CN118340250A - A salty taste enhancer and its application and salty taste enhancing method - Google Patents

Abstract

The invention provides a salty taste enhancer, application thereof and a salty taste enhancing method, and belongs to the technical field of large health foods. The salty taste enhancer comprises a polypeptide shown as SEQ ID NO. 1 or salt thereof, and the amino acid sequence of the polypeptide is SSDDK. The salty taste enhancer has high salty taste enhancing effect at low concentration, can achieve salt reduction effect without reducing salty taste at high concentration, and can achieve salt reduction of at least 25%. Thus, adding the salty taste enhancer of the present invention as a food additive, a seasoning, or the like to a salt can provide a salty food or beverage having a reduced salt content while having a high palatability.

Description

A salty taste enhancer and its application and salty taste enhancing method

Technical Field

The present invention relates to the technical field of health food, and in particular to a salty taste enhancer, an application thereof and a salty taste enhancing method.

Background technique

Humans can perceive five basic tastes, including "sour, sweet, bitter, salty and umami", and saltiness is one of them. Salt is the most widely used salting agent in food processing and has a vital impact on the flavor of food. Its main component is NaCl. However, excessive intake of sodium salt can lead to high blood pressure in the human body, which in turn causes cardiovascular and kidney diseases. The average sodium intake of adults worldwide is estimated to be 4.31g/d (10.78g/d of salt), which is more than twice the sodium intake recommended by the World Health Organization of <2g/d (<5g/d of salt). For this reason, the WHO has established a goal of reducing the average population salt/sodium intake by 30% by 2025, and governments around the world have also started salt reduction plans to protect public health safety. Therefore, there is an urgent need for salt substitutes or saltiness enhancers that can give food a salty taste like salt as a way to reduce salt intake.

A salt substitute refers to a material that can be used instead of salt and exhibits a salty taste itself. Potassium chloride is known as a salt substitute. However, in addition to the salty taste, potassium chloride also exhibits a bitter taste, thus reducing the palatability of food. In addition, excessive potassium intake is not good for human health.

In addition, salty taste enhancing peptides and salty peptides can be used as potential directions for salt reduction. In recent years, some bioactive peptides have been shown to have salt reduction effects. Three salty taste enhancing peptides, YDPNDPEK, DDWDEDAPR and DVPDGPPPE, were obtained from enzymatically hydrolyzed Agaricus bisporus protein by ultrafiltration, of which 0.4% YDPNDPEK can achieve a 30% salt reduction effect. In addition, a salty taste enhancing peptide EDEGEQPRPF was identified in commercial fermented tofu in China, and its salt reduction effect was confirmed by sensory evaluation: the saltiness of 0.4 mg/mL peptide mixed with 50 mmol/L NaCl solution was equivalent to the saltiness of 63 mmol/L NaCl solution. However, given the current situation, the intensity of the salty taste enhancement effect still needs further improvement.

Summary of the invention

The object of the present invention is to provide a salty taste enhancer and its application and salty taste enhancing method in view of the above-mentioned deficiencies in the prior art. The salty taste enhancer has a good salt reduction effect and can increase the saltiness of the solution when the concentration is low. When the concentration is high, the salt reduction effect can also be achieved without reducing the saltiness.

In order to achieve the above object, the present invention adopts the following technical solutions:

The first object of the present invention is to provide a salty taste enhancer, which comprises a polypeptide as shown in SEQ ID NO: 1 or a salt thereof, wherein the amino acid sequence of the polypeptide as shown in SEQ ID NO: 1 is SSDDK.

Furthermore, the polypeptide as shown in SEQ ID NO: 1 was molecularly docked with the salty taste receptor TMC4, and the key amino acids for its binding were ARG583, ARG330 and GLU284.

Furthermore, the polypeptide replaces no more than 25% of sodium chloride to enhance the saltiness.

Furthermore, the polypeptide replaces 50% to 75% of sodium chloride without reducing the saltiness.

The second object of the present invention is to provide a food additive comprising the above-mentioned polypeptide or a salt thereof.

The third object of the present invention is to provide a seasoning comprising the above polypeptide or a salt thereof.

Furthermore, the seasoning also includes sodium chloride.

The fourth object of the present invention is to provide a food comprising the above polypeptide or a salt thereof.

Further, sodium chloride is included.

A fifth object of the present invention is to provide a method for enhancing salty taste, characterized in that the salty taste enhancer is added to food or beverage containing salt.

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

The present invention provides a salty taste enhancer and its application and salty taste enhancing method. The salty taste enhancer comprises a polypeptide or a salt thereof as shown in SEQ ID NO:1, wherein the amino acid sequence of the polypeptide shown in SEQ ID NO:1 is SSDDK. The salty taste enhancer of the present invention has a high salty taste enhancing effect at low concentrations, and has no salty taste itself and no other peculiar smell or odor except salty taste. At high concentrations, the salt reduction effect without reducing the salty taste can be achieved, and at least 25% salt reduction can be achieved. Therefore, adding the salty taste enhancer of the present invention as a food additive, condiment, etc. to salt can provide a salty food or beverage with reduced salt content and high palatability.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a is a graph of pLDDT values corresponding to the TMC4 structural model;

Figure 1b is the Ramachandran plot of the TMC4 model;

Figure 1c is the Errat score graph of the TMC4 model;

Figure 1d is a diagram of the final structural model of the receptor of the TMC4 model;

FIG2 is a three-dimensional diagram of the interaction between SSDDK and TMC4 receptor molecules;

FIG3 is a two-dimensional diagram of the interaction between SSDDK and TMC4 receptor molecules;

FIG4 is a diagram of the hydrogen bond donor region and acceptor region (pink is the hydrogen bond donor region, green is the hydrogen bond acceptor region) of the interaction between SSDDK and the TMC4 receptor molecule.

FIG5a is a comparison of salinity values of 0.20 mg/mL NaCl and 0.05 mg/mL peptide + 0.15 mg/mL NaCl mixed solution;

FIG5 b is a graph showing the salt reduction effects of 25%, 50% and 75% of the peptide SSDDK verified by electronic tongue;

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

The salty taste enhancer of the present invention refers to a substance that has substantially no salty taste itself but makes the salty taste of salt (sodium chloride) strongly felt when used in combination with salt. Specific examples thereof include: a substance that, when coexisting with a salty substance, makes the subthreshold salty taste of the salty substance, which is no longer felt, felt; and a substance that has an effect of inducing a strong salty taste by its addition.

The salty taste enhancer provided by the present invention comprises a polypeptide as shown in SEQ ID NO: 1 or a salt thereof, wherein the amino acid sequence of the polypeptide as shown in SEQ ID NO: 1 is SSDDK, specifically Ser-Ser-Asp-Asp-Lys.

In some embodiments, the acceptable salt is a salt-forming reaction conventional in the art, for example, a salt is formed by a chemical reaction between a base and an acid, such as NH ₃ +H ₂ SO ₄ →(NH ₄ ) ₂ SO ₄ .

The salt may be a basic salt, an acidic salt, or a neutral salt. Basic salts produce hydroxide ions in water while acidic salts produce hydronium ions.

The above polypeptide SSDDK can form a salt of the polypeptide SSDDK of the present invention with a cation or anion between an anionic group or a cationic group, respectively. These groups can be located in the peptide part of the polypeptide SSDDK of the present invention.

The anionic group of the polypeptide SSDDK of the present invention may include a free carboxyl group of the peptide portion. The peptide portion generally includes a free carboxyl group at the C-terminus.

The cationic groups of the peptide moiety are not limited in the present invention and include the free amino group at the N-terminus (if present) and any free amino group of internal basic amino acid residues (eg, Lys).

In a specific embodiment, the analogs of the polypeptide SSDDK of the present invention are basic salts. These salts can be formed between anionic groups of the peptide portion and sodium or potassium cations.

In another specific embodiment, the analogs of the polypeptide SSDDK of the present invention are acidic salts. These salts can be formed between cationic groups of the peptide portion and chloride or acetate anions.

It should be noted that the polypeptide SSDDK of the present invention is screened out from crocodile hemoglobin, binds to the TMC4 salty taste receptor, is non-toxic and harmless as a protein peptide, and has certain nutritional value.

The screening process of peptide SSDDK is as follows:

(1) Virtual screening

Hemoglobin of jawed vertebrates is a heterotetramer composed of two α subunits and two β subunits. The amino acid sequences of crocodile hemoglobin α subunit (GenBank: BAN62838.1) and β subunit (GenBank: BAN62839.1) were obtained from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Pepsin (pH 1.3), chymotrypsin-low specificity (C item is [FYWML], not before P) and trypsin were selected in ExPASyPeptideCutter (http://web.expasy.org/peptide_cutter/) to simulate the gastrointestinal environment, and crocodile hemoglobin was virtually enzymatically hydrolyzed, and a total of 289 crocodile hemoglobin peptides were obtained.

The water solubility of crocodile hemoglobin peptides was predicted using an online peptide property calculator (http://www.innovagen.com/peptide-stability-storage-and-solubilisation). The water solubility prediction results showed that 39 peptides had good water solubility. The toxicity of the selected peptides with good water solubility was then predicted in Discovery Studio (DS) 2017R2 software, and the toxicity included Ames mutagenicity, developmental toxicity potential, and skin sensitivity. Finally, 24 peptides with good water solubility and no toxicity were screened out.

(2) AlphaFold2 builds the TMC4 structural model:

AlphaFold is a newly developed neural network calculation method that can predict protein structures with extremely high accuracy by inputting amino acid sequences. The amino acid sequence of TMC4 receptor protein was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/) (accession number: NP_001138775). According to the amino acid sequence of TMC4 protein, a total of 5 TMC4 protein structure models were predicted by the Alphafold2 program, and the model with the highest accuracy needs to be selected from them. The local distance difference test (lDDT) is a non-superposition score that evaluates the local distance differences of atoms in the model, and has been proven to be used to verify the rationality of structure prediction. The predicted local distance difference test (pLDDT) is an important confidence indicator for Alphafold2 to verify the accuracy of the predicted protein structure. The score ranges from 0 to 100, and the higher the score, the higher the confidence of the prediction. A pLDDT value of >90 indicates high-precision confidence, a value between 70 and 90 indicates high confidence, and a pLDDT value of >70 indicates that the backbone prediction is basically correct.

The pLDDT values of the five predicted TMC4 protein structure models are ranked from high to low as rank1=78.6, rank2=77.1, rank3=75.8, rank4=74.6, and rank5=73.9 (as shown in Figure 1a). The pLDDT value of rank1 is the highest and is >70. The Procheck and Errat programs in the SAVESv6.0 server (https://saves.mbi.ucla.edu/) are used to supplement the reliability of the model. The Ramachandran diagram (as shown in Figure 1b) obtained from the Procheck program identifies whether the protein structure is reasonable. It can be seen from the figure that 87.1% of the amino acid residues are located in the most favorable area, 9.1% are located in the additional allowed area, 2.9% are located in the large number of allowed areas, and only 0.8% are located in the unallowed area. 99.2% of the amino acids in the model are located in reasonable areas, and the model structure is reliable. Errat is a technique for analyzing the non-bonded interactions formed between different atomic types. The results will get a certain score. The higher the score, the higher the accuracy of the model. As shown in Figure 1c, the Errat score is 92.835, indicating that the model is highly accurate. In summary, the accuracy of the model is high enough for molecular docking, and the final model is shown in Figure 1d.

(3) Molecular docking

The 24 screened peptides and the constructed TMC4 structural model were molecularly docked in Discovery Studio (DS) 2017R2 software. The obtained TMC4 structural model was imported into the DS software, and after pretreatment by dehydration and hydrogenation, the active site for docking was determined according to previous studies. The amino acid sequence was entered into the DS software to construct the peptide, the energy of the peptide was minimized using the CHARMm force field, and the 2D structure of the peptide was converted to 3D. The pretreated peptide was docked with the TMC4 receptor protein in a semi-flexible manner using the CDOCKER program. The binding tightness of the bioactive peptide to the receptor protein was evaluated by comparing the "-CDOKER_ENERGY" value output by the software. The higher the -CDOKER_ENERGY value, the tighter the binding between the peptide and the protein. The -CDOKER_ENERGY values of the 24 peptides docked with TMC4 are shown in Table 1. The proven salty taste enhancing peptides SPE, NSE, NRTF, LSERYP and AHSVRFY were docked with TMC4 using the same method, and the -CDOKER_ENERGY values that can be used as positive controls were 53.1565, 73.4564, 86.9124, 96.6989 and 101.2340 kcal/mol, respectively. The results showed that the -CDOKER_ENERGY values of peptides SSDDK, GEAVK and CAYPQTK were higher than those of the positive control group, and the potential salt reduction effect may be better. The actual salt reduction effect still needs to be verified by electronic tongue analysis.

Table 1. Docking energy table of peptides and TMC4 receptor protein

The polypeptides SSDDK, GEAVK and CAYPQTK screened by the above method can be synthesized by solid phase synthesis or synthetic biology methods.

The products obtained by the above reaction can be separated and purified by conventional techniques, such as recrystallization, distillation, chromatography, etc.

The synthesis of the polypeptide or a salt thereof thus prepared can be confirmed by a known method, such as ¹ H-NMR measurement, ¹³ C-NMR measurement, or mass spectrometry (eg, electrospray ionization mass spectrometry (MS-ESI)).

The salty taste enhancer of the present invention exhibits a salty taste enhancing effect, which makes the salty taste of salt be strongly felt. The salty taste enhancing effect can be evaluated by an electronic tongue. Ordinary sensory evaluation requires members to be well-trained and is easily affected by human psychology. The electronic tongue is a relatively low-cost, fast, accurate and unbiased flavor evaluation method. The salt-reducing effect of the peptide was verified by the SA402B electronic tongue. A certain concentration of NaCl solution was used as a saltiness standard to measure the saltiness value, and then the peptide was used to replace part of the NaCl and the saltiness value of the mixed solution was measured. If the saltiness value does not change significantly or increases significantly, it is considered that the peptide has a salt-reducing effect and can replace the same amount of NaCl. The percentage of the replaced NaCl content to the original NaCl content is used to specifically express the salt-reducing effect of the peptide.

In order for the salty taste enhancer of the present invention to exert its effect, it is necessary to use salt in combination with the salty taste enhancer. In the field of health food applications, most foods and beverages that originally contain salt, such as condiments or soups, are generally required to reduce the salt content. Therefore, the salty taste enhancer exerts a salty taste enhancement effect by coexisting with the salt contained in the food and beverage. In addition, the salty taste enhancer of the present invention is almost not accompanied by peculiar smell and odor, is non-toxic and harmless as a protein peptide, and has certain nutritional value. Therefore, the salty taste enhancer of the present invention coexists with the salt in food and beverage to achieve salt reduction and better preservation of the texture and flavor of the food.

The present invention also provides a condiment comprising the above polypeptide or a salt thereof. The condiment of the present invention is not particularly limited as long as the condiment comprises the above polypeptide or a salt thereof and can be used for seasoning food. Specific examples include, but are not limited to: soy sauce, miso, sauce and ketchup; condiments comprising hydrolyzed animal and plant proteins, yeast extracts, amino acids, peptides, etc. as main ingredients; and soup powder, seasoning soy sauce, bean paste and seasoning.

From the viewpoint that the above polypeptide or its salt exerts a salty taste enhancing effect by coexisting with sodium chloride (salt), as a preferred aspect of the seasoning of the present invention, an embodiment further comprising sodium chloride is provided. Specifically, the seasoning comprises the above polypeptide or its salt (salty taste enhancer of the present invention) and sodium chloride; and the seasoning obtained by mixing such seasoning with additives such as excipients, pigments or spices that can be used in the manufacture of food and beverages as required.

The present invention also provides food and beverages comprising the above polypeptide or its salt. The types of food and beverages are not particularly limited, for example: condiments such as soy sauce, miso, sauces and ketchup; condiments containing hydrolyzed animal and plant proteins, yeast extracts, amino acids, peptides, etc. as main ingredients; seasoning products for seasoning of food, such as soup powder, seasoning soy sauce, bean paste and seasoning; processed cereal foods such as noodles, bread and snacks; processed meat and fish such as ham and sausage and fish paste; soup; pickles; and daily dishes. In addition, food and beverages include instant foods that can be cooked by adding hot water or water (for example, instant noodles powder and liquid soup, instant consommé, French soup, Chinese soup, miso soup, clear soup and soup instant noodles).

The amount of the salty taste enhancer of the present invention added to food and beverage is not particularly limited. Food and beverages that are required to have a reduced salt content are set according to the concentration when the food and beverage are ingested.

The salty taste enhancer of the present invention can be provided in the form of the above-mentioned polypeptide alone, or can be provided in the form of a solid composition or a liquid composition. When provided in the form of a composition, the salty taste enhancer can contain additives such as excipients, pigments or spices that can be used to manufacture food and beverages as needed, as long as the salty taste enhancement effect is not inhibited.

In some embodiments, the salty taste enhancer of the present invention can be provided in the form of a food additive comprising the above polypeptide or a salt thereof.

The present invention also provides a method for enhancing the salty taste of food and beverages. The method includes the step of adding the above-mentioned polypeptide or its salt to food and beverages. The specific technology of adding the above-mentioned polypeptide or its salt to food and beverages is not particularly limited. The above-mentioned polypeptide or its salt can be mixed in the form of one of the raw materials during the manufacture of food and beverages, or can be added to the food and beverage immediately before ingestion of food and beverages. The amount of the above-mentioned polypeptide or its salt added is not particularly limited.

Example 1

1. Study on the interaction between salty taste enhancing peptides and TMC4.

SSDDK, GEAVK and CAYPQTK were synthesized by solid phase synthesis. After structural confirmation, the molecular weights of the peptides SSDDK, GEAVK and CAYPQTK were 550.49, 502.55 and 809.92 Da, respectively, and the purities were 85.23%, 96.34% and 88.38%, respectively.

Referring to Figures 2 and 3, the peptide SSDDK binds to the amino acid residues GLU323, GLU284, GLU286, ARG424, ARG330, ARG580, SER290 and ALA283, forming 8 conventional hydrogen bonds, 5 charge interactions, 3 carbon-hydrogen bonds, 1 receptor interaction and 1 salt bridge. The results show that conventional hydrogen bonds are the main force for the binding of peptides to TMC4, which is consistent with previous research results. The H-bond donor and H-bond acceptor regions in the interacting peptides and amino residues are shown in Figure 4. Charge interactions and carbon-hydrogen bonds serve as secondary binding forces to assist in stabilizing the binding of peptides. In addition, when the amino acid residues ARG583 (3 conventional hydrogen bonds and 1 charge interaction), ARG330 (1 conventional hydrogen bond, 2 charge interactions and 1 salt bridge) and GLU284 (1 carbon-hydrogen bond, 1 charge interaction and 1 receptor interaction) bind to the peptide SSDDK, there are more than 2 binding forces, so these three amino acid residues can be considered as the key amino acid residues for binding.

Example 2

1. Evaluation of salt reduction effect.

The SA402B electronic tongue was used to evaluate the salt reduction effect.

First, the selected peptides were tested for 25% salt reduction effect, that is, 0.20mg/mL NaCl solution was used as the control group, and the saltiness values of the mixed solution of 0.05mg/mL peptide + 0.15mg/mL NaCl were compared to verify the 25% salt reduction effect. After that, the peptides with good salt reduction effect were further tested for 50% and 75% salt reduction effects. All solutions were analyzed 3 times.

0.20mg/mL NaCl solution was used as the positive control group and compared with the mixed solution of 0.05mg/mL peptide and 0.15mg/mL NaCl. The results are shown in Figure 5a. The differences in the saltiness of each component were statistically significant (P<0.05). The peptide SSDDK has a good salt reduction effect. After replacing 25% NaCl, the saltiness value not only did not decrease, but increased significantly. In contrast, the saltiness values of the peptides GEAVK and CAYPQTK decreased significantly and could not achieve the effect of salt reduction. The results of electronic tongue analysis are similar to the results of molecular docking energy. The reason why the peptides GEAVK and CAYPQTK cannot play a role in reducing salt may be that the binding between them and the TMC4 receptor is still not tight enough. Further research on the peptide SSDDK will continue to verify the 50% and 75% salt reduction effects. The results are shown in Figure 5b. After 0.05 mg/mL peptide SSDDK replaced 50% and 75% of NaCl, respectively, there was no significant difference in the saltiness value. Although it did not enhance the saltiness, it still replaced part of NaCl and played a role in reducing salt. This situation that the salt reduction effect continued to weaken as the ratio of synthetic peptide to NaCl concentration increased is consistent with previous studies. The proven salty taste enhancing peptide YDPNDPEK can achieve a 30% salt reduction effect when added to a 45 mmol/L NaCl solution at a concentration of 0.4%; another proven salty taste enhancing peptide DIQPEER can replace about 50% of NaCl compared to a 4.0 g/L NaCl solution. In summary, peptide SSDDK has a good salt reduction effect. It can enhance the saltiness of the solution at low concentrations, and can also achieve a salt reduction effect without reducing the saltiness at high concentrations.

The salty taste enhancing peptide SSDDK can be mixed with salt and added as a food additive to various foods or beverages, including but not limited to meat products, seafood products, puffed foods, soy sauce and sauces. It can replace up to 75% of the original NaCl content, achieving the effect of reducing sodium salt without reducing the saltiness. At a lower concentration, when it only replaces 25% of the original NaCl content, its saltiness is significantly higher than the saltiness of the original NaCl content, achieving the effect of enhancing the saltiness. When applied to actual food production, it can significantly reduce the use of sodium salt, reduce the risk of consumers suffering from cardiovascular and kidney diseases due to excessive sodium salt intake, and protect consumers' health. At the same time, it will not cause significant damage to the flavor of the product, reduce consumer preference, and maintain the product's strength.

For matters not mentioned above, the prior art applies.

Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, but will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A salty taste enhancer, characterized in that the salty taste enhancer comprises a polypeptide as shown in SEQ ID NO: 1 or a salt thereof, wherein the amino acid sequence of the polypeptide as shown in SEQ ID NO: 1 is SSDDK. 2. The salty taste enhancer according to claim 1, characterized in that the polypeptide as shown in SEQ ID NO: 1 is molecularly docked with the salty taste receptor TMC4, and the key amino acids for its binding are ARG583, ARG330 and GLU284. 3. The salty taste enhancer according to claim 2, characterized in that the polypeptide replaces no more than 25% of sodium chloride to enhance the salty taste. 4. The salty taste enhancer according to claim 2, characterized in that the polypeptide replaces 50% to 75% of sodium chloride without reducing the salty taste. 5. A food additive, characterized in that it comprises the polypeptide or a salt thereof according to claim 1. 6. A seasoning, characterized in that it comprises the polypeptide or a salt thereof according to claim 1. 7. The seasoning as claimed in claim 6, characterized in that it also includes sodium chloride. 8. A food, characterized in that it comprises the polypeptide or a salt thereof according to claim 1. 9. The food of claim 8, further comprising sodium chloride. 10. A method for enhancing salty taste, characterized in that the salty taste enhancer according to claim 1 is added to food or beverage containing salt.