J East Asian Soc Diet Life 27(3): 348 363 (2017) http://dx.doi.org/10.17495/easdl.2017.6.27.3.348 348 1 1 2 1 1, 2 Mixed Culture Characteristics of Fungi Strains isolated from Korean Traditional Nuruk Ji-Young Mun 1, Seong-Yeol Baek 1, Hyeon-Su Ro 2 and Soo-Hwan Yeo 1 1 Fermented Food Science Division, Dept. of Agrofood Resources, NIAS, RDA, Wanju 55365, Korea 2 Dept. of Microbiology and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea ABSTRACT We analyzed the mixed culture characteristics of isolated brewing fungi obtained from Korean traditional nuruk for their development as fermentation starters. In a previous study, we selected appropriate fungi strains based on their large fungi bodies and strong enzyme activities. These fungi strains were mixed cultured under various combination of ratios, phs and temperatures. As a result, total acidities of R. delemar mixed culture media were higher than those of A. oryzae cultured at 20 and 37. The phs of A. luchuensis 34-1 and A. oryzae 37-7 as well as R. delemar 58-8 and A. oryzae 37-7 mixed culture media gradually increased with the culture period. On the contrary, total acidities of the mixed culture media gradually decreased. α-amylase activities of R. delemar 26-4 and R. oryzae 82-7 mixed culture media were strong at 20 and ph 4, glucoamylase activities of R. delemar 58-8 and A. oryzae 37-7 were strong at 37, ph 3 and 1:2 ratio, and acidic protease activities of R. delemar 26-4 and A. oryzae 78-5 were strong at 20, ph 4 and 1:4 ratio. This study provides the optimal mixed culture conditions for fermentation starters based on changes in ph and total acidity, favorable enzyme activities, and fungi varieties. Key words : Korean traditional nuruk, fungi, mixed culture, enzyme activity, starter Corresponding author : Soo-Hwan Yeo, Tel: +82-63-238-3610, Fax: +82-63-238-3843, E-mail: yeobio@korea.kr,,,,.,,,, (Kwon YH 2012; Park CS & Lee TS 2002).,, (So MH 1999).,, (Park JH 2012; Im SY 2014; Jeong JH 2015; Kim MS 2015). ( ), Aspergillus luchuensis (So MH & Lee JW 1996)..,. ( ). 2014, (IIT 2014; http://www.bioin.or.kr), (So MH & Lee YS 2009). Rhizopus sp. (Cho HK 2012), Rhizopus sp. (So MH & Lee YS 2009). A. luchuensis A. oryzae (So MH 1991), Rhizopus sp. Aspergillus sp., (So MH & Lee JW 1996).
27(3): 348 363 (2017) 349, 2 (So MH 1991; So MH & Lee JW 1996),.,. (Mun JY 2016), 2 (Rhizopus sp., Aspergillus sp.),. 1. (Mun JY 2016) 6,. 6 Table 1, Table 2., (species), (genus) 2. (20, 28, 37 ) ph(3, 4, 6, 8),,,. 2. 5% (wheat extract broth). 20, 60 6. (Miracloth, Calbiochem 475855, EMD Biosciences, Inc., Germany), 1 N HCl 1 N NaOH ph 3, 4, 6, 8. ph (121, 15 ), (20, 28, 37 ). 3. 1) ( 70 ), PDA 5 µl. Rhizopus sp. 4, Aspergillus sp. 7 28. 10 ml spreader,. (4 ). 2) (Hemocytometer, DHC-N01, INCYTO Co. Ltd., Republic of Korea), 10 6 CFU/mL., 1:1, Aspergillus sp. ( ) 5, 5 Rhizopus sp. ( ), Rhi.:Asp. = 1:1, 1:2, 1:4, 1:8. 1%(v/v), 1%. 4. ph, ph ph meter(benchtop ph meter Orion Star A211, Thermo Scientific Co., USA). 5 ml Table 1. Fungi strains used in this study Sample no. Strains S4 Rhizopus delemar 26-4 S5 Rhizopus delemar 58-8 S6 Rhizopus oryzae 82-7 S7 Aspergillus luchuensis 34-1 S10 Aspergillus oryzae 78-5 S11 Aspergillus oryzae 37-7 S4 S5 S6 S7 S10 S11
350 Table 2. Various mixed culture fungi used in this study Experiment Mixed culture within fungi species Mixed culture between fungi species Mixed culture between fungi genus Mixed culture fungi Rhizopus delemar 26-4 and Rhizopus delemar 58-8 Aspergillus oryzae 78-5 and Aspergillus oryzae 37-7 Rhizopus delemar 26-4 and Rhizopus oryzae 82-7 Aspergillus luchuensis 34-1 and Aspergillus oryzae 78-5 Rhizopus delemar 26-4 and Aspergillus oryzae 78-5 Rhizopus delemar 58-8 and Aspergillus oryzae 37-7 2 3 0.1 N NaOH. Total acidity (%, v/v) = [(0.1 N NaOH(mL) 0.1 N NaOH factor (1.0) 0.006 (conversion factor)) 5 (sample volume)] 100 5. 1) α-amylase - ( ). 2% 40 µl 40 µl 50 30. 1 M 20 µl, 100 µl. 150 µl 96-well plate(96well plate SP32096, SPL Life Science Co. Ltd., Korea) 570 nm. 0.0317 g 0.1 g, 10 % 50 ml 1 L. 0 20 mg/ml, 570 nm. α-amylase 50 60 1 mg 1 ml 1 unit. Unit/mL = [(A 570 control A 570 sample) A 570 mg soluble starch] 2 (60 min 30 min) 0.04 (sample volume) 2) Glucoamylase DNS(3,5-dinitrosalicylic acid). 2% 1 ml 0.2 M 0.2 ml 40 5. 0.1 ml 40 20, 1 N 0.1 ml, 30 1 N 0.1 ml. 50 µl DNS 150 µl 100 5. 150 µl 96-well plate 550 nm. 0.2 M 0.2 M 0.2 M 1:2, ph 5.0. 0.2 2 mg/ml DNS, 550 nm. Glucoamylase 40 20 1 mg 1 ml 1 unit. Unit/mL = glucose (mg) 30 (1.5 ml 0.05 ml) 0.1 (sample volume) 3) Acidic Protease 0.5% 0.15 ml 0.1 ml 40 5, 0.05 ml 40 60. TCA(trichloroacetic acid) 0.3 ml,. 0.1 ml 0.4 M 0.5 ml (Folin & Ciocalteu s phenol reagent F9252, Sigma-Aldrich Co. LLC., USA) 0.1 ml 40 30, 660 nm. 0.2 M -2-0.1 M 1:4, ph 3.0. L- 20 100 µg/ml,. Acidic protease 40 60 1 µg 1 ml 1 unit. Unit/mL = tyrosine (µg) 6 (0.6 ml 0.1 ml) 0.05 (sample volume) 6. 3 ±
27(3): 348 363 (2017) 전통누룩에서 분리한 양조용 곰팡이의 혼합 배양에 따른 품질 특성 타내었다. 통계처리는 SPSS 12.0(SPSS Inc., Chicago, IL)을 이용하여 이원분산분석(two-way ANOVA)을 수행하였고, 유 의성 비교는 Duncan s multiple range test로 p<0.05 수준에서 검증하였다. 결과 및 고찰 1. 양조용 곰팡이 종(Species) 내 혼합 배양 특성 (A) R. delemar 26-4, 58-8 mixed culture media (20 ) (C) R. delemar 26-4, 58-8 mixed culture media (37 ) (E) A. oryzae 78-5, 37-7 mixed culture media (28 ) 1) 351 배양액의 ph 및 총산도 양조용 곰팡이 R. delemar 26-4와 R. delemar 58-8, A. oryzae 와 A. oryzae 37-7의 배양 조건(온도, ph)에 따른 혼합 배 양액의 ph 및 총산도를 측정하여 Fig. 1에 제시하였다. 배양 기간이 길어짐에 따라 총산도가 점차 감소하고, ph는 증가하 는 경향을 나타내었으나, R. delemar 26-4와 R. delemar 58-8 혼합 배양액(20 )에서는 배양 3일째 총산도가 ph 4, ph 6에 서 배양 2일째보다 각각 2.3배, 1.4배 증가하였고, ph는 감소 78-5 (B) R. delemar 26-4, 58-8 mixed culture media (28 ) (D) A. oryzae 78-5, 37-7 mixed culture media (20 ) (F) A. oryzae 78-5, 37-7 mixed culture media (37 ) Fig. 1. Total acidity and ph of mixed culture media within fungi species upon various temperature and ph. (A) (C) R. delemar 26-4 and R. delemar 58-8 mixed culture media, (D) (F) A. oryzae 78-5 and A. oryzae 37-7 mixed culture media.
352 [Fig. 1 (A)],. ph ph, 37 4 ph ph [Fig. 1 (C), (F)]. 20 37 R. delemar 26-4 R. delemar 58-8 A. oryzae 78-5 A. oryzae 37-7, Rhizopus, (Liao W 2007; So MH & Lee YS 2010).. (28, ph 8), 20 3 4. A. oryzae 36 48 (So MH 1993), 37 7. 2) α-amylase ph R. delemar 26-4 R. delemar 58-8 α-amylase Fig. 2, A. oryzae 78-5 A. oryzae 37-7 Fig. 3. A. oryzae 78-5 A. oryzae 37-7 37, ph 3 α-amylase 86.2 units/ ml, ph 4 86.2 units/ml (p<0.05). R. delemar 58-8 28, ph 4 86.1 units/ml, A. oryzae 37-7 37, ph 3 86.3 units/ml (p<0.05). R. delemar ph 3, 37 3 4 2.5, 4 ph 5.3 (Noh JM 2013). A. oryzae ph 5 6 (Noh JM 2013), A. oryzae ph 20, 28 37, ph. 3) Glucoamylase ph R. delemar 26-4 R. delemar 58-8 glucoamylase Fig. 4, A. oryzae 78-5 A. oryzae 37-7 Fig. 5. R. delemar 26-4 R. delemar 58-8 37, ph 6, 3,668.2 units/ml (p<0.05). R. delemar 26-4 28, ph 8 3,865.6 units/ml, A. oryzae 37-7 37, ph 3 3,132.4 units/ml (p<0.05). So MH & Lee YS(2009) Rhizopus sp. 28 48, Kim CJ (1985) R. oryzae 30, ph 3.5 4, 4) Acidic Protease ph R. delemar 26-4 R. delemar 58-8 acidic protease Fig. 6, A. oryzae 78-5 A. oryzae 37-7 Fig. 7. A. oryzae 78-5 A. oryzae 37-7 28, ph 3 acidic protease 51.1 units/ml (p<0.05), R. delemar 58-8 37, ph 8 47.9 units/ml, A. oryzae 78-5 20, ph 8 72.7 units/ml (p<0.05). R. delemar 37, A. oryzae 20, ph. 28 32, (So MH & Lee YS 2010; So MH 1993),. 2. (Species) 1) ph R. delemar 26-4 R. oryzae 82-7, A. luchuensis 34-1 A. oryzae 37-7 ph, Fig. 8. R. delemar 26-4 R. oryzae 82-7 [Fig. 8 (A)] 2 ph, 3 ph,, ph 3 4 ph 4, Rhizopus sp. (Liao W 2007; So MH & Lee YS 2010). A. luchuensis 34-1 A. oryzae 37-7 [Fig. 8 (B)], ph 7 ph 7 8. A. luchuensis
27(3): 348 363 (2017) 353 R. delemar 26-4, 58-8 (20 ) R. delemar 26-4, 58-8 (28 ) R. delemar 26-4, 58-8 (37 ) Fig. 2. α-amylase activity of mixed culture media within fungi species (R. delemar 26-4 and 58-8) upon various temperature and ph. Symbols: S4, R. delemar 26-4; S4+S5, R. delemar 26-4 + R. delemar 58-8; S5, R. delemar 58-8. * p<0.05.
354 A. oryzae 78-5, 37-7 (20 ) A. oryzae 78-5, 37-7 (28 ) A. oryzae 78-5, 37-7 (37 ) Fig. 3. α-amylase activity of mixed culture media within fungi species (A. oryzae 78-5 and 37-7) upon various temperature and ph. Symbols: S10, A. oryzae 78-5; S10+S11, A. oryzae 78-5 + A. oryzae 37-7; S11, A. oryzae 37-7. * p<0.05.
27(3): 348 363 (2017) 355 R. delemar 26-4, 58-8 (20 ) R. delemar 26-4, 58-8 (28 ) R. delemar 26-4, 58-8 (37 ) Fig. 4. Glucoamylase activity of mixed culture media within fungi species (R. delemar 26-4 and 58-8) upon various temperature and ph. Symbols : S4, R. delemar 26-4; S4+S5, R. delemar 26-4 + R. delemar 58-8; S5, R. delemar 58-8 * p<0.05.
356 A. oryzae 78-5, 37-7 (20 ) A. oryzae 78-5, 37-7 (28 ) A. oryzae 78-5, 37-7 (37 ) Fig. 5. Glucoamylase activity of mixed culture media within fungi species (A. oryzae 78-5 and 37-7) upon various temperature and ph. Symbols : S10, A. oryzae 78-5; S10+S11, A. oryzae 78-5 + A. oryzae 37-7; S11, A. oryzae 37-7. * p<0.05.
27(3): 348 363 (2017) 357 R. delemar 26-4, 58-8 (20 ) R. delemar 26-4, 58-8 (28 ) R. delemar 26-4, 58-8 (37 ) Fig. 6. Acidic protease activity of mixed culture media within fungi species (R. delemar 26-4 and 58-8) upon various temperature and ph. Symbols: S4, R. delemar 26-4; S4+S5, R. delemar 26-4 + R. delemar 58-8; S5, R. delemar 58-8. * p<0.05.
358 A. oryzae 78-5, 37-7 (20 ) A. oryzae 78-5, 37-7 (28 ) A. oryzae 78-5, 37-7 (37 ) Fig. 7. Acidic protease activity of mixed culture media within fungi species (A. oryzae 78-5 and 37-7) upon various temperature and ph. Symbols: S10, A. oryzae 78-5; S10+S11, A. oryzae 78-5 + A. oryzae 37-7; S11, A. oryzae 37-7. * p<0.05.
27(3): 348 363 (2017) 359 (A) R. delemar 26-4, R. oryzae 82-7 (ph 4, 20 ) (B) A. luchuensis 34-1, A. oryzae 37-7 (ph 3, 37 ) Fig. 8. Total acidity and ph of mixed culture media between fungi species. (A) R. delemar 26-4 and R. oryzae 82-7 at ph 4, 20, (B) A. luchuensis 34-1 and A. oryzae 37-7 at ph 3, 37. Symbols: S4, R. delemar 26-4; S4+S6, R. delemar 26-4 + R. oryzae 82-7; S6, R. oryzae 82-7; S7, A. luchuensis 34-1; S7+S11, A. luchuensis 34-1 + A. oryzae 37-7; S11, A. oryzae 37-7., A. oryzae (So MH 1991). A. luchuensis. 2) (α-amylase, Glucoamylase, Acidic Protease) R. delemar 26-4 R. oryzae 82-7 (20, ph 4) Fig. 9 (A) (C). α-amylase R. delemar 26-4 R. oryzae 82-7 (85.8 units/ml, p<0.05), glucoamylase R. delemar 26-4 (2,572.1 units/ml),. Acidic protease R. oryzae 82-7 30.5 units/ml (p<0.05). α-amylase, glucoamylase, acidic protease 2 3. A. luchuensis 34-1 A. oryzae 37-7 (37, ph 3) Fig. 9 (D) (F). α-amylase A. oryzae 37-7 (82.0 units/ml). Glucoamylase A. luchuensis 34-1 (3,032.7 units/ml, p<0.05), acidic protease A. luchuensis 34-1 A. oryzae 37-7 3 30.3 units/ml. 3. (Genus) 1) ph R. delemar 26-4 A. oryzae 78-5 (20, ph 4) ph Fig. 10 (A), R. delemar 58-8 A. oryzae 37-7 (37, ph 3) Fig. 10 (B). R. delemar 26-4 A. oryzae 78-5, ph ph 4. R. delemar 58-8 A. oryzae 37-7, ph ph 6 8. A. oryzae R. japonicus So MH & Lee JW(1996) A. oryzae ph, R. japonicus ph. R. delemar 58-8 A. oryzae 37-7 2 ph 58-8:37-7 = 1:8, 1:4, 1:2, 1:1, 3 1:1, 1:2 ph 1:4, 1:8. 2) (α-amylase, Glucoamylase, Acidic Protease) R. delemar 26-4 A. oryzae 78-5
문지영 백성열 노현수 여수환 360 東아시아 食生活學會誌 (B) R. delemar 26-4, R. oryzae 82-7 (ph 4, 20 ) (C) R. delemar 26-4, R. oryzae 82-7 (ph 4, 20 ) (D) A. luchuensis 34-1, A. oryzae 37-7 (ph 3, 37 ) (F) A. luchuensis 34-1, A. oryzae 37-7 (ph 3, 37 ) (A) R. delemar 26-4, R. oryzae 82-7 (ph 4, 20 ) (E) A. luchuensis 34-1, A. oryzae 37-7 (ph 3, 37 ) Fig. 9. Enzyme activities of mixed culture media between fungi species. (A) (C) R. delemar 26-4 and R. oryzae 82-7 at ph 4, 20, (D) (F) A. luchuensis 34-1 and A. oryzae 37-7 at ph 3, 37. Symbols: S4, R. delemar 26-4; S4+S6, R. delemar 26-4 + R. oryzae 82-7; S6, R. oryzae 82-7; S7, A. luchuensis 34-1; S7+S11, A. luchuensis 34-1 + A. oryzae 37-7; S11, A. oryzae 37-7. Values with different capital letters A D are significantly different among the culture periods in the same sample (p<0.05). Values with different small letters a c are significantly different among the samples in the same culture periods (p<0.05). (A) R. delemar 26-4, A. oryzae 78-5 (ph 4, 20 ) (B) R. delemar 58-8, A. oryzae 37-7 (ph 3, 37 ) Fig. 10. Total acidity and ph of mixed culture media between fungi genus. (A) R. delemar 26-4 and A. oryzae 78-5 at ph 4, 20, (B) R. delemar 58-8 and A. oryzae 37-7 at ph 3, 37. Symbols: S4, R. delemar 26-4; S4+S10, R. delemar 26-4 + A. oryzae 78-5; S10, A. oryzae 78-5; S5, R. delemar 58-8; S5+S11, R. delemar 58-8 + A. oryzae 37-7; S11, A. oryzae 37-7.
27(3): 348 363 (2017) 전통누룩에서 분리한 양조용 곰팡이의 혼합 배양에 따른 품질 특성 양액(20, ph 4)의 효소활성 결과를 Fig. 11 (A)(C)에, R. delemar 58-8과 A. oryzae 37-7 혼합 배양액(37, ph 3)의 효 소활성 결과를 Fig. 11 (D)(F)에 제시하였다. 효소별 유의 적(p<0.05)으로 가장 높은 활성이 나타난 조건은 α-amylase의 경우, R. delemar 58-8과 A. oryzae 37-7을 1:8의 비율로 3일간 배양했을 때 84.8 units/ml이었고, glucoamylase는 R. delemar 58-8과 A. oryzae 37-7을 1:2의 비율로 4일간 배양했을 때 3,393.0 units/ml이었으며, acidic protease는 R. delemar 26-4 와 A. oryzae 78-5를 1:4의 비율로 3일간 배양했을 때 89.5 units/ml로 나타났다. 전반적으로 acidic protease 활성은 R. delemar 26-4와 A. oryzae 78-5 혼합 배양액이 3090 units/ml 범위로 나타난 것과 달리, R. delemar 58-8과 A. oryzae 37-7 혼합 배양액은 2030 units/ml 범위에 그쳤다. A. oryzae 누 361 룩과 R. japonicus 누룩을 병용하여 발효시킨 연구에서 AO:RJ = 1:2 비율일 때 아미노산 함량이 가장 높았으며, 이는 누룩 병용으로 단백질 분해효소의 종류가 다양해진 결과라고 보 고되었다(So MH & Lee JW 1996). 본 연구에서도 단일 균주 로 배양했을 때보다 혼합 균주(R. delemar와 A. oryzae)로 배 양한 경우 protease 활성이 높았지만, So MH & Lee JW(1996) 가 보고한 결과와는 다소 차이가 있었다. 요약 및 결론 본 연구진의 선행연구(Mun JY 등 2016)에서 도출한 토착 곰팡이의 증식 속도, 균체 성장능 및 효소활성이 우수한 배 양조건을 바탕으로, 본 연구에서는 Rhizopus sp.와 Aspergillus (B) R. delemar 26-4, A. oryzae 78-5 (ph 4, 20 ) (D) R. delemar 58-8, A. oryzae 37-7 (ph 3, 37 ) (F) R. delemar 58-8, A. oryzae 37-7 (ph 3, 37 ) (A) R. delemar 26-4, A. oryzae 78-5 (ph 4, 20 ) (C) R. delemar 26-4, A. oryzae 78-5 (ph 4, 20 ) (E) R. delemar 58-8, A. oryzae 37-7 (ph 3, 37 ) Fig. 11. Enzyme activities of mixed culture media between fungi genus. (A) (C) R. delemar 26-4 and A. oryzae 78-5 at ph 4, 20, (D) (F) R. delemar 58-8 and A. oryzae 37-7 at ph 3, 37. Symbols: S4, R. delemar 26-4; S4+S10, R. delemar 26-4 + A. oryzae 78-5; S10, A. oryzae 78-5; S5, R. delemar 58-8; S5+S11, R. delemar 58-8 + A. oryzae 37-7; S11, A. oryzae 37-7. Values with different capital letters A D are significantly different among the culture periods in the same sample (p<0.05). Values with different small letters a f are significantly different among the samples in the same culture periods (p<0.05).
362 sp.,. R. delemar 20 37 A. oryzae, R. delemar 26-4 R. oryzae 82-7 2 3. A. luchuensis 34-1 A. oryzae 37-7, R. delemar 58-8 A. oryzae 37-7 ph,. α-amylase A. oryzae 37-7(37, ph 3, ), A. oryzae 78-5 A. oryzae 37-7(37, ph 3 4, ), R. delemar 58-8(28, ph 4, ), R. delemar 26-4 R. oryzae 82-7(20, ph 4, ). Glucoamylase R. delemar 26-4(28, ph 8, ), R. delemar 26-4 R. delemar 58-8(37, ph 6, ), R. delemar 58-8 A. oryzae 37-7(37, ph 3, 1:2 ), A. oryzae 37-7(37, ph 3, ). Acidic protease R. delemar 26-4 A. oryzae 78-5(20, ph 4, 1:4 ), A. oryzae 78-5(20, ph 8, ), A. oryzae 78-5 A. oryzae 37-7(28, ph 3, ), R. delemar 58-8(37, ph 8, ). ph,,, R. delemar 26-4 R. oryzae 82-7(20, ph 4), R. delemar 58-8 A. oryzae 37-7(37, ph 3, 1:2 ), R. delemar 26-4 A. oryzae 78-5(20, ph 4, 1:4 ).,,. ( PJ00999301),. REFERENCES Cho HK, Seo WT, Lee JY, Cho KM (2012) Quality characteristics of cereal Makgeolli rice Nuruk prepared Rhizopus oryzae CCS01. J Korean Soc Food Sci Nutr 41(7): 1002-1008. http://www.bioin.or.kr/board.do?cmd=view&bid=system&num =229722. Accessed on August 27, 2012. IIT (2014) Effect of Nagoya protocol on industry. Trade Brief, Korea. No. 59, Institute for International Trade, Seoul. pp 1-8. Im SY, Baek CH, Baek SY, Park HY, Choi HS, Choi JH, Jeong ST, Shin WC, Park HD, Yeo SH (2014) Quality characteristics of Takju according to different rice varieties and mixing ratio of Nuruk. Korean J Food Preserv 21(6): 892-902. Jeong JH, Chai HS, Lee YH, Kim JM, Lee JH (2015) Quality characteristics of Takju, Yakju, spirit made by cereal Nuruks. The Korean Journal of Culinary Research 21(1): 267-280. Kim CJ, Oh MJ, Lee JS (1985) Studies on digestion of raw starch by Rhizopus oryzae. Korean J Appl Microbiol Biocng 13(4): 329-337. Kim MS, Lee YS, Kim JS, Shin WC, Sohn HY (2015) In-vitro anti-thrombosis activity of R4-Nuruk made from Rhizopus oryzae KSD-815. Microbiol Biotechnol Lett 43(2): 169-174. Kwon YH, Lee AR, Kim JH, Kim HR, Ahn BH (2012) Changes of physicochemical properties and microbial during storage of commercial Makgeolli. Korean J Mycol 40(4): 210-214. Liao W, Liu Y, Frear C, Chen S (2007) A new approach of pellet formation of a filamentous fungus - Rhizopus oryzae. Bioresource Technol 98: 3415-3423. Mun JY, Baek SY, Park HY, Ro HS, Yeo SH (2016) Cultural characteristics of fungi strains isolated from Korean Nuruk. J East Asian Soc Diet Life 26(2): 125-140. Noh JM, Choi JH, Jung ST, Yeo SH, Park JW, Lee JW, Choi HS (2013) Mycelial production and amylase activity of fungi for brewing in different submerged culture conditions. J East Asian Soc Dietary Life 23(6): 833-838. Park CS, Lee TS (2002) Quality characteristics of Takju prepared by wheat flour Nuruks. Korean J Food Sci Technol 34(2): 296-302. Park JH, Yeo SH, Choi JH, Jeong ST, Choi HS (2012) Production of Makgeolli using rice treated with Gaeryang-Nuruk (for non-steaming process) extract. Korean J Food Preserv 19(1): 144-152. So MH (1991) Improvement in the quality of Takju by the combined use of Aspergillus kawachii and Aspergillus oryzae. Korean J Food & Nutr 4(2): 115-124.
27(3): 348 363 (2017) 363 So MH (1993) Conditions for the production of amylase and protease in making wheat flour Nuruk by Aspergillus oryzae L2. Korean J. Food & Nutr 6(2): 89-95. So MH, Lee JW (1996) Takju brewing by combined use of Rhizopus japonicus-nuruk and Aspergillus oryzae-nuruk. J Korean Soc Food Nutr 25(1): 157-162. So MH, Lee YS, Noh WS (1999) Improvement in the quality of Takju by a modified Nuruk. Korean J Food & Nutr 12(4): 427-432. So MH, Lee YS (2009) Effects of culture conditions of Rhizopus sp. ZB9 on the production of saccharifying amylase during the preparation of rice Koji. Korean J Food & Nutr 22(4): 644-649. So MH, Lee YS (2010) Effects of culture conditions of Rhi- zopus sp. ZB9 on the production of organic acid during the preparation of rice Koji. Korean J Food & Nutr 23(1): 70-75. Date Received Date Revised Date Accepted Nov. 16, 2016 Feb. 27, 2017 May 12, 2017