast peptide composition consists of the following peptides: a first peptide: phenylalanine-valine-alanine-leucine-proline, a second peptide: valine-leucine-isoleucine-leucine-lysine, and a third peptide: alanine-glycine-leucine-threonine-leucine.

2. The yeast peptide composition of claim 1, wherein, The mass ratio of the first peptide, the second peptide and the third peptide in the yeast peptide composition is 0.3-1:0.3-1:0.3-1.

3. The yeast peptide composition of claim 1, wherein, The mass ratio of the first peptide, the second peptide and the third peptide in the yeast peptide composition is 0.3-0.5:0.3-0.9:0.5-1.

4. A peptide, characterized in that, The peptide consists of the following sequence: phenylalanine-valine-alanine-leucine-proline.

5. A peptide, characterized in that, The peptide consists of the following sequence: valine-leucine-isoleucine-leucine-lysine.

6. A peptide, characterized in that, The peptide consists of the following sequence: alanine-glycine-leucine-threonine-leucine.

7. Use of the yeast peptide composition of any one of claims 1-3 or the peptide of any one of claims 4-6 in the preparation of a blood pressure lowering drug or a health care product for assisting blood pressure lowering.

8. A blood pressure lowering medicament, characterized in that, The blood pressure lowering drug contains the yeast peptide composition of any one of claims 1-3 or the peptide of any one of claims 4-6.

9. The antihypertensive medicament according to claim 8, wherein The content of the peptide in the yeast peptide composition in the blood pressure lowering drug is 65-85 wt%.

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