CN114766654B - Fish sauce flavor peptide, preparation method thereof, flavor intensity evaluation method and application - Google Patents
Fish sauce flavor peptide, preparation method thereof, flavor intensity evaluation method and application Download PDFInfo
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- CN114766654B CN114766654B CN202210429726.1A CN202210429726A CN114766654B CN 114766654 B CN114766654 B CN 114766654B CN 202210429726 A CN202210429726 A CN 202210429726A CN 114766654 B CN114766654 B CN 114766654B
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- fish sauce
- calcium ion
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6458—Fluorescence microscopy
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a fish sauce flavor peptide, a preparation method thereof, a flavor intensity evaluation method and application thereof. The invention uses membrane ultrafiltration, gel chromatography and reversed phase high performance liquid chromatography to separate fish sauce umami peptide, and Nano LC-Q-Orbitrap-MS/MS to identify the molecular weight and sequence of the peptide. And analyzing and predicting the flavor intensity of the flavor peptide to obtain the flavor peptide with the amino acid sequence shown in SEQ ID NO. 1-5, wherein the flavor peptide has good flavor and flavor enhancing property. The invention constructs a monoclonalized humanized T1R1/T1R3/G16gust44 stable transformation cell line, detects intracellular calcium ion fluorescence response caused by different concentrations of the umami peptide by using a calcium ion fluorescence imaging technology, calculates a relative freshness value by using sodium glutamate as a standard substance and the concentration of the sodium glutamate and the umami peptide when the strongest calcium ion fluorescence is changed, and relatively quantitatively detects the umami intensity of the umami peptide, thereby having good reproducibility, stability and reliability.
Description
Technical Field
The invention relates to the technical field of flavoring substances, in particular to a fish sauce flavor peptide, a preparation method thereof, a flavor intensity evaluation method and application thereof.
Background
The fish sauce is a liquid seasoning which takes marine low-value fish as a main raw material, carries out long-term fermentation decomposition on protein, fat and other components in the fish body through salt tolerance, halophilic microorganisms, protease of the fish body and the like under natural conditions after salting, and has unique flavor and delicious taste. The delicate flavor is one of five basic tastes, is a delicacy which is striven for in daily diet, has important contribution to the taste of food, and can make people feel comfortable and pleasant. The umami peptide is a small molecular polypeptide with the molecular weight of about 150-3000 Da, has the functions of enhancing freshness, flavoring and enhancing the taste of the whole food, and has good processing characteristics, heat stability and nutritional value. The umami peptide not only has the umami taste, but also can mask and weaken the bitter taste to improve the flavor of food, and can reduce the intake of salt and MSG by synergistically enhancing the freshness with other substances.
At present, the taste evaluation method of the umami peptide mainly comprises a sensory evaluation method, an electronic tongue analysis method and a taste biological sensor detection method.
Sensory evaluation is a common method for evaluating tastes by analyzing the characteristics of taste substances by using human taste organs, and comparing the stimulation signals of different chemical substances with standard substances to obtain the taste intensity of different substances. And carrying out threshold analysis and taste profile description on the umami peptide by adopting a taste dilution method and a quantitative description analysis method. Although sensory panel members are well trained and calibrated, subjective effects may still exist, such as psychological or physiological factors being an important contributor to the subjectivity of expert panel members. And sensory evaluation is only suitable for qualitative detection, and the quantitative analysis of the flavor intensity cannot be accurately performed.
The electronic tongue is a qualitative and quantitative analysis instrument based on the establishment of simulating human taste feeling by an electrochemical sensor technology. Compared with the sensory evaluation method, the electronic tongue analysis eliminates subjective influence, so that the obtained data is more accurate and has higher sensitivity. The electronic tongue can also objectively detect when the solution has a bad flavor or toxic substances. Because of its reliability and reproducibility, it has been widely used for the evaluation of umami taste intensity in various foods, and it is also dominant in the evaluation of umami peptides. However, the method cannot detect the delicate flavor at physiological level, cannot truly reflect the sensory result of human beings, and cannot accurately quantify the intensity of the delicate flavor.
Taste biosensing techniques based on electrochemical substances consist of biosensing elements (recognition elements), physicochemical sensors (e.g. oxygen electrodes, field effect transistors, light-sensitive tubes, piezo-electric crystals, etc.), and signal amplifying devices. Among other things, biological materials capable of producing sensitive and selective analytical signals are used as biological recognition elements, including microorganisms, enzymes, antibodies, antigens, cells, tissues, proteins, nerves and other bioactive substances. These functional biological recognition elements can not only react to taste stimuli, but can 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 biological sensor, and the Zoll er manufactures the umami biological sensor which takes the humanized T1R1/T1R3 protein as a detection element, but the detection efficiency is too low, and the detection efficiency can only be used for identifying and screening umami peptides, and the umami intensity of the umami peptides cannot be quantified.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a fish sauce umami peptide and a method for evaluating the umami intensity of the fish sauce umami peptide.
The first object of the invention is to provide a fish sauce umami peptide.
The second object of the invention is to provide a preparation method of fish sauce umami peptide.
The third object of the invention is to provide the application of the fish sauce umami peptide in preparing foods, food additives and/or health-care products.
A fourth object of the present invention is to provide a food, food additive and/or health product containing the fish sauce umami peptide.
The fifth object of the present invention is to provide a method for evaluating the flavor intensity of a test substance containing the fish sauce flavor peptide.
In order to achieve the above object, the present invention is realized by the following means:
therefore, the invention separates and identifies the umami peptide with the amino acid sequence shown as SEQ ID NO. 1-5 from fish gravy, and establishes a method for relatively quantifying the umami intensity on physiological level.
A fish sauce flavor peptide comprises flavor peptides with an amino acid sequence shown as one or more of SEQ ID NO. 1-5.
Preferably, the umami threshold of the umami peptide is 0.55-0.80 mmol/mL.
A method for preparing fish sauce umami peptide, comprising the following steps:
s11, separating the fish sauce fermentation stock solution by using an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and collecting the permeate to obtain a membrane ultrafiltration separation product.
S12, separating and purifying a product by using a membrane ultrafiltration method in the step S11 of Sephadex G-15 gel chromatography to obtain a gel chromatography separation product, wherein the gel chromatography separation product contains components of the fish sauce umami peptide;
the separation conditions are as follows: the specification of the chromatographic column is 1.6cm multiplied by 70cm, the loading amount is 2mL, the eluent is ultrapure water, the flow rate is 1mL/min, and the detection wavelength is 220nm.
Preferably, the product is separated by gel chromatography of reversed phase high performance liquid chromatography separation step S12;
separation conditions: the column was Spurisil 5 μm C18, 250×4.6mm; the sample injection volume is 20 mu L; mobile phase A is ultrapure water, mobile phase B is acetonitrile, detection wavelength 220nm, isocratic elution: the volume concentration is 7% B,93% A, the flow rate is 1mL/min, and the elution time is 20min;
obtaining the reversed-phase high performance liquid chromatography separation product.
Preferably, the reversed-phase high-performance liquid chromatography separation product sequentially contains umami peptides with amino acid sequences shown as SEQ ID NO. 1-5 from the early to the late according to the peak time.
The fish sauce umami peptide is applied to the preparation of foods, food additives and/or health-care products.
A food, food additive and/or health product containing the fish sauce flavor peptide.
The method for evaluating the flavor intensity of the object to be tested containing the fish sauce flavor peptide comprises the following steps:
mixing monoclonalized stable transformed cells which overexpress T1R1, T1R3 and G16gust44 proteins with a calcium ion fluorescent probe, collecting intracellular calcium ion fluorescent signals by using a fluorescent inverted microscope,
automatically performing image acquisition every 5s, adding an object to be detected containing the umami peptide and sodium glutamate serving as a freshness standard substance at 20s, continuously detecting, and continuously detecting for 240s in the whole detection process to obtain a calcium ion relative fluorescence change peak value corresponding to the umami peptide and sodium glutamate serving as the freshness standard substance;
the sodium glutamate freshness value was defined as 1. The calculation formula of the relative freshness value U is as follows:
U＝F sample /F MSG ，
wherein ,Fsample Maximum value representing peak value of calcium ion relative fluorescence change of the umami peptide, F MSG And (3) calculating the relative freshness value of the umami peptide by representing the maximum value of the calcium ion relative fluorescence change peak value of the sodium glutamate.
Preferably, the monocloned 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 end of GNA15 by 44 amino acids at the C-terminal end of GNA 3, 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 for collecting the fluorescence signal of the calcium ions in the cells is 490nm, and the emission wavelength is 514nm.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a fish sauce flavor peptide, a preparation method thereof, a flavor intensity evaluation method and application thereof. The invention separates the umami peptide in fish sauce by using a membrane ultrafiltration method, a gel chromatography method and a reverse phase high performance liquid chromatography, identifies the molecular weight and the sequence of the peptide by using Nano LC-Q-Orbitrap-MS/MS, analyzes ion fragments of main PEAKS by combining with manual sequencing from the head through PEAKS Studio 8.5 software, and obtains the amino acid sequence of the polypeptide. The biological activity of the small molecule peptide is analyzed on line by a BIOPEP database, the flavor development intensity of the flavor peptide is predicted based on the database, and the flavor peptide is respectively screened out from 5 components by combining the relative intensity of peptide fragments and scoring of peptide fragments, so that the flavor peptide with the amino acid sequence shown as SEQ ID NO. 1-5 is obtained, and has better flavor and flavor enhancing characteristics. The invention constructs a cell stably expressing the umami receptor T1R1/T1R3, namely a monoclonalized humanized T1R1/T1R3/G16gust44 stable transfer cell line, detects intracellular calcium ion fluorescence responses caused by umami peptides with different concentrations through a calcium ion fluorescence imaging technology, takes sodium glutamate as a standard substance, eliminates the influence of concentration, calculates relative freshness value by concentration when the sodium glutamate and the umami peptide cause the strongest calcium ion fluorescence change, can relatively quantitatively detect the umami intensity of the umami peptide on physiological level, and has good reproducibility, stability and reliability.
Drawings
FIG. 1 is a gel chromatography separation diagram.
FIG. 2 is a sensorgram of gel chromatographic separation components.
FIG. 3 is an electronic tongue radar chart of gel chromatography separation components.
FIG. 4 is a reversed phase high performance liquid separation chromatogram.
FIG. 5 is a secondary mass spectrum of 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 diagram of a sensory evaluation radar of synthetic peptides.
FIG. 11 is an electronic tongue evaluation radar chart of synthetic peptides.
FIG. 12 is a graph showing the synergistic freshening effect of synthetic peptides.
FIG. 13 shows the result of whole gene amplification, wherein a is T1R1, b is T1R2, and c is G16gust44.
FIG. 14 shows the results of vector cleavage, with M bands in order from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp and 250bp; 1. 3 and 5 represent: pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA vector cleavage products; 2. 4 and 6 represent: the vector was not digested.
FIG. 15 shows colony PCR identification results, wherein a is the colony PCR identification result of the recombinant plasmid pcDNA3.1-His-T1R 1; b is the PCR identification result of recombinant plasmid pcDNA3.1-Flag-T1R3 colony; c is the PCR identification result of recombinant plasmid pcDNA3.1-HA-G16gust44 colony; 1 is negative control (ddH 2O), 2 is negative control (empty self-connecting control group), 3 is positive control (GAPDH), M is Marker, and the sequence is 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp and 100bp from top to bottom; 4 to 9 represent colonies 1 to 6 in this order.
FIG. 16 shows the results of protein expression assay, wherein M is Marker;1 is HEK293-T1R1/T1R3/G16gust44 cells; 2 is HEK293 cells.
FIG. 17 is a graph showing the change in calcium ion relative fluorescence of cells caused by different concentrations of MSG.
FIG. 18 is a graph showing the peak changes in calcium ions relative to fluorescence of cells caused by different concentrations of MSG.
FIG. 19 is a graph showing the relative fluorescence change of calcium ions in cells caused by different concentrations of TLTDVEK.
FIG. 20 is a graph showing the change in calcium ion relative 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 different concentrations of LTLF.
FIG. 22 is a graph showing the change in calcium ion relative fluorescence of cells caused by different concentrations of AEEVEEERLK.
FIG. 23 is a graph showing the change in calcium ion relative fluorescence of cells caused by different concentrations of VLTHGEDKEGE.
FIG. 24 is a graph showing the peak changes in calcium ion relative fluorescence of cells caused by different concentrations of synthetic peptide.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 separation of umami peptides from fish gravy by Membrane ultrafiltration and gel chromatography
1. Experimental method
1. Membrane ultrafiltration method for separating umami peptide
Filtering the fish sauce fermentation stock solution with double-layer gauze and medium-speed filter paper, concentrating and desalting by a rotary evaporator, and storing at 4deg.C for use. Diluting the concentrated fish sauce with ultrapure water to the volume concentration of 1%, filtering with a 0.45 μm filter membrane, separating with a Sidoris ultrafiltration membrane with molecular weight cutoff of 3000Da, and vacuum freeze-drying the collected permeate to obtain the membrane ultrafiltration separation product.
2. Separation of umami peptide by gel chromatography
Separating the product by the ultrafiltration method of the membrane in the step 1, passing the product through a microporous membrane with the thickness of 0.45 mu m, and separating and purifying the product by using Sephadex G-15 gel chromatography. The separation conditions are as follows: the chromatographic column (1.6 cm. Times.70 cm), the loading amount is 2mL, the eluent is ultrapure water, the flow rate is 1mL/min, and the detection wavelength is 220nm. Collecting one tube every 3min, collecting according to different component peaks, concentrating, freeze-drying, performing sensory evaluation and electronic tongue evaluation, and selecting the component with strongest delicate flavor, and continuing to separate and purify.
2. Experimental results
The umami peptide is generally a polypeptide with molecular weight less than 3kDa, so that an ultrafiltration membrane with molecular weight cutoff of 3000Da is selected for separation, and permeate is collected for further gel chromatography separation. As shown in FIG. 1, a total of 4 component peaks (F1 to F4) were obtained by separation. The sensory evaluation results of the components are shown in fig. 2, the taste profile of each component is mainly fresh taste, and then sour taste, salty taste and sweet taste are obtained, so that the bitter taste is weak. There was a significant difference in the umami characteristics of the individual components, with the F2 component umami score being up to 4.6, followed by the F1 component, with a score of 3, and the F3 and F4 components umami scores being the same of 1.8. Analysis found that the trend of the change in the sourness and freshness in the profile of the taste was consistent, probably due to the presence of carboxyl groups in the R-or C-terminal residues of the polypeptides, whose dissociated hydrogen ions are important species responsible for the sourness. Among the umami peptides reported so far, many peptides have not only umami taste but also may exhibit a certain sour taste. The results of the electronic tongue analysis of the components are shown in fig. 3, the results of the electronic tongue analysis are not identical to the results of the sensory evaluation, but the general trend is identical, the umami taste of the F2 component is still the most prominent, and the F1 component, the F4 component and the F3 component are the next. Therefore, by combining the sensory evaluation and the electronic tongue analysis results, the F2 component has the strongest fresh flavor and the highest content and is easy to collect, and the F2 component is selected to continue to carry out reversed-phase high-efficiency liquid phase separation.
Example 2 separation and purification of umami peptide in fish gravy by reverse phase high performance liquid chromatography
1. Experimental method
The gel chromatography separation of example 1 gave 4 fractions (F1-F4), with the most umami fraction F2 prepared with primary water to a concentration of 5mg/mL, passing through a 0.45 μm microporous filter membrane, and further separating by reverse phase high performance liquid chromatography (RP-HPLC). Separation conditions for RP-HPLC: the column was Spurisil 5 μm C18, 250×4.6mm; the sample injection volume is 20 mu L; mobile phase A is ultrapure water, mobile phase B is acetonitrile, detection wavelength 220nm, isocratic elution: the volume concentration is 7% B,93% A, the flow rate is 1mL/min, and the elution time is 20min. The fractions corresponding to each separation peak were collected, concentrated and freeze-dried.
2. Experimental results
The components obtained by gel chromatography in example 1 are still mixtures, and the components are complex and cannot be directly identified in structure, so that further separation is required by reversed-phase high-performance liquid chromatography. The reversed-phase high-performance liquid chromatography is a chromatography for separating substances according to molecular hydrophobicity, has the advantages of simple operation, high efficiency, high speed, high sensitivity and good repeatability, and is usually used as the final step of separating and purifying proteins and polypeptides. The result of the reversed-phase high-efficiency liquid phase separation of the F2 component in ultrapure water is shown in fig. 4, and the result shows that the reversed-phase high-efficiency liquid phase purification has a good separation effect, 5 components are obtained through total separation, namely F2-1, F2-2, F2-3, F2-4 and F2-5, and the separated components are eluted in the first 10 minutes, so that the components have strong polarities.
Example 3 identification of Structure of fish dew umami peptide
1. Experimental method
And (3) carrying out reductive alkylation on the 5 components purified by the reversed-phase high-performance liquid phase in the embodiment 2, desalting the components, analyzing the treated sample by liquid chromatography-mass spectrometry (LC-MS/MS) to obtain a raw file of an original result of mass spectrum, and carrying out De novo analysis to obtain a peptide fragment sequence analysis result. The method comprises the following specific steps:
1. preparation of samples
A dithiothreitol solution was added to each of the 5 component samples to give a final concentration of 10mmol/L, and the mixture was reduced in a water bath at 56℃for 1 hour. The iodoacetamide solution was added to a final concentration of 50mmol/L and reacted in the dark for 40min. Desalting was performed using a self-packed desalting column, and the solvent was evaporated in a vacuum centrifugal concentrator at 45 ℃.
2. Capillary liquid chromatography conditions
Pre-column: 300 μm i.d. times. 5mm,packed with Acclaim PepMap RPLC C18,5 μm,
analytical column: 150 μm i.d. times. 150mm,packed with Acclaim PepMap RPLC C18,1.9 μm,
mobile phase a:0.1% formic acid; mobile phase B:0.1% formic acid, 80% acetonitrile; gradient elution procedure: 0-2 min 4-8% B, 2-45 min 8-28% B, 45-55 min 28-40% B, 55-56 min 40-95% B, and maintaining the mobile phase 95% B for 10min; flow rate: 600nL/min. The percentages are all volume concentrations.
3. Mass spectrometry conditions
Primary mass spectrometry parameters: resolution:70000 agc target:3e6, maximum IT:100ms, scan range:300to 1800m/z.
Secondary mass spectrometry parameters: resolution:17500, agc target:1e5, maximum IT:50ms, top N:20, NCE/stepped NCE:28.
de novo analysis is carried out to obtain the sequence analysis results of the peptide fragments of the components F2-1, F2-2, F2-3, F2-4 and F2-5, the peptide fragments are synthesized by adopting a solid phase synthesis method, and the polypeptide with the purity of more than 98% is obtained by carrying out desalting treatment and the like.
2. Experimental results
The 5 fractions (F2-1, F2-2, F2-3, F2-4, F2-5) obtained in the reverse phase high performance liquid phase separation and purification of example 2 were used to identify the molecular weight and sequence of the peptides by Nano LC-Q-Orbitrap-MS/MS. After separation and identification by Nano LC-MS/MS, the mass spectrum database is automatically searched, and the amino acid sequence of the polypeptide is obtained by analyzing the ion fragments of the main peak through PEAKS Studio 8.5 software and combining manual sequencing from the head. Although many impurities are removed by a series of separations and purifications, each sample component remains a mixture of polypeptides and cannot contain only one polypeptide. The mass spectrometer QExacte has ultra-high 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 intensity of the umami peptide based on the database, and screen one umami peptide from 5 components respectively by combining the relative intensity of peptide fragments and scoring of peptide fragments, wherein the main information is shown in table 1, and the secondary mass spectrogram of each umami peptide is shown in fig. 5-9.
TABLE 1 amino acid sequence identification of umami peptides
Example 4 analysis of taste Properties of umami peptides
1. Experimental method
1. Sensory evaluation
Sensory panel consisted of 12 normally tasted students (6 female 6 male, aged 20 to 25 years) and panelists all received sensory training to discern 5 basic tastes (sour, sweet, bitter, salty, fresh) according to GB/T16291.1-2012. The 5 basic taste acid, sweet, bitter, salty, fresh 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 sensory score of the standard solution was 5 minutes, the test took 10 minutes, with 0 indicating no taste and 10 indicating a strong taste. Components F1 to F4 separated by gel chromatography in example 1 are respectively prepared into solutions with the concentration of 0.2mg/mL, and synthetic peptides with the amino acid sequences shown in SEQ ID NO. 1 to 5 synthesized in example 3 are respectively prepared into solutions with the concentration of 1mmol/L for sensory evaluation by panelists.
The taste threshold of each synthetic peptide was determined by a triangle test method, and the synthetic peptides having the amino acid sequences shown in SEQ ID NO. 1 to 5 were dissolved in ultrapure water to a concentration of 5mmol/L, and the taste characteristics thereof were described by a sensory panel. Subsequently, 1 sample experimental group and 2 blank control groups were prepared according to the triangle experiment, diluted stepwise 1:1, and tasted sequentially from low concentration to high concentration by panelists. When just the sample solution can be distinguished from the two blank solutions, this concentration is the taste threshold of the peptide. The average of the evaluation results of all sensory panels was recorded as the final result.
Fresh-keeping effect of the synthetic peptide: taking 1mg/mL MSG solution as an delicious sensory standard substance, scoring for 5 minutes, adding synthetic peptide with an amino acid sequence shown as SEQ ID NO. 1-5 into the standard MSG solution, enabling the final concentration of the synthetic peptide in the composite solution to be 1mmol/L, performing sensory evaluation on the synthetic peptide solution to analyze the synergistic freshness-enhancing effect of the synthetic peptide, wherein the delicious taste is scored for 5-10 minutes when being stronger than the standard solution, and scored for 0-5 minutes when being weaker than the standard solution.
2. Electronic tongue evaluation
The electronic tongue system consists of 5 taste sensors: CA0 (acid), GL1 (sweet), C00 (bitter), AAE (fresh), CT0 (salty) and AE1 (astringent). For the first detection, all sensors are put into a reference solution (30 mmol/L potassium chloride and 0.3mmol/L tartaric acid mixed solution) to be activated for 24 hours, and after the sensors complete self-detection and signals are stable, the detection of the sample can be started. The components F1 to F4 separated by gel chromatography in example 1 are prepared into a solution with polypeptide concentration of 0.2mg/mL by ultrapure water, the peptides with amino acid sequences shown in SEQ ID NO. 1 to 5 synthesized in example 3 are respectively prepared into a solution with 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 total acquisition time is 4 times. All data are absolute output values taking reference liquid as a standard, and the state of the reference liquid is tested by the electronic tongue to simulate the state of only saliva in the oral cavity of a human. The standard solution is composed of potassium chloride and tartaric acid, and has taste value close to odorless, so that the non-taste point of sour taste is-13, the odorless point of salty taste is-6, and when the taste value of the sample is lower than the odorless point of the standard solution, the sample has no taste, otherwise.
2. Experimental method
The results are shown in FIG. 10. The synthesized 5 polypeptides have more obvious bitter taste except LTLF, have basically consistent taste profiles, have stronger delicate flavor as a whole, are sour, have certain sweet and delicate flavors and basically have no bitter taste. AEEVEEERLK has the strongest umami taste, followed by VLTHGEDKEGE, TLTDVEK, LPVDE and LTLF, but TLTDVEK and LPVDE have no significant differences in umami taste. And LTLF has a strong bitter taste, probably because of the increased intensity of bitter taste when hydrophobic amino acids (e.g., phenylalanine, proline, leucine, etc.) are in the C-terminal position of the peptide chain. The results of the electronic tongue analysis of the 5 synthetic peptides are shown in fig. 11, the flavor profile of the 5 synthetic peptides is similar to the results of sensory evaluation, and the most umami is AEEVEEERLK, the umami of VLTHGEDKEGE and TLTDVEK is no significant difference, then LPVDE, and the umami of LTLF is the least. It was found that the acidity of the synthetic peptides was significantly increased compared to components F1 to F3 separated by gel chromatography in example 1, probably because glutamic acid and aspartic acid were originally acidic amino acids or residual sodium acetate and amino acids during peptide synthesis. The taste profile and threshold values of the synthetic peptides are shown in table 2.
TABLE 2 taste profile and threshold for synthetic peptides
In order to more fully understand the taste characteristics of the synthetic peptide, the recognition threshold of the synthetic peptide is determined by adopting a taste dilution method, and a sensory evaluation personnel is required to carry out language description on the taste of the synthetic peptide, and the taste characteristics and the threshold of the synthetic peptide are shown in table 2. The threshold value of MSG is 1.77mmol/L, and the threshold values of the 5 synthetic peptides are lower than that of MSG, which indicates that the synthetic peptides have stronger delicate flavor. The sensory profile of the synthetic peptides remained essentially identical to the flavor profile analysis and electronic tongue analysis. When the concentrations of 5 synthetic peptides were consistent, the umami taste of AEEVEEERLK was strongest, while the synthetic peptide with the lowest threshold was VLTHGEDKEGE, indicating that there was no necessarily correlation between the umami intensity and the umami threshold, and that the umami taste of VLTHGEDKEGE was not more prominent than that of AEEVEEERLK in the sensory description.
To investigate the freshness-enhancing effect of synthetic peptides, sensory evaluation was performed by adding synthetic peptides to MSG solutions, and the results are shown in fig. 12. Both synthetic peptide AEEVEEERLK, VLTHGEDKEGE, TLTDVEK and LPVDE enhance the umami taste of MSG solutions, with AEEVEEERLK being the strongest umami effect, while LTLF mixed MSG solutions have slightly lower umami scores than MSG solutions, probably because LTLF has a more prominent unpleasant bitter taste, masking the umami taste, making the change in umami taste indistinct for sensory evaluation personnel.
EXAMPLE 5 construction of recombinant expression vectors
Human T1R1 (NM_ 138697), T1R3 (NM_ 152228), GNA15 (NM_ 002068) and GNAT3 (NM_ 001102386) gene sequences are queried through NCBI website, 44 amino acids at the C terminal end of GNA15 are replaced by 44 amino acids at the C terminal end of GNAT3 to obtain G16gust44, and genes T1R1-6His, T1R3 and G16gust44 are synthesized.
Designing a primer by using Prime primer software, respectively adding XhoI and KpnI restriction sites at the 5' end of a forward primer and a reverse primer of T1R1-6His, respectively adding XhoI and KpnI restriction sites at the 5' end of a forward primer and a reverse primer of T1R3, respectively adding XhoI and BamHI restriction sites at the 5' end of a forward primer of G16gust44, and designing the obtained primer as shown in Table 3:
TABLE 3 PCR primer sequences
And (3) taking the synthesized target gene as an amplification template to carry out PCR amplification. The reaction system shown in Table 4 was prepared, gently stirred and mixed, centrifuged briefly, and placed in a PCR instrument for reaction. The PCR amplification reaction condition is 98 ℃ for 5min;98 ℃,10s,55 ℃,10s,72 ℃,90s,30 cycles; 72℃for 8min. The amplified product was analyzed by agarose gel electrophoresis, and the result was observed by a gel imaging system, and the target band was recovered.
TABLE 4 PCR amplification System
Restriction enzymes of the plasmids pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA are XhoI/KpnI, xhoI/KpnI and XhoI/BamHI, respectively. The enzyme cutting system was prepared according to Table 5, gently stirred and mixed by a pipette, centrifuged briefly, and reacted at 37℃for 3 hours. And (3) carrying out agarose gel electrophoresis on the carrier enzyme digestion product, observing the result by a gel imaging system, and recovering the target band.
The T1R1-6His, T1R3 and G16gust44 gene sequences amplified by PCR are respectively connected to pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA carriers, a reaction system of the table 6 is prepared in an ice-water bath, a pipettor is used for gently blowing and mixing, short centrifugation is carried out, bubbles are avoided, the reaction is carried out for 30min at 37 ℃, and recombinant plasmids pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 and pcDNA3.1-HA-G16gust44 are constructed. Then placed in an ice-water bath for cooling for 5min and immediately converted. 10. Mu.L of the ligation reaction product was added to 100. Mu.L of E.coli Top10 competent cells, and the mixture was stirred well under the condition of a light bomb wall number and left on ice for 30min. Heat shock at 42 ℃ for 90s and ice-water bath incubation for 2min. 500. Mu.L of LB medium was added and the mixture was subjected to shaking culture at 37℃for 1 hour. And uniformly coating a proper amount of bacterial liquid on an LB plate containing ampicillin, and inversely culturing for 12-16 hours in a constant temperature incubator.
TABLE 5 vector cleavage reaction System
TABLE 6 ligation reaction System
2. Experimental results
After PCR amplification, target fragments of 2606bp, 2605bp and 1169bp in length are expected to be obtained respectively, and after agarose gel electrophoresis, the amplified bands are imaged as shown in FIG. 13 and are basically consistent with the expected sizes.
The vector digested products were subjected to agarose gel electrophoresis, as shown in FIG. 14, in which pcDNA3.1 was approximately 5.4kb, pcDNA3.1-Flag was approximately 5.5kb, and pcDNA3.1-HA was approximately 5.4kb, and the amplified bands were substantially identical to the expected sizes.
EXAMPLE 6 identification of recombinant expression vectors
1. Experimental method
The PCR reaction system was prepared according to Table 7, and the mixture was stirred and centrifuged briefly. In an ultra clean bench, single colonies cultured in example 6 were picked with a sterile tip into a 20. Mu. LPCR system, blown and mixed well, placed in a PCR apparatus for reaction, and detected by agarose gel electrophoresis. The primers of each reaction system are shown in Table 8. The PCR amplification reaction condition is 94 ℃ for 3min;94 ℃,30s,55 ℃,30s,72 ℃,30s,22 cycles; 72℃for 5min.
TABLE 7 colony PCR reaction System
TABLE 8 colony PCR primer sequences
And (3) adding the identified positive clone transformant into an LB liquid culture medium containing 0.1mg/mL ampicillin according to an inoculation proportion of 0.5-1% of volume concentration, performing shake culture for 12-16 h at 37 ℃ at 220rpm, 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.
2. Experimental results
As shown in FIG. 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 figures, indicate that the target gene is successfully inserted into the expression vector.
The sequence result of the Snapgene software shows that the gene sequence on the recombinant expression plasmid is 100% identical with the target gene sequence, and the successful cloning of the T1R1-6His, T1R3 and G16gust44 genes on pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA vectors is proved.
Example 7 determination of G418 killer concentration
1. Experimental method
Cloning exogenous DNA onto a vector with a certain resistance or selection gene, transfecting the vector into a host cell, integrating the vector into chromosome, and screening by using a selection marker contained in the vector to obtain a cell strain capable of stably expressing target protein. G418 (Geneticin ) is an amino sugar antibiotic, similar in structure to neomycin, gentamicin, kanamycin, which blocks protein synthesis by affecting 80S ribosomal function, and is toxic to cells, both prokaryotic and eukaryotic, including bacterial, yeast, plant and mammalian cells. Is the most commonly used selection reagent for stable transfection. When the neomycin gene is integrated into the genome of eukaryotic cells in a suitable place, transcription of the sequence encoded by the neomycin gene into mRNA can be initiated, thereby obtaining efficient expression of the resistant product aminoglycoside phosphotransferase, and allowing the cells to acquire resistance to grow in selective media containing G418. Since the resistance genes of the 3 plasmids pcDNA3.1, pcDNA3.1-Flag and pcDNA3.1-HA of example 5 were neomycin, G418 was used to select stable high-expression cell lines.
Each eukaryotic cell line is differently sensitive to G418, so the optimal screening concentration of G418 is determined prior to screening.
1. Selecting G418 to select the concentration range of 100 mug/mL-1 mg/mL. 40mg/mL G418 was diluted to 12 levels of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1100 μg/mL with no-diabody complete medium.
2. HEK293 cells were seeded on 96-well plates with complete medium at 37℃at 5% CO by volume 2 Culturing for 24h and adhering to the wall.
3. Discarding the culture medium of step 2, adding 200 μl of the G418 screening culture medium prepared in step 1 into each well, 3 wells each with a volume concentration of 5% CO at 37deg.C 2 Culturing.
4. The medium was changed every 24 hours. The lowest G418 concentration that caused all cell death within 10-14 days was selected as the screening concentration.
2. Experimental results
Screening experiments show that the lowest G418 concentration for the HEK293 cells to die completely within 10-14 days is 500 mug/mL, and stable expression strains are screened by the concentration.
EXAMPLE 8 pcDNA3.1-HA-G16gust44 plasmid transfection, pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 plasmid Co-transfection
1. pcDNA3.1-HA-G16gust44 plasmid transfection
Plasmid was extracted using OMEGA Endo-free Plasmid Mini Kit I kit according to Invitrogen Lipofectamine TM 3000 transfection reagent.
1. According to the inoculation proportion of 0.5% -1% of volume concentration, 1mL of glycerol bacteria with recombinant plasmid pcDNA3.1-HA-G16gust44 prepared in example 5 is inoculated in 100mL of LB liquid medium, the temperature is 37 ℃, the rpm is 220, and shaking culture is carried out overnight. 5mL of the bacterial liquid was used to extract pcDNA3.1-HA-G16gust44 recombinant plasmid using OMEGA Endo-free Plasmid Mini Kit I kit, and stored at-20 ℃.
2. HEK293 cells were inoculated in 6-well plates, added with 2.5mL of DMEM complete medium, and placed in CO 2 Culturing in a constant temperature incubator.
3. The cell growth state of step 2 was observed and transfection was performed when confluency reached 80%. Transfection procedure reference Lipofectamine TM 3000 transfection reagent Specification, to 750. Mu.L serum-free Opti-MEM Medium 45. Mu.L Lipofectamine was added TM 3000 reagent.
4. Then, 15. Mu.g of the recombinant plasmid (0.5 to 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, followed by 30. Mu. L P3000 TM The reagent is fully and evenly mixed.
5. Taking 750 mu L of diluted Lipofectamine TM 3000 reagent and 750. Mu.L of the recombinant plasmid pcDNA3.1-HA-G16gust44 diluted in step 4 were mixed uniformly (1:1 ratio). Incubating for 10-15 min at room temperature.
6. Step 5 250. Mu.L of DNA-liposome complex was added to each well and the cells were incubated at 37℃for 2 days. qRT-PCR analysis and stable strain selection of transfected cells were then performed.
2. pcDNA3.1-His-T1R1, pcDNA3.1-Flag-T1R3 plasmid Co-transfection
The cells transfected with pcDNA3.1-HA-G16gust44 plasmid were inoculated in 6-well plates, after overnight cell attachment was cultured, complete medium was aspirated, and the cells were screened with 500. Mu.g/mL G418 medium for 14 days, and then pcDNA3.1-His-T1R1 and pcDNA3.1-Flag-T1R3 plasmids were transfected, as shown by pcDNA3.1-HA-G16gust44 plasmid transfection, in which 15. Mu.g of the recombinant plasmid in step 4 was changed to 15. Mu.g of plasmid pcDNA3.1-His-T1R1 and 15. Mu.g of plasmid pcDNA3.1-Flag-T1R3.
EXAMPLE 9 establishment of monoclonal Stable strains
Cells transfected with pcDNA3.1-HA-G16gust44, pcDNA3.1-His-T1R1 and pcDNA3.1-Flag-T1R3 plasmids in example 8 were inoculated in 6-well plates, after overnight cell attachment, the complete medium was aspirated, and after screening with 500. Mu.g/mL G418 medium, resistant clones were seen to appear after about 14 days, followed by further screening of the monoclonal by limiting dilution.
Resistant clones were digested and diluted to 1X 10 4 cell/mL cell suspension 200 μl of cell suspension was added to A1 well in a 96-well plate, and 100 μl of complete medium was added to each remaining well. Then transferring 100 mu L of culture medium from the hole A1 to the hole B1, blowing and uniformly mixing, repeating the dilution step until the last hole H1 in the row, and sucking 100 mu L of culture medium in the hole H1 to ensure that the volumes of the culture mediums in the holes in the first row are the same. 100. Mu.L of culture was added with a row gun Mixing and blowing evenly the culture medium into each well in the first row, taking 100 mu L of the culture medium from the first row into each well in the second row, repeating the dilution step in the last row, sucking 100 mu L of the culture medium in the last row, and adding 100 mu L of the culture medium into all the wells to ensure that the final volume of all the wells is 200 mu L. 96-well plates were incubated at 37℃with 5% CO 2 Culturing for 48h. Then, the culture medium was changed to 500. Mu.g/mL G418 for each well, and the growth of single cells was observed by a microscope, and the formation of monoclonal cells was observed, usually for a period of 2 to 3 weeks. And (3) screening out a monoclonal with higher expression abundance by qRT-PCR and Western Blot for subsequent amplification culture to obtain a monoclonalized humanized T1R1/T1R3/G16gust44 stable-rotation HEK293 cell line (HEK 293-T1R1/T1R3/G16gust 44).
Example 10qRT-PCR detection of target Gene expression level Change
1. Experimental method
1. The monocloned human T1R1/T1R3/G16gust44 stable HEK293 cell line (HEK 293-T1R1/T1R3/G16gust 44) cultured in example 9 was removed, the cells were rinsed 2 times with PBS, 350. Mu.L of lysate was added to each well of the 6-well plate, and RNA was extracted according to the Ai Kerui company SteadyPure Universal RNA Extraction Kit kit. The concentration and purity of RNA are measured by a micro-spectrophotometer, and OD260 nm/280nm is 1.8-2.0, which indicates that the purity of RNA meets the requirement.
2. The RNA meeting the purity requirement is reversely transcribed into cDNA by using an Evo M-MLV RT Premix for qPCR reverse transcription kit of Ai Kerui company, and the reverse transcription reaction procedure is as follows: 37 ℃ for 15min;85 ℃,5s. The reverse transcription system is shown in Table 9.
TABLE 9 reverse transcription reaction system
3. GAPDH was used as an internal gene, and primers were designed based on the sequences of the humanized GAPDH, T1R1, T1R3, and G16gust44 genes as shown in Table 10. A qPCR reaction system was configured according to Ai Kerui company SYBR Green Premix Pro Taq HS qPCR Kit kit, the qPCR reaction procedure being: the first step: 95 ℃ for 30s; and a second step of: 95 ℃,5s,60 ℃,30s,40 cycles. The qPCR reaction system is shown in Table 11. The data processing is performed according to the following formula:
ΔΔCt＝(Ct target gene -Ct Reference gene ) HEK293-T1R1、T1R3、G16gust44 -(Ct Target gene -Ct Reference gene ) HEK293 ............(1)
Fold Change = 2 -ΔΔCt The expression level of the objective gene in the experimental group was expressed as a fold change relative to that in the control group.
TABLE 10 qPCR primer sequences
TABLE 11 qPCR reaction System
2. Experimental results
HEK293-T1R1/T1R3/G16gust44 cell mRNA obtained by monoclonal screening was extracted and subjected to qRT-PCR analysis, and the results were shown in Table 12, and 2 of T1R1, T1R3 and G16gust44 genes were calculated according to formula (1) -ΔΔCt 4871.00, 313.00 and 3236.01, respectively, the levels of gene expression indicated that the screened monoclonal cells stably expressed the 3 genes.
TABLE 12 qPCR Ct values for monoclonal stable strains
EXAMPLE 11 Western Blot detection of protein expression
1. Experimental method
The monocloned human T1R1/T1R3/G16gust44 cultured in example 9 was used to stably transform HEK293 cell line (HEK 293-T1R1/T1R3/G16gust 44), the culture broth was removed and washed once with PBS. mu.L of lysate was added to each well of the 6-well plate, and PMSF (phenylmethylsulfonyl fluoride, phenylmethanesulfonyl fluoride) was added to the lysate over a period of several minutes prior to use to give a final concentration of PMSF of 1mmol/L. The lysate and cells were contacted thoroughly by a gun blow number. And (3) centrifuging for 5min at 14000g after full pyrolysis, and taking the supernatant to perform subsequent electrophoresis and membrane transfer operation.
2. Experimental results
As a result, as shown in FIG. 16, the theoretical sizes of the G16gust44, T1R1, T1R3 and GAPDH proteins were 43kDa, 93kDa and 36kDa, respectively, and the bands were matched to the expected sizes, indicating successful overexpression of the G16gust44, T1R1, T1R3 in HEK293 cells from the protein expression levels.
EXAMPLE 12 construction of umami taste evaluation method based on HEK293-T1R1/T1R3/G16gust44 cell line
1. Experimental method
Sodium Glutamate (MSG) and synthetic peptides with the amino acid sequences shown in SEQ ID NOs 1-5 synthesized in example 3 were dissolved in HBSS buffer. MSG concentrations were set to 2.5, 5, 10, 20 and 40mmol/L, respectively, and synthetic peptide concentrations were set to 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 24-well plates and experiments were performed when the cells occupied about 70% of the plates. The culture medium was discarded, the cells were rinsed 2 times with HBSS, 200. Mu.L of 5. Mu. Mol/L of a calcium ion fluorescent probe was added to each well, incubated at 37℃for 30min, and then the cells were rinsed 2 times with HBSS again, and deesterified for 20min with 200. Mu.L of phenol red-free DMEM basal medium. The treated cells are placed under a fluorescence inversion microscope, the fluorescence signals of calcium ions in the cells are collected at the wavelengths of 490nm (excitation wavelength) and 514nm (emission wavelength), the image collection is automatically carried out every 5s, 50 mu L of sample synthetic peptide solution with different concentrations is added at the 20 th s, and the whole detection process lasts for 240s. MSG reaction final concentrations of 0.5, 1, 2, 4 and 8mmol/L, synthetic peptides reaction final concentrations of 0.25, 0.5, 1, 2 and 4mmol/L, blank added 50. Mu.L HBSS, and blank set 4 parallel to each concentration of the experimental group. And selecting taken fluorescence photographs, and detecting fluorescence values of at least 30 cell areas in each photograph. The calculation formula of the calcium ion relative fluorescence change is as follows:
Δf represents the relative fluorescence change value, and "F" represents the cell area real-time fluorescence value minus the background real-time fluorescence value; "F0" means "F" at 0 seconds.
MSG was used as a freshness standard, and the freshness value was defined as 1. The calculation formula of the relative freshness value U is as follows:
U＝F sample /F MSG ...................(3)
wherein ,Fsample Maximum value of peak value of fluorescence change of umami peptide calcium ion, F MSG The maximum value of the peak of the MSG calcium ion relative to the fluorescence change is shown.
2. Experimental results
The taste mechanism of umami is known to cause an increase in intracellular calcium ion concentration when umami peptide binds to the receptor T1R1/T1R 3. MSG is generally used as an umami standard and is able to bind to the receptor T1R1/T1R 3. Therefore, to verify whether the T1R1/T1R3/G16gust44 receptor overexpressed in the HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 had the 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.
As shown in FIG. 17, the results of the change in fluorescence of calcium ions in cells caused by different concentrations of MSG are shown, con represents the HBSS blank group, and if the relative fluorescence change value is greater than 0, it is shown that MSG can cause the response of cells, and the greater the relative fluorescence change value is, the stronger the response of calcium ion fluorescence is caused. As can be seen from FIG. 17, MSG stimulation of cells can cause an increase in intracellular calcium ion concentration, demonstrating that the T1R1/T1R3 receptor overexpressed in HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 has normal activity, and this cell can be used as a tool for detecting umami taste.
When the cells are stimulated with MSG with the minimum concentration of 0.5mmol/L, stronger calcium ion fluorescence change can still be caused, the threshold value of the cells capable of sensing MSG is between 0 and 0.5mmol/L, the sensory threshold of human to MSG is about 1.77mmol/L, and the HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9 can more sensitively identify MSG.
Comparing the intensities of the relative fluorescence changes of the calcium ions caused by the MSG with different concentrations, the peak value of the relative fluorescence changes caused by the MSG with different concentrations is shown in FIG. 18, it can be seen that when the concentration of the MSG is between 0.5 and 1mmol/L, the response of the calcium ions caused by the MSG increases with the increase of the concentration, the response of the calcium ions reaches the strongest value at 1mmol/L, the response of the calcium ions caused by the MSG does not reach the decrease of the response of the calcium ions at 1 to 8mmol/L, even at 8mmol/L, the response of the calcium ions increases with the increase of the concentration of the umami substance, but after the maximum concentration is reached, the response of the calcium ions decreases with the increase of the concentration of the umami substance, and when the taste receptor reaches saturation, the taste intensity remains unchanged after the maximum value is reached, even the inhibition effect is generated.
The results of changes in intracellular calcium ion response caused by different concentrations of umami peptide are shown in fig. 19 to 23. Both TLTDVEK, LPVDE, LTLF, AEEVEEERLK and VLTHGEDKEGE peptides can elicit cellular calcium response, and the intensity of the calcium fluorescence response varies with concentration, indicating that they all have umami taste. By comparing the intensities of the relative fluorescence changes of calcium ions caused by the different concentrations of the umami peptide, the relative fluorescence change peak value caused by the different concentrations of the umami peptide is shown as fig. 24, and it can be seen that the relative fluorescence change peak value of TLTDVEK is maximum at the concentration of 0.5mmol/L, the relative fluorescence change peak value of LPVDE is maximum at the concentration of 1mmol/L, the relative fluorescence change peak value of LTLF is maximum at the concentration of 1mmol/L, the relative fluorescence change peak value of AEEVEEERLK is maximum at the concentration of 2mmol/L, the relative fluorescence change peak value of VLTHGEDKEGE is maximum at the concentration of 2mmol/L, and the same trend as that of MSG, the calcium ion response intensity is enhanced with the increase of the concentration when the umami peptide is smaller than the concentration of the highest fluorescence response intensity, and the calcium ion response intensity is weakened with the increase of the concentration when the umami peptide is higher than the concentration of the highest fluorescence response intensity. As can be seen from FIG. 24, the maximum peak value of the relative fluorescence change of calcium ions has no necessary correlation with the corresponding concentration, and the relative fluorescence change of each peptide has different arrangement sizes at different concentrations, for example, the peak value of the relative fluorescence change of calcium ions is LPVDE, AEEVEEERLK, LTLF, VLTHGEDKEGE, TLTDVEK from large to small at a concentration of 1mmol/L, and the peak value of the relative fluorescence change of calcium ions is AEEVEEERLK, VLTHGEDKEGE, LTLF, TLTDVEK from large to small at a concentration of 2 mmol/L. Therefore, the peak of maximum calcium ion relative fluorescence change for each peptide was selected for comparison of 5 peptides for umami taste, ranging from large to small LPVDE, AEEVEEERLK, VLTHGEDKEGE, LTLF and TLTDVEK.
To more intuitively obtain the umami taste of the peptides, the umami taste of the peptides was quantified based on HEK293-T1R1/T1R3/G16gust44 cells constructed in example 9, and calculated according to formula (3), and the relative freshness values were LPVDE, AEEVEEERLK, VLTHGEDKEGE, LTLF and TLTDVEK in order from large to small as shown in Table 13.
TABLE 13 relative freshness value of synthetic peptides
Cosine similarity, also known as cosine similarity, is evaluated by calculating the cosine value of the angle between two vectors. Drawing the vectors into a vector space according to the coordinate values, obtaining 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 identical their directions are, the more similar. The sensory evaluation, electronic tongue analysis and relative freshness value method of 5 peptides were used as 3 matrices, respectively, and cosine similarity analysis was performed on each matrix, 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 with the freshness values measured by the other two methods, indicating that the relative freshness value method has reliability.
Sensory evaluation is somewhat subjective and is affected by other tastes, and subtle changes in umami taste are also difficult to perceive. The sensory evaluation and electronic tongue of example 4 were performed to detect umami taste at the same polypeptide concentration, but the umami taste of the taste-developing substances at different concentrations was inconsistent, and could not be uniformly evaluated. The method for evaluating the calcium ion relative fluorescence change delicate flavor based on HEK293-T1R1/T1R3/G16gust44 cells can eliminate subjective factors and concentration interference, and the detected delicate flavor intensity is more accurate.
Table 14 cosine values of three umami taste evaluation methods
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Sequence listing
<110> agricultural university of south China
<120> a fish sauce flavor peptide, a preparation method thereof, a flavor intensity evaluation method and applications thereof
<160> 5
<170> SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial Sequence)
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Thr Leu Thr Asp Val Glu Lys
1 5
<210> 2
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Leu Pro Val Asp Glu
1 5
<210> 3
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Ala Glu Glu Val Glu Glu Glu Arg Leu Lys
1 5 10
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Val Leu Thr His Gly Glu Asp Lys Glu Gly Glu
1 5 10
<210> 5
<211> 4
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Leu Thr Leu Phe
1
Claims (7)
1. The fish sauce umami peptide is characterized by being an umami peptide with an amino acid sequence shown in any one of SEQ ID NO. 1, 3 and 4.
2. The fish sauce umami peptide of claim 1, wherein the umami peptide has an umami threshold of 0.55 to 0.80 mmol/L.
3. The application of the fish sauce umami peptide in preparing foods, food additives or health products is characterized in that the fish sauce umami peptide is shown in any one of SEQ ID NO. 1-4.
4. A food, food additive or health product comprising the fish sauce umami peptide of claim 1.
5. The method for evaluating the flavor intensity of the to-be-tested object containing the fish sauce flavor peptide is characterized in that the fish sauce flavor peptide is the flavor peptide with an amino acid sequence shown in any one of SEQ ID NO. 1-4, and the method comprises the following steps:
mixing monoclonalized stable transformed cells which overexpress T1R1, T1R3 and G16gust44 proteins and a calcium ion fluorescent probe, collecting calcium ion fluorescent signals in the cells by using a fluorescent inverted microscope, automatically collecting images every 5 th s, adding an object to be detected containing the umami peptide and sodium glutamate serving as a freshness standard at the 20 th s, continuously detecting, and continuously maintaining 240 s in the whole detection process to obtain a calcium ion relative fluorescence change peak value corresponding to the umami peptide and sodium glutamate serving as the freshness standard;
The sodium glutamate freshness value is defined as 1, and the calculation formula of the relative freshness value U is as follows:
wherein ,F sample a maximum value indicating a peak value of a change in calcium ion relative to fluorescence of the umami peptide,F MSG and (3) calculating the relative freshness value of the umami peptide by representing the maximum value of the calcium ion relative fluorescence change peak value of the sodium glutamate.
6. The method for evaluating the umami taste intensity of an object to be tested of fish sauce umami taste peptide according to claim 5, wherein the monocloned stable transgenic cells overexpressing the T1R1, T1R3 and G16gust44 proteins are connected with recombinant expression vectors of 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 end of GNA15 by 44 amino acids at the C-terminal end of GNA 3, the NCBI accession number of the GNA15 gene is NM_002068, and the NCBI accession number of the GNAT3 gene is NM_001102386.
7. The method for evaluating the umami taste intensity of a test object of fish sauce umami peptide according to claim 5, wherein the excitation wavelength of the intracellular calcium ion fluorescence signal is 490 nm and the emission wavelength is 514 nm.
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