Peptide with ACE (angiotensin converting enzyme) inhibition and antioxidant activities as well as preparation method and application thereof
阅读说明：本技术 兼具ace抑制和抗氧化活性的肽及其制备方法和应用 (Peptide with ACE (angiotensin converting enzyme) inhibition and antioxidant activities as well as preparation method and application thereof ) 是由 党亚丽 郝丽 高新昌 潘道东 于 2019-10-21 设计创作，主要内容包括：本发明公开了一系列兼具ACE抑制和抗氧化活性的肽及其制备方法和应用,本发明兼具ACE抑制和抗氧化活性的肽,其氨基酸序列为以下三种：肽1：Cys-Cys-Asn-Lys；肽2：Ala-His-Ser-Val-Arg-Phe；肽3：His-Cys-His-Thr。兼具ACE抑制和抗氧化活性的肽的制备方法,鲜味肽溶液经过胃蛋白酶水解和胰蛋白酶水解后得到含兼具ACE抑制和抗氧化活性的肽的溶液。本发明兼具ACE抑制和抗氧化活性的肽能发挥较强的ACE抑制和抗氧化作用,非常适合用于制备降压药物和抗氧化功能药物,并可用于保健食品,药品或化妆品的研发。(The invention discloses a series of peptides with ACE inhibition and antioxidant activity, and a preparation method and application thereof, wherein the amino acid sequence of the peptide with ACE inhibition and antioxidant activity is as follows: peptide 1: Cys-Cys-Asn-Lys; peptide 2: Ala-His-Ser-Val-Arg-Phe; peptide 3: His-Cys-His-Thr. The preparation method of the peptide with both ACE inhibition and antioxidant activity comprises the steps of carrying out pepsin hydrolysis and trypsin hydrolysis on an umami peptide solution to obtain a solution containing the peptide with both ACE inhibition and antioxidant activity. The peptide with both ACE inhibition and antioxidant activity can play a strong role in ACE inhibition and antioxidant, is very suitable for preparing antihypertensive drugs and antioxidant drugs, and can be used for research and development of health-care foods, drugs or cosmetics.)
1. The peptide with ACE inhibition and antioxidant activity is characterized in that the amino acid sequence of the peptide is as follows:
peptide 1: Cys-Cys-Asn-Lys;
peptide 2: Ala-His-Ser-Val-Arg-Phe;
peptide 3: His-Cys-His-Thr.
2. The method for preparing the peptide with both ACE inhibitory and antioxidant activity according to claim 1, comprising the steps of:
1) preheating the fresh peptide solution in a water bath kettle at 33-40 ℃ for 5-30 min, adding HCl aqueous solution to adjust the pH value to 2-4 to obtain a solution to be hydrolyzed by pepsin, adding pepsin, uniformly mixing, hydrolyzing, and obtaining pepsin hydrolysate after the hydrolysis is finished;
2) and adjusting the pH value of the pepsin hydrolysate to 7-8 by using NaOH aqueous solution to obtain a solution to be subjected to trypsin hydrolysis, adding trypsin, uniformly mixing, hydrolyzing for 1-3 h, inactivating in a boiling water bath after reaching the time, and cooling to obtain a solution containing the peptide with ACE (angiotensin converting enzyme) inhibition and antioxidant activity.
3. The method of claim 1, wherein in step 1), the umami peptide solution is a first umami peptide solution, a second umami peptide solution or a third umami peptide solution;
the preparation of the first umami peptide solution comprises the following steps: dissolving ham-derived umami peptide with distilled water to enable the concentration of the peptide to be 0.5-2 mg/mL, wherein the amino acid sequence of the ham-derived umami peptide is peptide 4: Cys-Cys-Asn-Lys-Ser-Val to obtain a first umami peptide solution;
the preparation of the second umami peptide solution comprises the following steps: dissolving ham-derived umami peptide with distilled water to enable the concentration of the peptide to be 0.5-2 mg/mL, wherein the amino acid sequence of the ham-derived umami peptide is peptide 5: Ala-His-Ser-Val-Arg-Phe-Tyr to obtain a second umami peptide solution;
the preparation of the third delicious peptide solution comprises the following steps: dissolving the obtained ham-derived umami peptide with distilled water to ensure that the concentration of the peptide is 0.5-2 mg/mL, wherein the amino acid sequence of the ham-derived umami peptide is peptide 6: His-Cys-His-Thr-Asn to obtain the third delicious peptide solution.
4. The method for preparing a peptide with both ACE inhibitory activity and antioxidant activity according to claim 1, wherein in step 1), the peptide is preheated for 5-15 min in a water bath at 35-39 ℃.
5. The method for preparing the peptide with both ACE inhibitory activity and antioxidant activity according to claim 1, wherein in step 1), HCl aqueous solution is added to adjust the pH value to 2.5-3.5.
6. The method for preparing a peptide with both ACE inhibitory and antioxidant activity according to claim 1, wherein in step 1), the amount of pepsin added is 2-6% of the mass of the solution to be hydrolyzed by pepsin.
7. The method for preparing a peptide with both ACE inhibitory activity and antioxidant activity according to claim 1, wherein in step 2), the pH value of the pepsin hydrolysate is adjusted to 7.5-8.5 by NaOH aqueous solution.
8. The method for preparing a peptide with both ACE inhibitory and antioxidant activities according to claim 1, wherein in step 2), the amount of trypsin is added in a range of 2% to 6% to the solution to be subjected to trypsin hydrolysis.
9. The method of claim 1, wherein the umami peptide solution is a first umami peptide solution, and the peptide having both ACE inhibitory and antioxidant activities is peptide 1: Cys-Cys-Asn-Lys;
the umami peptide solution is a second umami peptide solution, and the peptide with both ACE inhibition and antioxidant activity is peptide 2: Ala-His-Ser-Val-Arg-Phe;
the umami peptide solution is a third umami peptide solution, and the peptide with ACE inhibition and antioxidant activity is peptide 3: His-Cys-His-Thr.
10. The use of the peptide of claim 1 having both ACE inhibitory and antioxidant activity in the preparation of a antihypertensive drug and a drug with antioxidant function.
Technical Field
The invention relates to the field of antihypertensive drugs and anti-oxidation drugs, and in particular relates to a series of peptides with ACE (angiotensin converting enzyme) inhibition and anti-oxidation activities as well as a preparation method and application thereof.
Background
Jinhua ham and Bama ham belong to dry-cured ham, are meat products prepared by using front and rear legs of pigs as raw materials and through the processing technologies of curing, fermentation and the like, and are the most unique traditional pork products in China and Mediterranean regions. The total annual output of the dry-cured ham in China is basically stabilized at 400500 thousands, wherein the annual output of the Jinhua ham is 300400 thousands, and the annual output of the Xuanwei ham reaches about 100 thousands. In addition, Jiangsu Rugao ham, Bingning ham and Hubei Enshi ham belong to the pork product with the traditional characteristic of the local prescription. The flavor of ham depends on the content and proportion of small molecular substances which generate flavor. The small molecular peptides and the free amino acids are core components of ham flavor substances, form the fresh flavor of ham, and greatly contribute to the flavor. Therefore, the research on the umami peptide in the ham is of great significance to the development of the traditional ham industry.
The umami peptide is also called flavor enhancing peptide, and is a peptide substance for supplementing or enhancing the original flavor of food. These peptides only enhance flavor when their levels or amounts are below their individual detection thresholds, and only produce umami taste when their levels or amounts are above their individual detection thresholds. Umami peptides do not affect other tastes (sour, sweet, bitter, salty) and enhance their respective flavor profiles. In general, umami peptides mostly contain acidic amino acids Glu or Asp, Glu, Asp, Gln, Asn in combination with each other or Thr, Ser, Ala, Gly, Met to produce umami taste. At appropriate concentrations, these peptides have synergistic or flavor enhancing effects with other flavor components (salt, monosodium glutamate, acidulant) at appropriate concentrations.
The umami peptide in the ham has certain effect on antioxidation. More and more studies show that anti-oxidation is an important step in preventing aging, because free radicals or oxidants break down cells and tissues, affect metabolic functions, and cause various health problems. If the excess oxidative free radicals can be eliminated, many diseases caused by free radicals and related to aging can be prevented. Such as common cancers, arteriosclerosis, diabetes, cataracts, cardiovascular diseases, senile dementia, arthritis, etc., which are considered to be associated with free radicals. Human beings should take enough antioxidants to delay the body's deterioration speed, prevent skin aging, and keep youthful spirit all the time. Antioxidant peptides are increasingly becoming the popular choice for antioxidants due to their natural harmlessness.
The delicious peptide in ham has certain effect on cardiovascular diseases. With the development of the world and the progress of society, the living standard of people is continuously improved, the living style and the dietary structure of the public are greatly changed, and the modern civilization diseases are also coming. Modern civilization diseases mainly include obesity, diabetes, hypertension, depression and the like. Among these common diseases, hypertension is one of the chronic diseases seriously harming human health, and is also a universal disease worldwide. According to WHO statistics, approximately 11.3 million adults worldwide have hypertension by 2018, resulting in 940 million deaths each year. Meanwhile, hypertension is a main controllable risk factor in the cardiovascular disease development process, and the risk of cardiovascular diseases is reduced by 16% when the systolic pressure of 5mmHg is reduced; sustained elevated blood pressure is highly likely to increase the risk of stroke, heart attack and renal failure. Hypertension has become one of the major diseases endangering human health. According to the prediction of the world health organization, non-infectious diseases account for 79% of death causes by 2020, and cardiovascular diseases such as hypertension account for the first place. Hypertension, one of the hallmarks of modern civilized disease, is commonly referred to as "non-predictive" disease. That is, the consequences of hypertension are always detected after significant damage to the body, often too late.
Generally, many of these peptides have relatively small molecular weight and are easily digested and absorbed in the human body as compared with proteins. The small peptides can not only provide nutrition required by the growth and development of human bodies, but also regulate the physiological functions of the human bodies, and play a role in preventing and even treating diseases. The food-derived bioactive peptides have various types, including Angiotensin Converting Enzyme (ACE) inhibitory peptide, antioxidant peptide, immunoregulatory peptide, anti-inflammatory peptide, antibacterial peptide, antithrombotic peptide, opioid bioactive peptide, calcium absorption promoting peptide, etc. Of these, ACE inhibitory peptides and antioxidant peptides have been closely associated with the treatment and prevention of hypertension and aging, respectively, and have attracted high attention from scientists and governments of various countries.
Disclosure of Invention
The invention aims to provide a series of peptides with both ACE inhibition and antioxidant activity and a preparation method and application thereof, wherein the peptides have both ACE inhibition and antioxidant activity, are a series of functional peptide sequences prepared by in vitro simulated digestion of umami peptides, have both ACE and antioxidant functions, and can be used for research and development of health-care food, medicines or cosmetics.
The peptide with both ACE inhibition and antioxidant activity has the following amino acid sequences:
peptide 1: Cys-Cys-Asn-Lys;
peptide 2: Ala-His-Ser-Val-Arg-Phe;
peptide 3: His-Cys-His-Thr.
Hereinafter, it can also be abbreviated as: peptide 1: CCNK (CK-4), peptide 2: AHSVRF (AR-6), peptide 3: HCHT (HT-4).
The ACE and antioxidant peptide sequence of the invention, including the active peptide sequence as a core, may be modified or modified accordingly.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) preparing fresh peptide in the ham;
peptide 4: Cys-Cys-Asn-Lys-Ser-Val,
peptide 5: Ala-His-Ser-Val-Arg-Phe-Tyr,
peptide 6: His-Cys-His-Thr-Asn. Hereinafter, it can also be abbreviated as: peptide 4: CCNKSV (CV-6), peptide 5: AHSVRFY (AY-7), peptide 6: HCHTN (HN-5).
(2) Solid phase synthesizing the ham-derived umami peptide with known sequence and functional peptide prepared from the umami peptide;
(3) determination of ACE inhibitory activity;
(4) measurement of antioxidant activity, including DPPH free radical scavenging activity, ABTS free radical scavenging activity;
(5) simulating gastrointestinal digestion in vitro (pepsin and pancreatin prepare functional peptides);
(6) amino acid sequence analysis of active peptides.
A preparation method of a peptide with ACE inhibition and antioxidant activity comprises the following steps:
1) preheating the fresh peptide solution in a water bath kettle at 33-40 ℃ for 5-30 min, adding HCl aqueous solution to adjust the pH value to 2-4 to obtain a solution to be hydrolyzed by pepsin, adding pepsin, uniformly mixing, hydrolyzing, and obtaining pepsin hydrolysate after the hydrolysis is finished;
2) and adjusting the pH value of the pepsin hydrolysate to 7-8 by using NaOH aqueous solution to obtain a solution to be subjected to trypsin hydrolysis, adding trypsin, uniformly mixing, hydrolyzing for 1-3 h, inactivating in a boiling water bath after reaching the time, and cooling to obtain a solution containing the peptide with ACE (angiotensin converting enzyme) inhibition and antioxidant activity.
In the step 1), the concentration of the umami peptide solution is 0.5-2 mg/mL (most preferably 1mg/mL), and the umami peptide solution is a first umami peptide solution, a second umami peptide solution or a third umami peptide solution;
the preparation of the first umami peptide solution comprises the following steps: dissolving ham-derived umami peptide with distilled water, wherein the amino acid sequence of the ham-derived umami peptide is peptide 4: Cys-Cys-Asn-Lys-Ser-Val, wherein the peptide concentration is 0.5-2 mg/mL (most preferably 1mg/mL), so as to obtain a first umami peptide solution;
the preparation of the second umami peptide solution comprises the following steps: dissolving ham-derived umami peptide with distilled water, wherein the amino acid sequence of the ham-derived umami peptide is peptide 5: Ala-His-Ser-Val-Arg-Phe-Tyr to make the peptide concentration 0.5-2 mg/mL (most preferably 1mg/mL) to obtain a second umami peptide solution;
the preparation of the third delicious peptide solution comprises the following steps: dissolving the obtained ham-derived umami peptide with distilled water, wherein the amino acid sequence of the ham-derived umami peptide is peptide 6: His-Cys-His-Thr-Asn, and the concentration of the peptide is 0.5-2 mg/mL (most preferably 1mg/mL), so as to obtain a third delicious peptide solution.
Preheating for 5-15 min in a 35-39 ℃ water bath kettle, and most preferably, preheating for 10min in a 37 ℃ water bath kettle.
The concentration of the HCl aqueous solution is 0.5-1.5 mol/L. Most preferably, the concentration of the aqueous HCl solution is 1 mol/L.
Adding HCl aqueous solution to adjust the pH value to 2.5-3.5. Most preferably, an aqueous HCl solution is added to adjust the pH to 3.
The adding amount of the pepsin is 2-6% of the mass of the solution to be hydrolyzed by the pepsin, and the most preferable adding amount of the pepsin is 4% of the mass of the solution to be hydrolyzed by the pepsin. The pepsin is from pig stomach, and the pepsin 1: 10,000U.
Hydrolyzing for 0.5-1.5 h, most preferably, hydrolyzing for 1 h.
In the step 2), the NaOH aqueous solution is 1 mol/L.
And adjusting the pH value of the pepsin hydrolysate to 7.5-8.5 by using NaOH aqueous solution, and most preferably adjusting the pH value of the pepsin hydrolysate to 8 by using NaOH aqueous solution.
The adding amount of the trypsin is 2 to 6 percent of the solution to be hydrolyzed by the trypsin. Most preferably, the trypsin is added in an amount of 4% of the solution to be trypsinized. The enzyme activity of the trypsin is 1: 250.
hydrolyzing for 1.5-2.5 h, most preferably, hydrolyzing for 2 h.
And after the time is up, inactivating in a boiling water bath for 5-15 min. Most preferably, the inactivation is carried out in a boiling water bath for 10min after the time.
The first umami peptide solution is hydrolyzed by pepsin and trypsin to obtain a solution containing Lys-Ser-Val (KSV) and Cys-Cys-Asn-Lys (CCNK, peptide 1). Namely, the umami peptide solution is a first umami peptide solution, and the peptide with both ACE inhibition and antioxidant activity is peptide 1: Cys-Cys-Asn-Lys.
The second umami peptide solution is hydrolyzed by pepsin and trypsin to obtain a solution containing Ala-His-Ser-Val (AHSV), Ala-His-Ser-Val-Arg-Phe (AHSVRF, peptide 2). Namely, the umami peptide solution is a second umami peptide solution, and the peptide with both ACE inhibition and antioxidant activity is peptide 2: Ala-His-Ser-Val-Arg-Phe.
The third delicious peptide solution is hydrolyzed by pepsin and trypsin to obtain a solution containing His-Cys-His-Thr (HCHT, peptide 3). Namely, the umami peptide solution is a third umami peptide solution, and the peptide with ACE inhibition and antioxidant activity is peptide 3: His-Cys-His-Thr.
The peptide with both ACE inhibition and antioxidant activity can play a role in strong ACE inhibition and antioxidant action, and is very suitable for preparing antihypertensive drugs and anti-oxidant drugs.
In the invention, the in vitro simulated digested umami peptide has ACE inhibitory activity and antioxidant activity, is equivalent to the umami peptide with biological activity, and has the advantages that: the peptide can be sensed to have delicate flavor in the oral cavity, is added with proper mouthfeel, shows ACE inhibitory activity and antioxidant activity after gastrointestinal digestion, and plays a role in preventing and even treating diseases. The biological functional activity of the polypeptide is researched by combining in vitro experiments with gastrointestinal tract digestion and absorption, and the method has important significance for the actual production and theoretical research of the functional active polypeptide.
Compared with the prior art, the invention has the following advantages:
the functional peptide prepared from the ham-derived umami peptide can enhance the umami of substances, and has potential medical value in ACE inhibitory activity and antioxidant activity.
The functional peptide has the functions of reducing blood pressure and resisting oxidation, has the characteristics of simple structure, safety, strong activity and the like, plays the roles of nutrition and health care, is expected to provide effective components for developing new medicaments with the functions of reducing blood pressure and resisting oxidation without toxic and side effects, and has wide application prospect.
Drawings
FIG. 1 is a graph showing ion peaks of mixed peptide fragments in a digested umami peptide Cys-Cys-Asn-Lys-Ser-Val (CV-6) solution measured by MALDI-TOF-TOF.
FIG. 2 is an ion peak diagram of the mixed peptide fragment in the digested umami peptide Ala-His-Ser-Val-Arg-Phe-Tyr (AY-7) solution measured by MALDI-TOF-TOF.
FIG. 3 is an ion peak plot of the mixed peptide fragments in post-digestion umami peptide His-Cys-His-Thr-Asn (HN-5) solution as measured by MALDI-TOF-TOF.
FIG. 4 is a diagram showing the identification of MALDI-TOF-TOF structure of Cys-Cys-Asn-Lys (CK-4).
FIG. 5 is a diagram showing the structural identification of MALDI-TOF-TOF of Lys-Ser-Val (KV-3).
FIG. 6 is a diagram showing the structural identification of Ala-His-Ser-Val (AV-4) by MALDI-TOF-TOF.
FIG. 7 is a diagram showing the structural identification of Ala-His-Ser-Val-Arg-Phe (AF-6) by MALDI-TOF-TOF.
FIG. 8 is a diagram showing the structural identification of His-Cys-His-Thr (HT-4) by MALDI-TOF-TOF.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.