JP2002171961A - New yeast and yeast extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an yeast extract, in which glutamic acid content is increased without adding glutamic acid as an additive, having thick taste and high potency of delicious taste. SOLUTION: An yeast extract comprising at least 3% (based on extract solid content) glutamic acid derived from glutamic acid intracellularly isolated is obtained by digesting an yeast containing free glutamic acid in an amount of >=15 mg per g dried yeast cell in the cell. The yeast extract in the present invention has remarkably thick taste, as compared with yeast extract to which glutamic acid is added as an additive.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel yeast extract and a method for producing the same. Yeast extract is used as a natural seasoning.

[0002]

BACKGROUND ART Industrial production of yeast extract is mainly performed by digesting baker's yeast, brewer's yeast, and yeast of the genus Candida, and is a natural seasoning comprising taste components of amino acids, peptides and nucleotides produced. Has been used as. These yeast extracts are used as a beef substitute or a secret taste for imparting a complex taste. These umami depend on the taste of nucleotide components such as peptide components, inosinic acid, and guanylic acid, and the umami titer is not so high. Its use has been limited because it is not possible to increase the amount added to the material. Further, the taste quality of yeast extract is mainly composed of peptides and nucleotides, and is characterized by a broad and relatively rich aftertaste, and lacks a clear and umami taste with a pre-taste.

[0003] Sodium glutamate and the like are added to the yeast extract as an additive in order to enhance the umami titer of the yeast extract and to achieve a complex seasoning characteristic comprising an amino acid having a so-called first taste and a nucleotide component having a aftertaste. (Hereinafter referred to as “external addition”) are also manufactured. However, in recent years, consumers who dislike seasoning to which sodium glutamate has been added have been increasing with the natural consciousness and health consciousness, and the seasoning industry has also tended to be a natural seasoning. From such a background, there has been a demand for a yeast extract having a high umami titer without externally adding sodium glutamate, that is, a yeast extract having a high glutamic acid content.

[0004] Industrial production methods of glutamic acid using prokaryotes, that is, coryneform bacteria, Escherichia coli, and the like have been widely known. For example, it has been reported that a large amount of glutamic acid is released and accumulated in a production medium by reducing the activity of 2-ketoglutarate dehydrogenase, which is one enzyme of the tricarboxylic acid cycle of the bacterium. However, it was not known whether the glutamate concentration in these bacterial cells increased. Furthermore,
Even if the KGD activity of the yeast cells is reduced, yeast, which is a eukaryote, is governed by a complex metabolic control system, and it has not been considered that glutamate is accumulated only by reducing the KGD activity.

On the other hand, it has been attempted to directly accumulate glutamate in yeast cells by culturing a mutant strain of yeast having resistance to glutamate analogs.
It is disclosed in EP 592785 that a yeast extract having a high glutamic acid content can be produced by digesting a yeast having a high intracellular free glutamic acid concentration. However, there is no description about the taste balance or yeast odor of the yeast extract, so Although the increase in price can be imagined, other effects cannot be confirmed.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have established a technique for producing a yeast extract having a high glutamic acid content without externally adding glutamic acid, and have studied with a view to confirming its effect on taste quality. Started.

That is, the present invention increases the content of glutamic acid, which is not externally added, preferably to about 3% or more based on the solid content of the extract, so that the umami has high umami taste and can be obtained when glutamic acid is simply externally added. Provided is a yeast extract having an unsavory taste.

[0008] The present invention further provides a method for producing the above yeast extract and a yeast containing 15 mg or more of free glutamine per gram of dry cells in cells used for the production of the above yeast extract.

The present invention further provides a method for obtaining the above yeast.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that (1) the activity of 2-ketoglutarate dehydrogenase (KGD) and / or glutamate decarboxylase (GAD) is reduced. Among the decreased yeasts, there are those having an increased concentration of free glutamic acid and free glutamine in the cells. (2) By digesting such yeasts in the presence of glutaminase, a yeast having a high glutamic acid content can be obtained. It was found that the extract can be produced, and (3) the taste of the yeast extract thus produced is surprisingly stronger in thickness than the conventional yeast extract with glutamic acid added externally. . Furthermore, (4) yeast having reduced glutamine synthetase (GS) activity together with KGD activity selectively accumulates glutamic acid in cells, but the yeast extract obtained therefrom is similar to that obtained by externally adding glutamic acid. The inventors confirmed that the thickness was small, and reached the present invention.

That is, the essence of the present invention is to find that when a yeast extract is produced from a yeast which accumulates free glutamine in cells, the "thickness" of the taste is specifically increased. Furthermore, another factor constituting the present invention is that the conversion of glutamine accumulated in cells to glutamic acid with glutaminase makes it possible to simultaneously increase the umami titer. Hereinafter, the present invention will be described in detail. The yeast that accumulates free glutamine may be any yeast that is acceptable as a food, and yeasts belonging to the genera Saccharomyces, Candida, Hansenula, Pichia, etc. may be used as the original strain. Although it is possible, a yeast capable of forming haploid spores of Saccharomyces and the like and capable of diploidization by male and female crossing is desirable in performing the breeding described below.

The yeast of the present invention having a high intracellular free glutamine concentration can be bred by introducing a mutation into a gene of an enzyme system involved in glutamic acid or glutamine metabolism. Examples of the gene into which the mutation is introduced include a 2-ketoglutarate dehydrogenase gene kgd1 and a glutamate decarboxylase gene gad1. However, even if a mutation is introduced into these genes, the amount of intracellular free glutamine does not increase in all yeasts, and it is important to select a yeast strain in which the amount of intracellular free glutamine increases due to the introduction of the mutation. It is.

With the recent advances in gene manipulation technology, it is easy to introduce a specific mutation into a specific yeast gene, and it is possible to use a gene disruption technique or a site-specific mutation technique to introduce the mutation. , Kgd1 and gad1 can be mutated to produce strains in which the activity of these gene products has been reduced or which have been deleted. In particular, Saccharomyces cere
visiae), the entire nucleotide sequence of the genome is known, and it is easy to use kgd1 or gad1 using PCR technology.
It is possible to clone the gene. A DNA fragment containing at least a part of the gene of interest is amplified by PCR and cloned using an E. coli host-vector system. After confirming the nucleotide sequence of the cloned fragment and confirming that it is a fragment of a gene into which a mutation is to be introduced, the fragment is used for introducing a mutation. Saccharomyces
Even when yeast other than S. cerevisiae is used, in some cases, the target gene can be amplified by performing PCR using primers designed based on the sequence of the Saccharomyces cerevisiae genome. If cloning by PCR is difficult, the desired gene product (enzyme) can be purified and its amino acid sequence can be examined to design cloning primers and probes to perform cloning with high accuracy. It is.

As a method for mutating these genes using cloned gene fragments containing kgd1, gad1, etc., gene disruption is convenient. That is,
A DNA fragment which is a part of the coding region of the gene of interest and does not contain portions corresponding to the N-terminal and C-terminal of the protein is cut out using a restriction enzyme or the like, and this is cut into a yIp type vector for yeast. Connecting. Using this, yeast is transformed and a gene-disrupted strain is selected. As a yeast transformation vector, a vector using a drug resistance gene such as an antibiotic as a marker is advantageous in selecting a transformed strain.

As a method other than gene disruption, site-specific mutation (Nucleic Acids Res. 10, 6487-6500 (1982))
Can also be used. Mutations such as deletion, insertion and substitution are introduced into the coding region of the cloned DNA fragment using a known standard method. In this case, in order to efficiently detect the strain into which the mutation has been introduced, co-transformation with a yEp-type or yRp-type plasmid having a drug-resistance marker is performed, and selection is made from drug-resistant colonies. It becomes possible to frequently select a strain into which the desired mutation has been introduced.

The transformation method can also be used in the step of introducing the DNA into which the mutation has been introduced into the genome of yeast. That is, yeast is transformed using the DNA fragment into which the mutation has been introduced, and yeast in which the mutant DNA has been replaced with genomic DNA is selected. For transformation, known methods such as protoplast method, alkali cation method and electroporation method can be used. Whether the target mutation has been introduced into the genome can be confirmed by PCR, and the activity of the gene product enzyme can be determined by a standard method (α-ketoglutarate dehyd
rogenase: J. Biol. Chem 249, 3660-3670 (1969), Advanc
e in Biophysics (Kotani, M., ed) Vol 9, pp.187-227, Uni
v. of Tokyo press, Glutamate decarboxylase: Meth.B
iochem.Anal. 4,285-306 (1957), Biochem.Biophys.Re
s. Commun. 28,525-530 (1967), Biochemistry 9,226-23
3 (1970), Glutamine synthetase: Anal.Biochem., 95, 2
75-285 (1979)).

As mentioned above, some yeasts have KGD.
There are those that accumulate glutamine and glutamic acid in cells at high concentrations by reducing GAD activity and those that do not, while using genetic engineering techniques, that is, using gene disruption or site-specific mutation. This makes it possible to easily discriminate these differences, which means that the yeast of the present invention can be bred with high accuracy by using the selected excellent strain as a breeding original strain.
Specifically, after introducing these mutations (gene disruption) using gene disruption or site-specific mutation, the yeast is cultured, and the free glutamine and free glutamic acid concentrations in the cells are measured using known methods. By doing so, a yeast that can be used in the present invention can be selected. The criterion for selecting a mutant (gene-disrupted strain) is that a large amount of glutamic acid or glutamine is accumulated in the cell. By digesting such a yeast in the presence of glutaminase, a yeast extract having a high glutamic acid content and a thick taste can be produced.

The intracellular glutamine and glutamic acid concentrations are measured as follows. The yeast cells are collected from the yeast culture by centrifugation, washed appropriately with water, and then freeze-dried. Lyophilized yeast cells (of known weight) are suspended in water, crushed with glass beads, and the centrifuged supernatant is subjected to hot water extraction. The glutamic acid concentration was measured by a known method such as amino acid analysis for both the glutamic acid reaction and the glutamic acid concentration before and after the glutaminase reaction, whereby the concentrations of intracellular free glutamic acid and free glutamine were determined. Is calculated. Glutamine and glutamic acid in the extract before the glutaminase reaction can be simultaneously quantified by changing the conditions of the amino acid analyzer.

When the kgd1 or gad1 mutation is introduced alone into yeast, 16 to 1 kg of yeast dry weight is required.
22 mg of free glutamine and 21 to 30 mg of free glutamic acid are accumulated, and a yeast extract containing 12 to 15% glutamic acid per solid can be produced by digesting this yeast. The taste characteristics of the yeast extract obtained in this way have a longer umami response time than those produced by simply adding the required amount of glutamic acid, and are accompanied by a kokumi taste, salting effect, etc. The taste is further added to the taste, and the whole is harmonious. This characteristic of taste is presumed to be of primordial significance in that at least 3% or more of glutamic acid in yeast extract is derived from intracellular free glutamine. Then, when this 3% or more free glutamine is converted into the amount in 1 g of dry yeast, it becomes at least 15 mg.

It is not clear why digestion of yeast in which glutamine accumulates in cells can produce a yeast extract with a rich taste, but the accumulation of glutamine balances intracellular nitrogen metabolism, that is, the balance between amino acids and organic acids. It is presumed that the balance changes, and as a result, a thick yeast extract is produced. In addition, by adding a step of a glutaminase reaction when digesting yeast, free glutamine can be efficiently converted to glutamic acid, and a yeast extract excellent in umami titer can be produced. In order to produce a yeast extract having a high umami titer, it is desirable that the total concentration of free glutamine and free glutamic acid in cells be 30 mg / g dry cells or more.

In the present invention, Saccharomyces cerevisiae IF is a specific example of a yeast in which intracellular glutamine is increased by reducing the activity of KGD and GAD.
O10150. This strain can be purchased by anyone from the Institute of Fermentation as a laboratory yeast. Kgd against Saccharomyces cerevisiae IFO10150
By performing one gene disruption, this strain accumulates more than 16 mg / g dry cells in cells and about 2.5 times the free glutamine of the original strain. When the gad1 gene was disrupted in this strain, the intracellular free glutamine content was 21 m
g / g dry cells. Furthermore, when both kgd1 and gad1 are destroyed, free glutamine of 24 mg / g dry cells can be accumulated.

On the other hand, even if the same Saccharomyces cerevisiae disrupts kgd1 with respect to YPH499, YPH500 and YPH501 strains (all of which can be purchased from Stratagene), the amount of intracellular free glutamine is 4.0 to 4.0. It is about 8 mg / g dry cells and hardly increases compared to the original strain. G for these strains
When the ad1 gene is disrupted, intracellular free glutamine slightly increases to 6.0-6.4 mg / g dry cells,
Destruction of both kgd1 and gad1 does not increase the amount of glutamine as compared to the gad1-disrupted strain.

Similarly, in practical yeasts, strains in which the amount of glutamine accumulated due to gene disruption changes and strains in which glutamine accumulation does not change can be selected. Normally, practical yeasts exist in a diploid form, and spores are formed from the diploids, and evaluation by gene disruption is performed in a haploid form. Among haploid yeasts obtained by sporulation, strains in which the amount of intracellular free glutamine increases due to gene disruption can be selected. For the selected haploid yeast, a site-specific mutation (Nucleic Acids Res.
10, 6487-6500 (1982)), or by carrying out mutagenesis using a conventional mutagen, and selecting a strain with reduced KGD or GAD activity, thereby breeding the yeast of the present invention. In general, haploid yeasts often grow at a slower rate than diploid yeasts. Therefore, the production of diploid yeasts based on breeding haploid yeasts can also be performed using hybridization and cell fusion techniques. It is possible.

Although yeast into which gene disruption or site-specific mutation has been introduced can be directly used for the production of yeast extract, yeast produced using recombinant DNA technology is used as a food material. Guidelines have not yet been established. Thus, it is substantially important to breed such yeasts without using recombinant DNA technology. An original strain selected by an operation such as gene disruption, that is, an original strain selected by site-directed mutation or gene disruption, that is, a strain capable of increasing the amount of intracellular glutamine is defined as an original strain, and these are irradiated with ultraviolet light or the like. By mutating with a mutagen and selecting a strain with reduced KGD or GAD activity, it is possible to breed a yeast having no problem in food hygiene. Fortunately, kgd1-disrupted strains often have a phenotype that cannot grow using ethanol, glycerin, pyruvic acid or the like as a single carbon source, and it is also possible to select a target mutant strain using this as an index. The target mutant can also be selected by directly measuring the intracellular glutamine concentration. The selected mutants often have poor growth rates and yields as they are, so it is possible to breed strains with good growth rates and yields and high intracellular glutamine concentrations by repeating crossing and cell fusion. it can. Preparation of yeast extract The method of culturing yeast is no different from known methods. The yeast is cultured in a medium containing sucrose, glucose, liquid sugar, molasses or the like as a carbon source and ammonia or urea as a nitrogen source. To suppress alcohol fermentation by yeast, it is necessary to supply oxygen sufficiently.However, if oxygen is supplied excessively, the accumulation of glutamic acid and glutamine may decrease. It is better to determine the amount. After completion of the culture, the yeast cells are collected by centrifugation or filtration.

Known methods can also be used to obtain a yeast extract from the collected yeast cells. As a typical example,
Water is added to the yeast, and the components contained therein are extracted by dissolving the cell wall of the yeast. Thereafter, the enzyme is degraded by the enzyme of the yeast and / or the enzyme added from the outside.

To self-digest with the enzyme of yeast, 4
The reaction may be performed at 0 to 55 ° C for 6 to 72 hours. When an enzyme is added for digestion, proteases are required for protein degradation, cell wall lysing enzymes are required for cell wall lysis, glutaminase is required for converting glutamine to glutamic acid, and deaminase and nuclease are required for producing delicious nucleic acids. Add according to. When adding the enzyme, the enzyme derived from the raw yeast may be present or may be inactivated.

The order of adding the enzymes is as follows. First, a cell wall lysing enzyme is added, and then, if necessary, a protease is added and reacted. Thereafter, glutaminase may be further added and reacted. The reaction with glutaminase is a preferred embodiment of the present invention because it increases the amount of glutamic acid to increase the taste titer of the yeast extract. Further, in order to improve the taste of the obtained yeast extract due to the nucleic acid component, it is preferable to extract yeast nucleic acid into the extract. In that case, the nucleic acid in the yeast can be sufficiently efficiently extracted into the extract by heating the reaction product at about 65 ° C. for about 2 hours at pH 8.5. It is preferable that the yeast extract thus obtained is further inactivated by, for example, heating at about 90 ° C. for about 30 minutes to deactivate enzymes in the cells and various added enzymes.

As the above various enzymes, commercially available enzymes can be used as they are. For example, as a protease, protease A “Amano” (Amano Pharmaceutical), protease N “Amano” (Amano Pharmaceutical), etc., and as a cell wall lysing enzyme, YL-15 (Amano Pharmaceutical), Chitarase M
(Kumiai Kasei), glutaminase, glutaminase Daiwa C100 (Yamato Kasei), glutaminase F "Amano" (Amano Pharmaceutical), etc., deaminase as deamizyme (Amano Pharmaceutical), and nuclease as nuclease "Amano" (Amano Pharmaceutical). Is mentioned. Usually, 0.01% to 5% (preferably 0.1 to 2%) of these enzymes is added to the raw materials. Protease reaction is performed at pH 4-7 (preferably 5-6) at a temperature of 40-60 ° C.
(Preferably 40 to 55 ° C.), and the reaction time is 1 to 7
2 hours (preferably 16 to 24 hours) is appropriate. The temperature of each other enzyme reaction is the optimal temperature and pH of each enzyme.
If it is a zone, there is no problem. Each reaction time is 0.5-8
Time (preferably 1 to 4 hours) is appropriate.

As a result, the amount of glutamic acid in the dry solid content of the extract obtained by digesting the yeast of the present invention is 12%.
That is all. After the enzymatic reaction, the enzyme is heat-inactivated, and the supernatant is obtained by removing the cell residue by centrifugation or filter press or the like. Depending on the purpose, the supernatant may be subjected to membrane filtration using UF, MF, or the like to clarify the supernatant. The obtained liquid is concentrated to a target solid content. Powdering may be performed in addition to concentration. For powderization, spray drying, freeze drying and the like may be appropriately selected.
An auxiliary may be used at the time of powdering. The yeast extract thus produced has a strong taste of glutamic acid and shows a good taste in combination with the complex taste of the yeast extract,
When glutamic acid is externally added, it has a thickness that cannot be seen, and exhibits a very favorable taste as a seasoning.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[0031]

EXAMPLES The following methods were used to prepare and confirm gene-disrupted strains. (1) 2-ketoglutarate dehydrogenase gene (k
gd1) Cloning and Gene Disruption Genome extraction kit (ISOPLAN) manufactured by Nippon Gene from Saccharomyces cerevisiae IFO10150
Genomic DNA was extracted using T). To about 100 ng of the genomic DNA, about 1 μg of the primer DNA shown in SEQ ID NO: 1 of the Sequence Listing and about 1 μg of the primer DNA shown in SEQ ID NO: 2 of the Sequence Listing were added. This was subjected to PCR in a volume of 50 μl using LA-Taq manufactured by Takara Shuzo. The PCR conditions were 95 ° C for 0.5 minute, 55 ° C for 1 minute, and 72 ° C for 1.5 minutes for 25 cycles. The amplification of a fragment of about 1.3 kb was confirmed, and this was purified using a DNA purification kit manufactured by QIAGEN. The PCR product was digested with the restriction enzyme SacI, and pKF
The fragment was inserted into the SacI cleavage point of No. 18 and introduced into Escherichia coli JM109 strain (purchased from Takara Shuzo) by electroporation. After preparing the recombinant plasmid, the DNA sequence of the inserted fragment was confirmed. As a result, it was confirmed that the inserted fragment had the DNA sequence shown in SEQ ID NO: 3 in the sequence listing. The inserted fragment was a portion corresponding to amino acid residues 80 to 497 in the coding region (encoding 1015 amino acid residues) of the kgd1 gene of Saccharomyces cerevisiae.

This inserted fragment was cut out again with SacI,
The recombinant plasmid p integrated at the SacI cleavage point of pAUR101 (Takara Shuzo), a yIp type shuttle vector
AUR101-KGD1 was produced. Since a restriction enzyme NruI cleavage point was present in the inserted fragment of this plasmid, yeast was transformed by electroporation using the plasmid cut with this restriction enzyme.
For transformants resistant to 0.5 μg / ml aureobasidin on YPD agar medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar), Shiio, I. and Ujigawa-Takeda, K., Ag
2-ketoglutarate dehydrogenase activity was measured by the method described in ric. Biol. Chem., 44, 1897-1904 (1980).

(2) Cloning and Disruption of Glutamate Decarboxylase Gene (gad1) SEQ ID NOS: 4 and 5 in the Sequence Listing as primers for PCR
DNA was used. Using yeast genomic DNA as a template, PCR was performed under the same conditions as above, and amplification of a fragment of about 1.0 kb was confirmed. After the amplified DNA was purified, it was digested with the restriction enzyme SacI and inserted into pKF18 at the SacI cleavage point. The DNA sequence of the inserted fragment is shown in SEQ ID NO: 6 in the sequence listing. This fragment contains g of Saccharomyces cerevisiae.
It was a portion corresponding to the 141st to 461st amino acid residues in the coding region of the ad1 gene (encoding 586 amino acid residues).

The inserted fragment was incorporated into pI-RED1 (Toyobo), a yeast yIp type vector, and the pI-R
ED1-GAD1 was obtained. This plasmid has the unique restriction enzyme NruI on the insert and the plasmid DN
A was digested and used to transform yeast by electroporation. 10 on YPD agar
For transformants resistant to 0 μg / ml cycloheximide, see Okada, Y. and Shimada, C., Brain. Res., 9
Glutamate decarboxylase activity was measured using the method described in 8, 202-206 (1975). However, the measurement of the amount of γ-aminobutyric acid performed in the activity measurement was quantified using an amino acid analyzer ALC-1000 manufactured by Shimadzu Corporation.

(3) Glutamine synthetase gene (g
Cloning and gene disruption of In1) SEQ ID NO: 7 and 8 in the Sequence Listing as primers for PCR
DNA was used. Using yeast genomic DNA as a template, PCR was performed under the same conditions as above, and amplification of a fragment of about 0.4 kb was confirmed. After the amplified DNA was purified, it was digested with the restriction enzyme SacI and inserted into pKF18 at the SacI cleavage point. The DNA sequence of the inserted fragment is shown in SEQ ID NO: 9 in the sequence listing. This fragment contains g of Saccharomyces cerevisiae.
It was a portion corresponding to the 92nd to 232nd amino acid residues in the coding region of the ln1 gene (encoding 351 amino acid residues).

This insert fragment was incorporated into pI-RED1, which is a yeast yIp type vector, and was inserted into pI-RED1-GL.
N1 was obtained. The plasmid DNA was digested with Eco0109I, which is the only restriction enzyme that this plasmid has on the insert, and the resulting plasmid was used to transform yeast by electroporation. 100μ on YPD agar
For transformants resistant to g / ml cycloheximide, see Stadtman, ER et al, Anal. Biochem., 95, 2
Glutamine synthetase activity was measured using the method described in 75-285 (1979).

(4) Measurement of Intracellular Free Glutamine and Free Glutamate Concentration A synthetic medium (6.7 g / LYeast Nitrogen)
Base (manufactured by Difco), 20 g / L glucose, and 50 ml of 0.5 μg / ml aureobasidin or 100 μg / ml cycloheximide, if necessary) in a 500 ml baffled flask containing 30 ml at 30 ° C. and 120 rpm. Shaking culture was performed for 18 hours using a rotary shaker. 1500 × yeast cells
The cells were collected by centrifugation at 4 ° C. for 5 minutes and washed twice with water, and the washed cells were collected. This was freeze-dried and the weight of the dried cells was measured. 1.5 ml of about 30 mg of dried cells
Into an Eppendorf tube, add 450 μl of distilled water and 480 mg of glass beads (BRAUN,
(0.45-0.50 mm), and the mixture was stirred at the maximum rotation speed for 2 minutes using a micro tube mixer manufactured by Tommy. This was cooled on ice for 2 minutes. This operation was repeated four times to disrupt the yeast cells. Centrifugation (10
(000 × g, 4 ° C., 5 minutes), and the supernatant was heated at 95 ° C. for 15 minutes to precipitate the protein. Take a portion of the reaction and add 10 mg / ml glutaminase
An equal amount of a solution of Daiwa C-100 was added and reacted at 37 ° C. for 20 minutes. Centrifugation (10000 xg, 4 ° C, 5 minutes)
To remove the precipitate. Samples before and after glutaminase reaction were used for amino acid analyzer ALC-1 manufactured by Shimadzu Corporation
Amino acid analysis was performed using 000, and the amount of glutamine was determined from the difference between the two. The amounts of intracellular free glutamine and free glutamic acid were determined from the analyzed amounts of glutamine and glutamic acid and the weight of the dried yeast cells used.

(5) Yeast culture After culturing yeast on a YPD plate medium for one day, one platinum loop was added to a synthetic medium (6.7 g / LYeast Nitrogen Ba).
se (manufactured by Difco), 20 g / L glucose, and 0.5 μg / ml aureobasidin or 1 as needed.
00 μg / ml cycloheximide) 10 m
1 and inoculated at 30 ° C. for 24 hours under the same conditions as in (4) above. Measure the OD at 660 nm of this culture solution with a spectrophotometer.
It was measured by UV-160A (Shimadzu Corporation). Next, this culture solution was used as a seed, and OD 660 nm
Inoculate so that the value is 0.063 ± 0.002, and inoculate 30
Cultured at 18 ° C for 18 hours. The obtained culture is centrifuged (1
(500 × g, 5 minutes), and the obtained cells were washed with physiological saline. After washing, the cells were centrifuged under the same conditions as above to collect the cells.

Example 1 NruI of the recombinant plasmid pAUR101-KGD1
Saccharomyces cerevisiae IFO10 using digest
150 transformations were performed. It was confirmed that colonies showing aureobasidin resistance had no 2-ketoglutarate dehydrogenase activity (0.62 nmol / mi).
After n / mg protein), the intracellular free glutamine and free glutamic acid concentrations were measured. As a control, IFO10150 transformed with pAUR101 was used. The concentrations of intracellular free glutamine and free glutamic acid were 7.4 mg / g dry cells, 1
While the cells were 0.6 mg / g dry cells, the gene-disrupted strains each had 16.2 mg / g dry cells and 21.5 m / g.
g / g dry cells.

Example 2 Nru of recombinant plasmid pI-RED1-GAD1
Saccharomyces cerevisiae IFO1 using I digest
0150 was transformed. After confirming that there was no glutamate decarboxylase activity (0.01 U / mg or less) in the colonies showing cycloheximide resistance, the intracellular free glutamine and free glutamic acid concentrations were measured. A transformant with the vector DNA was used as a control. The concentrations of intracellular free glutamine and free glutamic acid were 6.8 mg / g dry cells and 10.
3 mg / g dry cells, whereas the gene-disrupted strains showed 21.2 mg / g dry cells and 27.1 mg / g dry cells, respectively.
g dried cells.

Example 3 The GAD1 gene was disrupted from the transformant obtained in Example 1 by the same method as in Example 2.
Gene disruption was confirmed by confirming enzyme activity, and intracellular glutamine and free glutamic acid concentrations were measured. In the strain in which the gene was double disrupted, the concentrations of intracellular free glutamine and free glutamic acid were 24.2 mg each.
/ G dry cells and 29.7 mg / g dry cells.

Comparative Example 1 NruI of Recombinant Plasmid pAUR101-KGD1
Saccharomyces cerevisiae YPH49 using digest
Nine transformations were performed. After confirming that there was no 2-ketoglutarate dehydrogenase activity in colonies showing aureobasidin resistance, intracellular free glutamine and free glutamic acid concentrations were measured. As a control, pAU
YPH499 transformed with R101 was used. The concentrations of intracellular free glutamine and free glutamic acid were 4.6 mg / g dry cells and 7.4 mg / g dry cells in the control strain, respectively, whereas those in the gene-disrupted strain were 4.
4 mg / g dry cells and 6.7 mg / g dry cells.

Comparative Example 2 Nru of recombinant plasmid pI-RED1-GAD1
Saccharomyces cerevisiae YPH4 using I digest
99 transformations were performed. After confirming that there was no glutamic acid decarboxylase activity in colonies showing cycloheximide resistance, the intracellular free glutamine and free glutamic acid concentrations were measured. Vector DNA as control
Was used. The concentrations of intracellular free glutamine and free glutamic acid were 3.9 mg in the control strain, respectively.
/ G dry cells and 6.2 mg / g dry cells, whereas the gene-disrupted strains had 6.5 mg / g dry cells and 9.9 mg / g dry cells, respectively.

Comparative Example 3 The GAD1 gene was further disrupted from the transformant obtained in Comparative Example 1 by the same method as in Comparative Example 2.
Gene disruption was confirmed by confirming enzyme activity, and intracellular free glutamine and free glutamic acid concentrations were measured. In the strain in which the gene was double disrupted, the concentration of intracellular free glutamine and free glutamic acid was 7.8 m each.
g / g dry cells were 11.1 mg / g dry cells.

Example 4 Each of the yeasts obtained in Examples 1, 2 and 3 was used in an amount of 20 g per dry solid, water was added so that the solid content became 10%, and the mixture was sufficiently stirred and suspended. The suspension is treated with 40% NaOH
After adjusting the pH to 5.7 with a solution, the cell wall lysing enzyme YL-
15 (Amano Pharmaceutical) 0.1% per dry solid content,
The reaction was performed at 45 ° C. for 8 hours. After the reaction, the enzyme was inactivated by heating at 70 ° C. for 30 minutes and cooled to 37 ° C. Glutaminase Daiwa C-100 (Daiwa Kasei) at 1 per solid
%, And reacted at 37 ° C. for 1 hour. After the reaction, 7
After heating at 0 ° C. for 30 minutes, a supernatant was obtained by centrifugation.
The obtained supernatant was freeze-dried to obtain a yeast extract powder. The extract solids recovery was 52% to 56%. The glutamic acid content was 5.9% in an extract using a non-deficient yeast as a control, 12.6% in an extract using a 2-ketoglutarate dehydrogenase-deficient yeast, and 15.1 in an extract using a glutamic acid decarboxylase-deficient yeast. %, And 16.2% in an extract using yeast lacking both enzymes. The taste quality of the obtained extract was such that the umami of glutamic acid was felt very strongly in the extract using the defective yeast, and a unique thickness of taste was felt.

Example 5 Each of the yeasts obtained in Examples 1, 2 and 3 was used in an amount of 20 g per dry solid, water was added so that the solid content became 10%, and the mixture was sufficiently stirred and suspended. The suspension is treated with 40% NaOH
After adjusting the pH to 8.5 with the solution, the mixture was heated at 65 ° C. for 2 hours and then at 90 ° C. for 30 minutes. This solution was adjusted to pH 5.8 with 75% phosphoric acid, protease N “Amano” was added at 0.2% per substrate protein, and reacted at 52 ° C. for 12 hours. Heat at 70 ° C for 30 minutes to inactivate the enzyme,
Further, after adjusting the pH to 5.3 with 75% phosphoric acid, nuclease “Amano” (Amano Pharmaceutical) was added at 0.04% per dry solid content, and reacted at 64 ° C. for 4 hours. After the reaction with nuclease, the pH was adjusted to 5.5 with 40% NaOH solution.
Adjusted to 6. Deamizyme (Amano Pharmaceutical) was added at 0.04% per dry solid content, and reacted at 45 ° C. for 2 hours.
Thereafter, the mixture was heated at 70 ° C. for 30 minutes to deactivate the enzyme, and a supernatant was obtained by centrifugation. The obtained supernatant was freeze-dried to obtain a yeast extract powder. Extract solids recovery rate is 50% -5
2%. The glutamic acid content and the (inosinic acid + guanylic acid) content were 4.2% and 1.8% in the extract using the non-deficient yeast as a control, and 14.0% in the extract using the 2-ketoglutarate dehydrogenase-deficient yeast. Extracts using glutamate decarboxylase-deficient yeast, 17.4% and 1.8%, and extracts using yeast deficient in both enzymes, 19.1% and 1.8%. In the extract obtained using the defective yeast, the synergistic effect of umami by glutamic acid and (inosinic acid + guanylic acid) was strongly extracted, and umami was felt very strongly. This yeast extract also had a unique taste thickness.

Comparative Example 4 Eco-Production of Recombinant Plasmid pI-RED1-GLN1
Kgd1 obtained in Example 1 using the 0109I digest
The disrupted strain was further transformed. After confirming that the colonies exhibiting cycloheximide resistance had no glutamine synthetase activity (0.01 U / mg or less), the intracellular free glutamine and free glutamic acid concentrations were measured. kgd
In the strain in which 1 and gln1 were simultaneously disrupted, the intracellular free glutamine and free glutamic acid concentrations were 40.2 and 40.2, respectively.
mg / g dry cells and 4.4 mg / g dry cells.

From this yeast, a yeast extract was produced in substantially the same manner as in Example 4. The extract solids recovery was 53% and the glutamic acid content was 15.0%. In the obtained extract, although the umami of glutamic acid was strongly felt, the unique thickness of the taste was not felt.

Example 6 Regarding the effect of the intracellular free glutamine concentration on the taste thickness of yeast extract, a skilled panelist (a person whose threshold value for discriminating sodium glutamate is 100 ppm or less) 2
Five people compared the difference between the umami intensity and the thickness of the taste. For the sensory evaluation, the four yeast extracts obtained in Example 4, namely, those derived from the control strain (Extract A), those derived from the kgd1 disrupted strain (Extract B), and those derived from the gad1 disrupted strain (Extract C)
And a strain derived from a simultaneous disruption of kgd1 and gad1 (Extract D) and a strain derived from a simultaneous disruption of kgd1 and gln1 obtained in Comparative Example 4 (Extract E) were used.

The results of the sensory evaluation are shown in Table 1. As to the umami titer, Extracts B, C, D and E surpassed Extract A with a 1% risk factor. No significant difference was found between them. In addition, in comparison between extract B and extract D, extract D was evaluated to be excellent at a risk rate of 5%. On the other hand, regarding the thickness of the taste, B,
C and D were evaluated to be superior at a 5% risk factor, but extract E was not significantly different from extract A.

[0051]

[Table 1]

From the above data, it was confirmed that the thickness of the taste of the yeast extract was expressed when the concentration of free glutamine in the raw yeast cells was at least 15 mg / g or more. This value of intracellular free glutamine corresponds to 3% glutamic acid per solid extract when yeast extract is produced.

[0053]

By mutating genes involved in glutamic acid and glutamine metabolism in yeast, mothers can breed yeast which accumulates large amounts of free glutamine and free glutamic acid in cells. Further, by digesting the yeast in combination with glutaminase, it is possible to produce a yeast extract having a high glutamic acid content, an excellent taste titer, and a well-balanced taste imparted with a taste thickness that is not present in conventional yeast extracts. Became.

[0054]

[Sequence List] <110> Japan Tobacco Inc. <120> New yeast and yeast extract <130> 002663 <160> 9 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <400> 1 aagagctcaa cccaaagatt ccagctacaa aggc 34 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <400> 2 aagagctctg gcatctgtgt ggaacttatg tctc 34 <210> 3 <211> 1294 <212> DNA <213> Saccharomyces cerevisiae <400> 3 aagagctcaa cccaaagatt ccagctacaa aggcttttca ggctcctccc agtatcagta 60 actttcccca gggtaccgaa gcagctccct tagggaccgc aatgactggt tcagtagatg 120 agaacgtctc cattcatcta aaagtgcaat tgctatgtag agcttaccaa gttagaggtc 180 atttaaaagc ccatatagat cctttaggga tctcatttgg tagtaataaa aataaccctg 240 ttcctccgga attgactcta gactactacg gctttagcaa acacgatctt gataaagaaa 300 tcaacctagg acctggtatc ctgccaaggt ttgcaaggga cgggaaatct aaaatgtctc 360 tgaaagagat tgtggatcat ctagaaaagt tatattgttc ctcttatggg gtacaataca 420 cacatattcc atctaagcaa aagtgtgatt ggttaagaga gagaattgag attcctgaac 480 cttaccaata tacagtggac caaaagagac aaatcttaga tagattaa ca tgggccactt 540 cttttgagtc attcttatct acaaaatttc caaatgataa gaggttcggt ttagaaggtt 600 tggaaagtgt tgttccaggt attaaaactt tggttgatcg ttctgttgaa ttgggtgtag 660 aagatattgt tttgggtatg gctcaccgtg gtagattgaa cgttttatcc aatgtggtcc 720 gtaaaccaaa tgaatctatt ttttctgaat ttaagggttc gagcgctcgc gatgatattg 780 aaggatcggg tgatgtcaag taccatttgg gtatgaacta ccaaagacca actacgtctg 840 gtaagtacgt caatttatcg ctggtggcaa atccttctca tttagaatcc caagatccag 900 ttgttcttgg tagaactaga gctttattgc atgccaagaa cgatttgaag gaaaaaacaa 960 aggccttagg tgtgttatta catggtgatg ctgcttttgc tgggcagggt gttgtttatg 1020 aaaccatggg tttcttgacc ctaccagaat actctactgg tggtactatt catgttatta 1080 caaacaacca gatcggattc actacggatc caagatttgc aaggtccaca ccatatcctt 1140 ccgatttggc taaggccatt gatgccccaa ttttccatgt taacgctaat gacgtggaag 1200 ctgtgacctt tattttcaat ttagccgcag aatggagaca taagttccac acagatgcca 1260 gagacataag ttccacacag atgccagagc tctt 1294 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <400> 4 aagagctctc ccgatgaaga accaataggc tg 32 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <400> 5 aagagctcgg cacttctgga tattcttcgt gg 32 <210> 6 <211> 960 <212> DNA <213> Saccharomyces cerevisiae <400> 6 tcccgatgaa gaaccaatag gctgtgccac cacaggttct agtgaggcaa tcatgttggg 60 tggactcgcc atgaaaaaaa gatgggaaca cagaatgaag aatgctggta aagatgcttc 120 caagccgaac attataatgt cttctgcgtg ccaagtggca ttagagaagt ttacgagata 180 ttttgaagtg gaatgccgat tggttccggt atcccacaga agccatcata tgcttgaccc 240 agagtcgtta tgggattatg tagatgagaa cactattggc tgttttgtaa ttttaggaac 300 cacctacact ggccatttgg aaaatgtaga gaaagttgca gatgtcttgt cccaaattga 360 ggccaagcat cctgattgga gcaatactga tattccaatc catgcggatg gcgcttcagg 420 tgggtttatt atcccatttg gctttgaaaa agagcacatg aaagcttatg gcatggaacg 480 ttgggggttc aaccatccgc gtgtggttag tatgaacact agtggtcata agtttggctt 540 aaccactccc ggtctgggtt gggtgctatg gagagatgaa tccttactgg ctgatgaatt 600 gagattcaaa ctaaagtacc tcggtggcgt ggaagaaact ttcggtttga atttttcaag 660 acctggattt caagttgtcc atcaatactt caattttgtt tctctaggcc att cagggta 720 tagaacacaa ttccaaaatt ctctatttgt tgcaagagcg ttttctttcg aattattgaa 780 ttcgtcaaaa ttgcccggat gctttgaaat tgttagcagt atccatgaaa gcattgagaa 840 cgattccgcc cctaagtcag ttaaagacta ttgggaacac ccccaggctt acaaaccagg 900 tgtaccgctg gtagccttca aattgtccaa gaaattccac gaagaatatc cagaagtgcc 960 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <400> 7 aagagctctg ataccaaact cttctgccac tc 32 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <400> 8 aagagctcat tgttgtcttg gccgcatgtt ac 32 <210> 9 <211> 421 <212> DNA <213> Saccharomyces cerevisiae < 400> 9 attgttgtct tggccgcatg ttacaacaat gacggtactc caaacaagtt caaccacaga 60 cacgaagctg ccaagctatt tgctgctcat aaggatgaag aaatctggtt tggtctagaa 120 caagaataca ctctatttga catgtatgac gatgtttacg gatggccaaa gggtgggtac 180 ccagctccac aaggtcctta ctactgtggt gttggtgccg gtaaggttta tgccagagac 240 atgatcgaag ctcactacag agcttgtttg tatgccggat tagaaatttc tggtattaac 300 gctgaagtca tgccatctca atgggaattc caagtcggtc catgtaccgg tattgacatg 360 ggtgaccaat tatggatggc cagatacttt ttgcacagag tggcagaaga gtttggtatc 420a 421

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yukihiro Chujo 194-1 Mifuku Kanayama, Onito-cho, Tagata-gun, Shizuoka Prefecture F-term in the Japan Tobacco Inc. Food Research Center 4B018 LE03 LE05 MD81 MF01 4B024 AA03 AA05 BA74 BA80 CA04 GA14 HA01 4B047 LB03 LB06 LB07 LB09 LE06 LF02 LF08 LG15 LG56 LP18 4B064 CA06 CA19 CC24 CE02 DA10 4B065 AA80X AA80Y AB01 AC20 BA02 CA17 CA41

Claims (6)

[Claims] 1. 15 mg / g of dry cells in cells
A yeast containing the above free glutamine. 2. The yeast according to claim 1, wherein the yeast is a mutant strain in which one or both of 2-ketoglutarate dehydrogenase activity and glutamate decarboxylase activity are reduced. 3. The method according to claim 1, wherein 15 mg per 1 g of the dried cells is contained in the cells.
A method for producing a yeast extract containing at least 3% of glutamic acid derived from intracellular free glutamine with respect to the solid content of the extract, comprising a step of digesting the yeast containing free glutamine as described above. 4. The method according to claim 3, wherein the yeast to be digested is the yeast according to claim 2. 5. A yeast extract produced by the method according to claim 3 or 4. 6. A yeast extract comprising at least 3% of glutamic acid derived from intracellular free glutamine based on the solid content of the extract.