KR100771077B1 - Yeast nutrients for the culture medium of fermentation yeast - Google Patents

Abstract

Yeast nutrients are provided to effectively improve the glutathione productivity and yield of Saccharomyces cerevisiae FF-8(deposition no. KACC93023P) by optimizing supplying sources and supplying amounts of carbon, nitrogen, salt and amino acid. A yeast nutrient for a culture medium of fermentation yeast comprises 3.0 wt.% of glucose as a carbon source, 3.0 wt.% of a yeast extract as a nitrogen source, 0.06 wt.% of KH2PO4 as a salt source and 0.06 wt.% of L-cysteine as an amino acid source regarding the total weight of the culture medium.

Description

Yeast Nutrients for the culture medium of fermentation yeast}

1 is a graph showing the effect of glucose concentration on glutathione production capacity and dry cell weight.

Figure 2 is a graph showing the effect of yeast extract concentration on glutathione production capacity and dry cell weight.

3 is a graph showing the effect of KH ₂ PO ₄ concentration on glutathione production capacity and dry cell weight.

4 is a graph showing the effect of L-cysteine concentration on glutathione production capacity and dry cell weight.

Figure 5 is a graph showing the change in glutathione production capacity and dry cell weight in the yeast nutritional supplement of the present invention and conventional YM medium.

The present invention relates to a nutrient for culture medium of traditional strain fermented yeast strains, more specifically carbon, nitrogen, salts to improve the glutathione production capacity of fermented yeast strains such as Saccharomyces cerevisiae And it relates to a nutrient for the culture medium to improve the glutathione production capacity of the fermented yeast strain by optimizing the amino acid source and feed amount.

Since Korea has been developing fermentation and aging technology since ancient times, most of the traditional liquor has been manufactured using fermentation technology. Traditional liquor is divided into traditional distilled liquor and traditional fermented liquor according to the manufacturing method. In particular, traditional fermented liquor has unique taste and aroma and has unique functionalities according to added ingredients.

Traditional brewing yeasts such as Saccharomyces cerevisiae and Saccharomyces bynus are used in traditional fermentation, and it has been found that Saccharomyces cerevisiae produces glutathione.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is a well known thiol-containing tripeptide in animals, plants, and microorganisms, and glutathione is a very protective layer of ultraviolet light, heavy metals and various exogenous organic substances on cellular mechanisms. It is an important ingredient. Glutathione also serves as a sacrificial protective against oxidative damage of organisms. Glutathione is used as a drug, to prevent hepatotoxicity caused by acetoaminophene, vinyl ether or animal bromobenzene in health foods, and also in the cosmetic industry. The commercial use of glutathione is very wide.

As well as efforts to isolate and identify yeast strains that produce glutathione, which is a useful ingredient for the human body, many studies have been conducted to improve glutathione production in yeast strains known to produce glutathione.

In the past, yeast nutritional agents have been used to promote the cultivation and growth of yeast strains, a typical example being the addition of ammonium phosphate to the culture medium.

The present inventors have completed the present invention as a result of repeated studies to improve the glutathione production capacity of Saccharomyces cerevisiae strain in view of this point.

Accordingly, an object of the present invention is to provide a yeast nutrient for culture medium for improving glutathione production capacity of Saccharomyces cerevisiae yeast strain.

This object is achieved by supplying a culture medium of Saccharomyces cerevisiae yeast strain with yeast nutrients with optimized carbon, nitrogen, salt and amino acid sources and feed amounts, thereby improving the production capacity and yield of glutathione.

The present invention provides yeast nutrients optimized for carbon, nitrogen, salt and amino acid sources and feed amounts for improving glutathione production capacity and efficiency in the culture medium of fermented yeast strains.

Yeast nutrients for the culture medium of the preferred fermented yeast strains of the present invention are yeast nutrients containing glucose as a carbon source, yeast extract as a nitrogen source, KH ₂ PO ₄ as a salt source and L-cysteine as an amino acid source.

Yeast nutrient for the culture medium of the more preferred fermentation yeast strain of the present invention, 3.0% by weight of glucose as a carbon source, 3.0% by weight of yeast extract as a nitrogen source, 0.06% by weight of KH ₂ PO ₄ as a salt source and amino acid source in the total weight of the culture medium Yeast nutrient consisting of 0.06% by weight of L-cysteine.

Hereinafter, preferable embodiment of this invention is described focusing on an Example. However, this does not limit the scope of the present invention.

<Example>

Example  1: yeast culture

Saccharomyces cerevisiae FF-8 (KACC93023P) (hereinafter referred to as FF-8), a glutathione producing yeast strain, was obtained by 1.0 wt% glucose, 0.5 wt% peptone, 0.3 wt% yeast in a 500 mL flask. Growth was aerobic for 24 hours under conditions of pH 6.0 and 30 ° C. in 100 mL of YM medium consisting of the extract and 0.3 wt% malt extract. Subsequently, the cultured cells were placed in a 1 L flask each containing 200 mL of the same medium, and then incubated at 30 ° C. for 72 hours with stirring at 100 rpm. After incubation, the culture was centrifuged at 7,000 × g for 15 minutes, the supernatant was removed, and the yeast cells were washed three times with distilled water. Glutathione concentration and dry cell weight of cultured yeast cells were analyzed.

Example  2 : Glutathione  Assay and Cell Growth

The yeast cells cultured in Example 1 were suspended in 0.2 M phosphate buffer (pH 7.2) and pulverized by ultrasound. The pulverized cells were then removed by centrifugation, and the concentration of glutathione in the supernatant was measured by measuring the absorption capacity of the reaction solution at 412 nm with a spectrometer. The concentration of the sample was measured using a standard curve generated with a certain amount of glutathione. Cell growth was measured by measuring the dry weight of the cells. Wet cells were dried at 105 ° C. until their weight became constant to measure dry cell weight.

Example  3: Glutathione  Optimum Production Condition Measurement

Various carbon, nitrogen, salt and amino acid sources were tested to obtain optimal conditions for glutathione production and cell growth. The most important components were selected to optimize the concentration of each component.

1) Effects of various carbon sources

The glutathione production capacity of FF-8 was 90 mg / L under the optimal culture conditions of YM medium. The YM medium contained 0.5% by weight of peptone, 0.3% by weight of yeast extract and 0.3% by weight of malt extract, which was added three times in an amount of 1.0% by weight of various carbon sources, to give glutathione production capacity and cells of FF-8. The growth was observed and the effect is shown in Table 1. TABLE 1

Carbon source Glutathione Dry cell weight (mg / L) (%) (g / L) (%) Glucose 57.6 100.0 4.15 100.0 Galactose 54.6 94.8 4.05 97.6 Fructose 52.4 91.0 4.13 97.6 Lactose 18.8 32.6 2.10 50.0 Maltose 56.6 98.3 4.00 95.2 Sucrose 53.8 93.4 4.23 100.0

Glucose has been shown to be the most preferred carbon source for glutathione production and cell growth. Cell growth was similar, but glutathione production was 6.6% higher when glucose was used as the carbon source instead of sucrose. It has been known that glutathione production capacity and dry cell weight are greatest when glucose is used as the carbon source. When lactose was used, glutathione production capacity and cell growth were significantly lower by 67.4% and 50.0% than when glucose was used. The effect of glucose concentration on glutathione production and cell growth is shown in FIG. 1. Cell growth increased in proportion to glucose concentration. Glutathione production was highest when the glucose concentration was 3.0%.

2) Effects of Various Nitrogen Sources

The effect of nitrogen sources on glutathione production capacity and cell growth is shown in Table 2.

TABLE 2

Nitrogen source Glutathione Dry cell weight (mg / L) (%) (g / L) (%) peptone 66.4 100.0 4.60 100.0 Trypton 67.4 101.5 5.80 126.1 Yeast extract 88.4 133.1 5.63 121.7 Malt extract 46.2 69.6 4.85 106.5 Beef extract 44.4 66.9 3.77 82.6 Casein 36.0 54.2 3.94 84.8 Soybean Mill 64.2 96.7 4.53 97.8 NaNO ₃ 41.2 62.0 4.28 93.5 NH ₄ Cl 37.2 56.0 3.83 82.6 (NH ₄ ) ₂ SO ₄ 26.2 39.5 2.91 63.0

The medium contained 3.0 wt% glucose and was individually supplemented with 1.0 weeks wt% of various nitrogen sources. Yeast extract and tryptone were the best sources of nitrogen for glutathione production and cell growth by FF-8. Thus, yeast extract was chosen as the nitrogen source for further experiments. Glutathione production capacity and cell growth were highest when the concentration of yeast extract was 3.0 wt% and 4.0 wt%, respectively (see FIG. 2).

3) Effects of various salt sources

The effects of various salt sources on glutathione production capacity and cell growth are shown in Table 3.

TABLE 3

Salt source Glutathione Dry cell weight (mg / L) (%) (g / L) (%) Control 127.0 100.0 8.30 100.0 MgSO ₄ 135.8 106.9 8.30 100.0 K ₂ HPO ₄ 140.0 110.2 8.60 103.6 KH ₂ PO ₄ 144.8 114.0 8.68 104.8 NaCl 130.2 102.5 8.73 104.8 CaCl ₂ 128.6 101.3 8.73 104.8 FeSO ₄ 126.4 99.5 8.53 102.4 ZnSO ₄ 143.2 112.8 8.85 107.2 MnSO ₄ 140.0 110.2 8.73 104.8

To confirm the effect of the salt, 3.0% by weight glucose and 3.0% by weight yeast extract were contained in the medium and the salts were individually supplemented with 0.05% by weight. All salts except FeSO ₄ promoted glutathione production and cell growth compared to salt free medium. As KH ₂ PO ₄ was found to be the optimal salt source in terms of glutathione production capacity and cell growth, it was selected for further experiments. KH ₂ PO ₄ was found to be effective for FF-8 cell growth and glutathione production at a concentration of 0.06 wt% (see FIG. 3).

4) Effects of various amino acid sources

The effects of amino acid sources on glutathione production capacity and cell growth are shown in Table 4.

TABLE 4

Amino acid source Glutathione Dry cell weight (mg / L) (%) (g / L) (%) Control 158.0 100.0 8.40 100.0 L-glutamic acid 139.0 88.0 8.15 97.6 L-cysteine 200.4 126.8 8.28 98.8 Glycine 132.8 84.1 7.43 88.1 L-methionine 150.0 94.9 7.98 92.9 L-cystine 140.6 88.9 8.40 100.0 Taurine 134.0 84.8 7.90 94.0

The medium used for this experiment contained 3.0 wt% glucose, 3.0 wt% yeast extract and 0.06 wt% KH ₂ PO ₄ , and was supplied with an amino acid source of 0.05 wt%.

These results show that the addition of amino acid precursors and ATP can increase extracellular glutathione accumulation. L-cysteine, the amino acid precursor of glutathione, has been found to be the optimal amino acid for glutathione production. When the concentration of L-cysteine was 0.06% by weight, glutathin production capacity and cell growth peaked and decreased below that.

From these results, the optimal media composition for glutathione production by FF-8 was 3.0 wt% glucose as carbon source, 3.0 wt% yeast extract as nitrogen source, 0.06 wt% KH ₂ PO ₄ as salt source and L as amino acid precursor of glutathione. -Cysteine 0.06% by weight.

TABLE 5

Yeast Nutritional Supplement Concentration (% by weight) Glucose 3.0 Yeast extract 3.0 KH ₂ PO ₄ 0.06 L-cysteine 0.06

From these results, the concentration of glutathione as a result of using a yeast nutrient for fermentation yeast medium consisting of 3.0% by weight glucose, 3.0% by weight yeast extract, 0.06% by weight KH ₂ PO ₄ and 0.06% by weight L-cysteine, was used. Reached 204 mg / L, which was far superior to conventional results.

In the present invention, the culture medium of Saccharomyces cerevisiae FF-8, consisting of 3.0% by weight of glucose, 3.0% by weight of yeast extract, 0.06% by weight of KH ₂ PO ₄ and 0.06% by weight of L-cysteine By adding a yeast nutrient, the glutathione production capacity and efficiency of the FF-8 can be dramatically improved, thereby making it possible to contribute to the development in the fermentation industry using FF-8 as a fermentation yeast strain.

Claims (3)

To improve the glutathione production capacity and efficiency of Saccharomyces cerevisiae FF-8 (KACC93023P), a fermented yeast of traditional liquor, 3.0% by weight of glucose as carbon source, 3.0% by weight of yeast extract as nitrogen source, salt A yeast nutrient for culture medium of fermented yeast consisting of 0.06% by weight of KH ₂ PO _{4 as a} source and 0.06% by weight of L-cysteine as an amino acid source. delete delete

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