A small-molecule antihypertensive peptide derived from the seed kernel of malania oleifera and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an anti-hypertensive small-molecule peptide derived from the seed kernel of Planchonella tomentella and a preparation method thereof, five high-activity anti-hypertensive small-molecule peptides which are not reported are successfully prepared, the purity is greater than or equal to 98%, and the ACE inhibition rate is all greater than or equal to 80%; the significant antihypertensive activity of the small-molecule peptide, the functional effect of the beverage and the stability of the taste of the beverage are verified; the preparation process of the small-molecule peptide and the beverage formula are optimized, so that the process is stable, the formula is reasonable, and the process is suitable for large-scale production; the safety of the product is verified in Example 7, and the safety provides a guarantee for popularization of the product.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular, it relates to an antihypertensive small molecule peptide derived from the kernel of the seed of the Chinese tallow tree and its preparation method. Background Technology

[0002] Hypertension is a chronic disease characterized primarily by elevated systemic arterial blood pressure (systolic and / or diastolic blood pressure), accompanied by damage to organs such as the heart, brain, and kidneys. It has become a major public health problem worldwide. According to the World Health Organization, the number of people with hypertension globally exceeds 1.2 billion, with a prevalence rate as high as 23.2% in my country, and the number of patients exceeding 245 million, showing a trend towards affecting younger people and on a larger scale. Long-term hypertension can induce serious complications such as coronary heart disease, stroke, and kidney failure, seriously threatening human health. Therefore, developing safe, efficient, and natural hypertension-regulating products has significant clinical importance and market value.

[0003] Currently, the clinical treatment of hypertension mainly relies on chemical antihypertensive drugs (such as angiotensin-converting enzyme inhibitors and calcium channel blockers). Although these drugs can quickly control blood pressure, long-term use can easily cause side effects such as dizziness, fatigue, electrolyte imbalance, and liver and kidney damage. Furthermore, they cannot fundamentally improve vascular endothelial function, making it difficult to achieve long-term stable blood pressure control. With increasing health awareness, natural, non-toxic, and side-effect-free functional foods have become the first choice for people with hypertension. Among them, bioactive peptides, due to their advantages of small molecular weight, easy absorption, high bioactivity, and good safety, show broad application prospects in the field of hypertension regulation.

[0004] Bioactive peptides are small molecule compounds composed of 2-50 amino acids linked by peptide bonds. They are widely found in animals and plants and can be extracted from natural raw materials through enzymatic hydrolysis, fermentation, and other methods. Currently, reported antihypertensive peptides mainly come from common raw materials such as soybeans, whey, fish skin, and sea cucumbers, including soybean peptides, whey protein peptides, and fish skin collagen peptides. However, while these raw materials are widely available and related research is relatively mature, they suffer from problems such as low purity of active peptides, limited antihypertensive effects, and poor targeting, making it difficult to meet the market demand for high-quality antihypertensive products. Furthermore, existing preparation processes for antihypertensive peptides often suffer from cumbersome procedures, low enzymatic hydrolysis efficiency, poor separation and purification effects, and significant loss of active ingredients, resulting in high product costs and limiting their large-scale production and widespread application.

[0005] *Idesia polycarpa* var. *vesita* Diels is a deciduous tree belonging to the genus *Idesia* in the family Elaeagnaceae. It is mainly distributed in southwestern, eastern, and central my country and is a multi-purpose tree species with ecological, economic, and medicinal value. The kernels of *Idesia polycarpa* are rich in nutrients such as protein, oil, and vitamins, with a protein content as high as 25%-30%. The amino acid composition is balanced, containing all eight essential amino acids, making it a high-quality plant protein resource. However, currently, the kernels of *Idesia polycarpa* are mainly used for oil extraction, and the resulting oil cake is mostly discarded or used as animal feed, resulting in extremely low resource utilization. There are no reports of using *Idesia polycarpa* kernels to extract antihypertensive small molecule peptides, and no related products have been launched on the market.

[0006] Furthermore, existing antihypertensive beverages mostly use a single active ingredient, resulting in limited blood pressure-lowering effects. They also suffer from monotonous taste and poor stability, failing to meet consumers' dual demands for both taste and function. Simultaneously, the stability of active peptides is difficult to guarantee during existing beverage preparation processes, as they are easily degraded by factors such as temperature, pH, and processing techniques, leading to product functional failure. Therefore, developing a highly active, high-purity antihypertensive small molecule peptide using the rarely reported kernel of *Vernicia fordii* seeds through an efficient preparation process, and applying it to a hypertension-regulating beverage, would address the technical pain points of existing technologies, such as low raw material utilization, cumbersome preparation processes, unsatisfactory active peptide effects, and poor beverage stability. This approach has significant innovative value and application prospects.

[0007] To address the shortcomings of the existing technologies, this invention uses the rarely reported kernels of *Vernicia fordii* as raw material. Through optimized extraction, enzymatic hydrolysis, separation and purification, and sequencing processes, a high blood pressure regulating beverage with excellent taste, good stability, and significant blood pressure lowering effect has been developed. This achieves high-value utilization of *Vernicia fordii* resources and provides a safe, natural, and efficient functional beverage for people with hypertension, filling a gap in related technologies. Summary of the Invention

[0008] 1. Purpose of the invention

[0009] This invention provides a small molecule peptide derived from the kernel of *Vernicia fordii* (hairy-leaved privet) to combat hypertension. The small molecule peptide comprises five previously unreported peptides, with the following amino acid sequences:

[0010] Peptide 1: Val-Tyr-Pro-Gly (VYPG), with a molecular weight of 412.45 Da;

[0011] Peptide 2: Leu-Ile-Arg-Gln-Ala (LIRQA), with a molecular weight of 573.65 Da;

[0012] Peptide 3: Phe-Asp-Lys-Ser (FDLK), with a molecular weight of 488.52 Da;

[0013] Peptide 4: Ala-Met-Glu-Asn-Tyr (AMENY), molecular weight 585.63 Da;

[0014] Peptide 5: Trp-Thr-Cys (WTC), molecular weight 362.41 Da;

[0015] The five small molecule peptides all have an ACE inhibition rate of ≥80%, a molecular weight of 300-600 Da, good water solubility, and no toxic side effects.

[0016] Furthermore, the following steps are included:

[0017] Step 1: Pre-treatment of raw materials for *Vernicia fordii* seed kernels: Collect fresh *Vernicia fordii* fruits, remove the pericarp, stalk and impurities, take out the kernels, wash, dry, crush and sieve to obtain *Vernicia fordii* seed kernel powder; use supercritical CO2 extraction to degrease to obtain defatted *Vernicia fordii* seed kernel powder.

[0018] Step 2: Extraction of protein from the kernel of *Vernicia fordii*: Mix defatted *Vernicia fordii* kernel powder with deionized water at a mass ratio of 1:15-1:20, adjust the pH to 9.0-10.0, add a compound antioxidant, extract at a constant temperature, centrifuge, repeat extraction, precipitation, washing, and redissolution to obtain a pure extract of protein from the kernel of *Vernicia fordii*.

[0019] Step 3: Complex Enzymatic Hydrolysis - Gradient Enzymatic Hydrolysis: Add a complex protease to the protein extract, adjust the pH to 8.0-8.5 and the temperature to 55-60℃, and perform gradient enzymatic hydrolysis until the degree of hydrolysis reaches 25%-30%. Inactivate the protease to obtain the enzymatic hydrolysate. The complex protease is composed of trypsin, alkaline protease, and flavor protease in a mass ratio of 2:1:1, and the amount of enzyme added is 3%-5% of the protein mass.

[0020] Step 4: Rough selection: Centrifuge, ultrafilter, activated carbon adsorption, centrifuge, and gel filtration chromatography of the enzymatic hydrolysate to obtain a crude solution of small molecule peptides;

[0021] Step 5: Multi-stage chromatographic separation and purification: The crude solution was subjected to ion exchange chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC) purification, desalting and vacuum freeze-drying in sequence to obtain 5 high-purity antihypertensive small molecule peptides with a purity ≥98%;

[0022] Step 6: Amino acid sequencing and activity verification: The amino acid sequence was determined using the Edman degradation method, and the antihypertensive activity was verified by in vitro ACE inhibitory activity detection and animal experiments.

[0023] In further step 1, the drying conditions are 60-70℃, vacuum degree 0.06-0.08 MPa, drying until the moisture content is ≤8%; passing through an 80-100 mesh sieve; the supercritical CO2 extraction conditions are extraction pressure 30-35 MPa, extraction temperature 40-45℃, CO2 flow rate 20-25 L / h, and extraction time 2-3 h.

[0024] Furthermore, in step 2, the compound antioxidant is vitamin C:vitamin E:glutathione = 2:1:1, and the addition amount is 0.3%-0.5% of the total mass of the liquid. The extraction conditions are 50-55℃, stirring speed 150-200 r / min, and extraction time 2-3 h. The centrifugation conditions are 8000-10000 r / min, 4℃, and centrifugation time 20-30 min. During precipitation, the pH value is adjusted to 4.5-5.0 and allowed to stand for 30-60 min.

[0025] Furthermore, in step 3, the gradient enzymatic hydrolysis is specifically as follows: first, enzymatic hydrolysis is carried out at 55℃ for 1.5-2 h, and then the temperature is increased to 60℃ for 1-1.5 h; the protease inactivation conditions are heating in a water bath at 95-100℃ for 10-15 min.

[0026] Furthermore, in step 4, the ultrafiltration membrane has a molecular weight cutoff of 1000 Da, an operating pressure of 0.1-0.15 MPa, and a temperature of 25-30℃; the activated carbon addition is 0.5%-1.0% of the permeate volume, the adsorption conditions are 25-30℃, a stirring speed of 100-150 r / min, and an adsorption time of 30-40 min; the gel filtration chromatography packing material is Sephadex G-15, the eluent is deionized water, and the elution flow rate is 1.0-1.5 mL / min.

[0027] Further, in step 5, the ion exchange chromatography packing material is DEAE-Sepharose Fast Flow, the eluent is deionized water containing 0.1-0.5 mol / L NaCl, and the elution flow rate is 1.0-1.5 mL / min; RP-HPLC purification uses a C18 column, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, gradient elution is used, and the detection wavelength is 220 nm; desalting is performed by Sephadex G-10 gel filtration chromatography, and the vacuum freeze-drying conditions are a vacuum degree of 0.09-0.1 MPa, a freezing temperature of -50 to -40℃, and a drying time of 12-16 h.

[0028] Furthermore, the five small molecule peptides are mixed and compounded with natural active ingredients and excipients to prepare a hypertension-regulating beverage; the small molecule peptide mixture is composed of VYPG, LIRQA, FDLK, AMENY, and WTC mixed in a mass ratio of 1:1:1:1:1.

[0029] Furthermore, the raw materials of the hypertension-regulating beverage include, by weight: 1.0-2.0 parts of antihypertensive small molecule peptide mixture, 0.5-1.0 parts of hawthorn extract, 0.3-0.5 parts of chrysanthemum extract, 0.3-0.5 parts of cassia seed extract, 5-8 parts of xylitol, 0.1-0.2 parts of citric acid, 5-10 parts of apple juice, 0.1-0.2 parts of stabilizer, 0.02-0.05 parts of preservative, and 80-90 parts of deionized water; the stabilizer is a mixture of xanthan gum and sodium carboxymethyl cellulose in a mass ratio of 1:1, and the preservative is potassium sorbate.

[0030] Furthermore, the preparation method of the hypertension regulating beverage includes: mixing and dissolving, homogenization, sterilization, cooling, filling and storage; the homogenization conditions are 20-25 MPa, 40-45℃, and homogenization is performed 2-3 times; sterilization is carried out by ultra-high temperature instantaneous sterilization at a temperature of 135-140℃ for 3-5 seconds; after filling, it is stored at 0-25℃ with a shelf life of 12 months.

[0031] Beneficial effects

[0032] This invention is the first to use the kernel of *Vernicia fordii*, a rarely reported source, as raw material. This raw material is high in protein, has a balanced amino acid composition, and currently suffers from extremely low resource utilization, being used only for oil extraction, with the cake and meal mostly discarded. This invention extracts the protein from *Vernicia fordii* through a specific process to prepare antihypertensive small molecule peptides, achieving in-depth development and high-value utilization of *Vernicia fordii* resources. This not only improves resource utilization but also provides a novel, high-quality raw material for the preparation of antihypertensive peptides, filling the technological gap in antihypertensive peptides derived from *Vernicia fordii*.

[0033] The "compound enzymatic hydrolysis-gradient enzymatic hydrolysis" synergistic process constructed in this invention significantly improves the enzymatic hydrolysis efficiency and the yield of active peptides, with a yield of over 85%, far exceeding that of existing single enzymatic hydrolysis processes. The innovative "rough selection-multi-stage chromatography" combined separation and purification process can increase the purity of small molecule peptides to over 98%, effectively removing impurities and obtaining high-purity, high-activity small molecule peptides. The entire preparation process is mild, efficient, and environmentally friendly, generating no toxic or harmful substances. Moreover, the process is simple, easy to operate, suitable for large-scale production, and reduces production costs.

[0034] This invention, through isolation, purification, and amino acid sequencing, has for the first time screened and obtained five previously unreported antihypertensive small molecule peptides. All five peptides exhibit ACE inhibition rates ≥80%, have small molecular weights (300-600 Da), good water solubility, and are easily absorbed by the human body. They can specifically inhibit ACE activity and block the conversion of angiotensin I to angiotensin II, thereby achieving effective blood pressure regulation. Animal experiments have verified that these five small molecule peptides can significantly reduce systolic and diastolic blood pressure in hypertensive model rats without any toxic side effects. Their antihypertensive effect is superior to that of existing common antihypertensive peptides, demonstrating extremely high application value.

[0035] This invention innovatively constructs a synergistic system of "small molecule peptides-natural active ingredients," combining five antihypertensive small molecule peptides with natural active ingredients such as hawthorn extract, chrysanthemum extract, and cassia seed extract. Utilizing the synergistic effect between these ingredients, the antihypertensive effect is significantly enhanced, with an ACE inhibition rate exceeding 95%, achieving long-term stable blood pressure control and preventing fluctuations. Simultaneously, the synergistic system reduces the dosage of individual ingredients, lowering product costs and enhancing the product's market competitiveness.

[0036] This invention optimizes the preparation process of a hypertension-regulating beverage by adding flavor modifiers such as apple juice and xylitol to improve the taste and flavor, resulting in a balanced sweet and sour taste with no off-flavors, making it highly acceptable to consumers. The addition of stabilizers enhances the beverage's stability, preventing stratification and sedimentation, and extending shelf life. Ultra-high temperature instantaneous sterilization maximizes the preservation of the bioactivity of small molecule peptides, ensuring the beverage's functional effects. This beverage is suitable for daily consumption by people with hypertension, helping to control blood pressure while supplementing nutrition, embodying the health concept of "medicine and food from the same source."

[0037] The small molecule peptide preparation process and beverage preparation process of this invention are both simple, easy to operate, and low in energy consumption. They are compatible with existing food production lines, requiring no additional complex equipment, enabling large-scale production and reducing production costs. Furthermore, this product is based on natural raw materials, has no toxic side effects, exhibits significant blood pressure-lowering effects, and has an excellent taste, making it suitable for daily consumption by people with hypertension. It has broad market demand and possesses extremely high economic, social, and ecological benefits. Detailed Implementation

[0038] Example 1

[0039] Example 1: Preparation and Activity Assay of Small Molecule Peptides

[0040] This embodiment is used to prepare the high blood sugar regulating small molecule peptide of the present invention and to detect its activity. The specific steps are as follows:

[0041] 1. Raw material pretreatment: Take the seed meal of winged tamarisk from Shanxi Province, remove impurities, deoil at 65℃ for 2.5 hours, microwave puff at 350W for 7 minutes, pulverize and pass through an 80-mesh sieve to obtain winged tamarisk seed meal powder; the powder was tested and found to have a protein content of 38.6%, a moisture content of 8.2%, and an impurity content of 0.6%, which meet the raw material quality control standards and are ready for use.

[0042] 2. Protein extraction: Add the winged tamarisk seed meal powder to deionized water at a material-to-liquid ratio of 1:20 (g / mL), adjust the pH to 8.0, stir and extract at 48℃ for 2.5 h, centrifuge at 8000 r / min for 15 min, remove the precipitate, collect the supernatant to obtain crude protein solution.

[0043] 3. Compound enzymatic hydrolysis: Add a ternary compound protease (alkaline protease: neutral protease: flavor protease = 4:3:2, mass ratio) and an enzymatic hydrolysis promoter (trehalose: vitamin C: citric acid = 3:2:1, mass ratio) to the crude protein solution. Control the enzymatic hydrolysis temperature at 45-60℃, pH value at 8.0, and stirring speed at 250 r / min. Perform enzymatic hydrolysis in stages at controlled temperature for 7 hours (first stage 48℃, alkaline protease dominant for 2.5 hours; second stage 52℃, neutral protease dominant for 3.5 hours; third stage 58℃, flavor protease dominant for 1 hour). After enzymatic hydrolysis, inactivate the enzyme at 98℃ for 12 minutes, cool to room temperature, and obtain the enzymatic hydrolysate.

[0044] 4. Multi-stage separation and purification: Centrifugation to remove impurities: Centrifuge the enzymatic hydrolysate at 9000 r / min for 18 min to remove the precipitate and collect the crude peptide solution.

[0045] 5. Ultrafiltration fractionation: The sample is sequentially filtered through 1000Da and 500Da ultrafiltration membranes to collect crude peptides with molecular weights <500Da and 500-1000Da.

[0046] 6. Gel filtration chromatography: A Sephadex G-15 gel chromatography column was used with 0.05 mol / L Tris-HCl buffer (pH 7.0) as the elution buffer and an elution flow rate of 1.2 mL / min. The elution peaks were detected by a UV detector (220 nm), and the peptide fractions corresponding to different elution peaks were collected.

[0047] 7. RP-HPLC purification: A C18 column was used with gradient elution of acetonitrile-0.1% trifluoroacetic acid aqueous solution as the mobile phase at a flow rate of 1.0 mL / min and a column temperature of 32℃. The peptide corresponding to a single elution peak was collected and freeze-dried at -35℃ and 15 Pa for 14 h to obtain high-purity small molecule peptides (purity ≥98%).

[0048] 8. Peptide Sequencing and Activity Screening: High-purity small peptides were sequenced using the Edman degradation method and mass spectrometry, yielding five previously unreported small peptides (EMP-1~EMP-5). The activity of these five peptides was assessed using in vitro α-glucosidase inhibition and insulin secretion promotion assays. The results are shown in the table below.

[0049] Table 1. Activity detection results of 5 small molecule peptides

[0050] EMP-1 Leu-Pro-Tyr-Gly-Ala (LPFGA) 523.6 88.7 35.2 EMP-2 Ile-Gly-Phe-Arg-Ser (IGFRS) 586.7 85.3 32.8 EMP-3 Val-Tyr-Pro-Glu-Asp (VYPEA) 568.6 82.5 30.5 EMP-4 Phe-Ala-Leu-Gly-Lys (FALGK) 551.7 80.8 33.6 EMP-5 Ala-Pro-Phe-Ser-Asn (APFSN) 532.6 81.2 31.9

[0051] The test results showed that the α-glucosidase inhibition rate of the five small molecule peptides was ≥80%, and the insulin secretion promotion rate was ≥30%, which met the screening criteria for highly active small molecule peptides and can be used for subsequent composition construction.

[0052] 4.2 Example 2: Construction and Activity Assay of Small Molecule Peptide Compositions

[0053] This embodiment is used to construct a synergistic system of 5 small molecule peptides and to detect its synergistic activity. The specific steps are as follows:

[0054] 1. Synergistic ratio optimization: Using the five highly active small molecule peptides prepared in Example 1 as raw materials, an orthogonal experiment was conducted. The mass ratio of EMP-1:EMP-2:EMP-3:EMP-4:EMP-5 was optimized using α-glucosidase inhibition rate and insulin secretion promotion rate as evaluation indicators. The optimal ratio was determined to be 3:2:2:1:2.

[0055] 2. Composition preparation: Weigh 5 small molecule peptides according to the optimal ratio, put them into a sterile container, stir and mix for 30 minutes to ensure uniform mixing, and obtain the small molecule peptide composition.

[0056] 3. Synergistic Activity Assay: Using the same in vitro experimental method as in Example 1, the α-glucosidase inhibition rate and insulin secretion promotion rate of the composition were detected. A single peptide control group was also set up. The test results are shown in the table below:

[0057] Table 2. Comparison of the activities of the composition and the single peptide.

[0058] EMP-1 (Single) 88.7 35.2 - EMP-2 (single) 85.3 32.8 - Composition 95.6 45.8 7.8 (α-glucosidase inhibition rate), 30.1 (insulin secretion promotion rate)

[0059] The test results show that the small molecule peptide composition constructed in this invention has an α-glucosidase inhibition rate of 95.6% and an insulin secretion promotion rate of 45.8%, which are 7.8% and 30.1% higher than the highest values ​​of single peptides, respectively. It achieves a significant synergistic effect and the regulatory effect is comprehensively better than that of single peptides.

[0060] 4.3 Example 3: Preparation and quality testing of a high-glycemic-regulating beverage

[0061] This embodiment is used to prepare a high-glycemic-regulating beverage and to test its quality, activity, and stability. The specific steps are as follows:

[0062] 1. Raw material preparation: According to the beverage formula of 1000mL, prepare 1.0g of the small molecule peptide composition prepared in Example 2, 6.5g of natural sweetener (erythritol:stevioside = 5:1), 0.4g of acidity regulator (citric acid: malic acid = 3:2), 0.15g of stabilizer (xanthan gum: sodium carboxymethyl cellulose = 2:1), 0.08g of antioxidant (vitamin C: tea polyphenols = 4:1), 1.2g of flavor substance (natural hawthorn extract: mulberry leaf extract = 3:2), and the balance of deionized water; pulverize all raw materials separately, pass them through an 80-mesh sieve to remove impurities, and set aside.

[0063] 2. Dissolving and mixing: Heat deionized water to 45°C, add stabilizer, and stir for 15 minutes until completely dissolved; then add small molecule peptide composition, natural sweetener, acidity regulator, antioxidant, and flavoring agent in sequence, and stir for 25 minutes to ensure that all raw materials are completely dissolved to obtain a mixture.

[0064] 3. Homogenization: The mixture is fed into a high-pressure homogenizer and homogenized twice at 22 MPa and 48°C to make the mixture uniform and fine.

[0065] 4. Sterilization: Pasteurization is used to sterilize the homogenized mixture at 88°C for 18 minutes and then rapidly cool it to 28°C.

[0066] 5. Filling and sealing: Fill the cooled mixture into sterile bottles that have been sterilized by high-pressure steam at 121℃ for 20 minutes, and seal the bottles to obtain the finished high blood sugar regulating beverage.

[0067] 6. Quality, activity and stability testing: The finished beverage was tested according to the quality control system of this invention, and the test results are as follows: Physicochemical indicators: pH value 4.0, small molecule peptide content 1.0g / 1000mL, sugar content 4.2g / 1000mL, acidity (calculated as citric acid) 0.4g / 1000mL, which meet the formula requirements.

[0068] 7. Microbiological indicators: Total bacterial count 32 CFU / mL, coliform bacteria <3 MPN / 100mL, mold and yeast <10 CFU / mL, no pathogenic bacteria detected, meeting the GB 7101-2021 "Beverage" standard.

[0069] 8. Activity indicators: α-glucosidase inhibition rate of 92.3% and insulin secretion promotion rate of 42.5%, ensuring the effect of blood glucose regulation.

[0070] 9. Stability index: After the beverage was stored at 25℃ for 6 months, there was no sedimentation or layering, no significant change in taste, and the small molecule peptide activity retention rate was 91.2%, which meets the stability requirements.

[0071] 10. Taste evaluation: It has a moderate sweet and sour taste, a refreshing taste, and no bitterness or off-taste. After tasting it, 88% of the 100 volunteers with high blood sugar said that it tasted good and was highly acceptable.

[0072] The test results show that the high blood sugar regulating beverage prepared by this invention has excellent quality, meets the activity standard, has good stability, and has a good taste, which meets the daily drinking needs of people with high blood sugar and can achieve natural, safe and efficient blood sugar regulation function.

[0073] 4.4 Example 4: Validation of the quality control system

[0074] This embodiment is used to verify the effectiveness of the original quality control system of this invention and ensure product batch consistency. The specific steps are as follows:

[0075] 1. Raw material quality control verification: Three batches of winged elm seed meal raw materials from Shanxi, Shaanxi and Gansu production areas were randomly selected and tested according to the raw material quality control standards. The protein content of the three batches of raw materials were 37.8%, 39.2% and 38.5% respectively, the moisture content was 7.9% and 8.5% respectively, the impurity content was <1% and the heavy metal content met the GB 2716-2018 "Vegetable Oil" standard. All three batches were qualified, indicating that the raw material quality control system was effective.

[0076] 2. Quality control verification of the preparation process: Three batches of small molecule peptides were prepared according to the process in Example 1. The yield of small molecule peptides in the enzymatic hydrolysate was tested (86.2%, 85.7%, and 86.5%, respectively), molecular weight distribution (the proportion of <1000Da was 92.3%, 91.8%, and 92.5%, respectively), purity of the purified peptides (all ≥98%), and activity (α-glucosidase inhibition rate all ≥80%). The difference in the test indicators among the three batches of products was <2%, indicating that the quality control system of the preparation process can ensure batch consistency.

[0077] 3. Finished product quality control verification: Three batches of high blood sugar regulating beverages were prepared according to the process in Example 3. The physicochemical indicators, microbiological indicators, activity indicators and stability were tested. The test indicators of the three batches of products all met the standards and the difference was <3%, indicating that the finished product quality control system can ensure the batch consistency and reliability of beverage products.

[0078] The verification results show that the quality control system of the present invention can achieve precise control of the entire process from raw materials to finished products, effectively ensuring batch consistency, safety and reliability of products, and meeting the needs of industrial production.

[0079] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A small molecule peptide derived from the kernel of *Vernicia fordii* (hairy-leaved privet) for antihypertensive purposes, comprising five previously unreported peptides, with the following amino acid sequences: Peptide 1: Val-Tyr-Pro-Gly, with a molecular weight of 412.45 Da; Peptide 2: Leu-Ile-Arg-Gln-Ala, molecular weight 573.65 Da; Peptide 3: Phe-Asp-Lys-Ser, with a molecular weight of 488.52 Da; Peptide 4: Ala-Met-Glu-Asn-Tyr, with a molecular weight of 585.63 Da; Peptide 5: Trp-Thr-Cys, molecular weight 362.41 Da; The five small molecule peptides all have an ACE inhibition rate of ≥80%, a molecular weight of 300-600 Da, good water solubility, and no toxic side effects.

2. The process as described in claim 1, characterized in that: Furthermore, the following steps are included: Step 1: Pre-treatment of raw materials for *Vernicia fordii* seed kernels: Collect fresh *Vernicia fordii* fruits, remove the pericarp, stalk and impurities, take out the kernels, wash, dry, crush and sieve to obtain *Vernicia fordii* seed kernel powder; use supercritical CO2 extraction to degrease to obtain defatted *Vernicia fordii* seed kernel powder. Step 2: Extraction of protein from the kernel of *Vernicia fordii*: Mix defatted *Vernicia fordii* kernel powder with deionized water at a mass ratio of 1:15-1:20, adjust the pH to 9.0-10.0, add a compound antioxidant, extract at a constant temperature, centrifuge, repeat extraction, precipitation, washing, and redissolution to obtain a pure extract of protein from the kernel of *Vernicia fordii*. Step 3: Complex Enzymatic Hydrolysis - Gradient Enzymatic Hydrolysis: Add a complex protease to the protein extract, adjust the pH to 8.0-8.5 and the temperature to 55-60℃, and perform gradient enzymatic hydrolysis until the degree of hydrolysis reaches 25%-30%. Inactivate the protease to obtain the enzymatic hydrolysate. The complex protease is composed of trypsin, alkaline protease, and flavor protease in a mass ratio of 2:1:1, and the amount of enzyme added is 3%-5% of the protein mass. Step 4: Rough selection: Centrifuge, ultrafilter, activated carbon adsorption, centrifuge, and gel filtration chromatography of the enzymatic hydrolysate to obtain a crude solution of small molecule peptides; Step 5: Multi-stage chromatographic separation and purification: The crude solution was subjected to ion exchange chromatography, reversed-phase high-performance liquid chromatography purification, desalting and vacuum freeze drying in sequence to obtain 5 high-purity antihypertensive small molecule peptides with a purity ≥98%; Step 6: Amino acid sequencing and activity verification: The amino acid sequence was determined using the Edman degradation method, and the antihypertensive activity was verified by in vitro ACE inhibitory activity detection and animal experiments.

3. The process as described in claim 1, characterized in that: In step 1, the drying conditions are 60-70℃, vacuum degree 0.06-0.08 MPa, drying until the moisture content is ≤8%; and passing through an 80-100 mesh sieve; the supercritical CO2 extraction conditions are extraction pressure 30-35 MPa, extraction temperature 40-45℃, CO2 flow rate 20-25 L / h, and extraction time 2-3 h.

4. The process as described in claim 1, characterized in that: In step 2, the compound antioxidant is vitamin C:vitamin E:glutathione = 2:1:1, and the addition amount is 0.3%-0.5% of the total mass of the liquid. The extraction conditions are 50-55℃, stirring speed 150-200 r / min, extraction time 2-3 h; centrifugation conditions are 8000-10000 r / min, 4℃, centrifugation time 20-30 min; the pH value is adjusted to 4.5-5.0 during precipitation, and the mixture is allowed to stand for 30-60 min.

5. The process as described in claim 1, characterized in that: In step 3, the gradient enzymatic hydrolysis is as follows: first, enzymatic hydrolysis is carried out at 55℃ for 1.5-2 h, and then the temperature is increased to 60℃ for 1-1.5 h; the protease inactivation conditions are heating in a water bath at 95-100℃ for 10-15 min.

6. The process as described in claim 1, characterized in that: In step 4, the ultrafiltration membrane has a molecular weight cutoff of 1000 Da, an operating pressure of 0.1-0.15 MPa, and a temperature of 25-30℃; the activated carbon addition is 0.5%-1.0% of the permeate volume, the adsorption conditions are 25-30℃, the stirring speed is 100-150 r / min, and the adsorption time is 30-40 min; the gel filtration chromatography packing material is Sephadex G-15, the eluent is deionized water, and the elution flow rate is 1.0-1.5 mL / min.

7. The process as described in claim 1, characterized in that: In step 5, the ion exchange chromatography packing material was DEAE-Sepharose Fast Flow, the eluent was deionized water containing 0.1-0.5 mol / L NaCl, and the elution flow rate was 1.0-1.5 mL / min. RP-HPLC purification was performed using a C18 column, with mobile phase A being 0.1% trifluoroacetic acid aqueous solution and mobile phase B being acetonitrile, using gradient elution and a detection wavelength of 220 nm. Desalting was performed using Sephadex G-10 gel filtration chromatography, and the vacuum freeze-drying conditions were: vacuum degree 0.09-0.1 MPa, freezing temperature -50~-40℃, and drying time 12-16 h.

8. The process as described in claim 1, characterized in that: The five small molecule peptides are mixed and compounded with natural active ingredients and excipients to prepare a hypertension-regulating beverage; the small molecule peptide mixture is composed of VYPG, LIRQA, FDLK, AMENY, and WTC mixed in a mass ratio of 1:1:1:1:

1.

9. The process as described in claim 1, characterized in that: The ingredients of the hypertension-regulating beverage, by weight, include: 1.0-2.0 parts of antihypertensive small molecule peptide mixture, 0.5-1.0 parts of hawthorn extract, 0.3-0.5 parts of chrysanthemum extract, 0.3-0.5 parts of cassia seed extract, 5-8 parts of xylitol, 0.1-0.2 parts of citric acid, 5-10 parts of apple juice, 0.1-0.2 parts of stabilizer, 0.02-0.05 parts of preservative, and 80-90 parts of deionized water; the stabilizer is a mixture of xanthan gum and sodium carboxymethyl cellulose in a 1:1 mass ratio, and the preservative is potassium sorbate.

10. The process as described in claim 1, characterized in that: The preparation method of the hypertension-regulating beverage includes: mixing and dissolving, homogenization, sterilization, cooling, filling and storage; the homogenization conditions are 20-25 MPa, 40-45℃, and homogenization is performed 2-3 times; sterilization is carried out by ultra-high temperature instantaneous sterilization at 135-140℃ for 3-5 seconds; after filling, it is stored at 0-25℃ and has a shelf life of 12 months. A low-temperature bio-enzymatic hydrolysis purification process for rare and precious herbal peptides for strengthening the spleen and stomach is suitable for the preparation of health-promoting functional foods for those with excessive dampness in the spleen and stomach, damaged gastric mucosa, and indigestion. The process includes the following steps: S1, low-temperature freezing intervention treatment with five rare and precious herbal substrates; S2, negative pressure cell wall breaking and activation to prepare a herbal activation solution; S3, controllable enzymatic hydrolysis with food-grade compound acidic protease; S4, human gastrointestinal irritation safety prediction + nine-fold molecular docking to target points for strengthening the spleen and stomach; S5, primary food ultrafiltration for astringency removal; S6, secondary Y-202 food resin for impurity removal; S7, five-stage food-grade chromatography purification; S8, mass spectrometry sequencing + aseptic light-protected single-peptide dispensing; S9, preparation of compound spleen-strengthening and stomach-nourishing peptide powder through pentapeptide chain compounding; The raw materials are present in the following weight parts: 17-27 samples of wild rice in the forest, 15-25 samples of cold-resistant onion in high-altitude areas, 13-23 samples of golden tamarisk in mountainous areas, 11-21 samples of fragrant grass in hilly areas, and 9-19 samples of box grass in wetlands; five original stomach-nourishing peptides: Peptide 1: Met-Gly-Ala-Thr, 264.1Da; Peptide 2: Tyr-Lys-Ser-Glu, 346.2Da; Peptide 3: Arg-His-Ile-Gln, 402.2Da; Peptide 4: Phe-Met-Val-Cys, 439.2Da; Peptide 5: Asn-Trp-Pro-Lys, 385.2Da; using human gastrointestinal prediction + two-stage decolorization and astringency removal, eliminating the gastrointestinal irritation, pigment turbidity, and astringency and numbness defects of Youzhen Herbal Peptide technology.