DIABETES-ALLEVIATING OR ANTIOXIDANT COMPOSITION COMPRISING YEAST EXTRACT AND METHOD FOR PREPARING YEAST EXTRACT

Abstract

The present invention relates to a composition for ameliorating diabetes or enhancing antioxidant activity including a yeast extract and a method for preparing the yeast extract.

Claims

1. A method for preparing a yeast extract for ameliorating diabetes or enhancing antioxidant activity, comprising: culturing a yeast and separating the culture into biomass and a culture fluid by centrifugation; dissolving the biomass to produce a strain lysate and extracting the strain lysate and/or the culture fluid with an organic solvent to produce an extract from the strain lysate and/or an extract from the culture fluid, or dissolving the biomass in an organic solvent to produce an extract from the strain lysate and/or adding an organic solvent to the culture fluid to produce an extract from the culture fluid; and concentrating and drying the extract.

2. The method according to claim 1, wherein the organic solvent is but, of, chloroform, acetone, ethanol, ethyl acetate or hexane.

3. The method according to claim 1, wherein culturing is performed at a temperature of 20° C. to 40° C. for 12 hours to 7 days.

4. The method according to claim 1, wherein the yeast is Saccharomyces servazzii or Saccharomyces cerevisiae.

5. The method according to claim 4, wherein the Saccharomyces servazzii is deposited under the deposit number KCCM12157P.

6. A composition for ameliorating diabetes or enhancing antioxidant activity comprising a yeast extract prepared by the method according to claim 1,

7. The composition according to claim 6, wherein the composition comprises 1 wt % to 70 wt % of the yeast extract, based on the total solid content thereof.

8. The composition according to claim 6, wherein the composition is a pharmaceutical or food composition.

9. A composition for ameliorating diabetes or enhancing antioxidant activity comprising an extract from a strain lysate or culture fluid of a yeast.

10. The composition according to claim 9, wherein the organic solvent is butanol, chloroform, acetone, ethanol, ethyl acetate or hexane.

11. The composition according to claim 9, wherein the yeast is Saccharomyces servazzii or Saccharomyces cerevisiae.

12. A Saccharomyces servazzii strain (Ceb-kc-01.1), a culture fluid thereof or a strain lysate thereof, wherein the strain is deposited under the deposit number KCCM12157P.

Description

DESCRIPTION OF DRAWINGS

[0032] FIG. 1 compares the α-glucosidase inhibition activities of extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and acarbose as an α-glucosidase inhibitor in Example 3.

[0033] FIG. 2 shows the effects of extracts obtained by extraction of a strain lysate and a culture fluid with different solvents on the blood glucose levels of (2a) normal SD-rats and (2b) diabetes-induced SD-rats in Example 4 (CFP: strain lysate (phosphate buffer solution), CFR strain lysate (ethanol). CFEA: strain lysate (ethyl acetate),SPM: culture fluid, SPE: culture fluid (ethanol), SPEA: culture fluid (ethyl acetate)).

[0034] FIG. 3 compares the results of free radical scavenging assay for extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and ascorbic acid as an antioxidant in Example 5, demonstrating antioxidant activities of the extracts.

[0035] FIG. 4 compares the results of radical cation de-colorization assay for extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and ascorbic acid as an antioxidant in Example 5, demonstrating antioxidant activities of the extracts.

[0036] FIG. 5 compares the results of reducing power assay for extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and ascorbic acid as an antioxidant in Example 5, demonstrating antioxidant activities of the extracts.

[0037] FIG. 6 compares the a-glucosidase inhibition activities of extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and acarbose as an αglucosidase inhibitor in Example 3.

[0038] FIG. 7 compares the results of free radical scavenging assay for extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and ascorbic acid as an antioxidant in Example 5, demonstrating antioxidant activities of the extracts.

[0039] FIG. 8 compares the results of radical cation de-colorization assay for extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and ascorbic acid as an antioxidant in Example 5, demonstrating antioxidant activities of the extracts.

[0040] FIG. 9 compares the results of reducing power assay for extracts obtained by extraction of a strain lysate and a culture fluid with different solvents and ascorbic acid as an antioxidant in Example 5, demonstrating antioxidant activities of the extracts.

MODE FOR INVENTION

[0041] The present invention will be explained in more detail with reference to the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited thereto.

EXAMPLE 1

Preparation of Yeast Extracts

[0042] Sampling and Strain Isolation

[0043] A sample was taken from kimchi, a traditional Korean fermented food, diluted stepwise, plated on yeast extract peptone dextrose (YPD) supplemented with 1% sodium chloride, and cultured at 37° C. for 24 h. A dominant strain was isolated from the sample. Colonies were selected and passaged three times in fresh media. The pure cultured strain was placed in a medium supplemented with 20% glycerol and stored at ≤−70° C.

[0044] (2) Investigation of Taxological Properties

[0045] The isolated strain was identified. To this end, the taxological properties of the strain were analyzed by 18s rRNA partial sequencing. As a result, the strain was found to have the sequence set forth in SEQ ID NO: 1 and have a homology of 99% with Saccharomyces servazzii.

[0046] The newly isolated strain Saccharomyces servazzii Ceb-kc-011 was deposited with the Korean Culture Center of Microorganisms (120-861, Hongje-2ga-gil, Seodaemun-Gu, Seoul, Korea) on Nov. 10, 2017 and assigned accession number KCCM12157P.

[0047] The strain was freeze-dried and powdered.

[0048] (3) Preparation of Yeast Extracts 1

[0049] Saccharomyces servazzii Ceb-kc-011 was cultured by the following procedure.

[0050] 2 kg of dextrose, 1.5 kg of whole milk powder, 0.1 kg of peptone, and 0.1 kg of a commercial yeast extract were added to 100 liters of purified water. The mixture was sterilized in an autoclave at 121° C. for 15-30 min and cooled to around 35° C. Thereafter, the sterile mixture was inoculated with 0.2-0.4 liters of Saccharomyces servazzii Ceb-kc-011, followed by culture with supply of air at around 35° C. for 2-3 days.

[0051] Thereafter, the culture was separated into biomass and a culture fluid by centrifugation. The biomass and the culture fluid were extracted with the same amounts of different organic solvents (including ethanol, hexane, and ethyl acetate) by ultrasonic disintegration while maintaining a temperature of 25-30° C. for 2 h. Each of the organic solvent extracts was produced in an amount of 0 mg/g-200 mg/g (dry weight) depending on the extraction solvent.

[0052] The organic solvent extracts from the strain and the organic solvent extracts from the culture fluid were concentrated under reduced pressure at 45° C. and freeze-dried to prepare strain extracts (cell free) and culture fluid extracts (supernatant) as yeast extracts I,

EXAMPLE 2

Preparation of Yeast Extracts 2

[0053] Strain extracts and culture fluid extracts as yeast extracts 2 were prepared in the same manner as in (3) of Example 1, except that Saccharomyces cerevisiae was used as a yeast strain.

EXAMPLE 3

Evaluation of α-glucosidase Inhibition Activities

[0054] The α-glucosidase inhibition activities of yeast extracts I (Example 1) and 2 (Example 2) were evaluated by the following procedure.

[0055] The yeast extracts 1 and 2 were diluted to concentrations of 20, 40, 80, and 100 mg/ml. 20 μl of each of the diluted yeast extracts. 40 of a 0.1 M phosphate buffer (pH 7.0), 20 μl of a substrate (5 mM p-nitrophenyl-α-D-glucopyranoside, pH 7.0), and 20 μl of 1.0 unit/l of α-glucosidase (Sigma) were homogenized. The mixture was allowed to react in an incubator at 37° C. for 20 min. The reaction was quenched with 50 pi of Na.sub.2CO.sub.3 and the absorbance was measured at 405 nm. Inhibition (%) was calculated by (A-B/A)×100 (%), where A is the absorbance in the absence of the inhibitor and B is the absorbance in the presence of the inhibitor.

[0056] The α-glucosidase inhibitions of the extracts at different concentrations were calculated from regression curves plotted by entering data into the statistical program (SPSS 17.0) and were expressed as IC.sub.50 values. IC.sub.50 refers to the concentration of the extract required for 50% inhibition of enzyme activity. The results are shown in Tables 1 and 2 (see FIGS. 1 and 6).

TABLE-US-00001 TABLE 1 α-glucosidase inhibition activities of yeast extracts 1 (IC.sub.50, mg/mL) PB (only for cell) Ethyl acetate Hexane Acarbose  2.6 ± 0.6.sup.a — — Strain lysate 174.2 ± 21.3.sup.c 1.9 ± 0.1.sup.a 2.4 ± 0.3.sup.a Culture fluid 376.6 ± 6.9.sup.d 3.8 ± 1.6.sup.a 1.4 ± 0.2.sup.a

TABLE-US-00002 TABLE 2 α-glucosidase inhibition activities of yeast extracts 2 (IC.sub.50, mg/mL) PB (only for cell) Ethyl acetate Hexane Acarbose  2.6 ± 0.6 — — Strain lysate 421.2 ± 15.2 8.4 ± 1.2 6.9 ± 1.7 Culture fluid 915.1 ± 41.9 9.5 ± 2.3 7.8 ± 0.9

[0057] In Tables 1 and 2, PB represents treatment with phosphate buffer solution instead of the organic solvents.

[0058] The results in Tables 1 and 2 revealed that the strain lysate and the culture fluid per se were not active in inhibiting α-glucosidase but the ethyl acetate and hexane extracts were effective in inhibiting α-glucosidase.

EXAMPLE 4

Evaluation of Abilities to Lower Blood Glucose Levels: Rat Sugar Loading Test

[0059] 6 week old male SD-rats (180-200 g) as experimental animals were acclimated to the vivarium for 5 days with ad libitum access to food (20 g) and water while maintaining the temperature at 25° C. and the relative humidity (RH) at 60% under a 12 h light/dark cycle. The experimental animals were randomly divided into normal and diabetes-induced groups. The diabetes-induced groups were again divided into control and experimental groups. A solution of streptozotocin (STZ, 60 mg/kg, Sigma) as a diabetes inducing drug in a 0.1 M citrate buffer (pH 4.5) was once injected intraperitoneally into each fasted SD-rat. 3 days after injection, the blood glucose levels were measured using a glucometer. Only SD-rats whose blood glucose levels were more than 300 mg/dl were selected and used for experiments.

[0060] An experiment was conducted to investigate the abilities of yeast extracts 1 (Example 1) and 2 (Example 2) to inhibit increases in the blood glucose levels of the SD-rats. Each yeast extract (100 mg/kg), acarbose (20 mg/kg), and starch (3 g/kg) were orally administered and time-dependent changes in the blood glucose levels were measured.

[0061] The results are shown in FIGS. 2a (normal groups) and 2b (diabetes-induced groups).

[0062] The results in FIG. 2a revealed that SPEA (the ethyl acetate extract from the culture fluid). CFEA (the ethyl acetate extract from the strain lysate). CFE (the ethanol extract from the strain lysate), and SPE (the ethanol extract from the culture fluid) were effective in inhibiting increases in the blood glucose levels caused by digestion and uptake of the starch in the normal groups (normal rats) whereas SPM (the culture fluid) and CFP (the strain lysate the phosphate buffer solution) per se were not significantly effective in inhibiting increases in blood glucose levels.

[0063] The results in FIG. 2b revealed that the changes in the blood glucose levels of the diabetes-induced SD-rats were drastic compared to those of the normal groups. SPEA (the ethyl acetate extract from the culture fluid) and CFEA (the ethyl acetate extract from the strain lysate) caused the greatest reduction in blood glucose levels, and CFE (the ethanol extract from the strain lysate) and SPE (the ethanol extract from the culture fluid) inhibited marked initial increases in the blood glucose levels, whereas SPM (the culture fluid) and CFP (the strain lysate in the phosphate buffer solution) per se were not significantly effective in inhibiting increases in blood glucose levels.

EXAMPLE 5

Evaluation of Antioxidant Activities

[0064] The antioxidant activities of yeast extracts 1 (Example 1) and 2 (Example 2) were evaluated by the following procedures.

[0065] (1) Free Radical Scavenging Assay Using DPPH

[0066] A solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals in an organic solvent has a maximum absorbance at 515 nm. An antioxidant scavenges DPPH radicals, losing its original color and being made transparent.

[0067] The test sample was mixed with 2,2-diphenyl-1-picrylhydrazyl (DPPH, 100 μM) in a ratio of 1:20 and the mixture was stored at 37° C. for 30 min. After completion of the reaction, the absorbance was measured at 517 nm using a spectrophotometer. A lower absorbance indicates a higher antioxidant activity.

[0068] The DPPH scavenging activities of yeast extracts 1 and 2 were expressed as IC.sub.50 values (corresponding to the concentrations of the extracts required to scavenge 50% of the radicals). The results are shown in Tables 3 and 4 (see FIGS. 3 and 7).

TABLE-US-00003 TABLE 3 DPPH scavenging activities of yeast extracts 1 (IC.sub.50, mg/mL) AVERAGE STDEV Strain lysate 0.00 0.00 Strain lysate (ethyl acetate) 5.78 2.30 Culture fluid (ethanol) 2.32 0.61 Culture fluid (ethyl acetate) 0.62 0.03 Ascorbic acid 0.01 0.00

TABLE-US-00004 TABLE 4 DPPH scavenging activities of yeast extracts 2 (IC.sub.50, mg/mL) AVERAGE STDEV Strain lysate (ethyl acetate) 14.30 2.31 Culture fluid (ethanol) 8.65 0.85 Culture fluid (ethyl acetate) 1.55 0.07 Ascorbic acid 0.01 0.00

[0069] The results in Tables 3 and 4 revealed that each of the ethyl acetate extracts from the culture fluids showed the highest antioxidant activity comparable to that of ascorbic acid, and the ethanol extracts from the culture fluids and the ethyl acetate extracts from the strain lysates followed in this order. Each of the strain lysates per se showed no antioxidant activity.

[0070] (2) Radical Cation De-Colorization Assay Using ABTS

[0071] ABTS scavenging activities were measured as follows.

[0072] 7 mM ABTS was mixed with 2.45 mM potassium persulfate and incubated at room temperature for 16 h to create ABTS cations (ABTS.sup.+). The mixture was diluted until an absorbance of≤0.7 at 734 nm was reached. 100 μl of each sample was added to 100 μl of the ABTS solution. After 3 min, the absorbance was measured. The ABTS scavenging activities of yeast extracts 1 and 2 were expressed as IC.sub.50 values (corresponding to the concentrations of the extracts required to scavenge 50% of the cations). The results are shown in Tables 5 and 6 (see FIGS. 4 and 8).

TABLE-US-00005 TABLE 5 ABTS scavenging activites of yeast extracts 1 (IC.sub.50, mg/mL) AVERAGE STDEV Strain lysate 0.00 0.00 Strain lysate (ethanol) 13.12 2.61 Strain lysate (ethyl acetate) 8.68 0.27 Culture fluid (ethanol) 4.05 0.70 Culture fluid (ethyl acetate) 1.73 0.05 Ascorbic acid 0.26 0.03

TABLE-US-00006 TABLE 6 ABTS scavenging activites of yeast extracts 2 (IC.sub.50, mg/mL) AVERAGE STDEV Strain lysate 0.00 0.00 Strain lysate (ethanol) 32.50 4.10 Strain lysate (ethyl acetate) 22.80 2.30 Culture fluid (ethanol) 11.00 1.90 Culture fluid (ethyl acetate) 45.0 0.70 Ascorbic acid 0.26 0.03

[0073] The results in Table 5 revealed that the ethyl acetate extract from the culture fluid showed the highest antioxidant activity against ABTS comparable to that of ascorbic acid, and the ethanol extract from the culture fluid, the ethyl acetate extract from the strain lysate, and the ethanol extract from the strain lysate followed in this order. The results in Table 6 revealed that the ethyl acetate extract from the culture fluid showed the highest antioxidant activity against ABTS. Each of the strain lysates per se showed no antioxidant activity.

[0074] (3) Reducing Power Assay

[0075] The antioxidant activities of yeast extracts 1 and 2 were evaluated by reducing power assay. This assay was performed by the following procedure.

[0076] First, 50 μL of potassium ferricyanide and 20 μL of each sample were added to 50 μL of a 0.2 M phosphate buffer solution (pH 6.6) and the mixture was allowed to react at 50° C. for 20 min. After completion of the reaction, 50 μL of a 10% TCA solution was added, followed by centrifugation at 3000×g for 20 min. 100 μL of the supernatant, 200 μL of triple-distilled water, and 20 μL of 0.1% ferric chloride were added, and the absorbance was measured at 700 nm. Trolox and BHA were used as positive controls.

[0077] The results are shown in Tables 7 and 8 (see FIGS. 5 and 9).

TABLE-US-00007 TABLE 7 Reducing power activities of yeast extracts 1 Strain lysate Strain lysate Culture fluid Culture fluid (μg/mL) Strain lysate (ethanol) (ethyl acetate) (ethanol) (ethyl acetate) Ascorbic acid 15.625 0.002 ± 0.001 0.001 ± 0.001 0.002 ± 0.001 0.003 ± 0.002 0.004 ± 0.001 0.058 ± 0.001 62.5 0.001 ± 0.001 0.002 ± 0.001 0.004 ± 0.000 0.003 ± 0.001 0.015 ± 0.001 0.267 ± 0.002 250 0.001 ± 0.001 0.005 ± 0.001 0.019 ± 0.002 0.016 ± 0.001 0.060 ± 0.001 0.940 ± 0.005 1000 0.003 ± 0.001 0.021 ± 0.004 0.089 ± 0.001 0.079 ± 0.002 0.251 ± 0.004 1.146 ± 0.008

TABLE-US-00008 TABLE 8 Reducing power activities of yeast extract 2 Strain lysate Strain lysate Culture fluid Culture fluid (μg/mL) Strain lysate (ethanol) (ethyl acetate) (ethanol) (ethyl acetate) Ascorbic acid 15.625 0.000 ± 0.000 0.000 ± 0.001 0.000 ± 0.001 0.001 ± 0.000 0.001 ± 0.000 0.058 ± 0.001 62.5 0.000 ± 0.000 0.000 ± 0.001 0.001 ± 0.001 0.001 ± 0.001 0.004 ± 0.00 0.267 ± 0.002 250 0.001 ± 0.001 0.002 ± 0.001 0.005 ± 0.002 0.005 ± 0.002 0.020 ± 0.002 0.940 ± 0.005 1000 0.002 ± 0.001 0.005 ± 0.002 0.022 ± 0.003 0.021 ± 0.003 0.104 ± 0.004 1.146 ± 0.008