KR101682803B1 - 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

The present invention relates to salty peptides.

Salt (sodium chloride) plays an important role in seasoning and processing of food, such as imparting taste to food, improving preservation of food, and improving physical properties of food. Sodium salt and chlorine are essential nutrients of the human body.

However, excessive intake of sodium, a component of the salt, is considered to be a risk factor for many health problems, such as hypertension and heart disease and vascular diseases. In Japan and elsewhere in the developed world, there is a strong demand for a reduction in the intake of sodium chloride, especially sodium, as the number of elderly people susceptible to these diseases increases.

In order to reduce salt intake, it is the simplest way to reduce the amount of salt used in the seasoning and processing of food. However, reducing the amount of salt contained in food, whether home cooked or processed food, by more than 10% will generally damage the taste of the food.

A method of reducing the intake of sodium chloride, particularly sodium, without damaging the saltiness, that is, the method of infection in general, includes a method of using a substance which is itself a salt-like substance (hereinafter referred to as salt substitute substance) , And a method of using a substance that enhances the saltiness of the salt itself (hereinafter, referred to as a salt intolerance substance) when it coexists with salt, although the salt itself is not salty.

Examples of salt substitute materials include potassium salts, ammonium salts, basic amino acids, peptides composed of basic amino acids, and alkali metal salts of gluconic acid. It is not a substitute for salt, but it enhances the saltiness of the salt, thereby reducing the consumption of salt and enabling the reduction of salt.

Examples of non-saline intensifying substances include peptides (Patent Document 1) obtained by hydrolyzing collagen having a molecular weight of 50,000 daltons or less, thaumatin (sweetener protein) (Patent Document 2), aspergillus niger), and a decomposition solution obtained by digesting a mixture of Aspergillus oryzae emulsions (Patent Document 3).

In addition, in Korea, salty protein has been found from conventional soy sauce. However, the salty protein has a molecular weight ranging from 500 Da to 10,000 Da, and mannose and N-acetyl-glucosamine The specific sequence is not disclosed and it can be extracted after aging process, which complicates the manufacturing process [Patent Literature 4, 5].

Japanese Laid-Open Patent Publication No. 63-3766 Japanese Patent Application Laid-Open No. 63-137658 Japanese Unexamined Patent Application Publication No. 2-53456 Korean Patent No. 1126163 Korea Patent No. 2013-0057403

The inventors of the present invention have developed a new sweet peptide having a stable and high sweet taste by identifying a peptide exhibiting a salty taste in a part of peptides designed from a sequence of important sites revealed through structural and protein engineering studies of a sweet taste protein, .

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

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

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

The salty taste peptide according to the present invention is a short peptide consisting of 4 to 5 amino acids and has a salty taste even in the absence of the saccharification formula. The salty taste peptide of the present invention can be developed as a new salt substitute substitute for salt, and thus it is expected to be used as an excellent substitute for preventing adult diseases which is the biggest problem in modern diseases.

1 shows the mechanism (left) of carbohydrate sucrose and the mechanism (right) of synthetic sweetener saccharine different.
FIG. 2 shows the structure of TRPV1 (transient receptor potential cation channel subfamily V member 1).
Figure 3 shows sodium ion transport in the somatosensory taste receptor cells and an anterior saliva delivery model in the tongue.
Fig. 4 shows a three-dimensional structure of Brazazine.
Fig. 5 shows the important sites (residues 29 to 43) of bradylane.
Figure 6 shows the chemical structure of aspartame.
Figure 7 shows peptide purification using gel chromatography.
Figure 8 predicts the binding pocket of brassane and T1R2.
Figure 9 predicts the docking model of brassine receptors T1R2 and Brazeen.
10 predicts a docking model of sweet taste receptors T1R2 and BZ1.
Figure 11 predicts a docking model of sweet taste receptors T1R2 and BZ2.
12 predicts a docking model of sweet taste receptors T1R2 and BZ3.
13 predicts a docking model of the sweet taste receptors T1R2 and BZ4.
14 predicts a docking model of the sweet taste receptors T1R2 and BZ5.
15 predicts a docking model of the sweet taste receptors T1R2 and BZ6.
16 predicts a docking model of sweet taste receptors T1R2 and BZ7.
Figure 17 predicts a docking model of sweet taste receptors T1R2 and BZ8.
18 predicts a docking model of sweet taste receptors T1R2 and BZ9.
19 predicts a docking model of the sweet taste receptors T1R2 and BZ10.
20 shows a TRPV1 structure and a ligand docking model
Fig. 21 is a prediction of a docking model 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 the hydrogen bonding between the brassane and T1R2.
Figure 24 shows the interaction between salty peptides (BZ3-5) and TRPV1 [a: BZ3 (DKHAR); b: BZ4 (KKRAR); c: BZ5 (DEKR)].

The present invention relates to salty peptides.

The peptide comprises the sequence shown in SEQ ID NO: 3, 4 and / or 5.

SEQ ID NO: 3: DKHAR (derived from blazein)

SEQ ID NO: 4: KKRAR (artificial sequence)

SEQ ID NO: 5: DEKR (from Brazyn)

The peptide of the present invention is a short peptide consisting of 4 to 5 amino acids, unlike the conventional sugar-modified polymeric peptide (molecular weight 500 to 10,000 Da). Since it is easy to prepare and does not directly take the salt, And it has the advantage of being applicable to low calorie food because there is almost no calorie. Further, it was confirmed that the salty taste was exhibited in the present invention even in the state where the sugar was not added.

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

There is no particular limitation on the food to be subjected to the untreated salt substitute or food additive of the present invention, as long as it is a food containing no salt or a salt containing food. Foods such as soup such as soup, soup, soup, kochujang, soy sauce, soy sauce, dressing, mayonnaise, tomato ketchup, seasoning such as Japanese clear soup, Porridge and soup of soup and sauce, porridge, vegetables and rice for soup stock, shark fin soup, potage, miso soup, noodles (buckwheat noodles, udon noodles, Ehma is a food such as rice cooked food such as rice, ham, sausage, and cheese, processed fish products such as fish paste, salted fish, dried fish, salted fish and chinmi, vegetable processed products such as pickles, potato chips, , Cooked foods such as heated foods, fried foods, baked foods, and curries.

The salt intensifier and / or the food additive may contain a basic substance and / or succinic acid, and if necessary, various additives which can be used for foods such as inorganic salts, acids, amino acids, nucleic acids, sugars and excipients .

Examples of the inorganic salt include sodium chloride, 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 acid, and salts thereof. The salts include sodium and potassium salts. Examples of the amino acid include sodium glutamate, glycine and the like. Examples of the nucleic acid include sodium inosinate and sodium guanylate. Examples of the saccharides include sucrose, glucose and lactose. Examples of the excipient include starch hydrolyzate dextrin and various starches. The amount of these to be used 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.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

[ Example ]

I. Reference

1. Taste recognition mechanism

Taste is recognized in the taste papillae of the tongue (Mombaerts, 2004). The taste papilla can be divided into three parts: a circumvallate papillae on the back of the tongue, a foliate papillae on the posterior lateral margin, and a fungiform papilla on the sides of the tongue and tongue. The three flavor pods are composed of taste buds, and each taste bud consists of 50-150 taste receptor cells (TRCs).

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

The GPCRs expressed in TRCs consist of seven trans-membrane domains (7TM domains) of seven alpha-helical structures, with the N-terminal located outside the cell and the C-terminal located within the cell. The transmembrane region and the N-terminus bind to the ligand, while the third intracellular loop and the C-terminus bind to the G protein. Sweetness, umami taste, bitter taste are recognized by GPCR receptors (Kitagawa et < RTI ID = 0.0 > 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). Sweetness and umami taste are recognized by T1Rs, which correspond 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

Human sweet taste receptors are formed by two subunits belonging to class C GPCRs. Sweet receptors are expressed in Tasr2 and Tasr3 genes as T1R2 and T1R3, respectively. T1R2 / T1R3 consists of three domains: ATD (amino-terminal ectodomain; VFTM), cystein rich domain (CRD), and trans-membrane domain (TMD). The VFTM of ATR (T1R2, 1-484; T1R3, 1-462) of T1R2 and T1R3 binds to the ligand and the structure of CRD (T1R2, 491-544; T1R3, 495-547) And TMD (T1R2, 575-815; T1R3, 590-817) penetrates the membrane and serves to transfer structural changes to the G protein resulting from binding of the VFTM to the ligand.

The structure of T1R2 / T1R3 has not yet been elucidated and its function can be predicted from mGluR (glutamate receptor), which has the highest homology. mGluR acts as a heterodimer of T1R1 / T1R3 and causes a significant structural change when it forms a complex with the glutamic acid at the N-terminal site (Lopez Cascales et al ., 2010). m1-LBR, the ligand binding site of mGluR, is expected to form a free form I, in the absence of ligand, and form a complex of free form II, which is active when the ligand is bound (Temussi et < RTI ID = al . , 2002). Similarly, the sweet receptor T1R2 / T1R3 is expected to bind to the ligand to form an active complex.

When the ligand is the same, a structural change occurs by complexing with T1R2 / T1R3, and ATP is converted to cAMP by activating AC (adenylate cyclase) through isolated Gα _gust . Converted cAMP induces depolarization by inducing depolarization by direct cation through cyclic nucleotide-dependent ion channels, by activating PKA (protein kinase A), by phosphorylating the potassium channel of the basement membrane and by closing it to induce calcium ion influx .

On the other hand, the sweetness due to the non-sugar sweetener was obtained by hydrolyzing PIP2 (phosphatidylinositol 4,5-bisphosphate) with DAG (diacylglycerol) and IP3 (Margolskee, 2002), resulting in depolarization by releasing calcium ions from taste-cognized cells due to increased IP3 concentration (Ohkuri et < RTI ID = 0.0 > al ., 2009) (Fig. 1).

3. TRPV1 Is a salty taste

Many studies have shown that salty taste is recognized as sodium ions enter the sodium ion channel (ENaC) of taste-receptive cells. Electrophysiological studies using rodents have shown that the neuronal response to CTL (chorda tympani) to NaCl is significantly reduced in rats receiving oral amiloride, which is known to block ENaC. However, it was expected that the amyloride could not eliminate all of the CT response to NaCl, and thus another flavor transfer process (Smith et < RTI ID = 0.0 > al , 2012).

TRPV1 is a non-selective cation channel, capsaicin, heat, and acid-activated pain receptor. It has been shown that various types of TRPV1t exist in the taste bud (Liu et al. , 2001). The CT response to sodium, potassium, and ammonium ions was found to be increased by TRPV1 agonists, and the rodent blocking both ENaC and TRPV1 showed that the CT response to NaCl was erased. This electrophysiological study revealed that the TRPV1 channel is an important salty taste recognition pathway (FIG. 3). Maillard reacted peptides (MRPs) are proteins that are hydrolyzed and sugar-modified by heat and sugar. Studies by Katsumata et al. Have shown that CT neuronal response to NaCl in rats in which TRPV1 has been removed by MRPs is detected and that salty taste is induced by MRPs alone (Katsumata et < RTI ID = 0.0 > al . , 2008). Regarding this, in 2012, Dr. Ryumira of the Korea Food Research Institute found that KFRI-LHe, MRPs extracted from traditional soy sauce, showed salty taste through binding with TRPV1.

The higher order structure of TRPV1 was revealed by Liao et al . And revealed higher order and activated structures with a resolution of 3.4 Å using Cryo-EM (Liao et al ., 2013), and docking was performed using the TRPV1 structure revealed by the present invention. TRPV1 is composed of the same four subunits and four unit pieces are arranged around the ion channel. In one subunit, there is a pore loop between the transmembrane segment (S5-S6) and the S1-S4 potential receptors on the side, which are connected via the S4-S5 linker. In particular, the S4-S5 linker site interacts with the TRP site and is known to be involved in allosteric modulation. Regarding intracellular assembly of TRPV1, the arrangement of subunits is facilitated by the interaction of the intracellular N-terminal ankryin repeats, which are expected to provide a better understanding of the functional aspects of the TRP channel 2).

4. Protein Brazane

Brazzein is a member of the P. It is a sweet protein extracted from the fruit of brazzeana . It shows about 500 to 2000 times more sweet taste than sucrose. Brazeen is a major type and minor type, and most of the brassin extracted from plants are of major type. The major type has 54 amino acids including the pyroglutamic acid residue at the N-terminus whereas the minor type has 53 amino acids with the N-terminal pyroglutamic acid deficiency and shows about twice the sweetness than the major type. Brazene is the smallest sweetener protein and is a monomer with a molecular weight of about 6.5 kDa. The tertiary structure of the brassin revealed by NMR is composed of 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). Eight cysteine residues form four disulfide bonds and are highly stable against heat and pH (Gao et < RTI ID = 0.0 > al ., 1999).

In this study, 10 peptides were designed, sensory tests were performed based on the loop part of bradykinin, a sweet protein, and the results were analyzed by computer modeling.

II . Instruments and reagents

1. Instruments and reagents

BZ1, BZ2 and BZ6 were purchased from AbClon (Seoul, Korea) for peptides taste test, and BZ3-BZ5 and BZ7-BZ10 were purchased from Peptron (Daejeon, Korea). The synthesized peptides were purified using a Sephadex G-10 column from GE Healthcare Life Sciences (Pittsburgh, Pa., USA). To determine the concentration of the purified peptides, a UV / VIS spectrophotometer, HITACH Japan, 2000 product was used.

2. Structural Prediction and Computer Docking Programs

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

Ⅲ. Experimental Method

One. Of peptide  design

1.1 Peptides BZ1 , BZ2 Design of

Mutant studies have shown that the β-sheet structure (Figure 5) of the 29th to 43rd amino acid sequences of brassin is an important part of the brassin sweetness (Jin et al ., 2003). Therefore, BZ1 was designed based on this sequence, and the synthesized peptide sequence is composed of 15 amino acids as 'DKHARSGECFYDEKR'.

Various studies of the brassin variants have increased or decreased the sweetness. The Arg residue at position 29 of the Brazan was substituted with the Lys residue (Jin et al ., 2003), mutation of the His residue at position 31 to the Arg residue resulted in an increase in sweetness (Lee JW et al . , 2013), mutating the Glu residue at position 36 to an Asp residue (Do HD et al . , 2011), and even when the Glu residue at position 41 was mutated to Ala residue, the sweetness was increased compared to the wild type (Lee JW et al . , 2013). Based on the results of these mutant studies, residues 29, 31, 36 and 41 of BZ1 were designed to be modified as in the next 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 Peptides BZ3 - BZ6 Design of

Aspartame is a methyl ester form of a dipeptide composed of Asp residues and Phe residues (Figure 6) and is used as an artificial sweetener for beverages and foods (Ager et < RTI ID = 0.0 > al . , 1998). BZ3-BZ6 was designed by subdividing BZ1 and BZ2 in anticipation that short fragments of blazane would show sweetness, just as aspartame consisting of only two amino acids shows sweet taste. There are three loops in Brazeen. Loop I corresponds to positions 13 to 15, and loop II corresponds to positions 30 to 33. Finally, Loop III has a sequence of 40-43, and Boz II and Loop III exist in the sequence 29-43 of BZ1 (Fig. 5). Loop of such sweet protein is likely to be a 'sweet finger' model expected to show sweetness of sweet protein (Temussi et al . , 2002). Therefore, BZ3 was selected as the 'DKHAR' site 29-33 corresponding to Loop II of BZ1. BZ4 was designed so that the peptide is positive by changing Asp, the first residue of BZ3, to Lys like BZ2. As reported in previous studies, Glu41 of Loop III is close to Glu252 of T1R2 and Arg43 is an important residue (Yoon et al al . , 2011). BZ5 was selected as the region 40-43 corresponding to Loop III, and BZ6 was changed to BZ2 as shown in Table 2 by changing the Glu residue to Ala as in 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 Peptides BZ7 - BZ10 Design of

In BZ3 designed based on Loop II, by choosing BZ7 as 'KHAR', which is four sequences except for Asp which is equivalent to the sound residue, it is possible to change the sweetness according to the influence of the Loop II on the sweetness and the electrical properties, In order to investigate whether or not it appears.

On the other hand, BZ5 corresponding to Loop III was added with two residues on both sides to compare the flavor of BZ5 with that of 'FY' and 'VL', thereby confirming the role of 'FY' and 'VL' Respectively.

BZ9 and BZ10 were designed so that they can bind properly to the sweet taste receptor by maintaining the loop structure of BZ8. BZ9 is designed to retain the loop structure by inserting a Pro residue in the BZ8 sequence, the sequence of which is the same as that of 'FYDPEKRVL'. BZ10 allows the formation of a loop structure through a disulfide bond by adding a Cys residue at both ends of the sequence of BZ9, and its sequence is the same as that of '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 mutant 2 CFYDEKRVLC [SEQ ID NO: 10]

2. Of peptide  Synthesis and purification

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

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

3. Of peptide  Taste test

A total of 14 trained panels were tested for taste. The panels were selected as healthy men and women who do not smoke. To determine the correct taste, we performed the test at the meals and washed the mouth with the third distilled water before the test. Since the safety of the synthesized peptide was not known, it was immediately spit after taste and washed with distilled water. The results of the taste were expressed as blind test but freely expressed to eliminate psychological factors.

4. Of peptide  Structure prediction

The structure of the peptide synthesized through chemical synthesis will be greatly different from the structure of the corresponding bradine and thaumatin sequences. Therefore, the synthesized peptide predicts the structure with the most stable energy and compares it with the structure in the actual bradane and tau martin.

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

5. Peptides and  Prediction of binding of taste receptor

The Autodock vina program was used to predict the binding site and energy of the peptide and taste receptor (Trott et al ., 2010). This course was run on Intel (R) Core (TM) i3-3220 3.30 GHz CPU, memory 4 GB, Windows 7 specification computer. The structure of the T1R2 and T2R1 receptors, whose structures are not yet known, was predicted by the SWISS-MODEL web server provided by the Center for Molecular Biology at the University of Basel in Switzerland (Biasini et al ., 2014), and the web page (http://swissmodel.expasy.org/). The structure of T1R2 was predicted based on human glutamate receptor (PDB ID: 2E4U) corresponding to GPCR familly and T2R1 based on human delta opioid 7tm receptor (PDB ID: 4N6H). All pdb protein structure files were extracted using the AutoDockTools (ADT) program as a pdbqt file for Autodock vina docking (Morris et al ., 2009). Preparation using the ADT program includes the removal of water molecules, the addition of hydrogen atoms, the setting of a grid box to select the predicted binding site of the receptor, the choice of ligand torsion, and the charging process through the AMBER FF99 and Gasteiger-Marsili calculations. The selected range for the docking of the receptor and the peptide (grid box) is shown in Table 4. 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. Of peptide  Synthesis and purification

The 10 synthesized peptides designed from Brazane had a purity of greater than 95% for salty peptides, while the purity of HPLC purification was generally above 90% (Table 5).

In addition, Sephadex-G10 column was used to remove impurities and TFA which was used in synthesis and HPLC purification to interfere with the taste test. The purified peptides were quantitated by measuring the absorbance at a wavelength of 205 nm (Fig. 7). The peptide eluted fractions E1 to E5 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. Of peptide  Taste test

A panel of 14 peptides synthesized from BZ1 to BZ10 tested the taste. All the panels were free to express the tastes they felt in the response questionnaire. As a result, BZ3, BZ4 and BZ5, which showed salty taste, and BZ10, which had a bitter taste, showed no taste. In particular, BZ4 had a salty taste and a long tail, while BZ10 had a disgusting bitter taste (Table 6).

N ame 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 mutant 2 bitter +++++

3. Of peptide  Structure prediction

Designed through PEP-FOLD Web sever to identify structural differences through comparison of the synthesized 10 peptides with structures on actual proteins, and to identify binding models with taste receptors using predicted peptide structure files The structure of 10 peptides was predicted.

The results of the structural prediction of the peptide and the structure of the blaze phase show that BZ1 retains its original structure while BZ2 exists in a somewhat curved form. BZ3 and BZ4 retained the β-turn structure of the 29-33 sequence of the bradylanes very well and showed that BZ5 and BZ6 retained BZ6 better than the 40-43 sequence of bradyans (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 the respective blazane sequences (Table 8).

4. Peptides and  Prediction of binding of taste receptor

4.1 Peptides and  Sweet cognitive receptors T1R2 Combination with

Site-directed mutagenesis studies and chimeric formation studies of sweet receptors have shown that the VFT module of T1R2 is important for the acceptance of sweet substances (Assadi-Porte et al ., 2010). In addition, homology studies with mGlu receptors predicted binding of bradyin to T1R2 rather than T1R3 (Walters et < RTI ID = 0.0 > al ., 2006). In this study, we designed the binding sites and binding energy by combining 10 designed peptides and T1R2 through computer modeling. Also, prior to computer modeling, we predicted all binding pockets that could exist in T1R2 via CASTp Sever (Dundas et al . , 2006). To focus on the interaction of bradane with T1R2, 21 binding pockets were selected in 96 predicted binding pockets, excluding binding sites that were shorter than 35 Å in diameter (Table 9).

The binding sites of T1R2 and bradine were predicted in order to compare the binding sites of brassin and the peptide in the sweet taste. As a result, Brazenger showed accurate coupling to the center of the cleft portion located in the VFT module, corresponding to POC ID (pocket identification) 90 (black) and POC ID 92 (gray) A). The cross-section of the coupled model also shows that the N-terminal of the brassin and the L _{40 -43} region bind to T1R2 (Fig. 9B). The binding energy of bradine and T1R2 is -14.0 kcal / mol. Prediction sites of binding between BZ1 and T1R2 of BZ2 are shown in Fig. 10 and Fig. 11, respectively. As a result, it can be seen that both BZ1 and BZ2 bind to POC ID 90, which is the binding site of L- ₄₀ of L- _{40 of bradyin} in POC ID 90, 92 (FIG. 10 and FIG. 11). The bonding forces at this time are -12.4 kcal / mol and -11.4 kcal / mol, respectively.

Potential coupling pocket list of T1R2 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 A445, H447, Y469, H444, H444, H444, H444, H444, H454, P185, I327, Q328, S329, Y386, V330, Y386, S387, S390, L432, D433, H434, Q435, I436, F437, F438, D439, D443, 88 108 D32, Y33, L34, L89, R134, V135, V136, L399, L402, L403, D406, K407, K412, R413, V414, V415, 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, , 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 V36, K65, Y69, N70, Q73, S336, F338, R339, E340, W341, G342, P343, P349, R352, T353, S356, Y357, C359, N365, A369, T370, L371, S372, F373, 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 the total 92 potential binding pockets are longer than 35 Å which is the length of brazzein.

Prediction sites of BZ3 and BZ4 binding to T1R2 designed based on the region 29-33 of Brazane are shown in FIGS. 12 and 13, respectively. The synthesized and purified BZ3 showed a weak salty taste and the BZ4 showed a strong salty taste. Both BZ3 and BZ4 bind to the left side of the cleft and correspond to POC ID 92. At this time, the binding energies are -7.6 kcal / mol and -5.4 kcal / mol, respectively.

BZ5 and BZ6 were designed based on Brazeen's 40-43 sequence, BZ5 had a weak salty taste and BZ6 had no taste. The predicted binding sites of T1R2, BZ5 and BZ6 are shown in FIGS. 14 and 15, respectively. BZ5 binds to the upper left POC ID 85 and 92 of the cleft, and BZ6 binds to the right POC ID 90 of the cleft. The binding energies at this time are -8.3 kcal / mol and -7.6 kcal / mol, respectively.

The predicted binding site of BZ7 to T1R2 designed based on the 30-33 sequence of Brazane is shown in Fig. Predicted binding sites were combined with a binding energy of -6.9 kcal / mol on the left side of the cleft, POC ID 92.

BZ8, BZ9, and BZ10 were designed based on the 38-45 sequence of Brazane, and they did not show any sweetness. In the case of BZ8 and BZ9, the taste was irritating and very weak in intensity, but BZ10 showed a very strong bitter taste. The predicted binding sites for T1R2 are shown in Figs. 17, 18 and 19, respectively. Binding of BZ8 and BZ10 to POC ID 92 on the right side of the cleft and BZ9 on the left side of the cleft (POC ID 81). At this time, the binding force is -7.2, -6.9, and -7.1 kcal / mol in this order from BZ8 to BZ10.

4.2 Peptides and  Acupuncture channel TRPV1 Combination with

There are two pathways for recognition of salty taste through stimulation of ENaC, a sodium ion channel, and TRPV1, an acupuncture receptor. Since peptides do not cause depolarization through ENaC, they are likely to bind and increase the salty taste in combination with TRPV1, the channel for accepting the pain. TRPV1 used in this combined prediction program is TRPV1 of Rattus norvegicus whose structure is revealed by electron cryo-microscopy method (Liao et al ., 2013) The PDI ID is 3J5R (A in Fig. 20). All chains of TRPV1 composed of homo-tetramer were simultaneously docked.

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

This result suggests that capsaicin, which is a substance that binds to TRPV1 (transient receptor potential cation channel subfamily V member 1) and transmits pain, binds to the pore of the S3-S6 helix structure (Lee et al. al ., 2011), but in fact it is a similar result when combined with S3 and S4 helix. The results of the docking of the synthesized peptides showed binding between S1-S4 helix structures similar to capsaicin. However, unlike the results of capsaicin, all of the peptides show additional interaction with the S5 helix. This is expected to result from structural differences in peptide and capsaicin and is believed to further interact with the S5 helix to maintain stable binding. The peptides interact with the S1-S4 helix structure, S4-S5 linker and S5 helix, which, unlike capsaicin, appears to have a wide binding site, which is presumed to result in differences in taste (Fig.

V. Discussion

By analyzing the binding sites, the reason for not showing sweetness and the reason for showing salty and bitter taste were confirmed.

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

What can be expected from the synthesized peptide as the first reason for not having sweet taste is that the structure at the bradine is not completely maintained in the synthesized peptide. Predicted peptides from the PEP-FOLD structure prediction are comparable to those in Brazene, and the CD (circular dichroism) spectra of the actually synthesized peptides can also explain the predictability of the structure in practice there was. CD spectra of BZ1, BZ2, BZ6, and BZ7 of 10 peptides were measured using Jasco J-815 CD spectroscope from Inha University Joint Instrument for measuring CD spectra for validation of predicted structure 22).

The CDSSTR algorithm was used to predict the secondary structure from the CD results of BZ1, 2, 6, and 7 (Johnson, 1999). Based on the SMP56 reference set with reference to 56 proteins, the CD results showed that BZ1 and BZ2 had a ratio of β-sheet structure and BZ3 and BZ4 had a higher turn ratio (Sreerama et al ., 2004). Also, it can be seen that the ratio of β-sheet structure of BZ1 is higher than that of BZ2, and the structure of BZ4 is higher than that of BZ3, so that predicted results predict the structure of actual peptide well (Table 11) . However, even though the CD spectra analysis results and the PEP-FOLD structure predictions are correlated, the structure of the peptides obtained from PEP-FOLD structure prediction is not completely identical to the structure in Brazene, The number of peptide structures in which the rotation of the bond occurs can be equal to infinity. Thus, even if the peptides showing the sweet taste of bradine were synthesized, the probability of having the original structure in bradane was so low that it may not have been sweet.

A second reason for the expected sweetness in the designed peptides is that the multi-binding site that the brassin should bind is not met. The GPCR C-family receptor, T1R2 / T1R3, recognizes the sweet taste, which combines sugar and artificial sweeteners to deliver a sweet signal to the brain via the G protein. This means that the binding site and energy of the receptor and the ligand are important factors for the sweet taste transmission. The binding of T1R2 and bradine, known to bind to the sweet protein, among T1R2 and T1R3, was confirmed by binding to the entire cleft region of T1R2 as shown in Fig. 9, and the POC IDs at that time were 90 and 92, respectively. In addition, it was confirmed that binding at a high binding energy of -14 kcal / mol was confirmed. In addition, by analyzing the hydrogen bond of bradane and T1R2, residues participating in binding (Fig. 23) and residing in hydrogen bond were identified to show sweet taste (Table 12).

As shown in Table 12, it was confirmed that bradylanes bind strongly with 11 hydrogen bonds, and N-terminal and loop corresponding to residues 36-54 are involved in binding to T1R2. In addition, it can be seen that the POC ID 90, 92 portion of T1R2 corresponding to residues 46, 60, 255, 312-314, and 458 binds to bradane through strong hydrogen bonding. In contrast, BZ1 and BZ2 bind to the POC ID 90 bound to the brassin loop but not to the POC ID 92 to which the N-terminal binds. In contrast, BZ3, BZ4, and BZ5, which exhibited salty taste, bound to the binding site of POC ID 92 of brassin but did not bind to the loop binding site. Similarly, BZ6, BZ8, and BZ10 bind to the loop binding site but not to the N-terminal binding site. In addition, BZ7 and BZ9 showed binding at the N-terminal binding site, so none of the 10 designed peptides met both the N-terminal binding site and the loop binding site. The conclusion we can see from the results is that the brassin binds to T1R2 as a multiple binding site, and if one of them is not met, it can not be sweet.

The presence of a common amino acid of TRPV1 chain B involved in the interaction with the peptide was predicted through the docking results (Fig. 24). In the case of Glu513, Val567, Gln700, and Ile703, all three peptides showed an interaction-related amino acid. In particular, Val567 is an amino acid contained in S5 helix, and interaction with S5 helix is common, suggesting the possibility of salty taste when binding with Val567 of S5 helix and its surrounding amino acids. The interior of the S1-S4 helix pore structure in which the synthesized peptide is located is composed of polar amino acids. This is expected to contribute significantly to the binding stability of peptides containing a large number of polar amino acids, and the docking results showed that they bind to the S1-S4 helix pore with the lowest energy. In this regard, it can be assumed that the most salty taste of BZ4 is the most stable bond with the lowest energy.

Since the discovery of aspartame, numerous short peptides have been researched and developed. The peptides developed in the Tamura group are regarded as the dipeptide, tripeptide, and have an umami taste, which is thought to be the main cause of the umami taste of broth (Tamura et al . , 1989). It should be noted that the developed dipeptides and tripeptides were accompanied by not only an umami taste but also a salty taste, which consisted mainly of aspartic acid and glutamic acid with negative charge. The most salty KKRARs developed in this study mainly contain positively charged lysine and arginine, and the salty taste of the KKRAR is due to the charge opposite to that of the previously developed negative charge peptide. It can be inferred that it is irrelevant.

The results of docking the BZ3, BZ4, and BZ5 salty tastes and the anginal receptor TRPV1 suggest two important things for us. The first implication is that short peptides can show salty taste. The molecular weight of the salty protein found by the team of Dr. Ryumira of Korea Food Research Institute in 2012 is not only from 500 Da to 10,000 Da, but also the sugar-modified polymer of mannose and N-acetyl-glucosamine. Can be obtained by extraction (Rhyu et al ., 2012). On the other hand, the three peptides of BZ3 ~ 5 were short peptides composed of 4 ~ 5 amino acids and showed salty taste even without sugar modification. That is, it is possible to develop a peptide-based salty food additive capable of replacing salt. The second implication is that the type of signal delivered may vary depending on where the ligand binds to the TRPV1 receptor. Capsaicin, which delivers a spicy taste, is located in the S1-S4 helix pore while binding to S3 and S4 of TRPV1. BZ3-5 also binds to S3 and S4 but binds to the S1-S4 helix pore base and the additional interaction with S5 helix is the difference from capsaicin binding. In other words, when combined with the sides of S3 and S4 belonging to the voltage sensor domain, the result of docking can be expected to bind the spicy taste to the base and transmit the salty taste when interacting with the S5 helix.

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<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Salty peptide <130> P15U21C0062 <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 (5)

A salty peptide consisting of the amino acid sequence of SEQ ID NO: 3.
delete delete A salt substitute comprising the peptide of claim 1.
A food additive comprising the peptide of claim 1.

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