YEAST EXTRACT CONTAINING TRIPEPTIDE AND PREPARATION METHOD THEREFOR

Abstract

The present invention relates to a yeast extract having a high tripeptide content and a high dry matter content using a yeast cell containing tripeptide and a method for preparing the same.

Claims

1. A method of producing an yeast extract containing 0.5 to 20% by weight of glutathione based on solid content, comprising a step of obtaining a solvent extract by extracting yeast cells containing glutathione or suspension of the cells, with water at 60 to 90 C.

2. The method of claim 1, wherein the step of obtaining a solvent extract comprises obtaining a primary extract obtained by extracting yeast cells containing glutathione with a water at 60 to 90 C., and a secondary extract obtained by treating the cells recovered from the primary extract with a proteolytic enzyme and extracting with water.

3. The method according to claim 1, further comprising a step of obtaining a dried product by drying the solvent extract.

4. The method of claim 3, wherein the dry matter yield of the yeast extract is 20 to % by weight.

5. The method according to claim 3, wherein a time for extracting with water in the step of obtaining the solvent extract is 10 minutes to 120 minutes.

6. The method according to claim 1, wherein the cell concentration of the suspension of the cells has 50 to 500 of an optical density (OD) value measured at 600 nm.

7. The method of claim 1, wherein the yeast is Candida utilis.

8. The method of claim 2, wherein the proteolytic enzyme is endo-protease or mixed enzymes of endo-protease and exo-protease.

9. The method of claim 2, wherein the treated amount of the proteolytic enzyme is 5 ul/ml.

10. A yeast extract comprising a solvent extract using water at a temperature of 60 to 90 C. for yeast cells containing glutathione or suspension of the cells, and containing 0.5 to 20% by weight of glutathione based on solid content.

11. The yeast extract according to claim 10, wherein the solvent extract is obtained by extracting proteolytic enzyme-treated yeast cells or a suspension of the cells with water.

12. The yeast extract of claim 11, wherein the solvent extract comprises a primary extract obtained by extracting yeast cells containing glutathione or suspension of the cell with water at 60 to 90 C., and a secondary extract obtained by treating the cells recovered from the primary extract with a proteolytic enzyme and extracting with water.

13. The yeast extract of claim 11, wherein the proteolytic enzyme is endo-protease or mixed enzymes of endo-protease and exo-protease.

14. The yeast extract of claim 11, wherein the treated amount of the proteolytic enzyme is 0.5 to 5 ul/ml.

15. The yeast extract of claim 10, wherein the yeast is Candida utilis.

16. The yeast extract according to claim 10, wherein the yeast extract is a dried product obtained by drying the solvent extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows a tripeptide (glutathione) content pattern over the time passage during extraction using hot water of 90 C. according to an example of the present invention.

[0036] FIG. 3 shows a tripeptide (glutathione) content pattern over the time passage during extraction using hot water of 80 C. according to an example of the present invention.

[0037] FIG. 3 shows a tripeptide (glutathione) content pattern over the time passage during extraction using hot water of 70 C. according to an example of the present invention.

[0038] FIG. 4 shows a tripeptide (glutathione) content pattern over the time passage during extraction using hot water of 65 C. according to an example of the present invention.

[0039] FIG. 5 shows a tripeptide (glutathione) content pattern over the time passage during extraction using hot water of 60 C. according to an example of the present invention.

[0040] FIG. 6 is a graph showing the yield (w/w %) of the dried extract obtained content according to the type and concentration of the protease in an example of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The present invention will be explained 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: Preparation of Glutathione-Producing Microorganisms

[0042] 1-1: Microorganism Preparation

[0043] 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 24 hours 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.

[0044] The cell concentration was determined by measuring the absorbance at 600 nm for the cell culture, and the measured absorbance (cell optical density, OD) was 17.8.

[0045] 1-2: Glutathione Content Analysis

[0046] 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. The measured glutathione content was 112 mg/L.

[0047] 1-3: Measurement of Glutathione Content Per Cell Dry Weight (g)

[0048] 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.

[0049] 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.

[0050] After measuring the absorbance, glutathione was extracted from the cells in substantially the same manner as in Examples 1-2, 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) for the statin (productivity, GSH (%)/g-cell) was 1.6%.

[0051] 1-4: Alcohol Dehydrogenase (ADH) Activity Assay

[0052] 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 (AO) 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.

[0053] Therefore, the strain is a SYC-PR20 strain having a high OD value and high glutathione production, which is Candida utilis SYC-PR20 having accession number of KCCM 12777P. The strain has excellent properties such as a glutathione yield of 1.6% and an ADH activity of 0.26 m U/ml, and has the 18S rDNA sequence of SEQ ID NO: 1.

[0054] 1-5: Acquisition of Microbial Cells

[0055] As a seed culture, 3 mL of the cell culture was weighed and inoculated into an flask containing 50 mL of YPD medium. Shaking culture was performed at 30 C. for 48 hours, and 1 ml of the culture solution was sampled and centrifuged at 12,000 rpm for 5 minutes. The medium components of the supernatant were removed, the cells were washed with the same amount of distilled water, and cells were obtained by centrifugation at 12,000 rpm for 5 minutes.

Example 2: Extraction of Glutathione Depending Extraction Temperature

[0056] 2-1: Extraction Sample

[0057] As a method for recovering tripeptide (glutathione) in the cells, the cells were suspended with addition of distilled water to adjust the cell concentration to a certain concentration, and extraction was performed under various extraction temperature to determine the optimal extraction conditions for tripeptide (glutathione).

[0058] The glutathione-producing strain, Candida utilis SYC-PR20 obtained in Example 1 was used, and the cells were obtained in the same manner as in Example 1 and suspended in distilled water to adjust the OD600 value to 100 to prepare a diluted cell solution. The diluted cell solution was used as a sample for hot water extraction.

[0059] 2-2: Glutathione Extraction

[0060] The extraction sample was the prepared diluted cell solution that the cells were suspended in distilled water to adjust the OD600 value to 100. The glutathione extraction for the samples were carried out by using five waters of 90 C., 80 C., 70 C., 65 C., or 60 C.

[0061] Specifically, the extraction temperature was adjusted using a constant temperature water bath, and the extraction was performed by adjusting the amount of the extraction liquid to 100 mL. In the extraction process, samples to be analyzed were taken at intervals of 5 or 10 minutes and the content of glutathione contained in the extract was quantified by the DTNB color development method.

[0062] After centrifuging the extraction solution, the supernatant was taken and reacted at 40 C. for 20 minutes with 10 mM DTNB (5,5-Dithiobis-(2-Nitrobenzoic Acid)) dissolved in 0.5M potassium phosphate pH8.0 buffer, and absorbance at 412 nm was measured to determine the glutathione content, and the glutathione content (GSH mg/L). DTNB is mainly used for glutathione analysis and is known as Ellman's reagent designed to detect thiol compounds. The reaction between DTNB and GSH produces 2-nitro-5-benzoic acid in yellow color and GSSG, and the concentration of GSH can be calculated by measuring the OD value at 412 nm. GSSG is reduced to GSH by glutathione reductase and forms a recycling system that reacts with DTNB again. The analysis results of the glutathione content contained in the extract, depending on the temperature of hot water are shown in FIGS. 1 to 5, respectively. The maximum content of extracted glutathione and the time to reach the maximum content depending on the temperature of hot water shown in FIGS. 1 to 5 are represented in Table 1 below.

TABLE-US-00001 TABLE 1 Extraction time to reach the Extraction Maximum content of maximum content of temperature( C.) GSH (mM) GSH(minute) 90 1.10 5 80 1.23 5 70 1.12 10 65 1.14 40 60 0.90 190

[0063] According to the extraction results in Table 1 and FIGS. 1 to 5, the extraction time to reach the maximum GSH content at extraction temperatures of 80 C. and 90 C. was about 5 minutes, and the glutathione content slightly decreased as the extraction time passed. Therefore, it was preferable to extract glutathione at high temperature and for a short time. In addition, as the extraction temperature decreased, the extraction time to reach the maximum GSH content tended to increase, and the maximum glutathione extraction content gradually decreased at temperatures of 60 C. or lower.

[0064] Therefore, considering the maximum content of glutathione and the time to reach the maximum content of glutathione, and the cost of elevating temperature when applied industrially and the feasibility of controlling temperature when extracting at high temperature for a short time, it is recommended that the extraction temperature is 70 C. Under the condition that the extraction temperature was 70 C., the maximum extraction content of glutathione was shown when the extraction time was 10 minutes or more, and was almost constant until 20 minutes.

Example 3: Evaluation of the Dry Matter Yield Depending on the Cell Concentration

[0065] In this Example, optimal extraction conditions were set by measuring the dry matter content, dry matter yield, and GSH content of the yeast extract depending on the cell concentration of the sample used for extraction.

[0066] Specifically, in the process of preparing the extraction sample prepared in Example 2-1, the cells were suspended in distilled water to adjust the OD600 value to 100 to 500, to prepare six diluted cell solutions having different cell concentrations (Table 2).

[0067] Using the prepared diluted cell solutions, glutathione was extracted in substantially the same way as in Example 2-2 using hot water at 70 C., and PASTE was removed from the resulting extracted solution to obtain a crude extract solution, and the volume of the crude extract solution was measured and shown in table 2. The crude extract solution was freeze-dried to prepare a dry powder of the crude extract. 10 ml of the crude extract was freeze-dried in a speed vacuum concentrator, and the weight of the obtained dry powder was measured, and the dry weight (g/L) was shown in Table 2 below. Table 2 shows the total dry weight (g) of the dry matter obtained from the crude extract by multiplying the dry matter weight by the recovered liquid amount. The dry matter yield was obtained by inputting the obtained total dried weight (g) into Equation 1 below, and expressed as w/w % in Table 2 below. In Equation 1 below, OD is the optical density measured at a wavelength of 600 nm and means the cell concentration.

Yield of dry matter=Total weight of dry matter (g)/(OD*0.4)*100[Formula 1]

TABLE-US-00002 TABLE 2 Total Weight Recovered weight yield of of dry liquid of dry dry GSH Cell matter amount matter matter content OD g/L L g %(w/w) %(w/w) 100 13.96 0.91 12.73 31.83 5.85 200 29.16 0.85 24.73 30.91 6.53 250 39.32 0.81 32.01 32.01 6.23 300 48.80 0.77 37.38 31.15 6.26 400 69.87 0.70 48.77 30.48 6.13 500 75.95 0.63 47.85 23.92 6.93

[0068] As shown in Table 2, It was confirmed that the dry matter yield of the yeast extract was 23.92% (w/w) for 500 of OD600, and the dry matter yield of the yeast extract was the highest for 250 of the cell concentration OD600 value is 250. Therefore, since the total dry matter content and GSH content % increase as the cell concentration increases, the higher the cell concentration (OD) value is, the more advantageous it is. However, as the cell OD value increases, the amount of recovered liquid decreases and the dry matter yield decreases. Therefore, it is preferable to adjust the cell concentration appropriately by considering the yield of dry product. Accordingly, in consideration of the dry matter yield and GSH content as a result of the above experiments, extraction may be performed at an OD value of 50 to 500, or preferably 100 to 400.

Example 4: Glutathione Extraction Using Protease

[0069] In this Example, it was attempted to increase the extraction content of glutathione and increase the dry matter yield of yeast extract by treating with proteolytic enzyme (protease). Therefore, in the step before performing the hot water extraction, the diluted cell solution was treated with enzyme, and the GSH content extracted and eluted was determined as a measuring index in order to directly test the effect of the enzyme treatment. The use of protease accelerates cell degradation to quickly elute intracellular glutathione, and the eluted glutathione is degraded over time, resulting in a low content. Therefore, an enzyme with a large decrease in glutathione content depending on the time passage and increased amount of enzyme, should be determined in this test.

[0070] As proteolytic enzymes, 4 types in total including 2 types of Novozyme's Alcalase (trade name) and Flavourzyme (trade name), and 2 types of Multifect (trade name) and papain were selected and tested. The general characteristics of proteases act as two types of endo-type (alcalase) and exo-type (papain), Flavourzyme is a mixture of the two types.

[0071] Specifically, samples for enzyme treatment and extraction were prepared in the same manner as in Example 2-1. The prepared cell dilution sample was dispensed into a 1.5 ml E-Tube in a reaction volume of 1 ml, and the four enzymes were added to the dispensed cell dilution solution in an amount of 0, 1, 2, 5, 10, and 15 l/ml, respectively. The enzyme reaction was performed at a temperature of 50 C. for 120 minutes, with taking samples to be analyzed at 30 minutes, 60 minutes and 120 minutes, and was analyzed using the DTNB color development method in substantially the same manner as in Example 2-2. Table 3 shows the GSH content (uM) depending on the type and the used amount used (ul/ml) of the four enzymes.

TABLE-US-00003 TABLE 3 0 1 2 6 10 15 Enzyme minute ul/ml ul/ml ul/ml ul/ml ul/ml ul/ml Alcalase 30 0.87 0.67 0.60 0.45 0.34 0.27 Alcalase 60 0.65 0.38 0.30 0.17 0.10 0.07 Alcalase 120 0.31 0.03 0.03 0.02 0.01 0.01 Flavourzyme 30 0.80 0.76 0.71 0.60 0.45 0.33 Flavourzyme 60 0.65 0.57 0.52 0.40 0.25 0.16 Flavourzyme 120 0.28 0.19 0.15 0.08 0.05 0.04 Multifec 30 0.83 0.82 0.81 0.81 0.77 0.71 Multifec 60 0.64 0.63 0.62 0.62 0.57 0.49 Multifec 120 0.28 0.24 0.25 0.24 0.19 0.12 papain 30 0.83 0.82 0.86 1.31 1.99 2.56 papain 60 0.63 0.51 0.53 0.97 1.64 2.22 papain 120 0.29 0.10 0.10 0.35 0.91 1.46

[0072] When glutathione-containing cells were treated with 4 types of proteases, the change in GSH content depending on the change in protease concentration was investigated. In the case of Alcalase and Flavourzyme, the change in the GSH content was detected depending on the enzyme concentration, which indirectly confirmed their action on the cell wall. On the other hand, there was no change at enzyme concentration for multifec. Papain affected the GSH coloring method, and thus was excluded from the experiment. The two enzymes (Alcalase and Flavourzyme) with significant changes in GSH content were selected.

Example 5: Glutathione Extraction Using Protease and Hot Water Extraction

[0073] After performing solvent extraction of the cells using hot water, additional extraction was performed by treatment with a proteolytic enzyme for additional extraction of useful components in the cells, in order to increase the dry matter yield of yeast extract. The dry matter yield of the yeast extract mainly includes proteins and peptides, which are as water-soluble components eluted with water as an extraction solvent.

[0074] Specifically, samples for enzyme treatment and extraction were prepared by suspending cells in distilled water in the same manner as in Example 2-1 to adjust the OD600 value to 100 to prepare 100 mL of cell dilution samples. The prepared sample was dispensed into three 30 to 35 ml flacon tubes, respectively. In substantially the same manner as in Example 2, glutathione was extracted for 25 minutes using hot water from the cell dilution solution dispensed into the flacon tube at a temperature of 70 C. The extract was centrifuged and the supernatant was recovered to prepare a crude extract.

[0075] After adding 90 mL of distilled water to the cell paste remaining after recovering the supernatant, two kinds of Novozyme's Alcalase and Flavorzyme were added at an enzyme concentration of 1 ul/ml (0.001%), 5 ul/ml (0.005%), and 10 ul/ml (0.01%), respectively, and an enzyme reaction was performed at a temperature of 50 C. for 120 minutes.

[0076] The enzyme reaction product solution was secondarily extracted for glutathione for 25 minutes using hot water at a temperature of 70 C. The secondary extract was centrifuged and the supernatant was recovered to prepare a secondary crude extract.

[0077] The glutathione content contained in the extract sample obtained by mixing the first crude extract and the second crude extract was analyzed by the DTNB color development method in substantially the same manner as in Example 2-2.

[0078] In addition, the mixed sample was lyophilized to prepare a dry powder of the crude extract, and the weight of the dry powder obtained after freeze-drying 10 ml of the crude extract in speed vacuum concentrator was measured to obtain a dry weight (g/L). The total weight (g) of the dry matter of the crude extract was calculated by multiplying the dry weight with the recovered liquid amount. The total dry matter weight (g) obtained above was input into Equation 1 to obtain the dry matter yield (w/w %), which is shown in Table 4 as w/w %. In addition, the dry matter yield of the extract depending on the protease type and concentration is shown in FIG. 6.

TABLE-US-00004 TABLE 4 0 ul/ml 1 ul/ml 5 ul/ml 10 ul/ml of used of used of used of used Enzyme enzyme enzyme enzyme enzyme Flavourzyme 20%(w/w) 23%(w/w) 32%(w/w) 42%(w/w) Treatment Alcalase 20%(w/w) 36%(w/w) 51%(w/w) 63%(w/w) Treatment

[0079] As shown in Table 4, when the secondary extract was obtained by hot water extraction after treatment with a proteolytic enzyme, the dried yeast extract could be obtained in high yield advantageously without reducing the content of glutathione, and Alcalase was more preferable as a proteolytic enzyme. Within the experimental ranges, the dry matter yield of yeast extract increased as the amount of treated enzyme increased.