Medium-temperature protease EaPro48 and application thereof in preparation of crayfish umami peptide

Abstract

The invention discloses medium-temperature protease EaPro48 and application of the medium-temperature protease EaPro48 in preparation of a crayfish umami peptide. The amino acid sequence of the medium-temperature protease EaPro48 is as shown in SEQ ID NO. 1. The method comprises the following steps: inoculating Exiguobacterium antarctica DW2 into a protease-producing culture medium, culturing in an incubator to obtain a protease-containing bacterial suspension, centrifuging to take supernate, and purifying to obtain the medium-temperature protease EaPro48. The medium-temperature protease EaPro48 can efficiently hydrolyze the crayfish shell protein at 30 DEG C, and energy consumption in industrial application is remarkably reduced. By replacing a chemical process with enzymatic hydrolysis, use of toxic reagents is avoided, wastewater pollution is reduced, and green recovery and resource utilization of protein resources are realized. Sensory evaluation proves that the umami peptide obtained by the invention has pure umami and no bitter taste residue, and a novel core flavor substance is provided for development of natural seafood seasonings.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a mesothermal protease EaPro48 and its application in the preparation of crayfish umami peptides. Background Technology

[0002] Proteases are a class of enzymes that hydrolyze large proteins or peptides into amino acids or smaller peptides, playing a vital role in industry, medicine, and biotechnology. Mesophilic proteases, typically derived from bacteria in specific environments, have been described as possessing superior characteristics, such as high catalytic efficiency at room temperature and a flexible structure compared to thermophilic bacteria. Currently, mesophilic proteases have been found to have broad diversity and wide applications, including detergent additives in the cold washing industry, meat tenderizing, and other food processing applications. Due to their energy-saving properties, they hold great promise for future applications.

[0003] The production and processing of crayfish have been increasing year by year, especially in countries such as China, the United States, Australia, and Sweden. During processing, crayfish generate a large number of byproducts. While research on chitin recovery is relatively abundant, research on residual protein recovery is limited. Traditional protein extraction methods mainly rely on chemical processes, which are simple to operate and low in cost. However, this process is difficult to control, the products are unpredictable, and it generates a large amount of wastewater, leading to environmental pollution. Therefore, pollution-free biodegradation methods, especially protease hydrolysis, are particularly important in treating crayfish byproducts. The hydrolysate of crayfish shell protein can serve as a raw material for high-value-added seafood flavor extracts. Enzymatic protein recovery can not only further enhance the added value of byproducts but also significantly reduce solid waste generated during crayfish processing. In recent years, many studies have explored the application of proteases in the high-value utilization of food byproducts. For example, some researchers have used proteases to treat calf meat byproducts, successfully obtaining hydrolysates with antioxidant and anti-tyrosinase activities. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a mesophilic protease EaPro48 and its application in the preparation of crayfish umami peptides. The mesophilic protease EaPro48 of this invention is derived from *Microbacterium antarctica* (…). Exiguobacterium antarcticum DW2, this protease has medium-temperature catalytic properties. At low dosages, it can hydrolyze crayfish shell byproducts, improve umami flavor, and obtain umami peptides.

[0005] To achieve the above objectives, the technical solution designed in this invention is as follows: The present invention provides a mesothermal protease EaPro48, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The gene Ea encodes the above-mentioned protease pro 48, the gene EaPro48 The nucleotide sequence is shown in SEQ ID NO.2.

[0007] The aforementioned mesophilic protease EaPro48 is derived from Antarctic microbacteria ( Exiguobacterium antarcticum DW2, with accession number CCTCC NO:M 2019536, accession date July 10, 2019, address: Wuhan University, Wuhan, China; Antarctic Microbacterium ( Exiguobacterium antarcticum DW2 is disclosed in Chinese invention patent with publication number CN113862192B, entitled "Antarctic Microbacterium DW2 and Method for Preparing Chitosan Oligosaccharides".

[0008] The present invention also provides a method for preparing the above-mentioned mesophilic protease EaPro48. The method involves inoculating Antarctic Microbacterium DW2 into a protease-producing medium, then culturing it in an incubator to obtain a bacterial suspension containing the protease. The supernatant is then collected by centrifugation and purified to obtain the mesophilic protease EaPro48.

[0009] Furthermore, the composition of the protease-producing culture medium, by weight fraction, includes: skim milk powder: 1%, glucose: 0.5%, KH2PO4: 0.1%, K2HPO4: 0.1%, MgSO4·7H2O: 0.05%, seawater salt reagent: 3%, initial pH value of 7.8 (sterilized after adjustment), and the balance being water.

[0010] Furthermore, the temperature of the incubator is 30°C, and the incubation time is 3 days.

[0011] In practice, the purification steps are, in order, ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography.

[0012] The optimal pH for the mesophilic protease EaPro48 is 8.0, and the optimal temperature is 30°C.

[0013] The present invention also provides an application of the above-mentioned mesophilic protease EaPro48 in the degradation of crayfish shells.

[0014] The present invention also provides a method for hydrolyzing crayfish shells using the above-mentioned mesophilic protease EaPro48, comprising the following steps: 1) Dissolve the crayfish shells in Tris-maleate solution, homogenize thoroughly, centrifuge, and collect the precipitate; 2) Mix the precipitate into 10 times its volume of 20 mmol / L Tris-maleate solution, homogenize thoroughly, extract and centrifuge, and collect the supernatant; 3) Add EaPro48 solution of medium-temperature protease to the supernatant; enzymatically digest and inactivate; obtain the hydrolysis product.

[0015] Furthermore, the Tris-maleate solution has a concentration of 20 mmol / L and a pH of 7.0; it contains 0.05 mol / L NaCl. In step 3), the concentration of the mesophilic protease EaPro48 solution is 0.1 mg / mL; The enzymatic hydrolysis temperature was 30℃, the enzymatic hydrolysis time was 60 min, and the pH value was 8.0.

[0016] The present invention also provides a crayfish umami peptide prepared using the above-mentioned mesophilic protease EaPro48, wherein the amino acid sequence of the crayfish umami peptide is ADRLQKE.

[0017] The present invention also provides an application of the above-mentioned crayfish umami peptide in food processing.

[0018] The beneficial effects of this invention are: 1. The mesophilic protease EaPro48 provided by this invention is derived from a strain from a special Antarctic environment. It can efficiently hydrolyze crayfish shell protein at 30℃, overcoming the high energy consumption of traditional high-temperature proteases and significantly reducing energy consumption in industrial applications. By replacing chemical processes with enzymatic hydrolysis, the use of toxic reagents is avoided, wastewater pollution is reduced, and green recycling and resource utilization of protein resources are achieved.

[0019] 2. This invention obtains the umami peptide ADRLQKE by specifically enzymatically hydrolyzing crayfish by-products. It forms a stable interaction with the umami receptors T1R1 / T1R3. Sensory evaluation has confirmed that it has a pure umami flavor and no bitter residue, providing a novel core flavor substance for the development of natural seafood seasonings.

[0020] 3. The protease EaPro48 of the present invention maintains stable activity within a pH range of 5.5-10.0, and exhibits activity against Ca. 2+ The enzyme exhibits a response-enhancing effect with metal ions, making it adaptable to complex production environments. It demonstrates broad-spectrum hydrolytic ability against a variety of substrates, including casein and bovine serum albumin, making it suitable not only for processing crayfish byproducts but also for applications in the food industry such as meat tenderization and functional peptide preparation. Attached Figure Description

[0021] Figure 1 This is an SDS-PAGE image of purified Antarctic microbacterium protease EaPro48.

[0022] Figure 2 This is a schematic diagram showing the optimal temperature (A) and temperature stability (B) of the protease EaPro48.

[0023] Figure 3 This is a schematic diagram showing the optimal pH (A) and pH stability (B) of the protease EaPro48 in various 50mM buffer solutions at a temperature of 30°C.

[0024] In the diagram, ■ represents citrate-sodium citrate buffer (pH 4.5-6.5), ● represents disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6-8), ▲ represents Tris-HCl buffer (pH 7.0-9.0), and ▼ represents glycine-sodium hydroxide buffer (pH 8.5-10.5).

[0025] Figure 4 This is an SDS-PAGE analysis image of myofibrillar protein after EaPro48 treatment.

[0026] In the figure, MW refers to the standard molecular weight of the protein; 0–60 refers to the enzymatic digestion time (min).

[0027] Figure 5 This is a secondary mass spectrum of umami peptides.

[0028] Figure 6 The results of molecular docking between umami peptides and T1R1 and a magnified view of a portion thereof; Figure 7 The results of molecular docking between umami peptides and T1R3 and a magnified view of a portion thereof; Figure 6 and Figure 7 In this diagram, amino acid residues and their interactions are represented by two-dimensional graphics, with different residues represented by different colored spheres and interactions represented by dashed lines. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. The materials used in the following embodiments, such as reagents, cloning expression vectors, strains, and fermentation raw materials, are all commercially available. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0031] Example 1: Preparation of the mesophilic protease EaPro48 1) Antarctic microbacteria ( Exiguobacterium antarcticumDW2 (accession number CCTCC NO: M2019536) was inoculated into protease-producing medium and cultured at 30°C for 3 days to obtain a bacterial suspension containing protease. The protease-producing medium consisted of, by weight fraction, 1% skim milk powder, 0.5% glucose, 0.1% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4·7H2O, 3% seawater salt reagent, initial pH 7.8 (adjusted and then sterilized), with the remainder being water. 2) Centrifuge at 12000g for 10 min, collect the supernatant (crude enzyme solution), and purify it using ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography to obtain the target protein, which is predicted to be S8 protease. 3) SDS-PAGE electrophoresis was performed using a 12% separating gel and a 5% stacking gel. The target protein was observed using Comasassie Brilliant Blue R-250 staining. Figure 1 The molecular weight of the enzyme was analyzed using a protein marking kit (Solarbio, Beijing, China); the target protein had a molecular weight of approximately 48 kDa and an enzyme activity of 401 U / mg; it was named protease EaPro48. Sequencing by a biotechnology company revealed the amino acid sequence of protease EaPro48 as shown in SEQ ID NO.2, and the nucleotide sequence encoding protease EaPro48 as shown in SEQ ID NO.1.

[0032] The amino acid sequence of the mesophilic protease EaPro48 is shown in SEQ ID NO.2: MKQRPELLAPAGNLEKLKMAIHYGADAVYIGGQQFGLRSRAGNFSYDEMREGVAFAHARGAKVYVAANMVTHEGDIAGAGDFFRTLRDIGIDAVIVSDPAMIE TCLSEAEGLPVHLSTQASATNYETLRFWKEEGLERVVLAREVSMDEIVQMKQEVDVEIETFVHGAMCISYSGRCTLSNHMTLRDANRGGCAQSCRWKYGFFEEG DLLTEEMEARDEEPFSMSSVDLSMVRHIPDLVDAGVDSLKVEGRMKSIHYVATVCNVYRQVIDAYCEDPENFEFDPAWEEEIWKAAQRELASGFYYGIPTEKE QLFGKPRSIPQYAFAARVISYDGVSGMATLEQRNHFSIGEEVEFFGPGFVRFSQVIETIEDVDGQPLQSANQAMMTIRVKVDEPVATDFIMRKRKGVPARILV.

[0033] Example 2 Enzymatic properties of the mesophilic protease EaPro48 1. Method 1.1 Enzyme activity assay The Folin-Ciocalteu method was used to determine protease activity, as detailed below: First, the protease was dissolved and appropriately diluted in 50 mmol / L Tris-HCl buffer (pH 8.0) to prepare an enzyme solution. 1 mL of the enzyme solution was reacted with 1 mL of 1% casein solution at 30 °C for 10 min. After the reaction, 2 mL of 0.4 mol / L trichloroacetic acid solution was added to terminate the reaction. The mixture was then filtered, and 1 mL of the filtrate was added to 5 mL of 0.4 mol / L Na₂CO₃ solution and 1 mL of Folin-Ciocalteu solution. The mixture was incubated at 40 °C for 20 min, and the absorbance was measured at 680 nm. To plot the tyrosine standard curve, a series of tyrosine solutions were used instead of 1 mL of the filtrate, reacting with Na₂CO₃ solution and Folin-Ciocalteu reagent. Enzyme activity units (U) are defined as the amount of enzyme that releases 1 μg of tyrosine from casein per minute.

[0034] 1.2 Optimal Temperature and Temperature Stability Analysis ① Determination of optimal temperature: Using casein as a substrate, the enzyme activity of EaPro48 was determined in a 50 mmol / L Tris-HCl buffer system (pH 8) at 4℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃; the highest enzyme activity was defined as 100%.

[0035] ② Thermal stability analysis: EaPro48 was incubated in a 50 mmol / L Tris-HCl (pH 8) buffer system at different temperatures (20~60℃) for 30 min, and then the residual enzyme activity was measured.

[0036] 1.3 Optimal pH and pH stability analysis ① Optimal pH determination: The enzyme activity of EaPro48 was measured at 30℃ in buffer solutions (50 mmol / L) with different pH values ​​(4.5-10.5). The highest enzyme activity was defined as 100%. The buffer systems used in this example were citrate-sodium citrate buffer (pH 4.5-6.5), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.0-9.0), and glycine-sodium hydroxide buffer (pH 8.5-10.5) (Lv, Zhang, Ma, Oh, & Fu, 2022).

[0037] ② pH stability analysis: EaPro48 was incubated at 30℃ for 30 min at different pH (4.5-10.5), and then the residual enzyme activity was measured.

[0038] 1.4 Effects of Metal Ions on EaPro48 Enzyme Activity Different metal ions (Zn) were added to a 50 mmol / L Tris-HCl buffer system (pH 8) to a final concentration of 5 mmol / L. 2+ Ca 2+ K + Cu 2+ Ag + Ba 2+ Mg 2+ Fe 2+ Mn 2+ The enzyme was incubated at 30°C for 30 min to investigate the effect of metal ions on protease activity. The enzyme activity of the control group was defined as 100%.

[0039] 1.5 Substrate Specificity Various protein substrates (casein, ovalbumin, gelatin, and bovine serum albumin) were dissolved at a concentration of 1% in 50 mmol / L Tris-HCl buffer at pH 8.0, and enzyme activities were calculated. Activity against casein was used as a control.

[0040] 2. Conclusion 2.1 Enzyme activity The enzyme activity of EaPro48 protease is 401 U / mg.

[0041] 2.1 Optimal Temperature and Temperature Stability Analysis The optimal temperature for EaPro48 protease is 30°C. It still exhibits 17.77% of its maximum activity at a low temperature of 4°C. Figure 2 A). Temperature stability analysis further revealed that the protease retained over 80% of its activity after incubation for 30 minutes within the range of 20-40°C, and over 60% of its activity at 45°C. However, when the temperature was raised to 50°C, the activity decreased to below 20%. Figure 2 B).

[0042] 2.2 Optimal pH and pH stability The optimal catalytic pH for EaPro48 protease is 8.0. It retains over 40% of its relative activity over a wide pH range (5.5-10.0). Figure 3 A). pH stability results showed that after incubation in a buffer solution with a pH of 4.0-5.0 for 30 min, the enzyme activity decreased by 50%, while it remained relatively stable in an alkaline environment. Figure 3 B).

[0043] 2.3 Effects of metal ions on enzyme activity Metal ions generally act as cofactors in enzyme reactions, directly participating in the catalytic process and affecting enzyme activity and stability. Therefore, this example analyzes the effects of some metal ions on EaPro48.

[0044] As shown in Table 1: Ca 2+ K + Ba 2+ and Mg 2+ All of them had a promoting effect on the enzyme activity of EaPro48, among which Ca 2+ The promoting effect was most significant, increasing the enzyme activity of EaPro48 to 126.1%. At the same concentration, Zn... 2+ Cu 2+ Ag + Fe 2+ Mn 2+ Metal ions, however, inhibited the enzyme activity of EaPro48 to varying degrees.

[0045] Table 1. Effects of metal ions on the activity of the protease EaPro48 2.4 Substrate Specificity To investigate the substrate specificity of the EaPro48 protease, its hydrolysis of casein and other globular proteins was studied.

[0046] As shown in Table 2, the EaPro48 protease exhibits broad substrate specificity, showing significant hydrolytic activity not only on casein but also on bovine serum albumin, ovalbumin, and gelatin. Compared to gelatin, bovine serum albumin, and ovalbumin, this enzyme exhibits significantly higher hydrolytic activity towards casein.

[0047] Table 2. Substrate specificity assay of protease EaPro48 Example 3: Hydrolysis of crayfish shells using protease EaPro48 A method for hydrolyzing crayfish shells using the protease EaPro48 includes the following steps: 1) Add 10 times the volume of 20 mmol / L Tris-maleate solution (containing 0.05 mol / L NaCl, pH 7.0) to crayfish by-products (crayfish shells), homogenize thoroughly, centrifuge at 10000 r / min for 10 min, and collect the precipitate; 2) Mix the precipitate into 10 times the volume of 20 mmol / L Tris-maleate solution (containing 0.6 mol / L NaCl, pH 7.0), homogenize thoroughly, and extract for 1 h; finally, centrifuge at 10000 r / min for 10 min and collect the supernatant (protein solution). 3) Adjust the supernatant concentration to 2 mg / mL, then add an equal volume of 0.1 mg / mL proteinase EaPro48, adjust the pH to 8.0, and perform enzymatic hydrolysis at 30℃ for 60 min. Then, inactivate the enzyme by boiling in a water bath for 15 min to obtain the hydrolysis product.

[0048] During the enzymatic hydrolysis process described above, equal portions (100 μL) of the sample were taken from the test tube every 10 minutes to obtain a time-process sample for hydrolysis determination, so as to study the hydrolysis time process by SDS-PAGE.

[0049] like Figure 4As shown, EaPro48 exhibits high hydrolytic activity and reaction rate against crayfish myofibrillar proteins. During the incubation period from 0 to 60 minutes, the intensity of bands containing actin (36.13 kDa), troponin (23.49 kDa), and myosin light chain (17.59 kDa) significantly decreased or disappeared as the enzymatic reaction progressed. At 10 minutes, low-molecular-weight bands, possibly derived from the hydrolysates, were detected. However, as hydrolysis continued, a certain accumulation of low-molecular-weight protein bands was observed, indicating that high-molecular-weight proteins were effectively degraded into hydrolysates rich in peptides and amino acids. EaPro48's excellent hydrolytic ability against crayfish proteins further supports its application in crayfish byproducts.

[0050] Example 4: Obtaining umami peptides by hydrolyzing crayfish shells with protease EaPro48. 1. Method 1.1 Isolation and Mass Spectrometry Identification of Umami Peptides The hydrolysate obtained in Example 3 was centrifuged at 12000g for 15 min (to remove insoluble solids), and the supernatant was collected for further analysis. The hydrolysate was filtered through an ultrafiltration membrane (Millipore) to collect the fraction with a molecular weight less than 1 kDa for the identification of umami peptides.

[0051] Peptides smaller than 1 kDa were identified using nano-high performance liquid chromatography / mass spectrometry. Nano-HPLC-MS / MS analysis was performed on an EASY-nLC1200 (Thermo Fisher Scientific, USA) equipped with a C18 column (NanoViper C18, 75 μm × 50 cm) connected to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, USA).

[0052] like Figure 5 The mass of the umami peptide shown was determined by mass spectrometry to be 858.4 Da, and its amino acid sequence was ADRLQKE.

[0053] 1.2 Molecular docking The structures of the above-mentioned umami peptides and their umami taste receptors (T1R1 / T1R3) were established, and molecular docking was performed using MOE.

[0054] like Figure 6 As shown, this represents the molecular docking result between the umami peptide and the umami receptor T1R1. Figure 7 The figure shows the molecular docking results between the umami peptide and the umami receptor T1R3.

[0055] The above results indicate that the umami peptides can enter and bind to receptor protein domains to produce umami flavor.

[0056] 1.3 Solid-phase synthesis of peptides Umami peptides were synthesized using the Fmoc solid-phase method. After purifying the crude peptide to >98% purity, residual trifluoroacetic acid (TFA) was removed using an ion exchange resin, and the solution was freeze-dried to obtain the final product, which is the umami peptide, with the amino acid sequence ADRLQKE.

[0057] 1.4 Mouthfeel characteristics of synthetic peptides The taste characteristics of the aforementioned umami peptides were analyzed using a combination of electronic tongue and sensory evaluation. The taste characteristics of the samples were measured using the ASTREE electronic tongue system (Alpha MOS, France). Five test sensors were named AHS, CTS, NMS, ANS, and SCS, representing sourness, saltiness, umami, sweetness, and bitterness, respectively.

[0058] Before testing, the electronic tongue's sensors need to be activated, calibrated, and diagnosed. The sample solution to be tested is placed in a dedicated beaker for the electronic tongue, 100 mL per sample, and the test is performed by alternating between the sample and distilled water. Six parallel data points are collected for each sample's five basic tastes, and the three stable response values ​​are taken as valid data. The sample detection time is 10 seconds, and the test is conducted at room temperature of 25°C. Taste intensity data are collected using the electronic tongue's built-in data processing software.

[0059] Sensory evaluation employed quantitative descriptive analysis (QDA). Five reference solutions were used: monosodium glutamate (0.23%), citric acid (0.06%), quinine (0.002%), sodium chloride (0.56%), and sucrose (1.86%). The peptides were homogeneously dissolved in 2 mg / mL water, and the solutions were placed in odorless, transparent beakers and randomly numbered with three digits. Panel members were asked to describe the taste of the samples and rate each attribute on a scale.

[0060] The results of electronic tongue combined with sensory evaluation showed that the synthesized umami peptide (ADRLQKE) had a good umami taste at a concentration of 2 mg / mL and exhibited a certain degree of saltiness, but no bitterness.

[0061] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A mesophilic protease EaPro48, characterized in that: The amino acid sequence of the mesothermal protease EaPro48 is shown in SEQ ID NO.

1.

2. The gene Ea encoding the protease of claim 1 pro 48, characterized in that: The gene EaPro48 The nucleotide sequence is shown in SEQ ID NO.

2.

3. A method for preparing the mesophilic protease EaPro48 according to claim 1, characterized in that: The method involves inoculating Antarctic Microbacterium DW2 into a protease-producing medium, then culturing it in an incubator to obtain a bacterial suspension containing the protease. The supernatant is then collected by centrifugation and purified to obtain the mesophilic protease EaPro48.

4. The preparation method according to claim 3, characterized in that: The protease-producing culture medium comprises, by weight fraction, 1% skim milk powder, 0.5% glucose, 0.1% KH₂PO₄, 0.1% K₂HPO₄, and 0.1% MgSO₄·7H₂O. 0.05%, seawater salt reagent: 3%, initial pH value is 7.8, the remainder is water.

5. The preparation method according to claim 3, characterized in that: The temperature of the incubator is 30℃, and the incubation time is 3 days.

6. The application of the mesothermal protease EaPro48 as described in claim 1 in the degradation of crayfish shells.

7. A method for hydrolyzing crayfish shells using the mesophilic protease EaPro48 described in claim 1, comprising the following steps: 1) Dissolve the crayfish shells in Tris-maleate solution, homogenize thoroughly, centrifuge, and collect the precipitate; 2) Mix the precipitate into 10 times its volume of 20 mmol / L Tris-maleate solution, homogenize thoroughly, extract and centrifuge, and collect the supernatant; 3) Add EaPro48 solution of medium-temperature protease to the supernatant; hydrolyze and inactivate the enzyme; Hydrolysis products were obtained.

8. The method according to claim 7, characterized in that: The Tris-maleate solution had a concentration of 20 mmol / L and a pH of 7.0; it contained 0.05 mol / L NaCl. In step 3), the concentration of the mesophilic protease EaPro48 solution is 0.1 mg / mL; The enzymatic hydrolysis temperature was 30℃, the enzymatic hydrolysis time was 60 min, and the pH value was 8.

0.

9. A crayfish umami peptide prepared using the mesothermal protease EaPro48 described in claim 1, characterized in that: The amino acid sequence of the crayfish umami peptide is ADRLQKE.

10. The application of the crayfish umami peptide according to claim 9 in food processing.

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