JP2014158502A - Screening method of richness giving material, richness giving material, and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel screening method of richness giving material giving excellent richness with a small amount of the material, a richness giving material giving excellent richness with a small amount, an evaluation method of drinks and foods using the richness giving material as an indicator, and a detection kit of the richness giving material.SOLUTION: The screening method of the richness giving material is characterized by using an antibody against a carboxyalkyl amino acid and using reactivity against the antibody as an indicator.

Description

  The present invention relates to a screening method for a body taste imparting substance, a body taste imparting substance, a method for evaluating a food or drink or a manufacturing process management method, and a kit for detecting a body taste imparting substance.

  In the food and drink field, taste substances have been used for a long time. In particular, in addition to sweet taste, salty taste, sour taste, and bitter taste, a substance having five basic tastes in which a glutamate receptor (mGluR4) was found in a sensory cell in the taste bud of the tongue in 2000, or a substance that enhances these substances Widely used as a seasoning. Furthermore, there is “kokumi” as an important taste that cannot be expressed above, which is a combination of taste strength (drinking response), taste spread (thickness), and temporal change in taste (resonance).

  Conventionally, several substances for imparting a rich taste have been reported, such as glutathione derivatives, heated products of gelatin and tropomyosin, sulfone group-containing compounds, and peptide substances. In particular, as a method for enhancing the body taste, a peptide-like one is characterized in that an amino-carbonyl reaction product of a peptide having a molecular weight of 1000 to 5000 and a carbonyl compound is added to the food or drink. Is disclosed (Patent Document 1). It is also disclosed that a Maillard reaction product having a sugar chain prepared from a fraction of 1000 Da to 5000 Da has a rich taste imparting action (Non-patent Document 1).

  In addition, as an active ingredient, a seasoning having a rich taste imparting function containing a glycopeptide formed by binding a peptide with a sugar chain having a molecular weight of 1,000 to 30,000 is disclosed (Patent Document 2). However, there are only a few examples, and a satisfactory body taste imparting substance has not yet been obtained.

  Under such circumstances, development of a substance having a more excellent kokumi imparting action is desired, and therefore, a method capable of screening a kokumi imparting substance simply and with high sensitivity is desired. For example, a screening method (Patent Document 3) using calcium receptor activity as an index is disclosed. This evaluation index is an index related to biological function / disease pathogenesis basically performed in relation to calcium homeostasis. It is not enough as an indicator of “kokumi”. That is, it can be said that the lack of a good index of a component that contributes to taste or richness leads to difficulty in developing a richness imparting substance. Therefore, it is also necessary to provide a screening method for a body taste imparting substance that can present an excellent index.

Patent No. 3623753 International Publication WO2006 / 104022 Special table 2009-514791

Food Chemistry 99 (2006) 600-604

  An object of the present invention is to provide a novel screening method for a kokumi imparting substance that exhibits an excellent kokumi imparting action in a small amount, and a kokumi imparting substance that exhibits an excellent kokumi imparting action in a small quantity. Furthermore, it is another object of the present invention to provide a food / beverage product evaluation method and a production process management method using a body taste imparting substance as an index, and a kit for detecting a body taste imparting substance.

As a result of diligent research to solve the above-mentioned problems, the present inventors have found that an amino-carbonyl reaction (also referred to as a saccharification reaction or a Maillard reaction) of a side chain amino group in a protein or peptide is generated. -A glycation end product AGE (Advanced Glycation End Products) which is a carbonyl reaction product, more preferably a substance reactive with an antibody against carboxyalkyl amino acid (binding to the antibody) has an excellent body taste imparting action. I found it. Moreover, it discovered that the peptide of the molecular weight 10-20 kDa derived from wort boiling liquid has especially strong kokumi imparting effect | action among the substances reactive with the antibody with respect to a carboxyalkyl amino acid. Although described in the background art, the seasoning disclosed in International Publication WO2006 / 104022 contains a glycopeptide (molecular weight of 1,000 to 30,000), but is rich in taste obtained by the screening method of the present invention. Since the imparting substance is a terminal glycation product AGE that is an amino-carbonyl reaction product generated by an amino-carbonyl reaction (also referred to as a saccharification reaction or a Maillard reaction) between a side chain amino group in a protein or peptide and a carbonyl compound, It is a body taste imparting substance that is completely different from the glycopeptide in terms of structure. In addition, by adding the body taste imparting substance obtained by the screening method of the present invention to a food or drink, the body taste can be imparted to the food or drink, or the body taste of the food or drink can be enhanced. As an index, it discovered that quantitative evaluation, such as the rich taste of food-drinks, and the manufacturing process management of food-drinks could be performed.
The present inventors have further studied based on these findings, and have completed the present invention.
That is, the present invention includes the following inventions.

(1) A screening method for a body taste imparting substance, characterized by using an antibody against a carboxyalkyl amino acid and using the reactivity to the antibody as an index.
(2) The screening method according to (1), wherein the carboxyalkyl amino acid is carboxymethyllysine or carboxyethyllysine.
(3) The screening method according to (1) or (2) above, wherein the body taste imparting substance enhances at least one of sweet taste, salty taste, sour taste, bitter taste and umami taste.
(4) (a) contacting the antibody with the test substance, (b) detecting the binding of the antibody to the test substance, and (c) selecting the test substance bound to the antibody as a candidate substance. The method according to any one of (1) to (3), which is included in this order.
(5) The method according to any one of (1) to (4), wherein the antibody is a monoclonal antibody.
(6) The method according to any one of (1) to (5), wherein the test substance is derived from wort.
(7) A body taste imparting substance obtained by the method according to any one of (1) to (6).
(8) The richness imparting substance according to (7), which is derived from a malt boiling solution.
(9) The rich taste imparting substance according to (7) or (8), which contains carboxymethyllysine and is a peptide having a molecular weight of 10 to 20 kDa.
(10) A method for evaluating a food or drink or a manufacturing process management method using the body taste imparting substance according to any one of (7) to (9) as an index.
(11) The method according to (10), including a step of measuring the amount of the body taste imparting substance according to any one of (7) to (9).
(12) A kit for detecting a body taste imparting substance comprising an antibody against a carboxyalkyl amino acid for performing the screening method according to any one of (1) to (5).

  According to the present invention, a kokumi imparting substance that exhibits an excellent kokumi imparting action in a small amount can be effectively screened. Moreover, since the body taste imparting substance obtained by the screening method of the present invention has an excellent body taste imparting action, it is possible to effectively enhance the body taste of food and drink only by using a small amount of the substance. it can. Furthermore, by using the body taste imparting substance as an index, it is possible to effectively evaluate the quality and the like of the food and drink, and manage the food and drink in the manufacturing process.

FIG. 1 is a diagram showing the results of an indirect competitive ELISA in which wort and AGE antibody (CML antibody) are reacted. FIG. 2A is a diagram showing the results of analysis by SDS-PAGE of proteins contained in wort before and after boiling, and FIG. 2B shows AGE products (proteins subjected to Maillard reaction) contained in wort before and after boiling. It is a figure which shows the result detected by Western blotting. FIG. 3 is a diagram showing the dry matter weight in each fraction when proteins contained in boiled wort are separated by gel filtration. FIG. 4 is a diagram showing the amount of AGE product (protein subjected to Maillard reaction) in each fraction when proteins contained in boiled wort are separated by gel filtration. FIG. 5 is a diagram showing the results of a sensory test performed on each fraction when proteins contained in boiled wort are separated by gel filtration. FIG. 6 is a diagram showing the results of an indirect competitive ELISA in which each wine is reacted with an AGE antibody (CML antibody). FIG. 7 is a diagram showing the results of an indirect competitive ELISA in which each beer is reacted with an AGE antibody (CML antibody). FIG. 8 is a diagram showing the results of indirect competitive ELISA in which each miso extract and AGE antibody (CML antibody) were reacted. FIG. 9 is a diagram showing the results of indirect competition ELISA in which each sake and AGE antibody (CML antibody) are reacted. FIG. 10 is a diagram showing the results of an indirect competitive ELISA in which each cheese extract was reacted with an AGE antibody (CML antibody).

  The screening method for a body taste imparting substance of the present invention uses an antibody against an amino-carbonyl reaction product of a protein or peptide (a protein subjected to Maillard reaction), and is a carboxyalkyl amino acid (hereinafter also referred to as CM-modified amino acid), pentosidine. An antibody against AGE such as is used, and the reactivity to the antibody is used as an index. Preferably, an antibody against a carboxyalkyl amino acid is used, and the reactivity with the antibody is used as an index.

As the carboxyalkyl amino acid, preferably, the alkyl part of the carboxyalkyl amino acid is a lower alkyl having 1 to 4 carbon atoms, more preferably methyl or ethyl, and particularly preferably methyl. is there. The amino acid part of the carboxyalkyl amino acid is an amino acid having an amino group in the side chain, and lysine, arginine, asparagine, or glutamine is preferable. Particularly preferred is lysine.
For example, in a carboxyalkyl amino acid, carboxymethyl lysine (hereinafter also referred to as CML) is lysine (N-ε-carboxymethyl lysine) in which a side chain amino group is carboxymethylated (hereinafter also referred to as CM), Preferred as the carboxyalkyl amino acid in the present invention. Similarly, carboxyethyl lysine (hereinafter also referred to as CEL) in which the alkyl portion is ethyl, carboxymethyl arginine (hereinafter also referred to as CMA) in which the amino acid portion is arginine are suitable.

  The antibody in the present invention is preferably an antibody against carboxymethyllysine or carboxyethyllysine, and more preferably an antibody against carboxymethyllysine.

  The antibody against the above carboxyalkyl amino acid reacts with a free carboxyalkyl amino acid and is a carboxyalkylated amino acid that constitutes a protein or peptide and has an amino group in the side chain. Preferably, the antibody is also reactive with carboxyalkyl amino acids present in the form. That is, the antibody used in the present invention is an antibody having reactivity (binding) with a free carboxyalkyl amino acid, a protein containing a carboxyalkyl amino acid, and a peptide containing a carboxyalkyl amino acid. Among them, as the antibody against the carboxyalkyl amino acid used in the present invention, an antibody having reactivity (binding) with a protein containing carboxyalkyl amino acid and a peptide containing carboxyalkyl amino acid is more preferable.

  In the present invention, a protein containing a carboxyalkyl amino acid and a peptide containing a carboxyalkyl amino acid usually have an amino-carbonyl reaction (glycation reaction or Maillard reaction) between a side chain amino group such as a lysine residue in the protein or peptide and a carbonyl compound. (Also referred to as an amino-carbonyl reaction product) and is a kind of advanced glycation end product (AGE). A peptide containing a carboxyalkyl amino acid may contain two or more amino acids.

The antibody used in the method of the present invention may be a whole antibody molecule or a fragment thereof (for example, Fab or F (ab ′), which can bind to antigen carboxyalkyl amino acids, proteins containing carboxyalkyl amino acids, and peptides containing carboxyalkyl amino acids. ) ₂ fragments), which may be polyclonal antibodies or monoclonal antibodies. Preferably, it is a monoclonal antibody. Polyclonal and monoclonal antibodies can be produced by any of a variety of methods. Methods for producing such antibodies are well known in the art (see, eg, Sambrook, J et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)).

Commercially available monoclonal antibodies and polyclonal antibodies used in the method of the present invention can be purchased. For example, the AGE antibody supplied from the transgenic company is mentioned. It can also be produced using a protein containing a carboxyalkyl amino acid or a peptide containing a carboxyalkyl amino acid as an antigen. For example, antibodies against carboxymethyl amino acids are disclosed in JP 2000-219700, JP 2003-160599, JP 4012722, JP 2007-277263, JP 2007-326870, JP It can be produced according to the method described in, for example, 2009-96731. Antibodies against carboxyethyl amino acids can be produced, for example, according to the methods described in JP 2008-266271 A, JP 2000-7700 A, and the like.
Hereinafter, an example of a specific method for producing an antibody will be described by taking an antibody against a CM amino acid such as carboxymethyllysine (CML) as an example.

1. Preparation of CMized protein, CMized peptide and CMated amino acid The protein to be CMized may be a complex protein, a simple protein, a glycoprotein, a lipoprotein, or the like. These proteins, such as albumin (BSA, etc.), hemoglobin, keyhole limpet hemocyanin (KLH), human serum albumin (HSA), ribonuclease (RNase), beta ₂ microglobulin, histone, collagen, blood cell membrane proteins, or Examples thereof include low density or high density lipoprotein. In addition to proteins, peptides such as oligopeptides and polypeptides can also be used, and those synthesized by modification or degradation from proteins can also be used. For example, when an antibody against CML is produced, a protein, peptide, or lysine containing lysine is used as a CM.

In order to convert the side chain amino group in the above protein or peptide into CM, several tens mg / mL (for example, 5 to 50 mg / mL) of protein such as BSA and tens of mg / mL (for example, 10 to 100 mg / mL) are used. A method in which glyoxylic acid is reacted in a phosphate buffer solution (containing NaCNBH _{3 in} a molar amount 5 times that of glyoxylic acid, pH 7.4) at room temperature for about 24 hours is preferable. The CM protein obtained by the above method is purified by dialysis, liquid column chromatography, or the like, and then subjected to production of a polyclonal antibody or a monoclonal antibody.

  The CMized peptide and the CMated amino acid can be obtained by subjecting the CMized protein obtained as described above to a hydrolysis treatment with a protease such as trypsin, pepsin, papain, aprotinin or leupeptin, or an acid such as hydrochloric acid or sulfuric acid. For example, several tens of μL (for example, 10 to 50 μL) of the above-mentioned CM protein of tens of mg / mL (for example, 5 to 70 mg / mL) and tens of μL of trypsin solution of several tens of mg / mL (for example, 10 to 80 mg / mL) ( For example, 20 to 50 μL) is mixed and reacted in a 37 ° C. water bath for 3 hours. The reaction is stopped with 5 mM AEBSF, whereby the C-merized protein can be enzymatically treated to obtain a C-merized peptide and a C-merized amino acid. The trypsin hydrolysis reaction can be stopped by adding a trypsin inhibitor (for example, 4- (2-Aminoethyl) benzenesulfonylfluoride, HCL (AEBSF)) to the mixed solution.

2. Preparation method of antibody against CM protein (1) Preparation of monoclonal antibody against CM protein (i) Collection of antibody-producing cells Using the CM protein, CM peptide or CM peptide prepared as described above as an antigen Administration to animals such as rats, mice, rabbits and the like. The dose of the antigen per animal is 0.1 to 10 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and for example, immunization is preferably performed at intervals of several days to several weeks, more preferably at intervals of 2 to 5 weeks, preferably 1 to 10 times, more preferably 2 to 5 times. Then, antibody-producing cells are collected preferably 1 to 60 days later, more preferably 1 to 14 days after the final immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

(Ii) Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas. As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include P3X63-Ag. 8). Examples include mouse myeloma cell lines such as U1 (P3U1) and NS-I.

Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is preferably performed at about 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and about 2 × 10 ⁵ to about 1 × 10 ⁶ to 1 × 10 ⁷ cells / mL in animal cell culture media such as serum-free DMEM and RPMI-1640 medium. 2 × 10 ⁶ cells / mL myeloma cells are mixed (a cell ratio of antibody-producing cells to myeloma cells is preferably about 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of about 1000 to 6000 daltons (Da) can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(Iii) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As a method for this, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 3 × 10 ⁵ cells / well, a selection medium is added to each well, and thereafter The culture is carried out with the selective medium changed. As a result, cells that grow from about 10 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, the culture supernatant of the hybridoma that has grown is screened for the presence of antibodies that react with the CM protein. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay, radioimmunoassay or the like.

  Specifically, a hybridoma that is a cell that produces a monoclonal antibody that reacts with a plurality of types of CM-modified proteins by ELISA, etc., reacts with the former and does not react with the latter, using a plurality of types of non-CM-converted proteins as antigens. Establish. Cloning of fused cells is performed by the limiting dilution method or the like. Finally, a hybridoma that is a cell producing a monoclonal antibody that reacts with a plurality of types of CM proteins but does not react with non-CM proteins is established.

(Iv) Collection of monoclonal antibody As a method of collecting a monoclonal antibody from an established hybridoma, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the obtained hybridoma is cultured in a serum-free animal cell culture medium for 7 to 14 days under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration), and an antibody is obtained from the culture supernatant. To get.

In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

  In the present invention, a monoclonal antibody obtained by the above method can be suitably used. For example, as an antibody against CML, Mouse-Mouse hybridoma NF-1G (Independent Administrative Institution National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Japan), which is described in Patent No. 4012722 and Example 1 of JP-A-2003-160599, is disclosed. No. 1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture) was deposited as FERM P-18589 on November 6, 2001); Mouse-Mouse described in Example 1 of JP-A-2000-219700 hybridoma CMS-10 (incorporated administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center (1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan) deposited as FERM P-17153 on January 20, 1999 Prefer monoclonal antibodies produced by hybridomas such as It can be used for. More preferably, Mouse-Mouse hybridoma NF-1G (Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-1 1-1 Higashi, Tsukuba, Ibaraki, Japan) (Deposited as -18589). As an antibody against CEL, for example, the hybridoma KNH-30 (name: “Mouse-Mouse hybridoma KNH-30” described in Example 1 of JP-A-2000-7700, patented organism of the National Institute of Advanced Industrial Science and Technology) Monoclonal antibodies produced by the Deposit Center (deposited as FERM P-16842 on June 16, 1998) at Tsukuba City East 1-1-1 Tsukuba, Ibaraki, Japan, etc. can be suitably used.

(2) Preparation of polyclonal antibody against CM protein: The CM protein or peptide is used as an antigen and administered to a mammal such as a rat, mouse, rabbit or the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, or the like. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. And 6 to 60 days after the last immunization, the antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA), etc. On the day when the maximum antibody titer is shown, blood is collected to obtain antiserum.

  Thereafter, CM-modified proteins (such as BSA) are used, and the reactivity of polyclonal antibodies in the antiserum against these proteins is measured by ELISA or the like. By collecting fractions that react with the CM protein and do not react with the non-CM protein, a polyclonal antibody that reacts only with the CM protein can be obtained.

  In the above-described antibody production method, for example, when carboxyethyl (CE) protein, CE peptide, or CE amino acid is used instead of CM protein, an antibody against carboxyethyl amino acid can be produced.

  In the present invention, an antibody against the carboxyalkyl amino acid is used, and a body taste imparting substance is screened using the reactivity to the antibody as an index. The reactivity with the antibody usually means binding with the antibody. The method of the present invention is suitable, for example, for screening for a body taste imparting substance that enhances at least one of taste intensity (drinking response), taste spread (thickness), and temporal change in taste (resonance). In addition, when at least one of the strength of the taste (drinking response), the spread of the taste (thickness), and the change in taste over time (the reverberation) is enhanced in foods and beverages, etc. Alternatively, since two or more of these are enhanced, the method of the present invention is also suitable for screening for a body taste imparting substance that enhances at least one of sweet taste, salty taste, sour taste, bitter taste and umami taste.

  The method of the present invention includes, for example, (a) contacting an antibody against a carboxyalkyl amino acid with a test substance, (b) detecting the binding of the antibody to the test substance, and (c) a test bound to the antibody. It is preferable to include the process of selecting a substance as a candidate substance in this order. The method of the present invention may further include a step (d) of evaluating the body taste imparting effect of the selected candidate substance.

  The contact (reaction) between the antibody and the test substance in the step (a) is usually performed in a solution. In the step (a), the conditions for reacting the antibody and the test substance in the solution are not particularly limited. For example, the detection method used in the step (b) described later (for example, an immunodetection method such as an ELISA method) These conditions may be used.

In the step (b), the method for detecting the binding of the antibody to the test substance (the bond between the test substance and the antibody) is not particularly limited, and generally known immunodetection methods can be used. For example, in addition to a method using an enzyme-labeled antibody, a known method for detecting the binding between an antibody and an antigen can be suitably employed.
For example, using an enzyme-labeled antibody or the like, the degree of color development is compared between when the test substance is added and when the test substance is not added, and when the degree of color development when the test substance is added is large (or small), It is determined that the test substance has bound to the antibody.
As an example, for example, after reacting the test substance and the antibody in step (a), an enzyme-labeled antibody (secondary antibody) is added and reacted, and then a substrate for the enzyme is added to cause color development by the enzyme. The step of reacting, the step of measuring the degree of color development with a colorimeter, the step of comparing the degree of color development with the case where no test substance is added, the degree of color development when the test substance is added, When the test substance is larger than when no test substance is added, it is determined that the test substance is bound to the antibody.
As such a method, for example, after reacting the test substance with the antibody in step (a), an anti-mouse IgG antibody (secondary antibody) labeled with horseradish peroxidase (HRP) or the like is further reacted. It is possible to detect and quantify carboxyalkyl amino acids such as C-merized amino acids, C-merized peptides and C-merized proteins, or peptides or proteins containing the amino acids.

In addition, as a method using an enzyme-labeled antibody, for example, binding of an antibody to a test substance can be detected by a competitive ELISA method (preferably an indirect competitive ELISA method). The case of using an antibody against a CM-modified amino acid will be described as an example. In the indirect competitive ELISA method, a known CM-protein, CM-peptide or CM-amino acid (for example, the CM-amino acid used for the production of the above-described antibody, etc.) ) Is coated (immobilized) on a microplate, test tube, etc., a sample containing a test substance and an antibody (primary antibody) against the CM-modified amino acid are added to cause a competitive reaction, After washing away the test substance and the primary antibody that did not bind, a secondary antibody (for example, an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) or the like) is added to bind to the primary antibody. In the control, the same procedure is performed except that the sample containing the test substance is not added. Next, an enzyme substrate is added to cause a color reaction with the enzyme, the degree of color development is measured with a colorimeter or the like, and the degree of color development when the test substance is added and when the test substance is not added (control) is determined. Compare. When the color development degree is decreased as compared with the control due to the addition of the test substance, it is determined that the test substance is bound to the antibody against the CM amino acid.
The test substance in which the binding to the antibody against the C-merized amino acid is detected is selected as a candidate substance.

  In the method of the present invention, screening can be performed using a sample containing one or more test substances. Although the sample is not particularly limited, for example, foods and drinks, their raw materials, intermediate raw materials, etc. can be suitably used. For example, foods and drinks whose Maillard reaction has progressed through processes such as heating and aging, their raw materials, and intermediate raw materials are suitable. In addition to beer-taste beverages such as happoshu, beer, and their raw materials (wort, malt, etc.), stew, curry, ramen and protein hydrolyzed seasonings are also suitable for foods that are heated for a long time Can be used. Furthermore, aged fermented foods such as sake, wine, aged cheese, miso, soy sauce, and salted spices can also be suitably used. The sample is preferably wort, wine, beer, miso, Japanese sake, aged cheese, etc., and particularly preferably wort.

  Evaluation of the body taste imparting effect of the selected candidate substance can be performed, for example, by sensory evaluation. For example, the body taste imparting effect can be determined by preparing an aqueous solution of the selected candidate substance and performing sensory evaluation of the aqueous solution. In addition, for example, when the candidate substance is added to a food or drink and the body is enhanced compared to the case where the candidate substance is not added, it is determined that there is a body taste imparting effect. The body taste imparting substance in the present invention is preferably, for example, a substance that enhances at least one of taste strength (drinking response), taste spread (thickness), and taste change with time (roughness). For example, as compared with the case where the candidate substance is added to food and drink and the candidate substance is not added, at least one of the strength of taste (drinking response), the spread of taste (thickness), and the change in taste over time (afterglow) If it is enhanced, it is determined that there is a rich taste imparting effect. Candidate substances that have been confirmed to have a kokumi imparting effect are preferably used as kokumi imparting substances in the present invention.

  One or two or more rich taste imparting substances obtained by the screening method can be further purified by a known technique used for purification of proteins and peptides, for example, gel filtration. In addition, the structure, amino acid composition, and the like can be appropriately determined by known techniques used for protein and peptide analysis such as SDS-PAGE, two-dimensional electrophoresis, and amino acid analysis.

  The richness imparting substance obtained by the screening method of the present invention is also one aspect of the present invention. The body taste imparting substance of the present invention is usually a protein or peptide containing an amino acid that has undergone a Maillard reaction, or an amino acid that has undergone a Maillard reaction. Of these, proteins or peptides containing amino acids that have undergone Maillard reaction are preferred. Moreover, it is preferably a protein or peptide containing a carboxyalkyl amino acid, or a carboxyalkyl amino acid, and more preferably a protein or peptide containing a carboxyalkyl amino acid. Among them, preferred is a protein or peptide containing carboxymethyl amino acid or carboxymethyl amino acid, and particularly preferred is a protein or peptide containing carboxymethyl lysine.

  One of the preferable embodiments of the body taste imparting substance of the present invention is derived from a wort boiling liquid. The body taste imparting substance of the present invention is preferably a peptide containing carboxymethyllysine, and among them, a peptide having a molecular weight of 10 to 20 kDa is preferable.

  Such a carboxymethylated peptide is obtained by, for example, using a wort boiling solution as a sample, performing the above screening method using an antibody against carboxymethyllysine, and then analyzing the molecular weight of the selected candidate substance from the composition by SDS-PAGE or the like. It can be easily obtained by separating a peptide of about 10-20 kDa.

The above-mentioned richness imparting substance is also suitably used as an index in food and beverage evaluation methods and food and beverage manufacturing process management.
A method for evaluating a food or drink or a method for managing a production process using the above-described richness imparting substance as an index is also one aspect of the present invention. The richness imparting substance used as an index may be one kind or two or more kinds. In the method of the present invention, the amount of the richness imparting substance is preferably used as an index. Unlike the sensory evaluation, the richness can be quantitatively evaluated by using the amount of the richness imparting substance as an index.

  The method of the present invention preferably includes a step of measuring the amount of the richness imparting substance. The rich taste imparting substance in the present invention, that is, a carboxyalkyl amino acid such as a CM-modified amino acid, a CM-modified peptide or a CM-modified protein, or a peptide or protein containing the amino acid (preferably a carboxyalkyl amino acid such as a CM-modified peptide or a CM-modified protein) The amount of the peptide or protein to be contained can be quantified by a known method such as an enzyme immunoassay using the antibody used in the screening method described above. For example, by reacting the above-described monoclonal antibody or polyclonal antibody with a sample, and then using an enzyme-labeled antibody, a carboxyalkyl amino acid such as a CM-modified amino acid, a CM-modified peptide, or a CM-modified protein that is a rich taste imparting substance or the amino acid Peptides or proteins containing can be quantified. In addition, by performing competitive ELISA using the antibodies used in the above-described screening methods, CM rich amino acids, CMized peptides, CMized proteins, and the like, which are body taste imparting substances, can also be quantified.

  More specifically, the amount of the body taste imparting substance contained in the food or drink is measured by using the food or drink itself as a sample solution or a sample solution prepared from the food or drink, for example, carboxyalkyl amino acid. And (b) detecting the binding of the antibody to a carboxyalkyl amino acid in the sample solution, or a peptide or protein containing the antibody, and the like.

  As an example of such a quantification method, a method for quantifying a carboxyalkyl amino acid, a peptide or protein containing a carboxyalkyl amino acid by a competitive ELISA method (preferably an indirect competitive ELISA method) will be described. The case of using an antibody against a CM-modified amino acid will be described as an example. In the indirect competitive ELISA method, a known CM-protein, CM-peptide or CM-amino acid (for example, the CM-amino acid used in the production of the above-described antibody, etc. ) Is coated (immobilized) on a microplate, test tube, etc., and a sample solution and an antibody (primary antibody) against the above-mentioned CM amino acid are added to cause a competitive reaction, and it does not bind to the immobilized CM amino acid, etc. After washing and removing the sample solution fraction and the primary antibody, a secondary antibody (for example, an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) or the like) is added to bind to the primary antibody. In the control, the same procedure is performed except that the sample solution is not added. Next, an enzyme substrate is added to cause a color reaction by the enzyme, the degree of color development is measured with a colorimeter, etc., and the degree of color development when the sample solution is added and when the sample solution is not added (control) is determined. Compare. When the color development degree is decreased as compared with the control due to the addition of the sample solution, it is determined that the sample solution is bound to the CM-containing amino acid, the CM-containing amino acid-containing peptide or protein. That is, it can be confirmed that the sample solution contains one or more of CM-modified amino acids, peptides and proteins containing CM-ized amino acids. Further, in the competitive ELISA method, the degree of decrease in the degree of color development increases as the amount of the CM-containing amino acid, the peptide containing the CM-converted amino acid and the protein containing the CM-converted amino acid increases. Therefore, the amount of the CM-containing amino acid, the peptide containing the CM-converted amino acid, and the protein containing the CM-converted amino acid is measured by the degree of decrease in the degree of color development.

  In addition, for example, a carboxyalkyl amino acid having a known concentration, or a peptide solution containing the amino acid (or a protein solution containing the amino acid) is used instead of the sample solution, and the color development degree obtained by performing the same procedure as described above is used. From the ratio of the decrease in chromogenicity of the sample solution to the decrease, the carboxyalkyl amino acid, the peptide or protein concentration containing the amino acid can be obtained for the peptide solution (or protein solution) containing a known carboxyalkyl amino acid.

  In the method for evaluating a food or drink, the body taste or taste thickness of the food or drink can be quantitatively evaluated by measuring the amount of the body taste imparting substance in the food or drink. For example, when the amount (concentration) of the richness-imparting substance in a certain food or drink is large compared to other foods or drinks, the food or drink with a large amount of richness-imparting substance is superior in richness and has a thick taste. Evaluated as being. In addition, if the amount of the body taste imparting substance in the food or drink is a certain value or more, it can be evaluated that the food has a rich taste and has a thick taste, so the quality control of the food and drink can be easily and efficiently performed. It can also be done.

In the manufacturing process management method for foods and drinks, for example, at one or more points in the manufacturing process, a raw material, an intermediate product, or a final product is sampled, and the amount (concentration) of the body taste imparting substance in the sampled sample It is preferable to measure. Thereby, since the richness of a raw material, an intermediate product, or a final product can be evaluated quantitatively, the quality of a final product can be managed easily and efficiently. In addition, the quality of the product can be evaluated and managed by monitoring changes in the amount of the body taste imparting substance at each stage of the manufacturing process.
Thus, by managing the manufacturing process of food / beverage products, the food / beverage products of fixed quality can be manufactured efficiently.

The present invention also includes a kit for detecting a body taste imparting substance containing an antibody against a carboxyalkyl amino acid for performing the screening method. The antibody may be an antibody against the AGE product, and the above-described antibody against the carboxyalkyl amino acid is one of the preferable ones.
Such a kit is suitably used for the screening method, the evaluation method for food and drink, the manufacturing process management method, and the like described above.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples at all.

Reference example 1
European malt is dry pulverized with a commercially available cereal grinder, water four times its weight is added, and the mixture is stirred at 52 ° C. for 20 minutes and at 65 ° C. for 60 minutes, followed by centrifugation and filter paper filtration. (Sample 1). Sample 2 was obtained by heating Sample 1 with an autoclave at 100 ° C. for 90 minutes. Sample 3 was prepared by adding caramel dye (Ikeda Gakka Kogyo Co., Ltd.) to Sample 1 so that the absorbance at 430 nm was equal to Sample 2. In addition, evaluation by the color tone of wort is generally performed by measuring the light absorbency in 430 nm. The absorbance (430 nm) of samples 1 to 3 was measured with a spectrophotometer (manufactured by Shimadzu Corporation).

  Using samples 1 to 3 produced as described above, sensory evaluation was performed on the strength of the taste (drinking response), the spread of the taste (thickness), and the change with time of the taste (resonance) by 20 trained panelists. As a result, sample 2 had the highest evaluation, samples 1 and 3 had the same evaluation, and were lower than sample 2.

  From the above test, it was revealed that the color tone of wort (absorbance at 430 nm), which has been conventionally used as an index, does not serve as an index of taste and richness produced by heating wort.

Example 1
1. Wort preparation method European malt was dry pulverized with a commercially available cereal grinder, water four times its weight was added, and the mixture was stirred at 52 ° C. for 20 minutes and at 65 ° C. for 60 minutes, followed by centrifugation and filter paper. The wort was obtained by filtration. The liquid obtained by heating the obtained wort in an autoclave at 100 ° C. for 90 minutes was used as boiling wort.

2. Competition ELISA method An antibody against C-lysine (primary antibody) was prepared according to the method described in Example 1 of Japanese Patent No. 4012722.
A solution containing C-albumin obtained according to the method described in Example 1 of Japanese Patent No. 4012722 was added to a microplate, and allowed to stand for 2 hours to immobilize (coat) C-M protein and the like on the plate. After washing away the solution and uncoated CM protein, add a solution containing gelatin onto the plate and let stand for 1 hour. Fix the gelatin so that other components are not immobilized on the plate in the subsequent steps. (Blocking). After washing and removing the gelatin solution and uncoated gelatin, a sample solution containing the test substance (above boiling wort) and an antibody against the CM-modified amino acid (CM-lysine) (primary antibody) are added and allowed to stand for 1 hour. Then, a competitive reaction was performed. After washing and removing the test substance and the primary antibody that did not bind to the immobilized CMized amino acid or the like, an anti-mouse IgG antibody labeled with a secondary antibody (horseradish peroxidase (HRP)) was added to bind to the primary antibody. In the control, the same procedure was performed except that the boiling wort was not added. After washing and removing the secondary antibody that is not bound to the solution and the primary antibody, an enzyme substrate is added to cause a color reaction with the enzyme to confirm color development, and after color development is stopped with concentrated sulfuric acid, the degree of color development (at 492 nm) (Absorbance) was measured with a colorimeter or the like, and the degree of color development was compared between when boiling wort was added and when boiling wort was not added (control). In this method, it is determined that the test substance is bound to an antibody against the CM-modified amino acid when the color development degree is decreased as compared with the control due to the addition of the test substance. The addition of boiling wort decreased the degree of color development compared to the control, so it was determined that the substance contained in the boiling wort was bound to the antibody against the CM amino acid. The substance that bound to the antibody against the CM amino acid was selected as an AGE product (terminal glycation product).

  The concentration of the AGE product in the boiled wort is a sample solution containing a test substance from a known CM-modified protein (CM-albumin obtained according to the method described in Example 1 of Patent No. 4012722) having a known concentration. The concentration of the AGE product is determined from the ratio of the decrease in the color development degree of the sample solution to the decrease in the color development degree obtained by performing the same procedure as described above using (boiled wort) instead of the above-mentioned boiled wort. Obtained for a modified protein solution.

  The results are shown in FIG. The vertical axis of the graph in Fig. 1 represents the difference in color development from the control (absorbance difference at 492 nm: ΔAbs. @ 492 nm). The greater the AGE product content, the greater the difference in absorbance at 492 nm. From FIG. 1, it was found that the AGE product in the wort increased by boiling. In FIG. 1, the known AGE protein is AGE-HSA (protein concentration: 0.2 mg / mL).

3. SDS-PAGE and Western Blotting Pre-boiling wort and post-boiling wort were each concentrated and subjected to electrophoresis by SDS-PAGE. Two sets of samples and molecular weight markers were applied to the same gel, followed by electrophoresis, the gel was divided, one set was subjected to Coomassie blue staining, and the other set was subjected to Western blotting. Western blotting was performed according to a conventional method, and an AGE product (Maillard reacted protein) was detected using an anti-carboxymethyllysine antibody as a primary antibody and a peroxidase-labeled goat antibody as a secondary antibody.

FIG. 2A shows the result of SDS-PAGE. In FIG. 2A, STD is a molecular weight marker. (1) is the wort before boiling, and (2) is the wort after boiling.
FIG. 2B shows the results of Western blotting. In FIG. 2B, (1) is the wort before boiling, and (2) is the wort after boiling. The 15 kd band indicated by the arrow in FIG. 2B is the detected AGE product (Maillard reacted protein), and the AGE product (Maillard reacted protein) is specifically selected from many proteins in wort. I was able to detect it.

Example 2
Fractionation according to molecular weight of components in wort Boiled wort obtained in the same manner as in Example 1 was fractionated by molecular weight by gel filtration.
That is, the boiled wort was dialyzed for 2 days using a dialysis membrane (SPECTRUM LABS, Spectra / Por (registered trademark) 7, fractional molecular weight 10 kDa) to remove low molecular components from the wort. The obtained dialyzed wort was concentrated about 4 times by distilling off the solvent under reduced pressure, and then using a large column (length 30 cm, inner diameter 2 cm) of gel filtration fraction, water as the mobile phase and the molecular weight A fraction fraction was obtained. The fractions were 4 mL each, and the obtained fractions were No. 1-No. It was set to 10. This operation was repeated 10 times, and fractions with the same number obtained in each operation were combined and used for sensory experiments and competition ELISA. The amount of AGE product (Maillard reacted protein) in the fraction was measured by the competitive ELISA method of Example 1. The combined fractions were measured for dry solid weight by lyophilization.

  As a result, fraction no. 7, no. 9 and no. In 11 the protein was detected. The results are shown in FIG. The vertical axis in FIG. 3 represents the dry solid weight per fraction. Furthermore, the result of having measured the amount of Maillard protein contained in each fraction collected by competitive ELISA is shown in FIG. As can be seen from FIG. 4, the AGE product (Mailard-reacted protein) is fraction No. Only 9 were detected.

Example 3
Sensory evaluation Among the fractions obtained by the above gel filtration, the fraction No. contains a solid dry product but does not contain an AGE product (Maillard reacted protein). 7 and no. Fraction No. 11 containing 11 fractions and AGE product (Maillard reacted protein) Sensory evaluation was performed on 9. Specifically, a sample obtained by adding a solid dried product of each fraction to a commercial beer (Suntory liquor) so as to increase by 10 ppm is used as a sample. Thickness and amount of reverberation were evaluated. When the control value is 1, the response of each fraction is drunk, the thickness of the taste, and the amount of reverberation is higher than the control, the score is 2. The thing was evaluated.
The average sensory evaluation score is shown in FIG. It was found that the fraction containing a large amount of Maillard-reacted protein (Fraction No. 9) is more effective for imparting taste than the other fractions.

Example 4
Using the following wine, beer, miso extract, sake and cheese extract as specimens, the amount of AGE product (Maillard reacted protein) in each specimen was analyzed by competitive ELISA in the same manner as in Example 1. .

1. Wine Chateau Mouton Rothschild 2007 (Product name, Made in Chateau Mouton Rothschild (France))
Chateau Mouton Rothschild 1975 (Product name, Made in Chateau Mouton Rothschild (France))
Barolo Borgogne 2001 (Brand name, Borgogne (Italy))
Barolo Borgogne 1967 (Brand name, Borgogne (Italy))
Each of the above wines was subjected to a competitive ELISA.

2. Beer Cantillon Goose (trade name, manufactured by Cantillon Brewery, a blend of Belgian aged Lambic beer and aged young beer)
Cantillon Grand Cru Bruoxera (trade name, manufactured by Cantillon Brewery, beer using only Belgian beer aged 3 years or more)
Each of the above beers was subjected to a competitive ELISA.

3. Miso extract Ishii Miso 3 years old white (aged for 1 year) (trade name, manufactured by Ishii Miso)
Ishii Miso 3 years old red (aged 3 years) (trade name, manufactured by Ishii Miso)
15 g of water was added to 5 g of the sample (each miso), and the mixture was sufficiently stirred with a homogenizer at 10000 rpm for 2 minutes. Thereafter, centrifugation was carried out at 5000 rpm for 30 minutes at 4 ° C., and the supernatant was filtered through a 0.45 μm diameter filter (trade name: GL Chromatodisc Water System 0.45 μm, manufactured by Kurabo Industries) for competitive ELISA. Provided.

4). Japanese sake laurel wreath PREMIUM Yamada Nishiki Daiginjo (trade name, manufactured by laurel wreath)
Laurel wreath PREMIUM Omachi Junmai Ginjo (brand name, manufactured by laurel wreath)
Laurel Wreath Romantic Super Selected Treasured Liquor Decade (Product Name, Laurel Wreath)
Laurel Wreath Crown (trade name, manufactured by Laurel Wreath Co.)
Izumi Kido 25-year old sake (trade name, manufactured by Izumi Sake Brewery)
Masamune Tatsuma Chitose Fifteen Years Old Sake (Brand name, purchased from Shiraki Tsunesuke Shop)
Each of the above sakes was subjected to a competitive ELISA.

5. Cheese extract Boehmstar Gouda (Vlaskaas) (trade name, manufactured by CONO (Netherlands), no aging)
Boehm Star Gouda premier (trade name, manufactured by CONO (Netherlands), aged for 6 months)
Boehm Star Gouda classic (trade name, manufactured by CONO (Netherlands), 18-month aging type)
Boehm Star Gouda XO-ExtraOld (trade name, manufactured by CONO (Netherlands), 26-month aging type)
90 g of water was added to 30 g of each sample (each cheese described above), and the mixture was sufficiently stirred with a homogenizer at 15000 rpm for 2 minutes. Thereafter, centrifugation was performed at 3500 rpm for 15 minutes at 4 ° C. After centrifugation, the middle aqueous layer of the three layers was separated by decantation, and the aqueous layer was centrifuged again to obtain a supernatant. The supernatant was filtered through a 0.45 μm diameter filter (trade name: GL Chromatodisc aqueous 0.45 μm, manufactured by Kurabo Industries) and subjected to competitive ELISA.

  The amount of the AGE product in the sample was compared with the control (the difference in absorbance at 492 nm: ΔAbs. @ 492 nm) in the same manner as in Example 1. The difference in absorbance at 492 nm is a difference in absorbance measurement value that increases according to the content of the AGE product measured by the competitive ELISA method. That is, the greater the AGE product content, the greater the difference in absorbance at 492 nm.

  The results are shown in FIGS. The vertical axis of the graphs in FIGS. 6 to 10 represents the difference in color development from the control (difference in absorbance at 492 nm: ΔAbs. @ 492 nm), as in FIG.

  The wine, beer, miso, sake and cheese used for the samples are all foods and beverages produced through aging, but all samples contained AGE products. Furthermore, from FIGS. 6-10, it turns out that the amount of AGE products increases, so that maturing of food-drinks advances.

Example 5
Sensory evaluation was performed on each specimen of Example 4 by 5 trained panelists (panelists A to E). The evaluation criteria are as follows.
I did not feel the rich taste: 1, I did not feel the rich taste very much: 2, I felt the rich taste: 3, I felt a little bit rich, the taste was strong: 4, I felt a strong taste: I rated it with a rating of 5.

  The results of sensory evaluation are shown in Tables 1-5. The sensory evaluation results shown in Tables 1 to 5 coincided with the analysis results of the AGE product of Example 4. That is, it was determined that the higher the content of the AGE product, the higher the sensory point (evaluation point) of sensory evaluation and the rich taste. Therefore, the carboxyalkylated peptide, carboxyalkylated protein, etc. detected by the screening method using an antibody against carboxyalkylamino acid are actually substances effective for imparting rich taste, the carboxyalkylated peptide, carboxyalkyl It was confirmed that the richness of foods and beverages and the like can be quantitatively evaluated by measuring the amount of modified protein and the like.

  INDUSTRIAL APPLICABILITY The present invention is useful in the food field and the like as a screening method for a body taste imparting substance, a body taste imparting substance obtained by the screening method, a method for evaluating food or drink or a manufacturing process management method, and a kit for detecting a body taste imparting substance It is.

Claims (8)

  A substance that has reactivity with an antibody against a carboxyalkyl amino acid and contains carboxymethyllysine or carboxyethyllysine, and is a peptide having a molecular weight of 10 to 20 kDa.   The richness imparting substance according to claim 1, which is derived from wort, wine, beer, miso, sake, or cheese.   The richness imparting substance according to claim 1 or 2, which is derived from a malt boiling solution.   The food-drinks which added the savoriness imparting substance in any one of Claims 1-3.   The food or drink according to claim 4, which is beer.   The evaluation method or manufacturing process management method of food-drinks which used the richness imparting substance in any one of Claims 1-3 as a parameter | index.   The method of Claim 6 including the process of measuring the quantity of the richness imparting substance in any one of Claims 1-3.   A kit for detecting a body taste imparting substance comprising an antibody against a carboxyalkyl amino acid for performing a screening method for a body taste imparting substance using an antibody against a carboxyalkyl amino acid as an indicator of reactivity to the antibody. A kit wherein the imparting substance contains carboxymethyllysine or carboxyethyllysine and is a peptide having a molecular weight of 10 to 20 kDa.