JP2011160739A - Saccharomyces cerevisiae mutant and method for producing yeast highly containing sulfur-containing compound using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a Saccharomyces cerevisiae mutant having a high content of sulfur-containing compound such as glutathione, a method for producing yeast highly containing a sulfur-containing compound by using the mutant, a culture of the mutant, a culture extract and a food and beverage containing a sulfur-containing compound. <P>SOLUTION: The Saccharomyces cerevisiae mutant having a ≥2 wt.% sulfur-containing compound content per dried cell weight is obtained by (a) a step of mutating Saccharomyces cerevisiae YNN27 to give a yeast mutant having a mutant MET30 gene, (b) a step of mutating the yeast mutant obtained by the step (a) to give two or more yeast mutants having the mutant MET30 gene and a ≥1.5 wt.% sulfur-containing compound content per dried cell weight and (c) a step of crossing two mutants of the yeast mutants obtained by the step (b) to give a yeast mutant having a ≥2 wt.% sulfur-containing compound content per dried cell weight. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a Saccharomyces cerevisiae mutant having a high sulfur-containing compound such as glutathione, a method for producing a yeast containing a high sulfur-containing compound using the mutant, a culture of the mutant, and a yeast extract. .

  Typical sulfur-containing compounds in yeast cells include glutathione and S-adenosylmethionine. Glutathione is a very useful substance having liver function recovery, antioxidant activity, and the like, and has recently been expected to be used in a wide range of applications such as seasonings, health foods and other food and beverage additives, and cosmetic base materials. On the other hand, S-adenosylmethionine is known to act as a methyl group donor in various biological reactions. In addition, effects such as antidepressant action, alleviation of arthropathy, and recovery of liver function have been reported, and it is known that these sulfur-containing compounds play an important role for the living body.

  Sulfur-containing compounds are usually synthesized from transcription / translation products of many genes including MET genes (methionine synthesis genes) using sulfur-containing amino acids such as methionine and cysteine. Therefore, in order to obtain a yeast that produces a higher amount of sulfur-containing compounds, a mutation is caused in the genes related to the synthesis of these sulfur-containing compounds that the yeast has, and a yeast mutant containing a high sulfur-containing compound content is produced. Is widely practiced. As a method for obtaining such a mutant strain containing a high sulfur-containing compound, for example, (1) When the S-adenosylmethionine content in yeast is increased, transcription of the MET gene group is suppressed. Although the production amount decreases, the MET30 gene is probably involved in the transcriptional repression of this MET gene group through the interaction with the MET4 gene, which is probably the gene that regulates the transcription of other MET gene groups. (For example, refer nonpatent literature 1.).

  In addition, (2) a method of mutating yeast so as to accumulate a sulfur-containing compound at a high concentration using the MET4 gene mutated so as to enhance transcription activity, and retaining the mutated MET4 gene, glutathione Yeast with improved productivity is disclosed (for example, see Patent Document 1). In addition, (3) yeast having the ability to produce γ-glutamylcysteine, which is a precursor of glutathione, and holding the MET30 gene in which a specific amino acid is mutated has been disclosed (for example, see Patent Document 2). The MET30 gene is a gene involved in the ubiquitination of MET4. However, in this method, by retaining a specific mutant MET30 gene, the ability to produce γ-glutamylcysteine is reduced without deteriorating the growth ability of yeast. It is disclosed that it can be improved.

JP-A-10-33161 JP 2004-113155 A

Thomas, 5 others, Molecular and Cellular Biology, 1995, Vol. 15, No. 12, pages 6526-6534.

  However, in order to efficiently obtain a sulfur-containing compound such as glutathione industrially inexpensively, a yeast having a higher sulfur-containing compound content is desired.

  The present invention has been made in view of the above circumstances, and a Saccharomyces cerevisiae mutant strain capable of retaining a large amount of a sulfur-containing compound such as glutathione in a microbial cell, and a sulfur-containing compound-rich yeast using the mutant strain It is an object of the present invention to provide a production method, a culture of the mutant strain, a yeast extract, and a sulfur-containing compound-containing food or drink.

  As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention have developed a yeast mutant strain having a mutant MET30 gene obtained by mutating a common wild strain, Saccharomyces cerevisiae YNN27 strain, By mutating again, two types of yeast mutants having a higher sulfur-containing compound content than the strain before the mutation treatment were obtained, and by mating these yeast mutants, the sulfur-containing compound content was higher than the parent strain. The yeast mutant Saccharomyces cerevisiae ABYC1579, which has a large amount of yeast, was found and the present invention was completed.

(1) The present invention is a Saccharomyces cerevisiae mutation characterized in that the sulfur-containing compound content is 2% by weight or more per dry cell weight and can be obtained from the following steps (a) to (c). (A) obtaining a yeast mutant having a mutant MET30 gene by mutating the Saccharomyces cerevisiae YNN27 strain; and (b) the yeast obtained by step (a). (C) obtaining two or more yeast mutants having a mutant MET30 gene and having a sulfur-containing compound content of 1.5% by weight or more per dry cell weight by mutating the mutants; By multiplying two of the yeast mutants obtained in step (b), the sulfur-containing compound content was equivalent to the dry cell weight. Obtaining a yeast mutant is 2 wt% or more.
(2) The present invention also provides the Saccharomyces cerevisiae mutant strain according to (1) above, which is a Saccharomyces cerevisiae ABYC1579 strain (FERM BP-10923).
(3) Further, in the present invention, the sulfur-containing compound content is 2% by weight or more per dry cell weight, and the Saccharomyces cerevisiae mutant strain described in (1) or (2) is crossed with Saccharomyces cerevisiae YNN27. The present invention provides a Saccharomyces cerevisiae mutant characterized by being obtained by
(4) The present invention also provides a Saccharomyces cerevisiae mutant strain according to (3) above, which is a Saccharomyces cerevisiae ABYC1588 strain (FERM BP-10924).
(5) Moreover, this invention culture | cultivates the Saccharomyces cerevisiae mutant strain in any one of said (1)-(4), and contains 14 weight% or more of sulfur content per dry cell weight in the said mutant strain body. The present invention provides a method for producing a sulfur-containing compound-rich yeast characterized by containing a compound.
(6) Moreover, this invention provides the culture | cultivation of the Saccharomyces cerevisiae mutant in any one of said (1)-(4).
(7) Moreover, this invention provides the yeast extract prepared from the culture | cultivation of the Saccharomyces cerevisiae mutant in any one of said (1)-(4).
(8) Moreover, this invention provides the fraction containing the sulfur-containing compound obtained by fractionating the culture | cultivation of the Saccharomyces cerevisiae mutant in any one of said (1)-(4). is there.
(9) Further, the present invention provides the yeast extract according to (7) above, wherein the total content of oxidized glutathione and reduced glutathione is 8% by weight or more.
(10) Moreover, this invention provides the sulfur-containing compound containing food-drinks characterized by including the culture of said (6), or the fraction of said (8).
(11) Moreover, this invention provides the said sulfur-containing compound containing food-drinks of the said (10) characterized by the said sulfur-containing compound being glutathione.

  The Saccharomyces cerevisiae mutant of the present invention is a good yeast having a high content of sulfur-containing compounds such as glutathione. Therefore, by culturing the Saccharomyces cerevisiae mutant, a yeast containing a high sulfur-containing compound can be easily produced. In addition, since the culture of the Saccharomyces cerevisiae mutant has a sufficiently high content of sulfur-containing compounds, by using the culture, yeast extract and fractions containing a high content of sulfur-containing compounds, and these are contained. The sulfur-containing compound-containing food / beverage products to perform can be obtained simply.

FIG. 1 shows an S metabolism map in Saccharomyces cerevisiae.

  In the present invention, the dry cell weight means the weight after the cells are dried. As for the weight of the cells after drying, for example, the yeast culture is first centrifuged to collect the cells as a precipitate. This can be determined by measuring the weight of the collected cells after washing twice with a centrifugal separation and then drying at 105 ° C. for 5 hours.

  In the present invention, the sulfur-containing organic compound means a compound having an S (sulfur) -containing amino acid. Specific examples include glutathione, cysteine, homocysteine, methionine, adenosylmethionine, adenosylhomocysteine, cystathionine, and γ-glutamylcysteine described in the S metabolism map of Saccharomyces cerevisiae shown in FIG. . In the present invention, glutathione is preferable from the viewpoint of high useful physiological activity.

  In the present invention and the present specification, unless otherwise specified, glutathione means both oxidized glutathione and reduced glutathione, and the total glutathione content means the total content of oxidized glutathione and reduced glutathione. To do.

  The sulfur-containing compound content per dry cell weight of the Saccharomyces cerevisiae mutant of the present invention can be determined by a method usually performed when the sulfur-containing compound content in the microorganism is quantified. For example, the total glutathione content per dry cell weight of yeast can be measured according to the method of Titze et al. (Analytical Biochemistry, Vol. 27, p502, 1969).

  In the present invention, the mutant MET30 gene means a gene in which a part of the MET30 gene is mutated, that is, a gene in which a part of the base in the base sequence of the MET30 gene is substituted, deleted, or inserted. These mutations may be single nucleotide mutations, or may be mutations in a region in which a plurality of bases are continuous, such as mutations caused by chromosome translocation, inversion, duplication, and the like.

  In the present invention, the mutation treatment is not particularly limited as long as it is a treatment capable of mutating a part of a gene of an organism such as yeast, and is usually used when producing a mutant strain of a microorganism such as yeast. Any method used may be used. For example, by treating yeast with mutagen such as ultraviolet rays, ionizing radiation, nitrous acid, nitrosoguanidine, ethyl methanesulfonate (hereinafter abbreviated as EMS), etc., the yeast can be mutated. it can.

The Saccharomyces cerevisiae mutant of the present invention has a sulfur-containing compound content of 2% by weight or more per dry cell weight and can be obtained from the following steps (a) to (c): (a) Saccharomyces -Mutagenizing the cerevisiae YNN27 strain to obtain a yeast mutant strain having the mutant MET30 gene; (b) Mutating the yeast mutant strain obtained in step (a) A step of obtaining two or more yeast mutants having a MET30 gene and a sulfur-containing compound content of 1.5% by weight or more per dry cell weight, and (c) a yeast mutant obtained by the step (b) A step of obtaining a yeast mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight by multiplying two of them.
Thus, after repeating mutation and selecting a mutant strain having a high sulfur-containing compound content for each step, a mutant strain having a higher sulfur-containing compound content can be obtained by multiplying the selected strains. .

Hereinafter, it demonstrates for every process.
First, as a step (a), a Saccharomyces cerevisiae YNN27 strain is mutated to obtain a yeast mutant strain having a mutant MET30 gene. Specifically, the Saccharomyces cerevisiae YNN27 strain, which is a wild strain, is subjected to a mutation treatment, the nucleotide sequence of the MET30 gene of the yeast after the treatment is analyzed, and the yeast mutant strain in which the MET30 gene is mutated, A yeast mutant having the type MET30 gene is selected.

As the Saccharomyces cerevisiae YNN27 strain, those cultured by a conventional method can be used.
Further, the base sequence of the MET30 gene can be analyzed by a sequence method or the like usually used in the field of gene analysis.

  The yeast mutant selected in the step (a) is not particularly limited as long as it is a yeast mutant having a mutant MET30 gene, and in particular, the transcriptional repressing function of the other MET gene groups of the MET30 gene. A yeast mutant having a damaged or weakened mutant MET30 gene is preferred. This is because a yeast mutant having such a mutant MET30 gene is suitable as a raw material for producing a yeast mutant having a high sulfur-containing compound content such as glutathione.

  In addition, the selection of the yeast mutant strain having the mutant MET30 gene in the step (a) may be performed by analyzing and selecting the MET genes of all the yeasts subjected to the mutation treatment. Alternatively, a yeast having a relatively high sulfur-containing compound content, particularly a total glutathione content may be selected, and these MET genes may be analyzed to select a strain having a mutant MET30 gene. When the transcriptional repression function of another MET gene group possessed by the MET30 gene is inhibited due to a gene mutation or the like, the S metabolic gene group such as another MET gene is activated, and the content of sulfur-containing compounds in yeast, particularly total glutathione This is because the content is considered to be high. For example, the total glutathione content per dry cell weight is about 0.3% by weight in the Saccharomyces cerevisiae YNN27 strain, but may be about 1% by weight or more in the yeast mutant strain having the mutant MET30 gene.

  Next, as a step (b), the yeast mutant obtained in the step (a) is mutated to have a mutant MET30 gene, and the sulfur-containing compound content is 1.5 per dry cell weight. Two or more yeast mutants having a weight% or more are obtained. Thus, a yeast mutant having a higher sulfur-containing compound content than the yeast mutant before the mutation treatment can be obtained by subjecting the yeast mutant obtained in step (a) to repeated mutation treatment. .

  The yeast mutant obtained in the step (b) has a sulfur-containing compound content of 1.5% by weight or more per dry cell weight, and the sulfur-containing compound content is higher than that of the yeast mutant obtained in the step (a). It is a high yeast mutant. This may be because S metabolism was promoted by mutation of other S metabolism genes in addition to the MET30 gene by the mutation treatment.

  Furthermore, as a step (c), a yeast mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight is obtained by multiplying two of the yeast mutants obtained in the step (b). . In addition, crossing of yeast can be performed by a conventional method. In particular, a tetramolecular analysis (Tetrad Analysis) is performed on each parent strain, the sulfur-containing compound content of each spore is measured, and by multiplying the spores having the highest sulfur-containing compound content selected from each parent strain, A yeast mutant having a high sulfur-containing compound content of 2% by weight or more per dry cell weight can be easily obtained.

  Thus, it is not clear why a yeast mutant having a higher sulfur-containing compound content than the parent yeast mutant can be obtained by crossing the yeast mutants obtained in the step (b). The two strains obtained by the step (b) as the parent strain have mutant genes different from each other in addition to the MET30 gene. By multiplying these parent strains, the mutant strain possessed by each parent strain is also included. It is presumed that a high-sulfur compound yeast mutant can be obtained.

  Moreover, a yeast mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight can also be obtained by backcrossing the Saccharomyces cerevisiae mutant of the present invention with the parent Saccharomyces cerevisiae YNN27. Can do.

  Thus, the Saccharomyces cerevisiae mutant of the present invention is a yeast having a high content of sulfur-containing compounds having a mutant MET30 gene and having at least one mutant gene other than the MET30 gene. Such a Saccharomyces cerevisiae mutant can be obtained, for example, as follows.

1. Step (a): Preparation of yeast mutant strain having mutant MET30 gene First, a yeast mutant strain having a mutant MET30 gene was obtained by subjecting the wild strain Saccharomyces cerevisiae YNN27 to mutation treatment.
Saccharomyces cerevisiae strain YNN27 was cultured in a test tube containing YPD medium (glucose 2%, polypeptone 2%, yeast extract 1%) until the logarithmic growth phase. The cells were collected and subjected to mutation treatment using EMS according to a conventional method. Mutation treatment was performed under conditions such that the death rate was about 70%.
The strain subjected to the mutation treatment as described above was applied to a 2% agar-containing YPD plate medium, and statically cultured at 30 ° C. for 48 hours. The formed colonies were screened for yeast mutants with high total glutathione content by the nitroprusside method. Specifically, colonies were formed on a YPD agar plate medium, used as a master plate, copied from the master plate to a new 2% agar-containing YPD plate medium using a replica cloth, and cultured at 30 ° C. overnight. A colony was formed to produce a replica plate. On the replica plate, a 5% trichloroacetic acid solution containing 2% nitroplusside (Sodium Nitroplusside Dihydrate) was added, allowed to stand for 5 minutes, the nitroprusside-containing trichloroacetic acid solution was discarded, and 28% aqueous ammonia was added. This marked the red-stained colonies and picked up the corresponding strains from the master plate.
The nucleotide sequence of the MET30 gene of the picked-up strain was analyzed by a sequencing method, and one yeast mutant strain having a mutant MET30 gene was obtained. We named it 1 share.

2. Step (b): Production of a yeast mutant strain having a mutant MET30 gene and having a sulfur-containing compound content of 1.5% by weight or more per dry cell weight One strain was cultured until the logarithmic growth phase in a test tube containing YPD medium, and after mutation treatment using EMS in the same manner as in step (a), it was applied to a 2% agar-containing YPD plate medium at 30 ° C. The culture was stationary for 48 hours. The formed colonies were screened for yeast mutants with high total glutathione content by the nitroprusside method in the same manner as in step (a).
From the strains obtained by the screening, a yeast mutant strain having a mutant MET30 gene and having a sulfur-containing compound content of 1.5% by weight or more per dry cell weight was selected. Specifically, the total glutathione amount of each strain picked up from the master plate is measured according to the method of Titze et al. (Analytical Biochemistry, Vol. 27, p502, 1969), and divided by the dry cell weight of each strain. Thus, the total glutathione content per dry cell weight was calculated, and the yeast mutant strain (No. 2 strain) having a total glutathione content per dry cell weight of 2% by weight and the yeast mutant strain (2% by weight) ( No. 3 strain) and 2 strains were obtained.
No. No. 2 and No. When the nucleotide sequences of the three strains of the MET30 gene were analyzed by the sequencing method, It was a yeast mutant strain having the same mutant MET30 gene as one strain.

3. Step (c): Preparation of a yeast mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight.
No. No. 2 and No. By multiplying the three strains, a yeast mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight was prepared. Specifically, first, no. No. 2 and No. For each of the three strains, a tetramolecular analysis is performed, and the spore having the highest sulfur-containing compound content is selected, and by multiplying these spores, the total glutathione content per dry cell weight is 3% by weight. One strain of yeast mutant was obtained. The one strain was named ABYC1579 strain.

4). Bacteriological properties of ABYC1579 strain ABYC1579 strain is identical to the general mycological properties of Saccharomyces cerevisiae, except that the sulfur-containing compound content is 2% by weight or more per dry cell weight.
A colony obtained by culturing ABYC1579 strain on a YPD plate medium containing 2% agar at 30 ° C. for 2 days has the following morphological characteristics.
(1) Size: about 2 mm in diameter, (2) color tone: white to cream color, (3) shape: circular bulging half-lens shape on all edges, (4) surface shape: smooth, (5) transparency: opaque (6) Viscosity: butter-like.

  The ABYC1579 strain thus obtained is a Saccharomyces cerevisiae mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight, and is the Saccharomyces cerevisiae mutant of the present invention.

5). Acquisition of backcross strain Also, the obtained ABYC1579 strain was backcrossed with Saccharomyces cerevisiae YNN27 strain to obtain one yeast mutant strain having a total glutathione content of 3% by weight per dry cell weight. . The one strain was named ABYC1588 strain.

6). Bacteriological properties of ABYC1588 strain ABYC1588 strain is identical to the general mycological properties of Saccharomyces cerevisiae, except that the sulfur-containing compound content is 2% by weight or more per dry cell weight.
A colony obtained by culturing ABYC1588 strain on a YPD plate medium containing 2% agar at 30 ° C. for 2 days has the following morphological characteristics.
(1) Size: about 2 mm in diameter, (2) color tone: white to cream color, (3) shape: circular bulging half-lens shape on all edges, (4) surface shape: smooth, (5) transparency: opaque (6) Viscosity: butter-like.

  The ABYC1588 strain thus obtained is a Saccharomyces cerevisiae mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight, and is the Saccharomyces cerevisiae mutant of the present invention.

  In the present invention, the ABYC1579 strain is a Saccharomyces cerevisiae mutant newly produced by crossing yeast mutants obtained by mutation treatment. The ABYC1588 strain is a Saccharomyces cerevisiae mutant newly produced by backcrossing the ABYC1579 strain. Therefore, the applicant deposited the ABYC1579 strain and the ABYC1588 strain as new yeasts at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1-1-1 East, Tsukuba City, Ibaraki Prefecture). The accession numbers are FERM BP-10923 for the ABYC1579 strain and FERM BP-10924 for the ABYC1588 strain. In addition, the commission date is October 19, 2007.

  By culturing the Saccharomyces cerevisiae mutant of the present invention and containing 2% by weight or more of a sulfur-containing compound per dry cell weight in the mutant cell body, a yeast containing a high sulfur-containing compound content can be easily produced. Can do. The medium used for culturing the Saccharomyces cerevisiae mutant of the present invention is not particularly limited as long as it contains a carbon source, a nitrogen source, an inorganic salt, and the like, and the Saccharomyces cerevisiae mutant can grow. Any medium usually used for culturing yeasts such as Saccharomyces cerevisiae can be used.

  Examples of the carbon source contained in the medium for culturing the Saccharomyces cerevisiae mutant of the present invention include, for example, glucose, sucrose, acetic acid, ethanol, molasses, and sulfite pulp waste liquid that are used for normal microorganism culture. 1 or 2 or more types selected from the group which consists of can be used. The nitrogen source may be a nitrogen-containing inorganic salt or a nitrogen-containing organic substance. For example, one or more selected from the group consisting of urea, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, corn steep liquor (CSL), casein, yeast extract, and peptone can be used. In addition, as the inorganic salt, a phosphoric acid component such as lime superphosphate or phosphorous acid, a potassium component such as potassium chloride or potassium hydroxide, a magnesium component such as magnesium sulfate or magnesium hydrochloride, or the like can be used. In addition, inorganic salts such as zinc, copper, manganese, and iron ions may be used. Furthermore, vitamins, nucleic acid-related substances and the like may be appropriately added to the medium for culturing the Saccharomyces cerevisiae mutant of the present invention. Examples of such a medium include a YPD medium.

  The culture format is not particularly limited, and can be appropriately determined in consideration of the culture scale, the intended use of the obtained culture, and the like. For example, it may be applied to an agar plate medium and cultured, or may be cultured in a liquid medium. Examples of the culture format in the liquid medium include batch culture, fed-batch culture, and continuous culture. For example, in the case of subculture, since it is simple, it is preferable to culture on an agar plate medium or batch culture in an appropriate liquid medium. Moreover, when manufacturing yeast containing high sulfur-containing compounds industrially and mass-producing the culture, fed-batch culture or continuous culture is preferably performed.

  Moreover, the culture conditions of the Saccharomyces cerevisiae mutant of the present invention are not particularly limited, and can be cultured under conditions generally used when culturing Saccharomyces yeast. For example, the culture temperature is preferably 20 to 40 ° C, and more preferably 25 to 35 ° C. Moreover, it is preferable that pH of a culture medium is 3.5-8.0, and it is more preferable that it is 4.0-7.0. In particular, when industrially mass-producing cultures, it is preferable to periodically measure the pH in the medium and adjust it to maintain the pH at 4.0 to 7.0. In addition, the Saccharomyces cerevisiae mutant of the present invention is preferably cultured under aerobic conditions in the same manner as other Saccharomyces yeasts. For example, in batch culture, the aeration condition is preferably 0.5 to 2 vvm, and more preferably 0.8 to 1.5 vvm.

  For example, by performing batch culture for 20 hours or more using a YPD medium under the conditions of 25 to 35 ° C., stirring number 140 to 400 rpm, aeration rate 0.8 to 1.5 vvm, pH 4.0 to 5.0, The sulfur-containing compound content of the Saccharomyces cerevisiae mutant of the present invention can be 2% by weight or more per dry cell weight.

  By culturing the Saccharomyces cerevisiae mutant of the present invention, a culture having a sufficiently high sulfur-containing compound content can be obtained as compared with the case where a known strain of Saccharomyces cerevisiae is used. Therefore, by using the culture, a yeast extract having a high sulfur-containing compound content, particularly a yeast extract having a total glutathione content of 8% by weight or more and a high sulfur-containing compound content can be prepared.

  The preparation of the yeast extract from the culture of the Saccharomyces cerevisiae mutant of the present invention is not particularly limited, and any of the preparation methods commonly used for preparing the yeast extract may be used. Examples of the preparation method include a self-digestion method that solubilizes cells by using a proteolytic enzyme or the like inherent in yeast cells, and an enzyme decomposition that solubilizes cells by adding an enzyme preparation derived from microorganisms or plants. Method, hot water extraction method to solubilize cells by soaking in hot water for a certain period of time, acid / alkaline decomposition method to solubilize cells by adding various acids or alkalis, freezing and thawing once There are a freeze-thaw method for crushing bacterial cells by performing the above, a physical crushing method for crushing bacterial cells by physical stimulation, and the like. Examples of physical stimulation used in the physical crushing method include ultrasonic treatment, homogenization under high pressure, and grinding by mixing with a solid material such as glass beads.

  In addition, by subjecting the culture of the Saccharomyces cerevisiae mutant of the present invention to a drying treatment, dry yeast cells having a high sulfur-containing compound content can be obtained. The method for drying the culture is not particularly limited, and any of the methods commonly used for preparing dry yeast cells may be used. Examples of the preparation method include freeze-drying method, spray drying method, drum drying method and the like. Furthermore, by processing the obtained dry yeast cells into a powder form, a dry yeast powder having a high sulfur-containing compound content and excellent handleability can be obtained.

  In addition, a fraction containing a sulfur-containing compound may be obtained from the culture of the Saccharomyces cerevisiae mutant of the present invention. As a method for fractionating a fraction containing a sulfur-containing compound from a culture, any method may be used as long as it is a commonly used method. For example, the extract obtained by hot water extraction, extraction by cell disruption, etc. is fractionated using an affinity column carrying a substance having a high affinity for the sulfur-containing compound, thereby increasing the concentration of the sulfur-containing compound. It becomes possible to concentrate and purify the contained fraction.

  Preparations prepared from the cultures of the Saccharomyces cerevisiae mutant of the present invention itself, yeast extracts, dried yeast cells, fractions containing sulfur-containing compounds, etc. are food additives such as seasonings. Can be used particularly preferably. The food or drink to be added is not particularly limited, and examples thereof include alcoholic beverages, soft drinks, fermented foods, seasonings, soups, breads, and confectionery. In addition, the culture and the like can be consumed as a supplement or the like by processing into soft capsules, hard capsules, tableted tablets or the like.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1] Production 1 of yeast having a high total glutathione content
First, the ABYC1579 strain and the ABYC1588 strain were inoculated on a YPD flat plate medium containing 2% agar, respectively, and subjected to stationary culture overnight in a 30 ° C. incubator to prepare a subculture plate and stored at 4 ° C. .
One platinum loop colony was collected from the subculture plate, inoculated into 5 mL of YPD medium, and cultured overnight at 30 ° C. as a preculture solution. Thereafter, the preculture solution was added to 10 mL of YPD medium so as to have a final concentration of 5%, inoculated, and cultured at 30 ° C. at 160 rpm with stirring for 18 hours. The total amount of glutathione contained in each strain in the culture was measured in accordance with the method of Titze et al. (Analytical Biochemistry, Vol. 27, p502, 1969), and dried by dividing the dry cell weight of each strain. The total glutathione content per cell weight was calculated. As a result, the total glutathione content per dry cell weight of the cells in each culture was 2.7% by weight for the ABYC1579 strain and 2.8% by weight for the ABYC1588 strain.

[Example 2] Production 2 of yeast having a high total glutathione content
One platinum loop colony was collected from each subculture plate prepared in the same manner as in Example 1, inoculated into 50 mL of YPD medium, and cultured overnight at 30 ° C. as a preculture solution. Thereafter, 1.5 L of YPD medium is added to a 5 L jar fermenter (manufactured by Maruhishi Bioengineer), and a preculture solution is further added, so that the initial bacterial count becomes 1 × 10 ⁶ cells / mL. Inoculated in the manner described above, and batch culture was performed at 30 ° C., with a stirring rate of 250 rpm, and an aeration rate of 1 vvm. The pH in the medium during batch culture was maintained at 4.5 using aqueous ammonia. Similarly, a culture of a known Saccharomyces cerevisiae S288C strain was used as a control.
12 to 24 hours after the start of culture, 50 mL of the culture (ABYC1579 strain-containing YPD medium, ABYC1588 strain-containing YPD medium, or S288C strain-containing YPD medium) is collected and contained in the strain in each culture. The total glutathione content per dry cell weight was measured in the same manner as in Example 1.

  As a result, in the Saccharomyces cerevisiae mutant strains ABYC1579 and ABYC1588 of the present invention, the total glutathione content per dry cell weight was 2% by weight or more after 20 hours or more after the start of culture. On the other hand, the S288C strain, which is a known strain, is about 0.6% by weight, and it is clear that the Saccharomyces cerevisiae mutant of the present invention is a yeast having a very high content of sulfur-containing compounds such as glutathione.

[Example 3] Preparation of yeast extract Yeast extract was prepared using the culture of each strain after 24 hours of culture prepared in Example 2.
First, by centrifuging each culture, the contained yeast was recovered as a precipitate and washed with distilled water. Thereafter, an appropriate amount of distilled water was added so that the bacterial cell concentration was 10 to 15% to prepare a yeast suspension. The yeast suspension was heated at 85 ° C. for 70 seconds, rapidly cooled, and centrifuged to recover the extract. The collected extract was dried to prepare a yeast extract.
The total glutathione content in the obtained yeast extract was measured in the same manner as in Example 1. As a result, the yeast extract derived from the ABYC1579 strain culture was 8.4 wt%, and the yeast extract derived from the ABYC1588 strain culture was 8.1 wt%. Met. On the other hand, the S288C strain culture-derived yeast extract was 2.2% by weight.
From this result, it is clear that by using the Saccharomyces cerevisiae mutant of the present invention, a good yeast extract having a high content of sulfur-containing compounds having a total glutathione content of 8% by weight or more can be prepared.

  The Saccharomyces cerevisiae mutant of the present invention has a high sulfur-containing compound content such as glutathione, and by culturing the mutant strain, a yeast culture or yeast extract having a high sulfur-containing compound content can be easily produced. It can be used especially in the food field.

FERM BP-10923
FERM BP-10924

Claims (11)

A Saccharomyces cerevisiae mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight and obtainable from the following steps (a) to (c).
(A) obtaining a yeast mutant having a mutant MET30 gene by mutating Saccharomyces cerevisiae YNN27;
(B) Yeast having a mutant MET30 gene and having a sulfur-containing compound content of 1.5% by weight or more per dry cell weight by performing mutation treatment on the yeast mutant obtained in step (a) Obtaining two or more mutants;
(C) A step of obtaining a yeast mutant having a sulfur-containing compound content of 2% by weight or more per dry cell weight by multiplying two of the yeast mutants obtained in step (b).   The Saccharomyces cerevisiae mutant strain according to claim 1, which is Saccharomyces cerevisiae ABYC1579 strain (FERM BP-10923).   The Saccharomyces cerevisiae is characterized by having a sulfur-containing compound content of 2% by weight or more per dry cell weight and obtained by crossing the Saccharomyces cerevisiae mutant strain according to claim 1 or 2 with Saccharomyces cerevisiae YNN27. Mutant strain.   The Saccharomyces cerevisiae mutant strain according to claim 3, which is Saccharomyces cerevisiae ABYC1588 strain (FERM BP-10924).   A Saccharomyces cerevisiae mutant strain according to any one of claims 1 to 4 is cultured, and the mutant cell body contains 3% by weight or more of a sulfur-containing compound per dry cell weight. A method for producing a compound-rich yeast.   A culture of the Saccharomyces cerevisiae mutant according to any one of claims 1 to 4.   A yeast extract prepared from a culture of the Saccharomyces cerevisiae mutant according to any one of claims 1 to 4.   A fraction containing a sulfur-containing compound obtained by fractionating the culture of the Saccharomyces cerevisiae mutant according to any one of claims 1 to 4.   The yeast extract according to claim 7, wherein the total content of oxidized glutathione and reduced glutathione is 8% by weight or more.   A sulfur-containing compound-containing food or drink comprising the culture according to claim 6 or the fraction according to claim 8.   The sulfur-containing compound-containing food or drink according to claim 10, wherein the sulfur-containing compound is glutathione.

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