Glycosylated Proteins

Abstract

The purpose of the present invention is to provide: a substance that is useful for imparting a rich taste to a beverage or food; and a method for imparting a rich taste to a beverage or food. The present invention relates to a sugar-modified protein in which the side-chain amino group of lysine is glycated and wherein the side-chain amino group of lysine in a position corresponding to position 160 and / or position 189 of the amino acid sequence represented by SEQ ID NO: 1 of the proteins indicated in any one of (a1) to (a3) below is glycated. (a1) is a protein comprising the amino acid sequence represented by SEQ ID NO: 1. (a2) is a protein comprising an amino acid sequence in which 1-9 amino acids in regions other than the lysine at position 160 and / or position 189 are deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1. (a3) is a protein comprising an amino acid sequence that is at least 98% identical to the amino acid sequence represented by SEQ ID NO: 1 and in which the amino acid in the position corresponding to position 160 and / or position 189 of the amino acid sequence represented by SEQ ID NO: 1 is lysine.

Description

[Technical field]

[0001] The present invention relates to a glycomodified protein. More specifically, the present invention relates to a glycomodified protein in which the side chain amino group of lysine at a specific position in the protein is glycated. The present invention also relates to an agent for imparting kokumi. The present invention further relates to a method for imparting kokumi to food and drink. [Background technology]

[0002] With the recent diversification of consumer tastes, there is a demand for the development of beer-flavored alcoholic beverages and non-alcoholic beer-flavored beverages with a variety of flavor characteristics.

[0003] For example, Patent Document 1 discloses that alcohol components, sweet components, aldehyde components, etc. are used as flavor improvers for beer-like beverages in order to enhance the richness, etc., of beer-like beverages. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2016-214262 A Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a substance useful for imparting full body flavor to foods and beverages. Another object of the present invention is to provide a method for imparting full body flavor to foods and beverages. [Means for solving the problem]

[0006] The present inventors have investigated substances capable of imparting full body taste to foods and beverages, and as a result have found that a glycosylated protein or the like consisting of the amino acid sequence shown in SEQ ID NO: 1 in which the side chain amino group of lysine at position 160 and / or lysine at position 189 of the amino acid sequence is glycated is a substance that correlates with full body taste. The present inventors have also found that a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which the side chain amino group of lysine at the position corresponding to position 117 of the amino acid sequence is glycated is also a substance that correlates with full body taste.

[0007] That is, the present invention relates to the following glycosylated proteins, kokumi imparting agents, methods for imparting kokumi to foods and beverages, and the like, although not limited thereto. [1] A glycosylated protein in which the side chain amino group of a lysine is glycated, the side chain amino group of the lysine at the position corresponding to the 160th amino acid and / or the 189th amino acid in the amino acid sequence shown in SEQ ID NO:1 of a protein described in any one of (a1) to (a3) ​​below being glycated. (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine. [2] The glycosylated protein according to [1] above, wherein the protein according to (a1) to (a3) ​​above is a protein derived from barley. [3] A glycosylated protein in which the side chain amino group of a lysine is glycated, the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 of a protein described in any one of the following (b1) to (b3) being glycated: (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine. [4] The glycosylated protein according to [3] above, wherein the protein according to (b1) to (b3) above is a protein derived from barley. [5] A kokumi-imparting agent comprising the following glycosylated protein (A) and / or the following glycosylated protein (B): Glycosylated protein (A): A glycosylated protein in which the side chain amino group of lysine is glycated, the side chain amino group of lysine at the position corresponding to the 160th position and / or the side chain amino group of lysine at the position corresponding to the 189th position in the amino acid sequence shown in SEQ ID NO: 1 of a protein described in any one of (a1) to (a3) ​​below is glycated. (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine. Glycosylated protein (B): A glycosylated protein in which the side chain amino group of a lysine is glycated, the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 of a protein described in any one of (b1) to (b3) below is glycated. (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine. [6] The body flavor imparting agent according to the above [5], which is used to impart body flavor to a beer-flavored alcoholic beverage or a non-alcoholic beer-flavored beverage. [7] Use of the above-mentioned glycosylated protein (A) and / or the above-mentioned glycosylated protein (B) for imparting full-bodied taste to a food or drink. [8] The use according to [7] above, wherein the food or beverage is a beer-flavored alcoholic beverage or a non-alcoholic beer-flavored beverage. [9] A method for imparting full body to a food or drink, comprising adding the glycosylated protein (A) and / or the glycosylated protein (B) to the food or drink.

[10] The method according to [9] above, wherein the food or beverage is a beer-flavored alcoholic beverage or a non-alcoholic beer-flavored beverage. Effect of the Invention

[0008] According to the present invention, it is possible to provide a glycosylated protein useful for imparting full body taste to foods and beverages. Also, according to the present invention, it is possible to provide a method for imparting full body taste to foods and beverages. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 is a graph showing the ratio of the total LC-MS ion area value of the glycosylated Z4(142-174) peptide in which one hexose or two hexoses (dihexoses) are bound to K160 in a glycosylated protein purified from three different types of beer (B, C, D) to the LC-MS ion area value (ion area value of unmodified Z4(142-174) peptide) of the peptide fragment (unmodified Z4(142-174) peptide) consisting of amino acids 142 to 174 of the amino acid sequence of Serpin Z4 (sequence number 1). [Diagram 2] FIG. 2 is a graph showing the ratio of the total LC-MS ion area value of the glycosylated Z4(182-200) peptide in which one hexose or two hexoses (dihexoses) are bound to the 189th lysine (K189) in the peptide to the LC-MS ion area value of the unmodified Z4(182-200) peptide (ion area value of unmodified Z4(182-200) peptide) consisting of amino acids 182 to 200 of the amino acid sequence of Serpin Z4 (sequence number 1) in glycosylated proteins purified from three different types of beer (B, C, D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] A first aspect of the present invention is a glycosylated protein in which the side chain amino group of lysine is glycated, the side chain amino group of the lysine at the position corresponding to the 160th position and / or the 189th position of the amino acid sequence shown in SEQ ID NO:1 of a protein described in any of (a1) to (a3) ​​below is glycated. (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine. The above glycosylated protein is also referred to as the glycosylated protein of the first aspect of the present invention. The glycosylated protein of the first aspect of the present invention has an amino acid sequence in which the amino acid at the position corresponding to the 160th amino acid and / or the position corresponding to the 189th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is a lysine. The glycosylated protein of the first aspect of the present invention is a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 160th amino acid and / or the lysine at the position corresponding to the 189th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is glycated.

[0011] In the present specification, the position corresponding to the 160th position in the amino acid sequence shown in SEQ ID NO: 1 means the position corresponding to the 160th position (160th position) in the amino acid sequence shown in SEQ ID NO: 1 when the amino acid sequence (primary structure) of the protein (polypeptide) to be compared is aligned based on the homology of the amino acid sequence with the amino acid sequence shown in SEQ ID NO: 1 (reference amino acid sequence). Therefore, the amino acid at the position corresponding to the 160th position in the amino acid sequence shown in SEQ ID NO: 1 is the 160th lysine in SEQ ID NO: 1, but may not be the 160th position in a polypeptide having an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 1. The same applies to an amino acid other than the 160th position in the amino acid sequence shown in SEQ ID NO: 1, for example, a lysine at a position corresponding to the 189th lysine in the amino acid sequence shown in SEQ ID NO: 1. The same applies to a lysine at a position corresponding to the 117th lysine in the amino acid sequence shown in SEQ ID NO: 2 described later. In the present specification, the number of the amino acid is the number of the amino acid from the N-terminus. In the present specification, the position in the amino acid sequence corresponding to the 160th lysine in the amino acid sequence shown in SEQ ID NO: 1 is also referred to as "the position corresponding to the 160th position in SEQ ID NO: 1". Similarly, the position in the amino acid sequence corresponding to the 189th lysine in the amino acid sequence shown in SEQ ID NO:1 is also referred to as "the position corresponding to the 189th position in SEQ ID NO:1."

[0012] It is easy to identify the lysine at the position corresponding to position 160 and / or the lysine at the position corresponding to position 189 of SEQ ID NO:1 in any of the proteins (polypeptides) of (a1), (a2) or (a3) ​​above. Identification of the lysine at the position corresponding to the 160th position of SEQ ID NO: 1 and / or the lysine at the position corresponding to the 189th position of SEQ ID NO: 1 can be performed using a known sequence comparison program such as ClustalW or Protein Blast of NCBI. ClustalW can usually be used with default parameters. ClustalW can be accessed from websites of the Data Bank of Japan (DDBJ), the European Bioinformatics Institute (EBI), and the like. By inputting the amino acid sequences of two polypeptides to be compared and running the program, alignment based on the homology of the amino acid sequences can be performed. Here, by inputting the amino acid sequence of SEQ ID NO: 1 as one of the two polypeptides, it is possible to easily identify which position in the polypeptide of SEQ ID NO: 1 corresponds to a residue at a specific position of any polypeptide. For example, in the amino acid sequence shown in SEQ ID NO:2, the amino acid at the position corresponding to the 189th amino acid in SEQ ID NO:1 is the 190th lysine.

[0013] In the present invention, "the side chain amino group of lysine is glycated" means that sugar is bound to the side chain amino group of lysine. The sugar may be any of monosaccharides, disaccharides, trisaccharides or higher oligosaccharides, and polysaccharides, preferably monosaccharides, disaccharides, or trisaccharides, and more preferably monosaccharides or disaccharides. The monosaccharides include reducing sugars, including all monosaccharides, including glucose, fructose, galactose, mannose, rhamnose, arabinose, xylose. The monosaccharides are preferably in the D-form. Disaccharides include reducing sugars, such as maltose-type disaccharides such as maltose, isomaltose, and lactose. Examples of trisaccharides include maltotriose, isomaltotriose, certriose, fucosyllactose, and sialyllactose. In one embodiment, the sugar is preferably a sugar contained in beer. In one embodiment, the monosaccharide bonded to the side chain amino group of lysine is preferably, for example, glucose, arabinose, xylose, etc. The disaccharide is preferably maltose, isomaltose, etc. The trisaccharide is preferably maltotriose, isomaltotriose, etc.

[0014] The amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence of SerpinZ4 (also known as BSZ4, HorvuZ4, Major endosperm albumin, Serpin Z4, Serpin-Z4, Protein Z or Protein Z4) derived from barley (scientific name: Hordeum vulgare). In the protein described in (a1) above, the position corresponding to position 160 of SEQ ID NO: 1 is the 160th position of the amino acid sequence, and the position corresponding to position 189 is the 189th position of the amino acid sequence.

[0015] As used herein, the phrase "1 to 9 amino acids have been deleted, substituted, inserted, and / or added" in the amino acid sequence of a protein means that 1 to 9 amino acids have been deleted, substituted, inserted, and / or added at any one of 1 to 9 positions in the amino acid sequence within the same sequence, and two or more of the deletions, substitutions, insertions, and additions may occur simultaneously.

[0016] In the amino acid sequence of the protein described in (a2) above, the number of deleted, substituted, inserted and / or added amino acids is preferably 1 to 8, 1 to 7 or 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, particularly preferably 1 to 3, and most preferably 1 or 2.

[0017] In the above protein (a3), the identity (sequence identity) of the amino acid sequence to the amino acid sequence shown in SEQ ID NO:1 is preferably 99% or more. The identity of amino acid sequences can be calculated, for example, using analysis software such as BLAST with default parameters.

[0018] In one aspect, the glycosylated protein of the first aspect of the present invention is preferably a glycosylated protein in which the side chain amino group of the lysine at position corresponding to position 160 and / or the side chain amino group of the lysine at position corresponding to position 189 of the amino acid sequence shown in SEQ ID NO: 1 of the protein described in (a1) above (a protein consisting of the amino acid sequence shown in SEQ ID NO: 1) is glycated. This glycosylated protein can also be said to be a glycosylated protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which the side chain amino group of the lysine at position 160 and / or the side chain amino group of the lysine at position 189 of the amino acid sequence is glycated. In one aspect, the glycosylated protein of the first aspect of the present invention is preferably a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 160th amino acid in the amino acid sequence shown in SEQ ID NO:1 is glycated, in any one of the proteins described in (a1) to (a3) ​​above (preferably protein (a1)).

[0019] In the glycosylated proteins of the present invention, in the above proteins (a1), (a2) and (a3), the side chain amino group of a lysine other than the lysine at the position corresponding to the 160th position and / or the lysine at the position corresponding to the 189th position in the amino acid sequence shown in SEQ ID NO: 1 may be glycated.

[0020] The glycosylated protein of the first aspect of the present invention is not particularly limited in its production method or origin. In one aspect, the glycosylated protein of the first aspect of the present invention is preferably a cereal-derived protein in which the side chain amino group of the lysine at the position corresponding to the 160th position and / or the lysine at the position corresponding to the 189th position in the amino acid sequence of SEQ ID NO: 1 is glycated. Examples of cereals include barley, wheat, corn, rice, etc. Among them, barley is preferred. The above proteins (a1) to (a3) ​​are preferably proteins derived from barley.

[0021] A second aspect of the present invention is a glycosylated protein in which the side chain amino group of lysine is glycated, the side chain amino group of lysine at the position corresponding to amino acid 117 of the amino acid sequence shown in SEQ ID NO: 2 of a protein described in any one of (b1) to (b3) below is glycated. (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine. A glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 of the protein described in any of (b1) to (b3) above is glycated is also referred to as a glycosylated protein of the second aspect of the present invention. The glycosylated protein according to the second aspect of the present invention has an amino acid sequence in which the amino acid at the position corresponding to position 117 in the amino acid sequence shown in SEQ ID NO: 2 is lysine. The glycosylated protein according to the second aspect of the present invention is a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to position 117 in the amino acid sequence shown in SEQ ID NO: 2 is glycated. In the glycosylated protein according to the second aspect of the present invention, the sugar bonded to the side chain amino group of lysine and preferred aspects thereof are the same as those in the glycosylated protein according to the first aspect described above.

[0022] The position in the amino acid sequence corresponding to the 117th lysine in the amino acid sequence shown in SEQ ID NO:2 is also referred to as "the position corresponding to the 117th in SEQ ID NO:2." It is easy to identify the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 in any of the above proteins (polypeptides) (b1), (b2) or (b3). When identifying the lysine at the position corresponding to the 117th amino acid in SEQ ID NO: 2 in the above proteins (polypeptides) (b1), (b2) or (b3), it can be identified in the same manner as in the case of the glycosylated protein of the first aspect of the present invention, using a known sequence comparison program.

[0023] The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of barley-derived protein SerpinZ7 (also known as BSZ7, Serpin Z7 or HorvuZ7). In the protein described in (b1) above, the position corresponding to the 117th position in SEQ ID NO: 2 is the 117th position in the amino acid sequence.

[0024] In the amino acid sequence of the protein described in (b2) above, the number of deleted, substituted, inserted and / or added amino acids is preferably 1 to 8, 1 to 7 or 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, particularly preferably 1 to 3, and most preferably 1 or 2.

[0025] In one embodiment, the protein (b2) is preferably any of the proteins (b21) to (b24) below. The protein (b21) to (b24) is SerpinZ7 derived from barley (scientific name: Hordeum vulgare). (b21) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, in which the 173rd position is alanine. (b22) a protein having an amino acid sequence in which the 292nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is threonine (b23) a protein having an amino acid sequence in which the 303rd position in the amino acid sequence shown in SEQ ID NO: 2 is glutamic acid (b24) a protein having an amino acid sequence in which the 325th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is leucine

[0026] In the above protein (b3), the identity of the amino acid sequence to the amino acid sequence shown in SEQ ID NO:2 is preferably 99% or more.

[0027] In one embodiment, the glycosylated protein according to the second embodiment of the present invention is preferably a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 of the protein described in (b1) above (a protein consisting of the amino acid sequence shown in SEQ ID NO: 2) is glycated. This glycosylated protein can also be said to be a glycosylated protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which the side chain amino group of the lysine at the 117th amino acid in the amino acid sequence is glycated.

[0028] In the proteins (b1), (b2) and (b3), the side chain amino group of a lysine other than the lysine at the position corresponding to the 117th position in the amino acid sequence shown in SEQ ID NO:2 may be glycated.

[0029] The glycosylated protein of the second aspect of the present invention is not particularly limited in its production method or origin. In one aspect, the glycosylated protein of the second aspect of the present invention is preferably a cereal-derived protein in which the side chain amino group of lysine at the position corresponding to the 117th amino acid in the amino acid sequence of SEQ ID NO: 2 is glycated. Examples of cereals include barley, wheat, corn, rice, etc. Among them, barley is preferred. The above proteins (b1) to (b3) are preferably proteins derived from barley.

[0030] Hereinafter, the glycosylated protein according to the first aspect of the present invention and the glycosylated protein according to the second aspect of the present invention are also collectively referred to as the glycosylated protein of the present invention. The method for producing the glycosylated protein of the present invention is not particularly limited. The glycosylated protein of the present invention can be purified, for example, from grains or beer brewed using the grains as a raw material. The grains are preferably barley, and more preferably barley malt. In one embodiment, it is preferable to use North American malt.

[0031] The method for obtaining a glycosylated protein from beer is not particularly limited, and can be carried out according to a conventional separation and purification method. As the separation and purification method, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reversed-phase high performance liquid chromatography, dialysis, ultrafiltration, etc. can be used alone or in appropriate combination. For example, according to the method described in the Examples, beer is subjected to column chromatography using a cation exchange resin to recover a fraction containing a 40 kDa protein, ammonium sulfate is added to the fraction to perform salting out, and the supernatant is recovered to obtain a solution (supernatant) containing a glycosylated protein. The solution can be concentrated by a known method to obtain a purified product of the glycosylated protein of the present invention. Whether the amino group of lysine in the obtained protein has been glycated can be confirmed by colorimetric quantification of glycated lysine using the NBT (Nitroblue tetrazolium) method.

[0032] In the glycosylated protein of the first aspect of the present invention, the glycation of the side chain amino group of the lysine at the position corresponding to the 160th amino acid and / or the lysine at the position corresponding to the 189th amino acid in the amino acid sequence shown in SEQ ID NO:1 can be confirmed by fragmenting the purified protein by a known method and analyzing the obtained peptide fragments by LC-MS / MS.

[0033] In the glycosylated protein of the second aspect of the present invention, the glycation of the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO:2 can be confirmed by fragmenting the purified protein by a known method and analyzing the obtained peptide fragments by LC-MS / MS.

[0034] As shown in the Examples below, a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 160th position and / or the 189th position in the amino acid sequence shown in SEQ ID NO: 1 was glycated had an excellent effect of imparting kokumi. A glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 117th position in the amino acid sequence shown in SEQ ID NO: 2 was glycated had an excellent effect of imparting kokumi. The glycosylated protein of the present invention is useful for imparting full body taste to foods and beverages. When the glycosylated protein of the present invention is contained in foods and beverages, the higher the ratio of the protein, the more full body taste can be imparted. In the present invention, "kokumi" refers to a sensation that cannot be expressed by the five basic tastes, i.e., sweetness, saltiness, sourness, bitterness, and umami, and is expressed by taste strength (drinkability), spread of taste, depth of taste, change in taste over time (aftertaste or duration), etc. In the present invention, imparting kokumi includes, for example, imparting kokumi to a food or drink that does not have kokumi, and enhancing the kokumi of a food or drink that has kokumi. The presence or absence and the degree of kokumi can be evaluated by sensory evaluation.

[0035] The glycosylated protein of the present invention can be used by being contained in a food or drink. The content of the glycosylated protein of the present invention in a food or drink is not particularly limited, and can be appropriately set according to the type of food or drink, the desired degree of full-bodied taste, and the like. In one embodiment, the glycosylated protein of the present invention is preferably used to impart full-bodied taste to a beer-taste alcoholic beverage, a non-alcoholic beer-taste beverage, and the like. A beer-taste alcoholic beverage refers to an alcoholic beverage having a beer-specific flavor obtained by producing beer by brewing in a conventional manner. A non-alcoholic beer-taste beverage refers to a carbonated beverage having a beer-like flavor, and is a non-alcoholic type that does not substantially contain alcohol. The glycosylated protein of the present invention may be used alone or in combination of two or more kinds. The glycosylated protein of the present invention may be a purified protein.

[0036] The glycosylated protein of the present invention can be used as an active ingredient of a full-bodied flavor imparting agent used to impart full-bodied flavor to foods, beverages, and the like. The present invention also encompasses an agent for imparting kokumi flavor comprising the glycosylated protein of the first aspect of the present invention (glycosylated protein (A)) and / or the glycosylated protein of the second aspect of the present invention (glycosylated protein (B)). The body flavor imparting agent of the present invention comprises a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 160th amino acid and / or the 189th amino acid in the amino acid sequence shown in SEQ ID NO:1 of the protein described in any of (a1) to (a3) ​​above is glycated, and / or a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO:2 of the protein described in any of (b1) to (b3) above is glycated.

[0037] The kokumi-imparting agent of the present invention comprises at least one of the glycosylated proteins of the present invention, and typically comprises at least one of the glycosylated proteins as an active ingredient. In one embodiment, the kokumi-imparting agent preferably comprises the glycosylated protein of the first embodiment and the glycosylated protein of the second embodiment of the present invention. The kokumi-imparting agent may comprise the glycosylated protein of the first embodiment or the glycosylated protein of the second embodiment of the present invention. The glycosylated protein of the first embodiment of the present invention may be used alone or in combination of two or more kinds. The glycosylated protein of the second embodiment of the present invention may be used alone or in combination of two or more kinds. The glycosylated protein of the first embodiment of the present invention, the glycosylated protein of the second embodiment, and the preferred embodiments thereof are the same as those described above.

[0038] The body flavor imparting agent preferably comprises a glycosylated protein (also referred to as glycosylated protein (a1)) in which the side chain amino group of the lysine at the position corresponding to the 160th amino acid and / or the 189th amino acid in the amino acid sequence shown in SEQ ID NO:1 of the protein described in (a1) above is glycated, and / or a glycosylated protein (also referred to as glycosylated protein (b1)) in which the side chain amino group of the lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO:2 of the protein described in (b1) above is glycated, and more preferably comprises glycosylated protein (a1) and glycosylated protein (b1).

[0039] The body taste imparting agent of the present invention can be used, for example, to impart body taste to food and beverages. The body taste imparting agent of the present invention can be suitably used, for example, to impart body taste to beer-taste alcoholic beverages or non-alcoholic beer-taste beverages. The beer-taste alcoholic beverage is preferably beer. When imparting body taste to food and beverages using the body taste imparting agent of the present invention, the body taste imparting agent may be contained in the food and beverage. The method of adding the body taste imparting agent to the food and beverage is not particularly limited, and examples of the method include a method of mixing the body taste imparting agent in advance with the raw materials used in the production, and a method of adding the agent at any step in the production process of the food and beverage.

[0040] The present invention also encompasses the use of the glycosylated protein of the first aspect of the present invention (glycosylated protein (A)) and / or the glycosylated protein of the second aspect of the present invention (glycosylated protein (B)) for imparting full body flavor to foods and beverages. When the glycosylated protein of the first aspect of the present invention (glycosylated protein (A)) and / or the glycosylated protein of the second aspect of the present invention (glycosylated protein (B)) is contained in a food or beverage, the food or beverage can be imparted with a rich flavor. The present invention also encompasses a method for imparting full body flavor to a food or drink, which comprises adding the glycosylated protein of the first aspect and / or the glycosylated protein of the second aspect of the present invention to the food or drink. The above uses and methods use a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 160th position and / or the 189th position in the amino acid sequence shown in SEQ ID NO:1 of the protein described in any of (a1) to (a3) ​​above is glycated, and / or a glycosylated protein in which the side chain amino group of the lysine at the position corresponding to the 117th position in the amino acid sequence shown in SEQ ID NO:2 of the protein described in any of (b1) to (b3) above is glycated. The method of adding the glycosylated protein of the present invention to a food or drink is not particularly limited, and examples thereof include a method of mixing the glycosylated protein with the raw materials used in the production in advance, and a method of adding the glycosylated protein at any step in the production process of the food or drink. In a method of imparting full body taste to a food or drink, the above-mentioned full body taste imparting agent can be added to the food or drink. The food or drink is preferably a beer-taste alcoholic beverage or a non-alcoholic beer-taste beverage. In the above-mentioned uses and methods, at least one glycosylated protein of the present invention is used. The glycosylated protein of the first aspect of the present invention may be used alone or in combination of two or more kinds. The glycosylated protein of the second aspect of the present invention may be used alone or in combination of two or more kinds. In one embodiment, the above uses and methods preferably use the glycosylated protein of the first aspect of the invention and the glycosylated protein of the second aspect of the invention. The above uses and methods may use the glycosylated protein of the first aspect of the invention or the glycosylated protein of the second aspect of the invention. In one embodiment, the above uses and methods preferably use the glycosylated protein (a1) and / or the glycosylated protein (b1), more preferably use the glycosylated protein (a1) and the glycosylated protein (b1). EXAMPLES

[0041] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0042] <Example 1> North American malt was crushed to an appropriate size and placed in a brewing tank, where it was mixed with warm water. It was kept at a temperature suitable for the malt enzymes for a sufficient amount of time for saccharification, during which the enzymes in the malt converted the starch into sugar, producing a saccharified liquid. The saccharified liquid was then filtered, hops were added, and the liquid was boiled to produce hot wort in preparation for fermentation. The hot wort was cooled, and brewer's yeast was added to it. Over the course of a few days, the yeast broke down the sugar in the wort into alcohol and carbon dioxide, producing green beer. The green beer was then transferred to a storage tank and stored at low temperatures for several dozen days. During this time, the beer was aged, and its taste and aroma were harmonised. After aging was complete, the beer was filtered and bottled.

[0043] Glycosylated proteins were purified from beer brewed from North American malt using the above method (hereinafter also referred to as beer (I)) as follows.

[0044] (1) Fractionation by cation exchange resin 50 mL of the cation exchange resin SP Sepharose was placed in a free column. Beer (I) was adsorbed onto the resin. The adsorbed resin was then transferred to a column, washed with 20 mM sodium acetate buffer (pH 4.5), and then eluted with 20 mM sodium acetate (pH 4.5) + 0.5 M-NaCl, and fractions were collected. The fractions obtained were evaluated by SDS-PAGE, and fractions containing 40 kDa glycosylated protein (40 kDa protein) were collected and used as cation exchange resin-bound fractions.

[0045] (2) Ultrafiltration (buffer exchange) The cation exchange resin-bound fraction obtained in (1) was added to a water-washed ultrafiltration unit (Merck Amicon Ultra-15 30K), centrifuged (3500 rpm, 10 minutes, room temperature) and ultrafiltered to obtain a concentrate.

[0046] (3) Ammonium sulfate fraction 20 mM phosphate buffer (pH 7.0) + 2 M ammonium sulfate was placed in a beaker, and the concentrated solution obtained in (2) was added dropwise to it and stirred. The suspension was then centrifuged (2330 g, 10 minutes, room temperature). The supernatant was collected in a separate container. The collected solution was concentrated using an ultrafiltration unit. 20 mM sodium acetate (pH 4.5) was added to the concentrated solution, which was then centrifuged (2330 g, 10 minutes, room temperature) to concentrate, yielding a purified 40 kDa protein product (Bradford quantification (bovine serum albumin (BSA) equivalent), 20.4 mg / mL, 2.21 mL). The purity of the purified 40 kDa protein product obtained was confirmed by SDS-PAGE.

[0047] Analysis of the 40kDa protein and its modifications Analysis of the 40 kDa protein was carried out as follows. (1) Preparation of gel pieces 10 μL of the purified 40 kDa protein solution (concentration 1 mg / mL, 20 mM sodium acetate buffer (pH 4.5)) was electrophoresed on SDS-PAGE and stained with CBB (Coomassie Brilliant Blue), and the gel of the 40 kDa protein was excised.

[0048] (2) Enzymatic digestion of proteins The excised gel pieces were cut into small pieces, reduced with dithiothreitol (56°C, 1 hour), and carbamidomethylated with iodoacetamide (under dark, at room temperature, for 45 minutes). Next, 15 μL of 10 ng / μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium bicarbonate solution) containing 0.01% ProteaseMax, 15 μL of 5 mM calcium chloride, and 50 mM ammonium bicarbonate solution were added, and the mixture was incubated overnight, after which the enzyme digestion solution was collected. The collected solution was dried under reduced pressure and redissolved in 0.1% formic acid solution. This was used for LC-MS / MS analysis.

[0049] (3) Measurement by LC-MS / MS The LC-MS / MS measurements were carried out under the following conditions. Equipment used: Direct flow nanoLC system Easy-nLC 1000TM (Thermo Scientific) Trap column: Acclaim PepMap® (Thermo Scientific) Analytical column: NANO HPLC CAPILLARY COLUMN (Nikkyo Technos Co., Ltd.) Liquid chromatograph mass spectrometer: Q Exactive Plus (Thermo Scientific) Mobile phase: Solution A: 0.1% formic acid / water, Solution B: 0.1% formic acid / acetonitrile Flow rate: 300nL / min Gradient: 0-40%B / 0-30min, 40-60%B / 30-35min, 60-90%B / 35-37min, 90%B / 37-45min Injection volume: 10μL Ionization mode: ESI Positive Measurement range: MS1 (m / z 350-1750) Data Dependent Scan Mode

[0050] (4) Protein and modification analysis Protein identification and modification searches were performed under the following conditions. Search software: Proteome Discoverer 2.2.0.388 (ThermoFisher) Species: Barley (Hordeum vulgare), hops (Humulus), yeast (Saccharomyces cerevisiae) Search conditions:Digestive enzymes:Chymotrypsin Modification (Static): Carbamidomethyl (Cysteine) Modification (Dynamic): Oxidation (Methionine), Hex (K, R, Protein N-term), Hex (2) (K, R, Protein N-term), Hex (3) (K, R, Protein N-term), Acetyl (Protein N-term) Precursor ion mass error range: Monoisotopic, ±10 ppm Product ion mass error range: ±0.02 Da Maximum number of misscleavage: 5 Confidence level (Percolator): High (the most probable of the three levels of certainty) Database:SwissProt

[0051] As a result, the 40 kDa glycosylated protein was found to be glycosylated barley-derived Serpin Z4 (sequence coverage: 77.2%) and glycosylated barley-derived Serpin Z7 (sequence coverage: 72.8%). The amino acid sequence of barley-derived Serpin Z4 is shown in SEQ ID NO: 1, and the amino acid sequence of barley-derived Serpin Z7 is shown in SEQ ID NO: 2.

[0052] The results of modification analysis of barley-derived Serpin Z4 by LC-MS / MS are shown in Tables 1 to 8. Tables 1 to 4 show peptide fragments consisting of amino acids 142 to 174 of the amino acid sequence of barley-derived Serpin Z4 purified from beer (the amino acid sequence shown in SEQ ID NO: 1). The results of LC-MS / MS analysis of (EAVGQVNSWVEQVTTGLIKQILPPGSVDNTTKL (SEQ ID NO: 3)) (also referred to as Z4(142-174) peptide) are shown. Table 1 shows the m / z values ​​of precursor ions of peptide fragments by LC-MS. Tables 2 to 4 show the m / z values ​​of fragment ions of peptide fragments shown in Table 1 by LC-MS / MS. Table 2 shows the MH + This is the result of the analysis of the fragment ion of the peptide of 3521.885 Da. MH + The fragment ion of the peptide at 3521.885 Da is the fragment ion of the Z4(142-174) peptide without sugar modification. + The results of the analysis of the fragment ions of the peptide of 3683.935 Da are shown in Table 4. + This is the result of an analysis of fragment ions of the 3845.980 Da peptide.

[0053] Tables 5 to 8 show the results of LC-MS / MS analysis of a peptide fragment consisting of amino acids 182 to 200 of the amino acid sequence of Serpin Z4 derived from barley purified from beer (FKGAWDQKFDESNTKCDSF (SEQ ID NO: 4)) (also referred to as Z4(182-200) peptide). Table 5 shows the m / z values ​​of the precursor ions of the peptide fragments by LC-MS. Tables 6 to 8 show the m / z values ​​of the fragment ions of the peptide fragments by LC-MS / MS. Table 6 shows the m / z values ​​of the MH peptides shown in Table 5. + This is the result of the analysis of the fragment ion of the peptide of 2310.012 Da. MH + The fragment ion of the peptide at 2310.012 Da is the fragment ion of the Z4(182-200) peptide without sugar modification. + The results of the analysis of the fragment ions of the peptide of 2472.064 Da are shown in Table 8. + This is the result of an analysis of fragment ions of the 2634.118 Da peptide.

[0054] In Tables 2 to 4 and 6 to 8, No. is the number of the amino acid in the amino acid sequence shown in SEQ ID NO: 1. The bold characters surrounded by bold frames are the detected fragment ions. Regarding the modifications in the peptides shown in Table 1, K19 indicates that a modification of the 19th lysine in the Z4 (142-174) peptide was detected. Regarding the modifications in the peptides shown in Table 5, K8 indicates that a modification of the 8th lysine in the Z4 (182-200) peptide was detected.

[0055] [Table 1]

[0056] [Table 2]

[0057] [Table 3]

[0058] [Table 4]

[0059] [Table 5]

[0060] [Table 6]

[0061] [Table 7]

[0062] [Table 8]

[0063] The results shown in Tables 1 to 8 indicate that glycosylated Serpin Z4 includes those with a glycation structure in which the amino group of one or more lysines, lysine 160 (K160) and lysine 189 (K189), is modified with hexose. The Z4(142-174) peptide contains lysine 160 (K160) of Serpin Z4. From the m / z values ​​of No. 160 to 173 detected in the b+, b2+, and y+ columns of Table 2 and the m / z values ​​of No. 160 to 173 in the b+, b2+, and y+ columns of Table 3, MH + It was found that 162 Da, which corresponds to a hexose, was added to the lysine (K160) of the fragment ion No. 160 of the 3683.935 Da peptide. + It was found that the 3683.935 Da peptide contains a peptide having a modified sequence structure in which one hexose has been added to K160. From the m / z values ​​of No. 160 to 173 in the b+, b2+, and y+ columns in Table 2 and the m / z values ​​of No. 160 to 173 in the b+, b2+, and y+ columns in Table 4, it was found that MH + It was found that the lysine (K160) at No. 160 in the fragment ion of the 3845.980 Da peptide had a 324 Da addition corresponding to a dihexose. + The fragment ions of the 3845.980 Da peptide were found to contain a peptide with a modified sequence structure in which a dihexose was added to K160.

[0064] Tables 6 to 8 show the analysis results of the Z4 (182-200) peptide. The Z4 (182-200) peptide contains the 189th lysine (K189) of Serpin Z4. From the m / z values ​​of No. 189 to 199 in the b+ and y+ columns of Table 6 and the m / z values ​​of No. 189 to 199 in the b+, b2+, and y+ columns of Table 7, MH + It was found that 162 Da, which corresponds to a hexose, is added to the lysine (K189) of No. 189 of the fragment ion of the 2472.064 Da peptide. +The fragment ions of the 2472.064 Da peptide were found to include a peptide having a modified sequence structure in which one hexose was added to K189. From the m / z values ​​of No. 189 to 199 in the b+, b2+, and y+ columns of Table 6 and the m / z values ​​of No. 189 to 199 in the b+, b2+, and y+ columns of Table 8, MH + It was found that the lysine (K189) at No. 189 in the fragment ion of the 2634.118 Da peptide had a 324 Da addition corresponding to a dihexose. + The fragment ion of the peptide at 2634.118 Da was found to include a peptide with a modified sequence structure in which two hexoses were added to K189. Serpin Z4, in which the amino acid groups of one or more of the lysines at positions 160 (K160) and 189 (K189) in the amino acid sequence shown in SEQ ID NO: 1 are glycated, has not been reported so far. The hexose attached to the lysine of the sugar-modified Serpin Z4 was presumed to be glucose or xylose, and the dihexose was presumed to be maltose or isomaltose.

[0065] <Example 2> Glycosylated proteins were purified from three different types of beer, and the purified glycosylated proteins were added to beer and subjected to a sensory evaluation. The relationship between the glycation of lysine 160 and / or 189 in barley-derived Serpin Z4 and its effect of imparting kokumi flavor was investigated.

[0066] (Purification of 40 kDa glycosylated protein from three types of beer) In the same manner as in the purification of protein from beer (I) in Example 1, a 40 kDa glycosylated protein was purified from three types of beer (beers B, C, and D) to obtain a solution containing the glycosylated protein.

[0067] (Analysis of protein concentration and glycosylated proteins in glycosylated protein solutions purified from three types of beer) The protein concentration and glycated lysine concentration of the solutions containing glycosylated proteins purified from three types of beer (B, C, or D) were determined by the following method.

[0068] (Glycated Lysine Concentration) The concentration of lysine whose side chain amino group was glycated was colorimetrically quantified by the NBT (Nitroblue tetrazolium) method using a Fructosamine Assay kit (Abcam product, ab228558). The number of tests for each sample and blank was 2 (n=2), and the average value was used. The amount of glycated lysine amino groups (nmol / mL) in the solution containing the glycosylated protein purified from each beer was determined, and the protein concentration (mg / mL) in the solution containing the glycosylated protein purified from each beer was measured by the Bradford method (BSA equivalent). The amount of glycated lysine amino groups (nmol / mL) in a solution containing a glycosylated protein was divided by the protein concentration (mg / mL) to calculate the concentration of glycated lysine amino groups per weight of purified glycosylated protein (nmol / mg-protein).

[0069] (Analysis of purified glycosylated proteins) The analysis results of the concentration of glycated lysine amino groups and protein concentration (measured by the Bradford method (BSA equivalent)) in solutions containing glycosylated proteins purified from three types of beer (B, C, D) are shown in Table 9. The concentration of glycated lysine amino groups shown in Table 9 is the concentration of glycated lysine amino groups per weight of purified glycosylated protein.

[0070] [Table 9]

[0071] (Evaluation of the addition of richness to beer by glycosylated proteins purified from three types of beer) A solution containing glycosylated proteins purified from three types of beer (B, C, D) was added to commercially available beer A and subjected to sensory evaluation. In this case, the glycosylated protein solution purified from beer B, C, or D was added to beer A so that the concentration of glycated lysine in the sensory evaluation samples was the same, to obtain samples B, C, and D. Sample B is a sample to which a glycosylated protein solution purified from beer B was added, sample C is a sample to which a glycosylated protein solution purified from beer C was added, and sample D is a sample to which a glycosylated protein solution purified from beer D was added. The total purine concentration of commercially available beer A was 10.39 mg / 100 mL. The total purine concentration was measured by a degradation method at the Japan Food Research Center.

[0072] (Sensory evaluation) A sensory evaluation was carried out on commercially available beer A (control (no additives)) and samples B, C, and D. For the sensory evaluation, the body taste of commercially available beer A, which does not contain added glycosylated proteins, was set as a standard score of 1.5 points, and four expert panelists scored the samples in increments of 0.1 points. The scores were then averaged. The body taste was evaluated according to the following criteria. 0: Not felt at all 1 point: Somewhat felt 2 points: Clear feeling 3 points: Very strong feeling

[0073] The average scores are shown in Table 10. The control (no addition) in Table 10 is beer A to which no glycosylated protein was added. As a result, as shown in Table 10, sample C had the strongest full-bodied flavor, followed by sample D and sample B.

[0074] [Table 10]

[0075] (Analysis of the amount of lysine at positions 160 and 189 bound to the side chain amino group of glycosylated proteins purified from three types of beer) Modification analysis of glycosylated proteins purified from three types of beer (B, C, D) was performed in the same manner as in Example 1. The LC-MS ion area value of a peptide fragment (EAVGQVNSWVEQVTTGLIKQILPPGSVDNTTKL (SEQ ID NO: 3), unmodified Z4(142-174) peptide) consisting of amino acids 142 to 174 of the amino acid sequence of Serpin Z4 (SEQ ID NO: 1) and not glycosylated, and the LC-MS ion area value of the peptide fragment (glycosylated Z4(142-174) peptide) containing lysine 160 (K160) with sugar added to the side chain amino group were calculated. Furthermore, based on these area values, the ratio of (total ion area value of glycosylated Z4 (142-174) peptide) / (ion area value of unmodified Z4 (142-174) peptide) was calculated for the glycosylated proteins purified from the three types of beer, and the ratio of glycosylated Z4 (142-174) peptide in which the side chain amino group of K160 is glycated to unmodified Z4 (142-174) peptide was compared (Figure 1). (Considering the possibility that the LC-MS ionization efficiency is not the same for unmodified and modified peptides, the area ratios of each sample of the three types of beer were compared side by side.)

[0076] FIG. 1 is a graph showing the ratio of the total LC-MS ion area value of the glycosylated Z4(142-174) peptide in which one hexose or two hexoses (dihexoses) are bound to K160 in a glycosylated protein purified from three different types of beer (B, C, D) to the LC-MS ion area value (ion area value of unmodified Z4(142-174) peptide) of the peptide fragment (unmodified Z4(142-174) peptide) consisting of amino acids 142 to 174 of the amino acid sequence of Serpin Z4 (sequence number 1). The area ratio on the vertical axis in FIG. 1 is the ratio of the above area values ​​(total ion area values ​​of sugar-modified Z4(142-174) peptide) / (ion area value of unmodified Z4(142-174) peptide).

[0077] The "total ion area value of sugar-modified Z4(142-174) peptide" is the sum of the ion area of ​​the Z4(142-174) peptide in which a monosaccharide (Hex) is bound to the side chain of lysine at position 160 and the ion area of ​​the Z4(142-174) peptide in which a disaccharide (diHex) is bound to the side chain of lysine at position 160. The "ion area value of unmodified Z4(142-174) peptide" is the ion area of ​​the Z4(142-174) peptide in which no sugar is bound to the side chain of lysine at position 160 (the side chain of K160 is unmodified).

[0078] Furthermore, the LC-MS ion area values ​​of the peptide fragment (FKGAWDQKFDESNTKc(Carbamidomethyl)DSF, unmodified Z4(182-200) peptide) consisting of amino acids 182 to 200 of the amino acid sequence of Serpin Z4 (SEQ ID NO: 1) and not glycosylated, and the peptide fragment (glycosylated Z4(182-200) peptide) containing lysine 189 (K189) with sugar added to the side chain amino group were calculated. Based on these area values, the ratio of glycosylated Z4(182-200) peptide in which the side chain amino group of K189 is glycated to unmodified Z4(182-200) peptide was calculated based on the total ion area values ​​of glycosylated Z4(182-200) peptide / (ion area value of unmodified Z4(182-200) peptide) for the glycosylated proteins purified from the three types of beer was compared (Figure 2). (Considering the possibility that the ionization efficiency of LC-MS may not be the same for unmodified and modified peptides, the area ratios of the three beer samples were compared side by side.)

[0079] FIG. 2 is a graph showing the ratio of the total LC-MS ion area value of the glycosylated Z4(182-200) peptide in which one or two hexoses are bound to the 189th lysine (K189) in the peptide to the LC-MS ion area value of the unmodified Z4(182-200) peptide (ion area value of unmodified Z4(182-200) peptide) of the LC-MS ion area value of the unmodified Z4(182-200) peptide, which consists of amino acids 182-200 of the amino acid sequence of Serpin Z4 (sequence number 1) in glycosylated proteins purified from three different types of beer (B, C, D). The area ratio on the vertical axis in FIG. 2 is the ratio of the above area values ​​(total ion area values ​​of sugar-modified Z4(182-200) peptide) / (ion area value of unmodified Z4(182-200) peptide).

[0080] The "total ion area value of sugar-modified Z4(182-200) peptide" is the sum of the ion area of ​​Z4(182-200) peptide in which a monosaccharide (Hex) is bound to the side chain of lysine at position 189 and the ion area of ​​Z4(182-200) peptide in which a disaccharide (diHex) is bound to the side chain of said lysine. The "ion area value of unmodified Z4(182-200) peptide" is the ion area of ​​Z4(182-200) peptide in which no sugar is bound to the side chain of lysine at position 189 (the side chain of K189 is unmodified).

[0081] As a result, for peptide fragments containing lysine 160 (K160) with sugar bound to the side chain amino group, the ratio of modified peptide fragments (glycosylated Z4 (142-174) peptides) to unmodified peptide fragments (unmodified Z4 (142-174) peptides) was highest in beer C, followed by beer D and beer B. For peptide fragments containing lysine 189 (K189), the ratio of modified peptide fragments (glycosylated Z4 (182-200) peptides) to unmodified peptide fragments (unmodified Z4 (182-200) peptides) was highest in beer C, followed by beer D and beer B. These results were consistent with the sensory evaluation showing that sample C had the highest full-bodied taste. It was found that there is a correlation between the content of glycosylated proteins in which sugars are bound to the side chain amino groups of lysine 160 and lysine 189 and full-bodied taste.

[0082] 1 and 2, it was revealed that the richness is improved when the ratio of glycosylated proteins in which the side chain amino groups of lysine at positions 160 and / or 189 of the protein having the amino acid sequence of SEQ ID NO: 1 are glycated is high. It was revealed that the protein in which the amino groups of lysine at positions 160 and / or 189 are glycated has an excellent effect of imparting richness.

[0083] <Example 3> For detailed analysis of glycosylation of Serpin Z7, targeted monitoring was performed by LC-MS / MS. Samples for LC-MS / MS analysis were prepared in the same manner as in Example 1. LC-MS / MS measurement was performed in the same manner as in Example 1, except that the Data Dependent Scan mode was changed to target ion monitoring. The target ions used were m / z 524.788, m / z 605.814, m / z 686.840, and m / z 767.868.

[0084] The results of modification analysis of barley-derived Serpin Z7 by LC-MS / MS are shown in Tables 11 to 15. Tables 11 to 15 show the results of LC-MS / MS analysis of a peptide fragment (SLKPSFQEL (SEQ ID NO: 5)) (also referred to as Z7 (115-123) peptide) consisting of amino acids 115 to 123 of the amino acid sequence of barley-derived Serpin Z7 (amino acid sequence shown in SEQ ID NO: 2) purified from beer. Table 11 shows the m / z values ​​of the precursor ions of the peptide fragments by LC-MS. Tables 12 to 15 show the m / z values ​​of the fragment ions of the peptide fragments shown in Table 11 by LC-MS / MS. Table 12 shows the MH + This is the result of the analysis of the fragment ion of the peptide of 1048.566 Da. MH + The fragment ion of the peptide at 1048.566 Da is the fragment ion of the Z7 (115-123) peptide without sugar modification. + The results of the analysis of the fragment ions of the peptide of 1210.619 Da are shown in Table 14. + The results of the analysis of the fragment ions of the peptide of 1372.670 Da are shown in Table 15. + This is the result of an analysis of fragment ions of a peptide of 1534.729 Da.

[0085] [Table 11]

[0086] In Tables 12 to 14, No. is the number of the amino acid in the amino acid sequence shown in SEQ ID NO: 2. The bold characters enclosed in a bold frame are the detected fragment ions. Regarding the modifications in the peptides shown in Table 11, K3 indicates that a modification of the third lysine in the Z7(115-123) peptide was detected.

[0087] [Table 12]

[0088] [Table 13]

[0089] [Table 14]

[0090] [Table 15]

[0091] The results shown in Tables 11 to 15 indicate that the glycosylated Serpin Z7 includes those with a structure in which the amino group of the 117th lysine (K117) is modified with hexose (glycation). The Z7(115-123) peptide contains the 117th lysine (K117) of Serpin Z7. From the detected m / z values ​​in the b+, b2+ and y+ columns in Tables 12 and 13, MH + It was found that 162 Da, which corresponds to a hexose, is added to the lysine (K117) at No. 117 in the fragment ion of the 1210.619 Da peptide. + It was found that the fragment ion of the 1210.619 Da peptide contained a peptide having a modified sequence structure in which one hexose was added to K117. From the m / z values ​​detected in the b+, b2+ and y+ columns in Tables 12 and 13, MH + It was found that lysine (K117) at No. 117 in the fragment ion of the 1372.670 Da peptide had a 324 Da addition corresponding to a dihexose. + It was found that the fragment ions of the 1372.670 Da peptide contained a peptide with a modified sequence structure in which dihexose was added to K117. From the m / z values ​​detected in the b+, b2+ and y+ columns in Tables 12 and 13, MH +It was found that lysine (K117) at No. 117 in the fragment ion of the 1534.729 Da peptide had a 486 Da moiety corresponding to trihexose. + The fragment ions of the 1534.729 Da peptide were found to contain a peptide with a modified sequence structure in which trihexose was added to K117. Serpin Z7, in which the amino group of lysine 117 (K117) in the amino acid sequence shown in SEQ ID NO: 2 is glycated, has not been reported so far. The hexose attached to the lysine of the sugar-modified Serpin Z7 was presumed to be glucose or xylose, the dihexose was presumed to be maltose or isomaltose, and the trihexose was presumed to be maltotriose or isomaltotriose.

[0092] <Example 4> (Purification of 40kDa glycosylated protein from three types of beer) In the same manner as in the purification of protein from beer (I) in Example 1, a 40 kDa glycosylated protein was purified from three types of beer (beers E, F, and G) to obtain a solution containing the glycosylated protein.

[0093] (Analysis of protein concentration and glycosylated proteins in glycosylated protein solutions purified from three types of beer) Using the same method as in Example 2, the protein concentration (mg / mL) of the solution containing the glycosylated protein purified from three types of beer (E, F, or G) and the concentration of glycated lysine amino groups per weight of the purified glycosylated protein (nmol / mg-protein) were determined.

[0094] (Analysis of purified glycosylated proteins) The analysis results of the concentration of glycated lysine amino groups and protein concentration (measured by the Bradford method (BSA equivalent)) in solutions containing glycosylated proteins purified from three types of beer (E, F, and G) are shown in Table 16. The concentration of glycated lysine amino groups shown in Table 16 is the concentration of glycated lysine amino groups per weight of purified glycosylated protein.

[0095] [Table 16]

[0096] (Evaluation of the addition of richness to beer by glycosylated proteins purified from three types of beer) A solution containing glycosylated proteins purified from three types of beer (E, F, or G) was added to commercially available beer A, and a sensory evaluation was performed. In this case, the glycosylated protein solution purified from beer E, F, or G was added to beer A so that the concentration of glycated lysine in the sensory evaluation samples would be the same, thereby obtaining samples E, F, and G. Sample E is a sample to which a glycosylated protein solution purified from beer E was added, sample F is a sample to which a glycosylated protein solution purified from beer F was added, and sample G is a sample to which a glycosylated protein solution purified from beer G was added.

[0097] (Sensory evaluation) A sensory evaluation of commercially available beer A (control (no additives)) and samples E, F, and G was conducted by four expert panelists in the same manner as in the sensory evaluation of Example 2, except that the scores were given in increments of 0.05 points, and the full-bodied taste was evaluated. The average scores are shown in Table 17. The control (no addition) in Table 17 is beer A to which no glycosylated protein was added. As a result, as shown in Table 17, sample F had the strongest full-bodied flavor, followed by sample G and sample E.

[0098] [Table 17]

[0099] (Analysis of the amount of lysine with sugar bound to the side chain amino group) Modification analysis of glycosylated proteins purified from three types of beer (E, F, G) was performed in the same manner as in Example 1. The LC-MS ion area value of a peptide fragment (SLKPSFQEL (SEQ ID NO: 5), unmodified Z7(115-123) peptide) consisting of amino acids 115 to 123 of the amino acid sequence of Serpin Z7 (SEQ ID NO: 2) and not glycosylated, and the LC-MS ion area value of the peptide fragment (glycosylated Z7(115-123) peptide) containing lysine 117 (K117) with sugar added to the side chain amino group were calculated. Furthermore, based on these area values, the ratio of the combined ion area values ​​of the glycosylated Z7(115-123) peptide to the ion area value of the unmodified Z7(115-123) peptide for the glycosylated proteins purified from the three types of beer (combined ion area values ​​of the glycosylated Z7(115-123) peptide) / (ion area value of the unmodified Z7(115-123) peptide) was calculated, and the ratio of the glycosylated Z7(115-123) peptide in which the side chain amino group of K117 is glycated to the unmodified Z7(115-123) peptide was compared. (Considering the possibility that the ionization efficiency of LC-MS is not the same for unmodified and modified peptides, the area ratios of each sample of the three types of beer were compared side by side.) Table 18 shows the ratio of the sum of the ion area values ​​of the sugar-modified Z7(115-123) peptide to the ion area value of the unmodified Z7(115-123) peptide (ratio of the total area value of the LC-MS ions of the sugar-modified Z7(115-123) peptide). The "total ion area value of sugar-modified Z7(115-123) peptide" is the sum of the ion area of ​​the Z7(115-123) peptide in which a monosaccharide (Hex) is bound to the side chain of the 117th lysine, the ion area of ​​the Z7(115-123) peptide in which a disaccharide (diHex) is bound to the side chain of the lysine, and the ion area of ​​the Z7(115-123) peptide in which a trisaccharide (triHex) is bound to the side chain of the lysine. The "ion area value of unmodified Z7(115-123) peptide" is the ion area of ​​the Z7(115-123) peptide in which no sugar is bound to the side chain of the 117th lysine (the side chain of K117 is unmodified).

[0100] [Table 18]

[0101] The ratio of glycosylated peptide fragments (glycosylated Z7(115-123) peptide) to unmodified peptide fragments (unmodified Z7(115-123) peptide) was highest in beer F, followed by beers G and E. This result was consistent with the sensory evaluation showing that sample F had the highest full-bodied taste. All of the glycosylated protein solutions purified from beers E, F and G contained glycosylated proteins in which the side chain amino groups of lysine at positions 160 and / or 189 of the protein having the amino acid sequence of SEQ ID NO:1 were glycated. [Industrial Applicability]

[0102] By using the glycosylated protein of the present invention, it is possible to impart full body flavor to foods and beverages.

Claims

1. A glycosylated protein in which the side chain amino group of lysine is glycated, wherein glucose, maltose or isomaltose is bound to the side chain amino group of the lysine at the position corresponding to the 160th amino acid and / or the 189th amino acid in the amino acid sequence shown in SEQ ID NO:1 of a protein described in any of (a1) to (a3) ​​below. (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine.

2. The glycosylated protein according to claim 1, wherein the protein described in (a1) to (a3) ​​is a protein derived from barley.

3. A glycosylated protein in which the side chain amino group of lysine is glycated, wherein glucose, maltose, isomaltose, maltotriose or isomaltotriose is bound to the side chain amino group of lysine at a position corresponding to amino acid 117 of the amino acid sequence shown in SEQ ID NO:2 of a protein described in any of (b1) to (b3) below. (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine.

4. The glycosylated protein according to claim 3, wherein the protein described in (b1) to (b3) is a protein derived from barley.

5. A kokumi-imparting agent comprising the following glycosylated protein (A) and / or the following glycosylated protein (B). Glycosylated protein (A): A glycosylated protein in which the side chain amino group of lysine is glycated, and glucose, maltose or isomaltose is bound to the side chain amino group of the lysine at the position corresponding to the 160th position and / or the 189th position of the amino acid sequence shown in SEQ ID NO: 1 of a protein described in any one of (a1) to (a3) ​​below: (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine. Glycosylated protein (B): A glycosylated protein in which the side chain amino group of lysine is glycated, and glucose, maltose, isomaltose, maltotriose or isomaltotriose is bound to the side chain amino group of lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO:2 of a protein described in any one of (b1) to (b3) below. (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine.

6. The body flavor imparting agent according to claim 5, which is used to impart body flavor to a beer-taste alcoholic beverage or a non-alcoholic beer-taste beverage.

7. Use of the following glycosylated protein (A) and / or the following glycosylated protein (B) for imparting full body to a food or drink. Glycosylated protein (A): A glycosylated protein in which the side chain amino group of lysine is glycated, and glucose, maltose or isomaltose is bound to the side chain amino group of the lysine at the position corresponding to the 160th position and / or the 189th position of the amino acid sequence shown in SEQ ID NO: 1 of a protein described in any one of (a1) to (a3) ​​below: (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine. Glycosylated protein (B): A glycosylated protein in which the side chain amino group of lysine is glycated, and glucose, maltose, isomaltose, maltotriose or isomaltotriose is bound to the side chain amino group of lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO:2 of a protein described in any one of (b1) to (b3) below. (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine.

8. The use according to claim 7, wherein the food or drink is a beer-flavored alcoholic beverage or a non-alcoholic beer-flavored beverage.

9. A method for imparting full body to a food or drink, comprising adding the following glycosylated protein (A) and / or the following glycosylated protein (B) to the food or drink. Glycosylated protein (A): A glycosylated protein in which the side chain amino group of lysine is glycated, and glucose, maltose or isomaltose is bound to the side chain amino group of the lysine at the position corresponding to the 160th position and / or the 189th position of the amino acid sequence shown in SEQ ID NO: 1 of a protein described in any one of (a1) to (a3) ​​below: (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (a2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 160th and / or 189th lysine. (a3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 1 and in which the amino acid at the position corresponding to the 160th position and / or the position corresponding to the 189th position of the amino acid sequence shown in SEQ ID NO: 1 is lysine. Glycosylated protein (B): A glycosylated protein in which the side chain amino group of lysine is glycated, and glucose, maltose, isomaltose, maltotriose or isomaltotriose is bound to the side chain amino group of lysine at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO:2 of a protein described in any one of (b1) to (b3) below. (b1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 9 amino acids have been deleted, substituted, inserted and / or added in a region other than the 117th lysine. (b3) a protein having an amino acid sequence which has an identity of 98% or more to the amino acid sequence shown in SEQ ID NO: 2 and in which the amino acid at the position corresponding to the 117th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is lysine.

10. The method according to claim 9, wherein the food or drink is a beer-taste alcoholic beverage or a non-alcoholic beer-taste beverage.

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