JP4412658B2 - Thioredoxin-rich yeast and method for producing the same - Google Patents

Description

  The present invention relates to a thioredoxin-rich yeast and a method for producing the same, wherein the thioredoxin content in yeast cells is increased by culturing yeast under novel culture conditions.

  Thioredoxin (hereinafter referred to as “TRX”) is an approximately 12 kDa electron transfer protein having a highly conserved C—X—Y—C active center (in particular, X: Gly, Y: Pro), It is known to exist in the cells of most organisms (Non-Patent Documents 1 to 4). TRX has a redox activity of disulfide bonds existing in other proteins, and has a function of regulating the activity, function, and properties of the protein by the redox activity.

  Various stresses, especially oxidative stress, damages biological macromolecules such as lipids, genes, and proteins and harms cells and tissues, but it is known that the concentration of TRX in the body changes accordingly. . That is, it is considered that an increase in TRX concentration in the body can improve or prevent allergies caused by various stresses, protect or repair mucous membranes, protect skin, and the like.

Various techniques that apply the redox activity of TRX have been disclosed so far. For example, neutralization of various allergens by TRX (Patent Document 1), activation of transcription factor AP-1 by TRX (Patent Document 2), allergy prevention by TRX, cortical improvement, mucosal disorder protection (Patent Document 3), inflammatory diseases Prevention or treatment (Patent Document 4) is disclosed.
Japanese National Patent Publication No. 10-510244 Japanese Patent Laid-Open No. 10-191977 JP 2000-103743 A JP 2002-179588 A Laurent, T .; C. Moore, E .; C. & Reichard, P.A. (1964) J. Org. Biol. Chem. , 239, 3436-3444 Holmgren, A.M. (1989) Ann. Rev. Biochem. , 54, 237-271 Holmgren, A.M. (1989) J. Am. Biol. Chem. , 264, 13963-13966 Buchanan, B.H. B. Schurmann, P .; , Decotties, P .; & Lozano, R.A. M.M. (1994) Arch. Biochem. Biophys. , 314, 257-260 Inoue, Y .; , Trevanich, S .; Tsujimoto, Y. et al. , Miki, T .; , Miyabe, S .; , Sugiyama, S .; , Izawa, S .; & Kimura, A .; (1996) J. MoI. Sci. Food Agric. , 71 (3), 297-300 Tran, L.M. T. T. Inoue, Y .; & Kimura, A .; (1993) Biochim. Biophys. Acta. , 1164 (2), 166-172 Izawa, S .; Maeda, K .; , Sugiyama, K .; Mano, J .; Inoue, Y .; & Kimura, A .; (1999) J. MoI. Biol. Chem. , 274, 28459-28465 Kuge, S .; & Jones, N.M. (1994) EMBO J. et al. , 13 (3), 655-664 Inoue, Y .; & Kimura, A .; (1996) J. MoI. Biol. Chem. , 271 (42), 25958-25965 Inoue, Y .; Tsujimoto, Y. et al. & Kimura, A .; (1998) J. MoI. Biol. Chem. , 273 (5), 2977-2983 Kuge, S .; Jones, N .; & Nomoto, A .; (1997) EMBO J. et al. , 16 (7), 1710-1720

  It is considered that TRX can be used as a functional food material by utilizing the properties of TRX as described above. However, when manufacturing TRX in large quantities on an industrial scale, there are problems that must be overcome. That is, since TRX using a gene recombinant cannot be used for food, TRX must be extracted from a gene non-recombinant. However, TRX usually contains only about 10 to 100 μg per gram of yeast cells in a yeast cell. For example, in order to purify 1 mg of TRX, at least 1 to 1 is assumed even if the purification efficiency is assumed to be 100%. 10 kg yeast cells are required. In order to easily and efficiently recover and purify TRX from yeast extract, it is necessary to increase the TRX content in the yeast cell. Moreover, when ingesting yeast itself, it is preferable to use what contains TRX in high concentration. Accordingly, one of the objects of the present invention is to establish a novel yeast culture method that makes it possible to increase the TRX content in yeast cells, and to provide a thioredoxin-rich yeast produced by the method. That is.

  As a result of earnest research, the present inventors have been able to increase the content of thioredoxin (TRX) in yeast cells by cultivating yeast by adding a tea component to the medium and carrying out a load culture. As a result, the present invention has been completed. Hereinafter, the present invention will be described in detail.

(1) Method for producing yeast having a high TRX content According to the present invention, a method for producing a yeast having a high TRX content is provided, which includes stress loading in yeast culture. As used herein, the term “stress load” refers to giving a biological stimulus, biochemical stimulus, chemical stimulus, physical stimulus and the like different from physiological conditions to yeast. The TRX-rich yeast production method of the present invention is based on the discovery that the discovery of TRX is facilitated by activating transcription factors involved in TRX gene expression by stress loading. Here, transcription factors involved in TRX gene expression are not limited, but preferably include Yap1 and Skn7. As described in Example 7 described later, Yap1 normally shuttles between the cytoplasm and the nucleus, but is localized in the nucleus due to stress load and promotes the activity of TRX gene expression. Conceivable. Skn7 is usually localized in the nucleus.

  As one aspect of the present invention, in the method for producing a TRX-rich yeast of the present invention, the time for applying stress may be at the same time as the start of yeast culture, in the logarithmic growth phase, or in the stationary phase. As described in Example 6 described later, high expression of TRX is observed even when stress is applied at any of the above-mentioned times, but higher expression is obtained when stress is applied in the stationary phase. It was. In the present specification, the logarithmic period preferably means a range of OD610 = 0.1 to 4.0, more preferably OD610 = 0.5 to 2.0. The stationary phase means a state in which the OD 610 does not substantially change in the range of 15 to 40, for example, a state in which the culture is performed for 20 to 40 hours, more preferably 24 to 36 hours. Point to.

  As one aspect of the present invention, the stress load may be adding a tea component to the medium. Although the tea component used for the manufacturing method of this invention is not limited, A tea extract, catechin, and these mixtures are illustrated. Examples of the tea extract include the use of green tea, oolong tea, bancha, brown rice tea, etc. as raw materials, but the effect of increasing the TRX content in the cells and the cost for industrial production of such cells. Considering it, it is preferable to use a green tea extract as the tea extract. The “green tea extract” includes components such as catechins (eg, catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate). In the production method of the present invention, commercially available sunphenon BG (manufactured by Taiyo Kagaku Co., Ltd.) obtained by extracting and purifying green tea polyphenols (catechins) from green tea can also be used as described in the examples described later.

  In the present invention, the tea component can be added to the medium to produce a yeast with a high TRX content. When adding the tea component to the medium, the final concentration of the tea component is preferably 0.01-5.0%, more preferably 0.03-4.0%, even more preferably 0.1-3.0. %, Most preferably 1.0-2.0%. Moreover, the time for culturing the yeast after addition of the tea component is preferably 0.5 to 2.0 hours, more preferably 1.0 to 1.5 hours.

In the present invention, the culture medium used for yeast culture and the fed-batch solution used for fed-batch culture are not limited, but those skilled in the art can appropriately select from known ones according to the purpose. Preferred are H medium, SD medium, YPD medium, and molasses, more preferably H medium, SD medium, and most preferably H medium. In addition, these culture media may contain a carbon source, a nitrogen source, etc. suitably. Here, “H medium” used in the present specification means 1% glucose, 0.2% yeast extract, 0.5% peptone, 0.03% K ₂ HPO ₄ , 0.03% KH ₂ PO _4. Yeast medium containing 0.01% MgCl ₂ . “SD medium” refers to a yeast medium containing 2% glucose and 1% peptone. “YPD medium” refers to a yeast medium containing 2% glucose, 1% yeast extract, 2% peptone. When the medium is used as an agar medium, it can be prepared by adding 2% agar. Furthermore, the pH of the medium is important in the culture of yeast with a high TRX content of the present invention. The pH is preferably adjusted to 7.0 to 9.0, more preferably 7.0 to 8.0, even more preferably 7.4 to 7.8, and most preferably 7.6. In addition, when commercially available sunphenone BG is added to the medium, the pH of the medium is slightly lowered, so that the pH can be adjusted with, for example, a 1N aqueous sodium hydroxide solution.

  In one aspect of the present invention, TRX-rich yeast can be produced by performing stress loading with a green tea extract in yeast culture and then further culturing for a predetermined time under the culture conditions from which the stress loading has been removed.

  The yeast culture method is not limited, but examples include batch culture, fed-batch continuous culture, fed-batch culture and the like, and those skilled in the art can appropriately select them. Further, the culture can be suitably performed using a jar fermenter (Komatsugawa Kako Co., Ltd.). The culture conditions in the case of using a jar fermenter are not particularly limited and can be appropriately determined by those skilled in the art. For example, the culture temperature is about 28 to 33 ° C., and the culture time is about 1 to 120 hours. The pH can be adjusted within a range of about 6 to 9, the aeration amount is about 0 to 5 vvm, and the stirring speed is within a range of about 100 to 700 rpm.

  The production method of the present invention is based on activation of a transcription factor by stress load and promotion of TRX gene expression. The use of such a transcription factor is either an exogenous TRX gene or an endogenous TRX gene. Also good. However, since a recombinant into which an exogenous TRX gene has been introduced cannot be used for food, an endogenous TRX gene is preferred. Here, the “endogenous TRX gene” is a TRX gene that yeast originally has as a gene in the chromosome, and includes an exogenous TRX gene introduced as a foreign gene into yeast by genetic engineering techniques. Absent. Examples of the TRX gene include, but are not limited to, a gene encoding TRX2 consisting of 104 amino acid residues disclosed as GenBank accession number AAA85584. Some of these natural TRX contain one to multiple amino acid mutations due to differences in the varieties of species that produce them, genetic variations due to differences in ecosystems, or the presence of similar isozymes. It is well known that there are mutated proteins. It may also contain an allele of the TRX gene.

  As one embodiment of the present invention, the TRX content is significantly increased by stress load as compared to the case without stress load. More specifically, in Example 3 described later, when a gene recombinant was used, the activity of the TRX promoter was increased by at least about 6.5 times. Further, in Example 8, when the practical cells were used, the TRX content per 1 g of the wet cells increased by at least about 1.2 times and the maximum by about 1.6 times. Therefore, according to the method for producing a thioredoxin-rich yeast of the present invention, the TRX content is preferably 6.5 times or more, more preferably 5 times or more, even more preferably 2 as compared with the case where there is no stress load due to stress load. More than twice, even more preferably 1.6 times or more, and most preferably 1.2 times or more.

(2) TRX-rich yeast According to the present invention, a TRX-rich yeast produced by the production method described above is provided. The yeast used in the production method of the present invention is not limited, but an edible yeast obtained from a natural source that is easy to confirm safety is preferable in consideration of utilization as a functional food material. The edible yeast, Saccharomyces (Saccharomyces) genus Torulopsis (Torulopsis) genus Mikotorura (Mycotorula) genus Torulaspora (Torulaspora) genus Candida (Candida) genus Rhodotorula (Rhodotorula) genus Pichia (Pichia) sp cells of Is exemplified. The yeast used in the production of the yeast having a high TRX content of the present invention is preferably a yeast derived from the genus Saccharomyces (Saccharomyces cerevisiae). The yeast derived from the genus Saccharomyces is preferably baker's yeast, beer yeast, wine yeast, sake yeast, miso soy sauce yeast, and more preferably baker's yeast.

  The TRX content of the yeast with a high TRX content of the present invention is preferably 120 μg or more, more preferably 130 μg or more, still more preferably 140 μg or more, most preferably 180 μg / g wet cells or more and 200 μg / g wet cells or less. is there.

  The TRX-rich yeast of the present invention may be used in the form of viable bacteria when used for food applications, but can be used by preparing a crushed cell. The method for disrupting the bacterial cells is not particularly limited as long as the degree of disruption is such that there are no unbroken cells under microscopic observation, and those skilled in the art can appropriately select depending on the purpose. For example, a physical crushing process such as dyno mill or a chemical crushing process may be used. In addition, when using TRX high content yeast with the form of a crushing thing, the crushing thing may be the liquid thing which is not crushed and dried, or may be the dried substance dried after crushing.

  The TRX-rich yeast of the present invention or a crushed product thereof can be contained and used in foods or beverages. Although the mode of use is not limited, those skilled in the art can appropriately select depending on the purpose. For example, adding a predetermined amount of the yeast or crushed material to confectionery such as bread, biscuits and crackers, liquid foods, processed fishery products and meat products, beverages such as noodles, seasonings and juice, or various health foods and beverages Can do.

(3) Production of yeast with high TRX content in scale-up According to the present invention, TRX by yeast can be produced easily and in large quantities on an industrial scale. The scale-up from a small scale at a laboratory level to a large scale that can be produced industrially is not limited, but can be performed by a person skilled in the art by a known method. More specifically, as described in Example 8 described later, when 2.0% of sunphenone is added as a stress load, the TRX content of the practical cell is 197 μg / g wet cell, It can be increased by about 1.6 times compared with the case of no addition (126 μg / g wet cells).

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
Example 1 Yeast Culture and TRX Expression In the examples, for the purpose of examining TRX2 expression conditions at the laboratory level, the YPH250 strain into which the TRX2-lacZ reporter gene was introduced (hereinafter referred to as “recombinant YPH250 strain”). ) Was prepared.

  Specifically, first, a plasmid containing the TRX2-lacZ reporter gene was prepared by Kuge, S .; & Jones, N.M. (1994) EMBO J. et al. 13 (3), 655-664 (non-patent document 8). The plasmid has a structure in which the TRX promoter existing upstream of the TRX gene and the ORF of lacZ are combined. According to this configuration, activation of the TRX promoter by the addition of catechin can be replaced with expression of the downstream lacZ gene for reporter assay.

  Subsequently, a plasmid containing the TRX2-lacZ reporter gene was introduced into the YPH250 strain using a known electroporation method. Electroporation is described in, for example, Inoue, Y. et al. & Kimura, A .; (1996) J. MoI. Biol. Chem. 271 (42), 25958-25965 (Non-Patent Document 9), or Inoue, Y. et al. Tsujimoto, Y. et al. & Kimura, A .; (1998) J. MoI. Biol. Chem. , 273 (5), 2977-2983 (Non-patent Document 10). Specifically, Micro-Pulser manufactured by Bio-Rad was used, and general conditions (1.5 kV, 200Ω, 25 μF) were adopted for gene introduction into yeast.

To cultivate the recombinant YPH250 strain, 20 μg / ml in SD minimal medium (2% glucose, 0.67% Yeast nitrogen base w / o amino acids), pH 5.5) not containing uracil to prevent plasmid loss. L-Trp, L-His, L-Leu, L-Lys, and adenine added, and YPD medium (2% glucose, 1% yeast extract, 2% peptone) as needed, and H Medium (1% glucose, 0.2% yeast extract, 0.5% peptone, 0.03% K ₂ HPO ₄ , 0.03% KH ₂ PO ₄ , 0.01% MgCl ₂ ) (Non-patent Document 6) Was used. Note that NaOH was used to adjust the pH.

  As a stress load substance, a search was made for a substance that promotes the activation of the transcription factor Yap1, which is a substance approved for use as a food additive, and as a result, the green tea extract was focused. The grounds for this are the fact that the inventors previously conducted experiments using Escherichia coli, in which polyphenols in green tea components such as catechin and epigallocatechin gallate did not erase active oxygen, as is generally known. This is based on the finding that the reverse effect is shown in the presence of ions (Non-patent Document 5). The green tea extract used is commercially available Sanphenon BG (manufactured by Taiyo Kagaku Co., Ltd.). Catechin, epigallocatechin gallate, and gallic acid were purchased from Wakken Pharmaceutical Co., Ltd.

  In this test example, S.M. cerevisiae YPH250 strain (MATa trp1-Δ1 his3-Δ200 leu2-Δ1 lys2-801 ade2-101 ura3-52) (ATCC No. 204676) was obtained from Yeast Genetic Stock Center and used after planting.

  At 28 ° C., in order to prevent the plasmid from dropping off, after shaking culture in SD medium until OD610 = 1, 50 ml of the culture solution was transferred to a centrifuge tube to collect the cells, the supernatant was removed, and pH was adjusted in advance. The cells were suspended in a medium (50 ml). Thereafter, sunphenone powder or sunphenone previously dissolved in water was added to a final concentration of 0.1%, transferred to a 200 ml Erlenmeyer flask, and further cultured with shaking at 28 ° C. for 90 minutes. . It should be noted that the pH of the medium was adjusted because the pH slightly decreased when sunphenone was dissolved in water.

  After culturing, 50 ml of the culture broth was collected by centrifugation, and the cells were washed 2-3 times with 0.85% NaCl solution. Thereafter, the cells were transferred to a 1.5 ml Eppendorf tube, glass beads of approximately the same amount as the cells were added, and 250-300 μl of 10 mM potassium phosphate buffer (pH 7.0) was further added. This was shaken vigorously with Fast Prep (Qbiogen) for 30 seconds to disrupt the cells. Centrifugation (14000 rpm, 4 ° C., 10 minutes) was performed, and the supernatant (cell-free extract) was used as an enzyme. The β-galactosidase activity was measured according to a conventional method. Specific activity was expressed as 1000 × A420 / hour (h) / mg protein.

Among the methods for preparing the cell-free extract, a cell-free extract was obtained using 10 mM Tris-HCl buffer (pH 7.0) as a buffer for disrupting cells. After quantifying the protein concentration, SDS-PAGE (gel concentration: 15%) was performed according to a conventional method. The amount applied was adjusted so that about 50 μg of protein was loaded in each lane. Rabbit antiserum (non-patent document 7) immunized with TRX2 which was blotted on PVDF membrane according to a conventional method and expressed and purified in Escherichia coli as a primary antibody was used. As the secondary antibody, an anti-rabbit IgG antibody conjugated with HRP was used, and color was developed with 4-chloro-1-naphthol and H ₂ O ₂ to confirm the TRX band. It has been confirmed that the antibody used cannot distinguish between TRX1 and TRX2.

Example 2 Effect of TRX expression on pH change of medium The influence of TRX expression on the pH change of the medium when culturing yeast was examined. In the same manner as in Example 1, a strain having TRX2-lacZ was cultured in SD medium to OD610 = 1, and then the cells were suspended in H medium in which the pH was changed from 6.5 to 8.0. Sanphenone was added to 0.1%, and the mixture was cultured at 28 ° C. for 90 minutes, and β-galactosidase activity caused by TRX2-lacZ was measured. Little induction of β-galactosidase activity was observed on the acidic side (data not shown), but β-galactosidase activity was induced with increasing pH above pH 7, and maximum at pH 7.6 (about 112 U / mg protein) Thus, it was increased by about 6.5 times compared to the case where no sunphenone was added (FIG. 1). Moreover, the expression of TRX2-lacZ was not observed only by adjusting pH to the alkali side without adding sunphenone. Furthermore, as a result of examining the TRX level by Western blotting using an anti-TRX2 antibody, the expression of TRX protein was confirmed as in the detection of β-galactosidase activity (FIG. 2). Therefore, it can be said that the results obtained under the culture conditions for expressing the TRX2-lacZ reporter gene are reflected in the results of the TRX expression.

Example 3 Effect of Addition Concentration and Addition Time of Sunphenone on TRX Expression Cells cultured in SD medium to OD610 = 1 were suspended in H medium adjusted to pH 7.6, and each concentration of sunphenone was added to The culture was performed at 90 ° C. for 90 minutes. The expression of TRX2-lacZ was maximized when the concentration of added sunphenone was 0.1% (final concentration) (FIG. 3). In addition, an induction effect was observed even when a higher concentration of sunphenone was added, and precipitates were precipitated (data not shown). Therefore, in the following examples, the concentration of sunphenone was used as 0.1%.

  Furthermore, for the purpose of examining the addition time of sanphenone, the cells prepared above were suspended in H medium, and sanphenone was added so that the final concentration would be 0.1%. And incubated for 90 minutes. As a result, the expression of TRX2-lacZ reached a maximum at 60 minutes (FIG. 4).

Example 4 Influence on TRX Expression by Type of Medium The cells cultured in SD medium until OD610 = 1 were suspended in each of H medium, YPD medium, and SD medium adjusted to pH 7.6. Sanphenon BG was added to each medium so that the final concentration was 0.1%, and cultured at 28 ° C. for 45 minutes and 90 minutes. As a result, the maximum induction effect was observed when H medium was used, and almost no induction was observed with YPD medium (Table 1, FIG. 5). Therefore, in the following examples, H medium was used.

Unit: β-galactosidase (U / mg)
Example 5 Effect of phosphate concentration in H medium on TRX expression As compared with SD medium and YPD medium, H medium includes 0.03% K ₂ HPO ₄ and 0.03% KH ₂ PO _4. Contains phosphate. Therefore, in order to investigate the influence of phosphate, an H medium (pH adjusted to 7.6) was prepared and used as a potassium phosphate buffer so that the final concentrations were 0 mM, 1 mM, 10 mM, and 100 mM. Cells cultured in SD medium to OD610 = 1 were suspended in each H medium, and sanphenone was added to the final concentration of 0.1%, followed by incubation for 90 minutes. As a result, even in a medium containing no phosphate, β-galactosidase activity (about 85 U / mg protein) is present, and it is considered that the expression of TRX2-lacZ is less affected by potassium phosphate in the medium ( FIG. 6).

Example 6 Effect of Different Addition Times on TRX Expression It was examined whether the addition time of sunphenone to the medium affects TRX expression. A strain having the TRX2-lacZ reporter gene was cultured in SD medium, and cultured in H medium until log phase (OD610 = 1) and stationary phase (24 hours culture). NaOH was added dropwise to each cell culture solution to adjust the pH to 7.6, and then sanphenone was added so that the final concentration was 0.1%. Thereafter, the cells were further cultured for 90 minutes. As a result, the cells in the stationary phase were more induced (Table 2, FIG. 7).

Unit: β-galactosidase (U / mg)
Example 7 TRX2 gene expression control Generally, it is known that the expression of TRX2 gene is controlled by two transcription factors, Yap1 and Skn7. Skn7 is always localized in the nucleus, whereas Yap1 shuttles the cytoplasm and nucleus. Therefore, using Yap1 to which a GFP tag was added, the localization of Yap1 by catechin, epigallocatechin gallate, gallic acid, and sunphenone treatment was observed with a fluorescence microscope.

  Specifically, the fusion protein “GFP-Yap1” in which the GFP protein was fused to the N-terminal side of the Yap1 protein was used. Plasmids for GFP-Yap1 protein expression include Kuge, S .; Jones, N .; & Nomoto, A .; (1997) EMBO J. et al. , 16 (7), 1710-1720 (Non-patent Document 11), a plasmid “pRS cp-GFP HA YAP1” constructed according to the method described in FIG. The method for introducing the plasmid into the host cell was the same as the method for introducing the plasmid containing the TRX2-lacZ reporter gene described in Example 1.

  As a result of microscopic observation, the nuclear localization of Yap1 was observed with any drug (FIG. 8). Therefore, induction of TRX by sanphenone is caused by catechin and epigallocatechin gallate, which are the main components in green tea extract. It was considered a thing.

Example 8 Induced Expression of TRX Using Practical Strains at Industrial Level The above laboratory-level tests suggested that the green tea component is a TRX inducer. However, in order to use this green tea component at an industrial level, it is necessary to be able to induce TRX not only for the YPH250 strain but also for a practical strain. Furthermore, it is important to show a TRX induction effect even if the yeast concentration is high. Therefore, in this example, in order to examine whether or not TRX-induced expression is possible by using this sunphenone even in a practical strain, the following examination was performed using O-102 (accession number: FERM P-18568) which is a practical baker's yeast. Went.

  O-102 was inoculated with 1 platinum loop in SD medium (5 ml of 16φ test tube), cultured at 30 ° C. for 2 days, and then inoculated into H medium (Sakaguchi flask 100 mL) so that OD610≈0.05. After culturing at 30 ° C. for 14 hours (OD610≈4.5), the final concentration of green tea component sanphenone is 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0%. And then adjusted to pH 7.6 using 5N NaOH. Thereafter, shaking culture was performed at 30 ° C. for 90 minutes. Moreover, what adjusted pH 7.6 without the addition of sunphenone and the thing without pH adjustment were made into the control. The culture was collected and washed, and suspended in an appropriate amount of MilliQ water. After measuring the amount of wet cells in each sample, the suspension was taken into a 2 ml Eppendorf tube so that the wet cells would be 300 mg, centrifuged (8400 g, 2 minutes), and after collection, the resulting bacteria Protein was extracted from the body. Protein extraction was performed by adding 900 μl of Yeast Protein Extraction Reagent (PIERCE) (hereinafter referred to as YPER) to 300 mg of wet cells and leaving it at room temperature for 20 minutes after suspension. Further, the extract was centrifuged (35000 g, 15 minutes, 4 ° C.) to collect the water-soluble fraction, and subjected to heat treatment at 65 ° C. for 10 minutes to obtain a sample for measuring TRX concentration.

The TRX concentration in the sample was measured according to the following procedure.
1) Mix R1 (300 μL) and sample (20 μL) 2) Preheat 5 min at 25 ° C. 3) Add R2 (6 μL) and mix 4) Incubate 1 min at 25 ° C., measure Abs 340 decrease during that time (Here, R1 refers to 0.1 M Tris-HCl (pH 7.0), 2 mM EDTA, 0.8 mg / mL insulin, 0.16 mM NADPH solution. R2 refers to 0.3 U / mL) NTR (NADP-dependent thioredoxin reductase), 0.1 M Tris-HCl (pH 7.0) solution)
Table 1 shows the measurement results of the TRX content in the cells.

  The TRX content of the control was almost the same regardless of whether or not the pH was adjusted, and in the absence of sunphenone, adjusting the pH to 7.6 did not affect the intracellular TRX content. Moreover, the TRX content in the microbial cells showed a value almost equal to that of the control until the concentration of sunphenone was 0.5%. On the other hand, by increasing the concentration of sunphenone to 1.0% and 2.0%, the TRX content in the cells increased by about 1.2 times and 1.6 times, respectively, compared to the control (Table). 1 and FIG. 9). From these results, it was revealed that TRX can be induced by green tea extract even in practical strains.

  According to the present invention, it is possible to provide a TRX-rich yeast that is suitable as a food material, a crushed product of TRX-rich yeast, and a method for producing the TRX-rich yeast. Moreover, since the TRX-rich yeast of the present invention can be produced without relying on genetic recombination, it is highly safe and suitable for use as a food.

FIG. 1 shows the results of examining the effect of medium pH on the expression of TRX2-lacZ. A strain having TRX2-lacZ was cultured in SD medium until OD610 = 1, and then the cells were suspended in H medium in which the pH was changed from 7.0 to 8.0. Sunphenone was added to 0.1% as a green tea extract, and after culturing at 28 ° C. for 90 minutes, β-galactosidase activity caused by TRX2-lacZ was measured. FIG. 2 shows the results of confirming TRX expression in cells suspended in H medium having a pH changed from 7.0 to 8.0 by Western blotting using an anti-TRX2 antibody. FIG. 3 shows the results of examining the effect of added green tea extract concentration on the expression of TRX2-lacZ. Cells cultured in SD medium to OD610 = 1 are suspended in H medium adjusted to pH 7.6, and each concentration of sanphenone is added and cultured at 28 ° C. for 90 minutes. Was measured. FIG. 4 shows the result of examining the effect of the addition time of sunphenone on the expression of TRX2-lacZ. Cells cultured in SD medium to OD610 = 1 were suspended in H medium adjusted to pH 7.6. Sunphenone BG was added so that the final concentration was 0.1%, and after culturing at 28 ° C. for a predetermined time, β-galactosidase activity caused by TRX2-lacZ was measured. FIG. 5 shows the results of examining the influence of various media on the expression of TRX2-lacZ. Cells cultured in SD medium to OD610 = 1 were suspended in H medium, YPD medium, and SD medium adjusted to pH 7.6, respectively. Sunphenone BG was added so that the final concentration was 0.1%, and after culturing at 28 ° C. for 45 minutes or 90 minutes, β-galactosidase activity caused by TRX2-lacZ was measured. FIG. 6 shows the results of examining the influence of phosphate on the expression of TRX2-lacZ. Cells cultured in SD medium to OD610 = 1 were suspended in a medium prepared (pH 7.6) so that phosphate ions in H medium had a predetermined concentration. Sunphenone BG was added so that the final concentration was 0.1%, and after culturing at 28 ° C. for 90 minutes, β-galactosidase activity caused by TRX2-lacZ was measured. FIG. 7 shows the results of examining the effect of the addition time of sunphenone on the expression of TRX2-lacZ. A strain having the TRX2-lacZ reporter gene was cultured in SD medium, and cultured in H medium until log phase (OD610 = 1) and stationary phase (24 hours culture). NaOH was added dropwise to each cell culture solution to adjust the pH to 7.6, and then sanphenone was added so that the final concentration was 0.1%. Thereafter, the cells were further cultured for 90 minutes, and β-galactosidase activity caused by TRX2-lacZ was measured. FIG. 8 shows the result of observing the activation of Yap1 by adding a green tea extract and catechins with a fluorescence microscope. FIG. 9 shows the results showing the influence of the concentration of sunphenone on the TRX content of practical cells.

Claims (10)

A yeast containing high thioredoxin , comprising performing stress loading in culturing yeast, wherein the medium for culturing yeast is H medium, and the pH of the medium is 7.4 to 8.0 Manufacturing method.   The method for producing a thioredoxin-rich yeast according to claim 1, comprising activating a transcription factor of thioredoxin by stress loading.   The method for producing a yeast having a high thioredoxin content according to claim 1 or 2, wherein a stress load is applied simultaneously with the start of yeast culture, in the logarithmic growth phase, or in the stationary phase.   The method for producing a thioredoxin-rich yeast according to claim 3, wherein the stress load is adding a tea component to the medium.   The method for producing a yeast having a high thioredoxin content according to claim 4, wherein the tea component is added at a rate of 0.03-4.0%.   The method for producing a thioredoxin-rich yeast according to claim 4 or 5, wherein the tea component is a tea extract, catechins, or a mixture thereof. The method for producing a thioredoxin-rich yeast according to any one of claims 1 to 6 , wherein the pH of the medium is 7.6 . The method for producing a thioredoxin-rich yeast according to any one of claims 1 to 7 , wherein the yeast is an edible yeast. The method for producing a thioredoxin-rich yeast according to any one of claims 1 to 8 , wherein the thioredoxin is expressed from an endogenous thioredoxin gene. The method for producing a thioredoxin-rich yeast according to any one of claims 1 to 9 , wherein the thioredoxin content per gram of wet cells is increased by at least 1.2 times compared to a case where no stress load is applied.