A pea umami peptide and its preparation method and application

Abstract

The present invention belongs to the technical field of bioactive peptides, and in particular, relates to a pea umami peptide and a preparation method and application thereof. The pea umami peptide provided by the present invention contains at least one of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA. The above polypeptides all exhibit umami characteristics, have a low umami threshold, and have good application prospects in the fields of seasonings or food additives. The preparation method provided by the present invention first enzymatically hydrolyzes pea protein with pepsin and trypsin, and then obtains the product after further separation and purification. The operation is simple, the practicability is good, and it is easy to implement industrially. The obtained product contains the above four peptide segments, has a strong umami taste, and has a small bitter taste.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and in particular relates to a pea umami peptide and a preparation method and application thereof. Background Art

[0002] Umami is one of the five basic tastes. It is a pleasant and complex taste that is perceived by the brain when umami substances stimulate specific umami receptors on the human taste buds to produce a series of taste signals. Umami peptides are a class of polypeptides or their salts composed of two or more amino acids that can exhibit umami taste at a certain concentration. They are widely found in livestock and poultry meat, fish and shellfish, milk, beans and even some natural fermented foods. Umami peptides have multiple functional properties, such as imparting umami, reducing dependence on salt and providing nutritional benefits, and have huge market potential in the field of condiments.

[0003] Different umami peptides have different umami flavors and characteristics, which can enrich the types of seasonings and achieve different seasoning purposes. Therefore, it is necessary to develop more types of umami peptides to meet more seasoning needs. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a pea umami peptide and a preparation method and application thereof. The pea umami peptide provided by the present invention has an obvious effect of increasing freshness and reducing saltiness, and can be used in various fields such as seasonings and food additives; the preparation method of the pea umami peptide provided by the present invention is simple to operate, easy to implement, and suitable for the industrial production of pea umami peptide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a pea umami peptide, which contains at least one of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA; wherein the amino acid sequence of PGSVQEINRL is: Pro-Gly-Ser-Val-Gln-Glu-Ile-Asn-Arg-Leu (i.e., proline-glycine-serine-valine-glutamine-glutamic acid-isoleucine-asparagine-arginine-leucine, the sequence is shown in SEQ ID No. 1); the amino acid sequence of KAKLSSGDV is: Lys-Ala-Lys-Leu-Ser-Ser-Gly-Asp-Val (i.e., lysine-alanine-lysine-leucine-serine-serine-glycine-aspartic acid-valine, the sequence is shown in SEQ ID The amino acid sequence of the PDSRIQDD is: Pro-Asp-Ser-Arg-Ile-Gln-Asp-Asp (i.e., proline-aspartic acid-serine-arginine-isoleucine-glutamine-aspartic acid-aspartic acid, the sequence is shown in SEQ ID No.3); the amino acid sequence of the GHPVAITA is: Gly-His-Pro-Val-Ala-Ile-Thr-Ala (i.e., glycine-histidine-proline-valine-alanine-isoleucine-threonine-alanine, the sequence is shown in SEQ ID No.4).

[0007] The pea umami peptide provided by the present invention is derived from pea (Pisum sativum L.) protein. Pea is one of the main edible legume crops in China and has important value in nutrition and health. It tastes delicious, is rich in nutrition, and has a balanced amino acid ratio. Studies have shown that pea protein has a variety of physiological activities, including enhancing immunity, improving cardiovascular diseases, increasing satiety, and lowering blood lipids, and is deeply loved by people. However, there is no report on the preparation of umami peptides using pea protein.

[0008] The PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA in the pea umami peptide provided by the present invention are small molecule polypeptides with molecular weights of 1111.60, 903.50, 944.42 and 534.32 respectively. The hydrogen bond counts of the four polypeptides binding to T1R3 were 11, 11, 15 and 8, respectively, and the docking binding energies were -8.9 kcal / mol, -8.7 kcal / mol, -8.7 kcal / mol and -8.7 kcal / mol, respectively; the 20 amino acid residues of T1R3 that could be bound were Gln211, Gln222, Glu301, Asp219, Asp147, Asn150, Arg151, Arg277, Phe247, Ser109, Ser107, Ser276, Ser217, Ser148, Ser158, Lys155, Thr179, Thr154, Thr149 and Ala171. Experimental studies have shown that the above-mentioned polypeptides all exhibit umami characteristics, and the umami threshold is relatively low, at 0.23-0.41 mmol / L. Among them, PGSVQEINRL and PDSRIQDD also have sour and sweet tastes, KAKLSSGDV has a salty taste, and GHPVAITA has a sweet taste. Therefore, the above-mentioned polypeptides can each achieve different seasoning purposes, and can also be combined to increase the freshness and reduce the saltiness, and have good application prospects in the fields of seasonings or food additives.

[0009] Preferably, the total content of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA in the pea umami peptide is greater than or equal to 70%.

[0010] Preferably, the pea umami peptide contains PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA.

[0011] Further preferably, the mass ratio of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA is (1.8~2.2):(2.3~2.9):(1.3~1.5):(1.1~1.3).

[0012] Preferably, the PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA are chemically synthesized by solid phase synthesis.

[0013] The second aspect of the present invention provides a method for preparing pea umami peptide, which specifically comprises the following steps:

[0014] S1. dissolving pea protein in water to prepare a pea protein solution, adding pepsin and trypsin for enzymolysis to obtain a pea protein hydrolysate; the amount of pepsin and trypsin added is 19% to 21% of the mass of the pea protein;

[0015] S2, centrifuging the pea protein hydrolysate obtained in S1, taking the supernatant, filtering it with a 3500Da ultrafiltration membrane, collecting the filtrate with a molecular weight less than 3500Da, concentrating and drying it to obtain crude pea protein peptide;

[0016] S3. Dissolve the crude pea protein peptide obtained in S2 in ultrapure water, separate and purify it by gel chromatography, use an aqueous solution containing 0.14-0.16M NaCl and 18-22 mM sodium hydrogen phosphate as eluent, collect the effluent, and obtain a product containing the pea umami peptide.

[0017] The preparation method of the present invention uses pepsin and trypsin to enzymolyze pea protein, and the crude pea protein peptide obtained after centrifugation and ultrafiltration is mainly composed of small molecule polypeptides, and after further separation and purification by gel chromatography, the obtained product has strong umami taste, low bitterness, and contains the above four peptide segments. The preparation method is time-saving, simple to operate, good in practicality, and easy to implement industrially.

[0018] Preferably, before the enzymatic hydrolysis in S1, the pea protein solution is heat treated at 85-100° C. for 10-15 min to denature the pea protein, thereby making the subsequent enzymatic hydrolysis more complete.

[0019] Preferably, the enzymatic hydrolysis parameters in S1 are: pH 4.0-7.0, enzymatic hydrolysis temperature 36-38° C., and enzymatic hydrolysis time 230-250 min. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 90-95° C. for 10-20 min to obtain the pea protein hydrolysate.

[0020] Preferably, the centrifugal speed in S2 is 8000-10000 rmp / min, and the centrifugal time is 20-25 min.

[0021] Preferably, the drying method in S2 is freeze-drying.

[0022] Preferably, the chromatographic column of the gel chromatography in S3 is TK-col TA-GF30, and the detection wavelength of the ultraviolet detector is 214 nm.

[0023] Preferably, the concentration of NaCl in the eluent in S3 is 0.15 M, and the concentration of sodium hydrogen phosphate is 20 mM.

[0024] Further preferably, the sample volume in S3 is 1 mL, the flow rate is 0.5 mL / min, and the effluent after 110 min is collected.

[0025] More preferably, the effluent from the 112th to the 144th minute is collected. Exemplarily, 4 mL is collected per tube, and the effluent from the 14th to the 18th tube is collected.

[0026] Preferably, S3 further comprises concentrating and drying the effluent. The drying method may be freeze drying, low temperature reduced pressure drying or other low temperature drying methods.

[0027] The third aspect of the present invention provides the use of the above-mentioned pea umami peptide or the pea umami peptide prepared by the above-mentioned preparation method in the preparation of condiments or food additives.

[0028] The beneficial effects of the present invention are:

[0029] The pea umami peptide provided by the present invention mainly exhibits umami taste, and has a certain sweetness, with less unpleasant flavor. During the bonding process between the pea protein umami peptide and the human taste receptor T1R1 / T1R3, many amino acid residues such as Asn150, Ser148, Arg277 and Thr149 play a key role in its umami taste. The pea umami peptide comes from pea protein with a variety of physiological activities that are beneficial to the body, and the above four polypeptides are all oligopeptides and are easy to absorb. Therefore, the pea umami peptide has the functions of increasing freshness, reducing saltiness, and providing nutrition, and can be used in food fields such as condiments or food additives, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 1 is a high performance liquid chromatogram of the enzymatic hydrolysis product in Test Example 1 of the present invention; (a) is a high performance liquid chromatogram of the enzymatic hydrolysis product obtained by hydrolysis with pepsin in Comparative Example 1, (b) is a high performance liquid chromatogram of the enzymatic hydrolysis product obtained by hydrolysis with trypsin in Comparative Example 2, and (c) is a high performance liquid chromatogram of the enzymatic hydrolysis product obtained by hydrolysis with pepsin and trypsin in Example 9;

[0031] Figure 2 It is a radar chart of the electronic tongue test results of pea umami peptide in Test Example 2 of the present invention;

[0032] Figure 3 This is the gel chromatogram of the pea umami peptide in Test Example 3 of the present invention;

[0033] Figure 4 It is a conformation diagram of the umami receptor T1R1 / T1R3 in Test Example 3 of the present invention; (a) is a conformation diagram of T1R1 / T1R3-1EWK constructed by homology modeling of the present invention, and (b) is a Ramachandran diagram analysis of the T1R1 / T1R3 model constructed by using 1EWK as a template of the present invention;

[0034] Figure 5The electronic tongue radar diagram of the pea umami peptide in Test Example 4 of the present invention; Figure (a) is the electronic tongue radar diagram of different pea umami peptide solutions, and Figure (b) is the electronic tongue radar diagram of different pea umami peptide solutions containing 0.15M NaCl, NaCl and MSG;

[0035] Figure 6 It is a two-dimensional diagram of the binding of the pea umami peptide to the T1R3 active center in Test Example 5 of the present invention; Figure (a) is the binding mechanism of the polypeptide PGSVQEINRL of the present invention to the T1R3 active center, Figure (b) is the binding mechanism of the polypeptide KAKLSSGDV of the present invention to the T1R3 active center, Figure (c) is the binding mechanism of the polypeptide PDSRIQDD of the present invention to the T1R1 active center, and Figure (d) is the binding mechanism of the polypeptide GHPVAITA of the present invention to the T1R3 active center. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0037] Umami peptides are widely present in a variety of food raw materials or their processed products. They have the characteristics of imparting umami taste, reducing dependence on salt, and providing nutritional benefits, and have huge market potential. Obtaining peptides by enzymatic hydrolysis, separation, and purification of raw materials is a common way to produce peptide products. However, due to the complexity of raw material ingredients and protein structure, peptides obtained by conventional methods often have many components and different characteristics, and peptides with umami characteristics are not easily obtained.

[0038] The invention uses pea protein as a raw material to obtain a pea umami peptide, wherein the pea umami peptide contains at least one of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA, wherein the amino acid sequence of PGSVQEINRL is Pro-Gly-Ser-Val-Gln-Glu-Ile-Asn-Arg-Leu, the amino acid sequence of KAKLSSGDV is Lys-Ala-Lys-Leu-Ser-Ser-Gly-Asp-Val, the amino acid sequence of PDSRIQDD is Pro-Asp-Ser-Arg-Ile-Gln-Asp-Asp, and the amino acid sequence of GHPVAITA is Gly-His-Pro-Val-Ala-Ile-Thr-Ala. Pea protein has multiple physiological activities, including enhancing the body's immunity, improving cardiovascular diseases, increasing satiety and lowering blood lipids. The above four polypeptides are all oligopeptides and are easy to absorb. Therefore, the pea umami peptide has the functions of increasing freshness and reducing saltiness as well as providing nutrition, and has broad application prospects.

[0039] The present invention also provides a method for preparing pea umami peptide, which specifically comprises the following steps:

[0040] S1. dissolving pea protein in water to prepare a pea protein solution, adding pepsin and trypsin for enzymolysis to obtain a pea protein hydrolysate; the amount of pepsin and trypsin added is 19% to 21% of the mass of the pea protein;

[0041] S2, centrifuging the pea protein hydrolysate obtained in S1, taking the supernatant, filtering it with a 3500Da ultrafiltration membrane, collecting the filtrate with a molecular weight less than 3500Da, concentrating and drying it to obtain crude pea protein peptide;

[0042] S3. Dissolve the crude pea protein peptide obtained in S2 in ultrapure water, separate and purify it by gel chromatography, use an aqueous solution containing 0.14-0.16M NaCl and 18-22 mM sodium hydrogen phosphate as eluent, collect the effluent, and obtain a product containing the pea umami peptide.

[0043] The scheme of the present invention is described below through specific embodiments.

[0044] The enzymatic activity of pepsin used in the following examples is 30000 U / mg, and the enzymatic activity of trypsin is 2500 U / mg.

[0045] Unless otherwise specified, the raw materials, reagents, drugs or instruments used in the following examples are all conventional commercial products obtained from commercial channels. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0046] Example 1

[0047] This embodiment provides a pea umami peptide and a preparation method thereof.

[0048] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 90°C water bath for 15 min, and then pepsin and trypsin were added. The amount of each enzyme added was 20% of the mass of the pea protein powder. The pH value was adjusted to 4.5, and the solution was enzymatically hydrolyzed at 37°C for 4 h. The enzyme was then inactivated at 90-95°C for 15 min to obtain an enzymatic hydrolysis product.

[0049] (2) Dialysis: The enzymatic hydrolysate was centrifuged at 8000 rmp / min for 20 min, the supernatant was taken, and the supernatant was passed through a 3500 Da dialysis bag, and the filtrate was rotary evaporated at 40-45°C to obtain a concentrate, which was then freeze-dried to obtain pea protein crude peptide freeze-dried powder;

[0050] (3) The obtained pea protein crude peptide freeze-dried powder was dissolved in ultrapure water and separated and purified using a TK-col TA-GF30 gel chromatography column (1.6 cm × 60 cm; Chutian Microspheres, Changsha, China) with a sample volume of 1 mL and a flow rate of 0.5 mL / min. A solution containing 0.15 M NaCl and 20 mM sodium hydrogen phosphate was used as the eluent, and the eluent was collected and the absorbance value was measured at 214 nm. An automatic component collector was used to collect 4 ml per tube. Tubes 14 to 18 were collected, combined, concentrated and freeze-dried to obtain pea umami peptide.

[0051] Example 2

[0052] This embodiment provides a pea umami peptide and a preparation method thereof.

[0053] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a water bath at 85°C for 15 min, and then pepsin and trypsin were added. The amount of each enzyme added was 20% of the mass of the pea protein powder. The pH value was adjusted to 4.5, and the solution was enzymatically hydrolyzed at 37°C for 4 h. The enzyme was then inactivated at 90-95°C for 15 min to obtain an enzymatic hydrolysis product.

[0054] (2) Dialysis: same as in Example 1;

[0055] (3) Gel chromatography separation: same as in Example 1.

[0056] Example 3

[0057] This embodiment provides a pea umami peptide and a preparation method thereof.

[0058] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 100°C water bath for 10 min, and then pepsin and trypsin were added. The amount of each enzyme added was 20% of the mass of the pea protein powder. The pH value was adjusted to 4.5, and the solution was enzymatically hydrolyzed at 37°C for 4 h. The enzyme was then inactivated at 90-95°C for 15 min to obtain an enzymatic hydrolysis product.

[0059] (2) Dialysis: same as in Example 1;

[0060] (3) Gel chromatography separation: same as in Example 1.

[0061] Example 4

[0062] This embodiment provides a pea umami peptide and a preparation method thereof.

[0063] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 90°C water bath for 15 min, and then pepsin and trypsin were added. The amount of pepsin added was 19% of the mass of the pea protein powder, and the amount of trypsin added was 21% of the mass of the pea protein powder. The pH value was adjusted to 7.0, and the solution was enzymatically hydrolyzed at 37°C for 250 min, and then the enzyme was inactivated at 90-95°C for 15 min to obtain an enzymatic hydrolysis product.

[0064] (2) Dialysis: The enzymatic hydrolysate was centrifuged at a speed of 10,000 rmp / min for 20 min, the supernatant was taken, and the supernatant was passed through a 3,500 Da dialysis bag, and the filtrate was rotary evaporated at 40-45°C to obtain a concentrate, which was then freeze-dried to obtain pea protein crude peptide freeze-dried powder;

[0065] (3) Gel chromatography separation: same as in Example 1.

[0066] Example 5

[0067] This embodiment provides a pea umami peptide and a preparation method thereof.

[0068] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 90°C water bath for 15 min, and then pepsin and trypsin were added. The amount of pepsin added was 19% of the mass of the pea protein powder, and the amount of trypsin added was 21% of the mass of the pea protein powder. The pH value was adjusted to 6.0, and the solution was enzymatically hydrolyzed at 37°C for 250 min, and then the enzyme was inactivated at 90-95°C for 15 min to obtain an enzymatic hydrolysis product.

[0069] (2) Dialysis: The enzymatic hydrolysate was centrifuged at 8000 rmp / min for 25 min, the supernatant was taken, and the supernatant was passed through a 3500 Da dialysis bag, and the filtrate was rotary evaporated at 40-45°C to obtain a concentrate, which was then freeze-dried to obtain pea protein crude peptide freeze-dried powder;

[0070] (3) Gel chromatography separation: same as in Example 1.

[0071] Example 6

[0072] This embodiment provides a pea umami peptide and a preparation method thereof.

[0073] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 90°C water bath for 15 min, and then pepsin and trypsin were added. The amount of pepsin added was 21% of the mass of the pea protein powder, and the amount of trypsin added was 19% of the mass of the pea protein powder. The pH value was adjusted to 4.0, and the solution was enzymatically hydrolyzed at 37°C for 250 min, and then the enzyme was inactivated at 90-95°C for 15 min to obtain an enzymatic hydrolysis product.

[0074] (2) Dialysis: The enzymatic hydrolysate was centrifuged at 8000 rmp / min for 25 min, the supernatant was taken, and the supernatant was passed through a 3500 Da dialysis bag, and the filtrate was rotary evaporated at 40-45°C to obtain a concentrate, which was then freeze-dried to obtain pea protein crude peptide freeze-dried powder;

[0075] (3) Gel chromatography separation: same as in Example 1.

[0076] Example 7

[0077] This embodiment provides a pea umami peptide and a preparation method thereof.

[0078] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 100°C water bath for 10 min, and then pepsin and trypsin were added. The amount of each enzyme added was 21% of the mass of the pea protein powder. The pH value was adjusted to 4.5, and the solution was enzymatically hydrolyzed at 38°C for 230 min. The enzyme was then inactivated at 90-95°C for 20 min to obtain an enzymatic hydrolysis product.

[0079] (2) Dialysis: same as in Example 1;

[0080] (3) Gel chromatography separation: same as in Example 1.

[0081] Example 8

[0082] This embodiment provides a pea umami peptide and a preparation method thereof.

[0083] (1) Enzymatic hydrolysis: Pea protein powder was dissolved in water to prepare a 5% (W / V) pea protein solution, which was heat treated in a 100°C water bath for 10 min, and then pepsin and trypsin were added. The amount of each enzyme added was 19% of the mass of the pea protein powder. The pH value was adjusted to 4.5, and the solution was enzymatically hydrolyzed at 36°C for 250 min. The solution was then inactivated at 90-95°C for 10 min to obtain an enzymatic hydrolysis product.

[0084] (2) Dialysis: The enzymatic hydrolysate was centrifuged at 8000 rmp / min for 20 min, the supernatant was taken, and the supernatant was passed through a 3500Da dialysis bag, and the filtrate was rotary evaporated at 40-45°C to obtain a concentrate, which was then freeze-dried to obtain pea protein crude peptide freeze-dried powder;

[0085] (3) Gel chromatography separation: same as in Example 1.

[0086] Example 9

[0087] (1) Enzymolysis: same as Example 1;

[0088] (2) Dialysis: same as in Example 1;

[0089] (3) The obtained pea protein crude peptide freeze-dried powder was dissolved in ultrapure water and separated and purified using a TK-col TA-GF30 gel chromatography column (1.6 cm × 60 cm; Chutian Microspheres, Changsha, China) with a sample volume of 1 mL and a flow rate of 0.5 mL / min. A solution containing 0.15 M NaCl and 20 mM sodium hydrogen phosphate was used as the eluent, and the eluent was collected and the absorbance value was measured at 214 nm. An automatic component collector was used to collect 4 ml per tube. Tubes 14 to 26 were collected, combined, concentrated and freeze-dried to obtain pea umami peptide.

[0090] Comparative Example 1

[0091] This comparative example provides a pea umami peptide and a preparation method thereof.

[0092] The preparation method is basically the same as that of Example 9, except that pepsin is used to enzymolyze the pea protein, and the enzymolysis parameters are: pH 3.0, enzymolysis temperature 37° C., and enzymolysis time 4 h.

[0093] Comparative Example 2

[0094] This comparative example provides a pea umami peptide and a preparation method thereof.

[0095] The preparation method is basically the same as that of Example 9, except that trypsin is used to enzymolyze the pea protein, wherein the enzymolysis parameters are: pH 7.5, enzymolysis temperature 37° C., and enzymolysis time 4 h.

[0096] Comparative Example 3

[0097] This comparative example provides a pea umami peptide and a preparation method thereof.

[0098] The pea umami peptide is obtained by combining, concentrating and freeze-drying the 19th and 20th tubes of eluates separated by gel chromatography in the preparation process of Example 1.

[0099] Comparative Example 4

[0100] This comparative example provides a pea umami peptide and a preparation method thereof.

[0101] The pea umami peptide is obtained by combining, concentrating and freeze-drying the 21st and 22nd tube eluates separated by gel chromatography in the preparation process of Example 1.

[0102] Comparative Example 5

[0103] This comparative example provides a pea umami peptide and a preparation method thereof.

[0104] The pea umami peptide is obtained by combining, concentrating and freeze-drying the 23rd to 26th tube eluates separated by gel chromatography in the preparation process of Example 1.

[0105] Test Example 1

[0106] The protein peptide molecular weight of the enzymatic hydrolyzate of step (1) of Example 9, Comparative Example 1, and Comparative Example 2 was verified by high performance liquid chromatography. According to the molecular weight distribution characteristics of the polypeptide and the molecular exclusion requirements, a chromatographic column (LB7-TSkgelG2000SWXL, 7.8×300 mm) was selected as the detection column. The mobile phase was acetic acid: acetonitrile (85:15) solution (containing 0.5% trifluoroacetic acid), the mobile phase flow rate was controlled at 0.7 mL / min, the column temperature was 30°C, the detector temperature was 30°C, the UV detection wavelength was 214 nm, and the injection volume was 20 µL.

[0107] Example 9, Comparative Example 1, Comparative Example 2 The molecular weight distribution of the enzymatic hydrolysis product of step (1) measured by HPLC is as follows: Figure 1As shown in Table 1: Figure A is the molecular weight distribution diagram of the polypeptide obtained in Example 1. As shown in Table 1, most of the polypeptides are distributed below 3 kDa, accounting for 63.9148%, of which peptides less than 1500 Da account for 51.2328%; Figure B is the molecular weight distribution diagram of the polypeptide obtained in Example 2. As shown in Table 1, most of the polypeptides are distributed below 3 kDa, accounting for 89.6257%, of which peptides less than 1500 Da account for 78.3718%; Figure C is the molecular weight distribution diagram of the polypeptide obtained in Example 9. It can be seen that most of the polypeptides are distributed below 3 kDa, accounting for 97.8361%; of which peptides less than 1500 Da account for 96.9935%. It can be seen that the peptides obtained by enzymatic hydrolysis in Example 9 are mostly small molecule peptides, and the molecular weight of the large polypeptide segments is concentrated below 3000 Da, and more than half of the polypeptides have a molecular weight below 1500 Da. This phenomenon is more obvious in the components of Comparative Example 2 and Example 9.

[0108] Table 1 Different molecular weight distributions of Comparative Examples 1, 2 and Example 9

[0109] Group Protease <3 kDa content (%) 3 k-10 kDa content (%) >10 kDa content (%) Comparative Example 1 Pepsin 63.9148 33.9226 2.1626 Comparative Example 2 Trypsin 89.6257 9.6726 0.7017 Example 9 Pepsin, trypsin 97.8316 0 2.1639

[0110] Test Example 2

[0111] Asrree II / LS16 electronic tongue (Alpha MOS, France) was used to automatically detect the pea umami peptide solution (1 mg / mL) prepared in Example 9, Comparative Example 1 and Comparative Example 2 using a total of 7 sensor probes, namely AHS, CTS, NMS, SCS, ANS, PKS and CPS. AHS detected sourness, CTS detected saltiness, NMS detected umami, SCS detected bitterness, ANS detected sweetness, and PKS and CPS were universal detection probes. A blank model (tasteless model) containing 2.2365 mg / mL KCl and 0.045 mg / mL tartaric acid was used as a comparison. The sensor and reference probe were inserted into the sample solution and the relevant taste value was responded to by detecting changes in the membrane potential.

[0112] The results of sensory evaluation were as follows Figure 2 As shown in the figure, the pea umami peptide prepared in Example 9 has the strongest umami taste, with a score of 6.8, and its saltiness is also the strongest, with a score of 7.3. The lowest umami intensity is in Comparative Example 1, with a score of 4.9, and it has the highest score in bitterness; although the umami intensity of Comparative Example 2 is significantly higher than that of Comparative Example 1, its bitterness score is close to that of Comparative Example 1 and much higher than that of Example 9. This situation may be related to the molecular weight distribution and polypeptide composition of the peptides obtained by enzymatic hydrolysis of Comparative Example 1, Comparative Example 2 and Example 9.

[0113] Test Example 3

[0114] The gel chromatograms of the pea umami peptides obtained in Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 are as follows: Figure 3 shown.

[0115] LC-MS / MS amino acid sequence identification was performed on the pea umami peptides obtained in Example 1, Comparative Example 3, Comparative Example 4, and Comparative Example 5. The specific process of LC-MS / MS is as follows:

[0116] (1) After the sample was dissolved in the mobile phase, liquid phase separation was performed first, and then mass spectrometry detection was performed. Liquid A used in the liquid phase was 0.1% v / v formic acid in water, and liquid B was 0.1% v / v formic acid in acetonitrile (acetonitrile concentration was 84% ​​v / v). The liquid chromatography column was RP-C18 (0.15 mm×150 mm, Column Technology Inc.), equilibrated with 95% liquid A. The sample was loaded into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an autosampler, and then separated by a liquid chromatography column. The relevant liquid phase gradient was set as follows: 0 min → 50 min, liquid B linear gradient from 4% to 50%; 50 min → 54 min, liquid B linear gradient from 50% to 100%; 54 min → 60 min, liquid B maintained at 100%.

[0117] (2) The peptides were separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (ThermoFisher). The analysis time was 60 min. Detection method: positive ion. The mass-to-charge ratios of the peptides and their fragments were collected as follows: 10 fragment spectra (MS) were collected after each full scan. 2 scan).

[0118] (3) The raw files of mass spectrometry test were searched in the corresponding database using the software MaxQuant 1.5.5.1, and finally the protein identification and quantitative analysis results were obtained.

[0119] Two groups of amino acid sequences of human taste receptors were obtained from the Uniprot database, namely T1R1 (Q7RTX1) and T1R3 (Q7RTX0). Metabotropic glutamate receptor (P23385) was selected as the modeling template for homology modeling. The constructed model is shown in Figure 4 As shown in Figure A. The left subunit is T1R1, which is a closed conformation in the crystal structure, and the right subunit is T1R3, which is an open conformation in the crystal structure. The constructed T1R1 / T1R3 receptor must be verified and evaluated by Ramachandranplot of the SAVES server to ensure the reliability of the construction results. Figure 1 As shown in Figure B, 88.1% of the amino acid residues are located in the Ramachandran favorable region, 10.0% are located in the maximum allowed region, and 1.5% are located in the rotamer outlier region. The above results show that 99.6% of the amino acid conformations are acceptable, and according to the 90% key evaluation principle, the model is reasonable in terms of dihedral distribution and spatial collision. Molecular docking was performed using Auto dock, and the docking pocket coordinates were: x: 7.067, y:5.871 and z: 11.585, with a radius of 37.5 Å.

[0120] The pea umami peptides prepared in Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 were subjected to the above LC-MS / MS conditions to obtain peptide sequences, molecular weights and identification accuracy, and umami prediction scores were obtained by iUmami SCM. The docking energy was obtained by molecular docking of taste receptors T1R1 / T1R3. According to the rule that the smaller the docking energy, the better the docking effect, the polypeptides in each pea umami peptide were ranked from low to high according to the docking energy. The polypeptides with higher umami prediction scores in each sample are shown in Tables 2 to 5, respectively.

[0121] There are 18 peptides with high umami prediction scores in the pea umami peptides prepared in Example 1, and their peptide information and scoring results are shown in Table 2. The four main peptides are PGSVQEINRL, KAKLSSGDV, PDSRIQDD, and GHPVAITA, abbreviated as PG-10, KA-9, PD-8, and GH-8, respectively, with molecular weights of 1111.60, 903.50, 944.42, and 534.32, respectively, and their contents in the pea umami peptides are 20.03%, 26.10%, 13.67%, and 11.83%, respectively.

[0122] Table 2 Sequence information, umami prediction score and molecular docking results of Example 1 after LC-MS / MS identification

[0123] Peptide sequence length Molecular weight (Da) Identification accuracy (%) Umami prediction score Docking energy (kcal / mol) PGSVQEINRL 10 1111.6 128 596.33 -8.9 KAKLSSGDV 9 903.503 147.36 652.75 -8.7 PDSRIQ 8 944.42 128.69 615.5 -8.7 GHPVAITA 8 764.418 112.27 624.38 -8.7 LqCy 4 534.317 41.922 596.0 -8.6 VDRL 4 501.291 62.086 616.67 -8.4 SSVINNLPLD 10 1070.56 126.52 637.67 -8.3 LSRF 4 521.296 18.603 731.33 -8.2 EREEDEEQVDEE 12 1534.59 224.02 780.91 -7.8 LLQNSKSVAKTDAGE 15 1559.82 251.24 602.29 -7.7 SNKFGKL 7 792.449 112.37 620.43 -7.5 GAKF 4 421.233 35.819 614.0 -7.4 YKK 5 621.385 90.543 664.0 -7.3 LLED 4 488.248 89.046 588.75 -7.3 DKEEEQEEETSKQ 13 1607.68 438.45 736.75 -7.1 KVLLEEQEK 9 1114.62 186.78 649.62 -7.1 LKAL 4 443.311 38.261 590.67 -7.1 LKL 4 469.326 37.783 600.67 -7.0

[0124] There are 7 peptides with higher umami prediction scores in the pea umami peptides prepared in Comparative Example 3, and their peptide information and scoring results are shown in Table 3, including 1 pentapeptide, 1 tridecapeptide, 1 decapeptide, 1 nonapeptide, 2 tetrapeptides and 1 tripeptide, with molecular weights ranging from 1576Da to 217Da, and molecular docking energies of -6.8 and above.

[0125] Table 3 Sequence information, umami prediction score and molecular docking results of comparative example 3 after LC-MS / MS identification

[0126] Peptide sequence length Molecular weight (Da) Identification accuracy (%) Umami prediction score Docking energy (kcal / mol) EDV 4 474.23258 55.908 630.67 -6.8 LKL 4 459.30568 61.28 705.67 -6.6 GAA 3 217.10626 96.46 623.00 -6.6 DALEPDNRIE 10 1170.5517 127.68 590.00 -6.2 KVLLEEQEK 9 1114.6234 186.78 649.62 -5.8 LAQTFNTEEDTAKRL 15 1735.8741 245.94 595.86 -4.7 EEDEEKDDEKEGK 13 1578.6533 110.87 726.00 -4.7

[0127] There are 8 peptides with higher umami prediction scores among the pea umami peptides prepared in Comparative Example 4, and their peptide information and scoring results are shown in Table 4, including 1 tetradecapeptide, 2 dodecapeptides, 2 nonapeptides, 1 heptapeptide, 1 tetrapeptide and 1 tripeptide, with molecular weights ranging from 1454Da to 346Da, and molecular docking energies of -7.7 and above.

[0128] Table 4 Sequence information, umami prediction score and molecular docking results of comparative example 4 after LC-MS / MS identification

[0129] Peptide sequence length Molecular weight (Da) Identification accuracy (%) Umami prediction score Docking energy (kcal / mol) PEQKEQGSQ 9 1029.4727 206.23 643.38 -7.7 YQ 4 551.25913 66.306 590.0 -6.8 TAR 3 346.19647 64.547 600.0 -6.6 AQTFNTEEDTAK 12 1353.6048 99.568 606.0 -6.6 LEDAFNVNR 9 1076.5251 146.54 606.0 -6.3 RLAGTSSVINNLPL 14 1453.8253 99.568 603.62 -6.3 TVLKPDDRNSFN 12 1404.6997 99.653 599.09 -6.0 LTASSNL 7 704.37047 193.98 596.17 -5.7

[0130] Among the pea umami peptides prepared in Comparative Example 5, there are 13 peptides with higher umami prediction scores, and their peptide information and scoring results are shown in Table 5, including 2 pentapeptides, 1 tetradecapeptide, 2 tridecapeptides, 2 undecapeptides, 2 octapeptides, 1 heptapeptide, 1 tetrapeptide and 2 tripeptides, with molecular weights ranging from 1769Da to 373Da, and molecular docking energies of -7.1 and above.

[0131] Table 5 Sequence information, umami prediction score and molecular docking results of comparative example 5 after LC-MS / MS identification

[0132] Peptide sequence length Molecular weight (Da) Identification accuracy (%) Umami prediction score Docking energy (kcal / mol) LKENGTLKPVAIE 13 1410.8082 78.964 609.0 -7.1 SSVINNLPLDV 11 1169.6292 85.161 638.1 -7.0 ELEKEEEEEEE 11 1420.5729 200.56 859.4 -7.0 LDTSNKLN 8 903.46616 122.18 589.2 -6.9 ELL 3 373.22129 65.051 613.0 -6.9 NRLIKNQ 8 1012.6142 143.11 596.0 -6.5 FETF 4 542.23766 65.088 595.0 -6.5 EEDNVISQVQRPVKE 15 1768.8955 219.43 604.71 -6.2 VIRGEGR 7 785.45079 116.9 590.62 -6.1 GLRHSQVAQIKSN 13 1436.7848 218.15 592.0 -6.0 KVKESIIRKLQSPED 15 1769.0047 322.23 595.0 -5.8 LTF 3 379.21072 62.745 588.5 -5.0 VGRDSAISATPAEV 14 1371.6994 80.979 598.85 -4.5

[0133] The above umami prediction results and molecular docking results show that the pea umami peptides prepared in Comparative Examples 3 to 5 are not as good as the pea umami peptides prepared in Example 1 in terms of umami score and molecular docking energy.

[0134] Test Example 4

[0135] The umami taste of the pea umami peptides prepared in Comparative Example 3, Comparative Example 4, Comparative Example 5 and Example 1 was evaluated using an electronic tongue. The four samples were prepared into 1.10 mg / mL aqueous solutions with ultrapure water, and the sensor and reference probe were inserted into the sample solutions, and the changes in membrane potential were detected to respond to the relevant taste values. The results are shown in Figure 5 As shown in Figure A, it can be seen that the pea umami peptide prepared in Example 1 has the strongest umami taste, but the weakest saltiness, and its acidity is also significantly higher than other components, which may be related to the acidic amino acids it contains, such as aspartic acid (D) and glutamic acid (E). Since aspartic acid (D) and glutamic acid (E) are the main sources of umami, the pea umami peptide has the highest umami intensity and the acidity intensity is also enhanced.

[0136] It is generally believed that umami peptides can not only provide umami taste, but also enhance umami taste, thereby reducing the intake of MSG. Figure 5As shown in Figure B, in the presence of 0.15 M NaCl, there is no significant difference between the samples, but compared with the same concentration of NaCl, the saltiness intensity of each pea umami peptide is significantly lower than that of the NaCl solution. Compared with MSG, the umami intensity of each pea umami peptide is higher than that of the MSG solution (0.35%). This shows that each pea umami peptide has the effect of increasing umami and reducing saltiness.

[0137] Test Example 5

[0138] The interaction between the four main peptide segments PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA in the pea umami peptide prepared in Example 1 and the active residues of T1R3 was studied.

[0139] The interactions between the four peptides and the T1R3 active residues are described as Figure 6 The number and length of hydrogen bonds formed between the four peptides and the T1R3 subunit are shown in Figure 6 As shown, the hydrogen bond counts of subunits PGSVQEINRL, KAKLSSGDV, PDSRIQDD, and GHPVAITA bound to T1R3 are 11, 11, 15, and 8, respectively.

[0140] The results showed that the four peptides could bind to 20 amino acid residues of T1R3, namely Gln211, Gln222, Glu301, Asp219, Asp147, Asn150, Arg151, Arg277, Phe247, Ser109, Ser107, Ser276, Ser217, Ser148, Ser158, Lys155, Thr179, Thr154, Thr149, and Ala171. The results showed that Asn150, Ser148, Arg277, and Thr149 played a key role in the bonding process.

[0141] Example 10

[0142] This embodiment provides a pea umami peptide PGSVQEINRL.

[0143] The conventional solid phase synthesis method was used to synthesize the polypeptide according to the amino acid sequence of Pro-Gly-Ser-Val-Gln-Glu-Ile-Asn-Arg-Leu to obtain the pea umami peptide PGSVQEINRL.

[0144] Embodiment 11

[0145] This embodiment provides a pea umami peptide KAKLSSGDV.

[0146] The conventional solid phase synthesis method was used to synthesize a polypeptide according to the amino acid sequence of Lys-Ala-Lys-Leu-Ser-Ser-Gly-Asp-Val to obtain the pea umami peptide KAKLSSGDV.

[0147] Example 12

[0148] This embodiment provides a pea umami peptide PDSRIQDD.

[0149] The conventional solid phase synthesis method was used to synthesize the polypeptide according to the amino acid sequence of Pro-Asp-Ser-Arg-Ile-Gln-Asp-Asp to obtain the pea umami peptide PDSRIQDD.

[0150] Example 13

[0151] This embodiment provides a pea umami peptide GHPVAITA.

[0152] The conventional solid phase synthesis method was used to synthesize a polypeptide according to the amino acid sequence of Gly-His-Pro-Val-Ala-Ile-Thr-Ala to obtain the pea umami peptide GHPVAITA.

[0153] Embodiment 14

[0154] This embodiment provides a pea umami peptide, which is compounded by the pea umami peptides of Examples 10 to 13 in a mass ratio of 2.0:2.6:1.4:1.2.

[0155] Embodiment 15

[0156] This embodiment provides a pea umami peptide, which is compounded by the pea umami peptides of Examples 10 to 13 in a mass ratio of 1.8:2.3:1.3:1.1.

[0157] Example 16

[0158] This embodiment provides a pea umami peptide, which is compounded by the pea umami peptides of Examples 10 to 13 in a mass ratio of 2.2:2.9:1.5:1.3.

[0159] Test Example 6

[0160] This test example verifies the umami taste of pea umami peptides PGSVQEINRL, KAKLSSGDV, PDSRIQDD, and GHPVAITA (with a purity greater than 95%) prepared by solid phase synthesis in Examples 10 to 13.

[0161] The evaluation team consisted of 10 sensory evaluators (8 females and 2 males, aged 22-25 years old). All team members received sensory training based on GB / T16291.1-2012 to distinguish the five basic tastes (sour, sweet, bitter, salty, and umami). The team members performed a taste description analysis on the synthetic peptide solution and used the triangle test method to determine the umami threshold of each synthetic peptide: a synthetic peptide solution with an initial concentration of 2 mg / mL was prepared and gradually diluted in a 1:1 (v / v) ratio. The samples after the gradual dilution were distributed to the sensory officers for evaluation in the order of increasing concentration. The sensory officers were required to select samples with taste differences from 3 cups of solution (1 cup of sample solution and 2 cups of ultrapure water solution). The samples were randomly coded with three digits. The umami threshold of the synthetic peptide was the geometric mean of the individual thresholds of the team members.

[0162] The sensory evaluation results are shown in Table 6. All four synthetic peptides showed umami characteristics, among which PGSVQEINRL and PDSRIQDD also had sour and sweet tastes; KAKLSSGDV also had salty taste; GHPVAITA also had sweet taste; the umami thresholds of these four peptides ranged from 0.23 to 0.41 mmol / L.

[0163] Table 6 Taste description and umami threshold of pea umami peptides

[0164] Synthetic peptide sequences Taste Description Umami threshold ( mmol / L) PGSVQEINRL Sour, fresh, sweet 0.41 KAKLSSGDV Salty, fresh 0.23 PDSRIQ Sour, fresh, sweet 0.37 GHPVAITA Fresh and sweet 0.25

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A pea umami peptide, characterized in that: The pea umami peptide contains at least one of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA; wherein the amino acid sequence of PGSVQEINRL is: Pro-Gly-Ser-Val-Gln-Glu-Ile-Asn-Arg-Leu; the amino acid sequence of KAKLSSGDV is: Lys-Ala-Lys-Leu-Ser-Ser-Gly-Asp-Val; the amino acid sequence of PDSRIQDD is: Pro-Asp-Ser-Arg-Ile-Gln-Asp-Asp; the amino acid sequence of GHPVAITA is: Gly-His-Pro-Val-Ala-Ile-Thr-Ala.

2. The pea umami peptide according to claim 1, characterized in that The pea umami peptide contains PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA.

3. The pea umami peptide according to claim 2, characterized in that The total content of PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA in the pea umami peptide is greater than or equal to 70%.

4. The pea umami peptide according to claim 3, characterized in that The mass ratio of the PGSVQEINRL, KAKLSSGDV, PDSRIQDD and GHPVAITA is (1.8~2.2):(2.3~2.9):(1.3~1.5):(1.1~1.3).

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