CN116391846B

CN116391846B - Sweet taste enhancing peptide of steviol glycoside, composition containing sweet taste enhancing peptide and application of sweet taste enhancing peptide

Info

Publication number: CN116391846B
Application number: CN202310467580.4A
Authority: CN
Inventors: 张玉玉; 肖明华; 李沛; 李库; 熊建; 郭辉; 李志刚; 梁莉; 郝志林
Original assignee: Angel Yeast Co Ltd
Current assignee: Angel Yeast Co Ltd
Priority date: 2023-04-27
Filing date: 2023-04-27
Publication date: 2024-06-07
Anticipated expiration: 2043-04-27
Also published as: CN116391846A

Abstract

The present application relates to a sweet taste enhancing peptide of steviol glycoside, a composition comprising the same and uses thereof. The sequence of the sweet taste enhancing peptide of steviol glycoside is VIIGGGDTAT, and the sweet taste enhancing composition of steviol glycoside comprises a short peptide VIIGGGDTAT and a short peptide KVVITAPSS. The stevioside sweet taste enhancing peptide provided by the application can reduce the use amount of stevioside without reducing the sweet taste, reduce the bad taste caused by adding a large amount of stevioside, has better stevioside sweet taste enhancing effect, has better ACE enzyme activity inhibition, and reduces blood pressure to relieve cardiovascular and cerebrovascular diseases.

Description

Sweet taste enhancing peptide of steviol glycoside, composition containing sweet taste enhancing peptide and application of sweet taste enhancing peptide

Technical Field

The invention belongs to the field of sugar substitute sweetness enhancement, and particularly relates to a stevioside sweetness enhancement peptide, a composition containing the stevioside sweetness enhancement peptide and application of the stevioside sweetness enhancement peptide.

Background

Sweetness is one of the five basic tastes and can give a pleasant sensation. However, free sugar intake is liable to cause problems such as obesity, diabetes and caries, and reduction of sucrose content in foods has become an increasingly important product development trend. Currently, the consumer acceptance impact of sucrose reduction in foods can be overcome by the use of sweeteners. However, the addition of natural sweeteners or artificial sweeteners is too large to increase sweetness, but may result in unpleasant aftertaste and mouthfeel.

The stevioside is mainly present in leaves of stevia rebaudiana Bertoni of Compositae, the sweetness of the purified stevioside is 250-300 times that of sucrose, and the stevioside has afterbitter taste and green grass taste when the usage amount is large.

Therefore, how to reduce the usage amount of stevioside while maintaining sweetness without reducing taste is a technical problem to be solved in the art.

Disclosure of Invention

In view of the shortcomings of the prior art, one of the purposes of the present application is to provide a sweet taste enhancing peptide of steviol glycoside, the sequence of which is VIIGGGDTAT.

The sweet taste enhancing peptide of stevioside provided by the application can effectively reduce the usage amount of stevioside on the premise of not reducing sweet taste perception, and avoid unpleasant odors such as afterbitter taste, green grass taste and the like caused by adding a large amount of stevioside.

In addition, the short peptide VIIGGGDTAT has an ACE enzyme inhibition rate of 18.50%, and has a slight improvement effect on cardiovascular problems.

It is a further object of the present application to provide a use of the sweetness enhancer peptide according to one of the objects for enhancing the sweetness of steviol glycoside.

The sweet taste enhancing peptide and the stevioside are added into products requiring sweet taste, such as dairy products, beverages, jam, seasonings, essence and spices, and the like, so that the obtained food has richer sweet taste, can cooperate with the stevioside under the action of the sweet taste enhancing peptide, can keep good taste, simultaneously reduce the usage amount of the stevioside, and avoid bad taste caused by excessive stevioside.

The sweetness enhanced peptide provided by the application can be used as a sweetness providing ingredient of beverages, dairy products, coffee, jam and the like.

Preferably, the sweetness enhancing peptide is used at a concentration of 2.3-9.0 g sweetness enhancing peptide (e.g., 2.36g、2.65g、2.89g、3.33g、3.45g、3.78g、3.90g、4.12g、4.57g、4.98g、5.35g、5.55g、5.85g、6.48、6.92g、7.33g、7.55g、8.68g、8.90g, etc.) per gram steviol glycoside.

The application further provides a sucrose substitute, which is characterized by comprising 1 part by weight of steviol glycoside and 2.3-9.0 parts by weight of the sweetness enhancing peptide.

The sweetness of the sucrose substitute provided by the application in 100g of water can be equivalent to that of about 3.73-4.2 g of sucrose in 100g of water.

The sucrose substitute provided by the application can replace sucrose in products such as beverage, dairy products, coffee, jam and the like to provide sweetness

It is a fourth object of the present application to provide a sweet taste enhancing composition of steviol glycosides, which comprises short peptide VIIGGGDTAT and short peptide KVVITAPSS.

The sweetness enhancing composition has a synergistic effect, has a stronger adding effect on stevioside, and meanwhile, the short peptide KVVITAPSS has an ACE enzyme inhibition rate of 82.77%.

ACE enzyme is a substance that regulates blood pressure through the renin-angiotensin system, and is capable of converting angiotensin i into angiotensin ii, which is an vasoconstrictor and inactivating bradykinin, resulting in an increase in blood pressure.

The short peptide KVVITAPSS in the sweet taste enhancing composition provided by the application has obvious ACE enzyme inhibition activity, and can obviously reduce blood pressure and relieve cardiovascular and cerebrovascular diseases after long-term eating of substances containing ACE enzyme activity inhibition.

Namely, the sweet taste enhancing composition provided by the application can increase the sweet taste of stevioside, reduce the usage amount of stevioside, reduce the ingestion of sucrose, reduce the occurrence of problems such as obesity, diabetes and decayed teeth, inhibit ACE enzyme, reduce the incidence of cardiovascular diseases and improve the cardiovascular problems of users.

Preferably, in the sweet taste improving composition, the mass ratio of the short peptide VIIGGGDTAT to the short peptide KVVITAPSS is 3:1-1:1.

It is a fifth object of the present application to provide a use of the sweet taste improving composition according to the fourth object for improving the sweet taste of steviol glycosides.

The enhancing effect of the sweet taste enhancing composition on steviol glycosides is more pronounced than the short peptide VIIGGGDTAT alone.

The sweetness enhancing compositions provided herein are useful as sweetness-providing ingredients for beverages, dairy products, coffee, jams, and the like.

Preferably, the sweet taste enhancing composition is used in an amount of 0.58g to 10.20g of sweet taste enhancing composition per gram of steviol glycoside.

It is a sixth object of the present application to provide a sweet taste enhancing auxiliary peptide of steviol glycoside, which has a sequence KVVITAPSS.

Although its use alone does not significantly enhance sweetness, it can enhance the sweetness enhancing effect of the short peptide VIIGGGDTAT on steviol glycosides when used in admixture with the short peptide VIIGGGDTAT. Meanwhile, the sweet taste enhancement auxiliary peptide has 82.77% of ACE enzyme inhibition rate and has the function of improving cardiovascular problems. Namely, the short peptide KVVITAPSS has the effect of synergistically enhancing the sweetening effect of the short peptide VIIGGGDTAT on stevioside and also has the effect of synergistically improving cardiovascular problems.

The seventh object of the present application is to provide a use of the sweetness enhanced auxiliary peptide according to the sixth object, as any one or a combination of at least two of a health-care additive and a health food additive;

the health food comprises health food for inhibiting hypertension and/or health food for softening blood vessel.

In the short peptide provided by the application, T is threonine (Thr), P is proline (Pro), L is leucine (Leu), V is valine (Val), D is aspartic acid (Asp), and 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).

The short peptides VIIGGGDTAT and KVVITAPSS provided herein may be synthesized by standard methods, or may be prepared using several synthetic routes well known to those skilled in the art of peptide synthesis.

Illustratively, short peptides VIIGGGDTAT and KVVITAPSS may be synthesized using the methods described below as well as conventional techniques known to those skilled in the art of peptide synthesis or variations thereof as understood by those skilled in the art.

If the sweet taste enhancement peptide is synthesized by adopting a solid phase method, synthesizing the polypeptide from the C end to the N end, sequentially adding amino acid, adding Fmoc-protected proline and condensing agent according to the sequence of the target peptide, performing deprotection and condensation reaction of amino, repeating the deprotection-condensation process until all the amino acids are connected, and purifying.

Exemplary peptides VIIGGGDTAT (Val-Ile-Ile-Gly-Gly-Asp-Thr-Ala-Thr) are synthesized by the solid phase method as follows:

If a solid phase method is adopted to synthesize sweet taste enhancement peptide, fmoc-Val (pbf) -king resin is soaked and swelled and then washed, uncapping liquid (N, N-dimethylformamide+piperidine+acetic anhydride) is used for reacting for 15min and then washing is carried out, fmoc is removed, fmoc-Ile-OH amino acid is then added, HBTU (benzotriazol-N, N, N ', N' -tetramethyl urea hexafluorophosphate) and HOBT (1-hydroxybenzotriazole) are used as condensing agents, N, N-diisopropylethylamine and solvent N, N-dimethyl-4-pyridine amine are used as basic reagents, and condensation reaction is carried out for 40min at room temperature and then washing is carried out; adding uncapping liquid (N, N-dimethylformamide, piperidine and acetic anhydride) for reaction to remove fmoc and washing; repeating the steps to sequentially graft Ile, gly, gly, gly, asp, thr, ala to Thr, and eluting the peptide chain from the branch by trifluoroacetic acid after the target sequence is connected to obtain a crude product; and purifying the crude product by adopting an RP-HPLC C18 column, converting the trifluoroacetate into acetate, and freeze-drying to obtain a high-purity product.

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

(1) The application provides a stevioside sweetness enhancement peptide, which can reduce the usage amount of stevioside and reduce bad taste caused by adding a large amount of stevioside under the condition of not reducing sweetness.

(2) The application also provides a stevioside sweet enhancement composition, which is compounded with the short peptide VIIGGGDTAT and the short peptide KVVITAPSS, has better stevioside sweet enhancement effect, and simultaneously has better ACE enzyme activity inhibition, reduces blood pressure and relieves cardiovascular and cerebrovascular diseases.

(3) The sweet taste enhancing auxiliary peptide of stevioside provided by the application not only can assist the sweet taste enhancing peptide of stevioside to enhance the sweetness of stevioside, but also can improve the ACE enzyme activity inhibition and cardiovascular problems.

Drawings

FIG. 1 is an HPLC chromatogram of sweetness enhancing peptide VIIGGGDTAT;

FIG. 2 is a graph of a secondary mass spectrometry analysis of sweetness enhanced peptide VIIGGGDTAT;

FIG. 3 is an HPLC chromatogram of sweet enhancement helper peptide KVVITAPSS;

Fig. 4 is a graph of a secondary mass spectrometry analysis of the sweetness enhanced helper peptide KVVITAPSS.

Detailed Description

The following description of the present invention will further illustrate the technical solution of the present invention in conjunction with the specific embodiments, but should be construed as merely embodying the spirit and explanation of the technical solution of the present invention, and should not be construed as limiting the scope of the present invention.

Reagents and apparatus for the examples are commercially available and the detection methods are conventional and well known in the art.

Synthesis example 1

Sweet taste enhancing peptides VIIGGGDTAT were synthesized by the company Nanjing source peptide biotechnology limited.

The purity of the synthesized VIIGGGDTAT was checked by HPLC (apparatus model Agilent 1100) and was 98.38% as shown in fig. 1 (fig. 1 is an HPLC chromatogram of sweetness enhanced peptide VIIGGGDTAT).

Molecular weight and structure were determined using Easyn-LC-Orbitrap-Lumos (apparatus model Thermo FISHER SCIENTIFIC, wilmington, DE, USA), and as shown in FIG. 2 (FIG. 2 is a secondary mass spectrometry diagram of sweetness enhanced peptide VIIGGGDTAT), molecular weight was 903.47 Da and structure H-Val-Ile-Ile-Gly-Gly-Gly-Asp-Thr-Ala-Thr.

Synthesis example 2

Sweet taste enhancing helper peptides KVVITAPSS were synthesized by the company Nanjing source peptide biotechnology limited.

The purity of the synthesized KVVITAPSS was checked by HPLC (apparatus model Agilent 1100) and was 98.18% as shown in fig. 3 (fig. 3 is an HPLC chromatogram of the sweetness enhanced helper peptide KVVITAPSS).

Molecular weight and structure were determined using Easyn-LC-Orbitrap-Lumos (apparatus model Thermo FISHER SCIENTIFIC, wilmington, DE, USA), as shown in FIG. 4 (FIG. 4 is a secondary mass spectrometry plot of sweetness enhanced helper peptide KVVITAPSS), molecular weight 902.53Da, structure H-Lys-Val-Val-Ile-Thr-Ala-Pro-Ser-Ser.

Taste analysis:

10 members of the sensory panel with sensory evaluation experience were enrolled from the laboratory (5 females and 5 males, age 24-30 years) for physical health. First, the panelists were trained for 2 weeks in taste, the training content being: the six acids sweetness and bitterness were presented to the sensory evaluator as an aqueous solution of a salty and fresh astringent standard. The standard substances are food grade sucrose (37.5 g/L and 15.00 g/L), citric acid (3.00 g/L and 0.60 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 polyphenols (5.00 g/L and 1.00 g/L), respectively. Screening was performed based on whether the evaluator was able to correctly determine the difference between the two concentrations.

The method comprises the steps of preparing a sweet taste enhancement peptide VIIGGGDTAT and a sweet taste enhancement auxiliary peptide KVVITAPSS into 1g/L of evaluation solution (pure water serving as a solvent), carrying out gradient dilution on the evaluation solution by pure water according to a ratio of 1:1, wherein the concentration of the evaluation solution is 500.00 mug/mL, 250.00 mug/mL, 125.00 mug/mL, 62.50 mug/mL, 31.25 mug/mL, 156.25 mug/mL, 78.125 mug/mL and 39.0625 mug/mL respectively, and then presenting the evaluation solution to a sensory evaluation person to carry out taste sorting on the taste intensity of the evaluation solution. When the taste difference between the solution at a certain dilution level and the blank cannot be recognized by any evaluator, the dilution concentration of the sample is recorded as the threshold value of the sample in the blank solution (pure water) in the experiment.

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

The threshold of sweetness enhancer peptide VIIGGGDTAT in pure water was determined to be 749.05 μg/mL.

Example 1

A sugar substitute mixture comprising 7.14g of a sweetness enhancing peptide VIIGGGDTAT and 1g of steviol glycoside.

Example 2

A sugar substitute mixture comprising 5.36g of a sweetness enhancing peptide VIIGGGDTAT, 1.78g of a sweetness enhancing helper peptide KVVITAPSS, and 1g of steviol glycoside.

Example 3

A sugar substitute mixture comprising 3.57g of a sweetness enhancing peptide VIIGGGDTAT, 3.57g of a sweetness enhancing helper peptide KVVITAPSS, and 1g of steviol glycoside.

Comparative example 1

A sugar substitute mixture comprising 1.79g of a sweetness enhancing peptide VIIGGGDTAT, 5.36g of a sweetness enhancing helper peptide KVVITAPSS and 1g of steviol glycoside.

Comparative example 2

A sugar substitute mixture comprising 1g steviol glycoside.

Comparative example 3

A sugar substitute mixture comprising 7.14g of a sweetness enhancing auxiliary peptide KVVITAPSS and 1g of steviol glycoside.

Sugar substitution amount test:

(1) Preparing a sweet solution to be tested:

the sugar substitute mixture provided in the example was dispersed in pure water to obtain a sweet taste solution to be measured. The amount of sugar substitute mixture added in the sweet solution is 1.142 g/L.

And dispersing stevioside in pure water to obtain the sweet taste solution to be detected. The addition amount of steviol glycoside in the sweet solution is 0.142 g/L.

And dispersing the sweet taste enhancing auxiliary peptide KVVITAPSS and stevioside in pure water together to obtain the sweet taste enhancing auxiliary peptide solution to be detected. In the sweet taste improving peptide solution, the addition amount of stevioside is 0.142 g/L, and the addition amount of sweet taste improving peptide KVVITAPSS is 1 g/L.

(2) Preparing a 3.75wt% sucrose aqueous solution representing 5 minutes as a sweet standard solution by referring to GB/T19547-2004 ' sensory analysis method metric value estimation method ', selecting an evaluator of taste analysis ', tasting the sweetness of the sweet solution to be detected, scoring the sweet solution to be detected according to the standard solution, calculating the average value of the sweet solution to be detected, and marking the average value as X; the sugar substitute amount a is calculated by the following formula.

A g=3.75X/5

The measurement results are shown in Table 1.

Identification of ACE converting enzyme inhibition activity assay:

(1) Identification of inhibitory hypertension activity:

Salt-containing BBS buffer (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 (0.8766 g, relative molecular weight 58.44 g/mL), adding deionized water to constant volume, and preserving at 0deg.C;

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

preparing HHT (hippocampal-histidyl-leucine) solution: taking 5mg, dissolving in 10mL of salt-containing BBS buffer solution to prepare 2.5mg/mL of HIL solution, and preserving at-20 ℃;

preparing a peptide sample: the sugar substitute mixtures, the sweetness enhancer peptides VIIGGGDTAT and the sweetness enhancer auxiliary peptides KVVITAPSS provided in examples 1 to 3 and comparative examples 1 and comparative examples 2 to 3 were prepared as 2mmol/L solutions (the solvent was BBS buffer containing 0.9% NaCl) respectively, and were used as peptide samples;

And (3) testing: mixing 20 mu L of peptide sample with 100 mu L of HHT solution, preheating to 37 ℃, adding 20 mu LACE converting enzyme solution, carrying out metal bath for 1h at 37 ℃, adding 100 mu L of hydrochloric acid for inactivation after the temperature is restored to room temperature, and carrying out sample injection analysis on the reaction solution through an organic filter membrane with the thickness of 0.45 mu m.

To reduce errors, the reaction groups were set up for 2 replicates, each group was set up with a blank (20. Mu.L of peptide sample was mixed with 100. Mu.L of HHT solution and preheated to 37 ℃, 20. Mu.L of saline BBS buffer was added, the metal bath was carried out at 37℃for 2 hours, 100. Mu.L of hydrochloric acid was added to inactivate after the reaction was returned to room temperature, the reaction solution was subjected to sample injection analysis again through an organic filter membrane of 0.45. Mu.m), and to ensure the stringency of the experiment, a control group was again made (20. Mu.L of saline BBS buffer was mixed with 100. Mu.L of HHT solution and preheated to 37℃and added with 20. Mu.LACE converting enzyme solution, the metal bath was carried out at 37℃for 1 hour, 100. Mu.L of hydrochloric acid was added to inactivate after the reaction solution was returned to room temperature, and the reaction solution was subjected to sample injection analysis again through an organic filter membrane of 0.45. Mu.m).

The test shows that the ACE enzyme inhibition rate of the sweetness enhanced peptide VIIGGGDTAT is 18.50%, while the ACE enzyme inhibition rate of the sweetness enhanced auxiliary peptide KVVITAPSS is 82.77%.

The test results are shown in Table 1.

TABLE 1

Examples	Instead of sucrose amount	ACE enzyme inhibition rate
			Example 1	3.98 g	12.62%
Example 2	4.24 g	46.01%
			Example 3	3.86 g	55.33%
Comparative example 1	3.49 g	68.70%
			Comparative example 2	3.53 g	5.31%
Comparative example 3	3.45 g	74.93%

As can be seen from Table 1, the sweetness enhancer peptide VIIGGGDTAT provided by the application can enhance the sweetness of stevioside, and can replace 3.98g of sucrose after being mixed with stevioside according to the mass ratio of 7.14:1; in the sugar substitute mixture provided by the application, after the sweet taste enhancing peptide VIIGGGDTAT and the sweet taste enhancing auxiliary peptide KVVITAPSS are mixed according to the mass ratio of 3:1-1:1, the mixture is mixed with stevioside according to the mass ratio of 7.14:1, so that 3.86 g-4.24 g of sucrose can be replaced. The sweetness enhanced auxiliary peptide KVVITAPSS has obvious ACE enzyme inhibition rate, and can synergistically enhance the sweet taste of stevioside after being compounded with the sweetness enhanced peptide VIIGGGDTAT.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A sweet taste enhancing composition of steviol glycosides, wherein the sweet taste enhancing composition comprises sweet taste enhancing peptide VIIGGGDTAT and sweet taste enhancing helper peptide KVVITAPSS; in the sweet taste enhancing composition, the mass ratio of the sweet taste enhancing peptide VIIGGGDTAT to the sweet taste enhancing auxiliary peptide KVVITAPSS is 3:1-1:1.

2. Use of the sweetness enhancing composition of claim 1 for enhancing the sweetness of steviol glycosides.

3. The use according to claim 2, wherein the sweetness enhancing composition is used in an amount of 0.58g to 10.20g of sweetness enhancing composition per gram of steviol glycoside.

4. A sucrose substitute comprising 1 part by weight steviol glycoside and 0.58 to 10.20 parts by weight of the sweetness enhancing composition of claim 1.

5. A sweet taste enhancing helper peptide for steviol glycosides, wherein the sweet taste enhancing helper peptide has the sequence KVVITAPSS.

6. Use of a sweetness enhanced auxiliary peptide according to claim 5 for the preparation of any one or a combination of at least two of a nutraceutical additive, a health food additive;

The health care product comprises a health care product for assisting in reducing blood pressure.