More specifically, the enzymatic treatment is performed by an enzyme mixture in which pullulanase is added to the amylase.

The enzymatic treatment can be performed at 55 to 90° C. for 20 to 100 minutes.

Next, a medium is prepared by mixing water and vitamin C with the rice bran enzyme lysate (step b).

The medium may be adjusted specifically to pH 5 to pH 6, more specifically to pH 5.2 to pH 5.7.

Afterwards, a culture is obtained by culturing Aureobasidium pullulans (Aureobasidium pullulans SM2001, accession number KCCM 10307) in the medium (step c).

The culturing may be performed specifically by culturing firstly at 20 to 30° C. for 15 to 20 hours and then culturing secondly at 25 to 35° C. for 30 to 40 hours, more specifically by culturing firstly at 23 to 27° C. for 17 to 19 hours and then culturing secondly at 28 to 32° C. for 35 to 37 hours.

Finally, an Aureobasidium pullulans fermentation product is obtained by removing cells from the culture and concentrating the same (step d).

MODE FOR INVENTION

Hereinafter, specific examples are presented to help understand the present disclosure. However, the following examples only illustrate the present disclosure, and it is obvious to those skilled in the art that various changes and modifications can be made within the scope of the category and technical idea of the present disclosure and that those changes and modifications fall within the scope of the attached claims.

EXAMPLES
Preparation Example 1: First Culturing
(1) Preparation of Rice Bran Enzyme Lysate Using Single Enzyme

After mixing rice bran powder, sugar and water at 1:1.5:5, a rice bran enzyme lysate was prepared by adding 1.5 parts by weight of an amylase as a single enzyme based on 100 parts by weight of the rice bran powder and conducting enzymatic treatment at 70° C. for 1 hour.

(2) Preparation of First Medium

A first medium was prepared by mixing water with the enzyme lysate prepared using the single enzyme as described above and adding 2% (v/v) of vitamin C based on the total weight.

Preparation Example 2: Second Culturing
(1) Preparation of Rice Bran Enzyme Lysate Using Enzyme Mixture

After mixing rice bran powder, sugar and water at 1:1.5:5, a rice bran enzyme lysate was prepared by adding by weight of an enzyme mixture of 1.5 parts amylase and 1.5 parts by weight of pullulanase based on 100 parts by weight of rice bran powder and conducting enzymatic treatment at 70° C. for 1 hour.

(2) Preparation of the Second Medium

A second medium was prepared by mixing water with the rice bran enzyme lysate prepared using the enzyme mixture as described above and adding 2% (v/v) of vitamin C based on the total weight.

Example 1: Aureobasidium pullulans Fermentation Product Using First Medium

(1) Culturing of Aureobasidium pullulans Strain

For pre-culturing, Aureobasidium pullulans SM2001 (accession number KCCM 10307) was cultured in a solid medium for a predetermined period of time and inoculated into 100 mL of a medium composition (sugar 1% (v/v), yeast extract 0.2% (v/v), vitamin C 0.2% (v/v)) prepared by sterilizing in a 250-mL flask. Then, a seed culture was prepared by performing shaking culture at 24° C. and 200 rpm for 36 hours.

(2) Main Culturing

The Aureobasidium pullulans seed culture was sterilized in a 500-mL flask, inoculated to 50 mL of the first medium prepared in Preparation Example 1 at 5% (v/v), and cultured for 18 hours under the condition of 25° C. and 85 rpm with an air flow rate of 1 vvm. Subsequently, culturing was performed for 36 hours under the condition of 30° C. and 170 rpm with an air flow rate of 1 vvm.

(3) Removal of Cells and Concentration

The culture was centrifuged at 7000×g for 20 minutes, and the supernatant from which cells had been removed was recovered. The recovered supernatant was concentrated under reduced pressure, sterilized at 85° C. for 2 hours, and then freeze-dried to obtain an Aureobasidium pullulans fermentation product (T).

Example 2: Aureobasidium pullulans Fermentation Product Using Second Medium

An Aureobasidium pullulans fermentation product (TP) was prepared under the same conditions as in Example 1, except that the second medium prepared in Preparation Example 2 was used instead of the first medium prepared in Preparation Example 1 during the main culturing (2).

Comparative Example 1: Aureobasidium pullulans Fermentation Product Using Conventional Medium

An Aureobasidium pullulans fermentation product (P) was prepared under the same conditions as in Example 1, except that a conventional medium used for fermentation of Aureobasidium pullulans, containing 5 g/L K₂HPO₄, 1 g/L NaCl, 0.2 g/L MgSO₄·7H₂O, 0.6 g/L (NH₄)₂SO₄and 2.5 g/L yeast extract, was used instead of the first medium prepared in Preparation Example 1 during the main culturing (2).

EXPERIMENTAL EXAMPLES
Experimental Example 1: Assessment of Cytotoxicity

Drug-induced cytotoxicity was investigate using the MTT assay method. 200 μL of C2C12 cells were seeded onto a 96-well plate at a concentration of 1×10⁵cells/mL and cultured for 24 hours. After treating the C2C12 cells with the Aureobasidium pullulans fermented sample of Example 1 or 2 or Comparative Example 1 at 25, 50, 100, 400, 800 or 1000 μg/mL, followed by incubation for 24 hours, 5 mg/mL (DPBS) of MTT reagent diluted 5 times with a medium was dispensed at 100 μL/well and then reacted in an incubator for 1 hour. After removing the supernatant and dissolving formazan by treating with 100 μL of DMSO, absorbance was measured at 570 nm using a microplate reader (SpectraMaxi3, Molecular Devices, CA, USA). The result is shown in FIG. 1. (a) shows the result for Comparative Example 1 (P), (b) shows the result for the Aureobasidium pullulans fermentation product of Example 1 (T), and (c) shows the result for the Aureobasidium pullulans fermentation product of Example 2 (TP).

There was no effect on the survival rate of the C2C12 cells at all the tested concentrations. Since the cell survival rate at all the concentrations was 90% or higher, it was determined that there was almost no cytotoxicity. Therefore, the highest sample concentration was set to 200 μg/mL in the subsequent experiments.

Experimental Example 2: Analysis of Expression of Factors Related to Muscle Production and Muscle Cell Proteolysis (qPCR)

C2C12 cells were seeded onto a 6-well culture dish at 3×10⁵cells/well and cultured for 24 hours and was induced to differentiate for the next 6 days. The cells were treated with the Aureobasidium pullulans fermentation product of Examples 1 and 2 or Comparative Example 1 at a concentration of 100 or 200 g/mL 24 hours before inducing muscle loss. After the muscle loss was induced, the medium was removed. After washing with PBS, RNA was extracted using the RNeasy® mini kit (Aiagen, Hilden, Germany). The extracted RNA was quantified using a spectrophotometer (Nanodrop), and complementary DNA (cDNA) was synthesized from 1 μg of the RNA using the Maxima first strand cDNA synthesis kit for RT-qPCR (Thermo Scientific, Waltham, USA). 19 μL of a mixture containing 10 UL of PCR bio syGREEN blue mix (PCR Biosystems, Pennsylvania, USA) and 2 μL of primers and 1 μL of the cDNA were subjected to 40 cycles of polymerase chain reaction (PCR). The information of the primers used in the polymerase reactions is summarized in Table 1.

TABLE 1

Target
Primer sequences

MyoD
Forward
5′-GATGGCATGATGGATTACAG-3′

Reverse
5′-CTCCACTATGCTGGACAGG-3′

Myogenin
Forward
5′-AGTACATTGAGCGCCTACAG-3′

Reverse
5′-GACGTAAGGGAGTGCAGATT-3′

Atrogin-1
Forward
5′-CTGCCTGTGTGCTTACAACT-3′

Reverse
5′-TGCTCTCTTCTTGGGTAACA-3′

Myf5
Forward
5′-TGAGGGAACAGGTGGAGAAC-3′

Reverse
5′-AGCTGGACACGGAGCTTTA-3′

Myf6
Forward
5′-ATTCTTGCGGGTGCGGATTT-3′

Reverse
5′-ACGTTTGCTCCTCCTTCCTT-3′

MEF2
Forward
5′-TCCATCAGCCATTTCAACAA-3′

Reverse
5′-GTTACAGAGCCGAGGTGGAG-3′

FoxO3
Forward
5′-ACAAACGGCTCACTTTGTCC-3′

Reverse
5′-GTGCCGGATGGAGTTCTC-3′

Myostatin
Forward
5′-CTGTAACCTTCCCAGACCA-3′

Reverse
5′-GCAGTCAAGCCCAAAGTCTC-3′

MuRF1
Forward
5′-TGCCTACTTGCTCCTTGC-3′

Reverse
5′-CACCAGCATGGAGATGCAGT-3′

After differentiating C2C12 cells into myotubes, the expression of mRNA was analyzed by treating with the Aureobasidium pullulans fermentation product of Examples 1 and 2 or Comparative Example 1 24 hours before induction of amyotrophy, in order to confirm whether the Aureobasidium pullulans fermentation product of Examples 1 and 2 or Comparative Example 1 has an effect on muscle cell production and proteolytic factors in the cells in a dexamethasone-induced muscle atrophy model. The result of analyzing the mRNA expression level of MyoD, myogenin, MEF2, Myf5 and Myf6, which are transcription factors involved in muscle cell production, is shown in FIGS. 2 to 6, respectively. It was confirmed that the mRNA expression of MyoD, myogenin, MEF2, Myf5 and Myf6 increased significantly in a concentration-dependent manner in the Example 1 treatment group (T) or the Example 2 treatment group (TP) and, in particular, the muscle cell generation transcription factors were expressed at higher levels in the Example 2 treatment group (TP) (#p<0.05, ##p<0.01, ###p<0.001 vs. CTL; * p<0.05, ** p<0.01, *** p<0.001 vs. Dex group).

Also, the analysis result of the mRNA expression of MuRF1, atrogin-1, FoxO3 and myostatin, which are muscle cell proteolytic factors, is shown in FIGS. 7 to 10, respectively. It was confirmed that the expression level of MuRF1, atrogin-1, FoxO3 and myostatin decreased significantly in a concentration-dependent manner in the Example 1 treatment group (T) or the Example 2 treatment group (TP) and, in particular, the expression level was lower in the Example 2 treatment group (TP) (#p<0.05, ##p<0.01, ###p<0.001 vs. CTL; * p<0.05, ** p<0.01, *** p<0.001, vs. Dex group).

Experimental Example 3: Analysis of Expression of Factors Related to Muscle Cell Death (qPCR)

qPCR analysis was performed using the same method as in Experimental Example 2. The information of the primers used in polymerase reaction is summarized in Table 2.

TABLE 2

Target
Primer sequences

BCL-2
Forward
5′-GATTTCTCCTGGCTGTCTCT-3′

Reverse
5′-TGTGTGTGTGTGTTCTGCTT-3′

Bax
Forward
5′-CCTTTTTGCTACAGGGTTC-3′

Reverse
5′-TCCATATTGCTGTCCAGTTC-3′

BAD
Forward
5′-CGAAGGATGAGCGATGAGTT-3′

Reverse
5′-TAGAGTTCCGGGATGTGGAG-3′

Bid
Forward
5′-CAGGAAGAAATCATCCACAA-3′

Reverse
5′-GCTGCTTCACCTCATCAAG-3′

Caspase-3
Forward
5′-TGGTGATGAAGGGGTCATTT-3′

Reverse
5′-AGCCTCCACGGGTATCTTCT-3′

PARP
Forward
5′-ATTCCTAGCCGAAAGGAATGG-3′

Reverse
5′-TAGACAGGGGCTTGTCTGCT-3′

The measurement result of the mRNA expression level of BCL-2, which is a muscle cell anti-apoptotic factor, is shown in FIG. 11. BCL-2 was shown to increase significantly in the group (T) treated with the Aureobasidium pullulans fermentation product of Example 1 and the group (TP) treated with the Aureobasidium pullulans fermentation product of Example 2. In particular, it was measured at a higher level in the group (TP) treated with the Aureobasidium pullulans fermentation product of Example 2. Also, the mRNA expression level of Bax, BAD, Bid, caspase-3 and PARP, which are muscle cell pro-apoptotic factors, was measured and the result is shown in FIGS. 12 to 16, respectively. The expression of the muscle cell pro-apoptotic factors was generally lower in the group (T) treated with the Aureobasidium pullulans fermentation product of Example 1 and the group (TP) treated with the Aureobasidium pullulans fermentation product of Example 2. In particular, it was measured at a lower level in the group (TP) treated with the Aureobasidium pullulans fermentation product of Example 2 (#p<0.05, ##p<0.01, ###p<0.001 vs. CTL; * p<0.05, ** p<0.01, *** p<0.001 vs. Dex group).

Experimental Example 4: Analysis of Oxidative Stress

The content of the antioxidant glutathione (GSH) in C2C12 muscle cells was measured using a glutathione assay kit. Experiment was conducted by treating with the Aureobasidium pullulans fermentation product of Examples 1 and 2, or Comparative Example 1 at concentrations of 100 and 200 μg/mL, respectively, and the GSH content of the supernatant was measured using a glutathione assay kit (Cayman Co, USA). After adding 150 μL of an analytical cocktail in the kit to 50 μL of the supernatant and shaking with a microshaker in the dark, absorbance was measured at 405 nm using a microreader.

Meanwhile, in order to measure the level of reactive oxygen species (ROS) with oxidative power that attacks living tissue and damages cells in the C2C12 muscle cells, the cells were seeded onto a 24-well plate at 2×10⁵cells/mL and cultured for 24 hours. When the cells grew to 90%, differentiation was induced by replacing the medium with DMEM containing 2% HS and 1% P/S. The medium was exchanged every 2 days. On day 6 after the differentiation was completed, the Aureobasidium pullulans fermentation product of Examples 1 and 2 or Comparative Example 1 was treated at concentrations of 100 and 200 μg/mL, respectively, and the cells were washed with PBS 24 hours later and then treated with 10 UM dexamethasone to induce muscle atrophy. Then, the cells were washed with PBS, and after dispensing 1 mL of 10 UM DCF-DA to each well, were incubated in an incubator at 37° C. and 5% CO₂for 30 minutes. 30 minutes later, the cells were wash with PBS. After adding 1 mL of PBS to each well, the level of ROS in the cells was measured by measuring fluorescence at 485/20 nm for excitation and 528/20 nm for emission using a microplate reader.

The result of measuring the content of glutathione (GSH) is shown in FIG. 17, and the result of measuring the content of active oxygen species (ROS) is shown in FIG. 18. The glutathione (GSH) content was significantly higher level in the group treated with the Aureobasidium pullulans of Example 2 at 200 μg/mL as compared to the dexamethasone treatment group (Dex). And, the content of active oxygen species (ROS) was significantly lower in the groups treated with the Aureobasidium pullulans fermentation products of Examples 1 and 2 as compared to the dexamethasone treatment group (Dex) (#p<0.05, ##p<0.01, ###p<0.001 vs. CTL; * p<0.05, ** p<0.01, *** p<0.001, vs. Dex group).

Experimental Example 5: Analysis of Expression of Factors Related to Muscle Production and Muscle Cell Proteolysis (Western Blot)

The expression of proteins related to differentiation and muscle loss in C2C12 cells was measured by western blot. First, after treating C2C12 cells with the Aureobasidium pullulans fermentation product of Examples 1 and 2 or Comparative Example 1 at the nontoxic concentration of 100 or 200 μg/mL, dexamethasone was treated for 24 hours to induce muscle loss. After washing the cells 3 times with 1×PBS, they were lysed with 0.1 mL of a lysis buffer (50 mM HEPES, PH 7.4, 150 mM NaCl, 1% deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 μg/mL aprotinin). Proteins were separated by centrifuging the cell lysate at 12,000 rpm for 20 minutes. The concentration of the separated proteins was quantified with a protein assay solution, and then 30 μg of the proteins were mixed with a 5× sample buffer and separated by 8-15% SDS-PAGE. The separated proteins on the gel were transferred to an NC membrane and the membrane was blocked with 5% BSA for 1 hour at room temperature. After adding primary antibodies related to differentiation, apoptosis, and muscle loss to the membrane and incubating overnight at 4° C., the membrane was washed 3 times with TBS containing 0.05% Tween. After adding anti-igG conjugated HRP antibody to the membrane and incubating at room temperature for 1 hour, the membrane was washed 3 times with TBS (1×TTBS) containing 0.05% Tween and analyzed using a ChemiDoc™ Touch imaging system (BioRad, California, USA) using an ECL solution.

The result of measuring the expression level of MyoD and myogenin proteins, which are transcription factors involved in muscle cell production, is shown in FIG. 19. The expression level of myogenin was the highest in the group treated with the Aureobasidium pullulans fermentation product of Example 2 (TP), and the protein expression level of the MyoD was higher in the groups treated with the Aureobasidium pullulans fermentation product of Example 1 (T) and Example 2 (TP) as compared to the dexamethasone treatment group (Dex).

Also, the protein expression level of Foxo3a, MurF1 and PI3k, which are factors involved in signaling pathways related to protein synthesis and degradation, was measured after treating C2C12 myotube cells with the Aureobasidium pullulans fermentation product and dexamethasone for 24 hours in order to investigate the mechanism of action for improvement of muscle strength. The result is shown in FIG. 20 and FIG. 21. It was confirmed that the level of FOXO3α and MURF1 was increased significantly in the dexamethasone treatment group, and the level of Fox03a and MurF1, which are muscle cell proteolytic factors, was decreased significantly in the 200 μg/mL treatment groups of Example 1 (T) and Example 2 (TP). Also, it was confirmed that the degradation of muscle protein was inhibited in the treatment groups of Example 1 (T) and Example 2 (TP) because of increased p-PI3k in the PI3k pathway (#p<0.05, ##p<0.01, ###p<0.001 vs. CTL; * p<0.05, ** p<0.01, *** p<0.001, vs. Dex group).

Experimental Example 6: Analysis of Expression of Anti-Apoptotic Factors (Western Blot)

The protein expression level of caspase-3, cleaved-caspase-3, Bax, and Bcl-2 was measured according to the same western blot method as in Experimental Example 5. The result of measuring the caspase-3/cleaved-caspase-3 and Bax/Bcl-2 values is shown in FIG. 22.

It was confirmed that the caspase-3/cleaved-caspase-3 and Bax/Bcl-2 values that induce apoptosis were low in the treatment groups of Example 1 (T) and Example 2 (TP) and, in particular, they were lower in the Example 2 treatment group (TP), suggesting the effect of suppressing muscle cell death.

Experimental Example 7: Measurement of Beta-Glucan Content

Beta-glucan content was measured for the Aureobasidium pullulans