CN114766654A

CN114766654A - Fish sauce delicious peptide, preparation method thereof, delicious strength evaluation method and application thereof

Info

Publication number: CN114766654A
Application number: CN202210429726.1A
Authority: CN
Inventors: 高向阳; 张琦梦; 赵孟斌
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-07-22
Anticipated expiration: 2042-04-22
Also published as: CN114766654B

Abstract

The invention discloses a fish sauce delicious peptide, a preparation method thereof, an evaluation method of delicious strength and application. The invention uses a membrane ultrafiltration method, a gel chromatography method and a reversed-phase high performance liquid chromatography to separate the fish sauce flavor peptide, and a Nano LC-Q-Orbitrap-MS/MS identifies the molecular weight and the sequence of the peptide. Analyzing and predicting the flavor development strength of the umami peptide to obtain the umami peptide with an amino acid sequence shown as SEQ ID NO. 1-5, wherein the umami peptide has better umami and flavor enhancing characteristics. The invention constructs a monoclonal humanized T1R1/T1R3/G16gust44 stable cell line, detects the fluorescent response of calcium ions in cells caused by the umami peptides with different concentrations by a calcium ion fluorescent imaging technology, calculates a relative freshness value by taking sodium glutamate as a standard substance and the concentrations of the sodium glutamate and the umami peptides when the strongest calcium ion fluorescence changes, relatively quantitatively detects the umami intensity of the umami peptides, and has good reproducibility, stability and reliability.

Description

Fish sauce delicious peptide, preparation method thereof, delicious strength evaluation method and application thereof

Technical Field

The invention relates to the technical field of flavor development substances, in particular to a fish sauce flavor peptide, a preparation method thereof, a flavor intensity evaluation method and application.

Background

The fish sauce is a liquid seasoning which is prepared by taking low-value marine fish as a main raw material, salting the low-value marine fish, and fermenting and decomposing components such as protein, fat and the like in fish bodies for a long time through salt-tolerant and halophilic microorganisms, protease of the fish bodies and the like under natural conditions to form unique flavor and delicious taste. The delicate flavor is one of five basic tastes, is a delicious taste which people strive to pursue in daily diet, has important contribution to the taste of food, and can make people feel comfortable and pleasant. The umami peptide is a micromolecule polypeptide with the molecular weight of 150-3000 Da, has the effects of enhancing freshness, increasing aroma and enhancing the whole taste of food, and has good processing characteristics, thermal stability and nutritional value. The delicious peptide not only has delicate flavor, but also can cover and weaken bitter taste to improve the flavor of food, and can reduce the intake of salt and MSG by cooperating with other substances to enhance freshness.

At present, flavor evaluation methods of the delicious peptide mainly comprise a sensory evaluation method, an electronic tongue analysis method and a taste biosensor detection method.

Sensory evaluation is a common method for evaluating tastes, which utilizes human taste organs to analyze the characteristics of taste development substances, and uses taste buds to sense stimulation signals of different chemical substances, and compares the stimulation signals with standard substances to obtain the taste intensity of different substances. And performing threshold analysis and taste profile description on the umami peptide by adopting a taste dilution method and a quantitative description analysis method. Despite good training and calibration of sensory panelists, there may still be subjective effects such as psychological or physiological factors that are a significant contributor to the subjectivity of the panelists. And the sensory evaluation is only suitable for qualitative detection, and the umami intensity cannot be accurately and quantitatively analyzed.

The electronic tongue is a qualitative and quantitative analysis instrument established by simulating human taste sensation based on an electrochemical sensor technology. Compared with a sensory evaluation method, the electronic tongue analysis eliminates subjective influence, so that the obtained data is more accurate and has higher sensitivity. Electronic tongues can also be objectively tested when the solution has a bad flavour or toxic substances. Due to its reliability and reproducibility, it has been widely used for the evaluation of umami intensity in various foods, and also has a major role in the evaluation of umami peptides. However, the delicate flavor cannot be detected at a physiological level, the sensory result of human beings cannot be truly reflected, and the accurate quantification of the delicate flavor intensity is difficult to realize.

The taste biological sensing technology based on electrochemical substances consists of a biological sensing element (identification element), a physical and chemical sensor (such as an oxygen electrode, a field effect transistor, a photosensitive tube, a piezoelectric crystal and the like) and a signal amplification device. Among these, biomaterials capable of producing sensitive and selective analytical signals are used as biorecognition elements, including microorganisms, enzymes, antibodies, antigens, cells, tissues, proteins, nerves and other biologically active substances. These functional biorecognition elements not only respond to taste stimuli, but also transmit chemical signals. Taste cells can serve as functional biological elements of biosensors, but most presynaptic taste cells can respond to a variety of tastes. The umami receptor protein can also be used as a functional biological element of a biosensor, and Zoll manufactures the umami biosensor which takes human T1R1/T1R3 protein as a detection element, but the detection efficiency is too low, and the umami receptor protein can only be used for identifying and screening the umami peptide, and the umami intensity of the umami peptide cannot be quantified.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides fish sauce umami peptide and an evaluation method of umami intensity thereof.

The first purpose of the invention is to provide a fish sauce flavor peptide.

The second purpose of the invention is to provide a preparation method of the fish sauce flavor peptide.

The third purpose of the invention is to provide the application of the fish sauce umami peptide in preparing food, food additives and/or health care products.

The fourth purpose of the invention is to provide a food, food additive and/or health-care product containing the fish sauce flavor peptide.

The fifth purpose of the invention is to provide a method for evaluating the umami intensity of a test object containing the fish sauce umami peptide.

In order to achieve the purpose, the invention is realized by the following scheme:

therefore, the invention separates and identifies the umami peptide with the amino acid sequence shown in SEQ ID NO. 1-5 from the fish gravy, and establishes a method for relatively quantifying the umami intensity at a physiological level.

A fish sauce umami peptide comprises one or more umami peptides with amino acid sequences shown as SEQ ID NO 1-5.

Preferably, the umami threshold of the umami peptide is 0.55-0.80 mmol/mL.

A preparation method of fish sauce delicious peptide comprises the following steps:

s11, separating the fish gravy fermentation stock solution by using an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and collecting permeate to obtain a separated product by a membrane ultrafiltration method.

S12, separating and purifying the product of the step S11 by a Sephadex G-15 gel chromatography to obtain a gel chromatography separation product, wherein the gel chromatography separation product contains the components of the fish sauce delicious peptide;

the separation conditions were: the specification of the chromatographic column is 1.6cm multiplied by 70cm, the sample loading amount is 2mL, the eluent is ultrapure water, the flow rate is 1mL/min, and the detection wavelength is 220 nm.

Preferably, the product is separated by gel chromatography of step S12 using reverse phase high performance liquid chromatography;

separation conditions are as follows: the chromatographic column is Spursil 5 mu m C18, 250X 4.6 mm; the injection volume is 20 mu L; the mobile phase A is ultrapure water, the mobile phase B is acetonitrile, the detection wavelength is 220nm, isocratic elution is carried out: the volume concentration is 7% B, 93% A, the flow rate is 1mL/min, and the elution time is 20 min;

obtaining a reversed phase high performance liquid chromatography separation product.

Preferably, the reversed-phase high performance liquid chromatography separation product sequentially contains the delicious peptide with the amino acid sequence shown as SEQ ID NO. 1-5 from morning to night according to the peak-appearing time.

The fish sauce flavor peptide is applied to preparing food, food additives and/or health products.

A food, food additive and/or health product containing the fish sauce flavor peptide is provided.

The method for evaluating the umami intensity of a substance to be tested containing the fish sauce umami peptide comprises the following steps:

mixing the monoclonal stable cells over-expressing T1R1, T1R3 and G16gust44 proteins with a calcium ion fluorescent probe, collecting the intracellular calcium ion fluorescent signals by using a fluorescence inverted microscope,

automatically collecting images every 5s, adding the substance to be detected containing the delicious peptide and the freshness standard substance sodium glutamate at the 20 th s, continuously detecting for 240s in the whole detection process, and obtaining the relative fluorescence change peak value of calcium ions corresponding to the delicious peptide and the freshness standard substance sodium glutamate;

the sodium glutamate freshness value was defined as 1. The relative freshness value U is calculated by the formula:

U＝F_sample/F_MSG，

wherein ,F_sampleRepresents the maximum value of the peak of the change of calcium ion relative to fluorescence of the umami peptide, F_MSGAnd (4) representing the maximum value of the relative fluorescence change peak value of calcium ions of sodium glutamate, and calculating the relative freshness value of the delicious peptide.

Preferably, the monoclonal, stably transformed cells overexpressing the T1R1, T1R3, and G16gust44 proteins are linked to a recombinant expression vector of the T1R1, T1R3, and G16gust44 genes;

the NCBI accession number of the T1R1 gene is NM-138697, the NCBI accession number of the T1R3 gene is NM-152228, the G16gust44 gene is obtained by replacing 44 amino acids at the C terminal of GNAT3 with 44 amino acids at the C terminal of GNA15, the NCBI accession number of the GNA15 gene is NM-002068, and the NCBI accession number of the GNAT3 gene is NM-001102386.

Preferably, the excitation wavelength of the fluorescent signal of intracellular calcium ions is 490nm, and the emission wavelength is 514 nm.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a fish sauce delicious peptide, a preparation method thereof, an evaluation method of delicious strength and application. The method separates the delicate flavor peptide in the fish sauce by a membrane ultrafiltration method, a gel chromatography method and a reversed-phase high performance liquid chromatography, identifies the molecular weight and the sequence of the peptide by a Nano LC-Q-Orbitrap-MS/MS, and analyzes the ion fragment of a main peak by PEAKS Studio 8.5 software and combining with manual head sequencing to obtain the amino acid sequence of the polypeptide. BIOPEP database bioactive peptide database on-line analysis of the bioactivity of small molecular peptides, based on the database prediction of the flavor development strength of the flavor peptides, and combined with the relative strength of peptide fragments and the scoring of peptide fragments, the flavor peptides are respectively screened from 5 components, and the flavor peptides with amino acid sequences as shown in SEQ ID NO. 1-5 are obtained, and have better flavor and flavor enhancing properties. The invention constructs a cell stably expressing an umami receptor T1R1/T1R3, namely a monoclonal humanized T1R1/T1R3/G16gust44 stable cell line, detects the fluorescent response of calcium ions in cells caused by the umami peptides with different concentrations by a calcium ion fluorescence imaging technology, takes sodium glutamate as a standard substance, eliminates the influence of the concentration, calculates a relative freshness value by the concentration of the sodium glutamate and the umami peptides when the fluorescence of the strongest calcium ions is changed, can relatively and quantitatively detect the umami intensity of the umami peptides on a physiological level, and has good reproducibility, stability and reliability.

Drawings

FIG. 1 is a gel chromatography separation chart.

FIG. 2 is a sensory radar chart of gel chromatography separation of components.

FIG. 3 is a graph of the electroglossary of the fractions separated by gel chromatography.

FIG. 4 is a reversed-phase high performance liquid separation chromatogram.

FIG. 5 is a secondary mass spectrum of the synthetic peptide TLTDVEK.

FIG. 6 is a secondary mass spectrum of synthetic peptide LPVDE.

FIG. 7 is a secondary mass spectrum of synthetic peptide AEEVEEERLK.

FIG. 8 is a secondary mass spectrum of synthetic peptide VLTHGEDKEGE.

FIG. 9 is a secondary mass spectrum of synthetic peptide LTLF.

FIG. 10 is a radar chart for sensory evaluation of synthetic peptides.

FIG. 11 is an electronic tongue evaluation radar chart of synthetic peptides.

FIG. 12 is a graph of the synergistic freshening effect of synthetic peptides.

FIG. 13 shows the results of whole gene amplification, where a is T1R1, b is T1R2, and c is G16gust 44.

FIG. 14 shows the result of vector digestion, wherein the M bands are, from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp and 250 bp; 1. 3 and 5 represent: pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA vector enzyme digestion products; 2. 4 and 6 represent: the vector was not digested.

FIG. 15 shows the colony PCR identification result, in which a is the colony PCR identification result of recombinant plasmid pcDNA3.1-His-T1R 1; b is the result of colony PCR identification of the recombinant plasmid pcDNA3.1-Flag-T1R 3; c is the PCR identification result of the colony of the recombinant plasmid pcDNA3.1-HA-G16gust 44; 1 is a negative control (ddH2O), 2 is a negative control (no-load self-connection control group), 3 is a positive control (GAPDH), and M is Marker which is 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; 4 to 9 represent colonies No. 1 to 6 in this order.

FIG. 16 shows the results of protein expression assays, wherein M is Marker; 1 is HEK293-T1R1/T1R3/G16gust44 cells; HEK293 cells.

FIG. 17 is a graph showing the change in calcium ion versus fluorescence of cells caused by different concentrations of MSG.

FIG. 18 is a graph showing the peak of the change in calcium ion relative to fluorescence of cells caused by different concentrations of MSG.

FIG. 19 is a graph showing the change in calcium ion relative fluorescence of cells caused by different concentrations of TLTDVEK.

FIG. 20 is a graph showing the change in calcium ion versus fluorescence of cells caused by different concentrations of LPVDE.

FIG. 21 is a graph showing the change in calcium ion relative fluorescence of cells caused by LTLF at various concentrations.

FIG. 22 is a graph showing the change in calcium ion versus fluorescence of cells induced by different concentrations of AEEVEEERLK.

FIG. 23 is a graph of the change in calcium ion versus fluorescence of cells induced by different concentrations of VLTHGEDKEGE.

FIG. 24 is a graph showing the peak change in relative fluorescence of calcium ions in cells caused by different concentrations of synthetic peptides.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 separation of umami peptide from fish sauce by Membrane ultrafiltration and gel chromatography

First, experiment method

1. Membrane ultrafiltration method for separating delicate flavor peptide

Filtering the stock solution with double-layer gauze and medium-speed filter paper, concentrating and desalting by rotary evaporator, and storing at 4 deg.C. Diluting the concentrated fish gravy with ultrapure water until the volume concentration of solid matters is 1%, filtering with a 0.45-micrometer filter membrane, separating with a Saedodes ultrafiltration membrane with the molecular weight cutoff of 3000Da, collecting the permeate, and performing vacuum freeze drying to obtain the product separated by the membrane ultrafiltration method.

2. Separation of umami peptide by gel chromatography

And (2) filtering the product separated by the membrane ultrafiltration method in the step (1) through a 0.45 mu m microporous filter membrane, and separating and purifying by using Sephadex G-15 gel chromatography. The separation conditions were: a chromatographic column (1.6 cm. times.70 cm), a sample loading of 2mL, an eluent of ultrapure water, a flow rate of 1mL/min, and a detection wavelength of 220 nm. Collecting one tube every 3min, collecting according to peak of each component, concentrating, lyophilizing, performing sensory evaluation and electronic tongue evaluation, and selecting the component with strongest delicate flavor for further separation and purification.

Second, experimental results

The umami peptide is generally polypeptide with molecular weight less than 3kDa, so an ultrafiltration membrane with molecular weight cutoff of 3000Da is selected for separation, and permeate is collected for next step of gel chromatography separation. The separation results are shown in FIG. 1, and a total of 4 component peaks (F1-F4) were obtained. The sensory evaluation results of the components are shown in fig. 2, and the taste profile of each component is mainly fresh taste, secondly sour taste, salty taste and sweet taste, and is weaker in bitter taste. There was a significant difference in the umami character of the components, with the F2 component having an umami score of up to 4.6, followed by the F1 component having a score of 3, and the F3 and F4 components having the same umami score of 1.8. Analysis shows that the trend of sour taste and umami taste in the taste profile is consistent, which is probably because of the existence of carboxyl in R group or C terminal residue of the polypeptide, and the dissociated hydrogen ion is an important substance for causing sour taste. Among the delicious peptides reported so far, many peptides not only have delicious taste, but also may have a certain sour taste. The results of electronic tongue analysis of the respective components are shown in fig. 3, the results of electronic tongue analysis are not completely identical to the results of sensory evaluation, but the general trend is the same, the umami taste of the F2 component is still most prominent, followed by the F1 component, the F4 component and the F3 component. Therefore, according to the results of sensory evaluation and electronic tongue analysis, the F2 component has the strongest delicate flavor and the highest content and is easy to collect, and the F2 component is selected to be continuously subjected to reversed-phase high-performance liquid separation.

Example 2 separation and purification of umami peptide in fish sauce by reversed-phase high performance liquid chromatography

First, experiment method

The 4 components (F1-F4) are obtained after the gel chromatography separation in the embodiment 1, the component F2 with the strongest delicate flavor is prepared into a solution with the concentration of 5mg/mL by first-grade water, and the solution is filtered by a 0.45 mu m microporous membrane and further separated by reversed phase high performance liquid chromatography (RP-HPLC). Separation conditions for RP-HPLC: the chromatographic column is Spursil 5 mu m C18, 250 multiplied by 4.6 mm; the injection volume is 20 mu L; the mobile phase A is ultrapure water, the mobile phase B is acetonitrile, the detection wavelength is 220nm, isocratic elution is carried out: volume concentration 7% B, 93% A, flow rate 1mL/min, elution time 20 min. And (4) respectively collecting components corresponding to each separation peak, concentrating, and freeze-drying.

Second, experimental results

The components obtained by the gel chromatography separation in the embodiment 1 are still mixtures, the components are complex, and the structural identification cannot be directly carried out, so that the components need to be further separated by adopting a reversed-phase high performance liquid chromatography. The reversed-phase high performance liquid chromatography is a chromatography for separating various substances according to molecular hydrophobicity, has the advantages of simple and convenient operation, high efficiency, high speed, high sensitivity and good repeatability, and is usually used as the last step of separating and purifying proteins and polypeptides. The results of the reversed-phase high-performance liquid separation by redissolving the F2 component in ultrapure water are shown in FIG. 4, and it can be seen from the graph that the reversed-phase high-performance liquid separation has a good separation effect when used for purification, 5 components are obtained by total separation, namely F2-1, F2-2, F2-3, F2-4 and F2-5, and the separated components are eluted in the first 10min, which indicates that each component has strong polarity.

Example 3 identification of the Structure of Fish sauce flavor peptides

First, experiment method

5 components purified by the reversed-phase high-performance liquid phase in the example 2 are subjected to reductive alkylation, then the components are desalted, then a treated sample is analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) to obtain a raw file of an original result of a mass spectrum, and a peptide fragment sequence analysis result is obtained by De novo analysis. The method comprises the following specific steps:

1. preparation of samples

Dithiothreitol solution was added to each of the 5 fractions to give a final concentration of 10mmol/L, and the mixture was reduced in a 56 ℃ water bath for 1 hour. Adding iodoacetamide solution to make the final concentration 50mmol/L, and reacting for 40min in dark. Desalting with self-packed desalting column, and evaporating solvent in vacuum centrifugal concentrator at 45 deg.C.

2. Capillary liquid chromatography conditions

Pre-column: 300 μm i.d.. times.5 mm, packed with Acclaim PepMap RPLC C18,5 μm,

and (3) analyzing the column: 150 μm i.d.. times.150 mm, packed with Acclaim PepMap RPLC C18,1.9 μm,

a mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; gradient elution procedure: 0-2 min 4% -8% of B, 2-45 min 8% -28% of B, 45-55 min 28% -40% of B, 55-56 min 40% -95% of B, and keeping the mobile phase 95% of B to elute for 10 min; flow rate: 600 nL/min. The percentage contents are volume concentrations.

3. Conditions of Mass Spectrometry

Primary mass spectrum parameters: resolution: 70000, AGC target: 3e6, Maximum IT: 100ms, Scan range: 300to 1800 m/z.

Secondary mass spectrum parameters: resolution: 17500, AGC target: 1e5, Maximum IT: 50ms, Top N: 20, NCE/cascaded NCE: 28.

by De novo analysis, the sequence analysis result of the peptide fragments of the components F2-1, F2-2, F2-3, F2-4 and F2-5 is obtained, and accordingly, the peptide fragments are synthesized by a solid phase synthesis method and are desalted and the like to obtain the polypeptide with the purity of more than 98 percent.

Second, experimental results

The 5 fractions (F2-1, F2-2, F2-3, F2-4 and F2-5) obtained by reverse phase high performance liquid phase separation and purification in example 2 were used to identify the molecular weight and sequence of the peptide by Nano LC-Q-Orbitrap-MS/MS. After the Nano LC-MS/MS separation and identification, a mass spectrum database is automatically searched, and the ion fragments of the main peak are analyzed through PEAKS Studio 8.5 software and manual head sequencing, so that the amino acid sequence of the polypeptide is obtained. Although many impurities are removed through a series of separation and purification, each sample component is still a polypeptide mixture, and cannot contain only one polypeptide. The mass spectrometer Q active has ultrahigh resolution and sensitivity, and can identify substances with extremely low content in components, so that each component can detect a plurality of polypeptides. The BIOPEP database bioactive peptide database can analyze the bioactivity of small molecular peptides on line, predict the flavor development intensity of the umami peptides based on the database, and screen out one umami peptide from 5 components respectively by combining the relative intensity of peptide fragments and the scoring of the peptide fragments, wherein the main information of the umami peptide is shown in Table 1, and the secondary mass spectrogram of each umami peptide is shown in figures 5-9.

TABLE 1 amino acid sequence identification of umami peptides

Example 4 analysis of taste Properties of taste peptides

First, experiment method

1. Sensory evaluation

The sensory panel consisted of 12 normal tasting students (6 women, 6 men, aged 20 to 25 years), and panelists received sensory training to discern 5 basic tastes (sour, sweet, bitter, salty, fresh) according to GB/T16291.1-2012. The 5 basic taste sour, sweet, bitter, salty, and umami corresponding standards were 0.8mg/mL citric acid solution, 10mg/mL sucrose solution, 0.8mg/mL quinine solution, 3.5mg/mL sodium chloride solution, and 3.5mg/ML Sodium Glutamate (MSG) solution, respectively. The standard solution had a sensory score of 5 points and the test was rated at 10 points, with 0 points representing no taste and 10 points indicating a strong taste. The components F1-F4 separated by gel chromatography in example 1 were prepared into solutions with a concentration of 0.2mg/mL, and the synthetic peptides with amino acid sequences shown in SEQ ID NO 1-5 synthesized in example 3 were prepared into solutions with a concentration of 1mmol/L for the panelists to perform sensory evaluation.

Measuring taste threshold of each synthetic peptide by adopting a triangular test method, dissolving the synthetic peptide with an amino acid sequence shown as SEQ ID NO: 1-5 in ultrapure water to a concentration of 5mmol/L, and describing the taste characteristics by a small sensory group. Subsequently, 1 sample test group and 2 blank control groups were prepared according to the triangle test, and gradually diluted 1:1, and tasted by the panelists sequentially from low concentration to high concentration. This concentration is the taste threshold of the peptide when it is just possible to distinguish between the sample solution and the two blank solutions. The average of the evaluation results of all sensory groups was recorded as the final result.

The fresh-increasing effect of the synthetic peptide is as follows: taking 1mg/mL MSG solution as an umami sensory standard substance with the grade of 5, adding synthetic peptide with the amino acid sequence shown as SEQ ID NO: 1-5 into the standard MSG solution to enable the final concentration of the synthetic peptide in the composite solution to be 1mmol/L, and carrying out sensory evaluation on the synthetic peptide solution to analyze the synergistic freshening effect of the synthetic peptide, wherein the umami is 5-10 grades when being stronger than the standard solution, and the umami is 0-5 grades when being weaker than the standard solution.

2. Electronic tongue evaluation

The electronic tongue system consists of 5 taste sensors: CA0 (sour), GL1 (sweet), C00 (bitter), AAE (fresh), CT0 (salty), and AE1 (astringent). For the first detection, all sensors are activated in a reference solution (a mixed solution of 30mmol/L potassium chloride and 0.3mmol/L tartaric acid) for 24 hours, the sensors are subjected to self-detection, and the detection of a sample can be started after signals are stable. Components F1 to F4 separated by gel chromatography in example 1 are prepared into solutions with polypeptide concentration of 0.2mg/mL by using ultrapure water, peptides with amino acid sequences shown in SEQ ID NO:1 to 5 synthesized in example 3 are respectively prepared into solutions with polypeptide concentration of 1mg/mL, and the solutions are placed in an electronic tongue sample tray for data acquisition, wherein the data acquisition time of each sample is 120s, and the data acquisition is carried out 4 times in total. All data are absolute output values taking the reference liquid as a standard, and the state of the reference liquid for electronic tongue test simulates the state of saliva only in human oral cavity. The reference liquid is composed of potassium chloride and tartaric acid, the taste value is close to tasteless, so that the tasteless point of sour taste is-13, the tasteless point of salty taste is-6, and based on the taste value of the sample is lower than the tasteless point of the reference liquid, the sample is indicated to have no taste, and otherwise, the taste value is indicated to have nothing.

Second, Experimental methods

The results are shown in FIG. 10. The synthesized 5 polypeptides have more obvious bitter taste except LTLF, and the rest taste profiles are basically consistent, so that the whole polypeptide has stronger delicate flavor, second-order sour flavor, certain sweet taste and delicate flavor, and basically has no bitter taste. AEEVEEERLK had the strongest umami taste, followed by VLTHGEDKEGE, TLTDVEK, LPVDE and LTLF, but the umami taste of TLTDVEK and LPVDE was not significantly different. And LTLF has a strong bitter taste, probably because the strength of the bitter taste is increased when hydrophobic amino acids (e.g., phenylalanine, proline, leucine, etc.) are at the C-terminal position of the peptide chain. The results of electronic tongue analysis of 5 synthetic peptides are shown in fig. 11, the 5 synthetic peptides have similar taste profiles to those of the sensory evaluation, and the most umami was still AEEVEEERLK, secondly VLTHGEDKEGE and TLTDVEK did not differ significantly in umami, then LPVDE, LTLF was still the weakest in umami. It was found that the sourness of the synthetic peptides was significantly increased compared to fractions F1 to F3 isolated by gel chromatography in example 1, probably because glutamic acid and aspartic acid were either acidic amino acids or residual sodium acetate and amino acids during peptide synthesis. The taste characteristics and thresholds of the synthetic peptides are shown in table 2.

TABLE 2 taste Properties and thresholds of the synthetic peptides

In order to understand the flavor characteristics of the synthetic peptides more fully, the flavor dilution method was used to measure the recognition threshold of the synthetic peptides and the sensory evaluation personnel were asked to verbally describe their flavor, and the flavor characteristics and threshold of the synthetic peptides are shown in table 2. The threshold value of the MSG is 1.77mmol/L, and the threshold values of the 5 synthetic peptides are lower than that of the MSG, which indicates that the synthetic peptides have stronger delicate flavor. The sensory description of the synthetic peptides was essentially consistent with taste profile analysis and electronic tongue analysis. When the concentrations of 5 synthetic peptides were consistent, AEEVEEERLK showed the strongest umami taste, whereas the synthetic peptide with the lowest threshold was VLTHGEDKEGE, indicating that there was no necessary correlation between the umami intensity and the umami threshold, and that the umami taste of VLTHGEDKEGE was no more prominent than that of AEEVEEERLK in the sensory description.

To investigate the freshness-enhancing effect of the synthetic peptides, the synthetic peptides were added to the MSG solution for sensory evaluation, and the results are shown in fig. 12. Both synthetic peptide AEEVEEERLK, VLTHGEDKEGE, TLTDVEK and LPVDE enhanced the umami taste of MSG solution, with AEEVEEERLK being the most potent umami enhancing effect, while LTLF mixed MSG solution had an umami score slightly lower than MSG solution, probably because LTLF had a more pronounced unpleasant bitter taste, masking the umami taste, making changes in umami taste indistinguishable to sensory evaluators.

Example 5 construction of recombinant expression vector

Human T1R1 (NM-138697), T1R3 (NM-152228), GNA15 (NM-002068) and GNAT3 (NM-001102386) gene sequences are inquired through an NCBI website, the C-terminal 44 amino acids of GNA15 are replaced by the C-terminal 44 amino acids of GNAT3 to obtain G16gust44, and genes T1R1-6His, T1R3 and G16gust44 are synthesized.

Primers were designed by using Prime primer software, XhoI and KpnI cleavage sites were added to the 5 ' end of the T1R1-6His forward and reverse primers, XhoI and KpnI cleavage sites were added to the 5 ' end of the T1R3 forward and reverse primers, XhoI and BamHI cleavage sites were added to the 5 ' end of the G16gust44 forward and reverse primers, and the primers were designed as shown in Table 3:

TABLE 3 PCR primer sequences

The synthesized target gene is used as an amplification template to perform PCR amplification. Preparing a reaction system shown in the table 4, lightly blowing, uniformly mixing, centrifuging for a short time, and placing in a PCR instrument for reaction. PCR amplification reaction conditions are 98 ℃ and 5 min; 30 cycles of 98 ℃, 10s, 55 ℃, 10s, 72 ℃, 90 s; 72 ℃ for 8 min. And analyzing the amplification product by agarose gel electrophoresis, observing the result by a gel imaging system, and recovering a target band.

TABLE 4 PCR amplification System

The restriction enzymes of plasmids pcDNA3.1, pcDNA3.1-Flag, pcDNA3.1-HA were XhoI/KpnI, XhoI/BamHI, respectively. The enzyme digestion system was prepared according to Table 5, gently beaten by pipette, mixed well, centrifuged briefly, and reacted at 37 ℃ for 3 h. And (3) carrying out agarose gel electrophoresis on the vector enzyme digestion product, observing the result by a gel imaging system, and recovering a target strip.

The T1R1-6His, T1R3 and G16gust44 gene sequences amplified by PCR are respectively connected to pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA vectors, a reaction system in Table 6 is prepared in an ice water bath, a pipette is used for gently blowing and beating the vectors uniformly, the mixture is subjected to short-time centrifugation to avoid generating air bubbles, and the reaction is carried out for 30min at 37 ℃ to construct recombinant plasmids pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 and pcDNA3.1-HA-G16gust 44. Then placed in an ice water bath to cool for 5min before immediate transformation. Respectively adding 10 μ L ligation reaction product into 100 μ L Escherichia coli Top10 competent cell, flicking tube wall number, mixing, and standing on ice for 30 min. Heat shock at 42 deg.C for 90s, and incubating in ice water bath for 2 min. Adding 500. mu.L LB medium, and shaking-culturing at 37 deg.C for 1 h. And (3) uniformly coating a proper amount of bacterial liquid on an LB (lysogeny broth) plate containing ampicillin, and performing inverted culture in a constant-temperature incubator for 12-16 h.

TABLE 5 vector restriction reaction System

TABLE 6 ligation reaction System

Second, experimental results

After PCR amplification, the target fragments with the lengths of 2606bp, 2605bp and 1169bp are expected to be obtained respectively, agarose gel electrophoresis is carried out, imaging is carried out, and the amplified bands are basically consistent with the expected sizes as shown in figure 13.

The products of the digestion of the vector were subjected to agarose gel electrophoresis, as shown in FIG. 14, pcDNA3.1 was approximately 5.4kb, pcDNA3.1-Flag was approximately 5.5kb, and pcDNA3.1-HA was approximately 5.4kb, and the amplified band was substantially identical to the expected size.

Example 6 identification of recombinant expression vectors

First, experiment method

A PCR reaction system was prepared as shown in Table 7, shaken, mixed and centrifuged briefly. In a clean bench, the single colony cultured in example 6 was picked up by a sterile pipette tip into a 20 μ LPCR system, blown and mixed uniformly, placed in a PCR instrument for reaction, and detected by agarose gel electrophoresis. The primers for each reaction system are shown in Table 8. The PCR amplification reaction condition is 94 ℃ for 3 min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 30s, 22 cycles; 72 ℃ for 5 min.

TABLE 7 colony PCR reaction System

TABLE 8 colony PCR primer sequences

And taking a proper amount of bacterial liquid according to the inoculation proportion of the volume concentration of 0.5-1% of the identified positive clone transformant, adding the bacterial liquid into an LB liquid culture medium containing 0.1mg/mL ampicillin, performing shake culture at 37 ℃ and 220rpm for 12-16 h, and taking a proper amount of bacterial liquid for sequencing. The sequence of the target gene is compared with the sequence of the sequencing result by Snapgene software.

Second, experimental results

The image after agarose gel electrophoresis is shown in figure 15, the sizes of the bands amplified by using pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 and pcDNA3.1-HA-G16gust44 as templates are 923bp, 1003bp and 698bp respectively, which are consistent with the sizes of the bands in the figure, and the target gene is successfully inserted into an expression vector.

The sequencing result of the Snapgene software shows that the gene sequence on the recombinant expression plasmid is 100 percent consistent with the target gene sequence, and the T1R1-6His, T1R3 and G16gust44 genes are proved to be successfully cloned on pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA vectors.

Example 7 determination of G418 killing concentration

First, experiment method

Cloning exogenous DNA to carrier with certain resistance or selective gene, transfecting carrier to host cell and integrating it into chromosome, screening by selective marker contained in carrier, and screening to obtain cell strain capable of stably expressing target protein. G418 (Geneticin) is an aminosaccharide antibiotic with a structure similar to neomycin, gentamicin, kanamycin, blocks protein synthesis by affecting 80S ribosomal function, is toxic to both prokaryotic and eukaryotic cells, including bacterial, yeast, plant and mammalian cells. Is the most commonly used selection reagent for stable transfection. When the neomycin gene is integrated into a proper place of the genome of the eukaryotic cell, the neomycin gene coding sequence can be initiated to be transcribed into mRNA, so that the high-efficiency expression of a resistance product, namely aminoglycoside phosphotransferase can be obtained, and the cell can obtain resistance and can grow in a selective culture medium containing G418. Since the resistance genes of the 3 plasmids pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA of example 5 are neomycin, G418 was used to screen for stable high expression cell lines.

Eukaryotic cell lines differ in sensitivity to G418, so the optimal selection concentration of G418 is determined prior to selection.

1. The selection concentration range of G418 is 100 mu G/mL-1 mg/mL. 40mg/mL G418 was diluted to 12 grades with no dual antibody complete medium at 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1100 μ G/mL.

2. HEK293 cells were seeded in 96-well plates in complete medium at 37 ℃ with 5% CO by volume₂Culturing for 24 h.

3. Discarding the medium from step 2, adding 200. mu.L of G418 selection medium prepared in step 1 at each concentration, 3 wells per well, continuing at 37 ℃ and 5% CO by volume₂And (4) culturing.

4. The culture medium was changed every 24 h. The lowest G418 concentration at which all cells were killed within 10 to 14 days was selected as the screening concentration.

Second, experimental results

Screening experiments show that the lowest G418 concentration of all HEK293 cells dead within 10-14 days is 500 mug/mL, and stable expression strains are screened according to the concentration.

Example 8 transfection of pcDNA3.1-HA-G16gust44 plasmid, cotransfection of pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 plasmids

One, pcDNA3.1-HA-G16gust44 plasmid transfection

The Plasmid was extracted using the Endo-free Plasmid Mini Kit I Kit (OMEGA Co.) according to Lipofectamine (Invitrogen Co.)^TM3000 transfection reagent.

1.1 mL of the glycerol strain with the recombinant plasmid pcDNA3.1-HA-G16gust44 prepared in example 5 was inoculated into 100mL of LB liquid medium at 37 ℃ and 220rpm in an inoculation ratio of 0.5 to 1% by volume and cultured with shaking overnight. 5mL of the resulting bacterial solution was used to extract pcDNA3.1-HA-G16gust44 recombinant Plasmid using the Endo-free Plasmid Mini Kit I Kit (OMEGA corporation), and the Plasmid was stored at-20 ℃.

2. HEK293 cells were seeded in 6-well plates, 2.5mL DMEM complete medium was added, and placed in CO₂Constant temperatureCulturing in an incubator.

3. The growth state of the cells in step 2 was observed, and transfection was performed when the confluency reached 80%. Transfection procedure reference Lipofectamine^TM3000 transfection reagent Instructions, to 750. mu.L serum-free Opti-MEM Medium was added 45. mu.L Lipofectamine^TM3000 reagent.

4. Then, 15. mu.g of the recombinant plasmid (0.5-5. mu.g/. mu.L) pcDNA3.1-HA-G16gust44 extracted in step 1 was added to 750. mu.L of serum-free Opti-MEM medium, and 30. mu. L P3000 was added^TMAnd (5) fully mixing the reagents.

5. 750 μ L of diluted Lipofectamine was taken^TM3000. mu.L of the reagent and 750. mu.L of the recombinant plasmid pcDNA3.1-HA-G16gust44 diluted in step 4 were mixed well (1:1 ratio). And incubating for 10-15 min at room temperature.

6. Step 5 Add 250. mu.L of DNA-liposome complex per well and incubate cells for 2 days at 37 ℃. Then qRT-PCR analysis and stable strain screening of transfected cells were performed.

Secondly, pcDNA3.1-His-T1R1 and pcDNA3.1-Flag-T1R3 plasmids are cotransfected

The transfected cells from the pcDNA3.1-HA-G16gust44 plasmid were inoculated into 6-well plates, cultured overnight for cell attachment, the complete medium was aspirated, and screened with 500. mu.g/mL G418 medium for 14 days, followed by transfection of pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 plasmids, as indicated by the pcDNA3.1-HA-G16gust44 plasmid transfection, where 15. mu.g of recombinant plasmid in 4 steps was changed to 15. mu.g of plasmid pcDNA3.1-His-T1R1 and 15. mu.g of plasmid pcDNA3.1-Flag-T1R 3.

EXAMPLE 9 establishment of a stable monoclonal strain

The cells transfected with pcDNA3.1-HA-G16gust44, pcDNA3.1-His-T1R1 and pcDNA3.1-Flag-T1R3 in example 8 were seeded in 6-well plates, after overnight incubation for cell attachment, the complete medium was aspirated, and after about 14 days, resistant clones were observed by selection using 500. mu.g/mL G418, followed by further selection of single clones by limiting dilution.

Resistant clones were digested and diluted to 1X 10⁴cells/mL cell suspension, 200 μ L of cell suspension was added to a1 well in a 96 well plate, and 100 μ L of complete medium was added to each of the remaining wells. Then transferring 100 mu L from A1 well to B1 wellThe mixture was flushed through, the dilution step was repeated until the last well in the row H1, and 100. mu.L of medium was aspirated from the H1 wells to make the volume of medium in the first well equal. Add 100. mu.L of medium to each well of the first row using a row gun, mix and blow well, then take 100. mu.L of medium from the first row to the wells of the second row, repeat the dilution step to the last row, aspirate 100. mu.L of medium from the last row, and add 100. mu.L of medium to all wells to give a final volume of 200. mu.L in all wells. Mixing 96-well plate at 37 deg.C with 5% CO₂Culturing for 48 h. The wells were then screened by changing 500. mu.g/mL G418 medium and the growth of individual cells was observed microscopically, typically over a period of 2 to 3 weeks to observe the formation of single colonies. And (3) screening a monoclonal with high expression abundance by using qRT-PCR and Western Blot, and carrying out subsequent amplification culture to obtain a monoclonal humanized T1R1/T1R3/G16gust44 stable-transformed HEK293 cell line (HEK293-T1R1/T1R3/G16gust 44).

Example 10qRT-PCR detection of changes in expression levels of target genes

First, experiment method

1. The cultured monoclonal human T1R1/T1R3/G16gust44 cell line of HEK293 (HEK293-T1R1/T1R3/G16gust44) from example 9 was taken out, the cells were washed 2 times with PBS, 350. mu.L of lysate was added to each well of 6-well plate, and RNA was extracted according to SteadyPure Universal RNA Extraction Kit of Elekory. And (3) measuring the concentration and the purity of the RNA by using a micro spectrophotometer, wherein OD260 nm/280nm is between 1.8 and 2.0, which indicates that the RNA purity meets the requirement.

2. The method comprises the following steps of utilizing an Evo M-MLV RT Premix for qPCR reverse transcription kit of Ecori company to reversely transcribe RNA meeting the purity requirement into cDNA, wherein the reverse transcription reaction program is as follows: 15min at 37 ℃; 85 ℃ for 5 s. The reverse transcription system is shown in Table 9.

TABLE 9 reverse transcription reaction System

3. Primers were designed based on the sequences of human GAPDH, T1R1, T1R3, and G16gust44 genes using GAPDH as the reference gene, as shown in Table 10. The qPCR reaction system is configured according to an SYBR Green Premix Pro Taq HS qPCR Kit of Eikeri company, and the qPCR reaction program is as follows: the first step is as follows: at 95 ℃ for 30 s; the second step: 95 ℃, 5s, 60 ℃, 30s, 40 cycles. The qPCR reaction system is shown in table 11. The data processing is carried out according to the following formula:

ΔΔCt＝(Ct_{target gene}-Ct_{Reference gene})_{HEK293-T1R1、T1R3、G16gust44}-(Ct_{Target gene}-Ct_{Internal reference gene})_HEK293............(1)

Fold Change of difference 2^-ΔΔCtThe expression level of the objective gene in the test group is expressed as a fold change relative to the control group.

TABLE 10 qPCR primer sequences

TABLE 11 qPCR reaction System

Second, experimental results

The results of qRT-PCR analysis of the HEK293-T1R1/T1R3/G16gust44 cell mRNA obtained by monoclonal screening were shown in Table 12, and 2R 1, T1R3 and G16gust44 genes were calculated according to the formula (1)^-ΔΔCt4871.00, 313.00 and 3236.01, respectively, and the gene expression level indicates that the selected monoclonal cells stably express the 3 genes.

TABLE 12 Single clone Stable strain qPCR Ct values

EXAMPLE 11 Western Blot for detecting protein expression

First, experiment method

The monocloned human T1R1/T1R3/G16gust44 cultured in example 9 was taken to transfer the HEK293 cell line (HEK293-T1R1/T1R3/G16gust44) stably, the culture medium was removed and washed once with PBS. mu.L of lysis solution was added to each well of a 6-well plate, and PMSF (phenylmethylsulfonyl fluoride) was added to the lysis solution several minutes before use so that the final concentration of PMSF was 1 mmol/L. The lysate and cells were brought into full contact by several blows from a gun. After full lysis, 14000g of the solution is centrifuged for 5min, and the supernatant is taken, thus the subsequent electrophoresis and membrane transfer operations can be carried out.

Second, experimental results

The results are shown in fig. 16, the theoretical sizes of G16gust44, T1R1, T1R3 and GAPDH proteins are 43kDa, 93kDa and 36kDa, respectively, and the bands are in agreement with the expected sizes, indicating from the protein expression level that G16gust44, T1R1, T1R3 were successfully overexpressed in HEK293 cells.

Example 12 establishment of an Umami taste evaluation method based on the HEK293-T1R1/T1R3/G16gust44 cell line

First, experiment method

Sodium Glutamate (MSG) and synthetic peptides having amino acid sequences shown in SEQ ID NO: 1-5 synthesized in example 3 were dissolved in HBSS buffer. The MSG was brought to concentrations of 2.5, 5, 10, 20 and 40mmol/L, respectively, and the synthetic peptide was brought to concentrations of 1.25, 2.5, 5, 10 and 20mmol/L, respectively, for each sample. The calcium ion fluorescent probe was diluted to a concentration of 5. mu. mol/L with phenol red-free DMEM basal medium. HEK293-T1R1/T1R3/G16gust44 cells cultured in example 9 were seeded in a 24-well plate and experiments were performed when the cells occupied about 70% of the plate. Discarding the culture solution, rinsing the cells with HBSS for 2 times, adding 200 μ L of 5 μmol/L calcium ion fluorescent probe into each well, incubating at 37 deg.C for 30min, rinsing the cells with HBSS for 2 times, and adding 200 μ L phenol red-free DMEM basal culture solution for deesterification for 20 min. And (3) placing the treated cells under a fluorescence inverted microscope, collecting fluorescent signals of calcium ions in the cells at 490nm (excitation wavelength) and 514nm (emission wavelength), automatically collecting images once every 5s, adding 50 mu L of sample synthetic peptide solutions with different concentrations at the 20 th s, and keeping the whole detection process for 240 s. The final reaction concentrations of MSG were 0.5, 1, 2, 4 and 8mmol/L, the final reaction concentrations of synthetic peptide were 0.25, 0.5, 1, 2 and 4mmol/L, 50. mu.L HBSS was added to the blank group, and the blank group and the experimental group were set to 4 replicates per concentration. And selecting the well-taken fluorescence pictures, and selecting a minimum of 30 cell areas in each picture to detect the fluorescence value. The calculation formula of the change of calcium ions relative to fluorescence is as follows:

Δ F represents the relative fluorescence change value, "F" represents the real-time fluorescence value of the cellular region minus the background real-time fluorescence value; "F0" represents "F" at 0 second.

The freshness value was defined as 1 using MSG as the freshness standard. The relative freshness value U is calculated by the formula:

U＝F_sample/F_MSG...................(3)

wherein ,F_sampleRepresents the maximum value of the change peak of the umami peptide calcium ion relative to the fluorescence, F_MSGRepresents the maximum value of the MSG calcium ion relative to the peak of the fluorescence change.

Second, experimental results

As is known from the flavor mechanism of umami, binding of umami peptide to the receptor T1R1/T1R3 results in an increase in intracellular calcium ion concentration. MSG is commonly used as an umami standard and binds to the receptor T1R1/T1R 3. Therefore, to verify whether T1R1/T1R3 receptor over-expressed in the HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 has corresponding physiological activity, the cells were stimulated with 0.5, 1, 2, 4 and 8mmol/L MSG solutions, respectively, and whether the HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 could be used for umami taste detection was judged by observing the change in fluorescence of the cells.

The results of the change in calcium ion fluorescence of cells caused by MSG at different concentrations are shown in fig. 17, and con represents HBSS blank group, and if the relative fluorescence change value is greater than 0, this indicates that MSG can cause the response of cells, and the larger the relative fluorescence change value, the stronger the calcium ion fluorescence response. As can be seen from FIG. 17, MSG-stimulated cells can cause an increase in intracellular calcium ion concentration, which indicates that the T1R1/T1R3 receptor overexpressed in the HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 has normal activity, and the cells can be used as a tool for detecting umami taste.

When the cells are stimulated by MSG with the lowest concentration of 0.5mmol/L, the strong calcium ion fluorescence change can still be caused, the cell sensing MSG threshold value is between 0 and 0.5mmol/L, the human sensory threshold value for MSG is about 1.77mmol/L, and the HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 can more sensitively recognize MSG.

Comparing the relative fluorescence change intensity of calcium ions caused by different concentrations of MSG, the relative fluorescence change peak value caused by different concentrations of MSG is shown in FIG. 18, and it can be seen that when the concentration of MSG is 0.5-1 mmol/L, the calcium ion response caused by MSG increases with the increase of the concentration, and reaches the strongest at 1mmol/L, and when the concentration is 1-8 mmol/L, the calcium ion response caused by MSG decreases with the increase of the concentration, and even at 8mmol/L, the calcium ion response intensity cannot be caused, which shows that the calcium ion response intensity becomes stronger with the increase of the concentration of the umami substance, but after reaching the maximum concentration, the calcium ion response intensity becomes weaker with the increase of the concentration of the umami substance, and when the taste receptor is saturated, the taste intensity can be kept unchanged after reaching the maximum value, and even generates the inhibiting effect.

The results of the changes in intracellular calcium ion response caused by different concentrations of the umami peptide are shown in FIGS. 19-23. The 5 peptides TLTDVEK, LPVDE, LTLF, AEEVEEERLK and VLTHGEDKEGE can all cause cellular calcium ion response, and the fluorescence response intensity of calcium ions changes along with the change of concentration, which indicates that the peptides all have delicate flavor. By comparing the intensity of the relative fluorescence change of calcium ions caused by different concentrations of umami peptide, the peak value of the relative fluorescence change caused by different concentrations of umami peptide is shown in fig. 24, and it can be seen that the peak value of the relative fluorescence change of TLTDVEK is the largest at the concentration of 0.5mmol/L, the peak value of the relative fluorescence change of LPVDE is the largest at the concentration of 1mmol/L, the peak value of the relative fluorescence change of LTLF is the largest at the concentration of 1mmol/L, the peak value of the relative fluorescence change of AEEVEEERLK is the largest at the concentration of 2mmol/L, the peak value of the relative fluorescence change of VLTHGEDKEGE is the largest at the concentration of 2mmol/L, and the same trend as MSG, when umami peptide is less than the concentration of the highest fluorescence response intensity, the calcium ion response intensity increases with the increase of the concentration, and when umami peptide is higher than the concentration of the highest fluorescence response intensity, the calcium ion response intensity decreases with the increase of the concentration. It can be seen from fig. 24 that there is no necessary correlation between the maximum peak of the relative fluorescence change of calcium ion and the corresponding concentration, and the peak of the relative fluorescence change of calcium ion is different in different concentrations, for example, the peak of the relative fluorescence change of calcium ion is LPVDE, aeeveerlk, LTLF, VLTHGEDKEGE, TLTDVEK from large to small at a concentration of 1mmol/L, and the peak of the relative fluorescence change of calcium ion is AEEVEEERLK, VLTHGEDKEGE, LTLF, TLTDVEK from large to small at a concentration of 2 mmol/L. Therefore, the maximum peak value of the relative fluorescence change of calcium ions of each peptide is selected for comparing the umami taste of 5 peptides, and the umami taste is LPVDE, AEEVEEERLK, VLTHGEDKEGE, LTLF and TLTDVEK from large to small.

In order to more intuitively obtain the umami taste of the peptide, the umami taste of the peptide was quantified based on HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9, and calculated according to formula (3), and the results are shown in table 13, wherein the relative freshness values are LPVDE, aeeveerelk, vlthgedege, LTLF, and TLTDVEK in order from large to small.

TABLE 13 relative freshness values of the synthetic peptides

Cosine similarity, also called cosine similarity, is to evaluate the similarity of two vectors by calculating the cosine value of their included angle. And drawing the vectors into a vector space according to the coordinate values to obtain the included angles of the vectors, and obtaining cosine values corresponding to the included angles, wherein the cosine values can be used for representing the similarity of the two vectors. The smaller the angle, the closer the cosine value is to 1, and the more they coincide in direction, the more similar. The umami intensities obtained by sensory evaluation, electronic tongue analysis and relative umami value method of 5 peptides were respectively used as 3 matrixes, and cosine similarity analysis was performed on two of them, and the results are shown in table 14. As can be seen from Table 14, the cosine values of the sensory evaluation, the electronic tongue analysis and the relative freshness value method are all greater than 0.9, which indicates that the established relative freshness value method has higher similarity to the freshness values measured by the other two methods, and indicates that the relative freshness value method has reliability.

The sensory evaluation method is subjective and affected by other tastes, and it is difficult to feel a subtle change in umami taste. The sensory evaluation and the electronic tongue of example 4 were performed under the same polypeptide concentration, but the umami taste of the flavor-developing substances at different concentrations was inconsistent and could not be evaluated uniformly. The method for evaluating the umami taste based on the relative fluorescence change of the calcium ions of the HEK293-T1R1/T1R3/G16gust44 cells, which is established by the invention, can eliminate subjective factors and concentration interference, and the detected umami taste intensity is more accurate.

TABLE 14 cosine values of three umami taste evaluation methods

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> south China university of agriculture

<120> fish sauce umami peptide, preparation method thereof, umami intensity evaluation method and application

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Ala Glu Glu Val Glu Glu Glu Arg Leu Lys

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1

Claims

1. The fish sauce umami peptide is characterized by comprising one or more umami peptides with amino acid sequences shown as SEQ ID NO. 1-5.

2. The fish sauce umami peptide according to claim 1, wherein the umami threshold of the umami peptide is 0.55-0.80 mmol/mL.

3. A preparation method of fish sauce flavor peptide is characterized by comprising the following steps:

S12, separating and purifying the product of the membrane ultrafiltration separation method in the step S11 by using Sephadex G-15 gel chromatography to obtain a product of gel chromatography, wherein the product of gel chromatography contains the components of the fish gravy fresh taste peptide in claim 1;

4. The method for preparing the fish sauce umami peptide according to claim 3, wherein the gel chromatography separation product of step S12 of claim 3 is separated by reverse phase high performance liquid chromatography;

5. The method for preparing the fish sauce umami peptide according to claim 3, wherein the reverse phase high performance liquid chromatography separation product sequentially contains umami peptide with amino acid sequences shown in SEQ ID NO 1-5 from morning to evening according to peak emergence time.

6. Use of the fish sauce umami peptide of claim 1 for the preparation of a food, a food additive and/or a health product.

7. A food, food additive and/or health product comprising the fish sauce flavor peptide of claim 1.

8. The method for evaluating umami intensity of a test substance containing the fish sauce umami peptide of claim 1, comprising the steps of:

mixing monoclonal anti-metastatic cells over-expressing T1R1, T1R3 and G16gust44 proteins with a calcium ion fluorescent probe, collecting intracellular calcium ion fluorescent signals by using a fluorescence inverted microscope, automatically collecting images every 5s, adding a substance to be detected containing the umami peptide of claim 1 and a fresh standard substance sodium glutamate at the 20 th s, continuing detection, and continuing the whole detection process for 240s to obtain a calcium ion relative fluorescence change peak value corresponding to the umami peptide of claim 1 and the fresh standard substance sodium glutamate;

U＝F_sample/F_MSG，

wherein ,F_sampleThe maximum value of the peak of the change in the relative fluorescence of calcium ion, F, of the umami peptide of claim 1_MSGCalculating the relative freshness value of the umami peptide of claim 1, which represents the maximum value of the peak value of the relative fluorescence change of calcium ions of sodium glutamate.

9. The method for evaluating the umami intensity of a test substance of fish sauce umami peptide according to claim 8, wherein the monoclonal anti-metastatic cells overexpressing T1R1, T1R3 and G16gust44 proteins are connected with recombinant expression vectors of T1R1, T1R3 and G16gust44 genes;

10. The method of claim 8, wherein the excitation wavelength of the fluorescent signal of intracellular calcium ion is 490nm and the emission wavelength is 514 nm.