CN116507734A - Novel angiotensin I converting enzyme (ACE) inhibitory peptide - Google Patents

Abstract

The present invention provides fish-derived peptides having ACE inhibitory activity, and methods of producing peptide isolates comprising the fish-derived peptides. The invention also provides pharmaceutical products, dietary supplements and functional foods comprising the peptide isolate, and methods of reducing blood pressure in a subject by administering one or more fish-derived peptides to the subject.

Description

Novel angiotensin I converting enzyme (ACE) inhibitory peptide

Technical Field

The present invention relates to a process for preparing a fish-derived peptide having ACE inhibitory activity, which is useful as an active ingredient in pharmaceutical preparations, dietary supplements or food ingredients.

Background

Hypertension is defined as systolic pressure of 140mm Hg or more and diastolic pressure of 90mm Hg or more. It is considered to be the most common serious chronic health problem, one of the major risk factors for cardiovascular disease (CVD) including stroke, coronary artery disease, heart failure, atrial fibrillation and peripheral vascular disease. Global prevalence of hypertension is expected to be as high as 15.8 million adult patients in 2025 (WHO, 2011). Today, hypertension is mainly treated by changing lifestyle and using antihypertensive drugs (Hermansen, 2000). Although there are many causes of hypertension, it is well known that angiotensin I converting enzyme (ACE), a dipeptidyl carboxypeptidase (EC 3.4.15.1), plays an important role in the renin-angiotensin and kallikrein-kallikrein systems for regulating blood pressure and fluid and salt balance in mammals (Vercruysse et al 2005). It cleaves the dipeptide His-Leu in inactive decapeptide angiotensin I into the potent vasoconstrictor angiotensin II via the renin-angiotensin system, thereby raising blood pressure. In addition, it also converts the vasodilator bradykinin to an inactive peptide via the kallikrein-kallikrein system (Wang et al, 2008). Thus, inhibition of ACE activity is a major goal in reducing mortality in hypertensive patients. Although the ACE inhibiting efficacy of food-derived peptides is not as great as that of drugs commonly used for treating hypertension, they are considered to be milder, safer and free of side effects than drugs because they are derived from natural food proteins. Therefore, ACE inhibiting peptides of food protein source show great promise in developing new physiologically functional foods for the prevention of hypertension and therapeutic purposes.

To date, more and more ACE inhibiting peptides have been detected in protein hydrolysates prepared from animal and plant proteins (Iwaniak and dzibba, 2009; murray and FitzGerald, 2007). Among them, fish muscle proteins are well known as an excellent source of ACE inhibiting peptides (Charoenphun, youravong and Cheirsilp, 2013; chen, wang, zhong, wu and Xia, 2012; wijesekara, qian, ryu, ngo and Kim, 2011). In addition, there have been increasing attempts to identify and characterize ACE inhibiting peptides derived from various protein hydrolysates to identify their structure-activity relationship. However, since a variety of ACE inhibiting peptides having different amino acid sequences have been identified, the structure-activity relationship of ACE inhibiting peptides has not been established. To date, fish peptides have been obtained that exhibit ACE inhibiting and antihypertensive activity. Nakajima et al (2009) used pepsin, pancreatin and thermolysin to evaluate the ACE inhibitory activity of fish protein hydrolysates from fish including Atlantic salmon, silver salmon, alaska cod, and southern blue cod. Atlantic salmon and silver salmon, whose ACE was hydrolyzed by thermolysin, were inhibited with IC50 values of 0.078 and 0.138mg/mL, respectively. Wu et al (2008) disclose that shark meat hydrolysates obtained by digestion with protease SM98011 show a high ACE inhibitory activity (IC 50 value of 0.4 mg/mL) compared to untreated shark meat slurry (IC 50 value of 10.5 mg/mL). CF. EY and FE sequences proved to be novel ACE inhibitory peptides, and IC50 values were 1.96, 2.68 and 1.45 mu M respectively. All of these are dipeptides and have a hydrophobic amino acid residue Phe or Tyr at the C-terminal position. Balti et al (2010) showed ACE inhibitory activity of the VYAP (SEQ ID NO: 1), VIIF (SEQ ID NO: 2) and MAW (IC 50 values of 6.1, 8.7 and 16.32. Mu.M, respectively) sequences isolated from squid (Sepia officinalis) muscle hydrolysate.

Gastrointestinal (GI) digestion is particularly important for the bioavailability of ACE inhibiting peptides. Upon oral ingestion, gastrointestinal enzymes may break down peptides, thereby increasing or decreasing their activity. The purpose of the in vitro digestion model is to simulate in a simple way the digestion process occurring in the mouth, stomach and small intestine. There is a need for ACE inhibiting peptides that maintain bioactivity and stability after gastrointestinal tract treatment.

Drawings

FIG. 1 is a molecular exclusion chromatography of a hematogenous meat mix (RDM) hydrolysate eluted with deionized water.

FIG. 2 shows ACE inhibitory activity of 1mM leucine at the same concentration as the isolated peptide fraction.

FIG. 3 is a chromatogram of fraction B eluted with ACN containing 0.1% TFA.

FIG. 4 is a chromatogram of the B6 fraction eluted with ACN containing 0.1% TFA.

FIG. 5 shows the effect of in vitro Gastrointestinal (GI) digestion on the alpha-amino content of synthetic peptides NLLPHR (NR 6, SEQ ID NO: 3), VSVVQYSR (VR 8, SEQ ID NO: 4) and VIYSRINCR (VR 9, SEQ ID NO: 5).

FIG. 6 shows the effect of in vitro Gastrointestinal (GI) digestion on ACE inhibitory activity of synthetic peptides NLLPHR (NR 6, SEQ ID NO: 3), VSVVQYSR (VR 8, SEQ ID NO: 4) and VIYSRINCR (VR 9, SEQ ID NO: 5).

Disclosure of Invention

The present invention provides fish-derived peptides having ACE inhibitory activity, peptide isolates comprising the fish-derived peptides, and methods of producing the peptide isolates. The invention also provides medicaments, dietary supplements and functional foods comprising the fish-derived peptides, and methods of using the fish-derived peptides to reduce blood pressure.

As demonstrated in the examples of the present invention, the fish-derived peptides of the present invention have a strong angiotensin I converting enzyme (ACE) inhibitory activity. Specifically, the three peptides exhibiting the strongest ACE inhibitory potency were VIYSRINCR (SEQ ID NO: 5), VSVVQYSR (SEQ ID NO: 4) and NLLPHR (SEQ ID NO: 3) with IC50 (half inhibitory concentration) of 0.27 μg/ml (or 0.24 μM), 0.89 μg/ml (or 0.95 μM), 0.93 μg/ml (or 1.24 μM), respectively. These three novel peptides have a greater ACE inhibiting potential than the published ACE inhibiting peptides and commercial dietary supplements for ACE inhibition. In addition, gastrointestinal digestion of fish-derived peptide VIYSRINCR (SEQ ID NO: 5) resulted in peptides including VIYSR (SEQ ID NO: 6), INCR (SEQ ID NO: 7) and SRINCR (SEQ ID NO: 8). The fish-derived peptides having ACE inhibitory activity, including gastrointestinal digestion products of these peptides, are useful as antihypertensive agents in pharmaceuticals, dietary supplements and functional foods.

"Fish-derived peptide" refers to a peptide isolated from fish, or a digestion product (e.g., a product of gastrointestinal digestion) from a peptide isolated from fish. The fish are cold-blood aquatic vertebrates, including teleosts, cartilaginous and mandibular fish, typically having an elongated body terminating in a broad skegs, fin limbs (if any), and a two-chamber heart through which blood is delivered for oxygen supply. In certain embodiments, the fish is teleost fish. Teleosts belong to the teleosts class, including freshwater teleosts such as trout, weever, grouper, shuttle, limbic, sea bass, carp and certain salmon. Examples of salted teleoperational include salmon (e.g., atlantic salmon, knoop salmon, red salmon, silver salmon, pink salmon, and salmon), tuna (e.g., bonito, blue fin tuna, yellow fin tuna, and long fin tuna), and cod (e.g., atlantic cod and pacific cod), halibut, and loach.

In certain embodiments, the fish is tuna. Tuna refers to a salty water fish belonging to the tuna family, which is a subgroup of the mackerel family. The tuna family includes 15 species of five genera, including the following genera: the genus Allothunnus, known as elongated tuna; auxis genus, also known as guard-warship tuna; the genus Euthynnus, also known as tuna; katsuwonus genus, also known as bonito; tuna genus, including longfin tuna and true tuna. Tuna subgenera tuna (thunderus), also known as tuna blue fin, tuna subgenera (neochunnus), also known as tuna yellow fin. In some embodiments, the tuna belongs to the genus Katsuwonus (i.e., is bonito).

"fish" refers to any fish muscle tissue. In some embodiments, the meat is raw (i.e., uncooked). In some embodiments, the meat is a blood-mixed meat. The fish blood meat mixture refers to fish meat rich in myoglobin, and the texture and flavor of the fish meat can be affected when the fish blood meat mixture is cooked. For example, tuna blood meat is generally considered a low value tuna by-product of the tuna processing process because of its undesirable taste and texture. However, tuna blood meat is in an advantageous position as a source of protein or peptide extraction due to its abundance and low cost.

"ACE inhibitory activity" refers to the ability to inhibit the activity of angiotensin I converting enzyme (ACE). In some embodiments, a fish-derived peptide or peptide isolate described herein has an ACE inhibition IC50 value of 100 μg/ml or less. Methods for measuring ACE inhibitory activity include those described in example 5.

In certain aspects, the invention provides methods of producing a peptide isolate. These methods may include mixing fish meat with water to produce an aqueous mixture; adjusting the pH of the aqueous mixture to an alkaline pH (i.e., a pH greater than 7); the fish meat is hydrolyzed by adding alkaline protease to the aqueous mixture and incubating the aqueous mixture under conditions sufficient to produce a hydrolysate.

An "isolate" refers to a peptide extract or purified peptide derived from a cell or tissue, or a peptide extract or purified peptide product that has undergone enzymatic hydrolysis as defined herein.

In some embodiments, the aqueous mixture includes a meat to water ratio of about 1:1 to about 1:5. In some embodiments, the aqueous mixture comprises a meat to water ratio of about 1:2 to about 1:5, 1:2 to about 1:4, or about 1:2 to about 1:3.

In some embodiments, the aqueous mixture comprises a protein concentration in the range of about 1% w/v to about 20% w/v (g protein/ml water). In some embodiments, the aqueous mixture comprises a protein concentration ranging from about 1% w/v to about 15% w/v, from about 1% w/v to about 10% w/v, from about 5% (w/v) to about 20% (w/v), or from about 5% (w/v) to about 15% (w/v). In some embodiments, the aqueous mixture comprises a protein concentration of about 10% w/v. The protein concentration may be measured by techniques known in the art, for example by spectrophotometry to measure absorbance at 280 nm.

As previously described, the method may include adjusting the pH to an alkaline pH (i.e., a pH greater than 7). In some embodiments, the alkaline pH is in the range of about 7 to about 11. In some embodiments, the alkaline pH is in the range of about 7 to about 9 or about 8 to about 8.5.

As previously described, the method may include hydrolyzing fish meat by adding alkaline protease to the aqueous mixture and incubating the aqueous mixture under heat conduction sufficient to produce a hydrolysate.

"hydrolysate" refers to the product of an enzymatic hydrolysis. Enzymatic hydrolysis is the breakdown of a compound (e.g., in a cell or tissue sample) in the presence of an enzyme and water. A hydrolysate may refer to an enzymatic hydrolysate based on the addition of an exogenously provided enzyme or may refer to an enzymatic hydrolysate based on the enzymatic activity of an endogenous enzyme. Enzymatic hydrolysis based on the enzymatic activity of endogenous enzymes may be referred to as autolysis.

A "protease" is an enzyme that catalyzes the hydrolysis of a protein, which is the cleavage of peptide bonds, resulting in the breakdown of the protein into smaller polypeptides or individual amino acids. Proteases can be divided into seven classes: serine proteases that use serine alcohols as reactive nucleophiles; cysteine proteases using cysteine thiols as nucleophiles; threonine proteases using threonine secondary alcohols as nucleophiles; aspartic protease using aspartic carboxylic acid; glutamate protease, which uses glutamate carboxylic acid; metalloproteinases, which use a metal, typically zinc; and asparagine peptide lyase, which uses asparagine for an elimination reaction (without water).

"alkaline protease" refers to a protease that has enzymatic activity at alkaline pH (i.e., pH greater than 7). Alkaline proteases typically have activity at alkaline pH of up to about 11. Examples of alkaline proteases include proteinase K, subtilisin and glutelin.

In some embodiments, the alkaline protease comprises a serine protease. "serine protease" refers to an enzyme that cleaves peptide bonds in proteins, wherein serine acts as a nucleophilic amino acid at the active site (of the enzyme). Subtilisin is a serine protease obtainable from certain soil bacteria such as bacillus subtilis and bacillus licheniformis. The alkaline protease is a commercially available subtilisin from Bacillus licheniformis.

In some embodiments, the conditions sufficient to produce a hydrolysate include incubating the aqueous mixture with the alkaline protease for a period of at least about one hour. In some embodiments, the period of time is in the range of about 2 hours to about 5 hours.

In some embodiments, the conditions sufficient to produce a hydrolysate include incubating the aqueous mixture with an alkaline protease, including at a temperature in the range of about 40 ℃ to about 70 ℃.

In some embodiments, the method further comprises the step of removing cell debris from the hydrolysate after the hydrolyzing step.

By "removing cell debris" is meant separating soluble products, such as supernatant, from the cell extract. For example, cell debris can be removed by centrifugation and filtration. In some embodiments, removing cell debris includes centrifuging and filtering the hydrolysate to obtain a supernatant.

In some embodiments, the hydrolysis step further comprises terminating the hydrolysis reaction by heating the hydrolysate to a temperature in the range of about 80 ℃ to about 100 ℃ after incubating the aqueous mixture under conditions sufficient to produce the hydrolysate. In some embodiments, terminating the hydrolysis reaction includes incubating the hydrolysis product at a temperature in the range of about 80 ℃ to about 100 ℃ for a period of time in the range of about 10 minutes to about 20 minutes. For example, the hydrolysate can be heated to about 90 ℃ for about 15 minutes. After the hydrolysis reaction is terminated, the hydrolysis product may be cooled to a temperature below 40 ℃, for example to a temperature in the range of about 10 ℃ to about 30 ℃.

In some embodiments, the method further comprises the step of subjecting the hydrolysate to chromatography and collecting one or more chromatography fractions comprising peptides having ACE inhibiting activity.

"chromatography" refers to separation of a mixture by passing the mixture through a medium in which the different components move at different rates, either as a solution or suspension or as a vapor (as in gas chromatography). Size exclusion chromatography is a chromatographic method in which molecules in solution are separated by their size and, in some cases, by molecular weight. Reversed phase chromatography includes any chromatography method using a hydrophobic stationary phase.

In some embodiments, the method further comprises the step of enzymatically digesting the peptide isolate to produce one or more digestion products of the peptide. By "enzymatic digestion" is meant the breakdown of macromolecules into smaller compounds based on the activity of the enzyme.

In some embodiments, the enzymatic digestion comprises a computer simulated gastrointestinal digestion. "gastrointestinal digestion" refers to enzymatic digestion by enzymes naturally occurring in the gastrointestinal tract. Enzymes naturally occurring in the gastrointestinal tract include pepsin, trypsin and chymotrypsin. By computer-simulated gastrointestinal digestion is meant enzymatic digestion outside the gastrointestinal tract, e.g., in a test tube, using enzymes isolated from the gastrointestinal tract.

In some aspects, the invention provides formulations comprising a peptide isolate as described herein and other ingredients, such as a pharmaceutically acceptable carrier, diluent or excipient. Such products may be administered to or consumed by a subject and include both pharmaceuticals (also referred to as "pharmaceutical compositions") and non-pharmaceuticals (also referred to as "non-pharmaceutical compositions", e.g., food supplements and functional foods). The correct formulation of the product depends on the route of administration selected.

The formulation may contain different types of carriers, excipients or diluents, depending on whether it is to be administered in solid, liquid or aerosol form. The formulations (and any other active agents) described herein may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticular, intraprostatically, intrapleurally, intratracheally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intracapsularly, intramucosally, intrapericardially, intraumbilical, intraocular, orally, topically, inhalations (e.g., aerosol inhalations), injections, infusions, continuous infusions, direct local perfusion of target cells, by catheter, by lavage, by cream, by lipid compositions (e.g., liposomes), or by other methods known to those of ordinary skill in the art or any combination of the foregoing (e.g., see Remington's Pharmaceutical Sciences, 18 th edition Mack Printing Company, 1990). In particular embodiments, the formulation is formulated for oral administration.

A pharmaceutical product refers to a product for use in the treatment of a disease or disorder, or for use in the treatment of one or more symptoms of a disease or disorder. Non-pharmaceutical products refer to formulations other than pharmaceutical products, such as dietary supplements or functional foods.

"functional food" (also referred to as "nutraceutical") refers to a food product with additional functionality imparted by the addition of new ingredients (e.g., one or more peptide isolates as previously described) that are not normally or traditionally present in the food product.

In some aspects, the invention provides methods of using peptide isolates or formulations comprising peptide isolates as described previously.

"mammal" includes humans and domestic animals such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, rabbits) and non-domestic animals such as wild animals, and the like. In certain particular embodiments, the mammal is a human. In certain particular embodiments, the mammal is a pet, such as a canine or a feline.

The "subject" according to any of the above embodiments is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human.

"treating," "treating" or "ameliorating" refers to medical management of a disorder or disease in a subject (e.g., patient) to reduce or eliminate symptoms, shorten the duration of the disorder, or delay the onset or progression of the disorder or disease.

An "effective amount" refers to an amount of peptide isolate that provides a desired physiological change, such as lowering hypertension. In certain embodiments, the effective amount is a therapeutically effective amount. The desired physiological change may be, for example, a reduction in the symptoms of the disease, or a reduction in the severity of the symptoms of the disease, or may be a reduction in the progression of the symptoms of the disease. In certain embodiments, the desired physiological change is not related to the treatment of the disease.

In certain embodiments, the method comprises reducing hypertension in a subject comprising administering to the subject a peptide isolate as described above, a pharmaceutical product as described above, or a dietary supplement or functional food as described above.

"hypertension" refers to hypertension. When the heart beats, it creates pressure forcing blood through the vascular network, including arteries, veins, and capillaries. The pressure at which blood is forced through these vessels is a result of two forces: systolic pressure, which occurs when blood is pumped out of the heart and into an artery that is part of the circulatory system; and diastolic pressure, which is produced when the heart is resting between heartbeats. Methods of treating hypertension may include reducing elevated blood pressure or hypertension, and reducing the likelihood of developing elevated blood pressure or hypertension. In some embodiments, reducing elevated blood pressure or hypertension comprises reducing systolic blood pressure readings by at least 1mmHg, by at least 5mmHg, or by at least 10mmHg; and/or the diastolic blood pressure reading is reduced by at least 1mmHg, by at least 5mmHg, or by at least 10mmHg. Elevated blood pressure may refer to systolic pressure readings including 120mmHg or greater, diastolic pressure readings including greater than 80mmHg, or both. Hypertension may refer to systolic pressure readings including 130mmHg or greater, diastolic pressure readings including greater than 80mmHg, or both.

In certain embodiments, the method comprises promoting blood pressure in the health of a subject, comprising administering to the subject the peptide isolate of any one of claims 20 to 26, or the pharmaceutical product of claim 27, or the nutritional supplement or functional food of claim 28. Healthy blood pressure refers to systolic blood pressure readings below 120mmHg and diastolic blood pressure readings below 80mmHg.

Experimental studies of the present invention are shown below.

Examples

Example 1

Preparation of tuna blood-producing meat-synthesizing hydrolysate with ACE inhibitory activity

Tuna blood-producing meat-in-meat (RDM) was ground using a cutter with a 3mm cutting head. Ground tuna blood-producing meat (RDM) was prepared at a concentration of 10% w/v (g protein/ml water) and the pH was adjusted to 8.5 using sodium hydroxide (NaOH). 2.4L of alkaline protease was added to the mixture at a concentration of 4% (w/w) of protein substrate. Hydrolysis was carried out at 60℃for 4 hours. The hydrolysis mixture was heated at 95℃for 10 minutes and then centrifuged at 9000rpm for 20 minutes. The supernatant was called tuna blood-producing meat-in-meat (RDM) hydrolysate. The total solids content of the tuna blood-producing meat-in-meat (RDM) hydrolysate produced by this hydrolysis condition was about 8% and the protein recovery was about 75%. The IC50 of tuna blood-producing meat-in-meat (RDM) hydrolysate on ACE inhibitory activity was evaluated and showed excellent ACE inhibitory potency with an IC50 of 61.58. Mu.g/ml. The hydrolysis product of tuna blood-producing meat-in-meat (RDM) containing ACE inhibitory peptide is purified by column chromatography, i.e. size exclusion chromatography and reversed-phase high-efficiency chromatography.

Example 2

Purification of ACE inhibitory peptides

The tuna blood-producing meat-in-meat (RDM) hydrolysate was first purified by flash protein liquid chromatography. The peptides were eluted in isocratic mode with deionized water at a flow rate of 0.4 ml/min. The eluate was collected in 0.6-ml fractions and combined as shown in FIG. 1. The content of α -amino groups (expressed as leucine equivalent) was determined by the TNBS method (Adler-Nissen, 1979) for each pooled fraction, and ACE inhibitory activity was also analyzed at the same concentration of 1mM leucine equivalent. Tuna blood-producing meat-in-meat (RDM) hydrolysate was separated into 5 fractions (fractions a-E, fig. 1), and fraction B showed the highest ACE inhibitory activity (p <0.05, fig. 2), followed by fractions a and E, which showed similar ACE inhibitory activity (p >0.05, fig. 2). Fraction B was thus selected for a further purification step, namely reverse phase high performance liquid chromatography (RP-HPLC). Although many documents report that ACE inhibitory activity increases significantly with decreasing molecular weight of peptides (Byun and Kim, 2002; natesh et al, 2003; darewicz et al, 2014), the amino acid sequence and composition of peptides in hydrolysates may play a more important role in controlling ACE inhibitory activity.

A second purification, the lyophilized powder of fraction B, which was the most active, was dissolved in deionized water and isolated using a source 15rpc ST 4.6/150 column (GE Healthcare, piscataway, new jersey, usa) connected to an Agilent 1260 information HPLC system. Peptide elution was performed by a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid (0-100%) at a flow rate of 0.5 ml/min. As shown in FIG. 3, 0.5-ml fractions were collected and pooled. The alpha-amino content (expressed as leucine equivalent) and ACE inhibitory activity of each pooled fraction were determined. Fraction B was separated according to hydrophobicity and 6 main fractions were collected (B1-B6, FIG. 3). Based on Reversed Phase Chromatography (RPC), the polar protein/peptide is eluted first, whereas the non-polar protein/peptide binds to the column. Bound hydrophobic protein/peptide was eluted by increasing the concentration of organic solvent (Gaurav Pratap et al, 2016). Fraction B6, which has the highest hydrophobicity, shows the strongest ACE inhibitory activity based on the principle of Reverse Phase Chromatography (RPC) and specific inhibitory activity (see table 1). To obtain higher purity ACE inhibiting peptides fraction B6 was further purified on a Zorbax Eclipse Plus C18 flash column.

TABLE 1

ACE inhibitory Activity of the collected fractions

Note that: different letters from the same column indicate significant differences (p < 0.05).

The peptide content in the ACE inhibitory activity assay reaction is indicated.

For the third purification, fraction B6 (50. Mu.l) showing the highest ACE inhibitory activity was applied to a chromatographic column (3.5 μm particle size, 4.6X150 mm) connected to an HPLC system. Peptide elution was performed using a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid (0-100%) at a flow rate of 0.5 ml/min. As shown in FIG. 4, 0.5-ml fractions were collected and pooled. The alpha-amino content (expressed as leucine equivalent) and ACE inhibitory activity of each pooled fraction was determined. Fraction B6 was clearly separated into 3 fractions (fractions B6-I, B6-II and B6-III, FIG. 4). The B6-II fraction showed the strongest inhibitory activity when considering the specific inhibitory activity of the peptide fraction against ACE (see table 2).

TABLE 2

ACE inhibitory Activity of the collected fractions

Note that: different letters from the same column indicate significant differences (p < 0.05).

The peptide content in the ACE inhibitory activity assay reaction is indicated.

In order to obtain peptide sequences contained in the peptide fraction exhibiting excellent ACE inhibitory activity after purification, the purified peptide fractions selected for peptide sequencing using liquid chromatography tandem mass spectrometry (LC-MS/MS) were fractions B2 and B4 from purification 1, which have higher ACE inhibitory activity and high peptide yield, and fractions B6-II from purification 2, which have the highest ACE inhibitory efficacy.

Example 3

Identification of ACE inhibitory peptides

The amino acid sequences of the purified peptide components from fractions B2, B4 and B6-II were identified using liquid chromatography tandem mass spectrometry. To verify ACE inhibitory activity of the identified peptides and to better understand structure-activity relationships, peptides identified from LC-MS/MS were selected and chemically synthesized using solid phase peptide synthesis methods as shown in table 3. The purity of the synthetic peptide was determined by HPLC analysis to be greater than 98%. Manufacturers use liquid chromatography in combination with mass spectrometry to confirm the molecular weight of the synthetic peptides. ACE inhibitory activity of each synthetic peptide was measured. The amino acid sequence of the synthetic peptide was analyzed for computer-simulated ACE inhibitory activity using the BIOPEP database (http:// www.uwm.edu.pl/biochem/index. Php/en/biocep).

At the same peptide concentration of 1mg/ml, all synthetic peptides showed ACE inhibitory activity (see Table 3). Different arrangements of amino acid sequences in peptides lead to different ACE inhibiting potency. These peptides were found to be different small peptides with molecular weights in the range 600-1000Da and contained 5-10 amino acids in the peptide fragment.

Previous studies reported that most ACE inhibiting peptides are small peptides with 2-12 residues and a molecular weight of less than 3000Da, which may more easily adapt to ACE active sites and thus have inhibitory activity (Sun et al, 2019). Although di-or tripeptides with high ACE inhibiting activity have been widely reported, longer peptides were also found to have stronger ACE inhibiting activity. For example, FFGRCVSP from ovalbumin (SEQ ID NO: 9), FKGRYYP from chicken muscle (SEQ ID NO: 10), NGTWFEPP from human myofibrillar protein (SEQ ID NO: 11) and LKPNM from dried bonito muscle (SEQ ID NO: 12) have been found (Fujita et al, 2000; fujita and Yoshikawa, 1999; ghassle et al, 2011).

Among the synthetic peptides described herein, the first 3 peptides exhibiting the strongest ACE inhibitory activity were VIYSRINCR (SEQ ID NO: 5) with an IC50 of 0.27 μg/ml (or 0.24 μM), followed by VSVVQYSR (SEQ ID NO: 4) with an IC50 of 0.89 μg/ml (or 0.95 μM), and NLLPHR (SEQ ID NO: 3) with an IC50 of 0.93 μg/ml (or 1.24 μM). Notably, the inhibition of these 3 synthetic peptides was stronger than that of the bonito-derived peptides(ic50=144±8 μg/ml, liu et al 2012), a commercial dietary supplement for lowering blood pressure.

To assess the bioavailability of ACE inhibiting peptides, these 3 synthetic peptides were selected for further study on in vitro Gastrointestinal (GI) digestion. In addition, the amino acid sequences of the peptides shown in table 3 were compared to the bipep database, which has disclosed potential ACE inhibiting peptides from various protein sources. Most peptides contain potential ACE inhibiting peptides in their sequence. In addition, the peptides identified from the present invention were found to be novel ACE inhibiting peptides.

Although the structure-activity relationship of ACE inhibiting peptides has not been fully established, some common structural features of ACE inhibiting peptides have been disclosed. The C-terminus of peptides is considered to be a controlling factor for ACE inhibitory activity by interaction with the S1, S '1 and S'2 subsites of the ACE active site (Ondetti and Cushman, 1982), which generally contain hydrophobic amino acid residues. Furthermore, the N-terminal branched aliphatic amino acids have been reported to be most effective in increasing peptide binding activity of ACE (Byun and Kim, 2002). The present study found that peptides with Arg (R) at the C-terminal position may play an important role in ACE inhibitory activity, which is consistent with the results of Wang et al (2020). The positively C-charged amino acids (Lys and Arg) mean increasing the potency of ACE inhibiting peptides (Guang and Philips, 2009; topcham et al, 2015). Furthermore, the presence of hydrophobic amino acids with aromatic or branched chains at the N-terminus, including Gly (G), val (V), trp (W), leu (L), phe (F) and Met (M), appears to have a positive effect on ACE inhibitory activity of these synthetic peptides.

TABLE 3 Table 3

ACE inhibitory activity of synthetic peptides.

Note that: the synthetic peptides were tested for ACE inhibitory activity at the same concentration of 1 mg/ml. (-) = is not disclosed. (=) is disclosed in the bipep database.

Different letters from the same column indicate significant differences (p < 0.05).

Example 4

In vitro Gastrointestinal (GI) digestion of potent ACE inhibiting peptides

ACE inhibiting peptides must retain their activity in the gastrointestinal tract so that they can be absorbed into the blood and peptide inhibitors show their antihypertensive effect in the blood. In vitro gastrointestinal digestion provides a simple procedure to mimic the antihypertensive activity of peptides upon oral administration. The 3 synthetic peptides that exhibited the strongest ACE inhibitory effects, namely NLLPHR (NR 6, SEQ ID NO: 3), VSVVQYSR (VR 8, SEQ ID NO: 4) and VIYSRINCR (VR 9, SEQ ID NO: 5), were selected for assessing the stability of ACE inhibitory peptides to gastrointestinal enzymes (pepsin and pancreatin). According to Zhu et al (2008), some modifications were made to simulate in vitro gastrointestinal digestion. 1mg of each sample was dissolved in 0.5ml of 0.1M KCl-HCl and the pH was adjusted to 2.0 with 6M HCl. Pepsin (1% enzyme/substrate, w/w) was added and the mixture incubated in an oscillating water bath at 37 ℃ for 1h, then the pH was adjusted to pH 7.5. Subsequently, pancreatin (2% enzyme/substrate, w/w) was added and the mixture was further incubated in an oscillating water bath at 37 ℃ for 2 hours. The enzyme reaction was stopped by heating at 95℃for 10 minutes. The digestate was cooled to room temperature, the volume was adjusted to the same level and centrifuged at 8000 Xg for 20 min. The ACE inhibitory activity and the alpha-amino content of the polypeptides before and after in vitro gastrointestinal digestion were determined. After simulated gastrointestinal digestion, the α -amino content of all synthetic peptides was increased due to the release of small peptides by the action of pepsin and pancreatin (fig. 5). In addition, longer peptides can be easily digested by GI enzymes because of the higher α -amino content shown (fig. 5). ACE inhibitory activity of peptides NR6, VR8 and VR9 was reduced by about 30%, 19% and 14% respectively after gastrointestinal digestion (figure 6). Although all 3 peptides were digested by gastrointestinal enzymes and their inhibitory activity was reduced, their high ACE inhibitory potency still remained.

Example 5

Computer simulated Gastrointestinal (GI) digestion of effective synthetic peptides

To predict the amino acid sequence released by peptide fragments following gastrointestinal digestion, computer simulated Gastrointestinal (GI) digestion was performed on peptides NLLPHR (NR 6, SEQ ID NO: 3), VSVVQYSR (VR 8, SEQ ID NO: 4) and VIYSRINCR (VR 9, SEQ ID NO: 5) using the free web application FeptidDB. The application is for aiding in the discovery of bioactive peptides of food-derived compounds. This web-based information center allows the user to select the appropriate enzymes for computer-simulated enzymatic digestion (panyai et al, 2019). In this example, the GI enzymes selected for cleavage of peptide sequences in silico are pepsin, trypsin and chymotrypsin. Depending on the cleavage site of the GI enzyme, the hydrolysis products that may be obtained from the degradation of peptides NR6, VR8 and VR9 are shown in table 4. Predicted amino acid sequences were also searched against a database of bioactive peptides for ACE inhibitory peptides. Only peptide VIY is reported to be an ACE inhibitor. To verify the inhibitory activity of the predicted peptides, all predicted peptides released from VR9 were selected for the determination of ACE inhibitory activity. The results in table 5 show that not only peptide VIY, but also other computer-simulated digested peptides exert ACE inhibitory activity. Peptides VIYSR (SEQ ID NO: 6), INCR (SEQ ID NO: 7) and SRINCR (SEQ ID NO: 8) showed significantly stronger inhibitory potency than peptide VIY. This result suggests that the presence of the N-terminal hydrophobic amino acid and the C-terminal Arg (R) may enhance the inhibitory potency of the peptide.

TABLE 4 Table 4

The peptides NLLPHR (NR 6, SEQ ID NO: 3), VSVVQYSR (VR 8, SEQ ID NO: 4) and VIYSRINCR (VR 9, SEQ ID NO: 5) predicted by PEPTIDEDDB mimic gastrointestinal digestion in silico.

Note that: ACE inhibitory peptides as disclosed in the database.

TABLE 5

ACE inhibitory activity of synthetic peptides, wherein the amino acid sequence was obtained from a computer simulated gastrointestinal digestion of peptide VIYSRINCR (VR 9, SEQ ID NO: 5).

Note that: nd=undetected.

Different letters in the same column indicate significant differences (p < 0.05).

Determination of angiotensin I converting enzyme (ACE) inhibitory Activity

The measurement of ACE inhibitory activity was performed according to the methods of Cushman and Cheung (1971) and Wu et al (2002), with some modifications. A reaction mixture containing 50. Mu.l of hydrolysate or peptide, 150. Mu.l of 8.3mM hippocampal-L-histidyl-L-leucine (HIL) and 50. Mu.l of ACE (25 mU/ml) was incubated at 37℃for 1 hour. Subsequently, 250. Mu.l of 1M HCl was added to terminate the reaction. Released Hippuric Acid (HA) was extracted by adding 2.5ml ethyl acetate, and the mixture was vortexed and mixed for 1 minute, and then allowed to stand at room temperature for 1 hour. Then, 2ml of the supernatant was transferred to a beaker and dried at 80 ℃ to remove ethyl acetate. Finally, 1ml deionized water was added to dissolve Hippuric Acid (HA). Absorbance was measured at 228 nm. The percent ACE inhibition was calculated as follows:

wherein a is the absorbance at 228nm of the ACE containing reaction without the hydrolysis product; b is the absorbance at 228nm of a reaction containing ACE previously inactivated by addition of HCl in the absence of hydrolysis products; c is the absorbance at 228nm of the reaction in the presence of ACE and the hydrolysate; d is the absorbance at 228nm of the reaction containing ACE, which was previously inactivated by addition of HCl in the presence of the hydrolysate. IC50 is defined as the concentration of inhibitor required to inhibit 50% of ACE activity. The specific inhibitory activity was calculated as ACE inhibition (%) divided by total peptide content (mg).

Determination of alpha-amino content

The determination of the alpha-amino content was carried out according to Adler-Nissen (1979). Purified peptide fractions (50 μl) were mixed with 0.2125M phosphate buffer (0.5 ml) at pH 8.2 and 0.05% TNBS reagent (0.5 ml). The reaction mixture was incubated in a water bath at 50℃for 1 hour. Subsequently, 0.1M HCl (1 ml) was added to terminate the reaction. The mixture was left at room temperature for 30 minutes, and then the absorbance at 420nm was monitored. Leucine was used as a standard. The content of alpha-amino groups is expressed as leucine equivalent.

The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and/or listed in the application data sheet are incorporated herein by reference, in their entirety. If the concepts of the various patents, applications and publications need to be employed to provide yet another embodiment, different aspects of the embodiments can be modified.

These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the invention.

Claims (31)

1. A method of producing a peptide isolate comprising the steps of: mixing fish meat with water to produce an aqueous mixture;

adjusting the pH of the aqueous mixture to an alkaline pH;

the fish meat is hydrolyzed by adding alkaline protease to the aqueous mixture and incubating the aqueous mixture under heat conduction sufficient to produce a hydrolysate.

2. The method of claim 1, wherein the aqueous mixture comprises a meat to water ratio of about 1:2 to about 1:3.

3. The method of claim 1, wherein the aqueous mixture comprises a protein concentration in the range of about 5% (w/v) to about 15% (w/v) (g protein/ml water).

4. A method according to any one of claims 1 to 3, wherein the alkaline pH is in the range of from above 7 to about 11.

5. The method of any one of claims 1 to 4, wherein the alkaline protease comprises a serine protease.

6. The method of any one of claims 1 to 5, wherein the conditions sufficient to produce a hydrolysate comprise an incubation period in the range of about 2 hours to about 5 hours.

7. The method of any one of claims 1 to 6, wherein the conditions sufficient to produce a hydrolysate comprise incubation at a temperature in the range of about 40 ℃ to about 70 ℃.

8. The method of any one of claims 1 to 7, further comprising the step of removing cell debris from the hydrolysate after the hydrolyzing step.

9. The method of claim 8, wherein the step of removing cellular debris comprises centrifuging and filtering the hydrolysate to obtain a supernatant.

10. The method of any one of claims 1 to 9, wherein the hydrolyzing step further comprises terminating the hydrolysis reaction by heating the hydrolysate to a temperature in the range of about 80 ℃ to about 100 ℃ after incubating the aqueous mixture under conditions sufficient to produce the hydrolysate.

11. The method of claim 10, wherein terminating the hydrolysis reaction comprises incubating the hydrolysis product at a temperature in the range of about 80 ℃ to about 100 ℃ for a period of time in the range of about 10 minutes to about 20 minutes.

12. The method of claim 10 or 11, wherein the terminating the hydrolysis reaction further comprises cooling the hydrolysis product to a temperature in the range of about 10 ℃ to about 30 ℃ after heating the hydrolysis product.

13. The method of any one of claims 1 to 12, further comprising the step of subjecting the hydrolysate to chromatography and collecting one or more chromatography fractions comprising peptides having ACE inhibiting activity.

14. The method of claim 13, wherein the chromatography is selected from the group consisting of size exclusion chromatography and reversed-phase high-efficiency chromatography.

15. The method of claim 13 or 14, further comprising the step of enzymatically digesting the peptide isolate to produce one or more digestion products of the peptide.

16. The method of claim 15, wherein the enzymatic digestion comprises computer simulated gastrointestinal digestion.

17. The method of any one of claims 1 to 16, wherein the fish meat comprises blood-producing meat.

18. A method according to any one of claims 1 to 17, wherein the fish meat comprises minced fish meat.

19. The method of any one of claims 1 to 18, wherein the fish meat comprises tuna meat.

20. The method of any one of claims 1 to 19, wherein the fish meat comprises bonito fish meat.

21. A peptide isolate produced by the method of any one of claims 1 to 20.

22. A peptide isolate comprising one or more fish-derived peptides having ACE inhibiting activity.

23. The peptide isolate of claim 21 or 22, wherein the one or more fish-derived peptides having ACE inhibitory activity have an ACE inhibitory IC of 100 μg/ml or less ₅₀ Values.

24. The peptide isolate of any one of claims 21 to 23, wherein the one or more fish-derived peptides are selected from the group consisting of: GPLYHS (SEQ ID NO: 13), LIHAIL (SEQ ID NO: 14), SFLMRK (SEQ ID NO: 15), VIYSRINCR (SEQ ID NO: 5), VLMSQVFKQT (SEQ ID NO: 16), WTIHTP (SEQ ID NO: 17), LPPGKIV (SEQ ID NO: 18), IFERL (SEQ ID NO: 19), FDQFLPIH (SEQ ID NO: 20), NGPSGQTG (SEQ ID NO: 21), LLDHRANL (SEQ ID NO: 22), APPHIF (SEQ ID NO: 23), HFAASGK (SEQ ID NO: 24), LEQVSAGTT (SEQ ID NO: 25), NLLPHR (SEQ ID NO: 3), VVPAT (SEQ ID NO: 26), VEWKERATE (SEQ ID NO: 27), LLHAKPLN (SEQ ID NO: 28), VVQYSR (SEQ ID NO: 4), IKGERD (SEQ ID NO: 29), KKKLLEKKKK (SEQ ID NO: 30), GVGIHGS (SEQ ID NO: 31), QVGG (SEQ ID NO: 8633) and GPQGSHIGH (SEQ ID NO: MTGLPGPTGP).

25. The peptide isolate of any one of claims 21 to 24, comprising the digestion product of one or more fish-derived peptides.

26. The peptide isolate of claim 25, wherein the digestion product comprises at least one of: VIYSR (SEQ ID NO: 6), INCR (SEQ ID NO: 7) and SRINCR (SEQ ID NO: 8).

27. The peptide isolate of claim 25, wherein the digestion products comprise VIYSR (SEQ ID NO: 6), INCR (SEQ ID NO: 7) and SRINCR (SEQ ID NO: 8).

28. A pharmaceutical product comprising the peptide isolate of any one of claims 21 to 27 and at least one excipient.

29. A dietary supplement or functional food comprising the peptide isolate of any one of claims 21 to 27 and at least one additional ingredient.

30. A method of treating hypertension in a subject comprising administering to the subject a peptide isolate as claimed in any one of claims 21 to 27, or a pharmaceutical product as claimed in claim 28, or a dietary supplement or functional food as claimed in claim 29.

31. A method of promoting blood pressure health in a subject comprising administering to the subject a peptide isolate according to any one of claims 21 to 27, or a pharmaceutical product according to claim 28, or a dietary supplement or functional food according to claim 29.