Detection method for fresh substances of umami peptide
Through the combination of high-performance liquid chromatography-tandem mass spectrometry technology and umami peptide classification prediction model, the problem of inaccurate detection of umami peptides in food in the existing technology is solved, and more accurate umami peptide component identification and differential analysis are achieved. Provides more objective analysis results.
Patent Information
- Application Number
- CN202411552428.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-01
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art cannot accurately detect the existence and differences of umami peptides in food, and traditional sensory evaluation is highly subjective and cannot objectively reflect the differences of umami peptides.
Using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, samples were analyzed by online nanospray ion source, combined with the umami peptide classification prediction model for screening, and the umami peptide prepared by the cooking method and enzymatic method were determined, and the Hydrolysis difference analysis.
It improves detection sensitivity and accuracy, can more accurately identify and quantify umami peptide components, scientifically analyze differences in umami peptides, avoids strong subjective sensory evaluations, and provides more objective and quantifiable analysis results.
Smart Images
Figure CN119395180A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of detection technology, and in particular relates to a method for detecting umami peptide umami substances. Background Art
[0002] Oysters (Crassostrea ariakensis) are the world's largest farmed shellfish. They have delicious meat and are rich in a variety of umami substances. The protein content in the adductor muscle of oysters is high. Oyster sauce prepared by traditional boiling is popular among consumers around the world. Oyster umami peptides are mainly processed by steaming and enzymatic hydrolysis. The umami peptides prepared by the two methods have certain umami differences.
[0003] At present, the detection method of umami peptides in food cannot accurately determine the umami peptides in food; and the evaluation of umami differences is usually through sensory evaluation, which is highly subjective and cannot objectively reflect the umami differences of umami peptides. Summary of the invention
[0004] The object of the present invention is to provide a method for detecting umami peptide umami substances to solve the technical problems raised in the above background technology.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] A method for detecting umami peptide umami substances comprises the following steps:
[0007] S1 Sample preparation: Oysters were prepared by steaming and enzymatic hydrolysis to prepare sample solution;
[0008] S2 Peptide identification: The sample solutions of the cooking method and the enzymatic method were analyzed by an LC-MS / MS system equipped with an online nanospray ion source to obtain tandem mass spectra, and the tandem mass spectra were analyzed to obtain the total peptides of the cooking method and the enzymatic method respectively;
[0009] S3 Umami peptide screening: The total peptides are preliminarily screened, and then the umami peptide classification prediction model is used for secondary screening to determine the umami peptides obtained by the steaming method and the enzymatic hydrolysis method;
[0010] S4: The screened oyster umami peptides were compared with the total polypeptides obtained by cooking and enzymatic hydrolysis, and the precursor proteins with significant differences in umami peptides between enzymatic hydrolysis and cooking were analyzed for hydrolysis difference analysis.
[0011] Furthermore, in S1, the sample solution preparation method of the cooking method is: take a sufficient amount of closed shell muscle from fresh oysters and stir it into meat paste; take a certain mass of meat paste, add deionized water to the volume, place it in boiling water, wait for the meat paste to gather, pour out the floating foam liquid, add deionized water again to the previous volume, and then boil it in boiling water, stirring continuously during the period, and then fix the volume after the end to obtain the cooking method sample solution.
[0012] Furthermore, in S1, the preparation method of the sample solution of the enzymatic hydrolysis method is: taking a sufficient amount of closed shell muscle from fresh oysters and stirring it into meat paste; taking a certain mass of meat paste, adding deionized water to make up the volume, adding enzyme to hydrolyze, inactivating after hydrolysis, and centrifuging to obtain the enzymatic hydrolysis sample solution.
[0013] Furthermore, the enzyme is 0.2% trypsin, the hydrolysis temperature is 39°C, the time is 3h; the inactivation temperature is 100°C, the time is 10min; the centrifugation temperature is 4°C, and the time is 10min.
[0014] Furthermore, in S2, the sample solutions of the cooking method and the enzymatic method were taken and loaded onto the LC-MS / MS system, and the samples were separated with a gradient of 60 min, the column flow was controlled at 350 nL / min, the column temperature was 40°C, the electrospray voltage was 2 kV, the gradient started from 2.2% of the B phase, increased to 50% with a nonlinear gradient in 51 minutes, increased to 90% within 3.5 minutes, and maintained for 5.5 minutes.
[0015] Furthermore, the mass spectrometry parameters were set as follows: (1) MS: scanning range (m / z): 200-1500; resolution: 60000; Normalized AGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15000; Normalized AGC target: 50%; maximum injection time: 22 m; collision energy: 30%; dynamic exclusion time: 30 s.
[0016] Furthermore, in said S3, the preliminary screening method is: in the total polypeptides, a target peptide is selected whose amino acid content is less than 15 and the content of acidic amino acids (E, D) in the amino acid composition of the polypeptide exceeds 30%.
[0017] Furthermore, in S3, the secondary screening method is: the target peptides after the initial screening are subjected to secondary screening through multiple classification prediction models, and those that meet the model conditions are determined to be umami peptides obtained by the cooking method and the enzymatic method.
[0018] Furthermore, the classification prediction models are UMPred-FRL, Umami-MRNN, iUmami-SCM, TPDM and Umami_YYDS.
[0019] Furthermore, in S4, the umami peptide obtained in S3 is retrieved from the total polypeptide obtained in S2 through precursor protein, differential proteins are found, and are determined as marker proteins; the amino acid sequences of marker proteins are collected from the protein database, the positions of the umami peptides in the proteins are found, and the first three amino acids at the N-terminus and the last three amino acids at the C-terminus of the polypeptide are retained in the database, and finally the probabilities of these amino acids appearing at the enzyme cleavage positions are counted, which are the characteristics of protein hydrolysis by steaming and enzymatic hydrolysis.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] 1. The present invention improves the sensitivity and accuracy of detection by adopting high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology, and can more accurately identify and quantify umami peptide components; and then conducts secondary screening through primary screening and umami peptide classification model, which can accurately identify umami peptide components that produce umami.
[0022] 2. The present invention can scientifically and effectively analyze and evaluate the differences in umami peptides by performing hydrolysis difference analysis on the differences in umami peptides, thereby avoiding the subjectivity of traditional sensory evaluation and providing more objective and quantifiable umami difference analysis results.
[0023] 3. The present invention can perform differential analysis on umami peptides prepared by different methods, provide a scientific basis for umami peptide production, help select the optimal production process, and improve production efficiency and product quality. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is a diagram of the types of umami peptides screened by the cooking method of the present invention;
[0025] Figure 2 is a diagram of the types of umami peptides screened by the enzymatic hydrolysis method of the present invention;
[0026] Figure 3 It is the protein bubble diagram of the cooking method and the enzymatic hydrolysis method of the present invention;
[0027] Figure 4 It is the enzymatic hydrolysis point map of the cooking method of the present invention;
[0028] Figure 5 It is the enzymatic hydrolysis point map of the enzymatic hydrolysis method of the present invention. DETAILED DESCRIPTION
[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.
[0030] A method for detecting umami peptide umami substances comprises the following steps:
[0031] S1 Sample preparation: Oysters were prepared by steaming and enzymatic hydrolysis to prepare sample solution;
[0032] The sample solution preparation method of the cooking method is as follows: take a sufficient amount of closed shell muscle from fresh oysters and stir it into meat paste; take a certain amount of meat paste, add deionized water to make it up to volume, put it in boiling water, pour out the floating foam liquid after the meat paste gathers, add deionized water to make it up to the previous volume, and then boil it in boiling water, stirring continuously during the process, and then make it up to volume after the end, so as to obtain the sample solution of the cooking method;
[0033] The sample solution preparation method of the enzymatic method is as follows: take a sufficient amount of closed shell muscle from fresh oysters and stir it into meat paste; take a certain amount of meat paste, add deionized water to make up the volume, add 0.2% trypsin enzyme, hydrolyze at a temperature of 39°C for 3h, after hydrolysis, inactivate at a temperature of 100°C for 10min, and then centrifuge at a temperature of 4°C for 10min to obtain the enzymatic method sample solution;
[0034] S2 peptide identification: The sample solutions of the cooking method and the enzymatic method were analyzed by an LC-MS / MS system equipped with an online nanospray ion source. The samples were separated with 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, and increased to 50% in 51 minutes with a nonlinear gradient, and increased to 90% within 3.5 minutes, and maintained for 5.5 minutes; the mass spectrometry parameters were set as follows: (1) MS: scanning range (m / z): 200-1500; resolution: 60000; Normalized AGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15000; Normalized AGC target: 50%; maximum injection time: 22 m; collision energy: 30%; dynamic exclusion time: 30 s, and tandem mass spectra were obtained. The tandem mass spectra were analyzed to obtain the total peptides of the cooking method and the enzymatic method respectively;
[0035] S3 Umami peptide screening: The total peptides were preliminarily screened by selecting target peptides with an amino acid content of less than 15 and an acidic amino acid (E, D) content of more than 30% in the total peptides, and then using UMPred-FRL, Umami-MRNN, iUmami-SCM, TPDM and Umami_YYDS umami peptide classification prediction models for secondary screening. The umami peptides obtained by steaming and enzymatic hydrolysis were determined for those that met the model conditions.
[0036] S4 hydrolysis difference analysis: In the S4, the umami peptide obtained in S3 is retrieved from the total polypeptide obtained in S2 for precursor protein, and the differential protein is found and determined as the marker protein; the amino acid sequence of the marker protein is collected from the protein database, the position of the umami peptide in the protein is found, and the first three amino acids at the N-terminus and the last three amino acids at the C-terminus of the polypeptide are retained in the database, and finally the probability of these amino acids appearing at the enzyme cleavage position is counted, which is the protein hydrolysis characteristics of the cooking method and the enzymatic method, and the hydrolysis difference analysis of the cooking method and the enzymatic method is obtained.
[0037] The following is described by more specific embodiments:
[0038] Example 1
[0039] A method for detecting umami peptide umami substances comprises the following steps:
[0040] S1 Sample preparation: Oysters were prepared by steaming and enzymatic hydrolysis to prepare sample solution;
[0041] The sample solution preparation method of the cooking method is as follows: take a sufficient amount of closed shell muscle from fresh oysters and stir it into meat paste; take a certain amount of meat paste, add deionized water to make it 200ml (7:20, w:w), put it in boiling water until the meat paste gathers, pour out the floating foam liquid, add deionized water to make it to the previous volume again, and then boil it in a boiling water bath for 3h, stirring continuously during the period, and then make it 220ml after the end, so as to obtain the cooking method sample solution;
[0042] The sample solution preparation method of the enzymatic hydrolysis method is as follows: take a sufficient amount of closed shell muscle from fresh oysters and stir it into meat paste; take 50g of meat paste, add deionized water to make it 200ml, add 0.1g of 0.2% trypsin enzyme, hydrolyze at a temperature of 39°C for 3h, after hydrolysis, inactivate at a temperature of 100°C for 10min, and then centrifuge at a temperature of 4°C and a relative centrifugal force of 3922g for 10min to obtain the enzymatic hydrolysis sample solution;
[0043] S2 peptide identification: The sample solutions from the digestion method and the enzymatic method were analyzed by an LC-MS / MS system equipped with an online nanospray ion source. The entire system was an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. 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, and the gradient started from 2.2% B phase and increased to 50% in 51 min with a nonlinear gradient, and then 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: (1) MS: scan range (m / z): 200-1500; resolution: 60,000; 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, and tandem mass spectra were obtained. The tandem mass spectra were analyzed by PEAKS Studio version 10.6, the database was uniprot-Crassostrea ariakensis database, and the setting was none; the 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 was at least 1 unique peptide; peptide card value was -10lgP ≥ 20, and the total peptides obtained by the cooking method and enzymatic hydrolysis method were respectively obtained. Among them, the total peptides obtained by the cooking method were 3000 species, and the total peptides obtained by the enzymatic hydrolysis method were 3500 species;
[0044] S3 Umami peptide screening: The total peptides were preliminarily screened by selecting target peptides with an amino acid content of less than 15 and an acidic amino acid (E, D) content of more than 30% in the total peptides, and then using UMPred-FRL, Umami-MRNN, iUmami-SCM, TPDM and Umami_YYDS umami peptide classification prediction models for secondary screening. If the conditions of the model were met, the umami peptides of the cooking method and the enzymatic method were determined, such as Figure 1-2 As shown. 95 umami peptides were screened by the cooking method, and 117 umami peptides were screened by the enzymatic method;
[0045] S4 hydrolysis difference analysis: In said S4, the umami peptides obtained in S3 are searched for precursor proteins from the total polypeptides obtained in S2 to find out the differential proteins and determine them as marker proteins, such as Figure 3 As shown in the bubble diagram, it was found that myosin essential light chain and thermosensitive protein 70 had large bubbles under both enzymatic hydrolysis and cooking methods, indicating that the cooking method and enzymatic hydrolysis method produced more differential umami peptides in these two proteins. Among them, myosin essential light chain had 16 differential proteins in the enzymatic hydrolysis sample group and 10 differential proteins in the thermal hydrolysis sample group, a total of 26 (accounting for 7.74% of the total differential precursor proteins); thermosensitive protein 70 had 18 and 16 differential proteins in the enzymatic hydrolysis sample group and the thermal hydrolysis sample group, a total of 34 (accounting for The difference in umami peptides was 10.11% of proteins, indicating that the differential umami peptides were mostly concentrated in the essential light chain of myosin and the thermosensitive protein 70; the amino acid sequences of marker proteins were collected from the protein database to find out the location of the umami peptides in the protein, and the first three amino acids at the N-terminus and the last three amino acids at the C-terminus of the polypeptide were retained in the database. Finally, the probability of these amino acids appearing at the enzyme cleavage position was counted. The cleavage site of the protease was expressed as P3-P2-P1-P1'-P2'-P3', and the cleavage mainly occurred between the P1 and P1' residues, such as Figure 4-5 As shown in the figure, the enzymatic hydrolysis sites of the two treatment methods are mainly acidic amino acids Glu, followed by hydrophobic amino acids I (Ile), L (Leu) and F (Phe), and then polar amino acids T (Thr) and G (Gly). In the enzymatic hydrolysis method, the P1 and P1' positions are dominated by K () and E (), respectively, while the steaming method is dominated by D (Asp) and E (Glu), which indicates that lysine is a basic amino acid, and aspartic acid and glutamic acid are acidic amino acids. The combination and position of acidic and basic amino acids will affect the spatial structure, solubility and interaction with taste receptors of umami peptides, resulting in different umami perceptions.
[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for detecting umami peptide umami substances, characterized in that: The following steps are involved: S1 Sample preparation: Oysters were prepared by steaming and enzymatic hydrolysis to prepare sample solution; S2 Peptide identification: The sample solutions of the cooking method and the enzymatic method were analyzed by an LC-MS / MS system equipped with an online nanospray ion source to obtain tandem mass spectra, and the tandem mass spectra were analyzed to obtain the total peptides of the cooking method and the enzymatic method respectively; S3 Umami peptide screening: The total peptides are preliminarily screened, and then the umami peptide classification prediction model is used for secondary screening to determine the umami peptides obtained by the steaming method and the enzymatic hydrolysis method; S4: The screened oyster umami peptides were compared with the total polypeptides obtained by cooking and enzymatic hydrolysis, and the precursor proteins with significant differences in umami peptides between enzymatic hydrolysis and cooking were analyzed for hydrolysis difference analysis.
2. The method for detecting umami peptide umami substances according to claim 1, characterized in that: In S1, the preparation method of the sample solution of the cooking method is: take a sufficient amount of closed shell muscle from fresh oysters and stir it into meat paste; take a certain mass of meat paste, add deionized water to make up the volume, put it in boiling water until the meat paste gathers, pour out the floating foam liquid, add deionized water again to make up the volume to the previous volume, and then boil it in boiling water, stirring continuously during the process, and then make up the volume after the end, so as to obtain the cooking method sample solution.
3. The method for detecting umami peptide umami substances according to claim 1, characterized in that: In S1, the sample solution preparation method of the enzymatic hydrolysis method is as follows: take a sufficient amount of shell muscle from fresh oysters and stir it into meat paste; take a certain mass of meat paste, add deionized water to make up the volume, add enzyme to hydrolyze, inactivate after hydrolysis, and centrifuge to obtain the enzymatic hydrolysis sample solution.
4. The method for detecting umami peptide umami substances according to claim 3, characterized in that: The enzyme is 0.2% trypsin, the hydrolysis temperature is 39°C, the time is 3h; the inactivation temperature is 100°C, the time is 10min; the centrifugation temperature is 4°C, and the time is 10min.
5. The method for detecting umami peptide umami substances according to claim 1, characterized in that: In S2, the sample solutions of the cooking method and the enzymatic method were taken and loaded onto the LC-MS / MS system, and the samples were separated with a gradient of 60 min, the column flow was controlled at 350 nL / min, the column temperature was 40°C, the electrospray voltage was 2 kV, the gradient started from 2.2% of the B phase, increased to 50% with a nonlinear gradient in 51 minutes, increased to 90% in 3.5 minutes, and maintained for 5.5 minutes.
6. The method for detecting umami peptide umami substances according to claim 4, characterized in that: The mass spectrometry parameters were set as (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: 30 s.
7. The method for detecting umami peptide umami substances according to claim 1, characterized in that: In said S3, the preliminary screening method is: in the total polypeptides, a target peptide is selected whose amino acid content is less than 15 and the content of acidic amino acids (E, D) in the amino acid composition of the polypeptide exceeds 30%.
8. The method for detecting umami peptide umami substances according to claim 7, characterized in that: In S3, the secondary screening method is: the target peptides after the initial screening are subjected to secondary screening through multiple classification prediction models, and those that meet the model conditions are determined to be umami peptides obtained by the cooking method and the enzymatic method.
9. The method for detecting umami peptide umami substances according to claim 8, characterized in that: The classification prediction models are UMPred-FRL, Umami-MRNN, iUmami-SCM, TPDM and Umami_YYDS.
10. The method for detecting umami peptide umami substances according to claim 1, characterized in that: In S4, the umami peptide obtained in S3 is retrieved from the total polypeptide obtained in S2 through precursor protein, and differential proteins are found and determined as marker proteins; the amino acid sequences of marker proteins are collected from the protein database, the positions of the umami peptides in the proteins are found, and the first three amino acids at the N-terminus and the last three amino acids at the C-terminus of the polypeptide are retained in the database, and finally the probabilities of these amino acids appearing at the enzyme cleavage positions are counted, which are the characteristics of protein hydrolysis by steaming and enzymatic hydrolysis.