Crab flavor peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive peptides, and particularly relates to a crab flavor peptide as well as a preparation method and application thereof. The crab flavor peptide provided by the invention contains at least one of the following components: FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK and GVQPDKRPF. The crab flavor peptide provided by the invention can be used for preparing the crab flavor peptide. All the polypeptides have delicate flavor characteristics and have very wide application prospects in the fields of seasonings or nutritional supplements, fresheners and the like. According to the preparation method provided by the invention, the crab meat is firstly subjected to enzymolysis by protease, and then is further separated and purified to obtain the product, the operation is simple, the practicability is good, industrial implementation is easy, and the obtained product contains the peptide fragment, is strong in palatable taste and has certain palatable aftertaste.

Description

Technical Field

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

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

[0003] Aiming at the above technical problems, the present invention provides a crab flavor peptide, a preparation method thereof and an application thereof. The crab flavor peptide provided by the present invention has strong umami and umami aftertaste, and can be used in multiple fields such as condiments, nutritional fortifiers and flavor enhancers; the preparation method of the crab flavor peptide provided by the present invention is simple in operation, easy to implement, and suitable for industrial production of crab flavor peptides.

[0004] To achieve the above invention purpose, the present invention adopts the following technical scheme: In the first aspect of the present invention, a crab flavor peptide is provided. The crab flavor peptide contains at least one of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK, and GVQPDKRPF. 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 - glutamic acid - asparagine - tryptophan - threonine - lysine, and the sequence is as shown in SEQ ID No. 1). The amino acid sequence of 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, and the sequence is as shown in SEQ ID No. 2). The amino acid sequence of 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, and the sequence is as shown in SEQ ID No. 3). The amino acid sequence of 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, and the sequence is as shown in SEQ ID No. 4). The amino acid sequence of 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, and the sequence is as 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, and the sequence is as shown in SEQ ID No.as shown in Figure 6); the amino acid sequence of 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, and the sequence is as shown in SEQ ID No. 7); the amino acid sequence of GVQPDKRPF is: Gly-Val-Gln-Pro-Asp-Lys-Arg-Pro-Phe (i.e., glycine-valine-glutamine-proline-aspartic acid-lysine-arginine-proline-phenylalanine, and the sequence is as shown in SEQ ID No. 8).

[0005] The crab flavor peptide provided by the present invention is derived from crab meat. There is no report on the preparation of flavor peptides using crab meat as a raw material at present.

[0006] The docking energies of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK, and GVQPDKRPF in the crab flavor peptide provided by the present invention binding to the umami receptor T1R1 / T1R3 are -206.26, -196.76, -184.48, -183.06, -166.86, -164.32, -169.82, and -128.28 respectively. Their conformations binding to the T1R3 receptor are relatively stable and all exhibit umami characteristics.

[0007] Preferably, the crab flavor peptide contains at least one of FDQDDWENWTK, FDDDPFFGGHRS, VIKDLPNFYGK, and GVQPDKRPF. The results of the electronic tongue taste profile analysis show that FDDDPFFGGHRS, VIKDLPNFYGK, and GVQPDKRPF have umami taste, and FDQDDWENWTK has a fresh aftertaste. The results of the antioxidant activity investigation show that the above four polypeptides all have a certain antioxidant activity, and among them, FDQDDWENWTK has more excellent antioxidant activity.

[0008] 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).

[0009] More 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.

[0010] Preferably, the crab umami peptide contains VIKDLPNFYGK and GVQPDKRPF, and the mass ratio of VIKDLPNFYGK and GVQPDKRPF is (1.1~1.8):(1.2~1.3).

[0011] More preferably, the mass ratio of VIKDLPNFYGK and GVQPDKRPF is 1.1:1.3 or 1.8:1.2.

[0012] The crab umami peptide obtained by the above combination has better umami taste and antioxidant activity.

[0013] In the second aspect of the present invention, a preparation method of the above-mentioned crab umami peptide is provided, which specifically includes the following steps: S1. Crush the edible parts of Portunus trituberculatus or Eriocheir sinensis and disperse them in distilled water to make a crab meat homogenate, add protease for enzymatic hydrolysis to obtain a crab meat hydrolysate; the addition amount of the protease is 3%~8%wt of the crab meat homogenate; S2. Centrifuge the crab meat hydrolysate obtained in S1, take the supernatant, filter it with a 3000 Da ultrafiltration membrane, collect the filtrate, and dry it; S3. Dissolve the dried product obtained in S2 in ultrapure water, separate and purify it by gel chromatography, elute it with ultrapure water, collect the chromatography component peaks corresponding to each polypeptide respectively, concentrate and dry them to obtain a product containing the crab umami peptide.

[0014] This preparation method first enzymatically hydrolyzes with the protease that has the best effect on enzymatic hydrolysis of crab meat, then undergoes centrifugation and ultrafiltration to obtain a mixed peptide with a molecular weight less than 3000 Da. After further separation and purification by gel chromatography, the resulting product has a strong umami taste, little bitterness, and contains the above-mentioned peptide segments. This preparation method takes a short time, is simple to operate, has good practicability, and is easy to implement industrially.

[0015] Preferably, the edible parts described in S1 include body meat, hepatopancreas, foot meat, and claw meat.

[0016] Preferably, the mass ratio of the edible parts described in S1 to distilled water is 1:6.

[0017] Preferably, the protease described in S1 is selected from at least one of papain, trypsin, compound flavor enzyme, neutral protease, and alkaline protease. Further preferably, it is at least one of papain, trypsin, neutral protease, and alkaline protease. More preferably, alkaline protease or trypsin is used.

[0018] Different proteases have different action sites and hydrolysis modes on the substrate. Therefore, when different proteases are used to enzymatically hydrolyze crab meat, there are significant differences in the degree of hydrolysis of the hydrolysate. The above proteases can all obtain a relatively high degree of hydrolysis. Among them, the degree of hydrolysis of alkaline protease on the meat of Portunus trituberculatus is significantly higher than that of other enzymes during enzymatic hydrolysis, and the degree of hydrolysis of trypsin on the meat of Eriocheir sinensis is significantly higher than that of other enzymes during enzymatic hydrolysis.

[0019] Further preferably, the protease described in S1 is selected from papain, trypsin, compound flavor enzyme, neutral protease, or alkaline protease. Further preferably, it is papain, trypsin, neutral protease, or alkaline protease.

[0020] Further preferably, when the protease described in S1 is papain or neutral protease, the parameters of the enzymatic hydrolysis are: pH is 6.0 - 8.0, and the enzymatic hydrolysis temperature is 45 - 55 °C; when the protease described in S1 is trypsin, the parameters of the enzymatic hydrolysis are: pH is 6.0 - 8.0, and the enzymatic hydrolysis temperature is 37 - 50 °C; when the protease described in S1 is compound flavor enzyme, the parameters of the enzymatic hydrolysis are: pH is 5.0 - 7.0, and the enzymatic hydrolysis temperature is 50 - 55 °C; when the protease described in S1 is alkaline protease, the parameters of the enzymatic hydrolysis are: pH is 7.0 - 9.0, and the enzymatic hydrolysis temperature is 50 - 60 °C.

[0021] Preferably, the enzymatic hydrolysis time in S1 is 3 - 6 h; after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90 - 95 °C for 10 - 20 min. The further preferred enzymatic hydrolysis time is 4 h.

[0022] Preferably, the addition amount of the protease described in S1 is 4.4%wt of the crab meat homogenate.

[0023] Preferably, the rotation speed of the centrifugation in S2 is 4000 - 6000 rmp / min, and the centrifugation time is 15 - 25 min. More preferably, the rotation speed of the centrifugation is 5000 rmp / min, and the centrifugation time is 20 min.

[0024] Preferably, the drying method in S2 is freeze-drying.

[0025] Preferably, the chromatographic column for gel chromatography in S3 is Sephadex G-15, and the detection wavelength of the ultraviolet detector is 280 nm.

[0026] More preferably, the elution flow rate in S3 is 0.5 mL / min; when the raw material in S1 is Portunus trituberculatus, the eluate collected for 50 - 160 min contains FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, and WGDEHIPGSPFK; when the raw material in S1 is Eriocheir sinensis, the eluate collected for 50 - 275 min contains VIKDLPNFYGK and GVQPDKRPF.

[0027] Preferably, the drying method in S3 is freeze-drying.

[0028] Preferably, the preparation method further includes purifying the product obtained in S3 by high performance liquid chromatography. The eluate can be collected according to the elution time of each polypeptide to obtain the purified More preferably, the chromatographic conditions of the high performance liquid chromatography are as follows: RP-C18 chromatographic column, 150 mm×0.15 mm; Mobile phase: the aqueous phase is 0.1% formic acid aqueous solution, and the organic phase is 0.1% formic acid acetonitrile solution; Gradient elution: 0 - 50 min, the organic phase linearly changes from 4% to 50%; 50 - 54 min, the organic phase linearly changes from 50% to 100%; 54 - 60 min, the organic phase is maintained at 100%.

[0029] The third aspect of the present invention provides the application of the above-mentioned crab umami peptide or the crab umami peptide prepared by the above-mentioned preparation method in the preparation of seasonings, nutritional fortifiers, and / or flavor enhancers.

[0030] When preparing seasonings, nutritional fortifiers, and flavor enhancers with the above-mentioned crab umami peptide, each peptide segment can be chemically synthesized by solid-phase synthesis method or separated from the product obtained by the above-mentioned preparation method.

[0031] The beneficial effects of the present invention are as follows: The crab flavor peptide provided by the present invention mainly exhibits a fresh flavor or a lingering fresh aftertaste, has few off-flavors, and at the same time has a certain antioxidant activity. Moreover, it has a small molecular weight and is easy to be absorbed by the human body. Therefore, while enhancing the fresh flavor of food, it also has a certain health care effect, and has a relatively wide application prospect in food fields such as seasonings, nutritional fortifiers, and flavor enhancers. Description of the Drawings

[0032] Figure 1 It is the detection result of the degree of hydrolysis in Test Example 1 of the present invention (Portunus trituberculatus); Figure 2 It is the detection result of the degree of hydrolysis in Test Example 1 of the present invention (Eriocheir sinensis); Figure 3 It is the detection result of the sensory quality in Test Example 1 of the present invention (Portunus trituberculatus); Figure 4 It is the detection result of the sensory quality in Test Example 1 of the present invention (Eriocheir sinensis); Figure 5 It is the binding mechanism between the crab flavor peptide FDQDDWENWTK and the active center of T1R3 in Test Example 3 of the present invention Figure 6 It is the binding mechanism between the crab flavor peptide FDDDPFFGGHRS and the active center of T1R3 in Test Example 3 of the present invention Figure 7 It is the binding mechanism between the crab flavor peptide FSGMDSFDDMAK and the active center of T1R3 in Test Example 3 of the present invention; Figure 8 It is the binding mechanism between the crab flavor peptide ANGPSKEEFPMFE and the active center of T1R3 in Test Example 3 of the present invention; Figure 9 It is the binding mechanism between the crab flavor peptide VMWGDEHIPGSPF and the active center of T1R3 in Test Example 3 of the present invention; Figure 10 It is the binding mechanism between the crab flavor peptide WGDEHIPGSPFK and the active center of T1R3 in Test Example 3 of the present invention; Figure 11 It is the binding mechanism between the crab flavor peptide VIKDLPNFYGK and the active center of T1R3 in Test Example 3 of the present invention; Figure 12 It is the binding mechanism between the crab flavor peptide GVQPDKRPF and the active center of T1R3 in Test Example 3 of the present invention; Figure 13 It is the determination result of the taste profile of the synthetic peptide by the electronic tongue in Test Example 4 of the present invention, where Figure a is the taste radar chart of the synthetic peptide, and Figure b is the principal component analysis chart of the synthetic peptide; Figure 14DPPH radical scavenging rate of the synthetic peptide in Test Example 5 of the present invention; Figure 15 ABTS radical scavenging rate of the synthetic peptide in Test Example 5 of the present invention; Figure 16 Hydroxyl radical scavenging rate of the synthetic peptide in Test Example 5 of the present invention; Figure 17 Reducing power of the synthetic peptide in Test Example 5 of the present invention. Detailed implementation manners

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be 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 those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0034] Umami peptides have good flavor characteristics. Applying them to seasonings or flavor enhancers can not only enhance the flavor characteristics of foods, endow foods with delicious tastes, but also reduce the dependence on salt and provide nutritional benefits, having a broad market prospect. In the production of polypeptide products, common methods are to enzymatically hydrolyze, separate and purify raw materials to obtain polypeptides. However, due to the very complex raw material components and protein structures, different polypeptide components are complex and their properties are also different, and it is not easy to obtain umami peptides with ideal umami.

[0035] Crabs are rich in nutrients, but there are no reports on the development of umami peptides from crabs. The present invention uses crabs as raw materials to obtain a crab umami peptide, which contains at least one of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, WGDEHIPGSPFK, VIKDLPNFYGK and GVQPDKRPF. The conformations of the above polypeptides binding to the umami receptor T1R1 / T1R3 are relatively stable, all showing umami characteristics, and can be used to prepare seasonings, nutritional fortifiers and flavor enhancers.

[0036] The present invention also provides a preparation method of a crab umami peptide, specifically including the following steps: S1. Crush the edible parts of Portunus trituberculatus or Eriocheir sinensis and disperse them in distilled water to make a crab meat homogenate, add protease for enzymatic hydrolysis to obtain a crab meat enzymatic hydrolysate; the addition amount of the protease is 3% - 8%wt of the crab meat homogenate; S2. Centrifuge the crab meat enzymatic hydrolysate obtained in S1, take the supernatant, filter it through a 3000 Da ultrafiltration membrane, collect the filtrate with a molecular weight less than 3000 Da, and dry it. S3. Dissolve the dried product obtained in S2 in ultrapure water, separate and purify it by gel chromatography, elute it with ultrapure water, collect the chromatography component peaks corresponding to each polypeptide respectively, concentrate and dry them to obtain a product containing the crab umami peptide.

[0037] The following uses specific examples to illustrate the solution of the present invention.

[0038] In the following examples, the papain (enzyme activity is 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) are purchased from Nanning Donghenghuadao Biotechnology Co., Ltd., and pepsin (enzyme activity is 1.2×10 3 U / g) is purchased from Xiamen Moyihuai Food Co., Ltd.; Sephadex G-15 dextran gel is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0039] Unless otherwise specified, the raw materials, reagents, drugs or instruments used in the following examples are all conventional commercially available products obtained through commercial channels. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art.

[0040] Example 1 This example provides a crab umami peptide and its preparation method.

[0041] (1) Enzymatic hydrolysis: Rinse the swimming crab with running water, peel out 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 make a crab meat homogenate, add papain (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 7.0±0.5, and carry out enzymatic hydrolysis at 50±2°C for 4 h, and then inactivate the enzyme at 90-95°C for 15 min to obtain an enzymatic hydrolysate. (2) Dialysis: Centrifuge the enzymatic hydrolysate at a speed of 5000 rmp / min for 20 min, take the supernatant, and pass the supernatant through a 3000 Da dialysis bag to obtain a 3000 Da permeate and a 3000 Da retentate. Collect and freeze-dry the 3000 Da permeate for later use. (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). The sample loading volume was 1 mL, and the flow rate was 0.5 mL / min. Using ultrapure water as the eluent, the eluent from 50 to 160 min was collected, concentrated, and freeze-dried to obtain a crude crab umami peptide containing the above-mentioned peptide segments; (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 chromatographic column: 150 mm × 0.15 mm; the aqueous phase was a 0.1% formic acid aqueous solution, and the organic phase was a 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 eluent was collected according to the peak elution times of FDQDDWENWTK, FSGMDSFDDMAK, ANGPSKEEFPMFE, FDDDPFFGGHRS, VMWGDEHIPGSPF, and WGDEHIPGSPFK, concentrated, and freeze-dried to obtain a crab umami peptide containing the above-mentioned peptide segments.

[0042] Example 2 This example provides a crab umami peptide and its preparation method.

[0043] (1) Enzymatic hydrolysis: The swimming crab was rinsed clean with running water, and the body meat, hepatopancreas, leg meat, and claw meat were peeled off. The peeled crab meat was crushed and dispersed in 6 times the mass of distilled water to make a crab meat homogenate. Trypsin was added (the addition amount was 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, the pH value was controlled at 7.0 ± 0.5, and enzymatic hydrolysis was carried out at 45 ± 2 °C for 4 h, and then the enzyme was inactivated at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2) Dialysis: The same as in Example 1; (3) Gel chromatography separation: The same as in Example 1; (4) Purification: The same as in Example 1.

[0044] Example 3 This example provides a crab umami peptide and its preparation method.

[0045] (1)Enzymatic hydrolysis: Rinse the swimming crab with running water, remove the body meat, hepatopancreas, leg meat and claw meat. Crush the removed crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add compound flavor enzyme (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 6.0 ± 0.5, and carry out enzymatic hydrolysis at 50 ± 2 °C for 4 h, then inactivate the enzyme at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2)Dialysis: The same as Example 1; (3)Gel chromatography separation: The same as Example 1; (4)Purification: The same as Example 1.

[0046] Example 4 This example provides a crab umami peptide and its preparation method.

[0047] (1)Enzymatic hydrolysis: Rinse the swimming crab with running water, remove the body meat, hepatopancreas, leg meat and claw meat. Crush the removed crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add neutral protease (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 7.0 ± 0.5, and carry out enzymatic hydrolysis at 50 ± 2 °C for 4 h, then inactivate the enzyme at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2)Dialysis: The same as Example 1; (3)Gel chromatography separation: The same as Example 1; (4)Purification: The same as Example 1.

[0048] Example 5 This example provides a crab umami peptide and its preparation method.

[0049] (1)Enzymatic hydrolysis: Rinse the swimming crab with running water, remove the body meat, hepatopancreas, leg meat and claw meat. Crush the removed crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add alkaline protease (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 8.0 ± 0.5, and carry out enzymatic hydrolysis at 55 ± 2 °C for 4 h, then inactivate the enzyme at 90 - 95 °C for 10 min to obtain an enzymatic hydrolysate; (2)Dialysis: The same as Example 1; (3)Gel chromatography separation: The same as Example 1; (4)Purification: The same as Example 1.

[0050] Example 6 This example provides a crab umami peptide and its preparation method.

[0051] (1)Enzymatic hydrolysis: Rinse the swimming crab with running water, peel out the body meat, hepatopancreas, leg meat and claw meat. Crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add alkaline protease (the addition amount is 3%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 8.0 ± 0.5, and carry out enzymatic hydrolysis at 55 ± 2 °C for 6 h, then inactivate the enzyme at 90 - 95 °C for 10 min to obtain an enzymatic hydrolysate; (2)Dialysis: Centrifuge the enzymatic hydrolysate at a speed of 4000 rmp / min for 25 min, take the supernatant, and pass the supernatant through a dialysis bag with a molecular weight cut-off of 3000 Da to obtain a 3000 Da permeate and a 3000 Da retentate. Collect and freeze-dry the 3000 Da permeate for later use; (3)Gel chromatography separation: The same as in Example 1; (4)Purification: The same as in Example 1.

[0052] Example 7 This example provides a crab umami peptide and a preparation method thereof.

[0053] (1)Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel out the body meat, hepatopancreas, leg meat and claw meat. Crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add alkaline protease (the addition amount is 8%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 8.0 ± 0.5, and carry out enzymatic hydrolysis at 55 ± 2 °C for 3 h, then inactivate the enzyme at 90 - 95 °C for 20 min to obtain an enzymatic hydrolysate; (2)Dialysis: Centrifuge the enzymatic hydrolysate at a speed of 6000 rmp / min for 15 min, take the supernatant, and pass the supernatant through a dialysis bag with a molecular weight cut-off of 3000 Da to obtain a 3000 Da permeate and a 3000 Da retentate. Collect and freeze-dry the 3000 Da permeate for later use; (3)Gel chromatography separation: The same as in Example 1; (4)Purification: The same as in Example 1.

[0054] Example 8 This example provides a crab umami peptide and a preparation method thereof.

[0055] (1)Enzymatic hydrolysis: Rinse the Chinese mitten crab with running water, peel out the body meat, hepatopancreas, leg meat and claw meat. Crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add papain (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 7.0 ± 0.5, and carry out enzymatic hydrolysis at 50 ± 2 °C for 4 h, then inactivate the enzyme at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2) Dialysis: same as Example 1; (3) Gel chromatography separation: Dissolve the obtained 3000 Da permeate in ultrapure water, and separate and purify it with a Swphadex G-15 gel chromatography column (1.6 cm × 60 cm). The sample loading volume is 1 mL, and the flow rate is 0.5 mL / min. Using ultrapure water as the eluent, collect the eluent from 50 to 275 min, concentrate it and then freeze-dry it to obtain the crude crab flavor peptide containing the above peptide segments; (4) Purification: After desalting the crude crab flavor peptide obtained in step (3), separate it by high performance liquid chromatography. The chromatographic conditions are the same as in Example 1. Collect the eluent according to the elution times of VIKDLPNFYGK and GVQPDKRPF respectively, concentrate it and then freeze-dry it to obtain the crab flavor peptide containing the above peptide segments.

[0056] Example 9 This example provides a crab flavor peptide and its preparation method.

[0057] (1) Enzymatic hydrolysis: Rinse Eriocheir sinensis with running water, peel out the body meat, hepatopancreas, leg meat and claw meat. Crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add trypsin (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 7.0 ± 0.5, and carry out enzymatic hydrolysis at 45 ± 2 °C for 4 h, and then inactivate the enzyme at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2) Dialysis: same as Example 1; (3) Gel chromatography separation: same as Example 8; (4) Purification: same as Example 8.

[0058] Example 10 This example provides a crab flavor peptide and its preparation method.

[0059] (1) Enzymatic hydrolysis: Rinse Eriocheir sinensis with running water, peel out the body meat, hepatopancreas, leg meat and claw meat. Crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add compound flavor enzyme (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value to be 6.0 ± 0.5, and carry out enzymatic hydrolysis at 50 ± 2 °C for 4 h, and then inactivate the enzyme at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2) Dialysis: same as Example 1; (3) Gel chromatography separation: same as Example 8; (4) Purification: same as Example 8.

[0060] Example 11 This example provides a crab umami peptide and a preparation method thereof.

[0061] (1) Enzymatic hydrolysis: Rinse Eriocheir sinensis with running water, strip out the body meat, hepatopancreas, leg meat and claw meat. Crush the stripped crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add neutral protease (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value at 7.0 ± 0.5, and carry out enzymatic hydrolysis at 50 ± 2 °C for 4 h, then inactivate the enzyme at 90 - 95 °C for 15 min to obtain an enzymatic hydrolysate; (2) Dialysis: The same as Example 1; (3) Gel chromatography separation: The same as Example 8; (4) Purification: The same as Example 8.

[0062] Example 12 This example provides a crab umami peptide and a preparation method thereof.

[0063] (1) Enzymatic hydrolysis: Rinse Eriocheir sinensis with running water, strip out the body meat, hepatopancreas, leg meat and claw meat. Crush the stripped crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add alkaline protease (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value at 8.0 ± 0.5, and carry out enzymatic hydrolysis at 55 ± 2 °C for 4 h, then inactivate the enzyme at 90 - 95 °C for 10 min to obtain an enzymatic hydrolysate; (2) Dialysis: The same as Example 1; (3) Gel chromatography separation: The same as Example 8; (4) Purification: The same as Example 8.

[0064] Example 13 This example provides a crab umami peptide and a preparation method thereof.

[0065] (1) Enzymatic hydrolysis: Rinse Eriocheir sinensis with running water, strip out the body meat, hepatopancreas, leg meat and claw meat. Crush the stripped crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add trypsin (the addition amount is 3%wt of the mass of the crab meat homogenate) for enzymatic hydrolysis. During the enzymatic hydrolysis process, control its pH value at 7.0 ± 0.5, and carry out enzymatic hydrolysis at 45 ± 2 °C for 6 h, then inactivate the enzyme at 90 - 95 °C for 10 min to obtain an enzymatic hydrolysate; (2) Dialysis: Centrifuge the enzymatic hydrolysate at a speed of 4000 rmp / min for 25 min, take the supernatant, and pass the supernatant through a dialysis bag with a molecular weight cut-off of 3000 Da to obtain a 3000 Da permeate and a 3000 Da retentate. Collect and freeze-dry the 3000 Da permeate for later use; (3)Gel chromatography separation: same as Example 8; (4)Purification: same as Example 8.

[0066] Example 14 This example provides a crab umami peptide and its preparation method.

[0067] (1)Enzymolysis: Rinse Eriocheir sinensis with running water, peel out the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add trypsin (the addition amount is 8%wt of the mass of the crab meat homogenate) for enzymolysis. During the enzymolysis process, control its pH value to be 7.0 ± 0.5, and carry out enzymolysis at 45 ± 2 °C for 3 h, and then inactivate the enzyme at 90 - 95 °C for 20 min to obtain an enzymolysis solution; (2)Dialysis: Centrifuge the enzymolysis solution at a speed of 6000 rmp / min for 15 min, take the supernatant, and pass the supernatant through a dialysis bag with a molecular weight cut-off of 3000 Da to obtain a 3000 Da permeate and a 3000 Da retentate. Collect and freeze-dry the 3000 Da permeate for standby; (3)Gel chromatography separation: same as Example 8; (4)Purification: same as Example 8.

[0068] Comparative Example 1 This comparative example provides a crab umami peptide and its preparation method.

[0069] (1)Enzymolysis: Rinse Portunus trituberculatus with running water, peel out the body meat, hepatopancreas, foot meat and claw meat, crush the peeled crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add pepsin (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymolysis. During the enzymolysis process, control its pH value to be 2.5 ± 0.5, and carry out enzymolysis at 37 ± 2 °C for 4 h, and then inactivate the enzyme at 90 - 95 °C for 20 min to obtain an enzymolysis solution; (2)Dialysis: same as Example 1; (3)Gel chromatography separation: same as Example 1; (4)Purification: same as Example 1.

[0070] Comparative Example 2 This comparative example provides a crab umami peptide and its preparation method.

[0071] (1)Enzymolysis: Rinse Eriocheir sinensis thoroughly with running water, strip out the body meat, hepatopancreas, leg meat and claw meat. Crush the stripped crab meat and disperse it in distilled water with a mass 6 times that of the crab meat to make a crab meat homogenate. Add pepsin (the addition amount is 4.4%wt of the mass of the crab meat homogenate) for enzymolysis. During the enzymolysis process, control the pH value at 2.5±0.5, and carry out enzymolysis at 37±2°C for 4 h, then inactivate the enzyme at 90-95°C for 20 min to obtain an enzymolysis solution; (2)Dialysis: The same as Example 1; (3)Gel chromatography separation: The same as Example 8; (4)Purification: The same as Example 8.

[0072] Example 15 This example provides a crab umami peptide FDQDDWENWTK. Using the conventional solid-phase synthesis method, synthesize a polypeptide according to the amino acid sequence of phenylalanine-aspartic acid-glutamine-aspartic acid-aspartic acid-tryptophan-glutamic acid-asparagine-tryptophan-threonine-lysine, and the crab umami peptide FDQDDWENWTK is obtained.

[0073] Example 16 This example provides a crab umami peptide FSGMDSFDDMAK. Using the conventional solid-phase synthesis method, synthesize a polypeptide according to the amino acid sequence of phenylalanine-serine-glycine-methionine-aspartic acid-serine-phenylalanine-aspartic acid-aspartic acid-methionine-alanine-lysine, and the crab umami peptide FSGMDSFDDMAK is obtained.

[0074] Example 17 This example provides a crab umami peptide ANGPSKEEFPMFE. Using the conventional solid-phase synthesis method, synthesize a polypeptide according to the amino acid sequence of alanine-asparagine-glycine-proline-serine-lysine-glutamic acid-glutamic acid-phenylalanine-proline-methionine-phenylalanine-glutamic acid, and the crab umami peptide ANGPSKEEFPMFE is obtained.

[0075] Example 18 This example provides a crab umami peptide FDDDPFFGGHRS. Using the conventional solid-phase synthesis method, synthesize a polypeptide according to the amino acid sequence of phenylalanine-aspartic acid-aspartic acid-aspartic acid-proline-phenylalanine-phenylalanine-glycine-glycine-histidine-arginine-serine, and the crab umami peptide FDDDPFFGGHRS is obtained.

[0076] Example 19 This example provides a crab - flavored peptide VMWGDEHIPGSPF. Using the conventional solid - phase synthesis method, a polypeptide is synthesized in the amino acid sequence of valine - methionine - tryptophan - glycine - aspartic acid - glutamic acid - histidine - isoleucine - proline - glycine - serine - proline - phenylalanine, and thus the crab - flavored peptide VMWGDEHIPGSPF is obtained.

[0077] Example 20 This example provides a crab - flavored peptide WGDEHIPGSPFK. Using the conventional solid - phase synthesis method, a polypeptide is synthesized in the amino acid sequence of tryptophan - glycine - aspartic acid - glutamic acid - histidine - isoleucine - proline - glycine - serine - proline - phenylalanine - lysine, and thus the crab - flavored peptide WGDEHIPGSPFK is obtained.

[0078] Example 21 This example provides a crab - flavored peptide VIKDLPNFYGK. Using the conventional solid - phase synthesis method, a polypeptide is synthesized in the amino acid sequence of valine - isoleucine - lysine - aspartic acid - leucine - proline - asparagine - phenylalanine - tyrosine - glycine - lysine, and thus the crab - flavored peptide VIKDLPNFYGK is obtained.

[0079] Example 22 This example provides a crab - flavored peptide GVQPDKRPF. Using the conventional solid - phase synthesis method, a polypeptide is synthesized in the amino acid sequence of glycine - valine - glutamine - proline - aspartic acid - lysine - arginine - proline - phenylalanine, and thus the crab - flavored peptide GVQPDKRPF is obtained.

[0080] Example 23 This example provides a crab - flavored peptide, which is compounded from the crab - flavored peptides of Examples 15 - 20 according to the mass ratio of FDQDDWENWTK:FSGMDSFDDMAK:ANGPSKEEFPMFE:FDDDPFFGGHRS:VMWGDEHIPGSPF:WGDEHIPGSPFK = 2.0:1.2:1.3:1.1:1.3:1.4.

[0081] Example 24 This example provides a crab - flavored peptide, which is compounded from the crab - flavored peptides of Examples 15 - 20 according to the mass ratio of FDQDDWENWTK:FSGMDSFDDMAK:ANGPSKEEFPMFE:FDDDPFFGGHRS:VMWGDEHIPGSPF:WGDEHIPGSPFK = 1.1:2.2:2.0:1.2:1.2:1.4.

[0082] Example 25 This example provides a crab flavor peptide, which is compounded from the crab flavor peptides of Examples 15 to 20 according to the mass ratio of FDQDDWENWTK:FSGMDSFDDMAK:ANGPSKEEFPMFE:FDDDPFFGGHRS:VMWGDEHIPGSPF:WGDEHIPGSPFK = 1.4:1.3:1.2:2.4:2.2:1.3.

[0083] Example 26 This example provides a crab flavor peptide, which is compounded from the crab flavor peptides of Examples 21 to 22 according to the mass ratio of VIKDLPNFYGK:GVQPDKRPF = 1.1:1.3.

[0084] Example 27 This example provides a crab flavor peptide, which is compounded from the crab flavor peptides of Examples 21 to 22 according to the mass ratio of VIKDLPNFYGK:GVQPDKRPF = 1.8:1.2.

[0085] Test Example 1 The total nitrogen content in the enzymatic hydrolysate 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 hydrolysate was determined by the formaldehyde titration method: Take 1.0 mL of the enzymatic hydrolysate and add 10 mL of distilled water. Adjust the pH to 8.2 with 0.1 mol / L NaOH standard solution under magnetic stirring. Add 5 mL of neutral formaldehyde solution, adjust the pH to 9.2, and record the consumption volume of NaOH. Use distilled water as a blank control. The calculation formulas for the amino nitrogen content and degree of hydrolysis of the enzymatic hydrolysate are as follows:

[0086] Among them, v 0 is the volume (mL) of the NaOH standard solution consumed by the sample; v 1 is the volume (mL) of the NaOH standard solution consumed by the blank control; c is the concentration (mol / L) of the NaOH standard solution; v 2 is the total volume (mL) of the sample. In this experiment v 2 = 90.0 mL; V is the volume (mL) of the sample enzymatic hydrolysate taken. In this experiment 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.

[0087] The results are as Figure 1 andFigure 2 As shown, the degree of hydrolysis of alkaline protease hydrolyzing the meat of Portunus trituberculatus is significantly higher than that of other enzymes. Followed by neutral protease, trypsin and papain, the degree of hydrolysis of compound flavor enzyme is slightly lower, and pepsin has the worst hydrolysis effect on the meat of Portunus trituberculatus; The degree of hydrolysis of trypsin hydrolyzing the meat of Eriocheir sinensis is the highest, significantly higher than that of other enzymes. Followed by neutral protease, alkaline protease, compound flavor enzyme, papain, and pepsin has the worst hydrolysis effect on the meat of Eriocheir sinensis.

[0088] Test Example 2 Perform sensory evaluation on the enzymolysis solutions obtained in step (1) of Examples 1-5, Examples 8-12 and Comparative Examples 1 and 2.

[0089] Prepare the enzymolysis solution into an aqueous solution of 10 mg / mL. Select 10 sensory evaluation personnel (half male and half female) who have received sensory training. Use four solutions of 0.33 mg / mL citric acid, 0.42 mg / mL granulated sugar, 0.8 mg / mL salt, and 0.42 mg / mL monosodium glutamate as the evaluation standard products for sour, sweet, salty, and umami flavors respectively. With the standard product as 5 points, perform sensory evaluation on each sample enzymolysis solution from ten aspects of umami, sweetness, sourness, saltiness, bitterness, off-flavor, aftertaste, richness, overall taste, and the unique fishy aroma of crabs. Each sensory index is scored between 0-10 from strong to weak (the overall taste is scored from coordinated to uncoordinated). The scoring criteria are shown in Table 1.

[0090] Table 1 Sensory Evaluation Criteria

[0091] The results are as Figure 3 and Figure 4 shown. The umami, sweetness, aftertaste, richness, and the unique fishy aroma of crabs of the neutral protease hydrolysate of Portunus trituberculatus are the strongest, the off-flavor is the weakest, and the sourness and saltiness are weaker. The overall taste of the compound flavor enzyme hydrolysate is the most coordinated, but other sensory indexes are not prominent. The characteristics of various sensory indexes of the papain hydrolysate are relatively weak. The trypsin hydrolysate of Eriocheir sinensis has the strongest umami, richness, and the unique fishy aroma of crabs, and the overall taste is the most coordinated, with weaker bitterness and the best sensory evaluation.

[0092] Test Example 3 Perform molecular docking of each crab umami peptide in Examples 15-22 with the umami receptor T1R1 / T1R3.

[0093] The structural formula of the peptide was drawn using ChemDraw and converted into a three-dimensional structure in Chem3D, and then the structure optimization of the ligand was carried out. The processed umami receptor T1R1 / T1R3 was hydrogenated and CHARMM force field was added, and the possible binding sites of the receptor were predicted. The Minimization ligand protocol was used and the energy minimization of the peptide was carried out by adding CHARMM force field. The docking of the peptide with the umami receptor T1R1 / T1R3 was achieved using CDOCKER. The central coordinates of the active pocket are: x: 36.9691, y: -0.0196 and z: 36.1228, and the radius is 27 Å. The docking energies of each peptide and T1R1 / T1R3 and their activity prediction values in Peptide Ranker are shown in Table 2.

[0094] Table 2 Docking energies between peptides and T1R1 / T1R3

[0095] The molecular docking results of each peptide and the umami receptor T1R1 / T1R3 are as Figures 5 to 12 shown.

[0096] Test Example 4 Each crab umami peptide in Examples 15, 18, 21, and 22 was measured using an electronic tongue.

[0097] The synthetic peptide samples were prepared into an aqueous solution of 1.0 mg / mL, and after mixing, they were respectively filled into special beakers for the electronic tongue, and balanced and tested. The taste and aftertaste test time for each sample was 30 s, each sample was tested 4 times, and the first data was discarded, and the average value was taken as the test result.

[0098] The results are as Figure 13 shown. As shown in Figure a, except for FDQDDWENWTK, the taste profiles of the other three peptides are relatively similar, all having umami taste, accompanied by salty and sour tastes, and the taste abundance of the polypeptide is also enhanced accordingly. And these three peptides have no astringent taste and good taste characteristics. FDQDDWENWTK shows no umami taste, no salty taste either, but 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 samples of the three peptides except FDQDDWENWTK are very close and slightly overlapping on the PCA diagram, indicating that the samples of these three peptides have small differences and similar taste characteristics.

[0099] Test Example 5 The antioxidant activities of each crab umami peptide in Examples 15, 18, 21, and 22 were evaluated.

[0100] 1. Evaluation method 1.1 DPPH radical scavenging ability Prepare solutions of the enzymolysate at different concentrations. Take 100 μL of each and add it to a 96-well microplate, then add 100 μL of 0.1 mmol / L DPPH anhydrous ethanol solution. After reacting in the dark for 0.5 h, the absorbance measured at 517 nm is denoted as A 0 , and the absorbance measured with anhydrous ethanol solution replacing the DPPH anhydrous ethanol solution is denoted as A 1 , and the absorbance measured with water replacing the sample solution is denoted as A . Calculate the DPPH radical scavenging rate of the sample using the formula:

[0101] 1.2 ABTS radical scavenging ability Prepare a solution of 2.6 mmol / L K 2 S 2 O 8 and mix it with an equal volume of 7.4 mmol / L ABTS solution. After reacting in the dark for 14 h, dilute it with anhydrous ethanol to obtain the ABTS working solution. Add 40 μL of each concentration of the sample solution and 160 μL of the ABTS working solution to a 96-well microplate, react in the dark for 10 min, and the absorbance measured at 734 nm is denoted as A 0 , and the absorbance measured with water replacing the sample solution is denoted as A . Calculate the ABTS radical scavenging rate of the sample using the formula:

[0102] 1.3 Hydroxyl radical scavenging ability Prepare a 6.0 mmol / L FeSO 4 solution and a 6.0 mmol / L salicylic acid solution. Take 1.0 mL of each and add 1.0 mL of the sample solution, then add 1.0 mL of 6.0 mmol / L H 2 O 2 solution to initiate the reaction. Incubate in a 37 °C water bath for 0.5 h, and the absorbance measured at 510 nm is denoted as A 0 , and the absorbance measured with water replacing the H 2 O 2 solution is denoted as A 1 , and the absorbance measured with water replacing the sample solution is denoted as A . Calculate the hydroxyl radical scavenging rate of the sample using the formula:

[0103] 1.4 Reducing power Take 0.5 mL of the sample solution at each concentration in a test tube, add 2.5 mL of 0.2 mol / L PBS buffer with a pH of 6.6 and 1.0% K 3 Fe(CN) 6 solution. After mixing, react in a water bath at 50 °C 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% FeCl 3 solution. After mixing, keep it in the dark at room temperature for 10 min, and measure its absorbance at 700 nm.

[0104] 2. Results As Figures 14 to 17 shown, all four synthetic peptides have certain antioxidant activities. Among them, the DPPH, ABTS free radical and hydroxyl radical scavenging abilities of FDQDDWENWTK are significantly stronger than those of the other three peptides, and its reducing power is also slightly stronger. Although the free radical scavenging abilities of the other three peptides are not as good as FDQDDWENWTK, they still have certain antioxidant effects and are novel umami peptides with certain antioxidant activities.

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A crab umami peptide, characterized in that: The crab umami peptide 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; the amino acid sequence of FSGMDSFDDMAK is: Phe-Ser-Gly-Met-Asp-Ser-Phe-Asp-Asp-Met-Ala-Lys; the amino acid sequence of ANGPSKEEFPMFE is: Ala-Asn-Gly-Pro-Ser-Lys-Glu-Glu-Phe-Pro-Met-Phe-Glu; The amino acid sequence of DDPFFGGHRS is: Phe-Asp-Asp-Asp-Pro-Phe-Phe-Gly-Gly-His-Arg-Ser; the amino acid sequence of the VMWGDEHIPGSPF is: Val-Met-Trp-Gly-Asp-Glu-His-Ile-Pro-Gly-Ser-Pro-Phe; the amino acid sequence of the WGDEHIPGSPFK is: Trp-Gly-Asp-Glu-His-Ile-Pro-Gly-Ser-Pro-Phe-Lys; the amino acid sequence of the VIKDLPNFYGK is: Val-Ile-Lys-Asp-Leu-Pro-Asn-Phe-Tyr-Gly-Lys; the amino acid sequence of the 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 contains at least one of FDQDDWENWTK, FDDDPFFGGHRS, VIKDLPNFYGK and GVQPDKRPF; and / or 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); and / or 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).

3. The method for preparing the crab umami peptide according to claim 1 or 2, characterized in that: The specific steps include: S1. Crush the edible parts of the swimming crab or the Chinese mitten crab and disperse them in distilled water to prepare a crab meat homogenate, and add protease for enzymolysis to obtain a crab meat enzymolysate; the amount of the protease added is 3% to 8%wt of the crab meat homogenate; 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; 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.

4. The preparation method according to claim 3, characterized in that: The edible parts described in S1 include body meat, hepatopancreas, foot meat and claw meat; and / or The mass ratio of the edible part to distilled water in S1 is 1:6; and / or The protease in S1 is at least one selected from papain, trypsin, compound flavor enzyme, neutral protease and alkaline protease; and / or The enzymatic hydrolysis time in S1 is 3-6 h; after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90-95°C for 10-20 min; and / or The amount of protease added in S1 is 4.4%wt of the crab meat homogenate; and / or The centrifugal speed in S2 is 5000 rmp / min and the centrifugal time is 20 min; and / or The drying method in S2 is freeze drying; and / or The drying method described in S3 is freeze drying.

5. The preparation method according to claim 4, characterized in that: When the protease described in S1 is papain or neutral protease, the parameters of the enzymatic hydrolysis are: pH 6.0-8.0, and the enzymatic hydrolysis temperature is 45-55°C; when the protease described in S1 is trypsin, the parameters of the enzymatic hydrolysis are: pH 6.0-8.0, and the enzymatic hydrolysis temperature is 37-50°C; when the protease described in S1 is a composite flavor enzyme, the parameters of the enzymatic hydrolysis are: pH 5.0-7.0, and the enzymatic hydrolysis temperature is 50-55°C; when the protease described in S1 is an alkaline protease, the parameters of the enzymatic hydrolysis are: pH 7.0-9.0, and the enzymatic hydrolysis temperature is 50-60°C.

6. The preparation method according to claim 3, characterized in that: The chromatographic column of the gel chromatography described in S3 is Sephadex G-15, and the detection wavelength of the ultraviolet detector is 280 nm.

7. The preparation method according to claim 6, characterized in that: The elution flow rate in S3 is 0.5 mL / min; when the raw material in S1 is Swimming crab trituberculatus, the eluate is collected for 50 to 160 minutes; when the raw material in S1 is Chinese mitten crab, the eluate is collected for 50 to 275 minutes.

8. The preparation method according to any one of claims 3 to 7, characterized in that The preparation method also includes purifying the product obtained from S3 by high performance liquid chromatography.

9. The preparation method according to claim 8, characterized in that: The chromatographic conditions of the high performance liquid chromatography are: RP-C18 column, 150 mm × 0.15 mm; Mobile phase: aqueous phase is 0.1% formic acid aqueous solution, organic phase is 0.1% formic acid acetonitrile solution; Gradient elution: 0-50 min, the organic phase changes linearly from 4% to 50%; 50-54 min, the organic phase changes linearly from 50% to 100%; 54-60 min, the organic phase was maintained at 100%.

10. Use of the crab umami peptide according to claim 1 or 2 or the crab umami peptide prepared by the preparation method according to any one of claims 3 to 9 in the preparation of condiments, nutritional enhancers and / or flavor enhancers.

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