Primary taste bud cell sensitive to salty taste enhancing peptide as well as preparation method and application of primary taste bud cell

Abstract

The invention relates to a primary taste bud cell sensitive to salty taste enhancing peptide and a preparation method and application thereof. Specifically, the invention provides a primary taste bud cell line which is a primary taste bud cell TBC sensitive to taste regulatory substances, and is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2024314. The efficient and stable taste bud cell model is successfully constructed by optimizing the cell separation extraction and culture technology. The model has high cell viability, and can stably respond to the stimulation of taste peptide (mushroom peptide) and generate quantifiable taste signals. A powerful tool is provided for deeply researching the salty-increasing mechanism of the taste-active peptide, and a scientific basis is provided for improving the flavor and the quality of food.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a primary taste bud cell sensitive to salty taste enhancing peptide, and a preparation method and application thereof. Background Art

[0002] Flavor peptides, as a class of low molecular weight peptide compounds, have attracted widespread attention in the fields of food science and nutrition and health due to their unique flavor enhancement effects and potential health benefits. These peptides can not only enhance the flavor of food, but also improve the texture of food to a certain extent, and even have physiological activity, which has brought revolutionary changes to the food industry. However, in order to have a deeper understanding of the mechanism of action of flavor peptides, especially in the study of salt enhancement mechanism, it is necessary to use advanced biological models to simulate the real taste perception process.

[0003] Primary taste bud cell models, especially those from mice (or rats), are ideal tools for studying the mechanisms of taste perception. Taste bud cells are the basic units of taste perception and can directly respond to various taste stimuli, including salty taste. By establishing primary taste bud cell models, researchers can simulate the response of taste bud cells to flavor peptides in an in vitro environment, thereby revealing their saltiness-enhancing mechanisms.

[0004] The isolation and culture techniques of taste bud cells are relatively complex and full of difficulties, such as low cell activity and long culture cycle, which limit the application of taste bud cell models in taste research.

[0005] In the process of flavor peptide flavor enhancement research, electronic tongue and sensory evaluation experiments are generally used. During use, the electronic tongue has problems such as difficulty in truly reflecting sensory responses, and the electronic tongue sensor membrane is easily affected by temperature, easy to be contaminated, and has high environmental requirements. For sensory evaluation, the evaluator's personal preferences and experience can easily affect the evaluation results, which is too subjective, and human senses are easily fatigued during the evaluation process, which may reduce the accuracy of the evaluation results.

[0006] Therefore, there is an urgent need in this field to develop an objective, accurate and stable primary taste bud cell, which can provide a powerful tool for in-depth research on the saltiness-enhancing mechanism of flavor peptides and provide a scientific basis for enhancing food flavor and improving quality. Summary of the invention

[0007] In view of the above problems, the purpose of the present invention is to provide an objective, accurate and stable primary taste bud cell, which provides a powerful tool for in-depth research on the saltiness-enhancing mechanism of flavor peptides and provides a scientific basis for enhancing food flavor and improving quality.

[0008] In a first aspect of the present invention, a primary taste bud cell line is provided. The cell line is a primary taste bud cell TBC that is sensitive to taste modulating substances and is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024314.

[0009] In a preferred embodiment, the taste modulating substance comprises a taste peptide and a taste modulating substance selected from the group consisting of salty, sweet, bitter, sour and umami.

[0010] In a preferred embodiment, the flavor peptides include: salty peptides, sweet peptides, bitter peptides, sour peptides, and umami peptides.

[0011] In a preferred embodiment, the salty peptide comprises a salty taste enhancing peptide.

[0012] In a preferred embodiment, the cell line is a cell line sensitive to a salty taste enhancing peptide.

[0013] In a preferred embodiment, the salty taste enhancing peptide is selected from the group consisting of Volvariella volvacea peptide WPGFK (SEQ ID NO: 11), Volvariella volvacea peptide YFDWPGFK (SEQ ID NO: 12), and Volvariella volvacea peptide YFDWPGFKT (SEQ ID NO: 13).

[0014] In a preferred embodiment, the taste substance refers to a substance that does not cause cell damage to the primary taste bud cells.

[0015] In a second aspect of the present invention, a method for constructing a taste bud cell model is provided, comprising the following steps:

[0016] (1) Providing a cell fluorescent probe;

[0017] (2) The cell fluorescent probe is co-incubated with the primary taste bud cell line described in the first aspect of the present invention to obtain taste bud cells containing the cell fluorescent probe, namely, a taste bud cell model.

[0018] In a preferred embodiment, the cell fluorescent probe is a calcium ion fluorescent probe, preferably Fluo-4 AM.

[0019] In a preferred embodiment, the concentration of the cell fluorescent probe is 0.5-5 μM.

[0020] In a preferred embodiment, the incubation condition is 37° C. for 30-360 minutes, preferably 30-240 minutes, and more preferably 30-120 minutes.

[0021] In a preferred embodiment, the method further comprises the steps of: (3) stimulating taste bud cells containing cell fluorescent probes with taste substances, and observing the changes in fluorescence intensity of the taste bud cells as the stimulation time increases under a confocal microscope;

[0022] If the fluorescence intensity F of the taste bud cells stimulated with taste substances is compared with the fluorescence intensity F0 without stimulation, and the ratio F / F0 is ≥0.5 (preferably ≥1, more preferably ≥1.5), the taste bud cell model is considered to be successfully constructed.

[0023] In a preferred embodiment, the taste substance is selected from the group consisting of sodium chloride, monosodium glutamate or sucrose.

[0024] In a preferred embodiment, the taste substance is selected from the group consisting of 200 mM sodium chloride, 25 mg / mL monosodium glutamate or 25 mg / mL sucrose.

[0025] In a preferred embodiment, the stimulation time is 0s-500s; preferably 0s-400s, and more preferably 0s-200s.

[0026] In a third aspect of the present invention, a method for screening or evaluating salty taste enhancing peptides is provided, comprising the following steps:

[0027] (1) Providing the flavor peptide to be tested;

[0028] (2) stimulating the taste bud cell model described in the second aspect of the present invention with sodium chloride as a control group of cells;

[0029] (3) stimulating the control group cells with the flavor peptide to be tested to serve as the experimental group cells;

[0030] (4) observing the changes in fluorescence intensity of the control group cells in step (2) and the experimental group cells in step (3) as the stimulation time increases under a confocal microscope;

[0031] If the fluorescence intensity F produced by the experimental group cells is compared with the fluorescence intensity F0 produced by the control group cells, and the ratio F / F0 is ≥0.5 (preferably ≥1, more preferably ≥3), the taste peptide to be tested is considered to be a salty taste enhancing peptide.

[0032] In a preferred embodiment, the salty taste enhancing peptide is selected from the group consisting of Volvariella volvacea peptide WPGFK (SEQ ID NO: 11), Volvariella volvacea peptide YFDWPGFK (SEQ ID NO: 12), and Volvariella volvacea peptide YFDWPGFKT (SEQ ID NO: 13).

[0033] In a fourth aspect of the present invention, there is provided a use of the primary taste bud cell line described in the first aspect of the present invention or the taste bud cell model described in the second aspect of the present invention in screening or preparing taste regulating substances.

[0034] In a preferred embodiment, the taste modulating substance comprises a taste peptide and a taste modulating substance selected from the group consisting of salty, sweet, bitter, sour and umami.

[0035] In a preferred embodiment, the flavor peptides include: salty peptides, sweet peptides, bitter peptides, sour peptides, and umami peptides.

[0036] In a preferred embodiment, the salty peptide comprises a salty taste enhancing peptide.

[0037] In the fifth aspect of the present invention, there is provided a use of the primary taste bud cell line described in the first aspect of the present invention or the taste bud cell model described in the second aspect of the present invention in the study of the saltiness-enhancing mechanism of taste peptides.

[0038] In a sixth aspect of the present invention, a method for preparing the primary taste bud cells according to the first aspect of the present invention is provided, comprising the following steps:

[0039] Primary taste bud cells were prepared by digesting and separating hypolingual epithelial cells of non-human mammals using complex enzymes.

[0040] In a preferred embodiment, the complex enzyme comprises: 0.5-10 mg / mL dispase II solution; 0.5-10 mg / mL collagenase I solution and 0.25% trypsin-EDTA solution.

[0041] In a preferred embodiment, the method comprises the following steps: S1: digestion: taking sublingual epithelial tissue of a non-human mammal, digesting it with dispase II solution, collagenase I solution and 0.25% pancreatin-EDTA solution in sequence, sieving and centrifuging to obtain a sublingual epithelial tissue digestion solution;

[0042] S2: using complete culture medium to terminate the digestion of step S1 to obtain a taste bud cell suspension;

[0043] S3: Filter and centrifuge the taste bud cell suspension obtained in step S2 to obtain primary taste bud cells.

[0044] In a preferred embodiment, in step S1, the concentration of the dispase II solution is 0.5-10 mg / mL, preferably 0.5-5 mg / mL, and more preferably 1 mg / mL; the concentration of the collagenase I solution is 0.5-10 mg / mL, preferably 0.5-5 mg / mL, and more preferably 1 mg / mL; the pancreatin-EDTA solution is selected from a pancreatin-EDTA solution with a mass volume ratio of 0.25%.

[0045] In a preferred embodiment, in step S1, before digestion, the sublingual epithelial tissue is first cleaned with iodine tincture and PBS solution, and pre-cooled; then, it is chopped and digested in the dispase II solution for 2-10 minutes, preferably 5 minutes; digested in the collagenase I solution for 50-150 minutes, preferably 120 minutes; digested in the 0.25% trypsin-EDTA solution for 5-20 minutes, preferably 10 minutes; the filtration refers to sieving with 100 and 70 μm cell sieves in sequence; the centrifugation conditions are 100-600g, 5-10 minutes.

[0046] In a preferred embodiment, in step S2, the complete culture medium includes IMDM culture medium, MCDB-153 culture medium, fetal bovine serum (FBS), cell supplement (ITS), and gentamicin.

[0047] In a preferred embodiment, in step S3, the taste bud cell suspension obtained in step S2 is filtered using a 70 μm cell sieve and centrifuged at 100-600 g for 5-10 minutes to obtain primary taste bud cells.

[0048] In a preferred embodiment, the method further comprises step S4: verifying the expression of relevant specific proteins in the taste bud cells to ensure that the obtained primary taste bud cells are taste bud cells.

[0049] In a preferred embodiment, the specific protein expressed in the taste bud cells is selected from the following group: GLAST, α-gustducin, PLC-β2, NCAM and EnaCα.

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0051] Compared with the prior art, the present invention has the following technical effects:

[0052] (1) The present invention establishes a primary rat (or mouse) taste bud cell model suitable for studying the salt-enhancing mechanism of flavor peptides.

[0053] (2) The cell model established in the present invention not only ensures the high activity and stability of taste bud cells, achieves a stable response to taste peptide stimulation and accurately quantified taste signal output, but also further improves the accuracy and depth of the research.

[0054] (3) The cell model established by the present invention highly simulates the physiological environment, which enhances the reliability and physiological relevance of the experimental results; its excellent repeatability and versatility make it possible to widely explore different taste mechanisms and conduct cross-research verification;

[0055] (4) The use of the cell model established by the present invention for research can reduce the reliance on animal experiments, reduce experimental costs and ethical pressure. This innovative model provides strong support for scientific research and product improvement in the food and condiment industry, and is expected to promote the rapid transformation of taste science research into practical application fields, showing broad application prospects and far-reaching industry impact. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is an electron microscope image of taste bud cells in an embodiment of the present invention. Figure 1 A and B in the middle respectively indicate taste bud cells with obvious extension of the surrounding contour and obvious protrusions on the surface (the positions indicated by the arrows). Their morphology is more slender and there are protrusions on the top. C and D respectively indicate taste bud cells with fuller morphology and more protrusions on the top (the positions indicated by the arrows).

[0057] Figure 2 This is a diagram for identifying specific proteins and genes of taste bud cells in the examples of the present invention. Figure A shows the expression of specific proteins in primary hepatocytes and primary taste bud cells (TBC); Figure B detects the levels of specific genes in primary hepatocytes and primary taste bud cells; among them, GLAST marks type I taste cells, α-gustducin and PLC-β2 mark type II taste cells, NCAM marks type III taste cells, EnaCα marks salty taste-related taste cells, and β-actin represents an internal reference.

[0058] Figure 3 The image results of the primary taste bud cells responding to the basic taste substances in the embodiment of the present invention. The primary taste bud cells were stimulated with 200mM sodium chloride solution, 25mg / mL monosodium glutamate solution and 25mg / mL sucrose solution respectively. According to the changes in fluorescence intensity in the image, it can be seen that before the addition of the basic taste substances, i.e. at 0s, the cell fluorescence intensity was low. After the addition of the basic taste substances sodium chloride and monosodium glutamate solutions, the fluorescence intensity reached the highest at about 200s of stimulation, and the fluorescence intensity of the sucrose solution reached the highest at about 50s of stimulation. The results show that the primary taste bud cells are active and respond to the stimulation of the basic taste substance solutions.

[0059] Figure 4 The calcium imaging results of the enhanced sodium chloride response of taste bud cells induced by the mushroom peptides WPGFK, YFDWPGFK and YFDWPGFKT. In the figure, "∆F / F0" indicates the relative fluorescence change.

[0060] Figure 5 The sensory evaluation of the three peptides WPGFK, YFDWPGFK and YFDWPGFKT in sodium chloride solution showed that all three peptides had obvious salt-increasing effects.

[0061] Figure 6 Electronic tongue testing of the straw mushroom peptides WPGFK, YFDWPGFK and YFDWPGFKT in sodium chloride solution showed that all three straw mushroom peptides had significant salt-increasing effects.

[0062] the term

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0065] Flavor peptides

[0066] Flavor peptides refer to oligopeptides with a molecular mass less than 5000u that are extracted from food or synthesized from amino acids and have a certain contribution to food flavor, including characteristic flavor peptides and flavor precursor peptides.

[0067] Flavor peptides exist in various foods such as meat, fruits and vegetables, and are very important for the flavor of both processed and unprocessed foods. Flavor peptides can not only produce characteristic flavors, but also participate in the Maillard reaction as precursors of volatile flavor substances to form special aromatic odors. Therefore, the flavoring effect of various peptides is usually their comprehensive effect. There are five basic taste sensations of flavor peptides: salty, sweet, bitter, sour and umami.

[0068] Salty peptides

[0069] At present, the number of salty peptides reported in research is relatively small, mainly dipeptides, such as Orn-Tau·HCI, Lys-Tau·HC1, Orn-GlyHCI, LyS-Gly·HCI and Orn-β-Ala. These dipeptides, such as y-Glu-Tyr and y-Glu-Phe, do not have multiple taste characteristics such as salty, umami and sour. Salty taste, like sour taste, is mainly caused by cations, which are easily adsorbed by the phosphate groups of taste receptors located on the cell membrane and present a salty taste. The salty peptide taste channel receptor is a lipid that is not adjacent to the protein. Its salty taste is related to the degree of dissociation of its amino group and whether there are corresponding anions and cations. The ionized cations in the salty peptide enter the taste cells through the sodium channels located in the cell membrane to depolarize calcium ions. When the cell is positively charged, a small current is formed, followed by the release of the conduction medium, and the primary afferent neuron excitation is enhanced, giving the brain a "salty" taste. The amiloride molecule (EnaC) channel is a receptor for salty taste, but for humans, the EnaC channel cannot explain our complete sensitivity to salty taste, which indicates that there may be other taste channels that have not yet been confirmed, such as the transient receptor potential vanilloid subtype 1 (TRPV1) channel. In the development of functional foods, the discovery of salty peptides has potential value for special groups of people who need low-sodium foods, such as patients with diabetes and hypertension.

[0070] Salty taste enhancing peptides

[0071] At present, there are few studies on salty taste enhancing peptides, and they are not related to the length of the peptide chain. By observing the structure of salty taste enhancing peptides, it can be found that the long sequence salty taste enhancing peptides reported so far basically include the short peptides reported previously, and Arg, Asp, Glu and Ala residues appear frequently, which indicates that these short peptides or residues may play a role in regulating the taste characteristics of peptides in the composition of salty taste enhancing peptides. Salty taste enhancing peptides are generally derived from natural foods, mainly beans, meat, and fungi. When added with NaCl, they can enhance the perception of saltiness. Compared with the commonly used salt reduction strategies: direct salt reduction and alternative salt reduction, salty taste enhancing peptides can not only enhance the perception of saltiness, but also improve the flavor of the food itself without introducing odor. Unlike some mineral salts that cause hyperkalemia or arrhythmia, salty taste enhancing peptides are healthy, nutritious, easier to absorb and functional.

[0072] Sweet peptides

[0073] At present, the research on sweet peptides is relatively mature. For example, dipeptide derivatives such as aspartame (L-aspartyl-L-phenylalanyl methyl ester, Aspartame) and alitame (L-aspartyl-D-alaninamide, Alitame) have been added to various foods as functional sweeteners due to their high sweetness and low calories. At present, researchers from various countries are actively exploring the development of new oligopeptide sweeteners such as sweet lysine dipeptides (N-Ac-Phe-Lys, N-Ac-Gly-Lys), Monellin, Thaumatin, Pentadin, Curculin, Miraculin, Mabinlin and Brazzien from natural resources such as fruits and vegetables. Sweet taste receptors belong to the first family of taste receptors (TIR) ​​I531 Assadi-Porter et al. (54 Studies have shown that sweet peptides and low molecular weight sugars can produce sweetness because they have the same taste receptors T1R2 and T1R3.

[0074] Bitter peptides

[0075] Bitter peptides include dipeptides, tripeptides, hexapeptides, octapeptides and even peptides composed of dozens of amino acids. They exist in many natural foods and fermented products such as bean cakes, soy sauce, cheese and caviar. In particular, a large number of bitter peptides are produced during the fermentation process. Therefore, the bitterness of fermented products is often related to the bitter peptides formed by protein degradation during the fermentation process. Although bitterness is generally considered a bad flavor, it is a very important flavor characteristic in foods such as beer, coffee or cheese. In addition, some bitter peptides also have certain physiological functions and play a role in preventing chronic diseases.

[0076] Sour peptide

[0077] Sour taste is generally caused by hydrogen ions. Acidic or basic amino acid residues in peptides ionize enough hydrogen ions to enter taste bud cells through TRP ion channels, making peptides sour. It is generally believed that the receptor channel of sour peptides (HA) is phospholipids. The molecular structure of the peptide itself is very important for the sour taste. + It is a flavor base, anion A - It is a flavor enhancer. The sour taste of the peptide is related to the concentration of hydrogen ions and the anion ionization of the sour peptide. + The phospholipid heads of the receptors exchange with each other, and the hydrophobic amino acids in the peptides are adsorbed on the phospholipid membrane, increasing the membrane's affinity for H + affinity, thus causing a sour taste.

[0078] Umami peptide

[0079] The amino acid sequence of umami peptides generally contains hydrophilic amino acids such as Glu or Asp. Umami peptides do not affect other tastes (sour, sweet, bitter, salty), and enhance their respective flavor characteristics. Therefore, they have a good effect in enhancing the flavor of various vegetables, meat, poultry, milk, aquatic products and even wine.

[0080] Umami receptors belong to the G protein-coupled receptor family (GPCRS, T1RI+TIR3). The general structural formula of umami is -O-(C)nO-, n=3~9. Umami molecules need to have a lipid chain equivalent to 3 to 9 carbon atoms in length, and both ends are negatively charged. However, umami taste is strongest when n=4~6. Maintaining the negative charge at both ends of the molecule is crucial for umami taste. The flavor-fixing group of umami peptides is the negatively charged functional group -COOH at both ends of the molecule, and the flavor-enhancing group is α-L-NH 2 , -OH, etc. The umami taste of peptides is mostly due to the presence of acidic or basic amino acids. Studies have shown that the acidic and basic parts of peptides are the main parts that produce umami taste, and it is speculated that cations and anions are in close proximity. Umami molecules must have positively charged molecular groups (represented by AH), negatively charged molecular groups (represented by B) and molecular groups containing hydrophilic residues (represented by X). The three molecular groups are connected to the corresponding receptor positions to make people feel the umami taste. Therefore, from the amino acid sequence of beef spicy peptide, the first two amino acids (Lys-Gly) can be regarded as AH; the middle three amino acids (Asp-Glu-Ser) can be regarded as B; and the three amino acids at the tail end (Ser-Leu-Ala) can be regarded as X. These three molecular groups interact with TIR1+T1R3, which may be the reason why delicious peptides have umami taste. DETAILED DESCRIPTION

[0081] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, all the embodiments described are only partial embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0082] For the numerical ranges in the present invention, each intermediate value between the upper and lower limits of the range shall be included. Each smaller range between any stated value or intermediate value in the stated range, and any other stated value or intermediate value in the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range.

[0083] The raw materials used in the present invention, unless otherwise specified, are conventional commercial products. The methods used in the present invention, unless otherwise specified, are conventional methods in the art. The quality of each substance used in the present invention is the quality of conventional use.

[0084] Cell preservation

[0085] The cell of the present invention is classified and named Mus musculus., and is deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, deposit date: 2024.09.19, deposit number: CCTCC NO: C2024314.

[0086] Calcium ion fluorescent probe

[0087] Fluo-4 AM is one of the most commonly used fluorescent probes for detecting intracellular calcium ion concentration. 51 H 50 F 2 N 2 O 23 , molecular weight is 1096.95. Fluo-4 AM is improved on the basis of Fluo-3 AM, and its characteristics are faster loading and brighter fluorescence under the same conditions. Fluo-4 AM replaces the chlorine atom in the structure of Fluo-3 AM with a fluorine atom, causing its maximum excitation wavelength to deviate by about 12nm to a shorter wavelength. This wavelength is closer to the wavelength of argon laser 488nm, so when excited by argon laser, the fluorescence intensity of Fluo-4 will be twice that of Fluo-3. Since the affinity of Fluo-4 to calcium ions is similar to that of Fluo-3, its use is basically the same as Fluo-3. Fluorescence detection instruments such as laser confocal microscope and fluorescence microplate reader can be used to measure changes in intracellular calcium ion concentration.

[0088] Fluo-4 AM is the acetyl methyl ester derivative of Fluo-4, a fluorescent dye that can penetrate the cell membrane. The fluorescence of Fluo-4 AM is very weak, and its fluorescence does not increase with the increase of calcium ion concentration. After Fluo-4 AM enters the cell, it can be cleaved by the esterase in the cell to form Fluo-4, which is then retained in the cell.

[0089] It can bind to calcium ions and produce strong fluorescence after binding to calcium ions, with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 516nm. The recommended excitation wavelength for actual detection is around 488nm and the emission wavelength is 512-520nm.

[0090] The present invention aims to establish a primary rat (or mouse) taste bud cell model suitable for the study of the salt-enhancing mechanism of flavor peptides by optimizing cell separation and culture conditions. The model will have high cell activity, be able to stably respond to flavor peptide stimulation, and produce quantifiable taste signals. Through this model, the interaction between flavor peptides and taste bud cells can be deeply explored, the salt-enhancing mechanism can be revealed, and a scientific basis can be provided for the development and improvement of food condiments. In order to achieve the above purpose, the present invention provides a primary rat (or mouse) taste bud cell model for the study of the salt-enhancing mechanism of flavor peptides.

[0091] Example 1 Preparation of primary taste bud cells

[0092] The present invention separates and extracts primary taste bud cells, collects cell passages, freezes and extracts cell proteins and RNA, and identifies taste bud cells at the protein and gene levels. Specifically, protein blotting and QPCR experiments can be used for verification to ensure that the taste bud cells are obtained.

[0093] 1.1 Experimental Materials:

[0094] Dispase II solution: purchased from Yeasen, catalog number 40104ES80, used to separate single cells from different tissues or organs;

[0095] Collagenase I solution: purchased from Worthington, product number LS004196.

[0096] 1.2 Experimental steps:

[0097] S1.1: The iodine tincture, PBS and other solutions used to obtain tongue epithelium need to be pre-cooled, and the entire process should be performed on ice unless otherwise specified.

[0098] S1.2: When isolating the tongue epithelium of mice (or rats), digest it with 1 mg / mL dispase II solution for 5 minutes and then take the tongue epithelium; cut the tongue epithelium into pieces and centrifuge it, then add 1 mg / mL collagenase I solution and digest it for 120 minutes; sieve it with a 100μm cell sieve and centrifuge it, then add 0.25% trypsin-EDTA solution and digest it for 10 minutes; sieve it with a 70μm cell sieve and centrifuge it; the centrifugation conditions are 100-600g, 5-10 minutes.

[0099] S1.3: After digestion, the digestion was terminated with an equal amount of complete culture medium, wherein the complete culture medium components include IMDM culture medium (purchased from Adamas life, product number C8062), FBS (purchased from sigma, product number 12003C), MCDB-153 culture medium (purchased from Vivacell, product number C3170-0500), penicillin / streptomycin (P / S) (purchased from Adamas life, product number C8021), ITS (purchased from Adamas life, product number C8180; ITS is a cell supplement containing human recombinant insulin, human transferrin and sodium selenite), gentamicin (purchased from Bioss, product number C7011); after terminating the digestion, sieve with a 70μm cell sieve; after centrifugation, primary taste bud cells were obtained.

[0100] The morphology of taste bud cells after culture Figure 1 As shown, Figure 1 A and B in the middle respectively represent taste bud cells with obvious extension of the surrounding contour and protrusions on the surface, which are more slender in shape and have protrusions on the top. Figure 1 C and D in the middle represent taste bud cells with fuller morphology and more apical protrusions.

[0101] S1.4: Use Western blotting and QPCR experiments to verify the primary taste bud cells obtained in step S3 to ensure that the obtained cells are taste bud cells; the specific steps are as follows:

[0102] (1) Western blotting reaction specifically includes:

[0103] The mouse taste bud cells and primary hepatocytes prepared above were inoculated on culture dishes, cultured in an incubator for 3-5 days, and washed 2-3 times with PBS.

[0104] Add cell lysis buffer to the culture dish, collect the cell lysis buffer into a 1.5 mL microcentrifuge tube, sonicate for 30 min, and centrifuge at 12,000 rpm for 15 min. Take the supernatant and continue the experiment or store at -80°C. Avoid repeated freezing and thawing.

[0105] Use BCA kit to measure protein concentration, immediately add 6× loading buffer for quantification, boil at 95℃ for 10min, slowly return to room temperature, centrifuge slightly, and store at -80℃.

[0106] After assembling the separation gel and the concentrated gel into an electrophoresis device, add the electrophoresis buffer (TGS). Pipette an appropriate amount of sample or molecular weight standard Marker and slowly add it to the gel well. After completion, cover the electrophoresis tank lid, turn on the power, and stop the electrophoresis when the Maker reaches the bottom of the separation gel.

[0107] Take the glass plate out of the electrophoresis tank, place the PVDF membrane, blotting filter paper and sponge pad in order, and finally close the transfer clip, close the lock, put the assembled clip into the transfer electrophoresis tank, add pre-cooled 1× transfer buffer (TG), put in ice cubes, cover the lid, and transfer the membrane. After the transfer, take out the PVDF membrane, block it for 1 hour, immerse the membrane in the primary antibody working solution of the specific protein expressed by taste bud cells, incubate it overnight at 4℃, and incubate it with the secondary antibody working solution the next day. Add the prepared chemiluminescent solution to the PVDF membrane and gently drag it to make it completely soaked, and develop it with an electronic film imager.

[0108] The results are as follows Figure 2 As shown, from the bands in the Western blotting experiment, it can be seen that at the protein level, taste bud cells express relevant specific proteins, while primary hepatocytes do not contain the specific proteins; the relevant specific proteins expressed by taste bud cells include: GLAST, used to mark type I taste cells; α-gustducin and PLC-β2, used to mark type II taste cells; NCAM, used to mark type III taste cells; EnaCα, used to mark salty taste-related taste cells.

[0109] (2) QPCR reaction specifically includes:

[0110] The mouse taste bud cells and primary stem cells prepared above were inoculated on culture dishes, respectively, and cultured in an incubator for 3-5 days. TRIzol reagent was added at room temperature, and the cells were transferred to a 1.5 mL RNase-free test tube and incubated for 15 minutes.

[0111] Then add chloroform, shake vigorously for 10 seconds, react at room temperature for 3 minutes, centrifuge at 4°C, 12,000 rpm for 10 minutes, take the supernatant and transfer it to a new 1.5 mL RNase-free tube.

[0112] Add the same volume of isopropanol, mix gently, and incubate at room temperature for 10 minutes. Then centrifuge at 4°C, 12,000 rpm for 10 minutes. Discard the supernatant, add 800uL of 75% ethanol, mix gently, and centrifuge at 4°C, 12,000 rpm for 5 minutes. Discard the supernatant and air dry in a fume hood.

[0113] Dissolve all RNA precipitates in 20 µL enzyme-free water and continue the experiment or store at -80°C.

[0114] According to the instructions of the cDNA reverse transcription kit, total RNA was reverse transcribed into cDNA. Prepare the primer mixture, mix gently and add the calculated volume to the well plate, seal and centrifuge, place in the prepared thermal cycler and wait for total RNA to be reverse transcribed into cDNA, and store at -20°C until use.

[0115] The expression of selected genes was relatively quantified using a qPCR reaction system.

[0116] The primer sequences used are shown in Table 1 below:

[0117] Table 1

[0118]

[0119] The results are as follows Figure 2 As shown, from the relative mRNA expression levels of specific genes expressed in taste bud cells in the QPCR experiment, it can be seen that the relative RNA expression levels of specific genes related to taste bud cells are higher than those of primary hepatocytes. Therefore, the protein blotting and QPCR results verified that the primary taste bud cells isolated by the method described in this example expressed taste bud cell-specific proteins and genes, confirming that these primary taste bud cells are taste bud cells.

[0120] Example 2 Basic taste test

[0121] The taste bud cells obtained in Example 1 were subjected to a calcium imaging experiment with basic taste substances to test the cell's response to basic taste substances. This experiment was conducted by introducing a calcium ion fluorescent probe and observing changes in intracellular calcium levels under a confocal microscope. The specific process is as follows:

[0122] S2.1: Cell culture: Culture taste bud cells in confocal microplates until the growth density reaches 40-50%.

[0123] S2.2: Incubate the cells with fluorescent probe (Fluo-4 AM, purchased from Biotech, catalog number S1060) for 30-60 minutes, specifically including: incubating the taste bud cells with a solution containing 0.5-5μM Fluo-4 AM at 37°C for 30-60 minutes. After incubation, wash 3-5 times and continue culturing at 37°C for 30-60 minutes to ensure that Fluo-4 AM is fully converted into Fluo-4 in the cells.

[0124] S2.3: Observe the cells under a confocal microscope, and then add basic taste substances (such as 200 mM sodium chloride, 25 mg / mL monosodium glutamate or 25 mg / mL sucrose) for stimulation for 0s-200s;

[0125] S2.4: Observe and record the changes in fluorescence intensity of taste bud cells as the stimulation time increases.

[0126] The calculation formula for calcium imaging experiment is shown in Formula I:

[0127] Formula I

[0128] in," ” refers to the relative fluorescence ratio, and F refers to the actual fluorescence value, that is, the fluorescence value of the selected area after deducting the background. It refers to the fluorescence value "F" in the early stable stage.

[0129] The results are as follows Figure 3 As shown, taste bud cells were stimulated with 200 mM sodium chloride, 25 mg / mL monosodium glutamate or 25 mg / mL sucrose respectively. According to the changes in fluorescence intensity in the images, it can be seen that before the addition of basic taste substances, i.e. at 0 s, the cell fluorescence intensity was low; after the addition of basic taste substances sodium chloride and monosodium glutamate solution at around 200 s of stimulation, the fluorescence intensity reached the highest; the fluorescence intensity of the sucrose solution reached the highest at around 50 s of stimulation.

[0130] The results showed that primary taste bud cells were active and responded to stimulation by basic taste substance solutions; the changes in the fluorescence intensity of the images indicated that basic taste substances stimulated the taste bud cells, causing an intracellular calcium signal response, which means that the taste bud cells were active and responsive to basic taste substances.

[0131] Example 3 Peptide solution test

[0132] This example uses the above taste bud cells to study the saltiness-enhancing mechanism of flavor peptides: In order to study whether the mushroom peptides WPGFK (Trp-pro-gly-phe-lys, ≥98%, sequence number SEQ ID NO.11), YFDWPGFK (tyr-thr-asp-trp-pro-gly-phe-lys, ≥98%, sequence number SEQ ID NO.12), and YFDWPGFKT (tyr-thr-asp-trp-pro-gly-phe-lys-thr, ≥98%, sequence number SEQ ID NO.13) can enhance the cell sodium chloride reaction, that is, achieve the saltiness-enhancing effect, this experiment uses calcium imaging experiments, uses confocal microscopy to observe the fluorescence intensity of calcium in taste bud cells, and analyzes the relative fluorescence changes to explore the saltiness-enhancing effect of the above mushroom peptides. The specific experimental process is as follows:

[0133] S3.1: Taste bud cell culture, culture the cells in a confocal microplate until the growth density reaches 40-50%;

[0134] S3.2: Incubate the cells with fluorescent probe for 30-60 minutes, specifically including: incubate the calcium ion fluorescent probe at 37°C for 30-60 minutes, wash 3-5 times after incubation, continue culturing at 37°C for 30-60 minutes, and continue incubating for 30-60 minutes after washing.

[0135] S3.3 The cells were observed under a confocal microscope, with ultrapure water and 100 mM sodium chloride as the control group, and then 100 μM of the mushroom peptide WPGFK, YFDWPGFK, and YFDWPGFKT were added for stimulation;

[0136] S3.4 The cells were placed under a confocal microscope for observation, and a mixture containing 100 μM of WPGFK, YFDWPGFK, YFDWPGFKT and 100 mM sodium chloride was added for stimulation;

[0137] S3.5 Observe and record the changes in fluorescence intensity generated by the cells.

[0138] The results are as follows Figure 4 As shown, the relative fluorescence changes of taste bud cells were statistically analyzed. The results showed that 100 μM straw mushroom peptides WPGFK, YFDWPGFK, and YFDWPGFKT did not significantly cause a response in taste bud cells. A mixture containing 100 μM straw mushroom peptides WPGFK, YFDWPGFK or YFDWPGFKT and 100 mM sodium chloride could cause a reaction significantly greater than that of 100 mM sodium chloride solution. The results show that straw mushroom peptides WPGFK, YFDWPGFKT and YFDWPGFKT themselves have no salty taste, but have a significant salt-increasing effect, that is, straw mushroom peptides WPGFK, YFDWPGFK and YFDWPGFKT have a significant salt-increasing effect, and it can be seen from the figure that straw mushroom peptide WPGFK has the best salt-increasing effect.

[0139] Example 4 Sensory Evaluation

[0140] A sensory panel of 12 people (6 males and 6 females, aged 20-30 years) was selected to participate in the sensory evaluation experiment of this study. The experiment began after the panel members were screened and trained. The sensory evaluation experiment was conducted in a standard sensory laboratory (Sensory Laboratory of Shanghai University of Technology). Sucrose (50.00, 10.00 g / L), citric acid (5.00, 1.00 g / L), sodium chloride (15.00, 3.00 g / L), sodium glutamate (20.00, 4.00 g / L), and quinine (6.00, 1.00 g / L) were used as standard solutions. The judges were asked to score the intensity of the five basic tastes to screen whether the evaluators could correctly judge the intensity of the taste solutions.

[0141] After that, the evaluators received sensory training for two weeks, three times a week, 2 hours each time. A 100mM NaCl solution and a series of NaCl solutions containing 100μM mushroom peptides were prepared. At room temperature (20-25°C), the sensory evaluators took turns tasting the samples and scored the saltiness of the samples on a scale of 1 to 10 (1 for the weakest saltiness and 10 for the strongest saltiness). Each sample was evaluated three times. To avoid taste fatigue and carryover effects, the evaluators were given a reasonable rest time of 2 minutes between tasting different samples and rinsed their mouths with 50-60 mL of purified water.

[0142] The results are as follows Figure 5 As shown, the sensory scores of 100 mM sodium chloride solution and sodium chloride mixture containing 100 μM Volvariella peptide were statistically analyzed. The results showed that the sodium chloride mixture containing 100 μM Volvariella peptide was saltier than the 100 mM sodium chloride solution, which is similar to the calcium signal response results of primary taste bud cells provided by the present invention.

[0143] Example 5 Electronic tongue detection

[0144] The taste characteristics of the mushroom peptide were determined using the INSENT SA-402B electronic tongue system (SA-402B, Kanagawa Intelligent Sensor Technology Co., Ltd., Japan). Before the experiment, the electronic tongue instrument was activated, initialized, calibrated, and debugged. When the experiment started at an ambient temperature of about 25°C, a 100mM NaCl solution and a series of NaCl solutions containing 100μM mushroom peptide were prepared. The liquid to be tested was poured into the special beaker of the electronic tongue instrument and placed on the electronic tongue instrument automatic sampler alternately with the cleaning solution. The test conditions were set to clean for 5 minutes, test the sample for 30 seconds, measure the aftertaste for 30 seconds, and repeat the test 4 times.

[0145] The results are as follows Figure 6 As shown, the electronic tongue values ​​of 100mM NaCl solution and a series of NaCl solutions containing 100μM Volvariella peptides were observed. The data showed that the Volvariella peptides all had obvious salty taste enhancement effects, among which the Volvariella peptide WPGFK had the best salty taste enhancement effect. The salty taste enhancement effects of Volvariella peptides YFDWPGFK and YFDWPGFKT were also very obvious, but slightly worse than that of Volvariella peptide YFDWPGFKT. The electronic tongue detection results were consistent with the calcium signal response results of the primary taste bud cells provided by the present invention.

[0146] In summary, the present invention provides a primary rat (or mouse) taste bud cell model for the study of the salt-enhancing mechanism of flavor peptides, which can make up for the limitations of traditional research methods, become a driving force for in-depth exploration of the salt-enhancing mechanism of flavor peptides, and lay a solid scientific foundation for the innovative research and development of condiments in the food industry. With this novel model, researchers can more deeply analyze the complex interactions between flavor peptides and taste bud cells, thereby laying a solid scientific foundation for optimizing food flavor characteristics and improving product quality.

[0147] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A primary taste bud cell line, characterized in that: The cell line is a primary taste bud cell TBC that is sensitive to taste regulating substances and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024314.

2. The primary taste bud cell line according to claim 1, characterized in that The taste-modulating substances include flavor peptides and taste-modulating substances selected from the group consisting of salty, sweet, bitter, sour and umami.

3. The primary taste bud cell line according to claim 1, characterized in that The flavor peptides include salty taste enhancing peptides, and the salty taste enhancing peptides are selected from the following group: Volvariella volvacea peptide WPGFK (SEQ ID NO: 11), Volvariella volvacea peptide YFDWPGFK (SEQ ID NO: 12), and Volvariella volvacea peptide YFDWPGFKT (SEQ ID NO: 13).

4. A method for constructing a taste bud cell model, characterized in that: The following steps are involved: (1) Providing a cell fluorescent probe; (2) The cell fluorescent probe is co-incubated with the primary taste bud cell line as described in claim 1 to obtain taste bud cells containing the cell fluorescent probe, namely, a taste bud cell model.

5. A method for screening or evaluating salty taste enhancing peptides, characterized in that: The following steps are involved: (1) Providing the flavor peptide to be tested; (2) Stimulating the taste bud cell model described in claim 4 with sodium chloride as a control group of cells; (3) stimulating the control group cells with the flavor peptide to be tested to serve as the experimental group cells; (4) observing the changes in fluorescence intensity of the control group cells in step (2) and the experimental group cells in step (3) as the stimulation time increases under a confocal microscope; If the fluorescence intensity F produced by the experimental group cells is compared with the fluorescence intensity F0 produced by the control group cells, and the ratio F / F0 is ≥0.5 (preferably ≥1, more preferably ≥3), the taste peptide to be tested is considered to be a salty taste enhancing peptide.

6. Use of the primary taste bud cell line according to claim 1 or the taste bud cell model according to claim 4 in screening or preparing taste regulating substances.

7. Use of the primary taste bud cell line according to claim 1 or the taste bud cell model according to claim 4 in the study of the saltiness-enhancing mechanism of taste peptides.

8. A method for preparing primary taste bud cells as claimed in claim 1, characterized in that: The steps include: Primary taste bud cells were prepared by digesting and separating hypolingual epithelial cells of non-human mammals using complex enzymes.

9. The preparation method according to claim 8, characterized in that: The complex enzyme comprises: 0.5-10 mg / mL dispase II solution; 0.5-10 mg / mL collagenase I solution and 0.25% pancreatin-EDTA solution.

10. The preparation method according to claim 8, characterized in that: The steps include: S1: Digestion: Take the sublingual skin tissue of non-human mammals, digest it with dispase II solution, collagenase I solution and 0.25% trypsin-EDTA solution in sequence, sieve and centrifuge to obtain the sublingual skin tissue digestion fluid; S2: using complete culture medium to terminate the digestion of step S1 to obtain a taste bud cell suspension; S3: Filter and centrifuge the taste bud cell suspension obtained in step S2 to obtain primary taste bud cells.

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