CN114504097B

CN114504097B - Umami peptide, composition containing same and application

Info

Publication number: CN114504097B
Application number: CN202210408389.8A
Authority: CN
Inventors: 张玉玉
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2022-08-19
Anticipated expiration: 2042-04-19
Also published as: CN114504097A

Abstract

The present invention relates to an umami peptide, compositions comprising the same and uses thereof. The sequence of the umami peptide provided by the application is TPLVDR. The application provides an umami peptide with ACE enzyme inhibitory action, has better salty taste perception, can replace partial sodium chloride as the substitute salt, can reach and subtract the purpose that salt does not reduce salt, owing to have ACE enzyme inhibitory action, can play the effect of alleviating to cardiovascular disease simultaneously.

Description

Umami peptide, composition containing same and application

Technical Field

The invention belongs to the field of salt substitutes, and particularly relates to umami peptide, a composition containing the umami peptide and application of the umami peptide.

Background

Salty taste is the first of five tastes and is the favorite taste of people. At present, most of edible salt used by people is sodium salt, and excessive intake of sodium can cause hypertension, increase the incidence of cardiovascular and cerebrovascular diseases such as heart disease and stroke, but the reduction of the use of edible salt can greatly influence the flavor of food.

The data show that the umami peptide has the function of increasing the salty taste, so the proper use of the umami peptide as the food additive can achieve the function of reducing the salty taste but not reducing the salty taste. And the food additive is widely applied to instant noodles, meat products, chicken powder and seasonings, puffed and leisure foods, quick-frozen foods and the like.

There is a need in the art to develop a umami peptide with salt-substituting effect, which can improve the risk of hypertension or angiosclerosis diseases caused by sodium salt.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present application to provide an umami peptide having the sequence TPLVDR.

The umami peptide provided by the application has better salty taste perception, can be used as a substitute salt to replace partial sodium chloride, and can achieve the purpose of reducing salt without reducing salty taste. In addition, the umami peptide provided by the application also has a certain ACE enzyme inhibition effect.

ACE enzyme is key to cardiovascular therapy and plays an important role in regulating blood pressure through the renin-angiotensin system. ACE enzyme converts angiotensin i to angiotensin ii, which is a vasoconstrictor and inactivates bradykinin, a potent vasodilator, leading to elevated blood pressure.

The umami peptide provided by the application has obvious ACE enzyme inhibitory activity, and the long-term consumption of the umami peptide containing ACE enzyme inhibitory activity can obviously reduce blood pressure and relieve cardiovascular and cerebrovascular diseases.

That is, the umami peptide provided by the application not only can replace part of sodium chloride to achieve the effects of reducing salt and not reducing salt, reducing the intake of the sodium chloride and reducing the incidence of cardiovascular diseases, but also can inhibit ACE enzyme, reduce the incidence probability of the cardiovascular diseases caused by the sodium chloride which is not replaced, or improve the cardiovascular problems of a user per se.

Another object of the present application is to provide a use of the umami peptide according to one of the objects, as any one or a combination of at least two of health product additives and health food additives;

preferably, the health care product comprises a health food for inhibiting hypertension and/or a health food for softening blood vessels.

Preferably, the umami peptide is used at a concentration of 0.28-0.44 mg/mL, such as 0.30mg/mL, 0.33mg/mL, 0.35mg/mL, 0.37mg/mL, 0.39mg/mL, 0.40mg/mL, 0.42mg/mL, and the like.

It is a further object of the present application to provide a salt substitute mixture comprising the umami peptide of one of the objects and a second peptide; the sequence of the second peptide is AEINKILGN.

Preferably, the mixing molar ratio of the umami peptide and the second peptide is 1: 9-2: 3 (for example, 2:9, 1:3, 4:9, 5:9, etc.), and preferably 3: 7-3: 2.

The taste (such as bitter) that the umami peptide concentration of this application purpose is too high slightly to take bad taste, has restricted the addition of umami peptide, the umami peptide that this application provided with as substituting salt mixture after the second peptide mixes, can improve its sodium chloride's substitute quantity, can also keep ACE enzyme's inhibitory effect simultaneously.

A fourth object of the present application is to provide the use of a salt substitute mixture according to the third object as a salty taste additive.

Preferably, the salt substitute mixture is used as a salty additive for instant noodles, a salty additive for meat products, a salty additive for puffed and snack foods, a salty additive for quick-frozen foods, a salty additive for instant soups, a salty additive for thick soups or a salty additive for low-sodium seasonings.

Alternatively, the salt substitute mixture is used for a health food for inhibiting hypertension and/or a health food for softening blood vessels.

Preferably, the salt-substituting mixture is used in a concentration of 77-244 mg of the third or 5 salt-substituting mixture added per 100mL of water.

The fifth purpose of the present application is to provide a salty composition comprising 1.65-1.80 parts by weight of common salt and 1 part of the salt substitute mixture described in the third purpose.

It is a further object of the present application to provide a umami enhancing peptide for enhancing saltiness, which has the sequence AEINKILGN.

Preferably, the refreshing peptide is used as any one of or a combination of at least two of a health product additive and a health food additive.

Preferably, the freshening peptides are used as a health food for inhibiting hypertension and/or a health food for softening blood vessels.

Preferably, the use concentration of the freshness-enhancing peptide is 0.50-2.00 mg/mL.

In umami peptides, T is threonine (Thr), P is proline (Pro), L is leucine (Leu), V is valine (Val), D is aspartic acid (Asp), R is arginine (Arg); a is alanine (Ala), E is glutamic acid (Glu), I is isoleucine (Ile), N is asparagine (Asn), K is lysine (Lys), and G is glycine (Gly).

It is noted that the umami peptide of sequence TPLVDR and the umami peptide of sequence AEINKILGN provided herein can be synthesized by standard methods or by several synthetic routes well known to those skilled in the art of peptide synthesis to prepare the short-chain peptides of the invention.

Illustratively, the umami peptide of sequence TPLVDR and the umami peptide of sequence AEINKILGN can be synthesized using the methods described below and conventional techniques known to those skilled in the art of peptide synthesis or variations thereof as understood by those skilled in the art.

If a solid phase method is adopted to synthesize the delicious peptide or the flavor enhancing peptide, the Wang resin is taken as a solid phase carrier, the resin is soaked and swelled and then washed, proline and a condensing agent protected by Fmoc are added according to the sequence of the target peptide to carry out deprotection and condensation reaction of amino, the deprotection-condensation process is repeated until all amino acids are connected, and the target peptide is obtained after the resin is cut.

Exemplary of these are the umami peptide TPLVDR (Thr-Pro-Leu-Val-Asp-Arg), which can be synthesized by the following solid phase method:

if fresh peptide is synthesized by adopting a solid phase method, fmoc-Arg (pbf) -queen resin is soaked, swelled and washed, reacted for 15min by using a decapping solution (piperidine and N, N-dimethylformamide) and then washed to remove fmoc, then fmoc-Asp-OH amino acid is added, TBTU (O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate) is used as a condensing agent, N, N-diisopropylethylamine and a solvent N, N-dimethylformamide are used as an alkali reagent to carry out condensation reaction for 30min, and then washed; then adding a decapping solution (piperidine + N, N-dimethylformamide) to react and remove fmoc, and washing; repeating the steps to graft Val, Leu, Pro to Thr in sequence, finally performing a cutting reaction on the trifluoroacetic acid cutting fluid and the resin condensed with the amino acid, and adding ether into the filtrate to separate out the polypeptide to obtain a crude product; and then purifying the crude product by using ion exchange resin, converting trifluoroacetate into acetate, and freeze-drying to obtain a high-purity product.

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

(1) the application provides an umami peptide with ACE enzyme inhibitory action, has better salty taste perception, can replace partial sodium chloride as the substitute salt, can reach and subtract the purpose that salt does not reduce salt, owing to have ACE enzyme inhibitory action, can play the effect of alleviating to cardiovascular disease simultaneously.

(2) In the salty taste composition provided by the application, the delicious peptide can show bad taste (such as bitter taste) when being excessively concentrated, the addition amount of the delicious peptide is limited, and the delicious peptide provided by the application and the second peptide (flavor enhancing peptide) are mixed to be used as a salt substitute mixture, so that the substitution amount of sodium chloride can be increased, and the inhibiting effect of ACE enzyme can be kept.

Drawings

FIG. 1 is an HPLC chromatogram of umami peptide TPLVDR;

FIG. 2 is a graph of a second mass spectrometric analysis of the umami peptide TPLVDR;

fig. 3 is an HPLC chromatogram of the umami peptide AEINKILGN;

fig. 4 is a secondary mass spectrometry analysis of the freshener peptide AEINKILGN.

Detailed Description

The technical solution of the present invention is further explained with reference to the following embodiments, but it should be noted that the embodiments are only an embodiment and explanation of the spirit of the technical solution of the present invention, and should not be construed as a limitation to the scope of the present invention.

The reagents and instruments used in the examples are commercially available, and the detection method is a conventional method well known in the art.

Synthesis example 1

The Nanjing-derived peptide Biotechnology Ltd was entrusted with the synthesis of the umami peptide TPLVDR.

The purity of the synthesized TPLVDR was checked by HPLC (equipment model Agilent 1100), and as shown in fig. 1 (fig. 1 is an HPLC chromatogram of umami peptide TPLVDR), the purity was 98.72%.

The molecular weight and structure were determined by HPLC-MS (equipment model AB SCIEX X500R Q-TOF), and as shown in FIG. 2 (FIG. 2 is a secondary mass spectrometry analysis chart of umami peptide TPLVDR), the molecular weight was 699.39 Da and the structure was H-Thr-Pro-Leu-Val-Asp-Arg-OH.

Synthesis example 2

The peptide AEINKILGN was synthesized by Nanjing peptide Biotechnology Ltd.

The purity of the synthesized AEINKILGN was determined by HPLC (equipment model Agilent 1100), and as shown in fig. 3 (HPLC chromatogram of zenith peptide AEINKILGN in fig. 3), the purity was 98.96%.

The molecular weight and structure were determined by HPLC-MS/MS (equipment model AB SCIEX X500R Q-TOF), and as shown in FIG. 4 (FIG. 4 is a secondary mass spectrum analysis chart of Zengn peptide AEINKILGN), the molecular weight was 970.54Da and the structure was H-Ala-Glu-Ile-Asn-Lys-Ile-Leu-Gly-Asn-OH.

And (3) flavor analysis:

14 sensory evaluation panelists who were healthy (8 females and 6 males, aged 24 to 30 years) and had sensory evaluation experience were recruited from the laboratory. Firstly, taste training for 2 weeks is carried out on evaluation team members, and the training contents are as follows: and presenting the aqueous solutions of the six sour, sweet, bitter, salty, fresh and astringent taste markers to a sensory evaluator. The standards are food grade sucrose (50.00 g/L and 10.00 g/L), citric acid (5.00 g/L and 1.00 g/L), sodium chloride (15.00 g/L and 3.00 g/L), sodium glutamate (20.00 g/L and 4.00 g/L), quinine (0.006 g/L and 1.20 g/L) and tea polyphenol (5.00 g/L and 1.00 g/L), respectively. Screening is carried out according to whether the evaluator can correctly judge the difference between the two concentrations.

The method comprises the steps of taking the fresh taste peptide TPLVDR and the refreshing peptide AEINKILGN to respectively prepare 5g/L evaluation solution (a solvent is pure water), carrying out gradient dilution on the evaluation solution by the pure water according to the proportion of 1:1, wherein the concentrations are respectively 1250.00 mug/mL, 900.00 mug/mL, 800.00 mug/mL, 700.00 mug/mL, 625.00 mug/mL, 450.00 mug/mL, 312.50 mug/mL, 156.25 mug/mL, 78.125 mug/mL and 39.0625 mug/mL, and then submitting the solutions to sensory evaluation personnel for taste sorting of the taste intensity of the sensory evaluation personnel. When the difference in taste between a certain dilution level of the solution and the blank is not recognized by any one of the evaluators, the dilution concentration of a sample is recorded as the threshold value of the sample in the blank solution (pure water) in the experiment.

The threshold value determination experiment was repeated 5 times, and the threshold value results were averaged to obtain the threshold value of the sample in the blank solution (pure water).

According to determination, the threshold value of the umami peptide TPLVDR is 88.3883 mug/mL, and the threshold value of the freshness increasing peptide AEINKILGN is 707.1068 mug/mL.

Example 1

A salt substitute mixture comprising 1mol of the umami peptide TPLVDR and 9mol of the umami peptide AEINKILGN.

Example 2

A salt substitute mixture comprising 3mol of umami peptide TPLVDR and 7mol of umami peptide AEINKILGN.

Example 3

A salt substitute mixture comprising 1mol of umami peptide TPLVDR and 4mol of umami peptide AEINKILGN.

Example 4

A salt substitute mixture comprising 2mol of umami peptide TPLVDR and 3mol of umami peptide AEINKILGN.

Salt substitute quantity test:

(1) preparing a saline solution to be detected:

the salt substitute mixture provided in the example was dispersed in pure water together with sodium chloride to obtain a salty solution to be tested. In the salty solution, the content of sodium chloride was 0.3wt%, and the addition amount of the substitute salt mixture was 2 mmol/L.

Dispersing the umami peptide TPLVDR and sodium chloride in pure water to obtain the umami peptide solution to be detected. In the umami peptide solution, the content of sodium chloride is 0.3wt%, and the addition amount of the umami peptide TPLVDR is 0.6 mmol/L.

Dispersing the flavor enhancing peptide AEINKILGN and sodium chloride in pure water to obtain the flavor enhancing peptide solution to be detected. In the umami peptide solution, the content of sodium chloride is 0.3wt%, and the addition amount of the flavor enhancing peptide AEINKILGN is 2 mmol/L.

(2) Preparing sodium chloride solution with gradient concentrations of 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt% and 0.5wt% as standard solution, selecting an evaluator of 'taste analysis' to taste the salinity of the salty solution to be detected, judging the concentration of the standard solution close to the salinity of the salty solution to be detected, and calculating the average value of the concentration and marking as X (wt%); the salt substitution amount A is calculated by the following formula.

A%=(X-0.3wt%)/X

The results are shown in Table 1.

Assay for ACE converting enzyme inhibitory activity test:

(1) identification of antihypertensive activity:

preparation of saline BBS buffer (containing 0.9% dNaCl): transferring 100mL of 0.5M standard boric acid buffer solution (0.1M, pH 8.0) into a 500mL quantitative bottle, adding 0.015mol of NaCl solid, namely 0.8766g (the relative molecular mass is 58.44 g/mL), adding deionized water for constant volume, and storing at 0 ℃;

preparing an ACE enzyme (0.1U/mL) solution: dissolving 0.1U of ACE enzyme in 1mL of salt-containing BBS buffer solution (containing 0.03 MNaCl), and storing at-20 ℃ for later use;

preparing a HHL (equacyl-histidyl-leucine) solution: dissolving 5mg in 10mL of salt-containing BBS buffer solution to prepare a HHL solution of 2.5mg/mL, and storing at-20 ℃;

preparing a peptide sample: preparing the salt-substituting mixture, the umami peptide TPLVDR and the flavor enhancing peptide AEINKILGN provided by the embodiments 1-4 into 2mmol/L solutions (the solvent is BBS buffer solution containing 0.9% NaCl) respectively to be used as peptide samples;

and (3) testing: mixing 20 μ L of peptide sample with 100 μ L of HHL solution, preheating to 37 deg.C, adding 20 μ L of LACE converting enzyme solution, performing metal bath at 37 deg.C for 2 hr, adding 100 μ L of hydrochloric acid to inactivate when the temperature is returned to room temperature, and analyzing the reaction solution with 0.45 μm organic filter membrane.

In order to reduce errors, the reaction group is provided with 2 times of repeated experiments, and each group is provided with a blank group (20 mu L of peptide sample is taken to be mixed with 100 mu L of HHL solution and preheated to 37 ℃, 20 mu L of salt-containing BBS buffer solution is added, metal bath is carried out for 2h at 37 ℃, 100 mu L of hydrochloric acid is added for inactivation when the temperature is recovered to the room temperature, the reaction solution passes through a 0.45 mu M organic filter membrane and then is subjected to sample injection analysis), and in order to ensure the rigor of the experiment, a control group is made (20 mu L of salt-containing BBS buffer solution is taken to be mixed with 100 mu L of HHL solution and preheated to 37 ℃, 20 mu L of LACE converting enzyme solution is added, metal bath is carried out for 2h at 37 ℃, 100 mu L of hydrochloric acid is added for inactivation when the temperature is recovered to the room temperature, and the reaction solution passes through a 0.45 mu M organic filter membrane and then is subjected to sample injection analysis).

The test results are shown in Table 1.

TABLE 1

Examples of the invention	Amount of salt substitute	Inhibition rate
			Example 1	15	81.0%
Example 2	18	80.3%
			Example 3	20	81.1%
Example 4	22	80.4%
			Umami peptide TPLVDR	8	77.4%
Zengxian peptide AEINKILGN	5	74.4%

From table 1, the umami peptide TPLVDR provided by the application has a high ACE inhibition rate, and is matched with the freshening peptide AEINKILGN, so that the substitution amount of sodium chloride can be increased, and the inhibition effect of ACE enzyme can be maintained.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An umami peptide, wherein the sequence of the umami peptide is TPLVDR.

2. The use of the umami peptide of claim 1 as any one of or a combination of at least two of a nutraceutical additive, a health food additive;

the health care product comprises a health food for inhibiting hypertension and/or a health food for softening blood vessels.

3. The use of claim 2, wherein the umami peptide is used at a concentration of 0.28-0.44 mg/mL.

4. A primary salt mixture comprising the umami peptide of claim 1 and a second peptide;

the sequence of the second peptide is AEINKILGN.

5. The salt-substituting mixture of claim 4, wherein the mixing molar ratio of the umami peptide and the second peptide in claim 1 is 1: 9-2: 3.

6. Use of a salt substitute mixture according to claim 4 or 5 as a salty taste additive.

7. Use of the salt-substituting mixture according to claim 6 as a salty additive for instant noodles, for meat products, for puffed and snack foods, for quick-frozen foods, for instant soups or for low-sodium seasonings;

8. Use of a primary salt mixture according to claim 6 in a concentration of 77-244 mg per 100mL water of a primary salt mixture according to claim 4 or 5.

9. A salty composition comprising 1.65 to 1.80 parts by weight of common salt and 1 part of the salt substitute mixture according to claim 4 or 5.

10. A umami enhancing peptide for enhancing salty taste, wherein the sequence of the umami enhancing peptide is AEINKILGN.