RU2496864C2 - YEAST EXTRACT CONTAINING GAMMA-Glu-X OR GAMMA-Glu-X-Gly, AND METHOD OF ITS PRODUCTION - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: alternative methods are proposed to produce a yeast extract to give taste of "kokumi" to food products, containing peptide γ-Glu-X or γ-Glu-X-Gly. One version of proposed methods includes growing of yeast in a nutrient medium containing peptide selected from the group that consists of γ-Glu-X, γ-Glu-X-Gly and X-Gly, and preparation of the yeast extract from the produced cells. Another version includes interaction of γ-glutamiltransferase on the yeast extract containing X or X-Gly, produced from yeast grown in the nutrient medium, to which amino acid X or peptide X-Gly is added. X is an amino acid or its derivative, different from Cys and its derivatives. The yeast extract is described to give "kokumi" taste to food products and produced by the specified methods, containing peptide selected from the group that consists of γ-Glu-X and γ-Glu-X-Gly, in the amount of 0.005% or more of dry weight of the yeast extract, differing by the fact that X is amino acid or its derivative, different from Cys and its derivatives.

EFFECT: invention makes it possible to produce a yeast extract with improved properties.

24 cl, 8 dwg, 14 tbl, 13 ex

Description

Technical field

The present invention relates to a yeast extract containing γ-Glu-X-Gly or γ-Glu-X, and to a method for its preparation. The yeast extract according to the present invention is applicable in the field of food products such as seasonings and health products.

State of the art

Yeast extract gives atsumi food (density), umami flavor and is widely used as a seasoning in the food industry. It is known that especially glutathione (hereinafter also referred to as “GSH”), which is a tripeptide consisting of glutamic acid, cysteine and glycine, gives food the taste of “kokumi” (Ueda et al., Agric. Biol. Chem., 54, 163-169 (1990), Ueda et al., Biosci. Biotechnolo. Biochem., 61, 1977-1980 (1997)), and various condiments containing GSH are being developed.

Meanwhile, although it has been reported that a calcium-sensitive receptor (CaSR), a class C G protein, responds to GSH (Wang et al., Journal of Biological Chemistry, 281, 8864-8870 (2006)), its physiological role is not clear . In addition, this CaSR is also present in the cells of the tongue, and is believed to indicate its role in the sense of taste (Gabriel et al., Biochemical and Biophysical Research Communications, 378, 414-418 (2009)). Further, it has recently been shown that CaSR is involved in distinguishing between people the taste of “kokumi” (Ohsu et al., Journal of Biological Chemistry, 285, 1016-1022 (2010)). In this work, it was shown that not only GSH, which is already known as a “kokumi” flavored substance, but also, apparently, several γ-glutamyl compounds react with CaSR. In addition, it has been shown that these peptides having the general formula γ-Glu-X or γ-Glu-X-Gly (X is an amino acid or amino acid derivative other than Cys and its derivatives), for example, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val-Gly, etc. give the taste of kokumi (WO2007 / 055393). In addition, it was shown that the ester group of S- or O-carboxyalkylated peptides of γ-glutamyl or β-asparagil are also kokumi flavored compounds (WO2007 / 042288). Although these peptides give the food a “kokumi” flavor like GSH, unlike GSH, they do not have a reduced SH group. It is known that a substance having a reduced SH group, such as GSH, is generally unstable and its titer decreases as disulfide bonds form (WO2007 / 042288). However, γ-Glu-X, γ-Glu-X-Gly, etc., which are considered useful as peptides that give the taste of "kokumi" that do not have a reduced SH group in their composition, are stable.

Regarding foods containing a γ-Glu dipeptide, there are reports that various γ-Glu dipeptides are found in Gouda cheese, which has matured over an extended period, such as 44 weeks (Toelstede, S and Hofmann, T, J. Agric. Food. Chem., 2009). In this work, it was reported that various γ-Glu dipeptides, such as γ-Glu-Ala, γ-Glu-Glu and γ-Glu-Gln, were determined, and the content of γ-Glu dipeptides was determined - a maximum of 3590 μmol / kg. This value corresponds to 0.088% of dry matter.

However, any yeast extract containing γ-Glu-X or γ-Glu-X-Gly in an amount sufficient to impart flavor to “kokumi” is not known.

In addition, it is known that the synthesis and decomposition of glutathione, one of the γ-glutamyl compounds, is catalyzed by many enzymes called the enzymes of the γ-glutamyl cycle. In particular, it is known that γ-glutamyltranspeptidase transfers glutamate at the γ-position of GSH to another compound having an amino group, with the decomposition of GSH to cysteinylglycine (Protein Nucleic acid Enzyme, 1988-7, VOL. 33, NO. 9, ISSN 003909450, Special Issue "Epoch of glutathione research", pp. 1432-1433). It is believed that if a compound having an amino group is an amino acid in this rearrangement reaction, a γ-Glu-X dipeptide can be formed as a by-product. However, the search for microorganisms that efficiently produce γ-Glu-X has not been successful, in part because γ-Glu-X is a by-product.

Information regarding the γ-Glu-X dipeptide includes Micrococcus glutamicus culture fluid analysis (Ronald et al., Journal of Biological Chemistry, 240, p. 2508-2511 (1965)). This work reports that culture fluid was applied to various columns to separate peptides, etc., to isolate γ-Glu-Glu, γ-Glu-Val, and γ-Glu-Leu. However, they were discovered as a result of separation on different columns, and it is not known how much they were contained in the medium.

In addition, the GSH biosynthesis enzyme was isolated from Streptococcus agalactiae and Clostridium acetobutylicum and its substrate specificity was analyzed (Kino et al., JBB research communications, 352, pp. 351-359 (2007)). Two types of enzymes are usually involved in GSH biosynthesis, γ-glutamylcysteine synthetase, which combines Glu and Cys to form γ-Glu-Cys, and glutathione synthetase, which combines the resulting γ-Glu-Cys and Gly to form GSH. However, in the above two types of microorganisms, there is a unique enzyme corresponding to the hybrid of γ-glutamylcysteine synthetase and glutathione synthetase. There is evidence that according to the results of in vitro analysis, the recognition of the substrate by this enzyme is poorly expressed, for example, it also recognizes other amino acids (not only Cys) and, as a result, can form γ-Glu-X and γ-Glu-X-Gly. However, these are in vitro test results, and examples of the production by these microorganisms of detectable quantities of peptides, such as γ-Glu-X and γ-Glu-X-Gly, inside cells containing many compounds with an amino group in addition to the target X are described.

Yeast extracts are seasonings widely used in the food industry and are highly regarded by consumers. Therefore, as a carrier of γ-Glu-X-Gly or γ-Glu-X, a yeast extract form is more preferred. Examples of studies on the use of such extracts include work with mineral yeast. It is known that if metal is added to the medium, the yeast transports it into the cell (B. Volesky, H.A., Appl. Microbiol. BiotechnoL, 42; 797-806 (1995)). In particular, if trace amounts of elements such as zinc, iron, copper, manganese, selenium, molybdenum and chromium are added to the medium, yeast can be used as a source of the element, the enrichment of which is desirable for food products (Japanese Patent Laid-open (Kokai) No .2004-298014). In view of this fact, methods for producing mineral yeast are being developed (Japanese Patent Laid-open No.54-157890, Japanese Patent Laid-open No.60-75279, Japanese Patent Publication (Kokoku) No.6-16702).

In addition, enriching the yeast with these elements, you can get benefits related to taste. For example, Japanese Laid-open Patent Application No. 8-332081 describes yeast with a high manganese content. It describes that although there are foods on the market that are enriched with manganese by adding manganese salt, since it is an inorganic salt, the consequence of its addition is strong bitterness and astringent properties, and it is much more difficult to consume them regularly than natural manganese-containing foods, and it is disclosed technology for producing natural material by enriching yeast with manganese. As for nutritional value, the technology is disclosed in Japanese Patent Laid-Open No. 2008-99578. According to the data provided in the application, although zinc contributes to the improvement of taste sensations, the improvement of the generative function, etc., apparently, it cannot be considered that it is now consumed in sufficient quantities. If zinc is added during the cultivation of yeast, the yeast transports the zinc into the cells, but the water-soluble zinc does not accumulate in the yeast in its pure form, but binds to a protein or amino acid and accumulates in high concentrations in the form of amorphous zinc. Apparently, such amorphous zinc is more effectively absorbed in the body compared to crystalline zinc. As a result, by incorporating zinc into the yeast, improved absorption can be obtained compared to the consumption of pure zinc.

As described above, various benefits can be obtained by the absorption of the target substance by the yeast and the addition of yeast (in the form of a yeast or yeast extract) to food products. However, unlike minerals, which are important components of nutrition, the ability of yeast to absorb an amino acid or peptide is under complex control, and it is believed that it is difficult to simply apply the above technology to them.

Among studies of yeast dipeptides and tripeptides, many relate to the formation of GSH or γ-Glu-Cys. An example is the work that cites the evidence that GSH levels were increased by mutagenesis of Saccharomyces yeast and selection of a strain with increased resistance to zinc (Japanese Patent Application Laid-Open No. 02-295480), a message stating that expression suppression of the MET25 gene was depressed due to the mutant MET30 gene, as a result of which the intracellular content of γ-Glu-Cys was increased (Japanese Patent Application No. 2002-282743), etc. In addition, recent scientific discoveries include data on the transport of GSH via HGT1p. Although GSH and its dimer, GSSG, were transported into cells via HGT1p, even the presence of an excessive amount of amino acids, various dipeptides and tripeptides did not affect GSH transport by the HGT1p transporter. Therefore, it is believed that HGT1p is not a non-specific transporter, as previously thought, but a transporter specific for GSH (Bourbouloux et al., Journal of Biological Chemistry, 275, pp.13259-13265 (2000)). In addition, an active HGTIp site was also searched (Kaur et al., FEMS Yeast Res., 9, 849-866 (2009)).

As described above, although there is evidence of many discoveries related to GSH and its predecessor, there is virtually no evidence of yeast cells containing substances such as γ-Glu-X and γ-Glu-X-Gly, and a method for producing an extract from these cells.

SUMMARY OF THE INVENTION

The objectives of the present invention include the provision of a yeast extract containing γ-Glu-X or γ-Glu-X-Gly, and the provision of a method for producing said extract.

The aforementioned goals were achieved by the discovery of the fact that yeast transports γ-Glu-X and γ-Glu-X-Gly (X is a non-Cys amino acid or derivative thereof, the same applies to the description below) to the cell, and yeast an extract containing γ-Glu-X or γ-Glu-X-Gly can be obtained by preparing from said yeast cultured in a medium containing γ-Glu-X or γ-Glu-X-Gly. In addition, it was found that if the yeast is cultured in a medium containing γ-Glu-X or X-Gly, these compounds are transported to the cells, and γ-Glu-X-Gly can be formed in an enzymatic reaction within the cells. In addition, it was also found that a yeast extract containing γ-Glu-X or γ-Glu-X-Gly can be obtained by reacting γ-glutamyl transferase with a yeast extract to which an amino acid or peptide selected from the group consisting of X and X-Gly.

The aim of the present invention is the provision of a yeast extract containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, in an amount of 0.005% or more of the dry weight of the yeast extract, characterized in that X represents an amino acid or derivative thereof other than Cys and its derivatives.

Another objective of the present invention is the provision of the above yeast extract containing the peptide in an amount of 0.02% or higher.

It is also an object of the present invention to provide a yeast extract as described above, characterized in that X is Val.

It is also an object of the present invention to provide a yeast extract as described above, characterized in that said yeast is Saccharomyces cerevisiae.

Another objective of the present invention is the provision of a method for producing a yeast extract containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, including growing yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X, γ-Glu-X-Gly and X-Gly, and preparing from the obtained cells a yeast extract, wherein X is an amino acid or its derivative other than Cys and its derivatives.

It is also an object of the present invention to provide the method described above, characterized in that said culture medium contains 0.1 ppm (parts per million, 0.0001%) or more of said peptide, and said yeast extract contains a peptide selected from the group consisting of γ- Glu-X and γ-Glu-X-Gly, in an amount of 0.005% or more of the dry weight of the yeast extract.

It is also an object of the present invention to provide a method as described above, characterized in that X is Val.

It is also an object of the present invention to provide the method described above, characterized in that the yeast is modified in such a way that the ability to transport the indicated peptide to the cell in modified yeast is increased.

It is also an object of the present invention to provide the method described above, characterized in that the activity of HGT1p in said yeast is increased.

It is also an object of the present invention to provide the method described above, characterized in that said yeast is Saccharomyces cerevisiae.

It is also an object of the present invention to provide a method for producing a yeast extract containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, comprising reacting γ-glutamyl transferase with a yeast extract used as a feed to which added an amino acid or peptide selected from the group consisting of X and X-Gly, characterized in that X is an amino acid or its derivative, other than Cys and its derivatives.

It is also an object of the present invention to provide the method described above, characterized in that said amino acid or peptide is added in an amount of 1% or more of the dry weight of the yeast extract, and said yeast extract contains a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, in an amount of 0.005% or more of the dry weight of the yeast extract.

It is also an object of the present invention to provide a method as described above, characterized in that X is Val.

It is also an object of the present invention to provide the method described above, characterized in that said yeast is Saccharomyces cerevisiae.

According to the present invention, it is possible to obtain a yeast extract containing γ-Glu-X or / and γ-Glu-X-Gly. The yeast extract containing these peptides has an excellent kokumi flavor.

In addition, a yeast extract containing γ-Glu-X is also useful as a raw material for producing a yeast extract containing γ-Glu-X-Gly.

Brief Description of Drawings

Figure 1 shows mass chromatograms of standard samples of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly.

Figure 2 shows the structure of plasmid pUC19AOX-G418-BRI.

Figure 3 shows the structure of plasmid pKS-URA3-13.

Figure 4 shows the construction of the plasmid pKS-URA3-13-kanMX.

Figure 5 shows the construction of the plasmid pLoxP-PADH1-kanR5.

Figure 6 shows the construction of the plasmid pKS-URA3-PADH1-LR.

Figure 7 shows the construction of the plasmid pKS-URA3-P-ADH1.

Figure 8 shows a diagram of a DNA fragment used to replace a promoter.

DETAILED DESCRIPTION OF THE INVENTION

The yeast extract according to the present invention contains peptides selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, in an amount of 0.005% or more of the dry weight of the yeast extract, characterized in that X is an amino acid or its derivative other than Cys and its derivatives. The yeast extract according to the present invention contains the peptide, preferably in an amount of not less than 0.005%, more preferably not less than 0.02%, even more preferably not less than 0.1%, particularly preferably not less than 0.5% of the dry weight of the yeast extract.

The yeast used as a raw material for producing the yeast extract according to the present invention is the same yeast as used in the method of the present invention described below.

Glu and Gly peptides are glutamic acid and glycine, respectively. The symbol “-” denotes the peptide bond, “γ” in the designation γ-Glu means that another amino acid binds through the carboxy group of glutamic acid in the γ position.

“X” is any of the 19 naturally occurring amino acids or their derivatives, with the exception of Cys and its derivatives. Cys is cysteine, and examples of its derivatives include α-aminobutyric acid, β-aminobutyric acid, etc. The above amino acids, with the exception of Cys and its derivatives, include neutral amino acids such as glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ilé), series (Ser), threonine (Thr), methionine (Met), asparagine (Asn), glutamine (Gln) and proline (Pro), acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu), basic amino acids such as lysine (Lys), arginine (Arg) and histidine (His), and aromatic amino acids such as phenylalanine (Phe), tyrosine (Tur) and tryptophan (Trp). In particular, when the X-hydrophobic amino acid, the taste effect of the "kokumi" peptide is stronger, and such a peptide is preferred. Examples of such hydrophobic amino acids include Val, Ala, Leu, Phe, etc.

Examples of amino acid derivatives include, for example, norvaline (nVal), norleucine (nLeu), terleucin (tLeu), hydroxyproline (Hyp), etc.

Among the aforementioned peptides, γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly are most preferred.

In addition, all of the above amino acids and amino acid derivatives are L-isomers.

The form of the yeast extract of the present invention is not particularly limited, and may be in the form of a powder or solution. The yeast extract of the present invention can be used in the same way as traditional yeast extracts, for example, in the form of seasonings, food additives, health products, etc. The yeast extract of the present invention has an excellent kokumi flavoring property. Since the kokumi flavoring property is greatly enhanced when there is an umami flavor or a salty taste, umami flavoring agents, such as sodium L-glutamate and nucleotides, and / or salty substances, can be added to the yeast extract sodium chloride. In addition, umami flavoring agents can be added to seasonings, food additives or health products along with the extract of the present invention.

As shown in the Examples section, a yeast extract containing γ-Glu-Val-Gly, especially a solution containing yeast extract obtained by the method of the present invention described below, has more pronounced kokumi flavoring properties compared to a γ- solution Glu-Val-Gly with the same concentration of γ-Glu-Val-Gly. This proves the effectiveness of the yeast extract of the present invention.

The yeast extract of the present invention can be obtained, for example, by the methods of the present invention described below.

The first method according to the present invention is a method for producing a yeast extract containing a peptide selected from the group consisting of γ-Glu-X-Gly and γ-Glu-X, including growing yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X-Gly, γ-Glu-X and X-Gly, and preparing from the obtained cells a yeast extract, wherein X is an amino acid or derivative of an amino acid other than Cys and its derivatives.

There are no particular restrictions on the choice of yeast, provided that the yeast can transport γ-Glu-X, γ-Glu-X-Gly or X-Gly into the cell. Examples include yeast belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, belonging to the genus Candida, such as Candida utilis, belonging to the genus Pichia, such as Pichia pastoris, and belonging to the genus Schizosaccharomyces, such as Schizosaccharomyces pombe. Of these, Saccharomyces cerevisiae and Candida utilis, often used to produce yeast extract, are particularly preferred. The yeast of the present invention may be haploid or may have diploidy or higher polyploidy.

Yeast can be any wild-type strain or mutant strain, provided that the yeast can be transported into γ-Glu-X, γ-Glu-X-Gly or X-Gly cells and accumulated in γ-Glu-X and / or γ cells -Glu-X-Gly. Examples of mutant strains include a strain in which the activities of γ-glutamylcysteine synthetase (GSH1) and / or glutathione synthetase (GSH2) are enhanced. Yeast can also be modified to improve the transport of γ-Glu-X, γ-Glu-X-Gly or X-Gly to cells. The transport of γ-Glu-X, γ-Glu-X-Gly or X-Gly can be improved by increasing the activity of the protein involved in the transport of these peptides. Although HGT1p was thought to be a GSH-specific transporter, it was shown that with increasing HGT1p activity, γ-Glu-Val-Gly transport improves, as shown in the examples section. Therefore, it is possible that with increasing HGT1p activity, not only γ-Glu-Val-Gly, but also γ-Glu-X, γ-Glu-X-Gly and / or X-Gly improves cell transport. Methods to increase the activity of the aforementioned enzyme or protein include enhancing its expression by substituting the promoter or gene on the chromosome for a stronger promoter, enhancing its expression by inserting the target gene into the chromosome to increase its number to two or more, enhancing its expression by introducing into the yeast a plasmid containing the target gene, and the like.

As a promoter, a highly active type of traditional promoter can be obtained using a reporter gene, or known promoters that provide high expression, such as PGK1, PDC1, TDH3, TEF1 and HXT7, can be used. Alternatively, a plasmid with a CEN4 origin of replication or a multi-copy plasmid with a 2 μm DNA origin of replication can be used. In addition, transposon can be used to introduce the target gene into any region of the chromosome, or the target gene can be introduced using the target cDNA sequence present in the cell in 150 copies.

Enhanced activity of γ-glutamylcysteine synthetase is disclosed, for example, in US Pat. No. 7,553,638; Otake Y. et al., Bioscience and Industry, volume 50, No.10, pp. 989-994, 1992, etc. Although disruption of the glutathione synthetase gene is disclosed in US Pat. No. 7,553,638, the activity of glutathione synthetase can be enhanced in the same way as the activity of γ-glutamylcysteine synthetase. HGT1p activity can also be enhanced in a similar way.

The nucleotide sequences of the genes encoding GSH1 and GSH2 of Saccharomyces cerevisiae are disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). The nucleotide sequences of genes encoding GSH1 and GSH2 of Candida utilis are disclosed in U.S. Patent No. 7,553,638. The nucleotide sequence of the gene encoding GSH2 Saccharomyces cerevisiae is presented in the List of sequences under the number SEQ ID NO: 19. The amino acid sequence encoded by this nucleotide sequence is presented in the List of sequences under the number SEQ ID NO: 20.

The sequence of the gene encoding HGT1p can be obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org/). This nucleotide sequence can also be obtained as GSH11, ORT1 and the like, synonyms of HGT1p. The sequence disclosed as GSH11 of Saccharomyces cerevisiae is presented in the Sequence Listing under SEQ ID NO: 21. In addition, the amino acid sequence encoded by this nucleotide sequence is presented in the Sequence Listing under SEQ ID NO: 22.

A method of obtaining a yeast extract is explained below.

Yeast is grown in a nutrient medium containing a peptide. The composition of the culture medium is not particularly limited, provided that a medium is selected in which the yeast can propagate, and there are no restrictions on the SD medium described in the Examples, and the medium commonly used in production can be used.

When growing yeast, γ-Glu-X, γ-Glu-X-Gly or X-Gly is added to the abovementioned culture medium. You can add one of the types of these peptides or a mixture of two or more types. These peptides may be contained in the culture medium from the start of cultivation or may be added to the culture medium at any time during the cultivation process. When these peptides are added to the nutrient medium during the cultivation process, it is preferable to add them 0-50 hours before the end of cultivation (0 hours means that the cultivation is stopped immediately after addition), more preferably 0.1-24 hours before the end of cultivation, particularly preferably 0.5 -6 hours before the end of cultivation. In addition, when these peptides are added during cultivation, they can be added continuously.

Before cultivation in a nutrient medium containing these peptides, a preliminary cultivation can be carried out. The culture medium used for pre-cultivation may or may not contain these peptides.

These peptides are usually added to the nutrient medium in an amount of at least 0.1 ppm, preferably at least 0.5 ppm, more preferably at least 1 ppm, even more preferably at least 10 ppm, most preferably at least 50 ppm, in terms of the final concentration in the nutrient medium in moment of addition. Although the higher limits for the amount of these peptides are not particularly limited, an example is a value of not more than 100,000 or 50,000 ppm based on the cost of production, usually not more than 10,000 ppm, preferably not more than 1,000 ppm, more preferably not more than 500 ppm.

The cultivation conditions may be the same as those commonly accepted in the production of yeast extracts, they may be different depending on the yeast used. You can use any cultivation methods, such as cultivation in a batch culture, cultivation in a batch culture with the addition of a substrate and continuous cultivation. When the yeast used is Saccharomyces cerevisiae, preferably aerobic culture with shaking and the like. at 25-35 ° C, more preferably at 27-33 ° C, even more preferably at 28-32 ° C.

If the yeast is cultured as described above, γ-Glu-X or / and γ-Glu-X-Gly accumulate in the yeast cells. When γ-Glu-X or X-Gly is added to the culture medium, these peptides accumulate in the cells and, in addition, γ-Glu-X-Gly also accumulates. It is believed that the reason for this is that γ-Glu-X-Gly is formed from γ-Glu-X and X-Gly transported into the cell under the action of intracellular γ-glutamyl transferase. As shown in the Examples section, the content of γ-Glu-Val and γ-Glu-Val-Gly in yeast did not correlate with the content in GSH cells, and therefore, it is believed that yeast extracts obtained by traditional methods do not contain γ-Glu- X or γ-Glu-X-Gly in high concentrations, even if they are obtained from yeast containing GSH in high concentrations.

The yeast extract can be prepared from the obtained yeast in the same way as used in the traditional production of yeast extracts. The yeast extract can be obtained by extraction from cells with hot water and processing the extract, or by the method of cell destruction and processing the resulting product destruction. In addition, the obtained yeast extract can be concentrated or dried, if necessary, to obtain a powder.

In the manner described above, a yeast extract was obtained in which the content of γ-Glu-X or / and γ-Glu-X-Gly was increased. In a best embodiment of the invention, the yeast extract contains γ-Glu-X or / and γ-Glu-X-Gly in a total concentration of at least 0.005%, more preferably at least 0.02%, even more preferably at least 0.1%, particularly preferably at least 0.5% dry weight of yeast extract.

When γ-glutamyl transferase is exposed to a γ-Glu-X containing yeast extract obtained as described above, a yeast extract containing γ-Glu-X-Gly can be obtained.

The second method according to the present invention is a method for producing a yeast extract containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, including the action of γ-glutamyl transferase on a yeast extract to which an amino acid or peptide is added, selected from the group consisting of X and X-Gly, where X is an amino acid or derivative of an amino acid other than Cys and its derivatives.

When γ-glutamyl transferase acts on X or X-Gly, γ-Glu-X or γ-Glu-X-Gly is formed. Therefore, a yeast extract containing γ-Glu-X or γ-Glu-X-Gly can also be obtained by exposing a yeast extract containing X or X-Gly to γ-glutamyl transferase. Yeast extract containing X and / or X-Gly can be obtained from yeast grown in a nutrient medium containing X and / or X-Gly, or by adding X and / or X-Gly to the yeast extract used as a raw material .

The yeast extract used as a raw material may be any yeast extract obtained in a conventional manner.

One type X or X-Gly or a mixture of any two or more kinds thereof may be added to the yeast extract used as a raw material. X and / or X-Gly are added in a total amount of at least 1%, preferably at least 5%, more preferably at least 10% of the dry weight of the yeast extract used as raw material.

The reaction catalyzed by γ-glutamyl transferase is carried out in an aqueous solvent such as water or a buffer. Specifically, for example, the yeast extract used as a raw material is dissolved in an aqueous solvent and γ-glutamyl transferase is added. Reaction conditions are determined according to the γ-glutamyl transferase used. The reaction is usually carried out at a pH of 3-9 and a temperature of 15-70 ° C for 1-300 minutes, preferably at a pH of 5-8 and a temperature of 30-70 ° C for 5-150 minutes.

The concentration of yeast extract used as raw material can be determined by ease of handling. The indicated concentration is usually 0.1-50%, preferably 0.5-20%, of the dry weight of the yeast extract used as raw material.

Examples of γ-glutamyl transferase include glutaminase, γ-glutamyl transpeptidase (γ-GTP), etc. As for the amount of enzyme, in the case of γ-GTP, it is usually 0.001-1000 U / ml, preferably 0.005-100 U / ml, more preferably 0.01-25 U / ml, most preferably U / ml, where 1 U corresponds to activity, which, in 1 min, in a solution at pH 8.5 and a temperature of 25 ° C, 1.0 μM p-nitroaniline is released from γ-glutamyl-p-nitroanilide (determination is given in Sigma General Catalog, 2008-2009 Edition, p.917). The amount of glutaminase can also be determined in the same way as for γ-GTP.

After the enzymatic reaction, treatment may or may not be performed to inactivate γ-glutamyl transferase, for example, heat treatment at 80-100 ° C.

As a substrate for γ-glutamyl transferase, a γ-glutamyl compound, for example, GSH, can be added to the reaction mixture. GSH contained in the yeast extract can also be used as a substrate. In this case, you can use a yeast extract prepared from yeast in which the content of GSH is increased, for example, yeast in which the activity of GSH1 or / and GSH2 is enhanced. Although a higher GSH content in the yeast extract is more preferred, it is usually 1-50%, preferably 1-30%, and more preferably 5-20%, of the dry weight of the yeast extract.

In the manner described above, a yeast extract is obtained in which the content of γ-Glu-X or / and γ-Glu-X-Gly is increased. In a best practice of the invention, the yeast extract contains γ-Glu-X or / and γ-Glu-X-Gly in a total amount of at least 0.005%, more preferably at least 0.02%, even more preferably at least 0.1%, most preferably at least 0.5% dry weight of yeast extract.

The resulting yeast extract may, if necessary, be concentrated or dried to obtain a powder.

Examples

The present invention is described in more detail below with reference to the following Examples, which do not in any way limit the scope of the present invention.

Example 1: Determination of γ-Glu-Val-Gly and γ-Glu-Val in various yeast extracts

The contents of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly in various yeast extracts were measured. The measurement was carried out by obtaining fluorescent derivatives of peptides using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), and their determination using LC-MS / MS in accordance with the method described below. To 2.5 μl of each solution of various yeast extracts diluted to the appropriate concentration or to 2.5 μl of each standard solution containing γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly, respectively, 2.5 μl of Milli-Q water, 5 μl of 5 μM solution of internal standard substance (L-alanine-3,3,3-d3, Sigma (Ala-D3), DL-valine-2,3,4,4,4,5,5,5-d8, Sigma (Val-d8), both labeled with a stable isotope) and 30 μl of borate buffer (supplied with AccQ-Fluor (registered trademark) Reagent Kit, Nihon Waters). To each mixture was added 10 μl of an AQC reagent solution (prepared by dissolving the powder of the above reagent in 1 ml of acetonitrile). The resulting mixture was heated at 55 ° C for 10 min, and then 100 μl of a 0.1% aqueous formic acid solution was added to the mixture to prepare samples for analysis.

Then, prepared as described above, the sample was separated by reverse phase high performance liquid chromatography, described below, and then placed in a mass spectrophotometer. Separation conditions are described below.

(1) HPLC: Agilent 1200 Series

(2) Separation column: Unison UK-Phenyl, Inner Diameter: 2.0 mm, Length: 100 mm, Particle Size: 3 μm (Imtakt)

(3) Column temperature: 40 ° C

(4) Mobile phase A: an aqueous solution obtained by adjusting a 25 mM formic acid solution to pH 6.0 using aqueous ammonia

(5) Mobile phase B: methanol

(6) Flow rate: 0.25 ml / min

(7) Elution conditions: elution was carried out using a mixture of mobile phase A and mobile phase B. The ratio of mobile phase B to the mixture is as follows: 0 min (5%), 0-17 min (5-40%), 17-17.1 min ( 40-80%), 17.1-19 min (80%), 19-19.1 min (80-5%), 19.1-27 min (5%).

Then, the derivatives of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly, eluted under the aforementioned separation conditions, were placed in a mass analyzer and their quantity was determined by mass chromatography. The conditions for the determination were as follows.

(1) Mass analyzer: AB Sciex API3200 QTRAP

(2) Detection method: Monitoring of target ions (positive ion detection mode)

(3) Target ion: Table 1

Table 1 Derivative compound The first mass analyzer, (Q1) Second Mass Analyzer, (Q3) γ-Glu-Val-Gly 474.2 171.2 γ-Glu-Val 417.4 171.2 Val-gly 345.4 171.2 Ala-d3 263.0 171.2 Val-d8 298.0 171.2

The amount of derivatives of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly was determined using the Analyst ver program. 1.4.2 (AB Sciex). The internal compound Ala-d3 for γ-Glu-Val-Gly and γ-Glu-Val and the compound Val-d8 for Val-Gly, respectively, were used as an internal standard substance for quantification. The results are presented in Table 2. In this table, “ND” means that the quantity was less than the limit of determination (the same will be applicable later). The results of analysis (mass chromatograms) of derivatives of internal standard amino acids, γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly standard samples are shown in Figure 1.

table 2 γ-Glu-Val-Gly γ-Glu-Val Val-gly Brand a Nd 0.3 ppm 0.5 ppm Brand B Nd Nd Nd Brand s 2.2 ppm 19.2 ppm 11.1 ppm Brand D Nd Nd 0.4 ppm Brand E 3.1 ppm 25.9 ppm 33.1 ppm Brand f Nd Nd 0.4 ppm Brand g 4.6 ppm 38.3 ppm 0.4 ppm

As can be seen from Table 2, the content of γ-Glu-Val-Gly in various yeast extracts was a maximum of a few ppm, the content of γ-Glu-Val and Val-Gly was a maximum of several tens of ppm, and thus the studied extracts practically did not contain γ-Glu Val-Gly, γ-Glu-Val and Val-Gly.

Example 2: Determination of γ-Glu-Val-Gly and γ-Glu-Val in different yeast cells

In addition, the intracellular content of γ-Glu-Val-Gly and γ-Glu-Val in various yeasts was determined. As for the strains, the Saccharomyces cerevisiae S288C strain was used as the standard strain, and the Saccharomyces cerevisiae AJ14819 strain (deposited at international deposit as strain FERM BP-08502) was used as the standard strain. Strain S288C is hosted by an independent administrative agency. National Institute of Technology and Evaluation, Biological Resource Center (NBRC, NITE Biological Resource Center, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) under NBRC number 1136, where it can be obtained. This strain is also stored in the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, United States of America) under ATCC number 26108, where it can be obtained. Strain AJ14819 was obtained by mutagenesis of a monoploid yeast strain obtained from a commercially available strain of Saccharomyces cerevisiae with EMS and subsequent selection of a mutant strain in which the expression of the MET25 gene is not suppressed by methionine, the resulting strain was deposited with an independent administrative agency Agency of Industrial Science and Technology International Patent Organism Depository, Central 6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) September 11, 2002 under accession number FERM P-19007. Then, in accordance with the Budapest Treaty, the strain was internationally deposited on October 1, 2003 under accession number FERM BP-08502 (Japanese Patent No.4352877).

The above strains were inoculated in one loop of cell biomass into SD medium (50 ml of medium in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h with shaking at a speed of 120 rpm.

The composition of the SD environment:

Glucose 2%

Nitrogen bases 1-fold concentration

Nitrogen bases at a 10-fold concentration were obtained by dissolving a mixture of 1.7 g of Bacto Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Difco) and 5 g of ammonium sulfate in 100 ml of sterilized water, adjusted the pH of the solution to 5.2 and sterilized by filtration.

The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers) in such a quantity that the OD ₆₆₀ value at the beginning of cultivation was 0.01 (absorption was determined using DU640 SPECTROPHTOMETER, BECKMAN COULTER), cultivated at 30 ° C for 19 hours with shaking on a rotary shaker at a speed of 120 rpm. From the obtained culture, 400 OD U cells (1 OD U is defined as the number of cells contained in 1 ml of culture, the OD value of _{660 of} which is 1), cells were collected using centrifugation. The supernatant was removed as far as possible and the remaining cells were suspended in 45 ml of Milli-Q water. Cells were re-harvested using centrifugation and resuspended in 45 ml Milli-Q water. By repeating this operation three times, the cells were completely washed from the culture medium. The obtained washed cells were suspended in 1.5 ml of Milli-Q water, and the suspension was heated at 70 ° C for 10 min. At this stage, the extractable components contained in the cells were extracted. Then the extract and the remaining cells were separated using centrifugation.

Cell fragments were removed from the extract using centrifugation with a filtration membrane with a pore size of 10 kDa (Amicon Ultra - 0.5 ml 10K, MILLIPORE, Catalog Number UFC501096)), the obtained fraction was treated with AQC reagent as described in Example 1, and γ- Glu-Val-Gly and γ-Glu-Val by LC-MS / MS. Separately, the amount of GSH in the cell was determined by the traditional method (the GSH derivative in the extract was obtained using a fluorescent reagent ABD-F that binds to the thiol group, separated and its amount was determined using HPLC). In addition, the dry weight of the cells was determined after drying the washed cells at 104 ° C for 4 hours. Given the amount of γ-Glu-Val-Gly and γ-Glu-Val in the yeast extract prepared from cells contained in a specific culture volume, and measured, as described above, the dry cell weight was calculated the content of these peptides. The results are presented in Table 3.

Table 3 γ-Glu-Val-Gly, ppm γ-Glu-Val, ppm GSH,% S288C one 3 0.43 Aj14819 one 2 1.06

As a result, no correlation was found between the content of γ-Glu-Val-Gly or γ-Glu-Val and the content of GSH.

Example 3: Addition of γ-Glu-Val-Gly to strain S288C (1)

A loop of the biomass of S288C strain cells was inoculated into SD medium (50 ml in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h with shaking at a speed of 120 rpm. The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers) in such an amount that OD ₆₆₀ at the beginning of cultivation was 0.01, cultured at 30 ° C for 16 h with shaking on a rotary rocking chair at a speed of 120 rpm. To SD medium, γ-Glu-Val-Gly (Kokusan Chemistry) was added in advance to a final concentration of 100 ppm.

For the culture described above, the γ-Glu-Val-Gly content in the dried cells was calculated as described in Example 2. As a result, the γ-Glu-Val-Gly content was approximately 20,000 ppm (= 2%).

In addition, when the γ-Glu-Val-Gly content in the washing solution was measured after various washing steps, the content turned out to be 0.01 ppm, i.e. below the limit of determination, after triple centrifugation. The cells were washed again, and this ensured the almost complete removal of γ-Glu-Val-Gly during washing by fourfold separation and the prevention of its entry into the cell extract.

Example 4: Addition of γ-Glu-Val-Gly to strain S288C (2)

A loop of the biomass of S288C strain cells was inoculated into SD medium (50 ml in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h with shaking at a speed of 120 rpm. The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers) in such an amount that the OD ₆₆₀ at the beginning of cultivation was 0.01, cultured at 30 ° C for 19 h with shaking on a rotor rocking chair at a speed of 120 rpm. After 19 hours, aqueous solutions of γ-Glu-Val-Gly of various concentrations were added to obtain the desired concentrations and cultivation was continued for 1 h. For these cultures, the content of γ-Glu-Val-Gly in the dried cells was calculated as described in Example 2 In addition, the solids content in the extract and also the amount of γ-Glu-Val-Gly in solids were calculated. The results are presented in Table 4.

Table 4 The amount of γ-Glu-Val-Gly added to the medium (ppm) 0.01 0.1 one 5 10 one hundred The content of γ-Glu-Val-Gly in dried cells (ppm) 5 28 363 2647 5341 10191 The content of γ-Glu-Val-Gly in solids extract (ppm) 21 96 1110 10119 17408 32183

It can be seen from the presented results that the larger the amount of γ-Glu-Val-Gly added, the greater the content of γ-Glu-Val-Gly in the dried cells and in the dry matter of the extract.

Example 5: Obtaining a yeast extract containing γ-Glu-Val-Gly

A yeast extract was obtained from cells grown with γ-Glu-Val-Gly added to the medium. A loop of the biomass of S288C strain cells was inoculated into SD medium (50 ml in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h with shaking at a speed of 120 rpm. The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers, 12 flasks) in such a quantity that the OD ₆₆₀ value at the beginning of cultivation was 0.01, cultured at 30 ° C for 19 h shaking on a rotary shaker at a speed of 120 rpm. After 19 hours, an aqueous γ-Glu-Val-Gly solution was added to a final concentration of 50 ppm and cultivation was continued for 1 hour.

Cells were collected from the resulting culture and washed as described in Example 2. The washed cells in an amount of 400 OD Units were suspended in 1.5 ml Milli-Q water. This suspension was kept at 70 ° C for 10 min to obtain an extract from yeast cells. In addition, the suspension was centrifuged to remove cell debris and thus only the extract was collected. The concentration of γ-Glu-Val-Gly contained in this extract was determined (in the extract obtained in the experiment with the addition of γ-Glu-Val-Gly), it was approximately 300 ppm, and the solids content was approximately 1%. The concentration of γ-Glu-Val-Gly in the extract obtained in the same way, but without adding γ-Glu-Val-Gly to the culture fluid (in the extract obtained in the experiment without the addition of γ-Glu-Val-Gly) was below the limit definitions and a solids content of approximately 1%.

The extracts prepared as described above were evaluated for the presence of kokumi taste in the presence of MSG (monosodium glutamate) as follows. An aqueous solution containing 0.2% MSG and 0.5% NaCl was used as a control, and its organoleptic properties were evaluated as corresponding to 0.0. Then, an aqueous solution containing 0.2% MSG, 0.5% NaCl and 10 ppm γ-Glu-Val-Gly was used as the standard kokumi taste solution, and its organoleptic properties were evaluated as corresponding to 3.0. Using these two types of solutions as kokumi taste intensity standards, the kokumi taste intensity of yeast extract was evaluated. The taste intensity of kokumi extracts was evaluated using an aqueous solution containing yeast extract, 0.2% MSG and 0.5% NaCl as the test sample. The amount in the test sample of the extract obtained in the experiment with the addition corresponded to a concentration of γ-Glu-Val-Gly of 10 ppm. The amount in the test sample of the extract obtained in the experiment without addition was such that the solids content was the same as in the test sample of the extract obtained in the experiment with addition. So, the concentrations of γ-Glu-Val-Gly in the standard kokumi taste solution and in the test sample containing the extract obtained in the addition experiment were the same, and the solids content in the test sample containing the extract obtained in the experiment with addition, and in the test sample containing the extract obtained in the experiment without addition was the same.

Prepared as described above, test samples were tasted by kokumi by four members of a special jury. As a result, all four members of the jury concluded that the taste of the “kokumi” extract obtained in the addition experiment was stronger than in the sample with the same concentration of γ-Glu-Val-Gly. In addition, to evaluate the taste of “kokumi” as the initial and average taste of “kokumi” and the aftertaste of “kokumi”, grades used by four members of the jury for each type of taste were used. The results are presented in Table 5. The initial and average taste means the taste felt within 0-4 seconds after tasting, and the aftertaste means the taste felt after 5 seconds and later. As can be seen from the presented results, according to the jury, the initial and average taste of “kokumi” and the aftertaste of “kokumi” extract obtained with γ-Glu-Val-Gly during cultivation were stronger due to the fact that γ-Glu -Val-Gly was metabolized in a special way, or due to the synergy of γ-Glu-Val-Gly and some component of the yeast extract.

Table 5 Assessment of the initial and secondary taste of "kokumi" Kokumi aftertaste rating The control 0.0 0.0 Standard solution, (10 mM γ-Glu-Val-Gly, 0.2% MSG, 0.5% NaCl) 3.0 3.0 Extract obtained in the experiment with the addition of 4.0 ± 0.1 3.9 ± 0.3 The extract obtained in the experiment without adding 2.5 ± 0.9 1.8 ± 0.6

Example 6: Addition of γ-Glu-Val to strain S288C (1)

A loop of the biomass of S288C strain cells was inoculated into SD medium (50 ml in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h with shaking at a speed of 120 rpm. The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers) in such a quantity that the OD ₆₆₀ value at the beginning of cultivation was 0.01, cultured at 30 ° C for 24 hours with shaking on a rotary rocking chair at a speed of 120 rpm. Γ-Glu-Val (Bachem) was added to the SD medium in advance to a final concentration of 100 ppm.

For the culture described above, the content of γ-Glu-Val and γ-Glu-Val-Gly in the dried cells was calculated as described in Example 2. The results are presented in Table 6.

Table 6 γ-Glu-Val (ppm) γ-Glu-Val-Gly (ppm) No add 3 0 With the addition of γ-Glu-Val 7171 twenty

From the presented results it is seen that part of the γ-Glu-Val absorbed by the yeast cells was modified into γ-Glu-Val-Gly.

Example 7: Addition of γ-Glu-Val to strain S288C (2)

A loop of the biomass of S288C strain cells was inoculated into SD medium (50 ml in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h with shaking at a speed of 120 rpm. The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers) in such a quantity that the OD ₆₆₀ value at the beginning of cultivation was 0.01, cultured at 30 ° C for 19 h with shaking on rotary rocking chair at a speed of 120 rpm. After 19 hours, aqueous solutions of γ-Glu-Val of various concentrations were added to obtain desired concentrations and cultivation was continued for 1 hour. For these cultures, the content of γ-Glu-Val in the dried cells was calculated in the same manner as described in Example 2. In addition, calculated solids content in the extract and also the amount of γ-Glu-Val in solids. The results are presented in Table 7.

Table 7 The amount of γ-Glu-Val added to the medium (ppm) 0 25 one hundred 200 The content of γ-Glu-Val in dried cells (ppm) 2 1468 6369 10930 The content of γ-Glu-Val in the dry matter of the extract (ppm) 9 8491 27599 49474

It can be seen from the presented results that the larger the amount of γ-Glu-Val added, the greater the content of γ-Glu-Val in the dried cells and in the dry matter of the extract.

Example 8: Adding Val-Gly to strain S288C (1)

A loop of the biomass of cells of strain S288C was inoculated into SD medium (ammonium sulfate used in the preparation of SD medium was replaced with proline (final proline concentration was 0.1 g / L), 50 ml in 500 ml Sakaguchi flasks) and grown at 30 ° С for 24 h shaking at a speed of 120 rpm. The absorption of the obtained liquid culture was determined, the culture was inoculated into SD medium (400 ml in 2 L conical flasks with chippers) in such a quantity that the OD ₆₆₀ value at the beginning of cultivation was 0.01, cultured at 30 ° C for 24 hours with shaking on a rotary rocking chair at a speed of 120 rpm. Val-Gly (Bachem) was added to SD media in advance to a final concentration of 100 ppm.

For the culture described above, the γ-Glu-Val-Gly content in the dried cells was calculated as described in Example 2. The results are shown in Table 8.

Table 8 γ-Glu-Val-Gly (ppm) An experiment without adding Val-Gly 0 The experiment with the addition of Val-Gly 2

When Val-Gly was added to the medium, γ-Glu-Val-Gly accumulated in yeast cells.

Example 9: Yeast extract obtained with the addition of Val-Gly, which was affected by γ-glutamyltransferase

A 1% aqueous solution of yeast extract was prepared in which the GSH content was about 8% (in dry matter) and the pH was adjusted to 7.0 using NaOH. Powdered Val-Gly was added to this solution to final concentrations of 400 ppm, 800 ppm and 4000 ppm to prepare test samples. An aqueous solution of the yeast extract obtained without the addition of Val-Gly was also used as a control. Glutaminase (Glutaminase Daiwa SD-C100S, Daiwa kasei) was added to these test samples in an amount of 1 mg / ml and an enzymatic reaction was performed at 37 ° C for 120 min. In addition, instead of glutaminase, γ-GTP (horse kidney γ-glutamyl transpeptidase, Sigma, Code G9270-100UN) was added in an amount of 0.05 mg / ml and an enzymatic reaction was also carried out at 37 ° C for 120 min. The reaction mixtures were immediately cooled on ice after the reaction and the contents of γ-Glu-Val-Gly, GSH and Cys-Gly were determined.

As can be seen from the results presented in the following tables, γ-Glu-Val-Gly was almost not formed in the experiments without the addition of Val-Gly, but with an increase in the amount of added Val-Gly, the formation of γ-Glu-Val-Gly became visible. When γ-GTP was used, γ-Glu-Val-Gly production was especially noticeable.

Table 9 The concentration of components in the reaction mixture with glutaminase γ-Glu-Val-Gly (ppm) GSH (ppm) Cys-Gly (ppm) No add Before the reaction 0.0 835.6 6.3 After the reaction 0.0 3.1 701.6 400 ppm Before the reaction 0.0 862.4 9.9 After the reaction 1.4 3.5 715.0 800 ppm Before the reaction 0.0 851.5 6.7 After the reaction 3.8 3.6 705.7 4000 ppm Before the reaction 0.0 826.2 6.1 After the reaction 16.0 3.4 683.3

Table 10 The concentration of components in the reaction mixture with γ-GTP γ-Glu-Val-Gly (ppm) GSH (ppm) Cys-Gly (ppm) No add Before the reaction 0.0 813.6 5.2 After the reaction 0.8 432.4 335.1 400 ppm Before the reaction 0.0 824.6 4.5 After the reaction 150.9 333.2 388.2 800 ppm Before the reaction 0.0 836.9 6.1 After the reaction 157.9 316.3 417.3 4000 ppm Before the reaction 0.0 857.2 6.7 After the reaction 340.5 180.8 462.6

If, instead of Val-Gly, Val or nVal was added and the enzymatic reaction was carried out in a similar manner, γ-Glu-Val or γ-Glu-nVal were also formed.

Example 10: Addition of γ-Glu-Val to a strain with enhanced expression of GSH2

The following procedure can be used to enhance GSH2 expression. 1) Obtaining a uracil auxotrophic strain (ura3 mutant) from strain S288C.

The uracil auxotrophic strain can be obtained by plating the cells of a Saccharomyces cerevisiae strain treated with a mutagen using the traditional method on agar medium containing 5-FOA and selecting mutants from the grown ura3 strains (see, for example, METHODS IN YEAST GENETICS, 2000 EDITION, p .179). The uracil auxotrophic strain can also be obtained by introducing DNA adjacent to the URA3 gene that does not contain the URA3 gene to strain S288C followed by destruction of the URA3 gene, as shown below.

First, the region in front of the URA3 gene of 500 bp amplified by PCR using the primers shown in the Sequence Listing Numbers SEQ ID NO: 1 and SEQ ID NO: 2, and the chromosomal DNA of strain S288C as a template. In addition, the region after the URA3 gene of 500 bp also amplified by PCR using the primers shown in the Sequence Listing Numbers SEQ ID NO: 3 and SEQ ID NO: 4. The following conditions were used for PCR: 25 cycles: denaturation at 94 ° C for 10 sec, annealing at 55 ° C for 10 sec, elongation at 72 ° C for 1 min. Then, to obtain a 1 kb DNA fragment consisting of a region of 500 bp in front of the URA3 gene and regions after the 500 bp URA3 gene, PCR of the overlapping fragments was performed using the obtained two DNA fragments purified by ethanol precipitation as matrices and primers shown in the Sequence Listing Numbers SEQ ID NO: 5 and SEQ ID NO: 6. Strain S288C was transformed with this DNA fragment and then cultured overnight in SD medium supplemented with uracil, then the cells were plated on plates with 5-FOA medium. From the obtained transformants, a ura3Δ0 strain was obtained.

2) Obtaining a plasmid used as a template to replace the promoter.

First, the URA3 locus was amplified by PCR using primers SEQ ID NO: 7 and SEQ ID NO: 8 and the chromosomal DNA of the wild-type strain Saccharomyces cerevisiae as a template (25 cycles: denaturation: 94 ° C for 10 sec, annealing at 50 ° C within 10 seconds, elongation at 72 ° C for 1 min). The obtained DNA fragment was purified by ethanol precipitation, then it was digested with restriction enzymes SphI and EcoRI, the PCR product was inserted into the plasmid pUC19 at SphI-EcoRI sites to obtain pUC19-URA3. The PGK1 promoter region was then amplified using the chromosomal DNA of the wild-type strain Saccharomyces cerevisiae as the template and primers SEQ ID NO: 9 and SEQ ID NO: 10. This DNA fragment was treated with PstI restriction enzyme and inserted into pUC19-URA3 treated with PstI restriction enzyme treated with calf intestinal alkaline phosphatase PstI site to obtain pUC19-PGK1p-URA3. Similarly, the PGK1 promoter amplified using primers SEQ ID NO: 11 and SEQ ID NO: 12 was treated with AatII restriction enzyme and inserted into pUC19-PGK1p-URA treated with AatII restriction enzyme treated with calf intestinal alkaline phosphatase AatII site to obtain pUC19-PGK1 calf intestine -URA3-PGK1p. When the obtained plasmid was sequenced, the nucleotide sequence was expected, expected when the S288C strain was used as the chromosomal DNA template. Therefore, even when using the S288C strain chromosomal DNA as a template, a similar plasmid can be obtained instead of the wild-type chromosomal DNA.

3) Introduction of the PGK1 promoter into the GSH2 gene on the chromosome

PCR was performed using primer SEQ ID NO: 13, at the 5'-end of which is the sequence before the GSH2 gene, primer SEQ ID NO: 14, containing part of the open reading frame sequence starting with the start codon of the GSH2 gene, and C19-PGK1p-URA3 -PGK1p as a matrix (25 cycles: denaturation at 94 ° C for 10 sec, annealing at 60 ° C for 10 sec, elongation at 72 ° C for 4 min) to obtain a DNA fragment containing the URA3 gene between PGK1 promoters . The ura3Δ0 strain can be transformed with this DNA fragment, after which it is plated on plates with SD medium to obtain transformants, and a strain can be obtained from the transformants in which the GSH2 gene promoter is replaced by the construction of the PGK1 promoter –URA3 promoter PGK1.

4) Removal of the URA3 selective marker and replacement of the GSH2 gene promoter

A strain in which the GSH2 promoter is replaced by the construction of the PGK1 promoter-URA3 promoter PGK1 was grown overnight on SD with uracil, the corresponding volume of the resulting culture was plated on plates with 5-FOA medium. From the grown colonies, one can obtain a strain in which the URA3 gene is deleted due to homologous recombination between the introduced PGK1 promoters and the GSH2 promoter is replaced by the PGK1 promoter. In addition, by introducing into the resulting strain a DNA fragment amplified using the chromosome of the wild-type strain as the template and primers SEQ ID NO: 15 and SEQ ID NO: 16, it is possible to obtain a strain whose genotype is the same as the genotype of the strain ura3Δ0 except that the wild-type URA3 gene and the GSH2 promoter are replaced by the PGK1 promoter.

Using this strain as described above, a yeast extract was obtained and evaluated in the same manner as described in Examples 3, 4 and 6-8.

Example 11: Addition of γ-Glu-Val-Gly to a strain with enhanced expression of HGT1

A strain with enhanced expression of HGT1p can be obtained in the same manner as described in Example 10. Specifically, by PCR using pUC19-PGK1p-URA3-PGK1p as a template, primer SEQ ID NO: 17, at the 5'-end of which is the sequence before the GSH1 gene and primer SEQ ID NO: 18, containing a part of the open reading frame sequence starting with the start codon of the GSH11 gene, and introducing the obtained DNA fragment into sporulating yeast cells, cells were obtained in which the GSH11 promoter is replaced by the PGK1 promoter.

Using this strain as described above, a yeast extract was obtained and evaluated in the same manner as described in Examples 3, 4 and 6-8.

Example 12: Increased transport activity of γ-Glu-Val-Gly in an HGT1p or PKT2-modified strain.

The effect of HGT1p, which is considered specific for the GSH transporter, and the peptide transporter, PTR2, on the accumulation of γ-Glu-Val-Gly was evaluated as follows.

1) Obtaining a mutant auxotrophic for uracil (ura3 mutant) from strain S288C

The cassette with the geneticin resistance gene (G418) (P _ADH1 -kanR) was amplified by PCR using pUC19AOX-G418-BRI (Figure 2, SEQ ID NO: 23) as template and primers URA3Km-L1 (SEQ ID NO: 24 ) and URA3Km-R2 (SEQ ID NO: 25).

40 nucleotides at the 5'-end of the URA3Km-L1 primer are homologous to the region upstream of the URA3 gene, 40 nucleotides at the 5'-end of the URA3Km-R2 primer are complementary to the region downstream of the URA3 gene.

The obtained DNA fragment was used to transform S.cerevisiae S288C strain. Transformants were selected on plates with YPD medium containing 200 μg / ml G418. URA3 gene deletion was confirmed by PCR with primers ura3up2 (SEQ ID NO: 26) and ura3dn2 (SEQ ID NO: 27).

Got strain S288C ura3Δ0 :: P _ADH1 -kanR, which may also be referred to as S288C ura3Δ0. This strain was used to construct strains with increased expression of HGT1p or PTR2.

On the other hand, in order to use for construction of a strain with a deletion of HGT1p or PTR2, another uracil auxotrophic mutant was obtained as follows. A fragment containing the URA3 gene with the promoter and terminator regions was amplified in PCR using the chromosomal DNA of strain S288C as the template and the aforementioned primers ura3up2 and ura3dn2. This fragment was cloned at the Smal site in pBluescript II KS (+) and confirmed by sequencing. The plasmid pKS-URA3-13 was obtained (Figure 3, SEQ ID NO: 28).

Plasmid pKS-URA3-13 was treated with StuI and NcoI restriction enzymes and then treated with a Maple DNA polymerase I fragment to blunt the NcoI site. The resulting fragment is 4.38 kbp. ligated with HincII - HincII pUG6 fragment of 1.64 kbp [Gűldener, U., Heck, S., Fiedler, T., Beinhauer, J., and Hegemann, J.H. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research 24, 2519-2524] containing the loxP-kanMX-loxP module. The resulting plasmid is referred to as pKS-URA3-13-kanMX. The map and construction scheme of this plasmid is shown in FIG. 4, SEQ ID NO: 29.

Then, the kanMX gene flanked by fragments of the URA3 gene was amplified by PCR using the aforementioned primers ura3up2 and ura3dn2 and plasmid pKS-URA3-13-kanMX as a template. The resulting fragment was used to transform S. cerevisiae S288C strain, then transformants were selected on YPD medium containing 200 μg / ml G418. The result was a strain derived from S288C with a deletion of 227 nucleotides in the coding region of the URA3 gene. This deletion is referred to as ura3Δ227 :: loxP-kanMX-loxP. For deletion of the kanMX marker, the resulting strain was transformed with the plasmid pSH47 [Gűldener, U., Heck, S., Fiedler, T., Beinhauer, J., and Hegemann, J.H. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research 24, 2519-2524], containing the gene encoding Cre recombinase, under the control of the GAL1 promoter and the URA3 gene as a selective marker. Marker cutting was carried out after induction of the synthesis of Cre recombinase due to the transfer of cells from YPD medium to YPG medium (containing galactose). The resulting strain, from which the plasmid pSH47 was deleted, was named S288C ura3Δ227 :: loxP.

2) Obtaining a mutant strain with deletion of HGT1p

A DNA fragment containing an Agleu2-CaURA3-Agleu2 cassette was amplified by PCR using plasmid pAG61 [Goldstein, A.L., Pan, X., and McCusker, J.H. 1999. Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae. Yeast 15, 507-511] as the template and primers hgtl-pUG6u (SEQ ID NO: 30) and hgtl-pUG6d (SEQ ID NO: 31).

47 nucleotides at the 5'-end of the hgtl-pUG6u primer are homologous to the 5'-region of the HGT1p gene, 44 nucleotides at the 5'-end of the hgtl-pUG6d primer are complementary to the 3'-region of HGT1.

The resulting fragment was used to transform strain S288C ura3Δ227 :: loxP. Transformants were selected on plates with SD medium. The resulting strain S288C ura3Δ227 :: loxP HGT1Δ1981 :: Agleu2-CaURA3-Agleu2 with a nucleotide deletion from 1981 to 2400 of the coding region of the HGT1 gene was called S288C HGT1Δ. Deletion in the HGT1p gene was confirmed in PCR using primers hgt1-51 (SEQ ID NO: 32) and hgt1-33 (SEQ ID NO: 33).

3) Obtaining a mutant strain with deletion of PTR2

The P _ADH1-kanR module from plasmid pUC19AOX-G418-BRI was excised using restriction enzymes XbaI and SacI and subcloned at the same sites in plasmid pUG6. The resulting plasmid was named pLoxP-PADH1-kanR5 (Figure 5).

A DNA fragment containing the loxP-P _ADH1- kanR-loxP module was amplified in PCR using the plasmid pLoxP-PADH1-kanR5 as the template and primers ptr2-pUG6u (SEQ ID NO: 34) and ptr2-pUG6d (SEQ ID NO: 35 )

47 nucleotides at the 5'-end of the hgt1-pUG6u primer are homologous to the 5'-region of the PTR2 gene, 44 nucleotides at the 5'-end of the hgt1-pUG6d primer are complementary to the 3'-region of the PTR2 gene.

The resulting fragment was used to transform strain S288C ura3Δ227 :: loxP. Transformants were selected on plates with SD medium supplemented with 200 μg / ml G418. The obtained strain S288C ura3Δ227 :: loxP ptr2Δ967 :: loxP-P _ADH1 -kanR-loxP with a nucleotide deletion from 967 to 1806 of the coding region of the PTR2 gene was named S288C ptr2Δ.

Deletion in the PTR2 gene was confirmed in PCR using primers ptr2-51 (SEQ ID NO: 36) and ptr2-31 (SEQ ID NO: 37).

4) Obtaining mutant strains with a deletion of HGT1p or PTR2 from strain S288C

The strain S288C HCT1Δ ptr2Δ was constructed in the same manner as the strain S288C ptr2Δ, but the strain S288C HGT1Δ was used as a recipient strain for transformation.

5) Obtaining a strain with increased production of HGT1

The DNA fragment containing the ADH1 promoter was amplified by PCR using the chromosomal DNA of S. cerevisiae S288C strain as the adhlLl template and primers (SEQ ID NO: 38) and adhlRl (SEQ ID NO: 39). The resulting fragment was cloned at the SmaI site in plasmid pUC19 and the correct design was confirmed by sequencing. The resulting plasmid was named pUC19-PADH1-r (Figure 6).

A fragment of HindIII-HindIII plasmid pKS-URA3-13 of 1.16 kbp subcloned at the HindIII site in plasmid pBluescript II KS (+) to give pKS-URA3H-d. A fragment of the KpnI-SalI plasmid pUC19-PADH1-r was cloned at the SalI-KpnI sites in pKS-URA3H-d. The resulting plasmid pKS-URA3-P-ADH1 (Fig.7).

Then the plasmid pKS-URA3-PADH1 was treated with restriction enzymes ClaI and BsrGI. After restriction, the plasmid was treated with a Clenov fragment of DNA polymerase I so that the ends were blunt and ligated. The resulting plasmid, pKS-URA3-PADH1-5'd, was digested with EcoRI restriction enzyme, treated with a Maple fragment of DNA polymerase I, then digested with restriction enzyme XbaI. The resulting fragment was ligated with the P _ADH1 fragment excised from the plasmid pUC19-PADH1-r using restriction enzymes XbaI and BstZ17I. Thus, the plasmid pKS-URA3-PADH1-LR was obtained (Figure 6).

The DNA fragment containing the P _ADH1 -URA3-P _{ADH1 cassette} was amplified by PCR using the plasmid pKS-URA3-PADH1-LR as the template and primers PADH1-HGT1 (SEQ ID NO: 40) and HGT1-PADH1 (SEQ ID NO: 41).

40 nucleotides at the 5'-end of the PADH1-HGT1 primer are complementary to the 40 first nucleotides of the HGT1 coding region, 40 nucleotides at the 5'-end of the HGT1-PADH1 primer are homologous to the region before the HGT1 gene.

The DNA fragment containing the region upstream of the HGT1 gene was PCR amplified using the chromosomal DNA of S. cerevisiae S288C strain as the template and primers hgt53 (SEQ ID NO: 42) and hgt33 (SEQ ID NO: 43).

Then the fragments obtained as described above were combined using PCR (primers hgt53 and PADH1-HGT1 were used) as described in [Shevchuk, NA, Bryksin, AV, Nusinovich, YA, Cabello, FC, Sutherland M., Ladisch S. 2004 . Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res.32 (2): e19]. The resulting fragment was used to transform strain S288C ura3Δ0 :: P _ADH1 -kanR. Transformants were selected on plates with SD medium. As a result, the strain S288C ura3Δ0 :: P _ADH1- kanR P _ADH1 -URA3-P _ADH1 -HGT1 was obtained. The strain was named S288C P _ADH1 -HGT1.

The experimental design of the replacement of the promoter is shown in Fig. 8. Primer hgt53 corresponds to Primer 1, primer hgt33 corresponds to Primer 2, primer HGT1-PADH1 corresponds to Primer 3, primer PADH1-HGT1 corresponds to Primer 4.

6) Obtaining strain with increased production of PTR2

The replacement of the native promoter PTR2 was carried out in the same way as the replacement of the HGT1p promoter (Fig. 8).

Used primers:

ptr52 (SEQ ID NO: 44), corresponds to Primer 1;

ptr32 (SEQ ID NO: 45), corresponds to Primer 2;

PTR2-PADH1 (SEQ ID NO: 46), corresponds to Primer 3;

PADH1-PTR2 (SEQ ID NO: 7), corresponds to Primer 4;

40 nucleotides at the 5'-end of the PADH1-PTR2 primer are complementary to the 40 first nucleotides of the PTR2 coding region, 30 nucleotides at the 5'-end of the PTR2-PADH1 primer are homologous to the region in front of the PTR2 gene.

7) Assessment of mutants

Each strain was grown in 50 ml tubes with 5 ml SD medium supplemented with γ-Glu-Val-Gly to a final concentration of 100 ppm. The tubes were seeded with a night culture of the corresponding strain to an initial density of OD ₆₀₀ = 0.5 and placed on a shaker at 240 rpm, 30 ° C. Cultured for 7 hours. Then cells from 2 ml of the culture were collected using centrifugation and milliQ was washed three times with water. The washed cells were suspended in 1 ml milliQ-water and incubated at 70 ° C for 10 min in a water bath to extract intracellular contents. After separating the extract from the cell debris by centrifugation, the aqueous phase was transferred to a clean vial and vacuum dried. Thus, a dry extract was prepared. Γ-Glu-Val-Gly was then measured in the dry extract using the LC-MS / MS method. The procedure is described in detail below.

Equipment:

Mass Spectrometer: Triple Quadrupole Agilent 6410

HPLC: Agilent 1200

Columns: Thermo Hypersil-Keystone C18 100 mm * 2.1 mm * 5 μm

1. The sample was dissolved in 100 μl of water, then 100 μl of acetonitrile (MeCN) was added and centrifuged.

2. 190 μl of the supernatant was transferred to a new tube and dried.

3. The dried mass was dissolved in 40 μl of water.

4. To obtain the derivative, 20 μl of the resulting solution was mixed with 60 μl of buffer and 80 μl of the reagent to obtain the derivative and incubated at 55 ° C for 10 min.

5. After incubation, in order to stop the reaction to obtain the AQC derivative, 200 μl of a 0.1% solution of HCOOH in water was added.

40 μl of the resulting solution was used for HPLC and analyzed in γ-Glu-Val-Gly eluant using an MS / MS detector.

HPLC conditions:

Mobile phase A - 0.1% HCOOH in H ₂ O

Mobile phase B - 0.1% HCOOH in MeCN.

Flow rate - 0.250 ml / min

Table 11 Gradient conditions Time min Mobile phase A Mobile phase B 0 95 5 0.5 95 5 thirty 75 25 35 twenty 80 40 twenty 80 41 95 5

Table 12 Detection conditions on a mass spectrometer Compound Precursor ion (m / z) Ion Product (m / z) γ-Glu-Val-Gly 474.2 304.2 300.1 229.0

Using these data, the intracellular γ-Glu-Val-Gly content was calculated. The results are presented in Tables 13 and 14. It was shown that the HGT1p gene is essential for transport of γ-Glu-Val-Gly from the culture medium, and the PTR2 gene is not significant.

Table 13 Evaluation of strains with deletion of HGT1 and / or PTR2 Strain OD ₆₀₀ γ-Glu-Val-Gly (mg L ^-1 OD ₆₀₀ ^-1 ) S288C 3.3 1.93 S288CHGT1Δ 3.3 0.01 S288C ptr2Δ 3.8 1.84 S288C HGT1Δ ptr2Δ 3.4 0.01

Table14 Evaluation of strains with increased production of HGT1 or PTR2 Strain CD ₆₀₀ γ-Glu-Val-Gly (mg l ^-1 OD ₆₀₀ ^-1 ) S288C 3.40 3.3 S288C P _ADH1 -HGT1 1.64 28.6 S288C P _ADH1 -PTR2 2.64 2.6

Example 13: Effect of adding γ-Glu-Val-Gly to a strain with increased production of HGT1p

S288C P _ADH1- HGT1, the strain with increased HGT1p production obtained in Example 12, and its parent strain S288C were evaluated by their ability to transport γ-Glu-Val-Gly cells in the same manner as in Example 4. The amount added to the nutrient γ-Glu-Val-Gly medium - final concentration of 100 ppm. As a result, the intracellular content of γ-Glu-Val-Gly in S288C was about 0.9% by dry weight, and the intracellular content of γ-Glu-Val-Gly in S288C P _ADH1- HGT1 by dry weight was about 4.9%. This result confirmed the effect of increased HGT1p production.

Claims (24)

1. A method of obtaining a yeast extract to give the food taste "kokumi" containing the γ-Glu-X peptide, including growing yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X, γ-Glu-X -Gly and X-Gly, and preparing from the obtained cells a yeast extract, characterized in that X is an amino acid or its derivative, other than Cys and its derivatives. 2. The method according to claim 1, characterized in that said nutrient medium contains 0.1 ppm or more of said peptide, and said yeast extract contains γ-Glu-X peptide in an amount of 0.005% or more of the dry weight of the yeast extract. 3. The method according to claim 1 or 2, characterized in that X is Val. 4. The method according to claim 1, characterized in that the yeast is modified in such a way that the ability to transport the indicated peptide to the cell is enhanced in the modified yeast. 5. The method according to claim 4, characterized in that the activity of HGT1p is increased in said yeast. 6. The method according to claim 1, characterized in that said yeast is Saccharomyces cerevisiae yeast. 7. A method of producing a yeast extract to make kokumi taste food containing γ-Glu-X-Gly peptide, comprising growing yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X, γ-Glu -X-Gly and X-Gly, and preparing from the obtained cells a yeast extract, characterized in that X is an amino acid or its derivative, other than Cys and its derivatives. 8. The method according to claim 7, characterized in that said nutrient medium contains 0.1 ppm or more of said peptide, and said yeast extract contains γ-Glu-X-Gly peptide in an amount of 0.005% or more of the dry weight of the yeast extract. 9. The method according to claim 7 or 8, characterized in that X is Val. 10. The method according to claim 7, characterized in that the yeast is modified in such a way that the ability to transport the indicated peptide to the cell is enhanced in the modified yeast. 11. The method according to claim 10, characterized in that the activity of HGT1p is increased in said yeast. 12. The method according to claim 7, characterized in that said yeast is Saccharomyces cerevisiae yeast. 13. A method for producing a yeast extract to give a “kokumi” flavor to a food product containing a γ-Glu-X peptide, comprising exposing a yeast extract containing X or X-Gly obtained from yeast grown in a nutrient medium to γ-glutamyl transferase which added an amino acid or peptide selected from the group consisting of X or X-Gly, characterized in that X is an amino acid or its derivative, other than Cys and its derivatives. 14. The method according to item 13, wherein said amino acid or peptide is added in an amount of 1% or more dry weight of the yeast extract and said yeast extract contains γ-Glu-X peptide in an amount of 0.005% or more of the dry weight of the yeast extract. 15. The method according to item 13 or 14, characterized in that X is Val. 16. The method according to item 13, wherein said yeast is Saccharomyces cerevisiae. 17. A method of producing a yeast extract to flavor a “kokumi” food product containing a peptide, γ-Glu-X-Gly, comprising the action of γ-glutamyl transferase on a yeast extract containing X or X-Gly obtained from yeast grown in a nutrient medium to which an amino acid or peptide selected from the group consisting of X or X-Gly is added, characterized in that X is an amino acid or its derivative other than Cys and its derivatives. 18. The method according to 17, characterized in that said amino acid or peptide is added in an amount of 1% or more dry weight of the yeast extract and said yeast extract contains γ-Glu-X-Gly peptide in an amount of 0.005% or more of the dry weight of the yeast extract. 19. The method according to 17 or 18, wherein X is Val. 20. The method according to 17, characterized in that said yeast is Saccharomyces cerevisiae. 21. Yeast extract to make kokumi taste food containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly in an amount of 0.005% or more dry weight of the yeast extract, characterized in that X is an amino acid or its derivative, other than Cys and its derivatives, obtained by any of the methods according to claims 1, 7, 13 or 17. 22. The yeast extract according to item 21, containing the peptide in an amount of 0.02% or higher. 23. The yeast extract according to item 21, wherein X is Val. 24. The yeast extract according to item 21, wherein the said yeast is Saccharomyces cerevisiae.

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