2 전준영ㆍ이미향ㆍ정인학ㆍ정민정ㆍ김병목 Aspergillus kawachii (Kim et al., 215a),. ( ),.,, amylase protease, ph,, NaCl amylase protease.. 재료및방법 재료및종균 216. Aspergillus lu

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1 한수지 51(4), $, 218 Original Article Korean J Fish Aquat Sci 51(4),$,218 해조류첨가가쌀코지제조와효소활성에미치는영향 전준영 이미향 1 정인학 1 정민정 김병목 한국식품연구원, 1 강릉원주대학교해양식품공학과 Effects of Seaweeds on Rice Koji Production and Enzyme Activity Joon-Young Jun, Mi-Hyang Lee 1, In-Hak Jeong 1, Min-Jeong Jung and Byoung-Mok Kim Division of Strategic Food Research, Korea Food Research Institute, Gangneung 2544, Korea 1 Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung 25457, Korea This study investigated the effects of selected seaweeds on rice koji preparation (rice inoculated with Aspergillus luchuensis) and enzyme activity. Four types of rice koji were prepared using one of three seaweeds (.5% laver,.5% kelp and.5% green laver) or a control for 72 h. The changes in the moisture content, water activity, ph, total mold cell count, amylase and protease activities were measured. During preparation, there was no significant difference in the moisture content among the four kojis, whereas the ph in the kojis made with either laver or green laver decreased rapidly compared with the control (P<.5). This seemed to result from the seaweeds promoting the growth of mold cells. In terms of the activities of both amylase and protease, the koji with laver was superior. Subsequently, the amylase and protease activities of the koji made with laver were evaluated at various phs (3 to 9), temperatures (15-55 ), and NaCl concentrations (-1%). The activities of both enzymes decreased notably at ph 9 and the protease activity decreased at temperatures above 45. Although the activities of both enzymes decreased at greater than 2.5% NaCl, activity was present at 1% NaCl. Key words: Rice koji, Aspergillus luchuensis, Seaweed, Enzyme activity, NaCl 서론,,, (Mok et al., 25).,,,,,, (Kim and Lyu, 21).,, (Lim, 28; Park et al., 27). (koji), ( ).,, (Kim et al., 212). Aspergillus luchuensis (syn. A. kawachii) Aspergillus niger amylase, maltase, A. oryzae ph (Kim et al., 213). amylase protease (So and Lee, 21). (Kwon et al., 213), (Lee et al., 211), (Song and Lee, 213), (Kim et al., 215b), seed mash (Lee et al., 25), Aspergillus (Choi et al., 211), soybean koji rice koji (Jun et al., 216), Korean J Fish Aquat Sci 51(4), August 218 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 14 May 218; Revised 7 June 218; Accepted 19 June 218 Corresponding author: Tel: Fax: address: bmkim@kfri.re.kr Copyright 218 The Korean Society of Fisheries and Aquatic Science 1 pissn: , eissn:

2 2 전준영ㆍ이미향ㆍ정인학ㆍ정민정ㆍ김병목 Aspergillus kawachii (Kim et al., 215a),. ( ),.,, amylase protease, ph,, NaCl amylase protease.. 재료및방법 재료및종균 216. Aspergillus luchuensis (Ulsan, Republic of Korea)., 4 mesh.. 쌀코지제조 Jun et al. (216)., (1 kg) 16, 4 3.5% (w/w) , UV,.2% A. luchuensis 3. (85%) (3 ) 72. 시료의수집및조효소액추출 g,,, ph.. 2 g 1 ml 1, 3 3. (1, g, 4, 1 ). 수분함량및수분활성도 (925.45; AOAC, 25)., 5 g 2 1, (HP23-AW-A, Rotronic, Switzerland). ph 및곰팡이수 ph, 5 g 45 ml 1, (7, g, 15 ) ph meter (CH/Seven Easy-s2K, Mettler Toledo, Switzerland). (Park and Oh, 1995). 2 g.1% peptone water 18 ml (26 rpm, 5 ),. 1 ml potato dextrose agar (Difco; Becton Dckinson, Spark, MD, USA), colony (15-15) log CFU/g. Amylase 활성 amylase Bechman et al. (212) 3,5-dinitrosalicylic acid..1 M acetate buffer (ph 5.) 1% soluble starch 1 ml 1 ml ml, 96 mm 3,5-dinitrosalicylic acid.5 ml ml 15, 54 nm. maltose.2% maltose, standard curve. Amylase 1 g 1 mg maltose 1 unit (U). Protease 활성 Protease O-phthalaldehyde (Shakerian et al., 214)..21 M sodium phosphate buffer (ph 8.2).5% casein 1 ml 1 ml % trichloroacetic acid 4 ml, (7, g, 1 ).

3 해조류첨가쌀코지의효소활성 3 2 µl O-phthalaldehyde reagent 3 ml nm..5% serine standard curve. Protease 1 g serine 1 µg 1 U. O-phthalaldehyde reagent O-phthaladehyde 8 mg methanol 2% SDS 5 ml, 1 mm sodium tetraborate decahydrate 5 ml, 2-mercaptoethanol 3 µl 1 ml.. ph, 온도및 NaCl 농도가 amylase 와 protease 활성에미치는영향, ph, NaCl. ph 3, 5, 7, 9, 15, 25, 35, 45, 55, NaCl, 2.5, 5, 7.5, 1%. ph. ph 3,.1 M glycine-hcl buffer; ph 5,.1 M acetate buffer; ph 7,.1 M potassium phosphate buffer; ph 9,.1 M tris-hcl buffer. NaCl NaCl. ph (1 ) NaCl (%), NaCl (1 ) (35 ). amylase protease. 통계분석, IBM SPSS statistics program (Version 23; IBM Co., Amork, NY, USA) one-way ANOVA 95% Tukey s test. 결과및고찰 수분함량및수분활성도,.1-1% (72 ) amylase protease,,.5% (data not shown)..5%, Table 1.,,., 72 (P<.5). Aspergillus.65 (Stevenson et al., 215), %, ph 및곰팡이수 shochu A. kawachii ph ( ph 3.), citric acid (Mikami et al., 1987; Nagamine et al., 23). Kim et al. (213) A. oryzae citric acid, malic acid, lactic acid (dominant), A. kawachii citric acid, succinic acid Table 1. Changes in the moisture content and water activity of the rice kojis with different seaweeds during solid-state fermentation Incubation time (h) Control Laver Kelp Green laver Moisture (%) Water activity Moisture (%) Water activity Moisture (%) Water activity Moisture (%) Water activity 43.6±6.9 A ns.98±.1 A ns 49.5±3.6 A.98±.1 A 43.2±9.5 A.96±.1 A 39.1±3.6 A.99±.1 A ±7.5 AB ns.95±.3 AB ns 46.4±6.6 AB.98±.1 A 42.±2.6 A.97±.1 A 32.4±5.8 AB.99±.1 A ±4.3 ABC ns.89±.2 ABC a 4.±6. ABC.91±.2 B a 32.3±2.8 AB.9±.2 AB a 31.4±6.2 AB.84±.1 B b ±1.1 ABC ns.88±.5 ABC ns 36.±9.6 ABC.87±.3 BC 24.7±9.5 B.9±.5 AB 28.1±3.4 AB.83±.1 B ±6.8 BC ns.85±.8 BC ns 31.7±4.5 BC.84±.1 CD 24.8±6.7 B.88±.3 B 28.3±6. AB.79±.2 C 6 26.±1.3 BC ns.81±.1 C bc 29.1±3.2 C.83±.1 CD ab 23.7±2.6 B.86±.2 B a 26.3±3.7 AB.79±.1 C c ±3.6 C ns.81±.1 C ab 27.1±5.5 C.82±.2 D ab 22.1±2.1 B.84±.3 B a 24.2±5.5 B.78±.1 C b The different capital letters indicate significantly different values in the vertical column (P<.5). No significant difference in the horizontal column (P<.5). The different small letters indicate significantly different values in the horizontal column (P<.5). Data expressed as the mean±sd. in triplicate.

4 4 전준영ㆍ이미향ㆍ정인학ㆍ정민정ㆍ김병목 8 1 ph Mold cell count (log CFU/g koji) Fig. 1. Changes in the ph and mold cell count of the rice kojis with different seaweeds during solid-state fermentation. Symbols indicate:, control (without seaweed);, laver;, kelp;, green laver. Significant difference at P<.5 versus the control (ANOVA). Data expressed as the mean±sd. in triplicate. oxalic acid., ph. 225 ph Fig. 1. ph 2 (ph ), ph (2.8 )., (P<.5) ph..2% 1 (w/w) 6 log CFU/g (wet basis), , 7.6 log CFU/ g 8.2 log 12 CFU/g (P<.5) ph 12 Incubation time (h),, life cycle.,,, (Madigan and Martinko, 26), ph 2. Amylase activity (U/g koji) Amylase 및 protease 활성 Amylase activity (U/g koji) , ,,, 12. Bechman et al. (212), 1 A. oryzae 58 -amylase. 8 72, amylase 72 6 (Fig. 2A). Amylase 36, 4 (P<.5)., (P<.5), amylase 1 g 1,74 U, 1,733 U, 1,586 U, 1,378 U Amylase 72 Incubation time (h), protease 36 (Fig. 2B). 24, 36 (P<.5). 7% 9% (Choi, 2 21), protease,a a,b ab,bc ab,c b,c b amylase, A a, B a b 175, A b, B b, B b, B b. Aspergillus 15, Protease activity (U/g koji) Amylase activity (U/g koji)

5 해조류첨가쌀코지의효소활성 Amylase Amylase activity activity (U/g koji) (U/g koji) Incubation 36 time (h) Protease Protease activity activity (U/g koji) (U/g koji) Incubation 36time (h) Fig. 2. Changes in the amylase and protease activities of the rice kojis with different seaweeds during solid-state fermentation. Symbols indicate:, control (without seaweed);, laver;, kelp;, green laver. One unit of amylase activity was defined as the one milligram of maltose released from 1% soluble starch per a minute. One unit of protease activity was defined as the one microgram of leucine released from.5% casein per a minute. Significant difference at P<.5 versus the control (ANOVA). Data expressed as the mean±sd. in triplicate. Amylase Amylase activity activity (U/g koji) (U/g koji) ,C b,b a,a a,a a,a a,,c B ab,,b B a,,a B ab,,a A a,,a A a, b,, B AB a b,, A AB a c,, A AB a b, b, B b 15, B b 25, AB b 35, AB c 45, AB b 55, A b Temperature 35 ( ) Amylase Amylase activity activity (U/g koji) (U/g koji) ,A a,,a A aa, b, A b,b ab,,b B aba, b, B b,bc ab,,bc B aab, b, B b,c b,,c B ba, b, B b NaCl 5(%) 7.5 1,C b b,,c B ba, b c Temperature ( ) NaCl (%) Fig. 3. Effects of ph, temperature and NaCl concentration on the amylase activity of the rice koji with.5% laver. Symbols indicate:, ph 3;, ph 5;, ph 7;, ph 9. One unit of amylase activity was defined as the one milligram of maltose released from 1% soluble starch per a minute. The different capital letters indicate significantly different values in the horizontal column (P<.5). The different small letters indicate significantly different values in the vertical column (P<.5). Data expressed as the mean±sd. in triplicate. -amylase protease (Mikami et al., 1987; Siala 1 et al., 29). protease ph 1 9 8, 36 9 protease , Protease Protease activity activity (U/g koji) (U/g koji) ,E b, C b,d b,a c, A b,c c, B c ph, 1,A c,c b,c a. 1 9, A a, B a, C a, D a,,a A cb,,b B ba,,,b B bab, protease,c B b,,c B a 8 9,, A ca,, B ca,, C B ac,, C ca,, D ba, A b 48, B a, B ab, B b 7 8, A c, C c, C c, D b. Protease Protease activity activity (U/g koji) (U/g koji)

6 6 전준영ㆍ이미향ㆍ정인학ㆍ정민정ㆍ김병목 Protease activity (U/g koji) ,E b, C b, D b,d b, C a,a c, A b, A b ,C c, B c, AB b Protease activity (U/g koji) ,A c, A b, A c b, C c,c b, B b, C c ,C a, D a, D b Temperature ( ) NaCl (%) Fig. 4. Effects of ph, temperature and NaCl concentration on the protease activity of the rice koji with.5% laver. Symbols indicate:, ph 3;, ph 5;, ph 7;, ph 9. One unit of protease activity was defined as the one microgram of leucine released from.5% casein per a minute. The different capital letters indicate significantly different values in the horizontal column (P<.5). The different small letters indicate significantly different values in the vertical column (P<.5). Data expressed as the mean±sd. in triplicate. ph, 온도및 NaCl 농도가 amylase 활성에미치는영향 ph, NaCl,.5%, 48, ph, NaCl amylase (Fig. 3)., ph 3-9, amylase ph, ph. ph 9 amylase ph. amylase, 15 ph 5 7 ph 3 9, 25, 45, 55 ph 3, 5, 7 (P<.5). NaCl -1% amylase ph NaCl 2.5%, NaCl 1%., amylase ph NaCl, ph 9 NaCl ph 5 7 (P<.5). ph, 온도및 NaCl 농도가 protease 활성에미치는영향 ph protease amylase (Fig. 4). Amylase, protease ph 9 ph 35-45,. protease, 15 ph 5 ph 3, 7, 9, 25 ph 3 ph 5, 7, 9 (P<.5) ph 5 7 ph 3 9 (P<.5). NaCl protease ph 9 amylase. ph 9 NaCl., amylase protease ph 9,, 35.,. amylase protease, 1% NaCl. 사사 (E ),. References AOAC. 25. AOAC official methods of analysis, 18th ed.

7 해조류첨가쌀코지의효소활성 7 AOAC International publishing, Gaithersburg, MD, U.S.A. Bechman A, Phillips RD and Chen J Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage. J Food Sci 77, M318- M Choi I. 21. Physicochemical properties of rice cultivars with different amylose contents. J Korean Soc Food Sci Nutr 39, Choi JS, Jung ST, Kim JY, Choi JH, Choi HS and Yeo SH Quality characteristics of wheat nuruk and optimum condition of liquid starters for Aspergillus sp. Korean J Microbiol Biotechnol 39, Jun JY, Lim YS, Lee MH, Kim BM and Jeong IH Changes in the physiochemical quality of sailfin sandfish Arctoscopus japonicus sauces fermented with soybean koji or rice koji during storage at room temperature. Korean J Fish Aquat Sci 49, Kim BM, Lim JH, Jung JH, Jung MJ, Kim DS, Lee KP, Jun JY and Jeong IH. 215a. Quality changes and processing of fermented red snow crab Chionoecetes japonicus sauce using Aspergillus kawachii koji. Korean J Fish Aquat Sci 48, Kim JS, Lee JH, Chang YE, Kim GC and Kim KM The quality characteristics of rice mash by mixing ratios of rice and rice koji. J Korean Soc Food Sci Nutr 42, Kim JH, Kwon YH, Lee AR, Kim HR and Ahn BH Manufacture of koji using fungi isolation from nuruk and identification of koji molds. Kor J Mycol 4, doi.org/1.4489/kjm Kim MJ, Kim YH, Kim DW, Park HS and Kim MK. 215b. Brewing and quality characteristics of rice-wine using Basidionmycetes rice koji. J Agric Life Sci 46, Kim HS and Lyu ES. 21. Optimization of sulgidduk with green laver powder using a response surface methodology. Korean J Food Cook Sci 26, Kwon YH, Lee AR, Kim HR, Kim JH and Ahn BH Quality properties of makgeolli brewed with various rice and koji. Korean J Food Sci Technol 45, org/1.9721/kjfst Lee MK, Park JK and Lee SK. 25. The physicochemical properties of seed mash prepared with koji. Korean J Food Sci Technol 37, Lee SE, Suh HJ and Hwang JH Characteristics of rice doenjang prepared with brown rice koji. Korean J Food Preserv 18, kjfp Lim EJ. 28. Quality characteristics of cookies with added Enteromorpha intenstinalis. J Korean Soc Food Sci Nutr 21, Medigan MT and Martinko JM. 26. Brock biology of microorganisms, 11th edn. Pearson Education Inc, NJ, U.S.A., Mikami S, Iwano K, Shinoki S and Shimada T Purification and some properties of acid-stable α-amylases from shochu koji (Aspergillus kawachii). Agric Biol Chem 51, Mok JS, Park HY and Kim JH. 25. Trace metal contents and safety evaluation of major edible seaweeds from korean coast. J Korean Soc Food Sci Nutr 34, Nagamine K, Murashima K, Kato T, Shimoi H and Ito K. 23. Mode of α-amylase production by the shochu koji mold Aspergillus kawachii. Biosci Biotechnol Biochem 67, Park JM and Oh HI Changes in microflora and enzyme activities of traditional kochujang meju during fermentation. Korean J Food Sci Technol 27, Park YK, Kang SG, Jung ST, Kim DH, Kim SJ, Pak JI, Kim CH, Rhim JW and Kim JM. 27. Development of valueadded products using seaweeds. J Mar Biosci Biotechnol 2, Siala R, Sellami-Kamoun A, Hajji M, Abid I, Gharsallah N and Nasri M. 29. Extracellular acid protease from Aspergillus niger I1: purification and characterization. Afr J Biotechnol 8, So MH and Lee YS. 21. Effects of culture conditions of rhizopus sp. ZB9 on the production of protease during preparation of rice koji. J Korean Soc Food Sci Nutr 23, Song YC and Lee SP Evaluation in physicochemical properties of soy sauce fortified with soymilk residue (okara koji). Korean J Food Preserv 2, org/1.112/kjfp Shakerian M, Razavi SH, Khodaiyan F, Zial SA, Yarmand MS and Moayedi A Effect of different levels of fat and inulin on the microbial growth and metabolites in probiotic yogurt containing nonviable. Int J Food Sci Technol 49, Stevenson A, Cray JA, Williams JP, Santos R, Sahay R. Neuenkirchen N et al Is there a common water-activity limit for the three domains of life?. ISME J 9, doi.org/1.138/ismej

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DBPIA-NURIMEDIA 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