Yeast extract containing gamma-glu-x or gamma-glu-x-gly, and method of its production

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

 

The technical field

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

Prior art

Yeast extract gives food "atsumi" (thickness), the taste of "umami" and is widely used as a seasoning in food. It is known that especially glutathione (hereinafter also referred to as "GSH"), which is a Tripeptide consisting of glutamic acid, cysteine and glycine, makes food taste "kokumi" (Ueda et al., Agric. Biol. Chem., 54, 163-169 (1990), Ueda et al., Biosci. Biotechnolo. Biochem., 61, 1977-1980 (1997)), and developed a variety of seasonings containing GSH.

Meanwhile, though it was reported that celizicusually receptor (CaSR), which is G-protein class, 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 cells of the language, and believe that this is evidence of its role in the sensation of taste (Gabriel et al., Biochemical and Biophysical Research Communications, 378, 414-418 (2009)). Further, it was recently shown that CaSR is involved in the distinction between people of taste "kokumi" (Ohsu et al., Journal of Biological Chemistry, 285, 1016-1022 (2010)). In this work it is shown that not only GSH, which really is known as a substance with a taste of "kokumi", but apparently, several compounds γ-glutamyl react with CaSR. In addition, it is shown that these peptides having the General formula γ-Glu-X or γ-Glu-X-Gly (X represents an amino acid or derived amino acid other than Cys and its derivatives), for example, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val-Gly, etc. give taste "kokumi" (WO2007/055393). In addition, it is shown that the group of esters of S - or O-carboxyethylidene peptides γ-glutamyl or β-asparagine are also connections with taste "kokumi" (WO2007/042288). Although these peptides give food taste "kokumi" like GSH, in contrast to GSH, no part of the recovered SH-groups. It is known that the substance having the composition of the recovered SH group, such as GSH, as a rule, unstable, and its titer decreases as the formation of disulfide bonds (WO2007/042288). However, γ-Glu-X and γ-Glu-X-Gly, etc. that are considered useful as peptides, giving a taste of "kokumi", not having in its composition restored SH-group stable.

With regard to food products containing γ-Glu dipeptide, there is a message stating that the various γ-Glu dipeptides found in Gouda cheese, enjoy for a long period, such as 44 weeks (Toelstede, S and Hofmann, T., J. Agric. Food. Chem., 2009). In this work it is reported that defines the different γ-Glu dipeptides, such as γ-Glu-Ala, γ-Glu-Glu and γ-Glu-Gln, and defined the content of γ-Glu of dipeptides Mac is imum 3590 µmol/kg This value corresponds to 0.088% dry matter.

However, any yeast extract containing γ-Glu-X or γ-Glu-X-Gly in a quantity sufficient to impart flavor "kokumi" is not known.

In addition, it is known that the synthesis and degradation of glutathione, one of the γ-glutamyl compounds, catalyzed by many enzymes, called cycle enzyme γ-glutamyl. In particular, it is known that γ-glutamyltranspeptidase transports glutamate to γ-position of GSH to another compound with the amino group, with the decomposition of GSH to containerline (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 the compound having the amino group, in this rearrangement reaction is the amino acid, as a by-product can be formed dipeptide γ-Glu-X. However, the search for microorganisms, effectively producing γ-Glu-X, was not a success, partly due to the fact that γ-Glu-X is a by-product.

Information regarding the dipeptide γ-Glu-X, include an analysis of the culture fluid Micrococcus glutamicus (Ronald et al., Journal of Biological Chemistry, 240, p.2508-2511 (1965)). In this work provides information about what the culture fluid was applied on different columns for separation of peptides, etc. to select γ-Glu-Glu, γ-Glu-Val, γ-Glu-Leu. However, they were detected as a result of separation of the different columns, and you do not know what the number is E. they were contained in the environment.

In addition, the enzyme responsible for the biosynthesis of GSH, was isolated from Streptococcus agalactiae and Clostridium acetobutylicum and analyzed its substrate specificity (Kino et al., JBB research communications, 352, pp.351-359 (2007)). In the biosynthesis of GSH is usually involved two types of enzymes, γ-glutamylcysteine connecting Glu and Cys with the formation of γ-Glu-Cys, and glutathionylated connecting the resulting γ-Glu-Cys and Gly with the formation of GSH. However, in the above-mentioned two kinds of microorganisms has a unique enzyme, corresponding to the hybrid γ glutamylcysteine and glutathionylated. There is evidence that according to the results of in vitro assays, substrate recognition by this enzyme is poorly expressed, for example, he also learns other amino acids (not only Cys) and as a result can form γ-Glu-X and γ-Glu-X-Gly. However, this test results in vitro, and is not described examples of the products of these microorganisms defined quantities of peptides, such as γ-Glu-X and γ-Glu-X-Gly, inside cells that contain many compounds with amino group in addition to the target X.

Yeast extracts are spices, widely used in food, much appreciated by consumers. Therefore, as the carrier of γ-Glu-X-Gly or γ-Glu-X, shape yeast extract more preferable. Examples of studies on the use of such extracts include work with the miner who soderjasimi yeast. It is known that if the environment is to add metal, yeast transport it into the cell (B. Volesky, H.A., Appl. Environ. BiotechnoL, 42;797-806 (1995)). In particular, if trace amounts of such elements as zinc, iron, copper, manganese, selenium, molybdenum and chromium, are added in Wednesday, yeast can be used as the source of element enrichment which food is desirable (Japanese Patent Laid-open (Kokai) No.2004-298014). With this in mind develop methods of obtaining mineraloceramic yeast (Japanese Patent Laid-open No.54-157890, Japanese Patent Laid-open No.60-75279, Japanese Patent Publication (Kokoku) No.6-16702).

In addition, enriching yeast these items, you can obtain and benefits related to taste. For example, in patent application laid Japan No.8-332081 described yeast with a high content of manganese. It is described that although the market there are foods enriched with manganese by adding salts of manganese, as this inorganic salt, the result of add is strong bitterness and astringency, and regularly consume them much harder than natural mn containing foods, and disclosed a technology for production of natural material by enrichment of yeast manganese. As for nutritional value, the technology disclosed in patent application laid Japan No.2008-99578. As described in the application data, although zinc what is the contribution to the improvement of taste sensations, improving generative functions, and so, apparently, one cannot say that he is now consumed in sufficient quantities. If zinc is added in the process of culturing yeast, yeast zinc transport into cells, but water-soluble zinc does not accumulate in the yeast in pure form, and is associated with the protein or amino acid and accumulates in high concentrations in the form of amorphous zinc. Apparently, this amorphous zinc is more efficiently absorbed in the body in comparison with crystalline zinc. As a result, the inclusion of zinc in yeast can be obtained superior absorption compared with the consumption of zinc in its pure form.

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

Among studies of dipeptides and tripeptides yeast, many relating to the education of GSH or γ-Glu-Cys. As an example of work which provides evidence that the content of GSH increased by mutagenesis of the yeast Saccharomyces and oberstimm, highly resistant to zinc lined patent application Japan No.02-295480), a message indicating that the suppression of gene expression MET was derepression due to a mutant gene MET, resulting in increased intracellular content of γ-Glu-Cys (patent application Japan No.2002-282743), etc. in Addition, recent scientific discoveries include data about the transport of GSH through HGT1p. Although GSH and its dimer, GSSG, was transported into the cells through HGT1p, transport GSH Transporter HGT1p not influenced even the presence of excessive amounts of amino acids, various dipeptides and tripeptides. Therefore, it is believed that HGT1p is not a nonspecific conveyor, as previously thought, but by the conveyor, specific to GSH (Bourbouloux et al., Journal of Biological Chemistry, 275, pp.13259-13265 (2000)). In addition, also conducted a search of the active sites HGTIp (Kaur et al., FEMS Yeast Res., 9, 849-866 (2009)).

As described above, although there is information about many discoveries related to GSH and its precursor, there is almost no information about yeast cells containing substances such as γ-Glu-X and γ-Glu-X-Gly, and the method of obtaining the extract of these cells.

Brief description of the invention

The purpose 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 of obtaining the above-mentioned extract.

Videopom the mentioned objectives have been achieved by the discovery of the fact, that yeast is transported γ-Glu-X and γ-Glu-X-Gly (X is a non-Cys amino acid or its derivative, the same applies to the description below) in the cell, and a yeast extract containing γ-Glu-X or γ-Glu-X-Gly, can be obtained by preparation of these yeast cultured in a medium containing γ-Glu-X or γ-Glu-X-Gly. Also, found that if the yeast is cultured in a medium containing γ-Glu-X or X-Gly, these compounds are transported into the cells, and γ-Glu-X-Gly can be formed in enzymatic reactions inside cells. In addition, it also found that yeast extract containing γ-Glu-X or γ-Glu-X-Gly, can be obtained by the reaction of γ-glutamyltransferase with yeast extract, to which is added an amino acid or peptide, selected from the group consisting of X and X is 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 the amount of 0.005% or more of the dry weight of yeast extract, characterized in that X represents an amino acid or its derivative other than Cys and its derivatives.

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

Another objective of the present invention is pre the abandonment of the above-described yeast extract, wherein X is Val.

Another objective of the present invention is the provision of the above-described yeast extract, characterized in that these yeasts are Saccharomyces cerevisiae.

Another objective of the present invention is the provision of a method of producing a yeast extract containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, which includes the cultivation of yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly or X-Gly, and preparation obtained from cells yeast extract, characterized in that X represents an amino acid or its derivative other than Cys and its derivatives.

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

Also the aim of the present invention is the provision of the above-described method, wherein X is Val.

Also the aim of the present invention is the provision of the above-described method, wherein the yeast is modified so is the ability to transport the specified peptide into the cell in the modified yeast increased.

Also the aim of the present invention is the provision of the above-described method, wherein the activity of HGT1p in these yeast raised.

Also the aim of the present invention is the provision of the above-described method, wherein the indicated yeast is Saccharomyces cerevisiae.

Another objective of the present invention is the provision of a method of producing a yeast extract containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, which includes interaction of γ-glutamyltransferase from yeast extract, used as raw material, to which is added an amino acid or peptide, selected from the group consisting of X and X-Gly, wherein X represents an amino acid or its derivative other than Cys and its derivatives.

Also the aim of the present invention is the provision of the above-described method, characterized in that the amino acid or peptide is added in quantities of 1% or more of the dry weight of the yeast extract and the yeast extract contains a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, in the amount of 0.005% or more of the dry weight of the yeast extract.

Also the aim of the present invention is the provision of the above-described method, wherein X is Val.

Also the spruce of the present invention is the provision of the above-described method, characterized in that these yeasts are Saccharomyces cerevisiae.

According to the present invention, it is possible to get a yeast extract containing γ-Glu-X and γ-Glu-X-Gly. Yeast extract containing these peptides, has excellent taste "kokumi".

In addition, 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

The Figure 1 shows a mass chromatogram of a standard sample of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly.

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

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

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

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

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

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

The Figure 8 shows the scheme of obtaining the DNA fragment used to replace the promoter.

Detailed description of the invention

Yeast extract according to the present invention contains a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, in the amount of 0.005% or more of the dry weight of yeast extract, characterized in that X represents an amino acid or its derivative other than Cys and its derivatives. Yeast extract coz the ACLs present invention contains a peptide preferably 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 yeast extract.

Yeast is used as the raw material for producing yeast extract according to the present invention, the same yeast that is used in the following method of the present invention.

Glu and Gly peptide are glutamic acid and glycine, respectively. The symbol "-" indicates a peptide bond, "γ" in the designation of γ-Glu means that another amino acid linked via the carboxy group of the glutamic acid at the γ-position.

"X" represents any of the 19 natural amino acids or derivatives thereof, except for Cys and its derivatives. Cys is a cysteine, and examples of its derivatives include α-aminobutyric acid, β-aminobutyric acid, etc. of the Above-mentioned amino acids except Cys and its derivatives include neutral amino acids such as glycine (Gly), alanine (l), valine (Val), leucine (Leu), isoleucine (Il), 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 (Tight) and t is Aptian (TGR). In particular, when X is a hydrophobic amino acid, the effect of taste "kokumi" peptide stronger, and this 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), tartrazin (tLeu), hydroxyproline (Hyp), etc.

Among the above-mentioned peptides of the most preferred γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly.

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

The form of yeast extract of the present invention are not specifically limited, and it may be in the form of powder or solution. Yeast extract of the present invention can be used as a traditional yeast extracts, such as seasonings, food additives, health products, etc. Yeast extract of the present invention has an excellent property flavor "kokumi". Because the property of flavor "kokumi" is greatly enhanced in the presence of taste "umami or savory taste, yeast extract may be added substance, giving a taste of "umami", such as L-monosodium glutamate and nucleotides, and/or salty substances, such as sodium chloride. In addition, substances imparting taste "umami", can be added to the spices, dietary supplements or health products along with extras the drive of the present invention.

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

Yeast extract of the present invention can be obtained, for example, the following methods of the present invention.

The first method according to the present invention is a method of obtaining a yeast extract containing a peptide selected from the group consisting of γ-Glu-X-Gly and γ-Glu-X, which includes the cultivation of 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 preparation obtained from cells yeast extract, while X represents other than Cys and its derived amino acid or derived amino acids.

For selection of yeast there are no particular restrictions provided that the yeast can be transported into the cell of γ-Glu-X and γ-Glu-X-Gly or X-Gly. 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 the AK Schizosaccharomyces pombe. One of them is particularly preferred Saccharomyces cerevisiae and Candida utilis, often used to produce yeast extract. The yeast of the present invention can be haploid or can be deploymnet or higher polyploidal.

Yeast can be any strain wild-type or mutant strain, provided that these yeasts can be transported into the cells of γ-Glu-X and γ-Glu-X-Gly or X-Gly and accumulate in the cells of γ-Glu-X and/or γ-Glu-X-Gly. Examples of mutant strains include strain, which enhanced the activity of γ-glutamylcysteine (GSH1) and/or glutathionylated (GSH2). Yeast can also be modified to improve transport of γ-Glu-X and γ-Glu-X-Gly or X-Gly in cells. The transport of γ-Glu-X and γ-Glu-X-Gly or X-Gly can be improved by increasing the activity of a protein involved in the transport of these peptides. Although it was believed that HGT1p is a conveyor, it is specific to the GSH, it is shown that the increased activity of HGT1p, transport of γ-Glu-Val-Gly improved, as shown in the examples section. Therefore, there is a possibility that with increasing activity HGT1p improving transport in the cell is not only γ-Glu-Val-Gly, and γ-Glu-X and γ-Glu-X-Gly or X-Gly. Methods of increasing the activity of the above-mentioned enzyme or protein include increasing its expression by substitution on chromosome promoter of the gene encoding an enzyme or protein, bol is e strong promoter, the strengthening of its expression by inserting a target gene into the chromosome to increase the number of copies to two or more, the strengthening of its expression by introducing into the yeast plasmid containing the target gene, etc.

As a highly active promoter of the traditional type of promoter can be obtained using a reporter gene or can be used known promoters, providing a high expression, such as PGK1, PDC1, TDH3, TEF1 and NHT. On the other hand, you can use a plasmid origin of replication CEN4 or mnogoseriynuyu plasmid origin of replication 2 μm DNA. In addition, you can use the transposon for the introduction of a target gene in any region of the chromosome or the target gene can be introduced using as the target sequence of the cDNA present in the cell in 150 copies.

The increased activity of γ-glutamylcysteine disclosed, for example, in U.S. patent No.7,553,638; Y. Otake et al., Bioscience and Industry, volume 50, No.10, pp.989-994, 1992, etc. Although the destruction of the gene glutathionylated disclosed in U.S. patent No.7,553,638 activity glutathionylated can be enhanced in the same way as the activity of γ-glutamylcysteine. Activity HGT1p can also be amplified in a similar manner.

The nucleotide sequences of genes encoding GSH1 and GSH2 Saccharomyces cerevisiae, revealed in a database Saccharomyces Genome Database (http://www.yeatgenome.org/). The nucleotide sequences of genes encoding GSH1 and GSH2 Candida utilis, are disclosed in patinete USA No.7,553,638. Nucleotide posledovatelnoy gene encoding GSH2 Saccharomyces cerevisiae, are presented in the sequence Listing under the number SEQ ID NO: 19. Amino acid sequence encoded by this nucleotide sequence presented in the sequence Listing under the number SEQ ID NO:20.

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

A method of obtaining a yeast extract is explained below.

The yeast is grown in a nutrient medium containing the peptide. The nutrient medium composition are not specifically limited, provided that you select the environment in which the yeast can multiply, and there are no restrictions on the SD environment, described in the Examples, and can be used in the environment, typically used in production.

When growing yeast in the above-mentioned culture medium is dobavlaut γ-Glu-X, γ-Glu-X-Gly or X-Gly. You can add one of these peptides or a mixture of two or more species. These peptides may be contained in the culture medium from the beginning of cultivation, or may be added to the nutrient medium at any time during cultivation. When these peptides are added to the culture medium during cultivation, it is preferable to add them for 0-50 hours before the end of cultivation (0 hours means that the cultivation ceased immediately after add), more preferably for 0.1 to 24 hours before the end of cultivation, particularly preferably for 0.5-6 h before the end of the cultivation. In addition, adding these peptides in the process of cultivation, they can be added in a continuous mode.

Before culturing in a nutrient medium containing the indicated peptides may be carried out prior cultivation. The nutrient medium used for pre-cultivation, may contain or not contain these peptides.

These peptides are usually added to the nutrient medium in an amount not less than 0.1 ppm, preferably not less than 0.5 ppm, more preferably not less than 1 ppm, even more preferably at least 10 ppm, most preferably not less than 50 ppm, in terms of the final concentration in the medium at the time of adding. Although the higher p is edely number of these peptides are not specifically limited, as an example of a value not exceeding 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 can be the same as usually adopted in the production of yeast extracts, they can be different depending on the used yeast. You can use any of the methods of cultivation, such as the cultivation of periodical culture, cultivation of periodical culture with the addition of the substrate and continuous cultivation. When used the yeast is Saccharomyces cerevisiae, preferably aerobic cultivation with shaking, etc. at 25-35°C, more preferably at 27-33°C, even more preferably at 28-32°C.

If the yeast is cultivated, as described above, in yeast cells accumulate γ-Glu-X and γ-Glu-X-Gly. When γ-Glu-X or X-Gly added to the nutrient medium, these peptides accumulate in the cells, and, in addition, also accumulate γ-Glu-X-Gly. It is assumed 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, γ-Glu-Val-Gly in yeast did not correlate with the content of GSH in cells, and therefore, it is believed that the driven the s extracts, obtained by conventional methods do not contain γ-Glu-X or γ-Glu-X-Gly in high concentrations, even if they are derived from yeast containing GSH in high concentrations.

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

As described above obtained yeast extract, which increased the content of γ-Glu-X and γ-Glu-X-Gly. In the best method embodiment of the invention, yeast extract contains γ-Glu-X and γ-Glu-X-Gly in a total concentration of at least 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 yeast extract.

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

The second method according to the present invention is a method of obtaining a yeast extract containing a peptide selected the C group, consisting of γ-Glu-X and γ-Glu-X-Gly, including the effects of γ-glutamyl transferase in yeast extract, to which is added an amino acid or peptide, selected from the group consisting of X and X-Gly, where X represents an amino acid or derived amino acid other than Cys and its derivatives.

When exposed to γ-glutamyltransferase on X or X-Gly is formed of γ-Glu-X or γ-Glu-X-Gly. Therefore, yeast extract containing γ-Glu-X or γ-Glu-X-Gly, can also be obtained when exposed to γ-glutamyl transferase in yeast extract containing X or X-Gly. 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 adding X and/or X-Gly to the yeast extract used as raw materials.

Yeast extract is used as the raw material can be any yeast extract, obtained by a traditional method.

It is used as raw material of yeast extract may be added to one kind of X or X-Gly, or a mixture of any two or more kinds of them. X and/or X-Gly is added in a total amount of not less than 1%, preferably at least 5%, more preferably not less than 10% of the dry weight used as raw yeast extract.

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

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

Examples of γ-glutamyl transferase include glutaminase, γ-glutamyltranspeptidase (γ-GTP), etc. with regard to 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 the activity in which for 1 min in a solution at pH 8.5 and 25°C is released 1.0 μm p-nitroaniline from γ-glutamyl-p-nitroanilide (as defined in Sigma General Catalogue, 2008-2009 Edition, p.917). The number of glutaminase can also be defined in the same way as for γ-GTP.

After the enzymatic reaction can be carried out or not carried out processing for inactivation of γ-glutamyl transferase, for example, heat treatment is processing at 80-100°C.

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

This above described method are yeast extract, which increased the content of γ-Glu-X and γ-Glu-X-Gly. In the best mode for carrying out the invention, yeast extract contains γ-Glu-X and γ-Glu-X-Gly in a total amount of not less than 0.005%, more preferably not less than 0.02%, even more preferably not less than 0.1%, most preferably not less than 0.5% of the dry weight of yeast extract.

The obtained yeast extract, if necessary, can 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 restrict in any way the scope of the present invention.

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

Was measured the content of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly in various yeast extracts. The measurement was performed by obtaining fluorescent derivatives of peptides using 6-aminoquinolyl-N-hydroxysuccinimidyl (AQC), and their identification using LC-MS/MS as described below. To 2.5 μl of each solution of various yeast extracts, diluted to the appropriate concentration or 2.5 µl of each standard solution containing γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly, respectively, was added 2.5 μl of Milli-Q water, 5 μl of a 5 μm solution of the 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 to AccQ-Fluor (registered trademark) Reagent Kit, Nihon Waters). To each mixture was added 10 μl of a solution of AQC reagent (prepared by dissolving the powder of the above-mentioned reagent in 1 ml of acetonitrile). The resulting mixture was heated at 55°C for 10 min and then to the mixture was added 100 μl of 0.1% aqueous formic acid for the preparation of samples for analysis.

Then prepared as described above, the sample was separarely method of reversed-phase high-performance liquid chromatography, as described below, and then were placed in a mass spectrophotometer. Conditions of separation are described below.

(1) HPLC: gilent 1200 Series

(2) Separation column: Unison UK-Phenyl, inner diameter: 2.0mm, length: 100 mm, particle size: 3 μm (Imtakt)

(3) column Temperature: 40°C

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

(5) Mobile phase b: methanol

(6) flow Rate: 0.25 ml/min

(7) the Conditions of elution: elution was performed using a mixture of mobile phase a and mobile phase C. the mobile phase to a mixture of the following: 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, derivative compounds of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly, erwerbende at the above conditions of separation were placed in the mass analyzer and determined their number by the method of mass chromatography. Terms definitions were following.

(1) the Mass analyzer: AB Sciex API3200 QTRAP

(2) Method for detection: Monitoring of set ion detection mode positive ion)

(3) the Specified ion: table 1

Table 1
The derived connectionThe first mass analyzer (Q1)The second mass analyzer (Q3)
γ-Glu-Val-Gly474.2171.2
γ-Glu-Val417.4171.2
Val-Gly345.4171.2
Ala-d3263.0171.2
Val-d8298.0171.2

The number of derivatives of the compounds of γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly was determined using the program Analyst ver. 1.4.2 (AB Sciex). As an internal standard substance for the quantitative determination used a derived compound Ala-d3 in the case of γ-Glu-Val-Gly and γ-Glu-Val and the derived connection Val-d8 in the case of Val-Gly, respectively. The results are presented in Table 2. In this table, "ND" means that the number was less than the limit of detection (the same will apply hereinafter). The results of the analysis (mass chromato grams) derived internal standard amino acids, γ-Glu-Val-Gly, γ-Glu-Val and Val-Gly standard samples shown in figure 1.

Table 2
γ-Glu-Val-Glyγ-Glu-ValVal-Gly
Brand AndND 0.3 ppm0.5 ppm
BrandNDNDND
Brand2.2 ppm19.2 ppm11.1 ppm
Brand DNDND0.4 ppm
Brand E3.1 ppm25.9 ppm33.1 ppm
Brand FNDND0.4 ppm
Brand G4.6 ppm38.3 ppm0.4 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 a few tens of ppm, and thus the investigated extracts almost 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 the cells of various yeast

In addition, it was determined the intracellular content of γ-Glu-Val-Gly and γ-Glu-Val in various yeast. As for strains, in which the quality standard strain used the Saccharomyces cerevisiae strain S288C, and as a strain with a high content of GSH used the Saccharomyces cerevisiae strain AJ14819 (deposited at international Deposit as the strain FERM BP-08502). Strain S288C is stored in 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 number NBRC 1136 where to get it. This strain is also stored in the collection of the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, United States of America) under ATS 26108, where to get it. Strain J14819 was obtained by mutagenesis monolignol yeast strain obtained from commercially available Saccharomyces cerevisiae strain with EMS, and the subsequent selection of a mutant strain in which expression of the gene MET not inhibited by methionine, the resulting strain deposited at the 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) on September 11, 2002 under inventory number FERM P-19007. Then, in accordance with the Budapest Treaty was held an international Deposit of strain on 1 October 2003 under inventory number FERM BP-08502 (Japan patent No.4352877).

The aforementioned strains on a single loop biomass cells were inoculable in a nutrient medium SD (50 ml medium in Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at speed of 10 rpm

The composition of the medium SD:

Glucose 2%

Nitrogen base 1-fold concentration

Nitrogen bases in 10-fold concentration was 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, brought the solution pH to 5.2 and sterilized by filtration method.

Determined the absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips) in an amount such that the value OD660at the beginning of the cultivation was 0.01 (absorption was determined using DU640 SPECTROPHTOMETER, BECKMAN COULTER), and cultivated at 30°C for 19 h with shaking on a rotary shaker at 120 rpm From the obtained culture 400 OD Units of cells (1 OD Unit is defined as the number of cells contained in 1 ml of culture, the OD value660which is 1) collected cells using centrifugation. The supernatant was removed as much as possible and the remaining cells are suspended in 45 ml of Milli-Q water. Cells are again collected using centrifugation and resuspendable in 45 ml of Milli-Q water. By three-fold repetition of this operation cells completely washed from the nutrient medium. Received the washed cells are suspended in 1.5 ml of Milli-Q water, the suspension is warmed up to 70°C for 10 minutes At this stage extrage who was ovalis contained in the cells of the extracted components. Then extract and the remaining cells were separated using centrifugation.

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

Table 3
γ-Glu-Val-Gly, ppmγ-Glu-Val, ppmGSH, %
S288C13 0.43
AJ14819121.06

As a result of correlation between the content of γ-Glu-Val-Gly or γ-Glu-Val and GSH content was not found.

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

Loop biomass of cells of strain S288C was inoculable on Wednesday SD (50 ml Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at 120 rpm was Determined by absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips) in an amount such that the value D660at the beginning of the cultivation was 0.01, and cultivated at 30°C for 16 h with shaking on a rotary shaker at 120 rpm To a nutrient medium SD in advance was added γ-Glu-Val-Gly (Kokusan Chemistry) to a final concentration of 100 ppm.

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

In addition, when measured the content of γ-Glu-Val-Gly in detergent solution after various stages of the laundering, the content was 0.01 ppm, i.e. below the limit of detection, after triple centrifugation. Cells were washed again, and it guaranteed the almost complete destruction of the-Glu-Val-Gly in the process of washing by fourfold separation and preventing them from falling in the extract of cells.

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

Loop biomass of cells of strain S288C was inoculable on Wednesday SD (50 ml Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at 120 rpm was Determined by absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips) in an amount such that the value D660at the beginning of the cultivation was 0.01, and cultivated at 30°C for 19 h with shaking on a rotary shaker at 120 rpm After 19 h were added aqueous solutions of γ-Glu-Val-Gly different concentrations to obtain zadannykh concentrations and continued culturing for 1 h For these crops have calculated the content of γ-Glu-Val-Gly in the dried cells as described in Example 2. In addition, we calculated the content of dry substances in the extract and the amount of γ-Glu-Val-Gly dry substances. The results are presented in Table 4.

Table 4
The amount of γ-Glu-Val-Gly added to the medium (ppm)0.010.11510100
The content of γ-Glu-Vl-Gly in the dried cells (ppm) 5283632647534110191
The content of γ-Glu-Val-Gly dry substances of the extract (ppm)21961110101191740832183

The presented results show that the greater the number of added γ-Glu-Val-Gly, the more the content of γ-Glu-Val-Gly in the dried cells and in the dry matter of the extract.

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

Got yeast extract from cells grown with the addition on Wednesday, γ-Glu-Val-Gly. Loop biomass of cells of strain S288C was inoculable on Wednesday SD (50 ml Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at 120 rpm was Determined by absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips, 12 bulbs) in an amount such that the value D660at the beginning of the cultivation was 0.01, and cultivated at 30°C for 19 h with shaking on a rotary shaker at 120 rpm After 19 h was added an aqueous solution of γ-Glu-Val-Gly to the final end is Tracii 50 ppm and continued culturing for 1 o'clock

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

Extracts, prepared as described above were evaluated for the presence of taste "kokumi" in the presence of MSG (glutamate sodium) in the following way. As control was used an aqueous solution containing 0.2% MSG and 0.5% NaCl, and its organoleptic properties were rated 0.0. Then an aqueous solution containing 0.2% MSG, 0.5% NaCl and 10 ppm of γ-Glu-Val-Gly was used as a standard solution for a taste of "kokumi", and its organoleptic properties were rated 3.0. Using the ti two types of solutions as standards intensity of taste "kokumi", evaluated the intensity of taste "kokumi" yeast extract. The intensity of taste "kokumi" extracts was evaluated using a test sample of an aqueous solution containing yeast extract, 0.2% MSG and 0.5% NaCl. The number in the tested sample of the extract obtained in the experiment with the addition corresponded to the concentration of γ-Glu-Val-Gly 10 ppm. The number in the tested sample of the extract obtained in the experiment without the addition was such that the solids content was the same as in the tested sample of the extract obtained in the experiment with addition. Thus, the concentration of γ-Glu-Val-Gly in the standard solution for a taste of "kokumi" and the test sample containing the extract obtained in the experiment with the addition were the same, and the solids content of the test sample containing the extract obtained in the experiment with the addition, in the test sample containing the extract obtained in the experiment without added was the same.

Prepared as described above, the test samples were evaluated for taste "kokumi" four members of a special jury. As a result, all four members of the jury concluded that the taste "kokumi" extract obtained in the experiment with the addition, was stronger than in the sample with the same concentration of γ-Glu-Val-Gly. In addition, to evaluate the taste of the "kokumi" as primary and secondary taste "kokumi" and finish "kokumi" used evaluation exhibited four members of the jury for each type of taste. The results are presented in Table 5. Primary and secondary taste mean taste, feel within 0-4 sec after the tasting, and the finish means taste, feel in 5 seconds later. As can be seen from the presented results, according to the jury, primary and secondary taste "kokumi" and finish "kokumi" extract obtained by adding γ-Glu-Val-Gly in the process of cultivation, were stronger due to the fact that γ-Glu-Val-Gly metabolisable in a special way, or because of the synergism of γ-Glu-Val-Gly and some component of the yeast extract.

Table 5
Assessment of primary and secondary taste "kokumi"Assessment aftertaste "kokumi"
Control0.00.0
Standard solution (10 mM γ-Glu-Val-Gly, 0.2% MSG, 0.5% NaCl)3.03.0
The extract obtained in the experiment with added4.0±0.13.9±0.3
The extract obtained in the experiment without adding 2.5±0.91.8±0.6

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

Loop biomass of cells of strain S288C was inoculable on Wednesday SD (50 ml Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at 120 rpm was Determined by absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips) in an amount such that the value OD660at the beginning of the cultivation was 0.01, and cultivated at 30°C for 24 h with shaking on a rotary shaker at 120 rpm To a nutrient medium SD in advance was added γ-Glu-Val (Bachem) to a final concentration of 100 ppm.

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

Table 6
γ-Glu-Val (ppm)γ-Glu-Val-Gly (ppm)
Without adding30
With the addition of γ-Glu-Val717120

The presented results show that part of the absorbed etc is grevyi cells γ-Glu-Val was modified in γ-Glu-Val-Gly.

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

Loop biomass of cells of strain S288C was inoculable on Wednesday SD (50 ml Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at 120 rpm was Determined by absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips) in an amount such that the value OD660at the beginning of the cultivation was 0.01, and cultivated at 30°C for 19 h with shaking on a rotary shaker at 120 rpm After 19 h were added aqueous solutions of γ-Glu-Val of different concentrations to achieve a given concentration and continued culturing for 1 h For these crops have calculated the content of γ-Glu-Val in the dried cells as described in Example 2. In addition, we calculated the content of dry substances in the extract and the amount of γ-Glu-Val dry substances. The results are presented in Table 7.

Table 7
The amount of γ-Glu-Val, added on Wednesday (ppm)025100200
The content of γ-Glu-Val in the dried cells (ppm)2 1468636910930
The content of γ-Glu-Val in the dry matter of the extract (ppm)984912759949474

The presented results show that the greater the number of added γ-Glu-Val, the more 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)

Loop biomass of cells of strain S288C was inoculable on Wednesday, SD (used in the preparation of the environment SD ammonium sulfate was replaced by Proline (final concentration of Proline was 0.1 g/l), 50 ml Sakaguchi flasks 500 ml) and grown at 30°C for 24 h with shaking at 120 rpm was Determined by absorption of the liquid culture, the culture was inoculable on Wednesday SD (400 ml conical flasks with a volume of 2 l with rumble strips) in an amount such that the value OD660at the beginning of the cultivation was 0.01, and cultivated at 30°C for 24 h with shaking on a rotary shaker at 120 rpm To a nutrient medium SD in advance was added to Val-Gly (Bachem) to a final concentration of 100 ppm.

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

Table 8
γ-Glu-Val-Gly (ppm)
Experiment without adding Val-Gly0
Experiment with adding Val-Gly2

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

Example 9: Yeast extract, obtained by adding Val-Gly, which was affected by γ-glutamyltransferase

Prepared 1% aqueous solution of yeast extract, in which the GSH content was about 8% (dry matter basis), and brought the pH to 7.0 using NaOH. To this solution was added powdered Val-Gly to a final concentration of 400 ppm, 800 ppm and 4000 ppm for the preparation of test samples. An aqueous solution of yeast extract, obtained without adding Val-Gly, also used as control. These test samples were added glutaminase (Glutaminase Daiwa SD-C100S, Daiwa kasei) in an amount of 1 mg/ml and the enzymatic reaction was performed at 37°C for 120 minutes in Addition, instead of glutaminase was added γ-GTP (γ-glutamyltranspeptidase kidney horse, Sigma, Code G9270-100UN) in an amount of 0.05 mg/ml and perform the enzymatic reaction at 37°C for 120 min. the Reaction mixture cf the memory after the reaction was cooled on ice and determined the content of γ-Glu-Val-Gly, GSH and Cys-Gly.

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

Table 9
The concentration of the components in the reaction mixture with glutaminate
γ-Glu-Val-Gly (ppm)GSH (ppm)Cys-Gly (ppm)
Without addingTo reaction0.0835.66.3
After reaction0.03.1701.6
400 ppmTo reaction0.0862.49.9
After reaction1.43.5715.0
800 ppmTo reaction0.0851.56.7
After reaction3.83.6705.7
4000 ppmTo reaction0.0826.26.1
After reaction16.03.4683.3

Table 10
The concentration of the components in the reaction mixture with γ-GTP
γ-Glu-Val-Gly (ppm)GSH (ppm)Cys-Gly (ppm)
Without addingTo reaction0.0813.65.2
After reaction0.8432.4335.1
400 ppm To reaction0.0824.64.5
After reaction150.9333.2388.2
800 ppmTo reaction0.0836.96.1
After reaction157.9316.3417.3
4000 ppmTo reaction0.0857.26.7
After reaction340.5180.8462.6

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

Example 10: Add γ-Glu-Val to the strain with enhanced expression of GSH2

To enhance the expression of GSH2 you can use the following procedure. 1) Getting auxotrophic for uracil strain (ura3 mutant from strain S288C.

Auxotrophic for uracil strain can be obtained by seeding the cells of the strain Saccharomyces cerevisiae processed what utenam using traditional method on agar medium containing 5-FOA, and selection of strains grown ura3 mutants (see, for example, METHODS IN YEAST GENETICS, 2000 EDITION, p.179). Auxotrophic for uracil strain can also be obtained by injection adjacent to the gene URA3 DNA not containing the URA3 gene in strain S288C and subsequent destruction of the URA3 gene, as shown below.

First, the area before the URA3 gene size 500 BP amplified in PCR using the primers presented in the sequence Listing under the numbers SEQ ID NO:1 and SEQ ID NO:2, and chromosomal DNA of strain S288C as a matrix. Furthermore, the area after the gene URA3 value 500 BP also amplified in PCR using the primers presented in the sequence Listing under the numbers SEQ ID NO:3 and SEQ ID NO:4. Used the following conditions for PCR: 25 cycles: denaturation at 94°C for 10 sec, annealing at 55°C for 10 s, elongation at 72°C for 1 min. Then, to obtain the DNA fragment size 1 TPN, consisting of the area in front of the gene URA3 the size of 500 BP and the region after the gene URA3 the size of 500 BP, had overlapping PCR fragments using the obtained two DNA fragments purified by ethanol precipitation, as matrices and primers presented in the sequence Listing under the numbers SEQ ID NO:5 and SEQ ID NO:6. Strain S288C transformed with this DNA fragment, and then were cultured over night in medium SD with the addition of uracil, then cells were sown on a Cup with the environment 5-FOA. From the obtained transformants obtained ura3Δ0 strain.

2) Obtaining the plasmid is used as the matrix for the replacement of the promoter.

First, the URA3 locus amplified in PCR using primers SEQ ID NO:7 and SEQ ID NO:8, and the chromosomal DNA of the wild-type strain of Saccharomyces cerevisiae as a matrix (25 cycles: denaturation: 94°C for 10 sec, annealing at 50°C for 10 s, elongation at 72°C for 1 min). The obtained DNA fragment was purified by precipitation with ethanol, then treated with restrictase SphI and EcoRI, the PCR product was incorporated into plasmid pUC19 sites SphI-EcoRI to obtain a pUC19-URA3. Then amplified region PGK1 promoter using chromosomal DNA of strain wild-type Saccharomyces cerevisiae as a template and primers SEQ ID NO:9 and SEQ ID NO:10. This DNA fragment was treated with restriction enzyme > PST, and was built in pUC19-URA3 treated with restriction enzyme > PST processing site > PST alkaline phosphatase intestines of a calf, to obtain pUC19-PGK1p-URA3. Similarly the PGK1 promoter, amplificatory using primers SEQ ID NO:11 and SEQ ID NO:12, was treated with the restriction enzyme AatII and was built in pUC19-PGK1p-URA treated with restriction enzyme AatII processing AatII site of alkaline phosphatase intestines of a calf, to obtain pUC19-PGK1p-URA3-PGK1p. When the obtained plasmid sequenced, was confirmed by nucleotide sequence is alnost, expected in the case of using as the template the chromosomal DNA of strain S288C. Therefore, even when using as the template the chromosomal DNA of strain S288C instead of the chromosomal DNA of the wild-type strain can be obtained in this plasmid.

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

Spent PCR using primers SEQ ID NO:13, at the 5'end of which is a sequence before GSH2 gene, primers SEQ ID NO:14, containing part of the sequence open reading frame beginning with the start codon of the gene GSH2, and C19-PGK1p-URA3-PGK1p as matrix (25 cycles: denaturation at 94°C for 10 sec, annealing at 60°C for 10 s, elongation at 72°C for 4 min) to obtain a DNA fragment containing the URA3 gene between the PGK1 promoter. Strain ura3Δ0 can be transformed with this DNA fragment, after which it is sown on the Cup with the environment SD to obtain transformants, and transformants can be obtained strain, in which the promoter of the gene GSH2 substituted by design PGK1 promoter-URA3-PGK1 promoter.

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

Strain, in which the promoter of the gene GSH2 substituted by design PGK1 promoter-URA3-PGK1 promoter were grown over night on the environment SD with the addition of uracil, an appropriate volume of culture were sown on the Cup with the environment 5-FOA. From grown colonies m the should to obtain the strain, in which the URA3 gene is removed by homologous recombination between the introduced PGK1 promoters and promoter GSH2 replaced by the PGK1 promoter. In addition, by introducing into the resulting strain DNA fragment, amplified using the chromosome of the wild-type strain as template and primers SEQ ID NO:15 and SEQ ID NO:16, it is possible to obtain the strain genotype is the same as the genotype of the strain ura3Δ0 except that the URA3 gene of wild-type and promoter GSH2 replaced by the PGK1 promoter.

Using this strain as described above received yeast extract and appreciated it the same way as described in Examples 3, 4 and 6-8.

Example 11: Adding γ-Glu-Val-Gly to the strain with enhanced expression of HGT1

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

Using this strain as described above received yeast extract and appreciated it the same way as described in point is imarah 3, 4 and 6-8.

Example 12: an increase in the activity of transport of γ-Glu-Val-Gly in HGT1p or RCT-modified strain.

The influence of HGT1p, which is specific for GSH Transporter, and Transporter peptides, " 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 gene of resistance to geneticin (G418) (PADH1-kanR) amplified in 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 primer URA3Km-L1 homologous region before URA3 gene, 40 nucleotides at the 5'end of the primer URA3Km-R2 complementary region for the URA3 gene.

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

Got a strain S288C ura3Δ0::PADH1-kanR, which may be referred to and as S288C ura3Δ0. This strain was used to construct strains with increased expression of HGT1p or " PTR2.

On the other hand, to use to construct a strain with a deletion of HGT1p or " PTR2 one auxotrophic for uracil mutant was obtained in the following way. The fragment containing the URA3 gene regions of the promoter and terminator, amp shall have oficially in PCR using chromosomal DNA of strain S288C as a matrix and the above primers ura3up2 and ura3dn2. This fragment was cloned at the Smal site in pBluescript II KS (+) and confirm the correctness of a design by sequencing. Got plasmid pKS-URA3-13 (Fig 3, SEQ ID NO:28).

Plasmid pKS-URA3-13 was treated with restrictase StuI and NcoI and then processed by the fragment maple DNA polymerase I to blunt the NcoI site. The resulting fragment size of 4.38 TPN ligated to HincII - HincII fragment pUG6 value of 1.64 TPN [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], module containing loxP-kanMX-loxP. The resulting plasmid is referred to as pKS-URA3-13-kanMX. Map and diagram of the construction of this plasmid is shown in Figure 4, SEQ ID NO:29.

Then kanMX gene, flanked by fragments of the URA3 gene, amplified in PCR using the above primers ura3up2 and ura3dn2 and the plasmid pKS-URA3-13-kanMX as a matrix. The resulting fragment was used for transformation of S.cerevisiae strain S288C, then transformants were selected on YPD medium containing 200 μg/ml G418. The result was obtained strains derived from S288C, with a deletion of the 227 nucleotides of the coding region of the gene URA3. This deletion is referred to as ura3Δ227::loxP-kanMX-loxP. For deletions marker kanMX the resulting strain was transformed with 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 The-recombinases, p is on the control of the GAL1 promoter and the URA3 gene as a selective marker. Cut the marker was performed after the induction of the synthesis of She-recombinase due to migration of cells from the YPD medium on YPG medium (containing galactose). The resulting strain from which the deleted plasmid pSH47, called S288C ura3Δ227::l.

2) Obtaining a mutant strain with a deletion of HGT1p

The DNA fragment containing the cassette Agleu2-CaURA3-Agleu2, amplified in PCR using the 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 template and primers hgtl-pUG6u (SEQ ID NO:30) and hgtl-pUG6d (SEQ ID NO:31).

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

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

3) Obtaining a mutant strain with a deletion of the " PTR2

Module PADH1-kanR from plasmids pUC19AOX-G418-BRI cut using restricted bI and SacI and subclinically on the same sites in the plasmid pUG6. The obtained plasmid was named pLoxP-PADH1-kanR5 (Figure 5).

The DNA fragment containing loxP module-PADH1-kanR-loxP, amplified in PCR is the use of plasmid pLoxP-PADH1-kanR5 as template and primers " ptr2-pUG6u (SEQ ID NO:34) and " ptr2-pUG6d (SEQ ID NO:35).

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

The resulting fragment was used for transformation of strain S288C ura3Δ227::l. The transformants were selected on plates with medium SD with the addition of 200 μg/ml G418. The resulting strain S288C ura3Δ227::l ptr2Δ967::loxP-PADH1-kanR-loxP with a deletion of nucleotides from 967 in 1806 coding region of the gene called " ptr2 S288C tr2Δ.

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

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

Strain S288C 1Δ tr2Δ constructed similarly to that of strain S288C tr2Δ, but as the recipient strain for transformation used strain S288C HGT1Δ.

5) Obtaining strains with increased production of HGT1

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

Fragment HindIII-HindIII plasmid pKS-URA3-13 value of 1.16 TPN was subclinically the HindIII site in the plasmid pBluescript II KS(+) with a pKS-URA3H-d. Fragment Cloned-SalI digestion of plasmid pUC19-PADH1-r konyrov is whether the SalI-kpni restriction sites sites in pKS-URA3H-d. The obtained plasmid pKS-URA3-P-ADH1 (Fig.7).

Then the plasmid pKS-URA3-PADH1 were treated with restrictase lI and BsrGI. After restriction analysis of plasmid were treated with fragment maple DNA polymerase I to make ends were blunt and ligated. The resulting plasmid pKS-URA3-PADH1-5'd, was treated with restriction enzyme EcoRI, treated fragment maple DNA polymerase I, and then was treated with a restriction enzyme bI. The resulting fragment is ligated with a fragment of PADH1cut from the plasmid pUC19-PADH1-r using restricted bI and BstZ17I. Thus was obtained a plasmid pKS-URA3-PADH1-LR (6).

The DNA fragment containing the cassette PADH1-URA3-PADH1, amplified in 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 primer PADH1-HGT1 40 complementary to the first nucleotide coding region HGT1, 40 nucleotides at the 5'end of the primer HGT1-PADH1 homologous significant body before HGT1 gene.

The DNA fragment containing the area before the HGT1 gene, amplified in PCR using the chromosomal DNA of the strain S. cerevisiae S288C as 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 (the primers used were hgt53 and PADH1-HGT1) as described in [Shevchuk, N.A., Bryksin, A.V., Nusinovich, Y.A., Cabello, F.C., Sutherland M., Ladisch S. 2004. Construction of long DNA molecules using long CR-based fusion of several fragments simultaneously. Nucleic Acids Res.32(2):e19]. The resulting fragment was used for transformation of strain S288C ura3Δ0::PADH1-kanR. Transformants were selected on plates with medium SD. The result was obtained strain S288C ura3Δ0::PADH1-kanR PADH1-URA3-PADH1-HGT1. Strain called S288C PADH1-HGT1.

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

6) Obtaining strains with increased production of " PTR2

Replacement of the native promoter " PTR2 implemented as a replacement of the promoter of HGT1p (Fig).

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 the Primer 3;

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

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

7) Evaluation of mutants

Each strain was grown in test tubes with a volume of 50 ml with 5 ml of medium SD with the addition of γ-Glu-Val-Gly to a final concentration of 100 ppm. The tubes were seeded overnight culture of the appropriate strain to the initial density OD600=0.5 and placed on a shaker at 240 rpm and 30°C. were Cultured for 7 hours Then clickies 2 ml of culture were collected using centrifugation and washed three times milliQ water. The washed cells are suspended in 1 ml of milliQ water and incubated at 70°C for 10 min in a water bath for extraction of intracellular content. After separation of the extract from cell debris by centrifugation the aqueous phase was transferred into a clean vial and subjected to vacuum drying. Thus prepared dry extract. Then measured γ-Glu-Val-Gly dry extract using the method LC-MS/MS. The procedure is described in detail below.

Equipment:

Mass spectrometer: Triple Quadrupole Agilent 6410

HPLC:gilent 1200

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

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

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

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

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

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

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

The HPLC conditions:

The mobile phase A - 0.1% HCOOH in H2O

Mobile phase b - 0.1% HCOOH in MeCN.

Speed duct - 0.250 ml/mi is

Table 11
The terms of the gradient
Time minThe mobile phase AndThe mobile phase
0955
0,5955
307525
352080
402080
41955

Table 12
Conditions detection in the mass spectrometer
ConnectionIon-precursor (m/z)Ion-product (m/z)
γ-Glu-Val-Gly474.2304.2 300.1 229.0

Using these d is the R calculated intracellular content of γ-Glu-Val-Gly. The results are presented in Tables 13 and 14. It is shown that gene HGT1p essential for the transport of γ-Glu-Val-Gly from the culture medium, and " ptr2 gene is not significant.

Table 13
Evaluation of strains with a deletion of the HGT1 and/or " PTR2
StrainOD600γ-Glu-Val-Gly (mg l-1OD600-1)
S288C3.31.93
S288CHGT1Δ3.30.01
S288C ptr2Δ3.81.84
S288C HGT1Δ ptr2Δ3.40.01

Tablica
Evaluation of strains with increased production HGT1 or " PTR2
StrainCD600γ-Glu-Val-Gly (mg l-1D600-1)
S288C3.403.3
S288C PADH1-HGT11.6428.6
S288C PADH1- "PTR22.642.6

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

S288C PADH1-HGT1, a strain with increased production HGT1p, obtained in Example 12, and its parent strain S288C was assessed by their ability to transport into the cell of γ-Glu-Val-Gly in the same manner as in Example 4. The number added to the nutrient medium of γ-Glu-Val-Gly - final concentration of 100 ppm. In the intracellular content of γ-Glu-Val-Gly in S288C was about 0.9% on dry weight, and the intracellular content of γ-Glu-Val-Gly in S288C PADH1-HGT1 dry weight was approximately 4.9%. This result confirmed the influence of the increased production HGT1p.

1. A method of obtaining a yeast extract to make food taste “kokumi”containing peptide γ-Glu-X, which includes the cultivation of yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly or X-Gly, and preparation obtained from cells yeast extract, characterized in that X represents an amino acid or its derivative other than Cys and its derivatives.

2. The method according to claim 1, characterized in that the culture medium contains 0.1 ppm or more of the indicated peptide, and is shown yeast extract contains a peptide γ-Glu-X in the 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 such that in the modified yeast enhanced the ability to transport the specified peptide into the cell.

5. The method according to claim 4, characterized in that in said yeast increased activity of HGT1p.

6. The method according to claim 1, characterized in that these yeasts are Saccharomyces cerevisiae.

7. A method of obtaining a yeast extract to make food taste “kokumi”containing peptide γ-Glu-X-Gly, which includes the cultivation of yeast in a nutrient medium containing a peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly or X-Gly, and preparation obtained from cells yeast extract, characterized in that X represents an amino acid or its derivative other than Cys and its derivatives.

8. The method according to claim 7, characterized in that the culture medium contains 0.1 ppm or more of the indicated peptide and the indicated yeast extract contains a peptide γ-Glu-X-Gly in the 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 such that in the modified yeast enhanced the ability to transport the specified peptide into the cell.

11. The method according to claim 10, distinguish the different topics in these yeast increased activity of HGT1p.

12. The method according to claim 7, characterized in that these yeasts are Saccharomyces cerevisiae.

13. A method of obtaining a yeast extract to make food taste “kokumi”containing peptide, γ-Glu-X, including the effects of γ-glutamyl transferase in yeast extract containing X or X-Gly, obtained from yeast grown in a nutrient medium to which is added an amino acid or peptide, selected from the group consisting of X or X-Gly, wherein X represents an amino acid or its derivative other than Cys and its derivatives.

14. The method according to item 13, wherein the specified amino acid or peptide is added in quantities of 1% or more by dry weight of the yeast extract and the yeast extract contains a peptide γ-Glu-X in the 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 the specified yeast is the yeast Saccharomyces cerevisiae.

17. A method of obtaining a yeast extract to make food taste “kokumi”containing peptide, γ-Glu-X-Gly, including the effects of γ-glutamyl transferase in yeast extract containing X or X-Gly, obtained from yeast grown in a nutrient medium to which is added amino acids is or peptide, selected from the group consisting of X or X-Gly, wherein X represents an amino acid or its derivative other than Cys and its derivatives.

18. The method according to 17, characterized in that the amino acid or peptide is added in quantities of 1% or more by dry weight of the yeast extract and the yeast extract contains a peptide γ-Glu-X-Gly in the amount of 0.005% or more of the dry weight of the yeast extract.

19. The method according to 17 or 18, characterized in that X is Val.

20. The method according to 17, characterized in that these yeasts are Saccharomyces cerevisiae.

21. Yeast extract to make food taste “kokumi”containing peptide selected from the group consisting of γ-Glu-X and γ-Glu-X-Gly, in the amount of 0.005% or more by dry weight of yeast extract, characterized in that X represents 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. Yeast extract according to item 21, containing the peptide in the amount of 0.02% or higher.

23. Yeast extract according to item 21, wherein X is Val.

24. Yeast extract according to item 21, wherein the specified yeast is the yeast Saccharomyces cerevisiae.



 

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