Crab 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 crab umami peptide, a preparation method thereof, and an application thereof. The crab umami peptide provided by the present invention contains at least one of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK, and GVQPDKRPF. The above-mentioned polypeptides all exhibit umami characteristics and have broad application prospects in the fields of condiments, nutritional supplements, and flavor enhancers. The preparation method provided by the present invention first enzymatically hydrolyzes crab meat with protease, and then further separates and purifies the crab meat to obtain the product. The method is simple to operate, has good practicality, and is easy to implement industrially. The obtained product contains the above-mentioned peptide segments, has a strong umami flavor, and has a certain umami aftertaste.

Description

Technical Field

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

[0002] Umami is one of the five basic flavors, in addition to sour, sweet, bitter, and salty. Umami can play a coordinating role among other flavors, enhancing the salty taste and forming a synergistic effect of flavor enhancers. As an important flavor enhancer, umami peptides can not only directly enhance the umami of food and bring a delicious taste to the food, but also reduce its bitterness, playing a good auxiliary role. It is widely used in food as a condiment and flavor enhancer. Its sources are extremely wide, distributed in soybeans, meat, aquatic products and other ingredients with unique umami. The umami of umami peptides varies due to differences in the source of ingredients and preparation process. Therefore, it is necessary to develop more types of umami peptides to meet more people's needs. Summary of the Invention

[0003] To address the above technical issues, the present invention provides a crab umami peptide, its preparation method, and its application. The crab umami peptide provided by the present invention has a strong umami flavor and an umami aftertaste, and can be used in a variety of fields, such as condiments, nutritional supplements, and flavor enhancers. The preparation method of the crab umami peptide provided by the present invention is simple and easy to implement, making it suitable for industrial production of the crab umami peptide.

[0004] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention provides a crab umami peptide, which contains at least one of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK and GVQPDKRPF; wherein the amino acid sequence of FDQDDWENWTK is: Phe-Asp-Gln-Asp-Asp-Trp-Glu-Asn-Trp-Thr-Lys (i.e., phenylalanine-aspartic acid-glutamine-aspartic acid-aspartic acid-tryptophan-glutamate-asparagine-tryptophan-threonine-lysine, the sequence is as shown in SEQ ID No .1); the amino acid sequence of the FSGMDSFDDMAK is: Phe-Ser-Gly-Met-Asp-Ser-Phe-Asp-Asp-Met-Ala-Lys (i.e., phenylalanine-serine-glycine-methionine-aspartic acid-serine-phenylalanine-aspartic acid-aspartic acid-methionine-alanine-lysine, the sequence is shown in SEQ ID No. 2); the amino acid sequence of the ANGPSKEEFPMFE is: Ala-Asn-Gly-Pro-Ser-Lys-Glu-Glu-Phe-Pro-Met-Phe-Glu (i.e., alanine-asparagine-glycine-proline-serine-lysine-glutamic acid-glutamic acid-phenylalanine-proline-methionine-phenylalanine-glutamic acid, the sequence is shown in SEQ ID No. .3); the amino acid sequence of the FDDDPFFGGHRS is: Phe-Asp-Asp-Asp-Pro-Phe-Phe-Gly-Gly-His-Arg-Ser (i.e., phenylalanine-aspartic acid-aspartic acid-aspartic acid-proline-phenylalanine-phenylalanine-glycine-glycine-histidine-arginine-serine, the sequence is shown in SEQ ID No. 4); the amino acid sequence of the VMWGDEHIPGSPF is: Val-Met-Trp-Gly-Asp-Glu-His-Ile-Pro-Gly-Ser-Pro-Phe (i.e., valine-methionine-tryptophan-glycine-aspartic acid-glutamic acid-histidine-isoleucine-proline-glycine-serine-proline-phenylalanine, the sequence is shown in SEQ ID No. .5); the amino acid sequence of WGDEHIPGSPFK is: Trp-Gly-Asp-Glu-His-Ile-Pro-Gly-Ser-Pro-Phe-Lys (i.e., tryptophan-glycine-aspartic acid-glutamic acid-histidine-isoleucine-proline-glycine-serine-proline-phenylalanine-lysine, the sequence is shown in SEQ ID No.6); the amino acid sequence of the VIKDLPNFYGK is: Val-Ile-Lys-Asp-Leu-Pro-Asn-Phe-Tyr-Gly-Lys (i.e., valine-isoleucine-lysine-aspartic acid-leucine-proline-asparagine-phenylalanine-tyrosine-glycine-lysine, the sequence is shown in SEQ ID No. 7); the amino acid sequence of the GVQPDKRPF is: Gly-Val-Gln-Pro-Asp-Lys-Arg-Pro-Phe (i.e., glycine-valine-glutamine-proline-aspartic acid-lysine-arginine-proline-phenylalanine, the sequence is shown in SEQ ID No. 8).

[0006] The crab umami peptide provided by the present invention is derived from crab meat. Currently, there is no report on the preparation of umami peptide using crab meat as raw material.

[0007] The docking energies of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK and GVQPDKRPF in the crab umami peptides provided by the present invention binding to the umami receptors T1R1 / T1R3 are -206.26, -196.76, -184.48, -183.06, -166.86, -164.32, -169.82 and -128.28, respectively. The conformations bound to the T1R3 receptor are relatively stable and all exhibit umami characteristics.

[0008] Preferably, the crab umami peptide contains at least one of FDQDDWENWTK, FDDDPFFGGHRS, VIKDLPNFYGK, and GVQPDKRPF. Electronic tongue flavor profile analysis revealed that FDDDPFFGGHRS, VIKDLPNFYGK, and GVQPDKRPF exhibit umami flavor, while FDQDDWENWTK exhibits an umami aftertaste. Antioxidant activity testing revealed that all four peptides possessed moderate antioxidant activity, with FDQDDWENWTK exhibiting superior antioxidant activity.

[0009] Preferably, the crab umami peptide contains FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF and WGDEHIPGSPFK, and the mass ratio of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF and WGDEHIPGSPFK is (1.1~2.0):(1.3~2.2):(1.2~2.0):(1.1~2.4):(1.2~2.2):(1.3~1.4).

[0010] Further preferably, the mass ratio of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF and WGDEHIPGSPFK is 2.0:1.2:1.3:1.1:1.3:1.4, or 1.1:2.2:2.0:1.2:1.2:1.4, or 1.4:1.3:1.2:2.4:2.2:1.3.

[0011] Preferably, the crab umami peptide contains VIKDLPNFYGK and GVQPDKRPF, and the mass ratio of VIKDLPNFYGK to GVQPDKRPF is (1.1-1.8): (1.2-1.3).

[0012] Further preferably, the mass ratio of VIKDLPNFYGK to GVQPDKRPF is 1.1:1.3 or 1.8:1.2.

[0013] The crab umami peptide obtained by the above combination has better umami taste and antioxidant activity.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned crab umami peptide, which specifically comprises the following steps:

[0015] S1. Crush the edible parts of the swimming crab (Portunus trituberculatus) or the Chinese mitten crab (Eriocheir sinensis) and disperse them in distilled water to prepare a crab meat homogenate, and add protease for enzymatic hydrolysis to obtain a crab meat enzymatic hydrolysate; the protease is added in an amount of 3% to 8% by weight of the crab meat homogenate;

[0016] S2, centrifuging the crab meat enzymatic hydrolysate obtained in S1, taking the supernatant, filtering it with a 3000 Da ultrafiltration membrane, collecting the filtrate, and drying it;

[0017] S3. Dissolve the dry product obtained in S2 in ultrapure water, separate and purify it by gel chromatography, elute it with ultrapure water, collect the chromatographic component peaks corresponding to each polypeptide, concentrate and dry them, and obtain a product containing the crab umami peptide.

[0018] This preparation method first uses a protease that is optimal for crab meat enzymatic hydrolysis. This is followed by centrifugation and ultrafiltration to obtain a mixed peptide with a molecular weight of less than 3,000 Da. This product is then separated and purified by gel chromatography. The resulting product has a strong umami flavor, minimal bitterness, and contains the aforementioned peptides. This preparation method is time-efficient, simple to operate, and has excellent practicality, making it amenable to industrial implementation.

[0019] Preferably, the edible parts described in S1 include body meat, hepatopancreas, foot meat and claw meat.

[0020] Preferably, the mass ratio of the edible part to distilled water in S1 is 1:6.

[0021] Preferably, the protease in S1 is at least one selected from papain, trypsin, compound flavor enzyme, neutral protease, and alkaline protease. More preferably, it is at least one selected from papain, trypsin, neutral protease, and alkaline protease. Even more preferably, alkaline protease or trypsin is used.

[0022] Different proteases have different action sites and hydrolysis patterns on their substrates, resulting in significant differences in the degree of hydrolysis of crab meat. All of the above proteases achieved high degrees of hydrolysis, with alkaline protease achieving significantly higher hydrolysis degrees than other enzymes for Portunus trituberculatus crab meat, and trypsin achieving significantly higher hydrolysis degrees than other enzymes for Chinese mitten crab meat.

[0023] More preferably, the protease in S1 is selected from papain, trypsin, compound flavor enzyme, neutral protease or alkaline protease, and more preferably papain, trypsin, neutral protease or alkaline protease.

[0024] Further preferably, when the protease in S1 is papain or neutral protease, the enzymatic hydrolysis parameters are: pH 6.0~8.0, and enzymatic hydrolysis temperature 45~55°C; when the protease in S1 is trypsin, the enzymatic hydrolysis parameters are: pH 6.0~8.0, and enzymatic hydrolysis temperature 37~50°C; when the protease in S1 is a compound flavor enzyme, the enzymatic hydrolysis parameters are: pH 5.0~7.0, and enzymatic hydrolysis temperature 50~55°C; when the protease in S1 is alkaline protease, the enzymatic hydrolysis parameters are: pH 7.0~9.0, and enzymatic hydrolysis temperature 50~60°C.

[0025] Preferably, the enzymatic hydrolysis time in S1 is 3 to 6 hours; after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90 to 95°C for 10 to 20 minutes. A further preferred enzymatic hydrolysis time is 4 hours.

[0026] Preferably, the added amount of the protease in S1 is 4.4%wt of the crab meat homogenate.

[0027] Preferably, the centrifugal speed in S2 is 4000-6000 rmp / min, and the centrifugal time is 15-25 min. More preferably, the centrifugal speed is 5000 rmp / min, and the centrifugal time is 20 min.

[0028] Preferably, the drying method in S2 is freeze-drying.

[0029] Preferably, the chromatographic column of the gel chromatography in S3 is Sephadex G-15, and the detection wavelength of the ultraviolet detector is 280 nm.

[0030] Further preferably, the elution flow rate in S3 is 0.5 mL / min; when the raw material in S1 is swimming crab trituberculate, the eluate is collected for 50 to 160 min, which contains FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF and WGDEHIPGSPFK; when the raw material in S1 is Chinese mitten crab, the eluate is collected for 50 to 275 min, which contains VIKDLPNFYGK and GVQPDKRPF.

[0031] Preferably, the drying method in S3 is freeze drying.

[0032] Preferably, the preparation method further comprises purifying the product obtained in S3 by high performance liquid chromatography. The eluate is collected according to the peak time of each polypeptide to obtain the purified product.

[0033] Further preferably, the chromatographic conditions of the high performance liquid chromatography are:

[0034] RP-C18 column, 150 mm × 0.15 mm;

[0035] Mobile phase: aqueous phase is 0.1% formic acid in water, organic phase is 0.1% formic acid in acetonitrile;

[0036] Gradient elution: 0-50 min, organic phase linearly changed from 4% to 50%; 50-54 min, organic phase linearly changed from 50% to 100%; 54-60 min, organic phase maintained at 100%.

[0037] The third aspect of the present invention provides the use of the crab umami peptide or the crab umami peptide prepared according to the above preparation method in the preparation of condiments, nutritional enhancers and / or flavor enhancers.

[0038] When the crab umami peptide is used to prepare condiments, nutritional enhancers, and flavor enhancers, each peptide segment can be chemically synthesized by solid phase synthesis or separated from the product obtained by the above preparation method.

[0039] The beneficial effects of the present invention are:

[0040] The crab umami peptide provided by the present invention mainly presents umami or fresh aftertaste, has little unpleasant flavor, and has certain antioxidant activity. It has a small molecular weight and is easily absorbed by the human body. Therefore, it not only enhances the umami taste of food but also has certain health benefits. It has a broad application prospect in food fields such as condiments, nutritional enhancers and flavor enhancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The test results of the degree of hydrolysis in Test Example 1 of the present invention (Portunus trituberculatus);

[0042] Figure 2 The test results of the degree of hydrolysis in Test Example 1 of the present invention (Eriocheir sinensis);

[0043] Figure 3 The sensory quality test results of Test Example 1 of the present invention (Portunus trituberculatus);

[0044] Figure 4 The sensory quality test results of Test Example 1 of the present invention (Chinese mitten crab);

[0045] Figure 5 The binding mechanism of crab umami peptide FDQDDWENWTK and T1R3 active center in test example 3 of the present invention

[0046] Figure 6 The binding mechanism of crab umami peptide FDDDPFFGGHRS and T1R3 active center in test example 3 of the present invention

[0047] Figure 7 The binding mechanism of the crab umami peptide FSGMDSFDDMAK and the T1R3 active center in Test Example 3 of the present invention;

[0048] Figure 8 The binding mechanism of the crab umami peptide ANGPSKEEFPMFE and the T1R3 active center in Test Example 3 of the present invention;

[0049] Figure 9 The binding mechanism of the crab umami peptide VMWGDEHIPGSPF and the T1R3 active center in Test Example 3 of the present invention;

[0050] Figure 10 The binding mechanism of the crab umami peptide WGDEHIPGSPFK and the T1R3 active center in Test Example 3 of the present invention;

[0051] Figure 11 The binding mechanism of crab umami peptide VIKDLPNFYGK and T1R3 active center in Test Example 3 of the present invention;

[0052] Figure 12 The binding mechanism of the crab umami peptide GVQPDKRPF and the T1R3 active center in Test Example 3 of the present invention;

[0053] Figure 13 The results of the electronic tongue's measurement of the taste profile of the synthetic peptide in Test Example 4 of the present invention are shown in Figure a, which is a radar chart of the taste of the synthetic peptide, and Figure b is a principal component analysis chart of the synthetic peptide;

[0054] Figure 14 is the DPPH radical scavenging rate of the synthetic peptide in Test Example 5 of the present invention;

[0055] Figure 15 is the ABTS free radical scavenging rate of the synthetic peptide in Test Example 5 of the present invention;

[0056] Figure 16 is the hydroxyl radical scavenging rate of the synthetic peptide in Test Example 5 of the present invention;

[0057] Figure 17 This is to test the reducing power of the synthetic peptide in Example 5 of the present invention. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended 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 are within the scope of protection of the present invention.

[0059] Umami peptides possess excellent flavor characteristics. Their application in condiments or flavor enhancers not only enhances the flavor of food and imparts a savory taste, but also reduces salt dependence and provides nutritional benefits, promising broad market prospects. In the production of peptide products, a common method involves enzymatic hydrolysis, separation, and purification of the raw materials to obtain peptides. However, due to the complex composition and protein structure of the raw materials, the diverse composition of different peptides, and their varying properties, umami peptides with the desired umami flavor are difficult to obtain.

[0060] Crabs are rich in nutrients, but there have been no reports of developing umami peptides from them. This invention, using crab as raw material, has produced a crab umami peptide containing at least one of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK, and GVQPDKRPF. These peptides bind to the umami receptors T1R1 / T1R3 in a relatively stable conformation, exhibiting umami properties, and can be used in the preparation of seasonings, nutritional supplements, and flavor enhancers.

[0061] The present invention also provides a method for preparing crab umami peptide, which specifically comprises the following steps:

[0062] S1. Crush the edible parts of the swimming crab (Portunus trituberculatus) or the Chinese mitten crab (Eriocheir sinensis) and disperse them in distilled water to prepare a crab meat homogenate, and add protease for enzymatic hydrolysis to obtain a crab meat enzymatic hydrolysate; the protease is added in an amount of 3% to 8% by weight of the crab meat homogenate;

[0063] S2, centrifuging the crab meat enzymatic hydrolysate obtained in S1, taking the supernatant, filtering it with a 3000 Da ultrafiltration membrane, collecting the filtrate with a molecular weight less than 3000 Da, and drying it;

[0064] S3. Dissolve the dry product obtained in S2 in ultrapure water, separate and purify it by gel chromatography, elute it with ultrapure water, collect the chromatographic component peaks corresponding to each polypeptide, concentrate and dry them, and obtain a product containing the crab umami peptide.

[0065] The solutions of the present invention are described below through specific embodiments.

[0066] The papain used in the following examples (enzyme activity of 10×10 4 U / g), trypsin (enzyme activity is 10×10 4 U / g), compound flavor enzyme (enzyme activity is 10×10 4 U / g), neutral protease (enzyme activity is 20×10 4 U / g), alkaline protease (enzyme activity is 10×10 4 U / g) was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and pepsin (enzyme activity was 1.2×10 3 U / g) was purchased from Xiamen Moyihuai Food Co., Ltd.; Sephadex G-15 dextran gel was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0067] Unless otherwise specified, the raw materials, reagents, drugs, or instruments used in the following examples are all commercially available products. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0068] Example 1

[0069] This embodiment provides a crab umami peptide and a preparation method thereof.

[0070] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add papain (the addition amount is 4.4%wt of the crab meat homogenate mass) for enzymatic hydrolysis, and control the pH value to 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 50±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate.

[0071] (2) Dialysis: Centrifuge the enzymatic hydrolysate at 5000 rmp / min for 20 min, collect the supernatant, and pass the supernatant through a 3000 Da dialysis bag to obtain a 3000 Da permeate and a 3000 Da intercept. Collect the 3000 Da permeate and freeze-dry it for later use.

[0072] (3) Gel chromatography separation: The obtained 3000 Da permeate was dissolved in ultrapure water and separated and purified using a Swphadex G-15 gel chromatography column (1.6 cm × 60 cm) with a sample volume of 1 mL and a flow rate of 0.5 mL / min. Ultrapure water was used as the eluent, and the eluate from 50 to 160 min was collected, concentrated, and then freeze-dried to obtain the crude crab umami peptide containing the above-mentioned peptide segment;

[0073] (4) Purification: The crude crab umami peptide obtained in step (3) was desalted and then separated by high performance liquid chromatography. Chromatographic conditions: RP-C18 column: 150 mm × 0.15 mm; the aqueous phase was 0.1% formic acid aqueous solution, the organic phase was 0.1% formic acid acetonitrile solution, gradient elution: 0-50 min, the organic phase linearly changed from 4% to 50%; 50-54 min, the organic phase linearly changed from 50% to 100%; 54-60 min, the organic phase was maintained at 100%. The eluate was collected according to the peak time of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF and WGDEHIPGSPFK, concentrated and freeze-dried to obtain the crab umami peptide containing the above-mentioned peptide segments.

[0074] Example 2

[0075] This embodiment provides a crab umami peptide and a preparation method thereof.

[0076] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add trypsin (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 45±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate.

[0077] (2) Dialysis: Same as Example 1;

[0078] (3) Gel chromatography separation: same as in Example 1;

[0079] (4) Purification: Same as Example 1.

[0080] Example 3

[0081] This embodiment provides a crab umami peptide and a preparation method thereof.

[0082] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add compound flavor enzyme (addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, control the pH value at 6.0±0.5 during the enzymatic hydrolysis process, and perform enzymatic hydrolysis at 50±2℃ for 4 h, then inactivate the enzyme at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate;

[0083] (2) Dialysis: Same as Example 1;

[0084] (3) Gel chromatography separation: same as in Example 1;

[0085] (4) Purification: Same as Example 1.

[0086] Example 4

[0087] This embodiment provides a crab umami peptide and a preparation method thereof.

[0088] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add neutral protease (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value to 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 50±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate.

[0089] (2) Dialysis: Same as Example 1;

[0090] (3) Gel chromatography separation: same as in Example 1;

[0091] (4) Purification: Same as Example 1.

[0092] Example 5

[0093] This embodiment provides a crab umami peptide and a preparation method thereof.

[0094] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add alkaline protease (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 8.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 55±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 10 min to obtain the enzymatic hydrolyzate.

[0095] (2) Dialysis: Same as Example 1;

[0096] (3) Gel chromatography separation: same as in Example 1;

[0097] (4) Purification: Same as Example 1.

[0098] Example 6

[0099] This embodiment provides a crab umami peptide and a preparation method thereof.

[0100] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add alkaline protease (the addition amount is 3%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 8.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 55±2℃ for 6 h, and then the enzyme was inactivated at 90~95℃ for 10 min to obtain the enzymatic hydrolyzate.

[0101] (2) Dialysis: Centrifuge the enzymatic hydrolysate at 4000 rmp / min for 25 min, collect the supernatant, and pass the supernatant through a 3000 Da dialysis bag to obtain a 3000 Da permeate and a 3000 Da intercept. Collect the 3000 Da permeate and freeze-dry it for later use.

[0102] (3) Gel chromatography separation: same as in Example 1;

[0103] (4) Purification: Same as Example 1.

[0104] Example 7

[0105] This embodiment provides a crab umami peptide and a preparation method thereof.

[0106] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add alkaline protease (the addition amount is 8%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 8.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 55±2℃ for 3 h, and then the enzyme was inactivated at 90~95℃ for 20 min to obtain the enzymatic hydrolyzate.

[0107] (2) Dialysis: Centrifuge the enzymatic hydrolysate at 6000 rmp / min for 15 min, collect the supernatant, and pass the supernatant through a 3000 Da dialysis bag to obtain a 3000 Da permeate and a 3000 Da intercept. Collect the 3000 Da permeate and freeze-dry it for later use.

[0108] (3) Gel chromatography separation: same as in Example 1;

[0109] (4) Purification: Same as Example 1.

[0110] Example 8

[0111] This embodiment provides a crab umami peptide and a preparation method thereof.

[0112] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add papain (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value to 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 50±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate.

[0113] (2) Dialysis: Same as Example 1;

[0114] (3) Gel chromatography separation: The obtained 3000 Da permeate was dissolved in ultrapure water and separated and purified using a Swphadex G-15 gel chromatography column (1.6 cm × 60 cm) with a sample volume of 1 mL and a flow rate of 0.5 mL / min. Ultrapure water was used as the eluent, and the eluate from 50 to 275 min was collected, concentrated, and then freeze-dried to obtain the crude crab umami peptide containing the above-mentioned peptide segment;

[0115] (4) Purification: The crude crab umami peptide obtained in step (3) was desalted and then separated by high performance liquid chromatography. The chromatographic conditions were the same as those in Example 1. The eluates were collected according to the elution time of the VIKDLPNFYGK and GVQPDKRPF peaks, concentrated, and then freeze-dried to obtain the crab umami peptide containing the above-mentioned peptide segments.

[0116] Example 9

[0117] This embodiment provides a crab umami peptide and a preparation method thereof.

[0118] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add trypsin (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value to 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 45±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate;

[0119] (2) Dialysis: Same as Example 1;

[0120] (3) Gel chromatography separation: same as in Example 8;

[0121] (4) Purification: Same as Example 8.

[0122] Example 10

[0123] This embodiment provides a crab umami peptide and a preparation method thereof.

[0124] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add compound flavor enzyme (addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, control the pH value at 6.0±0.5 during the enzymatic hydrolysis process, and perform enzymatic hydrolysis at 50±2℃ for 4 h, then inactivate the enzyme at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate;

[0125] (2) Dialysis: Same as Example 1;

[0126] (3) Gel chromatography separation: same as in Example 8;

[0127] (4) Purification: Same as Example 8.

[0128] Example 11

[0129] This embodiment provides a crab umami peptide and a preparation method thereof.

[0130] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add neutral protease (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value to 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 50±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 15 min to obtain the enzymatic hydrolyzate;

[0131] (2) Dialysis: Same as Example 1;

[0132] (3) Gel chromatography separation: same as in Example 8;

[0133] (4) Purification: Same as Example 8.

[0134] Example 12

[0135] This embodiment provides a crab umami peptide and a preparation method thereof.

[0136] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add alkaline protease (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 8.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 55±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 10 min to obtain the enzymatic hydrolyzate.

[0137] (2) Dialysis: Same as Example 1;

[0138] (3) Gel chromatography separation: same as in Example 8;

[0139] (4) Purification: Same as Example 8.

[0140] Example 13

[0141] This embodiment provides a crab umami peptide and a preparation method thereof.

[0142] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add trypsin (the addition amount is 3%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, control the pH value at 7.0±0.5 during the enzymatic hydrolysis process, and perform enzymatic hydrolysis at 45±2℃ for 6 h, then inactivate the enzyme at 90~95℃ for 10 min to obtain the enzymatic hydrolyzate;

[0143] (2) Dialysis: Centrifuge the enzymatic hydrolysate at 4000 rmp / min for 25 min, collect the supernatant, and pass the supernatant through a 3000 Da dialysis bag to obtain a 3000 Da permeate and a 3000 Da intercept. Collect the 3000 Da permeate and freeze-dry it for later use.

[0144] (3) Gel chromatography separation: same as in Example 8;

[0145] (4) Purification: Same as Example 8.

[0146] Example 14

[0147] This embodiment provides a crab umami peptide and a preparation method thereof.

[0148] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add trypsin (the addition amount is 8%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 7.0±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 45±2℃ for 3 h, and then the enzyme was inactivated at 90~95℃ for 20 min to obtain the enzymatic hydrolyzate.

[0149] (2) Dialysis: Centrifuge the enzymatic hydrolysate at 6000 rmp / min for 15 min, collect the supernatant, and pass the supernatant through a 3000 Da dialysis bag to obtain a 3000 Da permeate and a 3000 Da intercept. Collect the 3000 Da permeate and freeze-dry it for later use.

[0150] (3) Gel chromatography separation: same as in Example 8;

[0151] (4) Purification: Same as Example 8.

[0152] Comparative Example 1

[0153] This comparative example provides a crab umami peptide and a preparation method thereof.

[0154] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add pepsin (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 2.5±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 37±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 20 min to obtain the enzymatic hydrolyzate.

[0155] (2) Dialysis: Same as Example 1;

[0156] (3) Gel chromatography separation: same as in Example 1;

[0157] (4) Purification: Same as Example 1.

[0158] Comparative Example 2

[0159] This comparative example provides a crab umami peptide and a preparation method thereof.

[0160] (1) Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel off the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in 6 times the mass of distilled water to prepare crab meat homogenate, add pepsin (the addition amount is 4.4%wt of the mass of crab meat homogenate) for enzymatic hydrolysis, and control the pH value at 2.5±0.5 during the enzymatic hydrolysis process. The enzymatic hydrolysis was carried out at 37±2℃ for 4 h, and then the enzyme was inactivated at 90~95℃ for 20 min to obtain the enzymatic hydrolyzate.

[0161] (2) Dialysis: Same as Example 1;

[0162] (3) Gel chromatography separation: same as in Example 8;

[0163] (4) Purification: Same as Example 8.

[0164] Example 15

[0165] This embodiment provides a crab umami peptide FDQDDWENWTK. The crab umami peptide FDQDDWENWTK is synthesized using a conventional solid-phase synthesis method according to the amino acid sequence of phenylalanine-aspartic acid-glutamine-aspartic acid-aspartic acid-tryptophan-glutamate-asparagine-tryptophan-threonine-lysine.

[0166] Example 16

[0167] This embodiment provides a crab umami peptide FSGMDSFDDMAK. The crab umami peptide FSGMDSFDDMAK is synthesized using a conventional solid-phase synthesis method in the amino acid sequence of phenylalanine-serine-glycine-methionine-aspartic acid-serine-phenylalanine-aspartic acid-aspartic acid-methionine-alanine-lysine to obtain the crab umami peptide FSGMDSFDDMAK.

[0168] Example 17

[0169] This embodiment provides a crab umami peptide ANGPSKEEFPMFE. The crab umami peptide ANGPSKEEFPMFE is synthesized using a conventional solid-phase synthesis method in the amino acid sequence of alanine-asparagine-glycine-proline-serine-lysine-glutamic acid-glutamic acid-phenylalanine-proline-methionine-phenylalanine-glutamic acid to obtain the crab umami peptide ANGPSKEEFPMFE.

[0170] Example 18

[0171] This embodiment provides a crab umami peptide FDDDPFFGGHRS. The crab umami peptide FDDDPFFGGHRS was synthesized using a conventional solid-phase synthesis method in the amino acid sequence of phenylalanine-aspartic acid-aspartic acid-aspartic acid-proline-phenylalanine-phenylalanine-glycine-glycine-histidine-arginine-serine.

[0172] Example 19

[0173] This example provides a crab umami peptide VMWGDEHIPGSPF. The crab umami peptide VMWGDEHIPGSPF was synthesized using a conventional solid-phase synthesis method in the amino acid sequence of valine-methionine-tryptophan-glycine-aspartic acid-glutamic acid-histidine-isoleucine-proline-glycine-serine-proline-phenylalanine.

[0174] Example 20

[0175] This embodiment provides a crab umami peptide WGDEHIPGSPFK. The crab umami peptide WGDEHIPGSPFK was synthesized using a conventional solid-phase synthesis method according to the amino acid sequence of tryptophan-glycine-aspartic acid-glutamic acid-histidine-isoleucine-proline-glycine-serine-proline-phenylalanine-lysine.

[0176] Example 21

[0177] This embodiment provides a crab umami peptide VIKDLPNFYGK. The crab umami peptide VIKDLPNFYGK is synthesized using a conventional solid-phase synthesis method according to the amino acid sequence of valine-isoleucine-lysine-aspartic acid-leucine-proline-asparagine-phenylalanine-tyrosine-glycine-lysine.

[0178] Example 22

[0179] This embodiment provides a crab umami peptide GVQPDKRPF. The crab umami peptide GVQPDKRPF is synthesized using a conventional solid-phase synthesis method in the amino acid sequence of glycine-valine-glutamine-proline-aspartic acid-lysine-arginine-proline-phenylalanine.

[0180] Example 23

[0181] This example provides a crab umami peptide, which is prepared by compounding the crab umami peptides of Examples 15 to 20 according to a mass ratio of FDQDDWENWTK:FSGMDSFDDMAK:ANGPSKEEFPMFE:FDDDPFFGGHRS:VMWGDEHIPGSPF:WGDEHIPGSPFK=2.0:1.2:1.3:1.1:1.3:1.4.

[0182] Example 24

[0183] This example provides a crab umami peptide, which is prepared by compounding the crab umami peptides of Examples 15 to 20 according to a mass ratio of FDQDDWENWTK:FSGMDSFDDMAK:ANGPSKEEFPMFE:FDDDPFFGGHRS:VMWGDEHIPGSPF:WGDEHIPGSPFK=1.1:2.2:2.0:1.2:1.2:1.4.

[0184] Example 25

[0185] This example provides a crab umami peptide, which is prepared by compounding the crab umami peptides of Examples 15 to 20 according to a mass ratio of FDQDDWENWTK:FSGMDSFDDMAK:ANGPSKEEFPMFE:FDDDPFFGGHRS:VMWGDEHIPGSPF:WGDEHIPGSPFK=1.4:1.3:1.2:2.4:2.2:1.3.

[0186] Example 26

[0187] This example provides a crab umami peptide, which is compounded by mixing the crab umami peptides of Examples 21 and 22 at a mass ratio of VIKDLPNFYGK:GVQPDKRPF=1.1:1.3.

[0188] Example 27

[0189] This example provides a crab umami peptide, which is compounded by mixing the crab umami peptides of Examples 21 and 22 at a mass ratio of VIKDLPNFYGK:GVQPDKRPF=1.8:1.2.

[0190] Test Example 1

[0191] The total nitrogen content in the enzymatic hydrolysates obtained in step (1) of Examples 1 to 5, Examples 8 to 12, and Comparative Examples 1 and 2 was determined by the Kjeldahl method, and the amino nitrogen content in the sample enzymatic hydrolysates was determined by formaldehyde titration:

[0192] Take 1.0 mL of enzymatic hydrolysate and add 10 mL of distilled water. Under magnetic stirring, adjust the pH to 8.2 with 0.1 mol / L NaOH standard solution. Add 5 mL of neutral formaldehyde solution and adjust the pH to 9.2. Record the volume of NaOH consumed and use distilled water as a blank control. The formula for calculating the amino nitrogen content and hydrolysis degree of the enzymatic hydrolysate is:

[0193]

[0194] in, v 0 is the volume of NaOH standard solution consumed by the sample (mL); v 1 is the volume of NaOH standard solution consumed by the blank control (mL); c is the concentration of NaOH standard solution (mol / L); v 2 is the total volume of the sample (mL). v 2 = 90.0 mL; V is the volume of sample enzymatic solution (mL). V =1.0 mL; 0.014 is the mass (g) of nitrogen equivalent to 1.0 mL of 1.0 mol / L NaOH standard solution.

[0195] The results are as follows Figure 1 and Figure 2 As shown in the results, the hydrolysis degree of swimming crab meat by alkaline protease was significantly higher than that by other enzymes, followed by neutral protease, trypsin and papain, and the compound flavor enzyme was slightly lower. Pepsin had the worst hydrolysis effect on swimming crab meat. The hydrolysis degree of Chinese mitten crab meat by trypsin was the highest, significantly higher than that by other enzymes, followed by neutral protease, alkaline protease, compound flavor enzyme and papain, and pepsin had the worst hydrolysis effect on Chinese mitten crab meat.

[0196] Test Example 2

[0197] Sensory evaluation was performed on the enzymatic hydrolysates obtained in step (1) of Examples 1 to 5, Examples 8 to 12, and Comparative Examples 1 and 2.

[0198] The enzymatic hydrolysate was prepared into a 10 mg / mL aqueous solution. Ten sensory assessors (half male and half female) who had undergone sensory training were selected to evaluate the taste of the enzymatic hydrolysates. Four solutions of 0.33 mg / mL citric acid, 0.42 mg / mL white sugar, 0.8 mg / mL salt, and 0.42 mg / mL monosodium glutamate were used as evaluation standards for sour, sweet, salty, and umami, respectively. The standard solution was scored as 5 points. The sensory evaluation of each sample enzymatic hydrolysate was conducted in ten aspects: umami, sweetness, sourness, saltiness, bitterness, off-flavor, aftertaste, richness, overall taste, and crab-specific fishy aroma. Each sensory index was scored from strong to weak (the overall taste was scored from harmonious to disharmonious) on a scale of 0-10. The scoring criteria are shown in Table 1.

[0199] Table 1 Sensory evaluation criteria

[0200]

[0201] The results are as follows Figure 3 and Figure 4 As shown, the neutral protease hydrolysate of the swimming crab (Portunus trituberculatus) exhibited the strongest umami, sweetness, aftertaste, richness, and crab-specific fishy aroma, while exhibiting the weakest unpleasant flavors, and relatively weak sourness and saltiness. The compound-flavor enzyme hydrolysate exhibited the most harmonious overall taste, but other sensory attributes were less prominent. The papain hydrolysate exhibited relatively bland sensory characteristics across all parameters. The trypsin hydrolysate of the Chinese mitten crab (Eriocheir sinensis) exhibited the strongest umami, richness, and crab-specific fishy aroma, as well as the most harmonious overall taste, with less bitterness, and the best sensory evaluation.

[0202] Test Example 3

[0203] The crab umami peptides of Examples 15 to 22 were docked with molecules of the umami receptor T1R1 / T1R3.

[0204] The peptide structure was drawn using ChemDraw and converted into a three-dimensional structure in Chem3D. Ligand optimization was then performed. The processed umami receptors T1R1 / T1R3 were hydrogenated and treated with the CHARMM force field. Potential receptor binding sites were predicted. Energy minimization of the peptides was performed using the Minimization ligand protocol and the CHARMM force field. Dockage of the peptides to the umami receptors T1R1 / T1R3 was performed using CDOCKER. The active pocket center coordinates are x: 36.9691, y: -0.0196, and z: 36.1228, with a radius of 27 Å. The docking energies of each peptide with T1R1 / T1R3 and their predicted activities using Peptide Ranker are shown in Table 2.

[0205] Table 2 Docking energy between peptides and T1R1 / T1R3

[0206]

[0207] The molecular docking results of each peptide with T1R1 / T1R3 umami receptor are shown in the figure. Figures 5 to 12 shown.

[0208] Test Example 4

[0209] The umami peptides of the crabs of Examples 15, 18, 21, and 22 were measured using an electronic tongue.

[0210] The synthetic peptide samples were prepared into 1.0 mg / mL aqueous solutions, mixed well, and placed into special beakers for electronic tongues for balancing and testing. The taste and aftertaste test time for each sample was 30 s. Each sample was tested four times, and the first data was discarded. The average value was taken as the test result.

[0211] The results are as follows Figure 13 As shown in Figure a, except for FDQDDWENWTK, the taste profiles of the other three peptides are relatively similar, all of which have umami, accompanied by salty and sour tastes, and the taste abundance of the peptides is therefore enhanced. In addition, these three peptides have no astringency and good taste characteristics. FDQDDWENWTK does not show umami, nor does it have salty taste, but it has a certain umami aftertaste. As shown in Figure b, the contribution rate of the first principal component (X-axis) of the sample is 91.65%, and the contribution rate of the second principal component (Y-axis) is 5.34%. The total contribution rate of the two principal components is greater than 96%, indicating that the principal component analysis diagram can effectively reflect the sample information. The three peptide samples except FDQDDWENWTK are very close to each other on the PCA diagram and slightly overlap, indicating that the samples of these three peptides are relatively different and have similar taste characteristics.

[0212] Test Example 5

[0213] The antioxidant activity of the crab umami peptides of Examples 15, 18, 21, and 22 was evaluated.

[0214] 1. Evaluation method

[0215] 1.1 DPPH free radical scavenging ability

[0216] The enzymatic hydrolysate was prepared into solutions of different concentrations, 100 μL of each solution was added to a 96-well microplate, and 100 μL of 0.1 mmol / LDPPH anhydrous ethanol solution was added. After the reaction was protected from light for 0.5 h, the absorbance measured at 517 nm was recorded as A 0, the absorbance measured by replacing DPPH anhydrous ethanol solution with anhydrous ethanol solution is recorded as A 1. The absorbance measured by replacing the sample solution with water is recorded as A The DPPH free radical scavenging rate of the sample was calculated using the formula:

[0217]

[0218] 1.2 ABTS free radical scavenging ability

[0219] Prepare equal volumes of 2.6 mmol / L K2S2O8 solution and 7.4 mmol / L ABTS solution and mix them in the dark for 14 h. Then dilute them with anhydrous ethanol to obtain ABTS working solution. Add 40 μL of each concentration sample solution and 160 μL of ABTS working solution to a 96-well microplate and react in the dark for 10 min. The absorbance measured at 734 nm is recorded as A 0, the absorbance measured by replacing the sample solution with water is recorded as A The ABTS free radical scavenging rate of the sample was calculated using the formula:

[0220]

[0221] 1.3 Hydroxyl radical scavenging ability

[0222] Prepare 6.0 mmol / L FeSO4 solution and 6.0 mmol / L salicylic acid solution, take 1.0 mL of each and add 1.0 mL of sample solution, then add 1.0 mL of 6.0 mmol / L H2O2 solution to start the reaction. Incubate in a 37 °C water bath for 0.5 h. The absorbance measured at 510 nm is recorded as A 0, the absorbance measured by replacing H2O2 solution with water is recorded as A 1. The absorbance measured by replacing the sample solution with water is recorded as A The hydroxyl radical scavenging rate of the sample was calculated using the formula:

[0223]

[0224] 1.4 Reduction Power

[0225] Take 0.5 mL of each concentration sample solution in a test tube, add 2.5 mL of 0.2 mol / L PBS buffer with a pH of 6.6 and 1.0% K3Fe(CN)6 solution, mix well, and react in a 50°C water bath for 20 min. After cooling, add 2.5 mL of 10% trichloroacetic acid and mix well. Take 1 mL and add it to a test tube, then add 1.0 mL of water and 0.2 mL of 0.1% FeCl3 solution. After mixing, protect from light at room temperature for 10 min and measure the absorbance at 700 nm.

[0226] 2. Results

[0227] like Figures 14 to 17As shown in the figure, all four synthetic peptides have certain antioxidant activity. Among them, FDQDDWENWTK has significantly stronger DPPH, ABTS and hydroxyl radical scavenging abilities than the other three peptides, and also has slightly stronger reducing power. Although the other three peptides are not as capable of scavenging free radicals as FDQDDWENWTK, they still have certain antioxidant effects and are new umami peptides with certain antioxidant activity.

[0228] 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 scope of protection of the present invention.

Claims

1. A crab umami peptide, characterized in that: The crab umami peptide is selected from at least one of FDQDDWENWTK and GVQPDKRPF; wherein the amino acid sequence of FDQDDWENWTK is: Phe-Asp-Gln-Asp-Asp-Trp-Glu-Asn-Trp-Thr-Lys; and the amino acid sequence of GVQPDKRPF is: Gly-Val-Gln-Pro-Asp-Lys-Arg-Pro-Phe.

2. The crab umami peptide according to claim 1, characterized in that The crab umami peptide is selected from FDQDDWENWTK and GVQPDKRPF.

3. Use of the crab umami peptide according to claim 1 or 2 in the preparation of condiments, nutritional enhancers and / or flavor enhancers.

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