JP2005245390A - Method for producing glutathione - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing glutathione comprising seeding a culture medium to which ethanol is added with a glutathione-producing yeast, making it possible that the glutathione produced by the yeast itself is discharged into the culture medium, when the glutathione-producing yeast is cultured in the natural culture medium. <P>SOLUTION: This method for producing the glutathione comprises seeding the culture medium to which the ethanol is added with the glutathione-producing yeast, wherein the glutathione is produced by seeding the natural culture medium for which a collagen peptide is used as a nitrogen source with the yeast and culturing the yeast. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing glutathione by inoculating a culture medium supplemented with ethanol with glutathione-producing yeast.

It is known that glutathione plays various physiological functions in vivo, such as an action as an enzyme or a coenzyme, a detoxification action, liver function enhancement, protection against radiation damage, and a cell membrane maintenance action.
This glutathione is present in yeast, cattle liver, sheep brain, etc., but as an industrial production method, a method of culturing yeast containing a large amount of glutathione and extracting glutathione therefrom is adopted. .

Patent Document 1 discloses that a glutathione-rich yeast can be obtained by aerobic culture of azeserine-resistant bacteria obtained by performing mutation treatment on a glutathione high-producing yeast introduced with a γ-glutamylcysteine synthetase gene. .
Patent Document 2 discloses that glutathione-producing yeast is produced in a large amount by aerobic shaking culture of glutathione-producing yeast in a medium containing malt extract and sodium glutamate under lactic acid bacteria fermentation.
However, even if the yeast is obtained from any method, glutathione is retained in the yeast, so the yeast is separated from the medium and used, for example, by a crusher disclosed in Patent Document 3. Thus, glutathione cannot be obtained unless the yeast cell membrane is destroyed.

Non-patent document 1 discloses a culture technique for allowing glutathione produced by yeast to flow out into a medium. That is, Candida, a glutathione-producing yeast, is added to a synthetic medium supplemented with ethanol.
When tropicalis Pk233 was inoculated, the cells changed to mycelium cells and ethanol acted on the yeast to promote glutathione outflow, which revealed that glutathione produced by the cells could be discharged into the medium.
Although it is extremely significant for yeast to cause the physiologically active substance to flow out into the medium, it cannot be used as it is because glutathione is discharged from a synthetic medium. Glutathione was forced to be separated from the medium by a complicated purification method, and a problem remained for industrial use.

The inventor studied the culture method for improving the culture technique proposed in Non-Patent Document 1 so that glutathione flowing out of the medium can be used as it is without undergoing a purification step.
First, using a known natural medium (commonly known as YPG medium) shown in Table 1 and adding 2% by volume (the same is true for the amount of ethanol added), glutathione-producing yeast is used. Known as Candida
Four types of yeast were cultured: tropicalis Pk233, Candida tropicalis IFO1400, Candida utilis and Pichia anomala.
(Table-1) Composition in 100 ml of YPG medium Glucose 5 g
Polypeptone 1g
Yeast extract 0.5g
Glucose and polypeptone manufactured by Wako Pure Chemical Industries, Ltd., and yeast extract manufactured by Takeda Pharmaceutical Company Limited were used.

In this culture, 20 ml of the medium was poured into a 100 ml flask, 0.1 ml of each yeast inoculated with 10 mg of each yeast was poured into the medium, and cultured with shaking at 30 ° C. for 100 hours. After that, the culture solution is kept in a centrifuge at 3000 rpm.
The glutathione content of the supernatant obtained over / 15 minutes and the culture supernatant once heated at 110 ° C. for 10 minutes by autoclave and then centrifuged at 3000 rpm / 15 minutes for the amount of glutathione Measured by the method.
In this DTNB method, as described in Non-Patent Document 1, 5,5′-dithiobis (2nitrobenzoic acid) (DTNB) is converted to nicotinamide adenine dinucleotide phosphate in the presence of glutathione reluctase and glutathione. Reduced type (NADPH ₂
) Is a method for measuring the amount of glutathione utilizing the fact that the reaction rate is proportional to the amount of glutathione present, and the increase in absorption at 412 nm after the start of the reaction was measured.
The amount of glutathione produced in the medium was as shown in Table 2.

(Table-2) Amount of glutathione produced in YPG medium (unit: μg / ml)

In the above YPG medium, glutathione flowed out into the medium was extremely small, and in the supernatant, there was almost no difference in the amount of flow between the one without ethanol and the one with 2% added.
In this medium, ethanol does not grow greatly in yeast itself, and inositol contained in polypeptone as a protein component strengthens the cell membrane of yeast, which is thought to suppress glutathione outflow by ethanol.
In addition, when the supernatant is heated, glutathione staying in the cells increases compared to when it is not heated. This is because the yeast produces glutathione while restricting its growth, It means that it had accumulated in the body.

JP-A-6-70752 JP 2003-102468 A JP-A-5-252894 Bulletin of Seirenjo Gakuin Junior College Vol. 12 “Fermentative production of physiologically active substances by Candida yeast (Part 2)-Production of glutathione by Candida tropicalis Pk233”

  The present invention relates to a method for producing glutathione by inoculating glutathione-producing yeast in a medium to which ethanol has been added, so that glutathione produced by the yeast can be discharged into the medium when the glutathione-producing yeast is cultured in a natural medium. This is an issue.

The first technical means for solving this technical problem is to use a collagen peptide as a nitrogen source of a natural medium.
Collagen peptide, which is a nitrogen source in natural media, is a highly purified peptide made by reducing the molecular weight of gelatin (collagen) with enzymes, and since it contains no inositol, yeast has a strengthened cell membrane. Absent. Since ethanol is added to the medium, glutathione produced by the yeast can be discharged from the cell membrane.
Therefore, yeast produces glutathione and causes it to flow out of the body. It is speculated that this also causes polysaccharides, proteins, vitamins and minerals to flow out at the same time.

When glutathione-producing yeast is cultured in a natural medium, glutathione produced by the yeast is allowed to flow out into the medium. As a result, there is an advantage that the culture solution can be taken out while yeast inoculation and culture are continued.
Yeast does not only produce glutathione produced during culture, but also polysaccharides and proteins, so it can be used as a raw material for food and medical treatment after removing the cells, or by concentrating or pulverizing as appropriate. In addition, there is an advantage that it can be used as a food or cosmetic material mainly composed of collagen remaining in the culture solution.

Candida, which is known as glutathione-producing yeast, is prepared with a natural medium having the composition shown in Table 3 with no ethanol added and with 2, 3, 4% by volume added.
tropicalis Pk233, Candida tropicalis
IFO1400, Candida utilis and Pichia
Anomala culture was performed.
20 ml of the medium was poured into a 100 ml flask, and 0.1 ml of each yeast inoculated with 10 mg of each yeast was poured into the medium and cultured with shaking at 30 ° C. for 100 hours.
(Table-3) Natural medium composition (in 100 ml)
Glucose 5g
KH ₂ PO ₄ 40mg
MgSO ₄ 7H ₂ O 20mg
Collagen peptide 4g
Vitamin B ₁ 12μg
Vitamin B ₂ 5μg
Vitamin B ₆ 50μg
PABA 25μg
Nicotinic acid 250μg
Pantothenic acid Ca 25 μg
Biotin 5μg
Glucose is manufactured by Wako Pure Chemical Industries, collagen peptide is manufactured by Nitta Gelatin Co., and others are manufactured by Nacalai Tesque.

After culturing, this culture solution was directly applied to a centrifugal separator at 3000 rpm / 15 minutes, and glutathione in the obtained supernatant was measured by the DTNB method.
The numbers in parentheses in the 0% and 3% columns in Table 4 indicate the amount of yeast grown (unit: mg dry / ml).
(Table-4) Amount of glutathione in unheated supernatant (unit: μg / ml)

On the other hand, the culture solution after culturing was heated at 110 ° C. for 10 minutes by autoclave, then subjected to 3000 rpm / 15 minutes in a centrifuge, and glutathione in the obtained supernatant was measured.
(Table-5) Amount of glutathione in the heated supernatant (unit: μg / ml)

The measurement results in Tables 4 and 5 above show the average values of the measurements performed on the culture solution performed three times under the same conditions.
As can be confirmed in Table-4, it can be understood that the growth rate of yeast is not increased when ethanol is added during the culture of yeast.
It can be seen from the measurement results that a large amount of glutathione has flowed into the medium by adding ethanol, but this does not mean that glutathione increases due to the growth of the yeast, while the yeast is inhibited from growing. This also means that a large amount of glutathione was produced and part of it was discharged into the culture medium.

From the measurement results, it can be easily understood that Candida tropicalis Pk233 has an insufficient amount of glutathione in the heated supernatant although the measured value of the supernatant when ethanol 4% is added is insufficient.
On the other hand, for Candida tropicalis IFO1400, Candida utilis and Pichia anomala, both the supernatant and the heated supernatant produced the largest amount of ethanol when 3% was added, and when 4% was added, the results were almost the same as when not added. ing. Therefore, it can be said that it is most desirable to add 2-3% of ethanol for these three yeasts.

In order to investigate the cause of the above difference in production, we examined changes in yeast morphology and found that Candida tropicalis
Only Pk233 was a mycelium type, and all other yeasts were found to be of the yeast type.
Therefore, the inventor made a natural medium without addition of ethanol and 2, 3, 4, 5, 6% added to the natural medium shown in Example 1, and poured 20 ml of this medium into a 100 ml flask. To Candida
A 10 ml dry / ml inoculated tropicalis Pk233 was poured into the medium, and 0.1 ml was poured into the medium and cultured with shaking at 30 ° C. for 160 hours.
Similarly to Example 1, the average value of the amount of glutathione measured by obtaining the supernatant of the culture solution and the heated supernatant obtained by performing the culture three times is shown in Table-6.

(Table-6) Glutathione content of Candida tropicalis Pk233 after 160 hours of culture

In the above measurement results, the amount of yeast decreases as the amount of ethanol added increases. In addition, the amount of glutathione produced in both the supernatant and the heated supernatant decreased rapidly when the amount of ethanol added increased to 6%.
With regard to tropicalis Pk233, glutathione production is actively carried out even when the amount of ethanol added is increased to 5%. However, when 6% is reached, it is considered that the survival and growth of bacterial cells will be limited due to the inhibitory action by ethanol.
In the case where ethanol is not added, the unheated supernatant is not suitable for continuous culture because the amount of glutathione is small.

  In Examples 1 and 2, the flow of glutathione remaining in the cell body was promoted when the medium was heated after the yeast culture was completed, and the amount of glutathione retained in the cell body was large. It is also revealed that it is discharged into the medium.

Claims (6)

A method for producing glutathione, which comprises inoculating a culture medium to which ethanol has been added with glutathione-producing yeast to produce glutathione, wherein the culture medium is inoculated with yeast in a natural medium using a collagen peptide as a nitrogen source. The method for producing glutathione according to claim 1, wherein the yeast is selected from Candida tropicalis Pk233, Candida tropicalis IFO1400, Candida utilis, and Pichia anomala. The method for producing glutathione according to claim 1 or 2, wherein 2 to 3% by volume of ethanol is added to the medium and the yeast is inoculated. The method for producing glutathione according to claim 1, wherein 2 to 5% by volume of ethanol is added to the medium and inoculated with Candida tropicalis Pk233. The method for producing glutathione according to claim 1, 2, 3, or 4, wherein yeast is shaken and cultured. A method for producing glutathione, comprising: completing culture by the method according to any one of claims 1 to 5; and heating the medium to discharge glutathione from yeast into the medium.