한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 α-galactosidase 를생산하는고온성 Bacillus coagulans KM-1 균주의생화학적특성 남기호 * 장미순

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1 한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 α-galactosidase 를생산하는고온성 Bacillus coagulans KM-1 균주의생화학적특성 남기호 * 장미순 박희연 이레나코네바 1 국립수산과학원식품안전과, 1 태평양수산연구소틴로센터 Biochemical Characterization of α-galactosidase-producing Thermophilic Bacillus coagulans KM-1 Ki Ho Nam*, Mi Soon Jang, Hee Yeon Park and Elena Koneva 1 Food and safety Research Division, National Fisheries Research and Development Institute, Busan , Korea 1 TINRO Centre, Pacific Scientific Research Fisheries Centre, Shevchenko Alley, Vladivostok , Russia A bacterium producing α-galactosidase (α-d-galactoside galactohydrolase, EC ) was isolated. The isolate, KM-1 was identified as Bacillus coagulans based on its 16S rrna sequence, morphology, and biochemical properties. α-galactosidase activity was detected the culture supernatant of B. coagulans KM-1. The bacterium showed the maximum activity for hydrolyzing para-nitrophenyl-α-d-galactopyranoside (pnp-αgal) at ph 6.0 and 50 C. It hydrolyzed oligomeric substrates such as melibiose, raffinose, and stachyose liberating a galactose residue, indicating that the B. coagulans KM-1 α-galactosidase hydrolyzed α-1,6 linkage. The results suggest that the decreased stachyose and raffinose contents in fermented soybean meal are due to the α-galactosidase activity. Key words: Bacillus coagulans KM-1, α-galactosidase, Stachyose, Raffinose, Fermented soybean meal 서론 (fish meal). (Kim et al., 2013)., sucrose, maltose, raffinose, stachyose arabinnan. stachyose raffinose -galactosyl -galactosidase ( -D-galactoside galactohydrolase, EC 3,2,1,22) raffinose stachyose,, (Rackis, 1981) (Reddy et al., 1980; Steggerda et al., 1968). (Yoo et al., 2007). (Li et al., 1991). -galactosidase (Min et al., 2002; Kim and Yoon, 1998; Choi et al., 1998; Kim et al., 2004; Irish et al., 1995) -galactosidase (park., 2011; Kim. et al., 2002; Kum and Yoon., 1998; Choi et al., 1998). stachyose raffinose., -galactosidase,,, galactose galactomannan Kor J Fish Aquat Sci 47(5) , October 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 22 September 2014; Revised 20 October 2014; Accepted 29 October 2014 *Corresponding author: Tel: Fax: address: dennis011@korea.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 516 pissn: , eissn:

2 Bacillus coagulans KM-1 의생화학적특성 517 melibiose, raffinose, stachyose galactomannan -1,6 -galactose (Dey and Campillo, 1984). (Engraulis japonica) -galactosidase. 배지및시약 재료및방법 Nutrient broth (NB, Difco, St. Louis, MO, USA) Nutrient Agar (NA, Difco) NA. glycerol 15% (V/V) -75 (IIshin DF8514l, Seoul, Korea). 균주의분리 (raw anchovy, Engraulis Japonica ) 10 g 40 ml 5 10 ml 100 ml NB 5 ml 50, 100 rpm (VISION SCI., VS-8480SR, Daejeon, Korea) ml NA 50 incubator colony. α-galactosidase 의활성측정 1 ml 12,000 g 15 -galactosidase. -galactosidase p-nitrophenyl- -D-galactopyranoside (pnp Gal) (Food Research International, 2009)., 700 L 100 mm potassium phosphate buffer (ph 6.5) 100 L 200 L 9.9 mm pnp Gal 5 50, 2 ml 0.2 M borate buffer (ph9.8) 405 nm -galactosidase. 미생물균주의최적발육조건분석 nm (Hitachi U-2900, Tokyo, Japan) ph NB ph nm. 주사전자현미경을이용한균주의관찰 Scanning Electron Microscopy (SEM, S-2400, Hitachi, JAPAN). SEM 0.1 M sodium phosphate buffer (ph ) 2.5% glutaraldehyde (TAAB Laboratoties Equipment Ltd, Berks, UK) M sodium phosphate buffer 2 1% osmium tetroxide (Sigma, St Louis, MO, USA) % 5 100% hexamethyldisilazane. SEM 20 kv 미생물균주의생화학적특성조사 API- ZYM (BioMèrieux, France) 19 -galactosidase API-50CH (BioMèrieux, France). 16S rrna 유전자염기서열과계통수 (phylogenetic tree) 분석 16S rdna., 16S rdna DNA PCR. DNA Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, USA), DNA sequencer (Applied Biosystems model 3730XL, USA). Ez- Taxon server 2.1. NCBI BLASTN 50 EMBL-EBI ClustalW2 Neighbor Joining (Saitou and Nei, 1987) phylogenetic tree. 난소화성탄수화물분해능측정 (Scalabrini et al., 1998) stachyose raffinose 10 g/l NB 5% (w/v) ml 12,000 g µl 750 L 0.2 m (membrance filter). Stachyose raffinose APS-2 hypersil column (thermo scientific; mm, 5 m, 120A)

3 518 남기호ㆍ장미순ㆍ박희연ㆍ이레나코네바 (column, 40 ;, 75% ACN;, 1.5 ml/min). RI (refractive index detector, Dionex). 미생물균주가생산하는 α-galactosidase 의최적반응온도측정 -galactosidase ,000 rpm 20. ammonium sulfate 35-85% ,000 g mm Tris-HCl (ph7.0) galactosidase p-nitrophenyl- -D-galactopyranoside (pnp Gal) (Sigma, St. Louis, MO, USA). 700 L 100 mm potassium phosphate buffer (ph 6.5) 200 L 9.9 mm pnp Gal 100 L , 2 ml 200 mm borate buffer (ph 9.8) 405 nm -galactosidase. 1 unit 1 1 mol p-nitrophenol, (specific activity) 1 mg 1 unit. 결과및고찰 미생물균주의분리및 α-galactosidase 활성 NA NB colony 20 -galactosidase. (Table 1) KM Unit/mL 19 stachyose raffinose. 배양시간에따른 KM-1 균주의발육조건 -galactosidase KM-1 Fig ph, ph ph 6.0. ph,, -galactosidase (Kim and Yoon, 1998) ph 주사전자현미경을이용한균주의관찰 Table 1. α-galactosidase activity of microorganisms isolated from raw anchovy O.D. (660 nm) Isolate Activity (U/mL) Isolate Activity (U/mL) KM KM KM KM KM KM KM KM KM KM KM KM KM KM KM KM KM KM KM KM Temperature ( ) Fig. 1. Effect of temperature on growth of Bacillus coagulans KM-1 after 12 hours cultivation. O.D. (660 nm) Fig. 2. Effect of ph on growth of Bacillus coagulans KM-1 after 12 hours cultivation. ph

Bacillus coagulans KM-1 의생화학적특성 519 20 -galactosidase KM-1 KM-1 (Gram staining)

4 Bacillus coagulans KM-1 의생화학적특성 galactosidase KM-1 KM-1 (Gram staining) (Spore staining), KM-1 (Fig. 3) m, m 2 (long rod). (catalase) (Bacillus sp.). API-ZYM 과 API-50CHL Kit 를이용한균주의특성 KM-1 API-ZYM (BioMèrieux, France) 19 (Table 2) KM-1 alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, phosphatase acid, naphthol-asbi-phosphohydrrolase, -galactosidase, -galactosidase -glucosidase. API-50CH (BioMèrieux, France) 50 (Table 3) glycerol, ribose, D-xylose, galactose, D-glucose, D- fructose, D-manose, N-acetyl glucosamine, maltose, melibiose, trehalose, starch, D-arabitol 5-keto-gluconate. 16S rrna 유전자염기서열분석과계통수 (phylogenetic tree) -galactosidase KM-1 16S rdna B. coagulans % (3/1478), B. acidiproducens % (40/1414), B. shackletonii % Fig. 3. Scanning electron microscopy image of Bacillus coagulans KM-1 ( 7,500). Table 2. Enzyme activities of Bacillus coagulans KM-1 by API- ZYM kit Enzyme Activity Enzyme Activity Control - Phosphatase acid + Phosphatase alcaline + Naphthol-ASBIphosphohydrrolase Esterase (C4) + α-galactosidase + Esterase lipase (C8) + β-galactosidase + Lipase (C14) - β-glucuronidase - Leucine arylamidase + α-glucosidase + Valine arylamidase - β-glucosidase - Cystine arylamidase + N-acetyl-βglucosaminidase - Trypsin - α-mannosidase - α-chymotrypsin - α-fucosidase - +, positive; -, negative. Table 3. Carbohydrate utilization pattern of Bacillus coagulans KM-1 by API-50CH kit Carbohydrate Activity Carbohydrate Activity Control - Esculin - Glycerol + Salicin - Erythritol - Celobiose - D-arabinose - Maltose + L-arabinose - Lactose - Ribose + Melibiose + D-xylose + Sucrose - L-xylose - Trehalose + Adonitol - Inulin - β-methyl-d-xyloside - Melezitose - Galactose + D-raffinose - D-glucose + Starch + D-fructose + Glycogen - D-manose + Xylitol - L-sorbose - β-gentiobiose - Rhamnose - D-turanose - Dulcitol - D-lyxose - Inositol - D-tagatose - Manitol - L-fucose - Sorbitol - D-arabitol + α-methyl-d-manoside - L-arabitol - α-methyl-d-glucoside - Gluconate - N-acetyl glucosamine + 2-keto-gluconate - Amygdalin - 5-keto-gluconate + Arbutin - +, positive; -, negative. +

난소화성탄수화물분해능 Fermentation time (h) 0 24 48 Raffinose 0.75* 0.56 0.39 Stachyose 0.49 0.25 0.16 Saccharose 0 0.23 0.42 galactose 0 0.33 0.49 *mg/100 g B. coagulans KM-1 (Table 4) stachyose raffinose 0.

5 520 남기호ㆍ장미순ㆍ박희연ㆍ이레나코네바 Fig. 4. Phylogenetic tree of Bacillus coagulans KM-1 based on 16S rdna sequencing. Table 4. Non-digestible carbohydrate resolution of Bacillus coagulans KM-1 Carbohydrate (62/1487). KM-1 B. coagulans KM-1 (Fig. 4) (NCBI) (JX569800, ) (KCCM P, ). 난소화성탄수화물분해능 Fermentation time (h) Raffinose 0.75* Stachyose Saccharose galactose *mg/100 g B. coagulans KM-1 (Table 4) stachyose raffinose 0.49 mg/100 g, 0.75 mg/100 g mg/100 g, 0.56 mg/100 g, mg/100 g, 0.39 mg/100 g. Stachyose raffinose saccharose galactose B. coagulans KM-1 -galactosidase stachyose raffinose. Kim and Yoon (1998) -galactosidase stachyose raffinose -galactosidase. 미생물균주가생산하는 α-galactosidase 의최적반응온도 B. coagulans KM-1 -galactosidase Fig. 5. B. coagulans KM-1 α-galactosidase activity (Unit/ ml) Temperature ( ) Fig. 5. Effect of temperature on α-galactosidase activity of Bacillus coagulans KM-1.

6 Bacillus coagulans KM-1 의생화학적특성 521 -galactosidase B. coagulans KM B. coagulans KM (Jang et al., 2013). 사사 (, RP-2014-FS-020). References Coombs J and Brenchley Characterization of two new glicosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA. Appl Environ Microbiol 67, Choi C, Choi KS, Lee SH, Hong SP, Lee HD and Bae DK Characteristics and action pattern of α-galactosidase from Scopulariopsis brevicaulis in Korean traditional Meju. ACB 41, Dey PM and Campillo ED Biochemistry of the multiple forms of glycosidases in plants. Adv Enzymol Relat Areas Mol Biol 56, Halstead JR, Fransen MP. Eberhart RY, Park AJ, Gilbert HJ and Hazlewood GP α-galactosidase a from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis. FEMS Microbiol Lett 192, Irish GG, Barbour GW, Classen HL, Tyler RT and Bedford MR Removal of the α-galactosidase of sucrose from soybean meal using either ethanol extraction or exogenous alpha-galactosidase and broiler performance. Poult Sci 74, Jang MS, Park HY, Nam KH, Han HS, Kim KW, Kim KD and Lee BJ Effect of extruded pellets containing fermented soybean meal as a partial substitute for fish meal on growth performance and muscle quality of olive flounder (Paralichthys olivaceus). J Agric Life Sci 47, Jindou S, Shuichi K, Emi F, Tsuchiyoshi F, Hidenori H, Tetsuya K, Kazuo S and Kunio O α-galactosidase Agar27A, an enzymatic component of the Clostridium josui cellulosome. J Bacteriol 184, Kim KW, Kim KD, Lee BJ, Lee JH, Han HS, Koo JW, Choi YH and Bai SC Dietary fermented soybean meal as a replacement for fish meal in juvenile olive flounder Paralichthys olivaceus. Kor J Fish Aquat Sci 46, Kim HS, Lee KS, Jae HS, Lee MS, Choi JH and Yoon KH Characterization of extracellular α-galactosidase produced by Bicillus licheniformis YB-42. Kor J Microbiol Biotechnol 32, Kim JS and Yoon S The Changes of α-galactosidase activities and stachyose and raffinose contents during fermentation of soybeans. Korea J Soc Food Sci 14, Li DF, Nelssen JL, Reddy PG, Blecha F, Klemm RD, Giesting DW, Hancock JD, Allee GL and Goodband RD Transient hypersensitivity to soybean meal in the early-weaned pig. J Anim Sci 68, Min BJ, Kim HI, Hong JW, Moon TH, Lee JH, Han YK, Kwon OS, Lee SH and Lee WB Influence of dietary carbohydrase on egg quality and nutrient digestibility in laying hens. Korea J Poult Sci 29, Rackis JJ Flatulence caused by soya and its control through processing. J Am Oil Chem Soc 58, Reddy NR, Salunkhe DK and Aharma RP Flatulence in rats following ingestion of cooked and germinated black and a fermented product of black gram and rice blend. J Food Sci 45, Silvestroni A, Connes C, Sesma F, Giori GS and Piard JC Characterization of the mel locus for α-galactosidase in Lactobacillus plantarum. Appl Environ Microbiol 68, Scalabrini, Rossi P, Spettoli M, Matteuzzi P Characterization of Bifidobacterium strains for use in soymilk fermentation. Int J food microbiol 39, Saitou N and Nei M The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, Steggerda FR, Richards EA and Rackis JJ Effects of various soybean products on flatulence in the adult man. Proc Soc Exp Biol Med 121, Sanada CTN, Karp SG, Spier MR, Portella AC, Gouvea PM, Yamaguishi CT, Vandenberghe LPS, Pandey A and Soccol CR Utilization of soybean vinasse for α-galactosidase production. Food Res Int 42, org/ /j.foodres Yoo JS, Cho JH, Chen YG, Kim HJ, Wang Q, Hyun Y, Ko TG, Park CS and Kim IH The Effects of environmentfriendly diets on the growth performance, nutrient digestibility, fecal excretion, nitrogen excretion and emission gases in manure for growing pigs. J Anim Sci Technol 49,

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