Method for extracting umami peptide from oysters

Abstract

The invention discloses a method for extracting umami peptide from oysters, which comprises the following steps: cleaning fresh and alive oyster meat, and stirring into meat paste; then carrying out enzymolysis, and cooking after enzymolysis; performing centrifugation, and performing low-temperature spray drying on centrifuged supernate to obtain oyster powder; dissolving the oyster powder, filtering the dissolved solution through a filter membrane, and retaining the filtrate; performing LC-MS / MS identification on the filtrate to obtain amino acid sequences of a series of peptides; and then iUmami-SCM and a molecular docking technology are used for screening to obtain the umami peptide. According to the method disclosed by the invention, two modes of enzymolysis and cooking are combined, the umami peptide is extracted, the deodorization of the oysters can be fully removed through an enzymolysis and cooking synergistic process, the oyster umami peptide with low bitterness and high freshness is extracted, and the extraction rate is 20% or above higher than that of pure enzymolysis and pure cooking. The extracted umami peptide has good stability and preservation performance, and has rich taste and higher extraction efficiency.

Description

Technical Field

[0001] This invention relates to the field of food flavor improvement technology, and more specifically, to a method for extracting umami peptides from oysters. Background Technology

[0002] Oysters (Crassostrea ariakensis) are the world's most farmed shellfish. Their meat is delicious and rich in various umami substances, earning them the nickname "milk of the sea." The adductor muscle of oysters is high in protein and also contains physiologically active complex phospholipids, EPA, DHA, and other nutrients. Oyster protein has a complete amino acid composition, containing not only 20 common amino acids but also various free amino acids, especially abundant natural taurine. Oysters are also rich in minerals and trace elements such as calcium, iron, zinc, manganese, and selenium, particularly zinc and selenium.

[0003] Umami peptides are small molecule peptides possessing umami flavor characteristics. Generally, umami peptides contain glutamic acid and aspartic acid and have a molecular weight of less than 3000 Da. Food-derived umami peptides have excellent umami-enhancing effects and can be used to prepare seasoning packets, condiments, and other flavored foods with a pronounced umami flavor. Glutamic acid and aspartic acid residues in umami peptides play a crucial role in enhancing their umami flavor. Traditional umami peptide screening methods involve hydrolyzing food proteins with commercially available proteases, followed by separation and purification to obtain the peptides, and then analyzing the structure of the repressive peptides using mass spectrometry.

[0004] After enzymatic hydrolysis, oysters produce volatile aldehydes and ketones from polyunsaturated fatty acids under the action of lipoxygenases. Aldehydes have a low threshold, and some aldehydes can significantly overlap with other components in flavor. Linear-chain saturated aldehydes, enaldehydes, and dienoals, produced by the oxidation of unsaturated fatty acids, are the main source of the fishy odor in oyster hydrolysate. This intensifies the fishy and bitter taste of the hydrolysate, deteriorating its flavor and becoming a bottleneck restricting its processing and utilization. Traditional boiling methods release less umami compounds from the oyster meat and contain some bitter peptides. Currently, there are no reports of extracting umami peptides from oysters using enzymatic hydrolysis and boiling methods. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for extracting umami peptides from oysters to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting umami peptides from oysters, comprising the following steps: (1) Wash the fresh oyster meat and stir it into a paste; (2) Place the minced meat in deionized water to make up the volume, and add enzymes for enzymatic hydrolysis; (3) Add the enzymatically hydrolyzed meat paste to deionized water and then steam it; (4) The steamed meat paste is centrifuged, and the supernatant after centrifugation is spray-dried at low temperature to obtain oyster powder; (5) Dissolve the oyster powder, filter the solution through a filter membrane, and retain the filtrate; (6) The filtrate was then identified by LC-MS / MS to obtain the amino acid sequences of a series of peptides; (7) Umami peptides were obtained by screening using iUmami-SCM and molecular docking technology.

[0007] Molecular docking refers to the process by which two or more molecules recognize each other through geometric and energy matching, with the aim of finding the optimal binding mode between ligand molecules and acceptor molecules.

[0008] Preferably, in step (1), the oyster meat is first deshelled, washed, drained, and weighed, and then the oyster is crushed and stirred into a paste.

[0009] Preferably, in step (2), the volume ratio of the meat paste to deionized water is 3-5:20.

[0010] Preferably, the enzyme is trypsin, and the amount of trypsin added is 0.1-0.2% of the mass of the homogenized and dispersed mixture, and the enzyme is hydrolyzed at 35-39°C for 10-15 minutes.

[0011] Preferably, in step (3), deionized water is added to the enzymatically hydrolyzed meat paste, and the amount of deionized water added is 2-3 cm above the meat paste. The paste is then placed in boiling water and kept for 2-3 hours, with continuous stirring during the process.

[0012] Preferably, the centrifugation is performed at 4°C and a relative centrifugal force of 3922g for 10-15 minutes.

[0013] Preferably, the amino acid sequence of the identified peptide is input into the iUmami-SCM website to predict the umami value, and peptides with low umami values ​​are removed to obtain umami peptides.

[0014] Preferably, the trypsin has an enzyme activity of 10,000 U / g or higher.

[0015] The present invention also provides an application of the umami peptide extracted by the method described above in directly edible foods.

[0016] The present invention further provides the application of the umami peptide extracted by the method described above in the preparation of various foods with oyster flavor and nutrition.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: This invention combines enzymatic hydrolysis and cooking to extract umami peptides. The combined enzymatic hydrolysis and cooking process effectively removes the fishy smell of oysters, extracting oyster umami peptides with low bitterness and high umami content. The extraction rate is more than 20% higher than that of enzymatic hydrolysis or cooking alone. The extracted umami peptides exhibit good stability and preservation, possessing a rich flavor and higher extraction efficiency. The nutritional value of oysters, rich in protein and various active substances, is preserved.

[0018] By using iUmami-SCM and molecular docking technology to screen umami peptides, the components that produce umami flavor can be accurately identified, thereby efficiently extracting the required umami peptides and improving production efficiency and product quality. Attached Figure Description

[0019] Figure 1 The results of electronic analysis of umami intensity are from Examples 2-3.

[0020] Figure 2 This is the result of electronic analysis of umami intensity in Example 1. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] A method for extracting umami peptides from oysters includes the following steps: (1) Wash the fresh oyster meat and stir it into a paste; (2) Place the minced meat in deionized water to make up the volume, and add enzymes for enzymatic hydrolysis; (3) Add the enzymatically hydrolyzed meat paste to deionized water and then steam it; (4) The steamed meat paste is centrifuged, and the supernatant after centrifugation is spray-dried at low temperature to obtain oyster powder; (5) Dissolve the oyster powder, filter the solution through a filter membrane, and retain the filtrate; (6) The filtrate was then identified by LC-MS / MS to obtain the amino acid sequences of a series of peptides; (7) The umami peptides were obtained by screening using iUmami-SCM and molecular docking technology. The amino acid sequence of the umami peptides is Pro-Gly-Ala-Asp-Glu.

[0023] In one embodiment, in step (1), the oyster meat is first deshelled, washed, and drained, and then the oyster is crushed and stirred into a paste.

[0024] In one embodiment, in step (2), the volume ratio of the meat paste to deionized water is 3-5:20.

[0025] In one embodiment, the enzyme is trypsin, and the amount of trypsin added is 0.1-0.2% of the mass of the homogenized and dispersed mixture, and the enzyme is hydrolyzed at 35-39°C for 10-15 minutes.

[0026] In one embodiment, in step (3), deionized water is added to the enzymatically hydrolyzed meat paste, and the amount of deionized water added is 2-3 cm above the meat paste. The paste is then placed in boiling water and kept for 2-3 hours, during which it is stirred continuously.

[0027] In one embodiment, the centrifugation is performed at 4°C and a relative centrifugal force of 3922g for 10-15 minutes.

[0028] In one embodiment, the amino acid sequence of the identified peptide is input into the iUmami-SCM website to predict the umami value, and peptides with low umami values ​​are removed to obtain umami peptides.

[0029] In one embodiment, the trypsin has an enzyme activity of 10,000 U / g or higher.

[0030] The present invention also provides an application of the umami peptide extracted by the method described above in directly edible foods.

[0031] The present invention further provides the application of the umami peptide extracted by the method described above in the preparation of various foods with oyster flavor and nutrition.

[0032] Example 1 Experimental materials Fresh oysters (Crassostrea gigas) were purchased from the local seafood market; adductor muscles were collected within 6 hours for the experiment. Trypsin (10000 U / g) and methanol were purchased from Merck. Standards and reagents required for liquid chromatography: tartaric acid, malic acid, lactic acid, citric acid, succinic acid, and trimethylamine oxide were purchased from Tanmo Quality Inspection. Potassium dihydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd. Cytosine nucleotides, adenine nucleotides, uracil nucleotides, guanine nucleotides, hypoxanthine nucleotides, and betaine were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Acetonitrile, potassium hydroxide, and perchloric acid were purchased from ACS. Potassium ion, sodium ion, phosphate, and chloride ion standards were purchased from Bolinda. Amino acid mixed standards were purchased from Wako Pure Chemical Industries Co., Ltd. Hydrochloric acid was purchased from Laiyang Kangde Chemical Co., Ltd. Take a sufficient amount of adductor muscle from fresh oysters, wash and drain the water, then stir into a meat paste; take 50g of meat paste, add deionized water to make up to 200ml, add 0.1g of 0.2% trypsinase, and hydrolyze at 39℃ for 3h. After hydrolysis, add deionized water to the enzymatically hydrolyzed meat paste, the amount of deionized water should be 2-3cm above the meat paste, and place it in boiling water for 2-3h, stirring constantly during the process. Centrifuge at 4℃ and a relative centrifugal force of 3922g for 10min to obtain the sample solution. The sample solution was analyzed by an LC-MS / MS system equipped with an online nano-spray ion source. The entire system was an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) with EASY-nanoLC1200 in series. A total of 2 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated by a gradient of 60 min. The column flow rate was controlled at 350 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, the gradient started from 2.2% B phase, increased to 50% in 51 min with a nonlinear gradient, increased to 90% in 3.5 min, and maintained for 5.5 min. The mass spectrometer was operated in data-dependent acquisition mode with the following parameters set: (1) MS: scan range (m / z): 200-1500; resolution: 60000; normalized AGC target: 300%; maximum injection Time: 25ms; (2) HCD-MS / MS: resolution: 15000; Normalized AGC target: 50%; maximum injection time: 22m; collision energy: 30%; dynamic exclusion time: 30s, obtained tandem mass spectrum, the tandem mass spectrum was analyzed by PEAKS Studio version 10.6, the database was uniprot-Crassostreaariakensis database, set none; search parameters fragment ion mass tolerance: 30.02Da, parent ion mass tolerance: 10ppm, variable modification: Oxidation (M) 15.99, Deamidation (NQ) 0.98, hydroxylation (KP) 16; protein card value is at least 1 unique peptide; peptide card value is -10lgP≥20, obtained the total peptide of this method, the total peptide has 3200 kinds.

[0033] Peptides with a molecular weight <1500 Da and a Glu(E) and Asp(D) content >30% were screened out. Then, a comprehensive screening was performed using an umami peptide classification model, and peptides that met all the above conditions were considered umami peptides. The iUmami-SCM database can be found at http: / / camt.pythonanywhere.com / iUmami-SCM. This method is a predictor of peptide umami flavor that uses the physicochemical properties of primary peptides, such as hydrophobicity, acidic amino acids, and low molecular weight.

[0034] Molecular docking of target peptides with umami receptors T1R1 / T1R3 was performed to screen for umami peptides. Specifically, the structures of umami receptors T1R1 / T1R3 were obtained using homology modeling, and molecular docking screening using the CDOCKER program in Data Science effectively screened peptides that bind to umami receptors T1R1 / T1R3. "-CDOCKER_ENERGY" indicates a higher affinity between the peptide and T1R1 / T1R3. Using "-CDOCKER_ENERGY" as an indicator, the amino acid sequence of the umami peptide was found to be Pro-Gly-Ala-Asp-Glu.

[0035] This embodiment successfully prepared oyster umami peptides with low bitterness and high umami content by combining enzymatic hydrolysis with segmented cooking and membrane filtration technology. Molecular weight distribution and amino acid analysis showed that the proportion of umami peptides (600-1200 Da) in the obtained product was significantly increased, and the content of characteristic umami substances was greatly improved compared with traditional processes, providing an effective solution for the intensive processing of oysters.

[0036] Example 2 Other methods are the same as in Example 1, except that: take a sufficient amount of adductor muscle from fresh oysters and stir it into a paste; take 50g of the paste, add deionized water to make up to 200ml (7:20, w:w), place it in boiling water and wait for the paste to gather before pouring off the foamy liquid, add deionized water again to make up to the previous volume and then boil in a water bath for 3 hours, stirring constantly during the process, and after the end, make up to 220ml to obtain the sample solution obtained by the cooking method.

[0037] Example 3 The other methods are the same as in Example 2, except that: sufficient adductor muscle is taken from fresh oysters and stirred into a paste; 50g of the paste is taken, deionized water is added to a final volume of 200ml, 0.1g of 0.2% trypsin is added, and the mixture is hydrolyzed at 39℃ for 3h. After hydrolysis, the mixture is inactivated at 100℃ for 10min, and then centrifuged at 4℃ and a relative centrifugal force of 3922g for 10min to obtain the enzymatic hydrolysis sample solution.

[0038] Electronic analysis The umami peptides from Examples 1-3 were used to detect sensory indicators using an electronic tongue (INSENT SA402B, Japan). Before use, the reference electrode was activated: 200 μL of internal fluid (3.33 M KCl + saturated AgCl solution) was injected into the sour, bitter, umami, salty, and aftertaste sensors using a syringe, ensuring the electrode core and lipid membrane were immersed in the fluid. The lipid membrane was pre-activated for over 24 hours using a reference solution (30.00 mM KCl + 0.30 mM tartaric acid). The internal solution (3.33 M KCl + saturated AgCl solution) was added to the ceramic reference sensor and pre-activated in 3.33 M KCl solution for 24 h.

[0039] Before analysis, the electronic tongue was cleaned and calibrated. The program settings were as follows: sample collection time, 30 seconds; aftertaste collection time, 30 seconds; cleaning time, 180 seconds; room temperature, 25°C. All samples were analyzed three times. Results of Examples 2-3 are as follows. Figure 1 The results of Example 1 are as follows Figure 2 As shown.

[0040] from Figure 1 , Figure 2 As can be seen, the electronic tongue analysis compared the umami intensity of umami peptides from the enzymatic hydrolysis-co-cooking method, the cooking method, and the enzymatic hydrolysis method. The results showed that the enzymatic hydrolysis-co-cooking method had a higher umami intensity, indicating that this method can effectively remove the fishy smell of oysters and extract oyster umami peptides with low bitterness and high freshness.

[0041] The yields of umami peptides obtained by the methods in Examples 1-3 were compared, and the results are shown in Table 1: The results in Table 1 show that the umami peptides obtained by the enzymatic hydrolysis combined with cooking method of the present invention have a lower yield (10-17%) than those obtained by simple cooking or simple enzymatic hydrolysis due to limitations in pyrolysis efficiency and protein conversion rate. However, by optimizing enzymatic hydrolysis pretreatment, cooking, and membrane filtration, the yield can be significantly increased to over 20%, while also improving the flavor and functional properties of the product.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting umami peptides from oysters, characterized in that, Includes the following steps: (1) Wash the fresh oyster meat and stir it into a paste; (2) Place the minced meat in deionized water to make up the volume, and add enzymes for enzymatic hydrolysis; (3) Add the enzymatically hydrolyzed meat paste to deionized water and then steam it; (4) The steamed meat paste is centrifuged, and the supernatant after centrifugation is spray-dried at low temperature to obtain oyster powder; (5) Dissolve the oyster powder, filter the solution through a filter membrane, and retain the filtrate; (6) The filtrate was then identified by LC-MS / MS to obtain the amino acid sequences of a series of peptides; (7) Umami peptides were obtained by screening using iUmami-SCM and molecular docking technology.

2. The method for extracting umami peptides from oysters according to claim 1, characterized in that, In step (1), the oyster meat is first deshelled, washed, drained, and weighed. Then the oysters are crushed and stirred into a paste.

3. The method for extracting umami peptides from oysters according to claim 1, characterized in that, In step (2), the volume ratio of the meat paste to deionized water is 3-5:

20.

4. The method for extracting umami peptides from oysters according to claim 1, characterized in that, The enzyme is trypsin, and the amount of trypsin added is 0.1-0.2% of the mass of the homogenized and dispersed mixture. Enzymatic hydrolysis is performed at 35-39℃ for 10-15 minutes.

5. The method for extracting umami peptides from oysters according to claim 1, characterized in that, In step (3), deionized water is added to the enzymatically hydrolyzed meat paste. The amount of deionized water added is 2-3 cm above the meat paste. The paste is then placed in boiling water and kept for 2-3 hours, with constant stirring during the process.

6. The method for extracting umami peptides from oysters according to claim 1, characterized in that, The centrifugation was performed at 4°C and a relative centrifugal force of 3922g for 10-15 minutes.

7. The method for extracting umami peptides from oysters according to claim 1, characterized in that, The amino acid sequence of the identified peptide is input into the iUmami-SCM website to predict the umami value. Peptides with low umami values ​​are removed to obtain the umami peptides.

8. The method for extracting umami peptides from oysters according to claim 4, characterized in that, The trypsin has an enzyme activity of 10,000 U / g or higher.

9. The use of a umami peptide extracted by the method according to any one of claims 1-8 in a directly edible food.

10. The application of a flavor peptide extracted by the method of any one of claims 1-8 in the preparation of various foods with oyster flavor and nutrition.

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