KR101682865B1 - Salty peptide - Google Patents

Abstract

The present invention relates to a salty taste peptide, and more particularly, a short peptide having 4 to 5 amino acids, which shows a salty taste, and it is expected that a salt food additive can be developed as a salt substitute.

Description

Salty peptide {Salty peptide}

The present invention relates to a salty peptide.

Salt (sodium chloride) plays an important role in seasoning and processing food, such as imparting taste to food, improving food preservation, and improving food properties. Salt gives foods a particularly tasty taste (salt taste), and its constituents, sodium and chlorine, are essential nutrients for the human body.

However, excessive intake of sodium, a constituent of salt, is considered to be a risk factor for many health problems, such as heart disease and vascular disease, such as high blood pressure. In Japan as well as in many developed countries, as the number of elderly people who are susceptible to these diseases increases, there is a strong demand for a decrease in the intake of salt, especially sodium.

In order to reduce the amount of salt intake, the simplest method is to reduce the amount of salt used in seasoning and processing of food. However, if the amount of salt contained in food, whether it is home cooked food or processed food, is reduced by 10% or more, the taste of the food is generally damaged.

The method of reducing the intake of salt, especially sodium, without damaging the salty taste, that is, generally referred to as an infection method, is a method of using a substance that has a salty taste by itself (hereinafter referred to as a salt substitute) and , A method of using a substance (hereinafter referred to as a salt taste enhancing substance) that enhances the salt taste when coexisting with salt, although itself does not have a salty taste, is known.

Examples of salt substitutes include potassium salts, ammonium salts, basic amino acids, peptides composed of basic amino acids, and alkali metal salts of gluconic acid. Salt taste enhancers cannot replace salt, but by enhancing the salt taste of salt, reducing the amount of salt used, it is a substance that enables infection.

As a salt taste enhancing material, for example, a peptide obtained by hydrolyzing collagen having a molecular weight of 50,000 Daltons or less (Patent Document 1), a sweet protein of thaumatin (Patent Document 2), and black bacterium (Aspergillus) having citric acid-producing ability. niger), and a decomposition solution (Patent Document 3) obtained by digesting a mixture of imperial black soup with Aspergillus oryzae, and the like are known.

In addition, in Korea, salty protein was discovered from conventional soy sauce, but this salty protein has a molecular weight ranging from 500 Da to 10,000 Da, and the sugars of mannose and N-acetyl-glucosamin are modified. As a resultant polymer, the specific sequence has not been identified, and the manufacturing process is cumbersome because it can be obtained only after the aging process has passed [Patent Documents 4 and 5].

Japanese Patent Application Laid-Open No. 63-3766 Japanese Patent Application Laid-Open No. 63-137658 Japanese Patent Application Laid-Open No. Hei 2-53456 Domestic Patent Registration No. 1126163 Domestic Patent Deduction No. 2013-0057403

In order to develop a new sweet peptide having a stable and high sweet taste, the present inventors the present invention by identifying a peptide that exhibits salty taste in some of the peptides designed from the sequence of an important site found through structural and protein engineering studies of a protein that exhibits sweet taste. Was completed.

Accordingly, an object of the present invention is to provide a newly designed salty peptide.

As a means for solving the above problems, the present invention provides a new salty peptide.

In addition, as another means for solving the above problems, the present invention provides a salt substitute and/or a salt food additive comprising the salty peptide.

The salty peptide according to the present invention is a short peptide consisting of 4 to 5 amino acids, and it was confirmed that the salty taste was exhibited even in a state where the sugar was not modified. Since it is possible to develop a new salt substitute that can replace salt by applying the salty peptide of the present invention, it is expected to be used as an excellent substitute that can prevent adult diseases, which is the biggest problem in modern diseases.

1 shows that the mechanism of carbohydrate sucrose (left) and that of the synthetic sweetener saccharin (right) are different.
Figure 2 shows the structure of TRPV1 (transient receptor potential cation channel subfamily V member 1).
3 shows a model of sodium ion transport in the solute-induced taste receptor cell and salty taste in the tongue in the front.
Figure 4 shows the three-dimensional structure of brazein.
Figure 5 shows the important site (29 to 43 residues) of brazein.
6 shows the chemical structural formula of aspartame.
7 shows peptide purification using gel chromatography.
Figure 8 predicts the binding pocket of brazein and T1R2.
9 is a prediction of a docking model of sweet receptor T1R2 and brazein.
Figure 10 predicts the docking model of sweet receptors T1R2 and BZ1.
Figure 11 predicts the docking model of sweet receptors T1R2 and BZ2.
12 predicts the docking model of sweet receptors T1R2 and BZ3.
13 predicts the docking model of sweet receptors T1R2 and BZ4.
14 predicts the docking model of sweet receptors T1R2 and BZ5.
15 predicts the docking model of sweet receptors T1R2 and BZ6.
16 predicts the docking model of sweet receptors T1R2 and BZ7.
17 predicts the docking model of sweet receptors T1R2 and BZ8.
18 predicts the docking model of sweet receptors T1R2 and BZ9.
19 predicts the docking model of sweet receptors T1R2 and BZ10.
Figure 20 shows the TRPV1 structure and ligand docking model
21 predicts the docking models of T2R1 and BZ10.
Figure 22 shows the CD spectra of the peptide [BZ1: solid line, BZ2: dotted line, BZ5: dashed line, BZ6: dot-dashed line].
23 shows hydrogen bonds between brazein and T1R2.
Figure 24 shows the interaction between the salty peptide (BZ3 ~ 5) and TRPV1 [a: BZ3 (DKHAR); b: BZ4 (KKRAR); c: BZ5 (DEKR)].

The present invention relates to a salty peptide.

The peptide consists of a sequence represented by SEQ ID NO: 3, 4 and/or 5.

SEQ ID NO: 3: DKHAR (from Brazzein)

SEQ ID NO: 4: KKRAR (artificial sequence)

SEQ ID NO: 5: DEKR (from Brazzein)

The peptide of the present invention is a short peptide composed of 4 to 5 amino acids, unlike conventional sugar-modified polymeric peptides (molecular weight 500 to 10000 Da). It can be reduced and has the advantage that it can be applied as a low-calorie food because there are few calories. In addition, in the present invention, it was confirmed that the salty taste was exhibited even in the state in which the sugar was not modified.

The present invention also provides a salt substitute and/or food additive comprising the salty peptide.

There is no particular limitation as long as foods that do not contain salt, foods containing salt, or foods that contain salt when eaten as a food subject to the salt taste substitute or food additive of the present invention. For example, soups such as soup, stew, soup, red pepper paste, cheonggukjang, miso, soy sauce, sauces, dressings, mayonnaise, seasonings such as tomato ketchup, clear soup such as Japanese clear soup, consomme soup, egg soup, Seaweed soup, shark fin soup, potage, soups such as miso soup, soups and sauces for noodles (buckwheat noodles, udon, ramen, pasta, etc.), porridge, porridge of vegetables and rice, and tea water Rice cooked foods such as rolled rice, processed livestock products such as ham, sausage and cheese, processed seafood products such as fish cake, dried fish, salted fish, and chinmi, processed vegetable products such as pickles, snacks such as potato chips, rice crackers, and cookies , Cooked foods such as heated food, fried food, baked food, and curry.

The salt flavor enhancer and/or food additive contains a basic substance and/or succinic acid, and if necessary, may contain various additives that can be used in food such as inorganic salts, acids, amino acids, nucleic acids, sugars, and excipients. .

Examples of the inorganic salt include table salt, potassium chloride, and ammonium chloride. Examples of the acid include carboxylic acids such as ascorbic acid, fumaric acid, malic acid, tartaric acid, citric acid and fatty acids, and salts thereof. Examples of the salt include sodium and potassium salts. Examples of the amino acids include sodium glutamate and glycine. Examples of the nucleic acid include sodium inosinate, sodium guanylate, and the like. Examples of the saccharides include sucrose, glucose, and lactose. Examples of the excipients include starch hydrolyzed dextrin and various starches. The amount of these can be appropriately set according to the purpose of use, for example, 0.1 to 500 parts by weight per 100 parts by weight of the peptide may be used.

Hereinafter, the present invention is described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

[ Example ]

I. Reference

One. Taste mechanism

Taste is recognized in the taste papillae of the tongue (Mombaerts, 2004). Taste nipples can be divided into three parts: circumvallate papillae on the back of the tongue, foliate papillae on the posterior lateral edge, and fungiform papilla on the tip of the tongue and on the side of the tongue. The three taste nipples are composed of taste buds, and each taste bud is composed of 50-150 special taste receptor cells (TRCs).

TRCs can be classified into 4 types from Type I to IV. Type I taste receptor cells express ecto-ATPase to prevent ATP from spreading to cells outside the group. Type II taste receptor cells are known as sensory receptor cells that express G protein coupled receptors (GPCRs), including taste receptors T1Rs and T2Rs, and are the only four types of taste receptor cells that are stimulated by ligands. Type III taste receptor cells form synapses, and Type IV taste receptor cells are located under the taste buds and serve as basal cells that can differentiate into other TRCs. One taste receptor cell is programmed to recognize only one taste.

GPCRs expressed in TRCs consist of seven alpha-helixed transmembrane domains (seven trans-membrane domains, 7TM domains), with the N-terminus outside the cell and the C-terminus in the cell. The transmembrane region and the N-terminus are bound to the ligand, and the G protein is bound to the third intracellular loop and the C-terminal region. Sweet, umami, and bitter tastes are recognized by the GPCR receptor (Kitagawa et al ., 2001). On the other hand, salty and sour tastes are detected through sodium/potassium ions and hydrogen ions through ion channels or ion exchangers located in the taste buds (Mombaerts, 2004). Sweet and umami tastes are recognized by T1Rs corresponding to class C GPCR receptors (Shi and Zhang, 2006), and bitter tastes are recognized by T2Rs belonging to class A GPCR receptors (Huang et al ., 2007).

2. Sweet receptor T1R2 / T1R3

The human sweet taste receptor works by forming a dimer of two subunits belonging to the class C GPCR. Receptors that recognize sweet taste are T1R2 and T1R3, which are expressed in Tasr2 and Tasr3 genes, respectively. T1R2/T1R3 consists of three domains: amino-terminal ectodomain (ATD), venus-flytrap binding domain (VFTM), cystein rich domain (CRD), and trans-membrane domain (TMD). VFTM of ATD (T1R2, 1-484; T1R3, 1-462) of T1R2 and T1R3 binds to the ligand, and CRD (T1R2, 491-544; T1R3, 495-547) maintains the structure by connecting ATD and TMD In addition, TMD (T1R2, 575-815; T1R3, 590-817) penetrates the membrane and plays a role of transmitting structural changes caused by binding of VFTM to the ligand to the G protein.

The structure of T1R2/T1R3 has not yet been revealed, and the function can be predicted from the glutamate receptor (mGluR) having the highest homology. mGluR acts as a heterodimer of T1R1/T1R3, and induces significant structural changes when it binds to glutamic acid at the N-terminus to form a complex (Lopez Cascales et al ., 2010). The m1-LBR, the ligand binding site of mGluR, is expected to have a form of free form I, which is a resting form in the absence of a ligand, and forms a complex of free form II, which is an active form when the ligand is bound (Temussi et al. , 2002). Similarly, the sweet receptor T1R2/T1R3 is also predicted to form an active complex by binding to a ligand.

In the case of the same day, a structural change occurs by forming a complex with T1R2/T1R3, and ATP is converted to cAMP by activating AC (adenylate cyclase) through the _{isolated Gα gust.} The converted cAMP directly induces depolarization by introducing cations through cyclic nucleotide-dependent ion channels, or by activating PKA (protein kinase A) and phosphorylating and closing the potassium channel of the basement membrane, thereby inducing the influx of calcium ions and causing depolarization. .

On the other hand, for sweetness caused by sweeteners other than sugar, Gq protein composed of Gqα and Gqβγ stimulates PLC β2 (phospholipase C β2) to hydrolyze PIP2 (phosphatidylinositol 4,5-bisphosphate) into DAG (diacylglycerol) and IP3. 2002), by releasing calcium ions from taste recognition cells due to the elevated IP3 concentration (Ohkuri et al ., 2009) (Fig. 1).

3. On TRPV1 by Is it salty?

Through many studies, it has been found that salty taste is recognized as sodium ions enter the sodium ion channel (ENaC) of taste receptor cells. According to the results of electrophysiological studies using rodents, it was found that the neurological response of CT (chorda tympani) to NaCl was significantly reduced in mice administered orally with amiloride, which is known to block ENaC. However, amiloride did not eliminate all CT responses to NaCl, and accordingly, another taste transfer process was expected (Smith et al , 2012).

TRPV1 is a non-selective cationic channel and is a nociceptor receptor activated by capsaicin, heat, and acid. It has been found that various types of TRPV1t exist in taste buds (Liu et al. , 2001). It was found that the CT reaction to sodium, potassium, and ammonium ions was increased by the agonist of TRPV1, and it was confirmed that the CT reaction to NaCl was eliminated in rodents that blocked both ENaC and TRPV1. Through this electrophysiological study, it was found that the TRPV1 channel is an important salty taste recognition pathway (FIG. 3). Maillard-reacted peptides (MRPs) are proteins that have been hydrolyzed and sugar-modified by heat and sugar. According to the results of Katsumata et al. , it was found that the CT neuronal response to NaCl in mice from which TRPV1 was removed by MRPs was detected, and that salty taste was induced by MRPs themselves (Katsumata et al., 2008). In this regard, in 2012, Dr. Mira Ryu's team at the Korea Food Research Institute discovered that KFRI-LHe, MRPs extracted from traditional soy sauce, exhibited salty taste through binding with TRPV1.

The higher order structure of TRPV1 was revealed by Liao et al ., and the higher order structure and activated structure at 3.4Å resolution were revealed using Cryo-EM (Liao et al., 2013), and docking was performed using the TRPV1 structure discovered through the present invention. Implemented. TRPV1 consists of four identical subunits, and four units are arranged around the ion channel. In one subunit, a pore loop exists between the transmembrane segment (S5-S6) and the S1-S4 translocation-receiving site on the side, and they are connected through an S4-S5 linker. In particular, it is known that the S4-S5 linker region interacts with the TRP region to participate in allosteric modulation. Regarding the intracellular assembly of TRPV1, the placement of subunits is promoted from the interaction of the N-terminal ankryin repeats, which is an intracytoplasmic region, and it is expected that more functional aspects of the TRP channel can be identified through these regions (Fig. 2).

4. Protein Brazzane

Brazzein is a West African P. It is a sweet protein extracted from the fruit of brazzeana, and shows about 500 to 2000 times sweeter taste when compared to sucrose. Brazein has a major type and a minor type, and most of the brazein extracted from plants exists as a major type. The major type has 54 amino acids including pyroglutamic acid residues at the N-terminus, whereas the minor type has 53 amino acids deficient in the N-terminal pyroglutamic acid, and has about twice the sweetness of the major type. Brazein is the smallest sweet protein, and is a monomer with a molecular weight of about 6.5 kDa. Brazein's tertiary structure revealed by NMR is one α-helix (residues 21-29) and three β-sheets (strand I, residues 5-7; strand II, residues 44-50, strand III, residues 34). -39) (FIG. 4), and 8 cysteine residues form 4 disulfide bonds, so stability against heat and pH is very high (Gao et al ., 1999).

In this study, to achieve the purpose of the study, 10 peptides were designed based on the loop portion of brazein, a sweet protein, and sensory tests were performed, and the results were analyzed through computer modeling.

II. Instruments and reagents

1. Instruments and reagents

For the taste test of the peptides, BZ1, BZ2, and BZ6 were synthesized by requesting AbClon (Seoul, Korea), and BZ3-BZ5 and BZ7-BZ10 by Peptron (Daejeon, Korea). The synthesized peptide was purified using a Sephadex G-10 column of GE Healthcare Life Sciences (Pittsburgh, PA, USA), and U- 2000 products were used.

2. Structure prediction and computer docking program

To visualize the virtual structure of the designed peptide, the Internet web server PEP-FOLD program operated by Diderot Universite (Paris, France) was used. In addition, the UCSF Chimera v1.7 program of the University of California and the Autodock Vina program of The Scripps Research Institute in the United States were used in conjunction with the UCSF Chimera v1.7 program to predict the binding structure of the ligand and the receptor.

Ⅲ. Experiment method

One. Peptide design

1.1 Peptide Design of BZ1, BZ2

Through the study of the variant, it was found that the β-sheet structure of the amino acid sequence 29-43 of brazein (Fig. 5) is an important part for the sweet taste of brazein (Jin et al ., 2003). Therefore, BZ1 was designed based on this sequence, and the sequence of the synthesized peptide is'DKHARSGECFYDEKR', which consists of a total of 15 amino acids.

Studies of various brazzein variants have either increased or decreased sweetness. Brazein was mutated from Arg residue 29 to Lys residue (Jin et al ., 2003) and His residue 31 to Arg residue to increase sweetness, respectively (Lee JW et al. , 2013), and Glu residue at 36 (Jin et al., 2003). Even when the Asp residue was mutated (Do HD et al. , 2011) and the 41st Glu residue was mutated to the Ala residue, the sweet taste was increased compared to the wild type, respectively (Lee JW et al. , 2013). Based on the results of this variant study, residues 29, 31, 36, and 41 of BZ1 were designed by modifying them as in the following sequence'KKRARSGDCFYDAKR' (Table 1).

Name Sub name Sequence BZ1 BZ 29-43 wild DKHARSGECFYDEKR (SEQ ID NO: 1) BZ2 BZ 29-43 mutant KKRARSGDCFYDAKR (SEQ ID NO: 2)

1.2 Peptide The design of the BZ3-BZ6

Aspartame is a methyl ester of dipeptide composed of Asp residues and Phe residues (Fig. 6), and is used as an artificial sweetener in beverages and foods (Ager et al. , 1998). Just as aspartame composed of only two amino acids exhibits sweet taste, a short fragment of brazein was expected to exhibit sweet taste, so BZ1 and BZ2 were subdivided to design BZ3-BZ6. There are three loops in Brazzein. Loop I is in the 13-15 region, and loop II is in the 30-33 sequence. Finally, Loop III is a sequence of 40-43, and Loop II and Loop III are present in the sequence of 29-43 selected as the sequence of BZ1 (FIG. 5). This loop of sweet protein has the potential to become a'sweet finger' model that is expected to represent the sweet taste of sweet protein (Temussi et al. , 2002). Therefore,'DKHAR' of area 29-33 corresponding to Loop II of BZ1 was selected as BZ3. BZ4 was designed so that the first residue of BZ3, Asp, was changed to Lys like BZ2 so that the peptide was positive. As reported in previous studies, since Glu41 of Loop III is close to Glu252 of T1R2 and Arg43 is an important residue (Yoon et al. , 2011),'DEKR', which is the region 40-43 corresponding to Loop III, is converted to BZ5. It was selected, and BZ6 was also made to neutralize the negative residue by changing the Glu residue to Ala like BZ2 (Table 2).

Name Sub name Sequence BZ3 BZ 29-33 wild DKHAR (SEQ ID NO: 3) BZ4 BZ 29-33 mutant KKRAR (SEQ ID NO: 4) BZ5 BZ 40-43 wild DEKR (SEQ ID NO: 5) BZ6 BZ 40-43 mutant DAKR (SEQ ID NO: 6)

1.3 Peptide The design of the BZ7-BZ10

In BZ3 designed based on Loop II,'KHAR', four sequences excluding Asp, which is a negative residue, was selected as BZ7. It was designed to study whether appears.

On the other hand, in BZ5 corresponding to Loop III, two residues were added to each side and compared with the taste of BZ5 to confirm the role of the surrounding residues'FY' and'VL'. Was selected.

By maintaining the loop structure of BZ8, we designed BZ9 and BZ10 so that it can bind to the sweet taste receptor correctly. BZ9 is designed to maintain the loop structure by inserting Pro residues in the BZ8 sequence, and the sequence is the same as'FYDPEKRVL'. In BZ10, a loop structure was formed through disulfide bonds by adding Cys residues at both ends of the sequence of BZ9, and the sequence is shown in “CFYDEKRVLC” (Table 3).

Name Sub name Sequence BZ7 BZ 30-33 wild KHAR [SEQ ID NO: 7] BZ8 BZ 38-45 wild FYDEKRVL [SEQ ID NO: 8] BZ9 BZ 38-45 mutant FYDPEKRVL [SEQ ID NO: 9] BZ10 BZ 38-45 mutant2 CFYDEKRVLC [SEQ ID NO: 10]

2. Peptide Synthesis and purification

BZ1, BZ2, and BZ6 were purified through high performance liquid chromatography (HPLC) with a purity of 80% or more by requesting AbClon to synthesize 5 mg each. BZ3~BZ5, BZ7~BZ10 were synthesized by 10 mg each by HPLC purification with a purity of 95% or more by requesting Peptron.

Peptides were purified using Sephadex G-10 (GE Healthcare) to remove TFA and impurities, which may be added during the synthesis and HPLC purification, and may cause a sour taste. Before purification, the sample was dissolved in 3 mg/ml using tertiary distilled water. The Sephadex G-10 column was washed until there was no change in absorbance at a wavelength of 205 nm by flowing tertiary distilled water at least 10 times the bed volume. After confirming that there was no change in absorbance, 1 ml of a sample previously dissolved in tertiary distilled water was injected into the column. When the surface of the bed is visible, the target peptide is eluted with third distilled water at a flow rate of 0.5 ml/min through free fall. Finally, the purified peptide solution was lyophilized using a freeze dryer.

3. Peptide Taste test

Taste tests were conducted on a total of 14 trained panels. Panels selected healthy men and women who do not smoke. It was carried out before meals to judge the exact taste, and the mouth was washed with 3rd distilled water before the test. Since the safety of the synthesized peptide is not known, after tasting it, spit it out immediately and wash the mouth with distilled water. The taste result was made by a blind test, but it was freely expressed in order to remove psychological factors.

4. Peptide Structure prediction

The structure of the peptide synthesized through chemical synthesis will differ greatly from that of the corresponding sequences in brazein and taumatin. Therefore, the structure of the synthesized peptide having the most stable energy was predicted and compared with the structure in actual brazein and taumatin.

PEP-FOLD ver. provided by Paris Diderot University, France. The structure of the designed peptide was predicted through 1.5.3 (http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD/) (Thevenet et al ., 2012). The pdb file created through the program is UCSF Chimera ver. 1.9 Implemented through the program (Pettersen et al ., 2004), and downloaded through the web page (https://www.cgl.ucsf.edu/chimera/download.htmlPEP-FOLD). The prediction program provided by PEP-FOLD can predict only 9 to 36 amino acid sequences. Therefore, in the case of BZ3-BZ8 consisting of 9 or less amino acids, Ala residues were added to the C-terminus to make 9 Ala residues, and then Ala residues added through the PEP-FOLD and USCF Chimera programs were removed. In addition, in the case of BZ10, a peptide containing a disulfide bond, a disulfide bond was introduced by selecting a disulfide bond option.

5. Peptide and Taste Predicting receptor binding

The Autodock vina program was used to predict the binding site and energy between peptides and taste receptors (Trott et al ., 2010). This course was operated on a computer with Intel(R) Core(TM) i3-3220 3.30 GHz CPU, 4 GB of memory, and Windows 7 specification. The structures of T1R2 and T2R1 receptors whose structures have not yet been identified were predicted through the SWISS-MODEL web server provided by the Center for Molecular Biology at Basel University in Switzerland (Biasini et al ., 2014), and the web page (http://swissmodel. It can be operated at expasy.org/). The structure of T1R2 was predicted based on the human glutamate receptor (PDB ID: 2E4U) corresponding to the GPCR familly, and T2R1 was predicted based on the human delta opioid 7tm receptor (PDB ID: 4N6H). All pdb protein structure files were extracted as pdbqt files for Autodock vina docking using the AutoDockTools (ADT) program (Morris et al ., 2009). The preparation process using the ADT program includes removing water molecules, adding hydrogen atoms, setting up a grid box for selecting a space for predicting receptor binding, selecting a ligand torsion, and assigning a charge through AMBER FF99 and Gasteiger-Marsili calculation method. Table 4 shows the range (Grid box) selected for docking of receptors and peptides. The ADT and Autodock vina programs were downloaded from the website (http://autodock.scripps.edu/downloads).

T1R2 T1R3 T2R1 TRPV1 center_x 24.73 26.03 -6.02 0.075 center_y -5.771 12.94 -73.57 0.000 center_z 53.92 51.58 85.02 7.475 size_x 59.77 63.12 34.50 120.0 size_y 64.78 68.50 47.78 126.0 size_z 74.49 65.61 67.12 100.0

IV. result

One. Peptide Synthesis and purification

The 10 synthesized peptides designed from brazein exhibited a high level of purity of more than 90% of HPLC purification, but more than 95% of salty peptides (Table 5).

In addition, using a Sephadex-G10 column was used to remove impurities and TFA, which was used for synthesis and HPLC purification, which interferes with the taste test. The purified peptide was quantified by measuring the absorbance at a wavelength of 205 nm (FIG. 7), and the fractions E1 to E5 from which the peptide was eluted were collected and lyophilized.

Peptide Purity( % ) Peptide Purity( % ) BZ1 80.04 BZ2 96.84 BZ3 97.32 BZ4 99.87 BZ5 96.79 BZ6 86.11 BZ7 97.39 BZ8 99.89 BZ9 98.59 BZ10 96.86

2. Peptide Taste test

Peptides synthesized from BZ1 to BZ10 were tested for taste by a panel of 14 people. All panels were allowed to freely express the taste felt on the subjective answer sheet. As a result, it did not show any taste except BZ3, BZ4, BZ5, which showed salty taste, and BZ10, which had a bitter taste. In particular, BZ4 had a strong salty taste and had a long tail, and a disgusting bitter taste was felt in BZ10 (Table 6).

Name Sub Name Taste Intensity BZ1 BZ 29-43 wild none - BZ2 BZ 29-43 mutant none - BZ3 BZ 29-33 wild salty ++ BZ4 BZ 29-33 mutant salty +++++ BZ5 BZ 40-43 wild salty ++ BZ6 BZ 40-43 mutant none - BZ7 BZ 30-33 wild none - BZ8 BZ 38-45 wild none - BZ9 BZ 38-45 mutant none - BZ10 BZ 38-45 mutant2 bitter +++++

3. Peptide Structure prediction

Designed through PEP-FOLD Web Server to confirm the structural difference by comparing the synthesized 10 peptides with the structure on the actual protein, and to confirm the binding model with the taste recognition receptor using the predicted peptide structure file. The structures of 10 peptides were predicted.

As a result of comparing the structure prediction result of the peptide and the structure of the brazein phase, it can be seen that BZ1 retains its original structure well, while BZ2 exists in a somewhat curved form. BZ3 and BZ4 very well maintain the β-turn structure of the sequence 29-33 of Brazein, and in the case of BZ5 and BZ6, it was found that BZ6 better maintains the bent shape of the sequence 40-43 of Brazein. Can (Table 7).

In the case of BZ7, BZ8 and BZ9, the structure is very well maintained for sequences 30-33 and 38-45 corresponding to each brazein sequence (Table 8).

4. Peptide and Taste Predicting receptor binding

4.1 Peptide and Sweet taste receptor With T1R2 Combination

It has been found that the VFT module of T1R2 is important for the acceptance of sweet substances through studies on the site-specific mutation method of the sweet receptor and the study of chimera formation (Assadi-Porte et al ., 2010). In addition, it was predicted that brazein would bind to T1R2 rather than T1R3 through homology studies with mGlu receptors (Walters et al ., 2006). Therefore, in this study, the binding site and binding energy were predicted by combining 10 designed peptides and T1R2 through computer modeling. In addition, before computer modeling, all binding pockets that may exist in T1R2 were predicted through CASTp Sever (Dundas et al. , 2006). In order to focus on the interaction between brazein and T1R2, 21 bonding pockets were selected from 96 expected bonding pockets, excluding bonding sites shorter than 35 Å, which is the diameter of brazein (Table 9).

In order to compare the binding site of brazein showing sweet taste and the binding site of peptide, the binding site of T1R2 and brazein was predicted. As a result, it was shown that Brazein was accurately combined in the center of the cleft part located in the VFT module (FIG. 8) corresponding to POC ID (pocket identification) 90 (black) and POC ID 92 (gray) (FIG. 9 A). From the cross section of the binding model _{, it can be seen that the N-terminus of brazein and the L 40-43} region bind to T1R2 (FIG. 9B). The binding energy between brazein and T1R2 is -14.0 kcal/mol. The binding prediction regions of BZ1 and BZ2 with T1R2 are shown in FIGS. 10 and 11, respectively. As a result, it can be seen that both BZ1 and BZ2 are bound to POC ID 90, which _{is a site to which L 40-43} of brazein is bound among POC ID 90 and 92, which are the binding sites of brazein (FIGS. 10 and 11). The bonding force at this time is -12.4 kcal/mol and -11.4 kcal/mol, respectively.

T1R2 potential combination pocket list POC ID ^a Length(Å) ^b The functionally relevant residues 73 36 M194, V195, M198, I206, V208, L223, V227, I232, C233, I234, V273, I299 74 40 M194, L197, M198, I299, A300, L323, I325, I450 77 35 L350, T353, S354, C359, N360, Q361, D364, N365, C366, L367 78 46 Y389, S390, Y393, A394, H397, K426, N428, I436, F437, F438 79 43 V208, V210, R217, G220, Q221, L223, G224, I234, Q237, E238, T239 80 54 D26, F27, Y28, Q55, E97, I98, V99, P110, Y113, F114 81 43 L54, Q55, M58, D100, V101, C102, I104, N106, N107, Q109, P110 82 58 V136, A137, P160, V395, A398, L399, Q419, L402, V415, Y416, P417, Q419, L420, E422, E423 83 54 N292, F293, T294, E315, L316, R317, H318, G320, T321, W453, W455, W483, H484, T485, I486 84 55 R202, N204, W205, D231, M494, C495, S496, Y506, V508, V512, C513, F515, Y533 85 64 D26, F27, L41, H42, F53, Q55, V56, I98, D100, P347, P348, S351 86 98 L158, P160, Q161, I162, K174, P178, A179, L180, L181, P417, W418, L420, L421, I424, W425, F438, D439, P440, Q441, G442 87 106 T183, T184, P185, I327, Q328, S329, Y386, V330, Y386, S387, S390, L432, D433, H434, Q435, I436, F437, F438, D439, D443, V444, A445, H447, Y469 88 108 D32, Y33, L34, L89, R134, V135, V136, L399, L402, L403, D406, K407, K412, R413, V414, V415, Y416, Q419 89 130 S40, E63, V66, I67, Y103, D142, N143, S144, S165, A166, I167, T184, Y215, P277, D278, L279, E302, S303, A305, I306, D307, T326, I327, E 382, R383 , V384 90 109 K65, I306, D307, P308, H311, N312, E315, I376, L377, R378, L379, S380, G381, E382, R383, W453, R457, S458, Q459, N460, F462 91 178 V64, K65, Y69, N70, Q73, S336, F338, R339, E340, W341, G342, P343, P349, R352, T353, S356, Y357, C359, N365, A369, T370, L371, S372, F373, N374, T375, I376, L377, R378, L379, S380 92 195 A43, N44, M45, K46, G47, K60, E61, Y62, E63, V64, K65, V66, Y103, I104, S105, N106, E145, S211, S212, D213, L240, P241, T242, L243, Q244, P245, N246, Q255, L257, P277, D278, L279, T280, Y282, H283, Q355, S356, T358

a. POC ID: pocket identification; b. All the selected ones among total 92 potential binding pockets are longer than 35Å which is the length of brazzein.

The binding prediction sites of BZ3 and T1R2 of BZ4 designed based on regions 29-33 of brazein are shown in FIGS. 12 and 13, respectively. Synthesized and purified BZ3 showed a weak salty taste, and BZ4 showed a strong salty taste. Both BZ3 and BZ4 were bound to the left part of the cleft and correspond to POC ID 92. The binding energy at this time is -7.6 kcal/mol and -5.4 kcal/mol, respectively.

BZ5 and BZ6 were designed based on the 40-43 sequence of Brazein, BZ5 had a weak saltiness and BZ6 had no taste. The binding prediction regions of T1R2, BZ5, and BZ6 are shown in FIGS. 14 and 15, respectively. It can be seen that BZ5 is coupled to POC ID 85 and 92, which is the upper left of the cleft, and BZ6 is coupled to POC ID 90, which is the right of the cleft. The binding energy at this time is -8.3 kcal/mol and -7.6 kcal/mol, respectively.

The predicted binding site of BZ7 to T1R2 designed based on the sequences 30-33 of brazein is shown in FIG. 16. As a result of predicting the binding site, it was bonded to POC ID 92, which is the left part of the cleft, with a binding energy of -6.9 kcal/mol.

BZ8, BZ9, and BZ10 were designed based on the sequence number 38-45 of Brazein, and none of them showed sweet taste. In the case of BZ8 and BZ9, the taste was irritating and the intensity was very weak, but the case of BZ10 showed a very strong bitter taste. The predicted regions of binding to T1R2 are shown in Figs. 17, 18, and 19, respectively. As a result of the binding prediction, BZ8 and BZ10 were bound to POC ID 92 corresponding to the right side of the cleft, and BZ9 bound to the left side (POC ID 81) off the cleft. The bonding force at this time is -7.2, -6.9, -7.1 kcal/mol in order from BZ8 to BZ10.

4.2 Peptide and Pain Receiving Channel With TRPV1 Combination

There are two pathways for the recognition of salty taste through stimulation of ENaC, a sodium ion channel, and TRPV1, a nociceptive receptor. Since the peptide does not cause depolarization through ENaC, it has the potential to transmit and increase the salty taste by binding to TRPV1, which is a nociceptive channel. The TRPV1 used in this binding prediction program is the TRPV1 of Rattus norvegicus whose structure was revealed by the electron cryo-microscopy method (Liao et al ., 2013), and the PDI ID is 3J5R (Fig. 20A). Docking was performed simultaneously for all chains of TRPV1 composed of homo-tetramer.

Among the 10 synthesized and purified peptides, the binding of BZ3, BZ4, BZ5 and TRPV1 showing salty taste was predicted (FIG. 20). As a result, it was bonded to the pore part between the S1-S4 helix structure at the membrane penetrating part of Chain B, and the binding energies were -7.2 kcal/mol, -8.0 kcal/mol, and -6.5 kcal/mol, respectively.

This result is a result of Lee et al . (Lee et al., 2011) showing the result of binding of capsaicin, a substance that transmits pain sensation by binding to TRPV1 (transient receptor potential cation channel subfamily V member 1), to the pores of the S3-S6 helix structure (Lee et al., 2011). It shows a difference with, but in fact, it can be said that it is a similar result when it is combined with S3 and S4 helix. When looking at the docking result of the synthesized peptide, it was bonded between the S1-S4 helix structures similar to capsaicin. However, unlike the results of capsaicin, it can be seen that all of the peptides interact additionally with S5 helix. This is expected to be a result of the structural difference between the peptide and capsaicin, and is believed to interact with the S5 helix to maintain a stable bond. Peptides appear to interact with the S1-S4 helix structure, S4-S5 linker and S5 helix, and appear to have a wide binding site unlike capsaicin, and it is estimated that a difference in taste appears due to this difference (FIG. 20).

V. Discussion

By analyzing the bonding site, it was possible to confirm the reason why the sweet taste did not appear and the reason that the salty taste and the bitter taste were expressed.

Ligand Receptor Binding energy
△ Gb (kcal/ mol ) Taste Binding Site
(POC ID) Brazzein T1R2 -14.0 sweet center of cleft
(90, 92) Aspartame T1R2 -5.6 sweet center of cleft
(90, 92) BZ1 T1R2 -12.4 none right side of cleft (90) BZ2 T1R2 -11.4 none right side of cleft (90) BZ3 T1R2 -7.6 salty left side of cleft (92) BZ4 T1R2 -5.4 salty left side of cleft (92) BZ5 T1R2 -5.6 salty left side of cleft (85, 92) BZ6 T1R2 -8.3 none right side of cleft (90) BZ7 T1R2 -7.6 none left side of cleft (92) BZ8 T1R2 -7.2 none right side of cleft (90) BZ9 T1R2 -6.9 none left side of VFT module (81) BZ10 T1R2 -7.1 bitter right side of cleft (90) BZ3 TRPV1 -8.4 salty TM1-4 pore BZ4 TRPV1 -8.3 salty TM1-4 pore BZ5 TRPV1 -8.1 salty TM1-4 pore BZ10 T2R1 -7.6 bitter N- terminal

The first reason why the synthesized peptide does not taste sweet is that the structure in brazein was not completely maintained in the synthesized peptide. The peptide predicted through PEP-FOLD structure prediction seems to be relatively similar to the structure in Brazein, and the CD (circular dichroism) spectra result of the actually synthesized peptide can also explain the effectiveness of the structure in practice. there was. For the CD spectra measurement process to confirm the validity of the predicted structure, a J-815 CD spectrascometer of Jasco, owned by Inha University's Institute of Technology, was used, and the CD spectra of BZ1, BZ2, BZ6, and BZ7 were measured among 10 peptides (Fig. 22).

The CDSSTR algorithm was used to analyze the secondary structure from the CD results of BZ1, 2, 6, and 7 (Johnson, 1999). As a result of analyzing the CD results based on the SMP56 reference set, which has 56 proteins as a reference, it was found that BZ1 and BZ2 had a high ratio of β-sheet structure and that of BZ3 and BZ4 had a high turn ratio (Sreerama et al ., 2004). In addition, from the fact that the ratio of the β-sheet structure of BZ1 is higher than that of BZ2, and that the turn structure of BZ4 is higher than that of BZ3, it can be seen that the predicted results well predict the actual peptide structure (Table 11). . However, although the CD spectra analysis result and the PEP-FOLD structure prediction result were shown to be correlated, the structure of the peptide obtained as a result of PEP-FOLD structure prediction was not completely identical to the structure in brazein. The number of cases in which the peptide structure in which the bond rotation occurs is equal to infinite. Therefore, even if a peptide showing the sweet taste of brazein is synthesized, the probability of having the original structure in brazein is very low, and the sweet taste may not be exhibited.

The second expected reason for the result of not showing sweet taste in the designed peptide is that the multi-binding site to which brazein must bind was not satisfied. T1R2/T1R3, a GPCR C-family receptor that recognizes sweet taste, binds to artificial sweeteners including sugar and transmits a sweet taste signal to the brain through the G protein. This suggests that the binding site and energy of the receptor and the ligand are important factors in sweet taste transmission. Among T1R2 and T1R3, the binding of T1R2 and brazein, which is known to bind to the sweet protein, was combined through computer modeling, and as a result, it was confirmed that it was bound to the entire cleft portion of T1R2 as shown in FIG.9, and the POC IDs at that time were 90 and 92. In addition, it was confirmed that it binds with high binding energy of -14 kcal/mol. In addition, by analyzing hydrogen bonds between brazein and T1R2, residues involved in bonding and hydrogen bonding were identified in order to show sweetness (FIG. 23) (Table 12).

As can be seen in Table 12, brazein has 11 hydrogen bonds and is strongly bonded, and it was confirmed that a loop corresponding to the N-terminus and residues 36-54 is involved in the bonding with T1R2. In addition, it can be seen that the POC ID 90 and 92 portions of T1R2 corresponding to residues 46, 60, 255, 312-314, and 458 are bonded to brazein through strong hydrogen bonds. In comparison, BZ1 and BZ2 bind to POC ID 90, which is bound by Brazein's loop, but not to POC ID 92, which is bound to the N-terminus. On the other hand, BZ3, BZ4, and BZ5, which showed salty taste, bound to the site to which the POC ID 92 of Brazein binds, but not to the site to which the loop binds. Likewise, BZ6, BZ8, and BZ10 bound to the site where the loop binds, but not the N-terminal linkage site. In addition, BZ7 and BZ9 showed the result of binding to the N-terminal binding site, and none of the designed 10 peptides satisfies both the N-terminal binding site and the loop binding site. The conclusion that can be drawn from this result is that brazein binds to T1R2 as multiple binding sites, and if even one of them is not satisfied, it cannot exhibit a sweet taste.

The presence of a common amino acid of TRPV1 chain B involved in the interaction with the peptide could be predicted through the docking result (FIG. 24). In the case of Glu513, Val567, Gln700, and Ile703, all three peptides were found to be amino acids involved in the interaction. In particular, Val567 is an amino acid contained in S5 helix, and it is believed that interaction with S5 helix occurs in common, suggesting the possibility of showing salty taste when it is combined with Val567 of S5 helix and its surrounding amino acids. The inside of the S1-S4 helix pore structure where the synthesized peptide is located is composed of polar amino acids. This is expected to contribute greatly to the binding stability of the peptide containing a large amount of polar amino acids, and the docking result showed that it binds to the S1-S4 helix pores with the lowest energy. In this regard, it can be estimated that the reason why BZ4 showed the most salty taste is because it has the most stable bonding with the lowest energy.

Since the discovery of aspartame, numerous short peptides have been researched and developed. Peptides developed by the Tamura group are dipeptide and tripeptide, which have umami taste and are thought to be the main cause of the umami taste of broth (Tamura et al. , 1989). Note that the developed dipeptides and tripeptides were accompanied by salty as well as umami taste, and these were mainly composed of negatively charged aspartic acid and glutamic acid. The salty KKRAR developed in this study mainly contains positively charged lysine and arginine, so it has an opposite charge from the previously developed negatively charged peptides. It can be inferred that it is irrelevant.

The saltiness of BZ3, BZ4, and BZ5 and the docking result with the nociceptor TRPV1 suggest two important points for us. The first implication is that short peptides can exhibit salty taste. In 2012, Dr. Mira Ryu's team at the Korea Food Research Institute found that the molecular weight of salty protein from conventional soy sauce ranged from 500 Da to 10,000 Da, as well as a sugar-modified polymer of mannose and N-acetyl-glucosamine. It can be obtained by extraction (Rhyu et al ., 2012). On the other hand, it was confirmed through this experiment that the three peptides of BZ3 to 5 are short peptides consisting of 4 to 5 amino acids, and that they exhibit salty taste even when sugar is not modified. In other words, it means that it is possible to develop a salty peptide food additive that can replace salt. The second implication is that the types of signals transmitted can vary depending on the site where the ligand binds to the nociceptor TRPV1. Capsaicin, which delivers spicy pain, was located in the middle of the S1-S4 helix pores while binding to S3 and S4 of TRPV1. BZ3~5 also binds to S3 and S4, but binds to the base of the pores of S1-S4 helix and the additional interaction with S5 helix is the difference from the binding pattern of capsaicin. In other words, it can be expected from the docking result that when combined with the side of S3 and S4 belonging to the voltage sensor domain, the spicy taste is transferred, and when the base is combined with the S5 helix, salty taste is transmitted.

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<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Salty peptide <130> P16U21C1818 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 15 <212> PRT <213> pentadiplandra brazzeana <400> 1 Asp Lys His Ala Arg Ser Gly Glu Cys Phe Tyr Asp Glu Lys Arg 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> BZ 29-43 mutant <400> 2 Lys Lys Arg Ala Arg Ser Gly Asp Cys Phe Tyr Asp Ala Lys Arg 1 5 10 15 <210> 3 <211> 5 <212> PRT <213> pentadiplandra brazzeana <400> 3 Asp Lys His Ala Arg 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> BZ 29-33 mutant <400> 4 Lys Lys Arg Ala Arg 1 5 <210> 5 <211> 4 <212> PRT <213> pentadiplandra brazzeana <400> 5 Asp Glu Lys Arg One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> BZ 40-43 mutant <400> 6 Asp Ala Lys Arg One <210> 7 <211> 4 <212> PRT <213> pentadiplandra brazzeana <400> 7 Lys His Ala Arg One <210> 8 <211> 8 <212> PRT <213> pentadiplandra brazzeana <400> 8 Phe Tyr Asp Glu Lys Arg Val Leu 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> BZ 38-45 mutant <400> 9 Phe Tyr Asp Pro Glu Lys Arg Val Leu 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> BZ 38-45 mutant2 <400> 10 Cys Phe Tyr Asp Glu Lys Arg Val Leu Cys 1 5 10

Claims (1)

A salty substitute comprising a salty peptide consisting of the amino acid sequence of SEQ ID NO: 5.

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