CN113248566B

CN113248566B - An active peptide with bitter taste blocking effect

Info

Publication number: CN113248566B
Application number: CN202110215489.4A
Authority: CN
Inventors: 于志鹏; 阚若彤; 赵文竹; 王莹雪
Original assignee: Bohai University
Current assignee: Bohai University
Priority date: 2021-02-10
Filing date: 2021-02-10
Publication date: 2022-02-11
Anticipated expiration: 2041-02-10
Also published as: CN113248566A

Abstract

The invention particularly relates to an active peptide with bitter blocking effect, the amino acid sequence of which is Asp-Asp-Asn-Lys (DDNK). The active peptide with the bitter blocking effect can be effectively combined with a bitter receptor T2R14, has a continuous and stable masking effect on bitter substances, and has the outstanding advantages of safety, no toxic or side effect, easiness in absorption, industrialization and the like. The active peptide DDNK can be used in food, medicine and health care products with bitter taste, and has wide application prospect and very important significance.

Description

Active peptide with bitter blocking effect

Technical Field

The invention belongs to the field of bioactive peptides, and particularly relates to a bioactive peptide with a bitter blocking effect.

Background

After digestion of the protein, it is absorbed directly, mainly in the form of a polypeptide. Protein peptides can be used in a variety of nutraceuticals or as flavor additives. However, various degrees of bitterness are generated during the process of preparing protein peptides. Studies have shown that insoluble proteins such as soy and fish, modified by protease treatment, while improving their solubility, thermal stability and anti-settling properties in acidic environments, produce bitter taste in their hydrolysates which can interfere with their use. The formation of bitter taste causes flavor defects in food products, limiting the use of protein hydrolysates. Therefore, masking and removing the bitter taste of protein hydrolysates has long been a challenge in the food, pharmaceutical and nutraceutical industries.

Bitter taste perception is mediated by bitter taste receptors (T2Rs), and these T2Rs are expressed on the human tongue and belong to the G protein-coupled receptor family. To date, almost 25 human bitter taste receptors have been identified, but most T2Rs still have no known ligands. These T2Rs have a single ligand binding pocket that is capable of recognizing many structurally different bitter tasting substances while maintaining high selectivity. T2R14 is one of the most widely regulated bitter receptors for a variety of bitter compounds and it recognizes a wide variety of natural or synthetic bitter compounds, including many drugs. Therefore, T2R14 can be used as an effective target for inhibiting bitter taste.

Conventional debittering methods typically involve selective separation and masking. However, the selective separation method has larger loss to the polypeptide, affects the nutrition and the activity of the polypeptide, and particularly has larger influence to substances taking bitter peptides as main active components; the masking method needs to add bitter masking agents such as cyclodextrin, modified starch and the like or other flavor substances, and the additional ingredients of the masking method do not accord with the novel healthy and natural diet concept. Pydi, S.P. et al by competitive Ca²⁺Kinetic analysis showed that two amino acid derivatives, aminobutyric acid (GABA) and N-bis (carboxymethyl) -L-lysine (BCLM), showed antagonist activity against T2R4 and were useful as bitter receptor blockers. However, since these two substances are chemically synthesized, they may cause some toxic and side effects to the human body. Wujianpin et al use protease to enzymolyze chicken protein to obtain hydrolysate, separate, purify and identify AGFAGDDAPR, FTQQIE, GDDAPR polypeptide with potential bitter taste receptor blocking activity. However, this conventional method is time consuming, expensive and results in some loss of highly active peptides.

The food-derived bioactive peptide has the advantages of safety, no toxic or side effect, multiple functions, easy modification and the like, and is widely concerned by the fields of food science, chemistry, biology and the like. There are also very limited reports of the types of natural T2Rs blockers in the prior art, and it is therefore of great interest to develop new natural T2Rs blockers. The egg white is a colloidal substance consisting of protein and water, the protein in the egg white mainly comprises 54% of egg white protein, 12% of ovotransferrin, 11% of ovomucoid and other proteins, and has wide biological activity. At present, the bioactive peptide separated and purified from egg white has been reported to have biological activities such as antihypertensive and antioxidant effects. Therefore, the bitter substance masking peptide which has good water solubility, biological activity and no toxicity and can be combined with T2R14 is screened from the egg white protein, the problem of bad bitter taste of food, medicine and health products can be solved, and the added value of the egg is improved.

The invention aims to provide an active peptide with bitter blocking effect, and the active peptide is applied to masking the bitter taste of bitter substances in foods, medicines and health-care products without damaging the flavor and the nutritional value of the products.

Disclosure of Invention

In view of the deficiencies of the prior art, the present invention aims to provide an active peptide with bitter taste receptor blocking effect, the amino acid sequence of which is Asp-Asp-Asn-Lys (DDNK).

The active peptide with the masking effect on the bitter substances acts on a target T2R14, competitively inhibits the combination of the bitter substances and T2R14, has a continuous and stable masking effect on the bitter substances, and has the characteristics of safety, no toxic or side effect, good water solubility and the like.

The active peptide having a masking effect on bitter substances of the present invention has a half Inhibitory Concentration (IC) against bitter substances₅₀) It was 0.14 mg/ml.

The active peptide with masking effect on bitter substances can be obtained by carrying out enzymolysis on ovalbumin, ovotransferrin and ovomucoid in egg white by utilizing pepsin, trypsin and chymotrypsin and purifying by multi-dimensional chromatography (gel filtration chromatography, affinity chromatography and semi-preparative liquid chromatography); can also be realized by a solid-phase chemical synthesis method. Preferably by solid phase chemical synthesis.

The form of the active peptide having a masking effect on bitter substances of the present invention is not particularly limited. For example, it is used by dissolving in water to prepare a liquid preparation, spray-drying to prepare a powder or granules, and freeze-drying or heat-drying to prepare a solid preparation. Preferably, when the active peptide is used in the form of a liquid preparation, the active peptide is contained in an amount of 7.5 to 25mg per 100ml of the liquid preparation.

In order to achieve the purpose, the invention adopts the following technical scheme:

egg white produced by gastrointestinal proteaseScreening active polypeptide generated by enzymolysis, and screening water solubility, biological activity and T2R14 binding capacity; and performing in vitro bitterness inhibiting activity IC on the selected active peptide₅₀Value determination, final screening of IC₅₀The active peptide with lower value is the bitter taste receptor blocking peptide. The method comprises the following specific steps:

(1) proteolysis and active peptide screening

According to the invention, egg albumin, ovotransferrin and ovomucoid in egg white are subjected to enzymolysis by virtue of an ExPASY PeptideC (http:// web. ExPASy. org/peptide _ cutter /) online virtual enzyme digestion tool, and are screened to obtain unreported dipeptide, tripeptide and tetrapeptide. The sequence of the active Peptide obtained above was subjected to prediction of toxicity and water solubility properties by the ADMET program in Discovery Studio (DS)2017 and Peptide Property Calculator. And (4) screening to obtain non-toxic dipeptide, tripeptide and tetrapeptide with good water solubility.

(2) Targeted screening

The crystal structure of the Bitter receptor T2R14 (Bitter DB ID: 14) was obtained from the BitterDB database (http:// BitterDB. agri. huji. ac. il/dbbitter. php) and used as a protein target. Active peptides capable of tightly binding to T2R14 were screened by molecular docking via the CDOCKER program of DS 2017. And screening to obtain the tetrapeptide DDNK with potential bitter receptor blocking activity by taking the CDOCKER-ENERGY' value, the number of formed hydrogen bonds and the number of acting key amino acids as indexes.

(2) Determination of in vitro bitter taste inhibitory Activity

The bitterness inhibitory activity of active peptide DDNK of a certain concentration was measured by electronic tongue. And respectively adding a certain mass of peptide DDNK into 30mL of water, uniformly mixing, adding 50mL of quinine (1mM) to prepare a solution to be detected, taking the quinine as a blank control group, and taking the peptides LELNQ and LEGSLE as positive control groups. After the cycle number, the data file name, the sample number, the sample name and the test method are set, the electrodes are installed, and the measurement is started.

The measurement process is as follows: (1) cleaning the sensor 90s in the positive and negative electrode solutions, and then cleaning the sensor 120s in the two reference solutions; (2) equilibrating the sensor in a conditioning solution for 30 s; (3) each sample was measured for 30 s; (4) after washing the sensor twice for 3s each, it was immersed in the reference liquid for 30s to measure the aftertaste value. Measurements were repeated four times per sample. After the measurement is completed, the data is processed.

Compared with the prior art, the invention has the following beneficial effects:

the active peptide DDNK with the masking effect on the bitter substances has the characteristics of safety, no toxicity, biological activity, good water solubility and the like, competitively inhibits the combination of the bitter substances and T2R14, and has a continuous and stable masking effect on the bitter substances without damaging the flavor, nutrition and efficacy of the product. Compared with other bitter taste masking agents such as saccharides and salts, the peptide is added to meet the novel diet concept of low sugar, low salt, health and naturalness. Therefore, the bitter taste masking agent can be used for masking bitter foods, medicines and health care products, and has wide application prospect.

Drawings

The invention is illustrated in figure 1, wherein:

FIG. 1 is a 2D graph of docking results of DDNK with T2R14 receptor;

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1.

This example screens and identifies a tetrapeptide DDNK with significant inhibitory effect on the binding of bitter tasting substances to T2R14 from egg white, comprising the steps of:

(1) and (3) performing virtual enzymolysis on egg white protein and performing primary screening.

The ovalbumin, ovotransferrin and ovomucoid in egg white were subjected to simulated enzymatic hydrolysis based on the online program ExPASy PeptideCutter using typical gastrointestinal digestive proteases, pepsin (EC 3.4.23.1), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1), the amino acid sequences of which were obtained via the NCBI website (https:// www.ncbi.nlm.nih.gov /), numbered AAB59956.1, CAA26040.1 and ACJ04729.1, respectively. 219 dipeptides and active peptides above the dipeptides are obtained. Selection of unreported di-, tri-and tetrapeptides toxicity and water solubility properties were predicted using the ADMET program and Peptide Property Calculator of DS 2017 software. And 86 nontoxic peptides with good solubility are obtained by screening.

(2) The 86 peptides with no toxicity and good solubility obtained above were subjected to molecular docking with T2R14 using DS 2017 software (fully known as Discovery Studio 2017R 2 Client), and the results of the successfully docked active peptides are shown in Table 1:

TABLE 1

As can be seen from the data in Table 1: the best docking result of the tetrapeptide DDNK is obtained by evaluating the binding result through an energy value.

Example 2 determination of blocking Activity of active peptides against bitter substances

The bitterness inhibitory activity of peptide DDNK at a certain concentration was measured by electronic tongue. And respectively adding a certain mass of peptide DDNK into 30mL of water, uniformly mixing, adding 50mL of quinine (1mM) to prepare a solution to be detected, taking the quinine as a blank control group, and taking the peptides LELNQ and LEGSLE as positive control groups. After the cycle number, the data file name, the sample number, the sample name and the test method are set, the electrodes are installed, and the measurement is started.

The results indicate that peptide DDNK can effectively inhibit the activity of bitter taste receptor T2R14, its IC₅₀The values are respectively 0.14mg/ml

Example 3 DS software analyzes the interaction pattern between peptide DDNK and T2R 14.

The two-dimensional plan between peptide DDNK and T2R14 is shown in fig. 1, from which it can be seen that: the tetrapeptide DDNK is capable of interacting with an Asp168 residue, a Trp89 residue, a Ser167 residue, a Gly158 residue, an Asn157 residue, a Cys165 residue, a Tyr159 residue, a Ser265 residue, an Ile262 residue and a Thr86 residue of T2R 14;

example 4 application of bitter taste blocking active peptide DDNK

In practical production, the bitter taste blocking active peptide DDNK can be prepared by solid phase chemical synthesis method, and can be added into food, medicine and health product with bitter taste in powder, granule or water-soluble liquid state. For example, active peptides are added to tea and coffee as a sachet to mask the bitter taste of the beverage while retaining the unique flavor of the beverage; the active peptide is added into the balsam pear to mask the bitter taste of the balsam pear to prepare balsam pear processed products such as dried balsam pear, balsam pear tea and the like, thereby eliminating the conflict feeling of people on the balsam pear and improving the edible and medicinal values of the balsam pear such as clearing heat and detoxicating, nourishing blood and nourishing liver, reducing blood sugar and the like; the active peptide is added into medicines, particularly Chinese herbal medicines, so that the bitter taste of medicinal components such as coptis chinensis, phellodendron, swertia chinensis and rhizoma atractylodis is masked, and people can take the medicines more pleasantly; the bioactive peptide is added into health-care food such as donkey-hide gelatin cakes, so that the bitter taste of the bioactive peptide is masked, the nutritive value of the health-care food can be improved, and a new idea for solving the problem of bitter taste is provided for the health-care product industry.

The technical solutions of the present invention are described in detail in the embodiments described above, it should be understood that the above are only specific embodiments of the present invention, and are not intended to limit the present invention, and it should be noted that: it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and these changes and modifications should also be considered as within the scope of the invention.

Claims

1. An active peptide with a bitter taste blocking effect, wherein the amino acid sequence of the peptide is Asp-Asp-Asn-Lys (DDNK).

2. Application of the active peptide of claim 1 in masking the bitterness of bitter substances in food, medicine and health care products.