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1 The Krean Jurnal f Micrbilgy, Vl. 42, N. 3, September 2006, p Cpyright 2006, The Micrbilgical Sciety f Krea β-galactsidase ³ p»á ù 2 Á 2 Á Á Áy 3 Á½ * w œw y œw l ƒm w œw œ f β-galactsidase (lactase) w ³ wš w β-galactsidase p w. X-gal sw MRS t ùkü 100 g ½e l w. ET-1 LA-12 ³ w, 16S rdna» mw ƒ ƒ Lactbacillus fermentum L. acidphilus Lactbacillus. ³ β-galactsidase activity w ùkü, w β-galactsidase y w, 55 C ƒ y ùkü, ph ET-1 ph 5.5 š y ùkü š, LA-12 ph 7.0 ƒ y. ³ ET-1 LA-12 w w ü ùkü. ³ œ 3 z» ³ w ³ yƒ š, w 0.3% xgall 24 z 1 lg cycle 10 8 CFU/ml ³ w. w k ³ ET-1 LA-12 t ƒ j š q. Key wrds ý β-galactsidase, Lactbacillus, ü, ü, ³ ³ ù w j w w. ü w ³ ³ y v w ww, ³ w bactericin j ü w³ ù w z (9). w w x e d, ³, g l w,, w, w y w z ƒ j w w (8, 11). s w, lactse galactse glucseƒ β(1 4) glycside w x, ƒ ww z lactase, ³ β- galactsidase. Lactase y v w š ü n w. w, ü w z k ƒ jš ü ph w w h ù w k. ü (lactse intlerance) wš šƒ. w ü 2/3 š, ù 84~ *T whm crrespndence shuld be addressed. Tel: , Fax: hkim@kribb.re.kr 86% š š š. w w w» w lactaseƒ w w, v wz œ w ü y j»y w v š (4). w w ƒ w ½e β-galactsidase (lactase) w w ü w ³ wš, ³ œ w ü w prbitic ƒ mw, ph w z y, w β-galactsidase y w w š w. β-galactsidase ³ β-galactsidase ³ w» w 1ml ½e 0.85% saline slutin w w z 32 µg/ml 5- brm-4-chlr-3-indlyl-d-galactpyranside (X-gal, Sigma, USA) ƒ sw MRS š (Difc, USA) 37 C, 3 w (16). x clny t š t ù kü clny 1 w (12). 1 clny Cutting (6) w β-galactsidase y d w. w clny 1,500 Miller Units/ml β-galactsidase y ùkü ³ 2 w. ³ ³ l Genmic DNA islatin kit (Quiagen, Hilden, Germany) w chrmsmal DNA w. PCR kit (Perkin Elmer C., CT, USA) 216

2 Vl. 42, N. 3 β-galactsidase ³ p 217 universal primer (9F, 5'-GAGTTTGATCCTGGCTCAG-3'; 536R, 5'-GWATTACCGCGGCKGCTG-3') w PCR ww, ; denaturatin 95 C, 5 min, annealing 60 C, 1 min, extensin 72 C, 1 min, final extensin 72 C, 10 min, 30 cycles. PCR z, 1% agarse gel (FMC, ME, USA) w s PCR y w. pgem T-easy vectr (Quiagen) E. cli DH5α cmpetent cell w transfrmatin ww. QIAprep spin miniprep kit (Qiagen) w plasmid DNA w z,» ww.» BLAST Search Prgram (NCBI) mw GenBank database w. β-galactsidase y d ³ 1 ml 8,000 g 2 w z, e ³ 0.85% saline slutin 2z w, Z buffer (Na 2 HPO 4 Á7H 2 O 60 mm, NaH 2 PO 4 40 mm, KCl 10 mm, MgSO 4 Á7H 2 O 1 mm, β-mercaptethanl 50 mm, ph 7.0) 1 ml xkw. ONPG (-nitrphenyl-β-d-galactpyranside, Sigma)» 4.0 mg/ml ƒw 30 C 5 w z 1 M sdium carbnate g. w Ÿ 420 nm 550 nm d w β-galactsidase y w, z y 1 ml Miller unit ùkü (6). Buffer ONPG β-galactsidase y d w. ph 7.0 Z buffer (cntaining β-mercaptethanl) acetate buffer (ph 4.0), phsphate buffer (ph 7.0, mitting β- mercaptethanl) w z y w, ONPG w z y, 4.0 mg/ml 16 mg/ml ONPG w z y w. w, ³ q z y w. Tluene y snicatr (Snic & Materials Inc., USA) w s q w z, z y w. Snicatin 50/60 Hz 30 30z, š w z y yw s q w, tluene 1ml tluene 10 µl ƒw sq z, y d w. z MRS w 37 C 18 w z, 1% (v/v) w. 37æ, 18 w z 1 ml w saline slutin 2z w z w. 1) MRS medium, 2) 0.85% saline slutin, 3) (4 C), 4) (30 C) 4ƒ ww 20 ³ ³ d w, z β-galactsidase y d w ùkü. ph z y β-galactsidase y y w» w ph 6.5 ONPG slutin w 25 C~65 C ww. z prewarming y w β- galactsidase y yw. ph β-galactsidase y y w» w ph 4.0 ~ph 9.0 w 0.1 M phsphate buffer w š, z y d 37 C 55 C w z y w. HPLC ³ w β-galactsidase lactse w y w HPLC ww. S10 NH 2 clumn (Waters Spherib C.) w, mbile phase acetnitrile DW (65 : 35, v/v) w š, flw rate 1.7 ml/min. RI dectectr (Shdex, RI-71) w. ³ üy w ü üy ³ ü w m š A B t Lactbacillus ³ ƒ 1 ³ w ü, w. MRS pepsin 1% (w/v) ƒwš 1N HCl ph 2.0, 2.5, 3.0 w œ w w ü w (14, 17). œ 37 C, 24 w 1ml 8,000 g, 5 w ³ z w. ³ œ 1:1 (v/v) ƒw 3 w z MRS š 37 C, 48 w ³ d mw œ w ü ùkü. w ü w» w MRS 37 C, 24 w z 1 ml w ³ z w. œ 3 w z 0.3% xgall (Difc) ƒ œ (ph 6.5) 1:1 (v/v) ƒw 37 C 24 w w ü d w (15). š β-galactsidase ³ Klaenhammer et al.(13) w ³ ³ xk w ü ƒ, clny xk rugh type clny smth type w ü w š š. ½e l w ü ƒ β-galactsidase y w ³ w» w 2% X-gal ƒ MRS l t š t ù kü 100 clny 1 w, clny Cutting (6) w β- galactsidase y d w. 100 clny 1,500 Miller Unit/ml β-galactsidase y ùkü 6 ³ 2 w, 2 ³ MRS w z,

3 218 Yung-Ki Lee et al. Kr. J. Micrbil 4 C 7 w, ³ d w ³ š, z y w ET-1 LA-12 ³ w. ³ w 16S rdna» ww ³ w. 2 ³, ET-1 LA-12 ƒƒ Lactbacillus fermentum L. acidphilus Lactbacillus (Table 1). m i p A B t l w ³ β-galactsidase y w. ³ ET-1 LA-12 2,600~2,700 Miller Unit/ml z y q A B ³( 1,600~1,800 Miller Unit/ ml) w 50% β-galactsidase y ùkü (data nt shwn). (viability) z β-galactsidase ³ y ³ z ƒ w wù. x β-galactsidase ³ ƒ mw» w ³ ³ w β- galactsidase w x ww. ³ β-galactsidase x ( ) (MRS saline slutin) ww 20 w d w. ET-1, 4 C, MRS (4M) w, 16 ¾ 10 8 CFU/ml ³ w. z, saline slutin (RS) w w, 5 ¾ β-galactsidase y ƒw w ù, 9 z w ùkü. w 4M, 7 ¾ û z y ù, w z y 1,100 Miller Units/ml z y w (Fig. 1, panel A). LA-12 ³, 4 C, saline slutin (4S) w w, 20 z ³ (10 CFU/ml) ù 7 kü. ù z y, saline slutin w y, 20 z 800 Miller Units/ml û y w. ³ LA- 12 z š w 4 C, MRS w, saline slutin 7 ¾ û y ùkü 9 z z y yƒ š w z y w (Fig. 1, panel B). k ³ w z w» w 4 C, MRS w Fig. 1. Viability f selected lactic acid bacteria during the preservatin with different cnditins. Several experiments were perfrmed, and typical results are shwn. Slid lines and dt lines represent viable cell number and β-galactsidase activity, respectively. Panel A, Lactbacillus fermentum ET-1; panel B, Lactbacillus acidphilus LA- 12. RS, saline slutin at rm temperature; RM, MRS medium at rm temperature; 4S, saline slutin at 4 C; 4M, MRS medium at 4 C. w 4 C, saline slutin w w ƒ ƒ z. ù ³ y z mw ƒe š w 4 C, saline slutin w q. β-galactsidase y m β-galactsidase endenzyme w z w» z s š. w d z y ùkü (12). x ³ z y ƒ ET-1 w buffer, Table 1. 16S rdna sequencing analysis f the selected lactic acid bacteria Strain Identificatin (%) Clset strain Accessin number ET-1 466/472 (98%) Lactbacillus fermentum AY LA /500 (100%) Lactbacillus acidphilus AB

4 Vl. 42, N. 3 β-galactsidase ³ p 219 ONPG», š s q z y w. ET-1 (» ³ 10 CFU/ml) 9 phsphate buffer (ph 7.0), acetate buffer (ph 4.5) Z buffer (ph 7.0) w w z snicatin mw s q w. w ³ e w z 4.0 mg/ml 16 mg/ml ONPG w z y d w. Buffer phsphate buffer w y d w (Table 2), Z buffer y acetate buffer w 16%~50% z y. ONPG, z ONPG» z y ƒ y (Table 2). s q z y y w» w tluene (6) snicatin w s q w z z y w. Snicatin q m w ³ s q w z y negative y psitive effect ùkü š š (11). Phsphate buffer ³ xkw snicatin s q w, 3,000 Miller Units/ml z y d š, snicatin w d z y tluene w 15% y ƒw (Table 2). l ³ ET-1 β-galactsidase y phsphate buffer (ph 7.0) w snicatin (50/60 Hz, 30 sec, 30 times) s q w z, 4.0 mg/ml ONPG» w 30 C 5 min w z y. ph z y β-galactsidase y ph, š w w w w (10). x ET-1 LA- 12ƒ w β-galactsidase w ph y w. ³ z 55 C d z y 37 C z y w 20~40% ƒ y w (Fig. 2). ³ ph β-galactsidase y y 55 C Fig. 2. Optimum temperature fr β-galactsidase activity f lactic acid bacteria ET-1 and LA-12. The activity represented as a relative activity (%) against that at 50 C. Square (þ) and diamnd (ü) represent LA-12 and ET-1, respectively. w, ET-1, ph 5.5 z y ƒ, ph 7.0 w ùkü. LA-12 ET-1 ph 7.0 z y ùkü, z w (Fig. 3). Fig. 2 Fig. 3 l ET-1 LA-12ƒ w β-galactsidase, 55 C ƒ y ùkü š, ph 5.5 ET-1 š y ùkü š, LA-12 ph 7.0 ƒ y. ¾ ONPG» w β-galactsidase y d w. ³ β-galactsidaseƒ lactse galactse glucse ww y w» w HPLC w w. Lactse 0.5 g cntrl w ww, retentin time (RT) 3.72 min lactse cntrl peakƒ, ET-1 LA-12 RT 3.12 min 3.37 min ƒ ƒ galactse glucse w w peak ƒ w ùkù y w (Fig. 4). l ³ ƒ w β-galactsidase lactse w w ƒ y w, ƒ y w. Table 2. Effects f buffer, ONPG cncentratin, and cell disruptin methd n β-galactsidase activity f ET-1 Buffer Fractin Activity (Miller Units/ml) 1 ONPG c 4 ONPG Snicatin d Tluene d Sup a 1,316 1,139 1,616 Phsphate buffer Pel b 1, ,092 Ttal 2,989 2,519 2,989 Sup Acetate buffer Pel Ttal Sup 1,457 1,237 1,457 Z buffer Pel Ttal 2,562 2,017 2,562 Sup, supernatant; b Pel, pellet. 1 ONPG means the cncentratin f 4.0 mg/ml. Cells were disrupted by snicatin and tluene methd, respectively. a c d

5 220 Yung-Ki Lee et al. Kr. J. Micrbil Fig. 3. Optimum ph fr β-galactsidase activity f lactic acid bacteria ET-1 and LA-12. The activity represented as a relative activity (%) against that at ph 7.0. Square ( þ ) and diamnd ( ü ) represent LA- 12 and ET-1, respectively. üy ³ ü w ph 1.4~2.0 w ü phƒ w s k. ù, ³ ü w» {w» w ( 2~3 ) û ph w ü ƒ w (1, 15, 17). ³ (ET-1 LA-12) ph 2.0, ph 2.5, ph 3.0 w œ w ü x ww š, q t w ³ w. ET-1 3 z» ³ (10 10 CFU/ml) w 1 lg cycle ùkü š, LA-12 ³ yƒ q ³ A B w ü w ƒ y w (Table 3). mw ³ w w ü ƒ w š (2, 3). x œ w z œ w q t w ³, w. ET-1 LA % Table 3. Effects f artificial gastric juice a n the viability f lactic acid bacteria. G (Viable cell, lg N. CFU/mL) Strains ET-1 ph Incubatin time (min) LA A b B b a Artificial gastric juice is prepared by adding pepsin t MRS medium at 1% (w/v) (adjusted ph by 1N HCl). b A and B are the lactic acid bacteria islated frm the cmmercial prducts Fig. 4. HPLC analysis f β-galactsidase activity prduced by the selected lactic acid bacteria. Panel (A), cntrl (lactse 0.5 g); panel (B), ET-1; panel (C), LA-12. Table 4. Effects f bile n the viability a f lactic acid bacteria. (Viable cell, lg N. CFU/ml) Incubatin time (hr) Strains Cntrl b ET-1 Bile c Cntrl LA-12 Bile Cntrl A d Bile Cntrl B d Bile a The viability f lactic acid bacteria was examined during the incubatin in MRS medium with and withut xgall fr 24 hr at 37 C after treated with artificial gastric juice fr 2 hr at 37 C. b Cntrl means the MRS medium withut xgall. c Bile is prepared by adding xgall t MRS medium at 0.3% (w/v). d A and B are the lactic acid bacteria islated frm the cmmercial prducts.

6 Vl. 42, N. 3 β-galactsidase ³ p 221 xgall 8 z, cntrl (withut xgall) w» ³ 10 9 CFU/ml ³ w. 24 z 10 8 CFU/ml ³ w y, q ³ B ³ 1 lg cycle w y (Table 4). x mw ³ ET-1 LA-12 w ü w q ³ A, B³ w w ù w y ty ü prbitics» {w ƒ. l x w ³ ET-1 LA-12 w z y (β-galactsidase) ùký ü» {w», x m š t l ³ w w ü w w» ƒœ t y»k t ty ƒeƒ w š ƒ. w œ» f KRIBB» š w w. š x 1. Ahn, Y.-Y., Y.-H. Kim, E.-J. Jung, H.-T. Lim, H.-J. Kang, and H.- U. Kim Resistance f Lactbacilli and Bifidbacteria islated frm fermented milk prducts t lw ph and bile acid. Kr. J. Anim. Sci. 41, Berrada, N., J.F. Lemeland, G. Larche, P. Thuvent, and M. Piaia Bifidbactrium frm fermented milk: Survival during gastric transit. J. Dairy Sci. 74, Chung, S.H., H.J. Suh, and H. Lee Utilizatin f sybean curd whey as a medium fr Lactbacillus acidphilus and acid and bile-tlerance f cultured strains. J. Kr. Sc. Fd Sci. Nutr. 26, Chung, C.-W Ptential crrelatin between lactse intlerance and cancer ccurrence. J. Kr. Ass. Cancer Preventin. 4, Cnway, P., L. Grbach, and B.R. Gldin Survival f lactic acid bacteria in the human stmach and adhesin t intestinal cells. J. Dairy Sci. 70, Cutting, S.M. and P.B. Vander Hrn Genetic analysis, p In C. R. Harwd and S. M. Cutting (ed.), Mlecular bilgical methds fr Bacillus. Jhn Wiley, Chichester. 7. Dare, R., J.T. Magee, and G.E. Mathisn In vitr studies n the bactericidal prperties f natural and synthetic gastric juices. J. Med. Micbil. 5, De man, J.C., M. Rgsa, and M.E. Sharpe A medium fr the cultivatin f Lactbacilli. J. Appl. Bacteril. 23, Giannella, R.A., S.A. Britman, and N. Zamchick Gastric acid barrier t ingested micrrganisms in man : studies in viv and in vitr. Gut. 13, Hd, S.K. and E. A. Zttla Effect f lw ph n the ability f Lactbacillus acidphilus t survive and adhere t human intestinal cells. J. Fd Sci. 53, Jasewics, L. and A.E. Wasserman Quantitative determinatin f lactase. J. Dairy Sci. 44, Kilara, A. and K.M. Shahani Lactase activity f cultured and acidified diary prducts. J. Dairy Sci. 59, Klaenhammer, T.R. and E.G. Kleeman Grwth characteristics, bile sensitivity and freeze damage in clnial variants f Lactbacillus acidphilus. Appl. Envirn. Micrbil. 41, Kbayashi, Y., K.GThyama and T.GTerashimaUG1974. Tlerance f the multiple antibitic resistant strain, L. casei PSR 3002, t artificial digestive fluids. Jap. J. Micrbil. 29, Lee, S.-H. and M.-J. N Viability in artificial gastric and bile juice and antimicrbial activity f sme Lactic acid bacteria islated frm Kimchi. Kr. J. Appl. Micrbil. Bitechnl. 25, Park, J.-W., J.-S. Y, and D.-H. Rh Identificatin f nvel psychrtlerant bacterial strain and prductin f β-galactsidase. Kr. J. Micrbil. 42, Shah, N. and P. Jelen Survival f lactic acid bacteria and their lactase under acidic cnditins. J. Fd Sci. 55, (Received July 3, 2006/Accepted August 19, 2006) ABSTRACT : Characterizatin and Selectin f Lactic Acid Bacteria Prducing β-galactsidase Yung-Ki Lee, Susanna Chi 2, Yung-Il Park 2, Chan-sun Park, Byung-Dae Yn, Yun-Sik Hwang 3, and Hee-Sik Kim* (Envirnmental Bitechnlgy Research Center, KRIBB, Daejen , Krea, 2 Department f Bitechnlgy, Cathlic University f Krea, Seul , Krea, 3 Anichem Cmpany Ltd., KRIBB BVC, Daejen , Krea) This study was carried ut t select the lactic acid bacteria prducing β-galactsidase (lactase) and investigate the prperties f the β-galactsidase. Abut 100 strains f lactic acid bacteria shwing blue clny n the MRS agar medium cntaining X-gal were islated frm several kinds f Kimchi. Amng them, 2 strains were selected as ptential β-galactsidase prducers. The selected strains, ET-1 and LA-12, were identified as Lactbacillus fermentum and L. acidphilus, respectively by the analysis f 16S rdna sequences. They shwed relatively high β-galactsidase activity and cellular viability. Their β-galactsidase shwed the highest activity at 55 C. And the ptimum phs f the enzymes prduced by ET-1 and LA-12 were ph 5.5 and ph 7.0, respec-

7 222 Yung-Ki Lee et al. Kr. J. Micrbil tively. They were als highly resistant t artificial gastric juice and bile. Tw selected strains shwed little change f viable cell number fr 3 hr incubatin in artificial gastric juice, and maintained the viable cell number at 10 8 CFU/ml fr 24 hr in 0.3% xgall after incubatin fr 2 hurs in artificial gastric juice. Based n these results, ET-1 and LA-12 are expected t be applied in dairy industry.