MICROORGANISM WITH HIGH TRIPEPTIDE PRODUCTIVITY AND USE THEREOF

Abstract

The present invention relates to a Candida utilis strain having high tripeptide-producing ability and/or alcohol lyase activity and its use.

Claims

1. A Candida utilis strain having alcohol dehydrogenase (ADH) activity and alcohol tolerance, and glutathione production ability.

2. The strain according to claim 1, wherein the strain has an ADH activity of 0.18 mU/ml or higher per cell dry weight (g), and a glutathione production of 0.8% by weight or more per cell dry weight (g).

3. The strain according to claim 1, wherein the strain has an alcohol-tolerance being capable of growing under the condition of ethanol concentration of 2 to 15% (v/v).

4. The strain according to claim 3, wherein the strain has an optical density (OD) value of 120 to 200%, based on 100% of an optical density (OD) value for Candida utilis deposited with an accession number of KCCM 11355, when cultured under the condition of an alcohol concentration of 6 to 15% (v/v).

5. The strain according to claim 1, wherein the Candida utilis strain has accession number of KCCM 12777P.

6. A composition for a composition for preventing, improving or treating oxidative stress-related diseases, comprising at least one selected from the group consisting of a cell of the Candida utilis strain of claim 1, a culture of the strain, a lysate of the cell, a disruption of the cell, and an extract thereof.

7. The composition of claim 6, wherein the oxidative stress-related diseases are selected from the group consisting of AIDS, diabetes mellitus, macular degeneration, congestive heart failure, cardiovascular disease, coronary artery restenosis, lung disease, inflammatory disease, asthma, RNA virus infection, DNA virus infection, sepsis, osteoporosis, bone disease, microbial infection, toxin exposure, radiation exposure, burn trauma, prion disease, neurological disease, blood disease, blood corpuscle disease, arterial disease and muscle disease.

8. A composition for decomposing alcohol, comprising at least one selected from the group consisting of a cell of the Candida utilis strain of claim 1, a culture of the strain, a lysate of the cell, a disruption of the cell, and an extract thereof.

9. The composition for decomposing alcohol according to claim 8, wherein the strain has an alcohol dehydrogenase (ADH) activity of 0.18 mU/ml or more per cell dry weight (g).

10. A composition for relieving, improving, alleviating or preventing a hangover, comprising at least one selected from the group consisting of a cell of the Candida utilis strain of claim 1, a culture of the strain, a lysate of the cell, a disruption of the cell, and an extract thereof.

11. A method for increasing glutathione production by culturing the Candida utilis strain according to claim 1.

12. The method of claim 11, wherein the culturing the strain is performed by using sucrose or glucose as a carbon source.

13. The method of claim 11, wherein the culturing of strain is performed by adjusting a carbon source supply rate with measuring an ethanol content in fermentation solution.

14. The method according to claim 11, wherein the strain is cultured with adding cysteine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1 is a graph showing glutathione productivity of candidate strains primarily selected according to the present invention.

[0057] FIG. 2 is a graph showing the concentration of NADH produced per unit time of the candidate strains primarily selected according to the present invention.

[0058] FIG. 3 is a graph showing a change rate of glutathione content depending on the sucrose supplying rate according to the present invention.

[0059] FIG. 4 is a graph showing an ethanol content in the culture medium depending on the sucrose supplying rate according to the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0060] The present invention will be described in more detail with reference to the following examples, but the present invention is not intended to be limited to the following examples.

Example 1: Isolation of Microorganism Producing Glutathione

1-1: Microorganism Culture

[0061] To screen microorganisms producing glutathione, rice wine (Makgeolli) koji, and traditional sources were purchased at traditional markets across the country and were used them as samples. 1 g of the sample was suspended in 10 mL of 0.85% NaCl, and 100 l of the suspension was plated on a YPD (Yeast extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L) agar plate and the solid culture was performed at 30 C. for 2 days. Among the colonies grown on the solid medium, 150 colonies were isolated by selecting those with different shapes and sizes, and then cultured at 30 C. for 2 days in a test tube containing YPD broth (yeast extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L) with agitation, to obtain the cell culture.

1-2: Measurement of Cell Growth Degree (Absorbance)

[0062] The cell concentration was determined by measuring the absorbance at 600 nm for the cell culture, and the result was represented as the cell optical density (OD) value.

1-3: Measurement of Glutathione Content in the Culture

[0063] The cell culture solution was centrifuged to remove the supernatant, and the microbial cells were collected by washing with distilled water once. The collected cells were added with 4070% ethanol, and the intracellular glutathione was extracted using a fine mixer for 1030 minutes. After centrifugation of the extraction solution, the supernatant was taken and reacted with 10 mM DTNB (5,5-Dithiobis-(2-Nitrobenzoic Acid) dissolved in 0.5 M potassium phosphate at pH8.0 buffer for 20 min at 40 C., and the glutathione content (GSH mg/L) was determined by measuring the absorbance at 412 nm. DTNB, commonly used for glutathione content analysis, is known as Ellman's reagent designed to detect thiol compounds. The reaction of GSH with DTNB produces y2-nitro-5-benzoic acid and GSSG, and the concentration of GSH can be calculated by measuring the OD value at 412 nm. GSH is prepared from GSSG by glutathione reductase, and reacts with DTNB again, to form a recycling system.

1-4: Measurement of Glutathione Content Per Cell Dry Weight (g)

[0064] The cell culture solution was centrifuged to remove the supernatant, and the microbial cells were collected by washing with distilled water once. The absorbance was measured for the collected cells, and the dried cell weight was calculated according to the absorbance. Specifically, in order to measure the cell dry weight (g), the cell culture solution was centrifuged to remove the supernatant, and the microbial cells were collected only. The collected cells were washed with 0.9% NaCl and diluted with distilled water to prepare samples with 0.1 to 1 of absorbance.

[0065] The absorbance was measured at 600 nm for the diluted cell samples, and the dry cell weight was calculated according to the absorbance. After measuring the absorbance, the cells were filtered under reduced pressure with a 0.2 m filter paper. The filter paper with cells remaining was dried at 60 C. for more than 12 hours, left in a desiccator containing silica gel for more than 6 hours, and then the cell weight was measured. The amount of dried cells was determined by calculating the weight difference between the empty filter paper and the filter paper with the cells filtered out. Therefore, it was possible to determine the dry cell concentration (g/L) according to the absorbance value.

[0066] After measuring the absorbance, glutathione was extracted from the cells in substantially the same manner as in Examples 1-3, and the extraction solution was centrifuged to take the supernatant, and then the produced amount of glutathione (g/L) was measured. The measured amount of glutathione was divided by the calculated dry cell weight (g/L) and then multiplied by 100 to calculate GSH % per dry cell weight (g). The glutathione content per dry cell weight (g) was measured, and shown in Table 1 as GSH (%)/g-cell.

[0067] As a result of measuring the glutathione productivity of 150 strains by the method, the glutathione production per cell dry weight (g) (GSH (%)/g-cell) ranges about 0.3 to 2% by weight. The top seven (70 candidate strains were isolated based on the glutathione content per sugar. As a result of the analysis of the selected 7 strains, the cell OD of Example 1-2, the glutathione content of Example 1-3, and the glutathione content per cell dry weight (g) are shown in Table 1, and the cell dry weight (g) Glutathione content per sugar is shown in FIG. 1.

[0068] As a control, the same experiment was performed on the standard strain of C. utilis KCCM 11355, and the results are shown in Table 1 and FIG. 1.

TABLE-US-00001 TABLE 1 glutathione glutathione Cell content productivity Sample growth(O.D) (GSH mg/L) GSH(%)/g-cell Control group 18.0 46 0.6 SYC-A 18.0 49 0.7 SYC-7D 14.3 74 1.3 SYC-P1 19.9 73 0.9 SYC-P3 17.5 135 1.9 SYC-JH 20.9 65 0.8 SYC-PR9 18.0 112 1.6 SYC-PR19 17.0 110 1.6 SYC-PR20 17.8 112 1.6

[0069] As shown in Table 1, as a result of analyzing cell growth and glutathione production in an ethanol-free medium, the glutathione contents of the seven selected strains were higher than that of the standard strain C. utilis KCCM 11355, specifically about 0.7 to 2% by weight. The six strains except for SYC-7D strain, had the same or higher degree of cell growth than that of the standard strain, and thus had more desirable characteristics.

[0070] Therefore, considering glutathione production (mg/L) and glutathione production per dry cell weight (GSH (%)/g-cell) as strain selection criteria, SYC-7D, SYC-JH, SYC-P1, SYC-P3, SYC-PR9, SYC-PR19, and SYC-PR20 could be selected. Preferably SYC-7D, SYC-JH, SYC-P1, SYC-P3, SYC-PR9, SYC-PR19, and SYC-PR20 having 0.8% by weight or more of the glutathione production per cell dry weight (g) (GSH (%)/g-cell), or more preferably, SYC-PR9, SYC-PR19, and SYC-PR20 having 1.5% by weight or more of the glutathione production per cell dry weight (g) (GSH (%)/g-cell) could be selected.

[0071] In particular, when considering industrial mass production, it is more preferable to consider both the cell OD value and the produced amount of glutathione per dry cell weight (GSH (%)/g-cell). From this point of view, it was confirmed that the strains SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20 having higher cell OD values and higher glutathione production than the standard strains were more preferable.

Example 2: Alcohol Dehydrogenase (ADH) Activity Assay

[0072] The ADH activity was measured by culturing the seven (7) strains with high glutathione content selected in Example 1.

[0073] Specifically, the ADH activity was analyzed using an ADH Activity Assay Kit (Abcam). After culturing for 24 to 48 hours at 30 C. in YPD medium, the cells were collected to 110.sup.6 CFU/ml. The collected cells were washed with distilled water, and then were added with ADH assay buffer, to disrupt the cell walls using a bead beater. As the composition of the reaction solution, 50 l of sample or NADH standard material was mixed with 82 l of ADH assay buffer, 8 l of Developer, and 10 l of Isopropanol for each concentration. After reacting at 37 C. for 3 minutes, the absorbance of the experimental group (A0) and the control group was measured at 450 nm. After an additional reaction at 37 C. for 30 minutes, the change in absorbance at 450 nm was measured, and the amount of NADH produced per unit time (minute) was calculated to compare the ADH activity of each strain. The amount of NADH production for each strain measured is shown in FIG. 2. It was confirmed that the 7 strains had NADH production in the range of 24 to 160 nmol. As a control, the same experiment was performed for the standard strain of C. utilis KCCM 11355, and the results are shown in Table 2.

TABLE-US-00002 TABLE 2 sample ADH Activity (mU/ml) Control group 0.16 SYC-A 0.14 SYC-7D 0.08 SYC-P1 0.24 SYC-P3 0.03 SYC-JH 0.04 SYC-PR9 0.22 SYC-PR19 0.18 SYC-PR20 0.26

[0074] As shown in Table 2, the ADH activity of the 7 selected strains ranged from 0.03 to 0.26 mu/ml, and the strains having higher ADH activity that that of the standard strain C. utilis KCCM 11355 (control) were SYC-P1, SYC-PR9, SYC-PR19 and SYC-PR20, and the strain showing the highest activity was identified as SYC-PR20. Accordingly, in terms of ADH activity, SYC-P1, SYC-PR9, SYC-PR19, and SYC-PR20 could be selected secondarily. Preferably, SYC-P1, SYC-PR9, and SYC-PR20 having an ADH activity of 0.20 (mU/ml) or more could be selected. In consideration of combining the glutathione content per dry cell weight (g) shown in Table 1 of Example 1, and the ADH activity analysis results in Table 2, the strains with high glutathione production and ADH activity are preferred. Accordingly, SYC-PR9, SYC-PR19, and SYC-PR20 can be selected as secondary candidate strains.

Example 3: Evaluation of Cell Growth and GSH Production Depending on Culture Time

[0075] For SYC-PR20 selected in Example 1, cell growth and GSH production were evaluated according to the culture time.

[0076] YPD (yeast extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L) medium was prepared as a medium for culturing the SYC-PR20 strain. 3 ml of medium was dispensed into a test tube, and the culture was performed for a culture time of 24 to 60 hours at 30 C. with agitation. The culture solution was taken at 24 hours, 36 hours, 48 hours, and 60 hours, respectively, and cell OD value, glutathione content, and Glutathione content per cell dry weight (g) were measured, and the results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Glutathione Culture glutathione content productivity time(hour) Cell growth(O.D) (GSH mg/L) GSH(%)/g-cell 24 12 69 1.4 36 15 82 1.4 48 18 111 1.6 60 19 120 1.6

[0077] As a result of measuring the cell O.D value and GSH production from 24 hours to 60 hours of incubation time of the strain, the cell O.D increased from 12 at 24 hours, 15 at 36 hours, 18 at 48 hours, and 19 at 60 hours. It was confirmed that the production of GSH was 69 at 24 hours, 82 at 36 hours, 111 at 48 hours, and 120 mg/L at 60 hours. Therefore, it was confirmed that the GSH % per g of cell dry weight increased from 1.4% at 24 hours to 1.6% at 60 hours.

Example 4: Evaluation of Microorganism's Tolerance to Ethanol

[0078] For SYC-PR20 selected in Example 1, cell growth was evaluated in an ethanol-containing medium.

[0079] Specifically, a 5YPD (yeast extract 50 g/L, Peptone 100 g/L, Dextrose 100 g/L) medium was prepared and mixed with 100% ethanol to make the final ethanol concentration in the medium to be 0, 2, 4, 6, 8, 10, or 15 v/v %. 3 ml of the medium containing ethanol for each concentration was dispensed into a test tube and cultured with shaking at 30 C. for 60 hours to obtain culture solution. For the culture solution, the cell concentration was analyzed by measuring the absorbance at 600 nm in substantially the same manner as in Example 1-2, and the results are shown in Table 4 below.

[0080] As a control, the same experiment was performed on the standard strain of C. utilis KCCM 11355, and the results are shown in Table 4.

TABLE-US-00004 TABLE 4 Ethanol(v/v %) 0 2 4 6 8 10 15 OD value of control 18.0 13.9 12.2 9.3 2.8 1.2 0.5 group Relative OD value of 100.0% 77.2% 67.8% 51.7% 15.6% 6.7% 2.8% control group to ethanol-free medium OD value of SYC- 19 15.8 13.6 11.0 4.2 2.1 0.8 PR20 Relative OD value of 100.0% 81.0% 69.7% 56.4% 21.5% 10.8% 4.1% SYC-PR20 to ethanol-free medium Relative OD value of 108.3% 113.7% 111.5% 118.3% 150.0% 175.0% 160.0% SYC-PR20 to control group (%)

[0081] Saccharomyces cerevisiase, known as a strain with high alcohol tolerance, can grow at ethanol concentrations of 7-11% (v/v). The SYC-PR20 strain, which was secondly selected according to Examples 2 and 3, had high alcohol tolerance and, in particular, exhibited high cell growth in an alcohol-containing medium compared to the standard strain of C. utilis KCCM 11355 (control group), particularly high cell growth of 110% or more at an ethanol concentration of 0.5 to 15 v/v %.

[0082] In Table 4, the SYC-PR20 strain having a relatively high ADH activity had a higher cell concentration at the same ethanol concentration than the control group. Accordingly, it was confirmed that the SYC-PR20 strain had excellent glutathione production ability, ADH activity and alcohol tolerance.

Example 5: Strain Identification

[0083] In Example 4, the SYC-PR20 strain showed the most preferable characteristics among the 7 selected strains with a glutathione yield of 1.6% and an ADH activity of 0.26 mU/ml.

[0084] The SYC-PR20 strain was subjected to 18S rRNA sequence analysis by using universal primers ITS1 (SEQ ID NO: 2: 5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (SEQ ID NO: 3: 5-TCCGTAGGTGAACCTGCGG-3). The 18S rDNA sequence of the SYC-PR20 strain is shown in SEQ ID NO: 1. As a result of strain identification based on the 18S rDNA sequence, it was identified as Candida utilis (Pichia jadinii).

[0085] The Candida utilis SYC-PR20 strain was deposited at the Korean Culture Center of Microorganism, located at 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, on Aug. 7, 2020, and received accession number of KCCM 12777P.

Example 6: Evaluation of Glutathione Productivity on Fermenter Culture

[0086] In the flask culture conditions such as Examples 1-4 for the isolation and characterization of microorganisms, it is difficult to increase the agitation speed beyond a certain level due to mechanical limitations, and to control a constant culture environment (air amount, pH, etc.). On the other hand, in industrial scale culture, by supplying air to the culture medium and performing agitation, medium components and oxygen are uniformly distributed in all spaces, and temperature, pH, etc. can be adjusted, thereby enabling cell culturing under optimized conditions. Accordingly, the fermenter culturing shows a higher cell growth rate than the flask culturing, and thus, the consumption of the added sugar occurs quickly and the productivity increases. Therefore, fermenter culturing experiments for industrial application of the microorganism are required.

[0087] Specifically, the colony formed on YPD agar plate inoculated with the Candida utilis SYC-PR20 strain according to Example 5 was inoculated into 3 mL of YPD (Yeast extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L) broth, cultured for 24 hours at a temperature of 30 C. and 240 rpm to obtain the seed culture, and 3 mL of the previously cultured seed culture was inoculated into 100 mL of the same YPD broth and cultured under the same conditions to prepare a seed culture for a 5 L fermenter.

[0088] In order to evaluate cell growth and glutathione production according to carbon sources, 5 L fermenter culturing was performed. Specifically, a glucose-based culture medium and a sucrose-based culture medium were prepared in a final 2 L culture volume in a 5 L fermenter with the medium composition shown in Table 5, respectively. The prepared seed culture for 5 L fermenter culturing was inoculated into a 5 L fermenter and cultured while stirring under the conditions shown in Table 6.

TABLE-US-00005 TABLE 5 glucose-based culture medium sucrose-based culture medium component Content (g/L) component content (g/L) Glucose 30 Sucrose 30 MgSO4-7H2O 0.5 MgSO4-7H2O 0.5 Yeast extract 3.75 Yeast extract 3.75 Corn steep Powder 5.25 Corn steep Powder 5.25 Methionine 4 Methionine 4 (NH4)Cl2 10 (NH4)Cl2 10 KH2PO4 2 KH2PO4 2 K2HPO4 2 K2HPO4 2 NaCl 0.5 NaCl 0.5

TABLE-US-00006 TABLE 6 Primary Culture seed Seed Main condition culture culture culture Volume 3 mL 100 mL 2 L Temperature 30 C. 30 C. 28 C. Agitation speed 240 rpm 240 rpm 500 rpm Culturing pH Not control Not control 5.0 Culturing time 24 hr 24 hr 29 hr

[0089] Cell concentration and glutathione production of the obtained culture solution were measured in substantially the same manner as in Example 1, and the measurement results are shown in Table 7 below.

TABLE-US-00007 TABLE 7 GSH content GSH content (mg/L) on (mg/L) on Cell saccharide saccharide Culturing Culturing O.D.(600 concentration concentration conditions time(hr) nm) of 30 g/L of 1 g/L glucose-based 29.0 28.4 182.3 6.08 culture medium sucrose-based 29.0 31.9 223.3 7.44 culture medium

[0090] As shown in the results of Table 7, the time when the initial input saccharides was almost consumed was the end of the culture at 29 hours for both glucose and sucrose-based cultures, and OD value at 600 nm as the cell concentration at that time was 28.4 for glucose-based culture and 31.9 for sucrose-based culture, to show increased cell yield for sucrose-based culture by 112%. The glutathione content was 223.3 mg/L in the sucrose-based culture, which was about 122% higher than that of the glucose-based culture in the same time culture.

Example 7: Evaluation of Glutathione Production According to Culture Conditions

[0091] The colony formed on the YPD agar plate inoculated with the Candida utilis SYC-PR20 strain according to Example 5 was inoculated into 3 mL of YPD (Yeast extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L) broth, cultured for 24 hours at a temperature of 30 C. and 240 rpm to obtain the seed culture, and 3 mL of the previously cultured seed culture was inoculated into 100 mL of the same YPD broth and cultured under the same conditions to prepare a seed culture for a 5 L fermenter.

[0092] In order to evaluate high-concentration cells and glutathione production, the seed culture and 5 L fermenter culture were prepared as in Example 5. In batch culture, when the accumulated ethanol concentration after initial saccharide consumption was 5 g/L or lower, Fed-batch culture is performed by continuously supplying saccharide solution containing sucrose of 600 g/L to maintain high-concentration cell culture and glutathione productivity.

[0093] As a result, according to the sucrose feeding rate, the amount of change in glutathione content in the culture solution was measured over time and shown in FIG. 3, and the ethanol content in the culture solution was measured over time and shown in FIG. 4. In addition, the change in cell concentration according to the sucrose feeding rate was measure over time and shown in FIG. 5. In FIGS. 3, 4 and 5, the white sphere means a sucrose feeding rate of 4.5 g/L.Math.h1, and the black sphere means a sucrose feeding rate of 6 g/L.Math.h1.

[0094] When supplied at a sucrose feeding rate of 6 g/L.Math.h1, the ethanol content in the culture medium was maintained at 3 g/L or more, and accordingly, the glutathione content change rate was almost constant. On the other hand, at a low sucrose feeding rate (4.5 g/L.Math.h1), the ethanol content was maintained at low and the intracellular glutathione content decreased. This fact indicates that when the sucrose feeding rate is high, the energy required for glutathione biosynthesis is sufficiently supplied by sufficient saccharide supply in the culture medium, but when the sucrose feeding rate is low, the energy supply is not sufficient, resulting in limited cell growth and decreased glutathione content.

[0095] Therefore, when Candida utilis SYC-PR20 strain according to the present invention is supplied at a saccharide feeding rate of 6 g/L.Math.h1, ethanol is accumulated in the culture medium as a metabolite of the cells due to sufficient saccharide supply, and glutathione is increased to the level of batch culture in the high concentration culture section. content can be maintained, which is more preferable.

[Accession Number]

[0096] Name of depository authority: Korean Culture Center of Microorganisms [0097] Accession number: KCCM12777P [0098] Accession date: 20200807