KR101167345B1 - Mutant yeast, method of producing glutathione-rich yeast, culture thereof, fraction thereof, yeast extract and glutathione-containing foods and drinks - Google Patents

Abstract

It provides a yeast capable of maintaining a large amount of glutathione in cells. A yeast mutant strain having a cadmium resistance or a macrolide antibiotic resistance and capable of containing a large amount of glutathione.

Yeast mutant, glutathione, cells, Candida utility, culture

Description

Production method of yeast mutant strain, high glutathione-containing yeast, its culture, fractions, yeast extract and food products containing glutathione AND DRINKS}

The present invention relates to a yeast mutant strain of the genus Candida sp. More specifically, mutants of Candida utilis, which may contain a large amount of reduced or oxidized glutathione in the cells, cultures, fractions, yeast extracts obtained by culturing the mutants, and food and drink using them It is about.

Glutathione is a tripeptide consisting of three amino acids, glutamic acid, glycine, and cysteine, and is widely distributed in tissues such as the liver and muscle of yeasts and animals, and is involved in detoxification and redox equilibrium in vivo. In view of this physiological activity, glutathione has been widely used as a medicine for treating liver diseases such as alcoholic fatty liver medicine.

On the other hand, as health functional foods have been in the spotlight recently from the viewpoint of health orientation, foods containing the same substance have attracted attention in the food field based on the physiological activity of glutathione.

As an industrial manufacturing method of glutathione, the method of extracting from yeast is common, but in this case, acquisition of yeast with a high glutathione content rate becomes a requirement.

Conventionally known methods for producing glutathione-containing yeast include a method of adding amino acids to a medium (Patent Document 1), a method of limiting zinc ions (Patent Document 2), and a method of culturing at a lower culture temperature than usual (Patent Document 3). ). In addition, there are ethionine-sulfite-resistant strains (Patent Document 4), polyene-based antibiotic-resistant strains (Patent Document 5), etc. by mutant treatment.

However, in order to obtain glutathione industrially at low cost, further glutathione-containing yeast is desired.

Patent Document 1: Japanese Patent Application Laid-open No. 53-94089

Patent Document 2: Japanese Patent Application Laid-open No. Hei1-141591

Patent Document 3: Japanese Patent Application Laid-open No. 60-156379

Patent Document 4: Japanese Patent Laid-Open No. 59-151894

Patent Document 5: Japanese Patent Laid-Open No. 2003-284547

An object of the present invention is to provide a glutathione-containing food and drink using yeast and the same yeast capable of maintaining a large amount of glutathione in cells under the above technical background.

MEANS TO SOLVE THE PROBLEM As a result of earnestly examining in order to solve the said subject, the present inventors discovered that the yeast mutant strain which has cadmium resistance or macrolide antibiotic resistance can contain glutathione in a larger amount of cell than a general wild yeast. Thus, the present invention has been completed.

That is, the present invention is as follows.

(1) A yeast mutant strain having cadmium resistance or macrolide antibiotic resistance and capable of containing a high concentration of glutathione.

(2) The yeast mutant strain as described in (1) whose content in glutathione cells of said yeast mutant strain is 0.3 wt% or more.

(3) The yeast mutant according to (1), wherein the yeast mutant having cadmium resistance is Candida utility ABYC1560 (Accession No. FERM P-20094).

(4) The yeast mutant strain according to (1), wherein the macrolide antibiotic is rapamycin.

(5) The yeast mutant strain according to (1), wherein the yeast mutant strain having macrolide antibiotic resistance is Candida utility ABYC1561 (Accession No. FERM P-20095).

(6) A method for producing a high glutathione-containing yeast, wherein the yeast mutant strain according to any one of (1) to (5) is cultured aerobicly to accumulate glutathione at a high concentration in the yeast cells.

(7) A culture obtained by culturing the yeast mutant strain according to any one of (1) to (5) under aerobic conditions.

(8) A fraction of said culture comprising glutathione obtained by culturing the yeast mutant strain according to any one of (1) to (5) under aerobic conditions.

(9) A yeast extract produced using a culture obtained by culturing the yeast mutant strain according to any one of (1) to (5) under aerobic conditions.

(10) A dry yeast cell produced using a culture obtained by culturing the yeast mutant strain according to any one of (1) to (5) under aerobic conditions.

(11) A glutathione-containing food and drink comprising a culture obtained by culturing the yeast mutant strain according to any one of (1) to (5), and a fraction of the culture comprising glutathione.

EFFECTS OF THE INVENTION [

The novel yeast mutant strain of the present invention can contain a high concentration of glutathione in the cells, and the new yeast mutant strain of the present invention can be cultured aerobicly to accumulate glutathione in the yeast cell at a high concentration.

Thus, the novel yeast mutant strain of the present invention can be cultured under aerobic conditions to obtain a culture or fraction containing high concentration of glutathione.

In addition, the yeast extract of the present invention can be cultured under aerobic conditions to obtain a yeast extract containing high concentration of glutathione.

In addition, a glutathione-containing food and drink can be obtained by containing a culture obtained by culturing the novel yeast strain of the present invention under aerobic conditions, a fraction of the culture containing glutathione or the culture or fraction treated by heat treatment.

Hereinafter, the present invention will be described in detail.

The novel yeast mutant of the present invention is a yeast mutant having cadmium resistance or macrolide antibiotic resistance and capable of containing a high concentration of glutathione.

In addition, Cd-resistant yeast mutant strains of the present invention, such as Candida and utility strains 15-10 strains as parent strains, and mutant strains with higher heavy metal resistance than the parent strains by mutation treatment. Obtained by selection.

Specifically, mutagenesis is carried out using mutations such as ultraviolet rays, ionizing radiation, nitrous acid, nitrosoguanidine, and ethyl methanesulfonate (hereinafter abbreviated as 'EMS'), and include heavy metals such as cadmium. The mutants that are recognized for growth on the medium are selected.

By measuring the amount of glutathione contained in the cells of the selected mutant strains, a further mutant strain having an increased amount of glutathione is selected, and a mutant strain capable of containing a high concentration of glutathione is searched. At this time, cadmium, mercury, etc. are used as a heavy metal to be used. Thus, in this invention, Candida utility ABYC1560 which is a new variant strain was obtained.

Variant Candida® utility ABYC1560 of the present invention accumulates at least 0.3 wt% glutathione per dry weight in the cells. The glutathione in this invention means the total glutathione amount of glutathione of a reduced type and an oxidation type.

In addition, the general mycological properties of the novel variant Candida® utility ABYC1560 strain of the present invention are exactly the same as the general mycological properties of Candida utility except for cadmium resistance. The novel variant Candida® utility ABYC1560 strain of the present invention was deposited as 'Accession No. FERM P-20094'.

The yeast mutant of the present invention may have any resistance to macrolide antibiotics, and furthermore, any type of yeast mutant may contain oxidized and reduced glutathione in cells. Specifically, Candida utility ABYC1561 obtained by a mutation process using Candida utility ABYC1560 as a parent.

Macrolide antibiotics are antibiotics having a large ring-shaped lactone, and the macrolide antibiotics of the present invention include, for example, rapamycin, leinamycin, and lancaacidin C. Can be mentioned.

In order to obtain a yeast mutant strain having a macrolide antibiotic resistance of the present invention, specifically, agar medium containing a macrolide antibiotic using a mutation source such as ultraviolet rays, ionizing radiation, nitrous acid, nitrosoguanidine, EMS, etc. Select the state in which the growth is recognized. By measuring the amount of glutathione contained in the cells of the selected mutant strains, a further mutant strain having an increased amount of glutathione is selected, and a mutant strain capable of containing a high concentration of glutathione is searched. Thus, in this invention, Candida utility ABYC1561 which is a new variant strain was obtained.

Variants Candida utility ABYC1561 having a macrolide-based material resistance of the present invention accumulates more than 0.5wt% glutathione per dry weight in the cells. The glutathione in this invention means the total glutathione amount of glutathione of a reduced type and an oxidation type.

In addition, the general mycological properties of the novel variant Candida® utility ABYC1561 strain of the present invention are exactly the same as the general mycological properties of Candida utility, except for macrolide antibiotic resistance. The novel variant Candida® utility ABYC1561 strain of the present invention was deposited as 'Accession No. FERM P-20095'.

In the present invention, there is also provided a method for producing glutathione-containing yeast for aerobic culture of mutant strains.

In the present invention, there is also provided a method for producing glutathione-containing yeast for aerobic culturing the yeast mutant strain. In order to carry out the present invention, the cadmium resistant mutant strain or macrolide antibiotic resistant mutant strain obtained by the above-mentioned method may be cultured aerobicly in a medium containing a carbon source, a nitrogen source, an inorganic salt, and the like.

As the medium composition of these strains, one or two or more selected from the group consisting of glucose, sucrose, acetic acid, ethanol, molasses and sulfite pulp waste liquid used for cultivation of ordinary microorganisms as a carbon source is used. As the nitrogen source, a group consisting of urea, inorganic salts such as ammonia, ammonium sulfate, ammonium chloride or ammonium phosphate, and nitrogen-containing organic substances such as corn steep liquor (CSL), casein, yeast extract or peptone, etc. One or two or more kinds selected from are used. Moreover, you may add a phosphoric acid component, a potassium component, and a magnesium component to a medium, As these, normal industrial raw materials, such as lime superphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, and magnesium hydrochloride, may be sufficient. In addition, inorganic salts such as zinc, copper, manganese and iron ions may be used. In addition, vitamins and nucleic acid-related substances may be added.

As a culture type, any of batch culture, oil value culture, or continuous culture may be used, but industrially, oil value culture or continuous culture is employed.

The culture temperature may be in accordance with general yeast culture conditions, for example, 20 to 40 ° C, preferably 25 to 35 ° C, and the pH is preferably 3.5 to 8.0, particularly 4.0 to 6.0.

By the method of the present invention, a culture containing high concentration of glutathione in yeast cells is obtained, but a fraction containing glutathione may be obtained from the culture. As a method of fractionating a glutathione-containing fraction from a culture, any method may be used as long as it is a conventional method, but hot water extraction, extraction by cell breakdown, etc. are mentioned. Furthermore, by supporting the obtained extract on a carrier, it is possible to concentrate the fraction containing glutathione at a high concentration.

Moreover, you may prepare yeast extract from the culture cultured by the said method. As the method for preparing the yeast extract, any method may be used as long as it is usually performed. However, a self-extinguishing method, an enzyme decomposition method or an alkali extraction method is employed industrially.

In addition, dry yeast cells may be prepared from the culture cultured by the above method. As a method for producing a dry yeast cell, any method may be used as long as it is usually carried out, but industrially, a lyophilization method, a spray dry method, a drum dry method and the like are employed.

The present invention also relates to a culture product obtained by culturing the yeast mutant strain under aerobic conditions, and a food and drink containing a fraction of the culture containing glutathione.

As these food-drinks, any food-drinks to which dry yeast and yeast extract can be normally added may be sufficient, For example, alcoholic drinks, soft drinks, fermented food seasonings, soups, general foods, confectionery, etc. are mentioned.

In order to manufacture the food-drinks of the present invention, in the production process of the food-drinks, a culture obtained by culturing the yeast mutant strain under aerobic conditions, and a fraction of the culture containing glutathione may be added.

Therefore, according to this invention, the food-drinks containing a high concentration of glutathione can be manufactured efficiently.

Hereinafter, an Example is given and described this invention. The present invention is not limited to these examples.

On the other hand, the cell weight was measured from the weight after the culture solution was washed twice with a centrifuge and dried at 105 ° C. for 5 hours. Quantitative determination of total glutathione combined with reduced and oxidized glutathione was determined according to Titze's method (Analytical Biochemistry, Vol. 27, p. 502, 1969). The total glutathione amount (% (w / w)) in the dry cells was calculated as a percentage by dividing the obtained total glutathione by the dry cell weight.

Example 1

Candida utility was cultured in vitro to a logarithmic growth stage containing YPD medium (2% glucose, 2% polypeptone, 1% yeast extract). Thereafter, the cells were recovered and subjected to mutation using EMS in accordance with the conventional method. The mutation treatment was carried out under the condition that the mortality was about 70%.

The 15-10 strains subjected to the mutation treatment as described above were inoculated into YPD medium containing 0.2 mg / ml of cadmium, and left to incubate at 30 ° C for 48 hours. As a result, 200 strains of cadmium resistant mutants were isolated. The total amount of glutathione was measured for these cadmium resistant mutant strains to obtain ABYC1560 strain (Accession No. FERM P-20094) in which the amount of glutathione in cells was enhanced.

Example 2

ABYC1560 strain (Accession No. FERM P-20094) and its parent strain 15-10 strains which obtained cadmium resistance were incubated 18 hours before in YPD medium, then inoculated in YPD medium, and main culture was performed at 30 ° C. for 18 hours. . After the cultured cells were collected by centrifugation, washed twice with water, and then boiled in a boiling water bath for 5 minutes, the glutathione in the cells was extracted, and the total glutathione amount was measured according to the above-described method. Was carried out. The total amount of glutathione in the dried cells was 1.00 wt% in the cadmium resistant strain (ABYC1560 strain (Accession No. FERM P-20094)), compared to 0.52 wt% in the parent strain (15-10).

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Example 3

Candida utility was incubated in vitro with logarithmic growth stages containing YPD medium (2% glucose, 2% polypeptone, 1% yeast extract). The cells were recovered and subjected to mutant treatment using EMS according to the conventional method. The mutation treatment was carried out under the condition that the mortality was about 70%.

As described above, the ABYC1560 strain subjected to the mutation treatment was inoculated into YPD agar medium containing 1 µg / ml rapamycin, and left to incubate at 30 ° C for 48 hours. As a result, 745 rapamycin resistant mutants were isolated. About these resistant strains, the total amount of glutathione was measured by the method mentioned above, and the ABYC1561 strain (accession number FERM P-20095) with which the amount of glutathione in cells was enhanced was obtained.

Example 4

ABYC1561 strains that obtained rapamycin resistance and ABYC1560 strains, the parent strains thereof, were cultured 18 hours before in YPD medium, then inoculated in YPD medium, and main culture was performed at 30 ° C. for 18 hours. After the cultured cells were collected by centrifugation, washed twice with water, and then boiled in boiling water bath for 5 minutes, the glutathione in the cells was extracted, and the total amount of glutathione was measured according to the above-described method. The total amount of glutathione in the dried cells was 1.82% in the rapamycin resistant strain (ABYC1561 (Accession No. FERM P-20095)), compared to 1.00% in the parent strain (ABYC1560).

Industrial applicability of this invention exists in the following points.

The novel yeast mutant strain of the present invention can contain a high concentration of glutathione in the cells, and the new yeast mutant strain of the present invention can be cultured aerobicly to accumulate glutathione in the yeast cell at a high concentration.

Thus, the novel yeast mutant strain of the present invention can be cultured under aerobic conditions to obtain a culture or fraction containing high concentration of glutathione.

In addition, the yeast extract of the present invention can be cultured under aerobic conditions to obtain a yeast extract containing high concentration of glutathione.

Further, a glutathione-containing food and drink can be obtained by containing a culture obtained by culturing the novel yeast strain of the present invention under aerobic conditions, a fraction of the culture containing glutathione, or the culture or fraction subjected to heat treatment.

Claims (11)

Candida-utility ABYC1560 (Accession No. FERM BP-10759) yeast mutant strain that is cadmium resistant and capable of containing a high concentration of glutathione. delete delete Candida® utility ABYC1561 (Accession No. FERM BP-10760) Yeast Variant with cadmium resistance and rapamycin resistance and capable of containing high concentrations of glutathione. delete A method for producing a high glutathione-containing yeast, wherein the yeast mutant strain according to claim 1 or 4 is cultured aerobicly to accumulate glutathione at a high concentration in the yeast cells. A culture obtained by culturing the yeast mutant strain according to claim 1 or 4 under aerobic conditions. A fraction of said culture comprising glutathione obtained by culturing the yeast mutant strain of claim 1 or 4 under aerobic conditions. The yeast extract produced using the culture obtained by culturing the yeast mutant strain of Claim 1 or 4 in aerobic conditions. The dry yeast cell produced using the culture obtained by culturing the yeast mutant strain of Claim 1 or 4 in aerobic conditions. A glutathione-containing food and drink comprising a culture obtained by culturing the yeast mutant strain according to claim 1 or 4 under aerobic conditions, and a fraction of the culture comprising glutathione.