KR20120127153A - Yeast Strain Expressing Glutathione Highly and Use thereof - Google Patents

Abstract

PURPOSE: A yeast strain which highly expresses glutathione(GSH) is provided to improve liver function activity and to immunity and to enhance productivity of cultured fishes. CONSTITUTION: A BK-34(KCTC 11635BP) yeast strain highly expresses glutathione. A method for producing yeast containing high content of glutathione comprises a step of culturing BK-34(KCTC 11635BP) strain using a medium containing 0.5-4% of carbon sources and 5-20% of nitrogen source at 25-35 Deg. C., 100-600 rpm, and pH 4.5-5.5 for 12-20 hours. The carbon source is glucose or molasses. The nitrogen source is yeast extract or corn steep liquor(CSL). The method also comprises a step of adding 0.03-0.06% of amino acids such as L-cysteine, glycine, L-glutamic acid, L-methionine, or taurin. A feed additive composition for fishes contains the yeast as an active ingredient.

Description

Yeast Strain Expressing Glutathione and Its Use {Yeast Strain Expressing Glutathione Highly and Use}

The present invention relates to a yeast strain that expresses glutathione (GSH), and more particularly to a BK-34 strain (KCTC 11635BP) strain that expresses glutathione.

Glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) was isolated from yeast by Hopkins in 1929 and is a tripeptide consisting of three amino acids such as glutamic acid, cysteine and glycine. It accounts for 90% of the total nonproteinaceous part of the cell. Glutathione is mostly present in reduced form and is present in small amounts as GSS-protein (PrSSG) and glutathione reductase (GR) mixed with oxidized and disulfide.

Glutathione is a feed additive for fish, and has been used steadily to improve liver function and immunity. However, existing products currently on sale are too expensive to supply as much as necessary.

The present invention contributes to the development of the aquaculture industry by improving the health and productivity of aquaculture fish through low-cost supply of glutathione required by the aquaculture industry through low-cost production of yeast containing high concentration glutathione. The purpose.

 The present invention relates to a yeast strain that expresses glutathione (GSH), and more particularly to a BK-34 strain (KCTC 11635BP) strain that expresses glutathione.

The BK-34 strain was selected as the yeast strain having the highest yield of glutathione (GSH) by the present inventors among several yeast strains isolated from yeast, fermented food, traditional liquor, etc. It was deposited on February 8 (Accession No .: KCTC 11635BP).

In addition, the present invention relates to a method for producing a yeast comprising a high content of glutathione (GSH) culturing the BK-34 strain using a nutrient medium comprising a carbon source and a nitrogen source.

In the present invention, the carbon source is the most important element in the microbial medium, and in general, when it is necessary to control the fermentation process, monosaccharides including glucose are mainly used, but most commercial uses do not use glucose or sugar for economic reasons. Cheap starch and molasses are often used. In the present invention, glucose (Glucose) or molasses (Molasses) may be preferred as a carbon source.

In the present invention, the nitrogen source is required for synthesis of various amino acids as a material for protein synthesis. In large scale processes, ammonium salts, urea, ammonia gas and CSL are used as nitrogen sources. In the present invention, it may be preferable to use a yeast extract (Yeast extract) or corn steep liquor (CSL) as a nitrogen source.

The carbon source and the nitrogen source are not particularly limited in the concentration contained in the medium, it is preferable to include a carbon source 0.5 to 4% concentration in a nitrogen source 5 to 20% concentration, most preferably 4% carbon source at a concentration of 15% nitrogen source It may be included in a concentration.

In addition, the present invention may further add amino acids during the process of culturing the yeast in order to further increase the production of glutathione (GSH).

The amino acid is not particularly limited, but is composed of L-cysteine, Glycine, L-glutamic acid, L-methionine, and Taurin. It is preferably one selected from the group, more preferably L-cysteine (L-cysteine), glycine (Glycine) may be.

In the present invention, the amino acid is not particularly limited in addition time, in order to maximize the production of glutathione is preferably added 4 to 18 hours after the culture, more preferably 12 hours after the culture.

In addition, the addition amount of the amino acid is not particularly limited, but may be preferably added at a concentration of 0.03 to 0.06%, more preferably at a concentration of 0.05%.

Method for producing a yeast containing a high content of glutathione (GSH) of the present invention, in order to maximize the production of glutathione, incubation temperature 25 ~ 35 ℃, stirring speed 100 ~ 600 rpm, initial pH 4.5 ~ 5.5 and incubation time 12 It is preferable to incubate at a culture condition of 120 hours, more preferably, the culture temperature of 30 ℃, agitation speed 100 rpm, the initial pH 5 and the culture time 72 hours culture conditions.

Another aspect of the present invention relates to a yeast comprising a high content of glutathione produced by the above culturing method. The yeast of the present invention contains glutathione in high content, and vitamins and amino acids are contained in the yeast itself, which reduces the stress of the fish, and is characterized by an excellent effect of improving the liver function and immune enhancement of the fish.

Another aspect of the invention relates to a feed additive composition for fish comprising a yeast comprising a high content of glutathione as an active ingredient. The feed additive composition for fish may include a yeast containing a high content of glutathione alone, or may be included with various nutrients such as vitamins, amino acids, minerals, antioxidants, antibiotics, antibacterial agents, and other additives. have. As the shape of the fish feed additive of the present invention, it may be prepared in a suitable state such as powder, granules, pellets, suspension, and the like. When the feed additive of the present invention is supplied, it may be supplied alone to the fish or mixed in the feed. May be supplied.

According to the present invention, since it contains glutathione (GSH) in a high content, it can significantly improve liver function and immunity of fish. Especially, yeast containing expensive glutathione, which has been dependent on imports, can be manufactured at low cost by domestic technology. It can be supplied in large quantities, and it is possible to supply enough to fish farms, thereby improving the health and productivity of farmed fish, and contributing to the development of domestic aquaculture industry.

1 shows BK-34 and Saccharomycetes sp. (Refer to the BK-34 shown in the drawing is a model at the time of testing, and since the model name was changed to BK-34 at the time of deposit, the BK-34 of FIG. 1 and the present invention) BK-34 is the same).
Figure 2 shows a graph of the dry weight and glutathione production of BK-34 over the incubation time.
Figure 3 shows a graph of the dry weight and glutathione production of BK-34 according to the culture temperature.
Figure 4 shows a graph of the dry weight and glutathione production of BK-34 according to the stirring speed.
Figure 5 shows a graph of the dry weight and glutathione production of BK-34 according to the initial pH.
Figure 6 shows a graph of the dry weight and glutathione production of BK-34 according to the concentration of Glucose and molasses (molasses).
Figure 7 shows a graph of the dry weight and glutathione production of BK-34 according to the concentration of Yeast extract and Corn steep liquor.
Figure 8 shows a graph of the dry weight and glutathione production of BK-34 according to the concentration of glucose for the optimization of carbon and nitrogen sources.
Figure 9 shows a graph of the dry weight and glutathione production of BK-34 according to the Permanent batch culture.
Figure 10 shows a graph of the dry weight and glutathione production amount of BK-34 according to the incubation time of one embodiment according to the present invention.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely examples for describing the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Experimental Method

1-1. Strain screening

Separators for separating yeast strains were used to purchase commercial yeast, fermented food, traditional liquor and the like. Nuruk was added to 2 g of Nuruk by adding 18 ml of brine and ground using a mixer. The fermented food and traditional liquor were directly used as the stock solution.

Pure strains of yeast strains were separated by spread-plate and streak-plate methods. Serial dilution with saline and spreading on YM Agar (Yeast extract 3.0 g, Malt extract 3.0 g, Peptone 5.0 g, Glucose 10 g, Agar 20 g) and incubated for 48 hours at 30 ℃. Single colony estimated as yeast by colony shape and microscopy was streaking to YM agar. And all the isolates were stored at -70 ℃ using YM broth (Yeast extract 3.0 g, Malt extract 3.0 g, Peptone 5.0 g, Glucose 10 g) added 30% glycerol.

Screening of strains was performed by injecting 100 ml of YM broth into a 500 ml Erlenmeyer flask and inoculating a loop of purely isolated strains with shaking culture (30 ℃, 100 rpm) for 72 hours to investigate dry weight and GSH production.

1-2. Selection of excellent strains

Selection of excellent strains was carried out by regenerating 10 kinds of strains with excellent GSH production in yeast strains stored. 100 ml each of YM broth was inoculated into a 500 ml shake Erlenmeyer flask, and the inoculated strain was inoculated at 2% for preculture (30 ° C., 100 rpm) for 24 hours. And inoculated 2% of the pre-culture solution in the shaking Erlenmeyer flasks prepared in the same manner and incubated for 72 hours under the same conditions, the dry weight and GSH production was investigated.

Yeast strains with the best GSH production in the selection culture were commissioned by Solgent Co., Ltd. to analyze the ITS1 sequence. 16S ribosomal RNA sequences of the analyzed strain were compared with similarity using BLAST database (http: //www.ncbi.nlm.nih.gob/blast/) and EMBOSS Align (www.ebi.ac.uk/). It was.

1-3. Initial culture condition

Incubation time, incubation temperature (25, 30, 35, 40 ℃), stirring speed (0, 50, 100, 150, 200 rpm), initial pH (2, 3, 4, 5, 6) , 7, 8), etc., were examined for dry weight and GSH production.

1-4. Badge optimization

Most microorganisms use carbohydrates as carbon or energy sources as chemically synthesized heterotrophs. Carbon sources are the most important element in microbial media. In general, when it is necessary to control the fermentation process, glucose and other monosaccharides are mainly used. However, most commercial applications use cheap starch and molasses without economical glucose or sugar.

Most microorganisms grow using organic and inorganic nitrogen sources. Nitrogen sources are required for the synthesis of various amino acids as the material for protein synthesis. In large scale processes, ammonium salts, urea, ammonia gas and CSL are used as nitrogen sources. Some microorganisms do not grow or become very slow in their amino acid requirements without one or more specific amino acids. In this case, the specific amino acid required should be added.

① Carbon source selection

In this example, glucose and molasses were used as the carbon source for the mass culture of the high concentration of GSH-containing yeast. The molasses was heat-treated at 105 ° C. for 30 minutes as a pretreatment process, and then used for 15 minutes at 7000 rpm. Carbon source selection experiments were performed by YM broth without glucose, Yeast extract 0.3%, Malt extract 0.3%, Peptone 0.5%, glucose at 1% intervals in the range of 1-5%, molasses in the range of 2-10% The experiment was performed by adding at intervals.

② Nitrogen source selection

In this embodiment, among the nitrogen sources containing amino acids or peptides and used as the organic nitrogen source, Yeast extract and corn steep liquor (CSL), which are organic nitrogen sources mainly used for mass processing, were used. CSL was used by centrifugation at 7,000 rpm for 15 minutes as a pretreatment process. Nitrogen source concentration was determined by adding yeast extract at 2.5% interval from 0.5% to 15.0% and CSL at 1.0% to 30.0% at 5.0% interval. Glucose 1.0% was added to the medium as a carbon source.

③ Optimization of carbon and nitrogen sources

Glucose was added at concentrations of 0%, 0.1%, 1.0%, 2.0%, 4.0%, 6.0%, 8.0%, and 10%, respectively, to investigate the blending ratio of the carbon and nitrogen sources that produced the maximum GSH. At this time, the nitrogen source CSL with the highest increase in GSH production was fixed at a concentration of 15%.

④ Amino Acid Optimization

In this example, L-glutamic acid, L-cysteine, Glycine, L-methionine and Taurin were tested to investigate the amino acids required for GSH yeast culture. Each amino acid addition ratio was added to each concentration of 0.05% to perform the experiment.

1-5. Investigation of Glutathione (GSH) Production According to Cysteine Administration Time

In order to investigate the production of GSH according to the addition time of cysteine, L-Cysteine (0.1%) was administered at different times (culture 4, 8, 12, 24, 48 hours) to investigate the production of GSH. The medium was used by adding glucose 4%, CSL 15%, Glycine and L-Cysteine at a concentration of 0.05%.

1-6. Fermenter batch culture

In order to investigate the dry weight of yeast (BK-34, Accession No .: KCTC 11635BP) and the production amount of GSH according to the culture apparatus, batch culture was performed using a 5 L fermenter. The volume of the optimum medium was 2 L, and the culture conditions were set to initial pH 5.0, culture temperature 30 ℃, stirring speed 200 rpm, air supply 1.5 L / min. Samples were inoculated at 2% preculture and taken at 8 hour intervals for 72 hours to investigate dry weight and GSH production.

1-7. Dry weight of yeast

The recovery of each experiment was investigated by measuring dry weight. In a flask cultured for 72 hours, 1 ml each was sampled into a microtube, centrifuged at 13,000 rpm for 3 minutes, washed three times with distilled water, dried in a drying oven (60 ° C.), and weighed with a precision scale. Was measured.

1-8. GSH content analysis

GSH content analysis according to each experiment to wash the cultured sample three times to remove the media components, the content of ultrapure water was added, and then extracted GSH by boiling in hot water for 15 minutes at 95 ℃. The extracted sample was stored at -70 ° C until measurement. GSH analysis was measured using Sigma's Glutathione assay kit (CS0260).

2. Experimental results

2-1. Strain screening

A total of 213 yeasts were isolated from yeast, fermented food, and traditional liquor. As a result of examining the dry weight and GSH production of the isolated yeast, the dry weight of yeast was 1.1-6.2 g / L, the GSH production was 0.3-58.1 mg / L, and the GSH content was 0.2-18.5 mg / g. Among the isolated strains, the dry weight was the best with BK-26 strain (6.2 g / L) and the highest yield with 58.1 mg / L (BK-05 strain). GSH content of BK-28 was the highest at 18.5 mg / g.

Frozen storage strains were regenerated and used in experiments, there was a difference in the activity of the strain and the production capacity of GSH. Therefore, excellent strain selection tests were performed on 10 strains with high GSH content in the isolated yeast.

2-2. Selection of excellent strains

In the first screening, 10 strains with excellent GSH production were recovered and pre-cultured, and YC broth was inoculated with the pre-incubation solution. The yeast dry weight of BK-43 and BK-34 was the best at 3.3 g / L. The production and content of, GSH were the highest at 42.5 mg / L for BK-34.

16S ribosomal RNA sequences of the experimental strain were compared with BLAST database and EMBOSS Align . Saccharomycetes sp. It showed 97.8% similarity with HZ178 18S ribosomal RNA gene [GU213443] (FIG. 1).

The present inventors deposited the BK-34 strain on February 8, 2010 with the Korea Research Institute of Bioscience and Biotechnology (Accession Number: KCTC 11635BP).

2-3. Initial culture condition

① Culture time

The dry weight of BK-34 and the production of GSH according to the incubation time were investigated up to 120 hours at 12 hour intervals. The dry weight of the cells rapidly grew to 2.1 g / L at 12 hours of cultivation, and gradually increased from 24 hours to 4.0 g / L at 120 hours. The production of GSH was rapidly increased to 37.0 mg / L at 36 hours of culture, and then increased from 48 hours to 48.0 mg / L at 120 hours (FIG. 2).

② Culture temperature

To investigate the dry weight and GSH production according to the culture temperature of BK-34, 2% inoculation of YM broth was incubated for 72 hours at 25, 30, 35, and 40 ° C. The dry weight was the highest at 2.9 g / L and the production of GSH was 36.6 mg / L. Dry weight and GSH production at 25 ° C and 35 ° C were 2.0 g / L and 27.4 mg / L, 2.0 g / L and 32.7 mg / L, respectively. Therefore, the highest growth of BK-34 and production of GSH was set to 30 ℃ culture temperature (Fig. 3).

③ Stirring Speed

The dry weight and GSH production of BK-34 according to the stirring speed were investigated.The result was 1.2 g / L (12.7 mg / L) at 0 rpm, 3.7 g / L (42.4 mg / L) at 100 rpm and 2.2 at 200 rpm. g / L (40.4 mg / L). BK-34 is the highest dry weight and production of GSH at 100 rpm, the optimum stirring condition was set to 100 rpm (Fig. 4).

④ initial pH

The dry weight of BK-34 and GSH production according to the initial pH were the highest at 4.0 g / L (52.2 mg / L). As the acidity or alkalinity increased at pH 5, the growth and yield decreased, which was 3.1 g / L (26.0 mg / L) at pH 2 and 3.4 g / L (37.4 mg / L) at pH 8 (FIG. 5).

2-4. Badge optimization

① Carbon source selection

To select the carbon source, Glucose and Molasses were added by concentration. The dry weight of BK-34 according to the carbon source concentration was 3.8 ~ 4.2 g / L and 3.9 ~ 6.2 g / L in Glucose and Molasses, respectively. The production of GSH was 45.3 ~ 51.6 mg / L and 40.2 ~ 54.9 mg / L in Glucose and Molasses, respectively. Molasses showed better growth of BK-34 than glucose, but there was no significant difference in the production of GSH. In addition, Molasses was selected for the Glucose as a carbon source for the optimization of the medium, because it requires a pretreatment process such as heat treatment and centrifugation to use as a medium (Fig. 6).

② Nitrogen selection

Yeast extract and CSL were added to each concentration to select nitrogen source. The dry weight of BK-34 was 3.5-5.6 g / L and 4.8-6.4 g / L in Yeast extract and CSL, respectively. The production of GSH was 43.9 ~ 55.2 mg / L and 44.4 ~ 68.7 mg / L, respectively. The dry weight of CSL added group was highest at 6.4 g / L at 0.5% CSL and GSH production was highest at 68.7 mg / L at 15% CSL. In addition, the CSL 15% addition group showed a high value of 6.1 g / L also in the growth of BK-34, so that CSL 15% was selected as the nitrogen source (FIG. 7).

③ Optimization of carbon and nitrogen sources

Optimization of carbon source and nitrogen source of BK-34 was carried out by fixing CSL 15% concentration selected as nitrogen source and adding Glucose selected as carbon source by concentration. Glucose non-additives had a dry weight of 4.4 g / L and increased dry weight by the addition of Glucose. The dry weight was highest at 11.1 g / L in the Glucose 0.5 addition.

The production of GSH of BK-34 was 90.6 mg / L in the non-Glucose group and increased by Glucose addition to 134.1 mg / L in the 4% group. However, GSH production decreased gradually in the addition of glucose more than 6%, the lowest yield was 69.9 mg / L in 10% Glucose addition (Fig. 8).

2-5. Amino acid optimization

The dry weight of BK-34 and the production of GSH were investigated with the addition of amino acids. The dry weight and the production of GSH of the control without the amino acid were 8.6 g / L and 87.1 mg / L, respectively. The growth of BK-50 was increased by amino acid addition, the highest at 12.4 g / L in the Glycine supplementation. The production of GSH by amino acid addition was 91.8 mg / L and 92.2 mg / L in L-cysteine and Glycine supplemented groups, respectively.

2-6. Amino Acid Dosing Time

In order to improve the production of GSH, the amino acid L-cysteine was administered during the experiment. The dry weight and GSH production of the control group were determined to be 7.6 g / L and 90.0 mg / L, respectively.

The dry weight by L-cysteine addition was 7.8 g / L in the experimental group added at 12 hours of incubation. The dry weight was lower than that of the control in all experimental groups except this one, and the lowest was 5.8 g / L in the experimental group added after 4 hours of incubation.

In the production of GSH, all of the experimental groups containing cysteine showed higher yield than the control, and the highest concentration of 128.8 mg / L was added to the experimental group after 12 hours of incubation.

2-7. Fermenter batch culture

The dry weight and GSH production according to the cultivation apparatus were investigated using optimized conditions and media for the basic investigation for scale-up. As a result of batch culture of BK-34 in a 5 L fermenter, the dry weight increased to 8.7 g / L after 8 hours of incubation, and continued to grow to 28.5 g / L at 72 hours after the end of the experiment. . On the other hand, the production of GSH increased to 76.5 mg / L at 40 hours of culture but decreased to 62.2 mg / L at 48 hours of culture, the value was maintained for 72 hours (Fig. 9).

Example 1

The medium was prepared by preparing a medium such as Glucose 4%, CSL 15%, L-cysteine 0.05%, Glycine 0.05%, and adjusting the pH to 5.0 with 4N HCl or 4N NaOH. BK-34 was inoculated in this medium at a concentration of 2% and precultured for 24 hours. BK-34 preculture was inoculated in 2 L of the same medium at a concentration of 2% and cultured using a 5 L fermenter for 48 hours at a culture temperature of 30 ° C., DO 20%, agitation speed of 200 to 600 rpm, and air supply of 1 vvm. . Cysteine 2 mM was added at 4 hours of culture, and L-glutamic acid, Glycine and L-cysteine were added at a concentration of 10 mM at 12 hours of culture.

As a result of culturing as described above, yeast containing 1.47% GSH was obtained at 20 hours of culture. (DCW 42.1 g / L, GSH 624.5 mg / L) (FIG. 10)

Korea Biotechnology Research Institute KCTC11635BP 20100208

Claims (12)

BK-34 (KCTC 11635BP) strain that expresses glutathione. A method for producing a yeast comprising glutathione having a high content of culturing the strain of claim 1 using a nutrient medium comprising a carbon source and a nitrogen source. The method of claim 2,
The carbon source is characterized in that the glucose (Glucose) or molasses (Molasses). The method of claim 2,
The nitrogen source is a yeast extract (Yeast extract) or corn steep liquor (corn steep liquor, CSL) characterized in that. The method of claim 2,
The carbon source and the nitrogen source are 0.5 to 4% of the carbon source at a concentration of 5 to 20% of the nitrogen source. Method comprising a concentration. The method of claim 2,
And further adding amino acids. The method according to claim 6,
The amino acid is characterized in that any one selected from the group consisting of L-cysteine (L-cysteine), glycine (Glycine), L-glutamic acid (L-glutamic acid), L-methionine (L-methionine) and Taurin (Taurin) How to. The method according to claim 6,
The amino acid is characterized in that the addition of 4 to 18 hours after incubation. The method according to claim 6,
The amino acid is added at a concentration of 0.03 to 0.06%. The method of claim 2,
Culture temperature 25 ~ 35 ℃, stirring speed 100 ~ 600 rpm, initial pH 4.5 ~ 5.5, incubation time 12 to 120 hours culturing in culture conditions. A yeast comprising a high content of glutathione produced by any one of claims 2 to 10. Feed additive composition for fish comprising the yeast of claim 11 as an active ingredient.

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