WO2022242339A1 - 一种寡肽及其制备方法与应用 - Google Patents

一种寡肽及其制备方法与应用 Download PDF

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WO2022242339A1
WO2022242339A1 PCT/CN2022/085017 CN2022085017W WO2022242339A1 WO 2022242339 A1 WO2022242339 A1 WO 2022242339A1 CN 2022085017 W CN2022085017 W CN 2022085017W WO 2022242339 A1 WO2022242339 A1 WO 2022242339A1
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oligopeptide
separation
angiotensin
converting enzyme
tyr
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PCT/CN2022/085017
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English (en)
French (fr)
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乔子骄
何英
谢奎
宁亚丽
颜卫彬
曹庸
汪家琦
潘丽娜
戴智勇
刘果
安彦君
侯艳梅
李辉宇
滕爽
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澳优乳业（中国）有限公司
海普诺凯营养品有限公司
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Publication of WO2022242339A1 publication Critical patent/WO2022242339A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of goat milk enzymolysis, and in particular relates to an oligopeptide, a preparation method and application thereof.
  • cardiovascular diseases such as hypertension, stroke, rheumatic heart disease and coronary artery disease has increased significantly.
  • cardiovascular disease is the number one cause of death in the world, and hypertension is considered to be the most important cause of cardiovascular disease. risk factors. Therefore, preventing hypertension is considered an important way to reduce the risk and morbidity of cardiovascular diseases.
  • Angiotensin-converting enzyme is a key enzyme in regulating blood pressure. It can convert angiotensin I into effective angiotensin II, thereby causing vasoconstriction to increase blood pressure and inactivating bradykinin, which has a hypotensive effect. Therefore, inhibition of ACE is considered to be an important way to treat hypertension.
  • Various synthetic angiotensin-converting enzyme inhibitors such as caputolil, benazepril, enalapril, and arapril can effectively treat hypertension. But these synthetic ACE inhibitor drugs also exhibit significant side effects, such as headache, cough, dizziness, fatigue, and diarrhea. In addition to the side effects of drugs, the treatment and prevention of hypertension also brings a huge economic burden to society, prompting people to turn their attention to natural functional foods without side effects.
  • the present invention provides an oligopeptide and its preparation method and application.
  • the oligopeptide is obtained by separation and purification after enzymatic hydrolysis of goat milk casein, and has an inhibitory effect on angiotensin-converting enzyme. Antihypertensive effects of food or drugs provide the basis.
  • the first aspect of the present invention provides an oligopeptide, which has the amino acid sequence shown in SEQ ID NO.1: Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr.
  • the IC 50 of the oligopeptide against angiotensin-converting enzyme is 24.447 ⁇ g/mL.
  • the oligopeptide is derived from goat milk casein.
  • the research on milk-derived bioactive peptides mainly focuses on milk protein and milk products.
  • Goat's milk is one of the important milk sources. Compared with cow's milk, goat's milk has low allergenicity, low lactose content, and protein is easier to digest.
  • As a product of whey protein production goat casein is a waste of resources if sold at a low price or discarded as waste. Therefore, research on enzymatic hydrolysis of goat casein can increase its application value.
  • the second aspect of the present invention provides a method for preparing an oligopeptide, comprising the following steps:
  • the oligopeptide raw material obtained in step S1 is gradually separated and purified to obtain an oligopeptide having an inhibitory effect on angiotensin-converting enzyme.
  • the separation and purification in the step S2 sequentially includes primary separation on a C18 preparative column, secondary separation on a C18 preparative column, three separations on a C18 hydrophilic preparative column, and separation and purification by reversed-phase high performance liquid chromatography.
  • goat milk casein is used as a raw material, after enzymolysis by trypsin, guided by angiotensin-converting enzyme inhibitory activity, an oligopeptide is obtained through repeated reverse liquid chromatography separation and purification, and it is found through amino acid sequence determination that the The oligopeptide has the amino acid sequence of Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr, the molecular weight is 1218.355Da, and it is found through the determination of angiotensin-converting enzyme (Angiotensin-converting enzyme, ACE) inhibitory activity that the oligopeptide
  • ACE angiotensin-converting enzyme
  • the third aspect of the present invention provides the application of an oligopeptide in the preparation of food or medicine.
  • the invention provides an application of an oligopeptide in the preparation of an angiotensin-converting enzyme inhibitor.
  • the invention also provides the application of the oligopeptide in the preparation of blood pressure lowering medicine.
  • the fourth aspect of the present invention provides a composition comprising the oligopeptide described above.
  • the present invention provides an angiotensin-converting enzyme inhibitor, including the oligopeptide described above.
  • angiotensin-converting enzyme inhibitor including the oligopeptide described above.
  • other excipients that can be used in the field of blood pressure reduction can also be included, and the medicine can be prepared into forms such as granules, capsules and tablets.
  • the present invention also provides a drug for lowering blood pressure, including the oligopeptide described above.
  • a drug for lowering blood pressure including the oligopeptide described above.
  • other excipients that can be used in the field of blood pressure reduction can also be included, and the medicine can be prepared into forms such as granules, capsules and tablets.
  • the present invention uses goat milk casein as raw material, separates and purifies to obtain an oligopeptide with hypotensive effect, the oligopeptide has the amino acid sequence of Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr, and the molecular weight is 1218.355Da , the oligopeptide with hypotensive effect prepared by the present invention has good activity, is safe and non-toxic, and found through the measurement of angiotensin-converting enzyme (ACE) inhibitory activity that the oligopeptide has a significant inhibitory effect on ACE, and can be applied to reduce blood pressure. blood pressure field, and is conducive to resource utilization of goat milk casein.
  • ACE angiotensin-converting enzyme
  • Fig. 1 is a C18 preparative column one-time separation segmented sampling diagram
  • Figure 2 is the inhibitory activity of the C18 preparative column for the primary separation sample to ACE
  • Fig. 3 is C18 preparative column secondary separation segmentation drawing
  • Figure 4 is the inhibitory activity of the secondary separation sample of the C18 preparative column to ACE
  • Fig. 5 is a three-time separation segmented sampling diagram of C18 hydrophilic preparative column
  • Figure 6 shows the inhibitory activity of the three separation samples of the C18 hydrophilic preparative column to ACE
  • Fig. 7 is a RP-HCLP separation segment connection sample diagram
  • Fig. 8 is the inhibitory activity of RP-HCLP separation sample to ACE
  • Fig. 9 is the secondary mass spectrogram of component F3;
  • Fig. 10 is the PPSQ sequencing figure of component F3;
  • Fig. 11 Inhibitory activity of component F3 and synthetic oligopeptide on ACE.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features.
  • “plurality” means two or more, unless otherwise specifically defined.
  • references to the terms “one embodiment,” “some embodiments,” “exemplary embodiments,” “example,” “specific examples,” or “some examples” are intended to mean that the implementation A specific feature, structure, material, or characteristic described by an embodiment or example is included in at least one embodiment or example of the present disclosure.
  • schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
  • Goat micellar casein concentrate 60-63 (gMCC60-63) provided by AoYo Dairy (China) Co., Ltd., that is, goat milk casein, with a protein content of 60-63%.
  • Reagents trypsin; sodium hydroxide, hydrochloric acid, boric acid (H 3 BO 3 ), borax, sodium chloride, calcium chloride, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and disodium hydrogen phosphate are all analytically pure Acetonitrile, trifluoroacetic acid, chromatographically pure; hippuric acid (hippuric acid, Hip), American Sigma Company; hippuryl-histidyl-leucine (N-hippuryl-His-Leu tetrahydrate, HHL), American Sigma Company ; Angiotensin-converting enzyme (ACE), 1U, American Sigma Company.
  • trypsin sodium hydroxide, hydrochloric acid, boric acid (H 3 BO 3 ), borax, sodium chloride, calcium chloride, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and disodium hydrogen phosphate are all analytically pure Acetonitrile, tri
  • HHL Under the catalysis of ACE, HHL is rapidly decomposed to produce hippuric acid (Hip) and dipeptide (His-Leu, HL). Hippuric acid has a maximum absorption at 228nm.
  • Hip hippuric acid
  • His-Leu dipeptide
  • Hippuric acid has a maximum absorption at 228nm.
  • R the inhibitory rate (%) of ACEI sample to ACE
  • A The peak area of hippuric acid in the control group
  • A0 The peak area of hippuric acid in the blank tube.
  • IC 50 is defined as the inhibitor concentration required to inhibit half of the ACE enzyme activity under certain conditions. Since there is not a linear relationship between the inhibitory rate and the concentration of the preparation, it is necessary to first draw a curve of the relationship between the inhibitory concentration and the inhibitory rate, and then find out the IC 50 from the curve.
  • the temperature of the enzymolysis solution was raised to 90°C in a water bath, and kept for 20 minutes to inactivate the enzyme to obtain the oligopeptide raw material.
  • the oligopeptide raw material was prepared into a 150 mg/mL solution with ultrapure water, and after passing through a 0.45 ⁇ m filter membrane, the goat milk antihypertensive peptide was preliminarily separated by means of preparative high performance liquid chromatography (LC-8A, Shimadzu). The injection was repeated, and the eluted peaks were collected, concentrated in vacuo, and freeze-dried.
  • LC-8A preparative high performance liquid chromatography
  • Chromatographic conditions the chromatographic column is a self-packed glass column (20mm ⁇ 450mm, C18 filler (10 ⁇ m, Macherey Nagel, France)); mobile phase: pump A is ultrapure water (containing 0.1% TFA), pump B is acetonitrile (containing 0.1% TFA); flow rate: 10mL/min; injection volume: 5mL; detection wavelength: 214nm , 280 nm; elution program (concentration of mobile phase B): 10%-48% (0.01-66.00min), 48%-90% (66.00-66.01min), 90%-90% (66.01-71.00min).
  • the component Y3 with the highest ACE inhibitory activity in the previous step was prepared into a 25 mg/mL solution, passed through a 0.45 ⁇ m filter membrane, and then re-separated and prepared by using a C18 preparative column.
  • the chromatographic column model was Shimadzu PRC-ODS (K) column (30mm ⁇ 250mm, 15 ⁇ m, Shimadzu), the preparation conditions are: mobile phase A is primary water (containing 0.1% TFA), mobile phase B is acetonitrile (containing 0.1% TFA), the flow rate is 10mL/min, and the injection volume is 4 mL, monitored at 214nm and 280nm, the elution program (concentration of mobile phase B) was 25%-60% (0-70min).
  • the component Y3-3 with the highest ACE inhibitory activity in the previous step was prepared into a 10 mg/mL solution, passed through a 0.45 ⁇ m filter membrane, and then re-separated and prepared by using a C18 hydrophilic preparation column.
  • the chromatographic column model is ECOSIL C18 steel column (300mm ⁇ 20mm, 10 ⁇ m, Germany), the preparation conditions are: mobile phase A is primary water (containing 0.1% TFA), mobile phase B is acetonitrile (containing 0.1% TFA), the flow rate is 10mL/min, and the injection volume is 3 mL, monitored at 214nm and 280nm, elution program (concentration of mobile phase B): 30%-43% (0.01-66.00min), 43%-90% (66.00-66.01min).
  • the separation and detection spectrum of attached drawing 5 it can be seen that according to the peak conditions and the degree of separation, it is divided into M1, M2, M3, M4, M5 and M6. There are 6 sections for sampling, and these 6 components are collected respectively and concentrated. After lyophilization, the inhibitory activity of each component on ACE enzyme was measured. According to the measurement results of the inhibition rate in Fig. 6, after further separation, when the sample concentration is 1 mg/mL, the inhibition rate of the M4 component is the highest, as high as 70.17%. In order to clarify the highly active peptides that play a major role, the M4 fraction was further separated and analyzed.
  • the component M4 with the highest ACE inhibitory activity in the previous step was prepared into a 10mg/mL solution, passed through a 0.22 ⁇ m filter membrane, and then re-separated by RP-HCLP.
  • the column model was ECOSIL C18 (260mm ⁇ 4.6mm, 5 ⁇ m) , the analysis conditions are: mobile phase A is primary water (containing 0.1% TFA), mobile phase B is chromatographic grade acetonitrile (containing 0.1% TFA), the flow rate is 1mL/min, the injection volume is 20 ⁇ L, at 214nm and 280nm Monitoring, elution program (concentration of mobile phase B): 25%-30% (0.01-60min).
  • the molecular weight of the purified peptide was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MOLDI-TOF-MS/MS), and the mass unit of the attached proton was subtracted from the charge-to-mass ratio to obtain the The relative molecular mass of peptide F3 is 1218.355Da. Then use the amino acid sequencer (PPSQ) to determine the amino acid composition of the peptide, and PPSQ can measure the absolute sequence of the N-terminal of the oligopeptide.
  • PPSQ amino acid sequencer
  • oligopeptide F3 determines the sequence of oligopeptide F3 to be Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr (FPQYLQYPY) .
  • FPQYLQYOY lowering blood pressure oligopeptide F3
  • the oligopeptide has high ACE inhibitory activity.
  • hypotensive oligopeptide F3 In order to carry out reverse verification of hypotensive oligopeptide F3 and characterize its inhibitory effect on ACE in vitro, high-purity hypotensive oligopeptide F3 (purity>98%) was synthesized by Nanjing Jiepei Biotechnology Co., Ltd., and compared with The activity of the separated F3 fraction and the hydrolyzate produced by the goat whey protein by-product after trypsin hydrolysis were compared, and the results are shown in Figure 11.
  • the ACE inhibitory activity evaluation test was carried out according to the conventional ACE inhibitory activity assay method, and the IC 50 value obtained from the test was 24.447 ⁇ g/mL.
  • hypotensive oligopeptide F3 isolated by the present invention has good ACE inhibitory activity, and it is reversely verified by means of artificial synthesis that this oligopeptide does have the effect of lowering blood pressure.
  • the ACE inhibitory activity of synthetic oligopeptides is better than that of isolated ones, which has a greater relationship with the purity of oligopeptides. This peptide is expected to be used in the field of lowering blood pressure, and lays the foundation for the application of natural product antihypertensive peptides in the future.

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Abstract

本发明提供了一种寡肽及其制备方法与应用，该寡肽的氨基酸序列为Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr，对血管紧张素转化酶的IC 50为24.447μg/mL。本发明通过对羊乳酪蛋白酶解、分离纯化后，得到对血管紧张素转化酶具有抑制作用的寡肽，为今后天然产物降压肽的应用奠定了基础。

Description

一种寡肽及其制备方法与应用 技术领域
本发明属于羊乳酶解技术领域，具体涉及一种寡肽及其制备方法与应用。
背景技术
近年来，高血压、中风、风湿性心脏病和冠状动脉疾病等心血管疾病的发病率显著上升，目前心血管疾病是世界第一的致死原因，而高血压被认为是导致心血管疾病最重要的危险因素。因此，预防高血压被认为是降低心血管病风险和发病率的重要途径。
血管紧张素转换酶(ACE)是调节血压的关键酶。它能将血管紧张素I转化为有效的血管紧张素II，从而使血管收缩血压升高，并使有降压作用的缓激肽失活。因此，抑制ACE被认为是治疗高血压的重要途径。卡普托利、贝那普利、依那普利、阿拉普利等多种人工合成的血管紧张素转换酶抑制剂均能有效治疗高血压。但这些合成的血管紧张素转换酶抑制剂药物也表现出明显的副作用，如头痛、咳嗽、眩晕、疲劳和腹泻。除药物具有的副作用外，高血压的治疗和预防也给社会带来巨大的经济负担，促使人们将目光转移到天然无副作用的功能食品上。
发明内容
为了克服现有技术中的问题，本发明提供一种寡肽及其制备方法与应用，所述寡肽通过羊乳酪蛋白酶解后分离纯化得到，对血管紧张素转化酶具有抑制作用，为制备具有降血压作用的食品或药物提供了基础。
为了实现上述目的，本发明通过以下技术方案实现：
本发明第一方面提供一种寡肽，所述寡肽具有SEQ ID NO.1所示的氨基酸序列：Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr。
在一个优选的实施方案中，所述寡肽对血管紧张素转化酶的IC 50为24.447μg/mL。
在一个优选的实施方案中，所述寡肽来源于羊乳酪蛋白。乳源生物活性肽的研究主要集中在牛乳蛋白和牛乳制品方面。羊奶是重要的乳源之一，与牛乳相比，羊乳致敏性低，乳糖含量低，并且蛋白更易消化。羊乳酪蛋白作为生产乳清蛋白的一种产物，以低价出售或以废弃物丢弃十分浪费资源，因此研究酶解羊乳酪蛋 白可提高其应用价值。
本发明第二方面提供一种寡肽的制备方法，包括以下步骤：
S1、将羊乳酪蛋白用胰蛋白酶酶解2～3h，酶解完成后升温使胰蛋白酶失活，得到寡肽原料；
S2、以血管紧张素转化酶的抑制活性为导向，将步骤S1得到的寡肽原料进行逐步分离纯化，得到对血管紧张素转化酶具有抑制作用的寡肽。
在一个优选的实施方案中，所述步骤S2的分离纯化依次包括C18制备柱一次分离、C18制备柱二次分离、C18亲水制备柱三次分离和反相高效液相色谱分离纯化。
本发明以羊乳酪蛋白为原料，经过胰蛋白酶酶解后，以血管紧张素转化酶抑制活性为导向，通过多次反向液相色谱分离纯化得到一种寡肽，经氨基酸序列测定发现，该寡肽具有Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr氨基酸序列，分子量为1218.355Da，经血管紧张素转化酶(Angiotensin-converting enzyme，ACE)抑制活性的测定发现，该寡肽对ACE具有显著的抑制作用，对羊乳酪蛋白的再利用及降血压肽的应用有一定参考价值。
本发明第三方面，提供一种寡肽在制备食品或药物中的应用。
本发明提供一种寡肽在制备血管紧张素转化酶抑制剂中的应用。
本发明还提供一种寡肽在制备降血压药物中的应用。
本发明第四方面提供一种组合物，包含上面所描述的寡肽。
本发明提供了一种血管紧张素转化酶抑制剂，包括上面所描述的寡肽。为提高药物的适用范围，还可以包括其他可应用于降压领域的辅料，该药物可制备成颗粒剂、胶囊剂和片剂等形式。
本发明还提供一种降血压药物药物，包括上面所描述的寡肽。为了提高药物的适用范围，还可以包括其他可应用于降压领域的辅料，该药物可制备成颗粒剂、胶囊剂和片剂等形式。
本发明的有益效果如下：
本发明以羊乳酪蛋白为原料，分离纯化得到一种降血压作用的寡肽，该寡肽具有Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr的氨基酸序列，分子量为1218.355Da，本发明制备的具有降血压作用的寡肽，活性好，安全无毒，经血管紧张素转化酶 (ACE)抑制活性的测定发现，该寡肽对ACE具有显著的抑制作用，可以应用于降血压领域，且有利于羊乳酪蛋白的资源化利用。
附图说明
图1为C18制备柱一次分离分段接样图；
图2为C18制备柱一次分离样品对ACE的抑制活性；
图3为C18制备柱二次分离分段接样图；
图4为C18制备柱二次分离样品对ACE的抑制活性；
图5为C18亲水制备柱三次分离分段接样图；
图6为C18亲水制备柱三次分离样品对ACE的抑制活性；
图7为RP-HCLP分离分段接样图；
图8为RP-HCLP分离样品对ACE的抑制活性；
图9为组分F3的二级质谱图；
图10为组分F3的PPSQ测序图；
图11位组分F3与人工合成寡肽对ACE的抑制活性。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
实施例1降血压肽的分离
(1)材料与试剂
材料：澳优乳业(中国)有限公司提供的Goat micellar casein concentrate 60-63(gMCC60-63)，即羊乳酪蛋白，蛋白质含量为60-63％。
试剂：胰蛋白酶；氢氧化钠、盐酸、硼酸(H 3BO 3)、硼砂、氯化钠、氯化钙，硫酸镁、磷酸氢二钾、磷酸二氢钾、磷酸氢二钠均为分析纯；乙腈、三氟乙酸，色谱纯；马尿酸(hippuric acid，Hip)，美国Sigma公司；马尿酰-组氨酰-亮氨酸(N-hippuryl-His-Leu tetrahydrate，HHL)，美国Sigma公司；血管紧张素转化酶(ACE)，1U，美国Sigma公司。
(2)ACE抑制活性的测定方法
2.1反应液的制备
分别向样品管(B)和空白管(A)加入10μLACE溶液(0.2U/mL，ACE酶溶于pH7的磷酸钾缓冲液中)，样品管加入10μL血管紧张素转化酶抑制剂(ACEI)，空白管10μL缓冲液，37℃温育5min后加入30μL HHL溶液(6.5mmol/mL，HHL溶于pH8.3的0.1mol/L硼酸缓冲液，含0.3mol/LNaCl)，在37℃条件下反应1h后，后加入80μL 1.0mol/L HCl终止反应，得到反应液。
2.2色谱条件
色谱柱：ECOSIL C18(260mm×4.6mm，5μm)。
流动相与色谱条件：乙腈：超纯水＝25：75(含0.1％(v/v)TFA)，流速：1mL/min；检测波长：228nm；柱温：30℃；进样量：20μL。
2.3结果计算
原理：HHL在ACE的催化下快速地分解产生马尿酸(Hip)和二肽(His-Leu，HL)，马尿酸在228nm处有最大吸收。当加入ACEI样品时，ACE酶的活性受到抑制，马尿酸生成量减少，所以可通过高效液相色谱测定马尿酸的生成量来评估ACEI对ACE活性的抑制率。
计算公式为：
Figure PCTCN2022085017-appb-000001
式中：R：ACEI样品对ACE的抑制率(％)；
A：对照组中马尿酸的峰面积；
B：添加ACEI组中马尿酸的峰面积；
A0：空白管中马尿酸的峰面积。
其中，IC 50的定义为在一定条件下抑制ACE酶活性一半时所需要的抑制剂浓度。由于抑制率与制剂浓度之间不是一个线性的关系，因此必须首先绘制抑制剂浓度与抑制率关系的曲线，再从曲线上查出IC 50。
(3)羊乳酪蛋白酶解工艺
将羊乳酪蛋白溶于水中至其浓度为5％，水浴加热至50℃时用10％pH调节剂调节pH为8.0，加入0.4％的胰蛋白酶酶解2.5h，酶解过程中用10％NaOH维持溶于pH＝8.0。酶解结束后水浴升温酶解液至90℃，保持20min灭酶，得到寡肽原料。
(4)C18制备柱一次分离
将寡肽原料用超纯水配置成150mg/mL溶液，并过0.45μm滤膜后，借助制备型高效液相色谱(LC-8A，Shimadzu)对羊乳降压肽进行初步分离。重复进样，收集洗脱峰后真空浓缩，冷冻干燥。
色谱条件：色谱柱为自装玻璃柱(20mm×450mm，C18填料(10μm，
Figure PCTCN2022085017-appb-000002
Macherey Nagel，France))；流动相：A泵为超纯水(含0.1％TFA)，B泵为乙腈(含0.1％TFA)；流速：10mL/min；进样量：5mL；检测波长：214nm，280nm；洗脱程序(流动相B的浓度)：10％-48％(0.01-66.00min)，48％-90％(66.00-66.01min)，90％-90％(66.01-71.00min)。
根据附图1的分离检测图谱可以看出，在寡肽常用检测波长214nm下，根据出峰时间及峰形相似度，一共分为Y1、Y2、Y3、Y4、Y5、Y6，共6段接样，分别收集这6个组分，然后即刻进行浓缩及真空冻干，减少寡肽降解，随后测量6个组分对ACE酶的抑制活性。根据附图2的抑制率测定结果可知，样品浓度为1mg/mL时，Y3组分的抑制率最高，达46.13％，选择其进行下一步的分离和分析。
(5)C18制备柱二次分离
将上一步中对ACE抑制活性最高的组分Y3配制成25mg/mL的溶液，过0.45μm滤膜，采用C18制备柱对其进行再分离制备，色谱柱型号为Shimadzu PRC-ODS(K)柱(30mm×250mm,15μm,Shimadzu)，制备条件为：流动相A为一级水(含 0.1％TFA)，流动相B为乙腈(含0.1％TFA)，流速为10mL/min，进样量为4mL，于214nm和280nm处监测，洗脱程序(流动相B的浓度)为25％-60％(0-70min)。
根据附图3的分离检测图谱可以看出，按照出峰情况及分离度，一共分为Y3-1、Y3-2、Y3-3、Y3-4和Y3-5，共5段接样，分别收集这5个组分，浓缩冻干后测量各组分对ACE酶的抑制活性。根据附图4的抑制率测定结果可知，经过进一步分离，当样品浓度为1mg/mL时，Y3-3组分的抑制率最高，高达62.58％。为了明确其中起主要作用的高活性肽段，对Y3-3组分进行下一步的分离和分析。
(6)C18亲水制备柱三次分离
将上一步中对ACE抑制活性最高的组分Y3-3配制成10mg/mL的溶液，过0.45μm滤膜，采用C18亲水制备柱对其进行再分离制备，色谱柱型号为ECOSIL C18钢柱(300mm×20mm,10μm,Germany)，制备条件为：流动相A为一级水(含0.1％TFA)，流动相B为乙腈(含0.1％TFA)，流速为10mL/min，进样量为3mL，于214nm和280nm处监测，洗脱程序(流动相B的浓度)：30％-43％(0.01-66.00min)，43％-90％(66.00-66.01min)。
根据附图5的分离检测图谱可以看出，按照出峰情况及分离度，一共分为M1、M2、M3、M4、M5和M6，共6段接样，分别收集这6个组分，浓缩冻干后测量各组分对ACE酶的抑制活性。根据附图6的抑制率测定结果可知，经过进一步分离，当样品浓度为1mg/mL时，M4组分的抑制率最高，高达70.17％。为了明确其中起主要作用的高活性肽段，对M4组分进行下一步的分离和分析。
(7)反相高效液相色谱(RP-HCLP)的分离纯化
将上一步中对ACE抑制活性最高的组分M4配制成10mg/mL的溶液，过0.22μm滤膜，利用RP-HCLP进行再分离制备，色谱柱型号为ECOSIL C18(260mm×4.6mm，5μm)，分析条件为：流动相A为一级水(含0.1％TFA)，流动相B为色谱级乙腈(含0.1％TFA)，流速为1mL/min，进样量为20μL，于214nm和280nm处监测，洗脱程序(流动相B的浓度)为：25％-30％(0.01-60min)。
根据附图7所示的HPLC图谱可以看出，根据出峰情况，一共分为F1、F2、F3、F4、F5、F6和F7，共7个峰接样，收集这7个样品，经旋蒸浓缩和冷冻干燥后测定各组分对ACE的抑制率。根据附图8的抑制率测定结果可知，组分F3(1mg/mL)的ACE抑制率达到71.9％，显著高于其它6个组分(p<0.05)。故对F3的 寡肽进行详尽的后续研究。
(8)降血压寡肽结构的确定
为了研究F3降血压肽的组成，利用基质辅助激光解吸电离飞行时间质谱仪(MOLDI-TOF-MS/MS)鉴定纯化肽的分子量，由荷质比减去附着的质子的质量单位，得出寡肽F3的相对分子质量为1218.355Da。再运用氨基酸测序仪(PPSQ)测定肽的氨基酸组成，PPSQ可测出寡肽的N端绝对序列。根据二级质谱图(附图9)和氨基酸测序仪分析图(附图10)，最终确定寡肽F3的序列为Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr(FPQYLQYPY)。这是首次从食物源天然产物中分离出FPQYLQYOY(降血压寡肽F3)，且寡肽具有高ACE抑制活性。
实施例2
降血压寡肽的人工合成以及ACE的抑制活性评价
为对降血压寡肽F3进行反向验证，并详细表征其在体外对ACE的抑制作用，由南京杰肽生物科技有限公司合成高纯度的降血压寡肽F3(纯度>98％)，并与分离得到的F3组分，以及羊乳乳清蛋白副产物在胰蛋白酶酶解后产生的酶解物的活性进行对比，结果如图11所示。按照常规的ACE抑制活性的测定方法进行ACE的抑制活性评价试验，试验所得IC 50值为24.447μg/mL。
通过附图11可以看出，本发明分离得到的降血压寡肽F3具有较好的ACE抑制活性，且通过人工合成的手段反向验证这种寡肽确实具有降血压的功效。但人工合成寡肽的ACE抑制活性比分离的要好，这与寡肽的纯度有较大关系。此多肽有望应用于降血压领域，为今后天然产物降压肽的应用奠定基础。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只限于这些说明。对于本发明所属领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (9)

  1. 一种寡肽，其特征在于，所述寡肽为SEQ ID NO.1所示的氨基酸序列：Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Pro-Tyr。
  2. 根据权利要求1所述的一种寡肽，其特征在于，所述寡肽对血管紧张素转化酶的IC 50为24.447μg/mL。
  3. 根据权利要求1所述的一种寡肽，其特征在于，所述寡肽来源于羊乳酪蛋白。
  4. 根据权利要求1-3任一所述的寡肽的制备方法，其特征在于，包括以下步骤：
    S1、将羊乳酪蛋白用胰蛋白酶酶解，酶解完成后升温使胰蛋白酶失活，得到寡肽原料；
    S2、以血管紧张素转化酶的抑制活性为导向，将步骤S1得到的寡肽原料进行逐步分离纯化，得到对血管紧张素转化酶具有抑制作用的寡肽；
    所述步骤S2的分离纯化依次包括C18制备柱一次分离、C18制备柱二次分离、C18亲水制备柱三次分离和反相高效液相色谱分离纯化。
  5. 根据权利要求1-3任一所述的寡肽在制备食品或药品中应用。
  6. 根据权利要求1-3任一所述的寡肽在制备血管紧张素转化酶抑制剂或降血压药物中的应用。
  7. 一种组合物，其特征在于，包含权利要求1-3任一所述的寡肽。
  8. 一种血管紧张素转化酶抑制剂，其特征在于，包含权利要求1-3任一所述的寡肽。
  9. 一种降血压药物，其特征在于，包含权利要求1-3任一所述的寡肽。
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