DBPIA-NURIMEDIA

Korean Society for Biotechnology and Bioengineering Journal 32(2): 153-159 (2017) http://dx.doi.org/10.7841/ksbbj.2017.32.2.153 ISSN 1225-7117 / eissn 2288-8268 Research Paper 한국인장관에서분리한유산균의항균활성및프로바이오틱스특성연구 강창호 *, 김용경, 한설화, 정율아, 백남수 Antibacterial Activity and Probiotic Properties of Lactic Acid Bacteria from Korean Intestine Origin Chang-Ho Kang*, YongGyeong Kim, SeulHwa Han, Yulah Jeong, and Nam-Soo Paek Received: 10 May 2017 / Revised: 15 June 2017 / Accepted: 20 June 2017 2017 The Korean Society for Biotechnology and Bioengineering Abstract: The purpose of this study was to investigate the probiotic properties of lactic acid bacterial strains isolated from human feces. The properties were tested on the basis of guideline for probiotic selection protocol such as tolerance for acid or bile salt, autoaggregation, antibiotic resistance, and antimicrobial activity. Total 25 lactic acid bacteria were isolated from human feces, and their antibacterial activity was tested against Staphylococcus aureus, Escherichia coli, E. coli O157:H7, Vibrio parahaemolyticus, V. alginolyticus using an agar diffusion assay. Among them, 4 selected strains were identified by analysis of their 16S rrna, as Lactobacillus rhamnosus MG316, L. acidophilus MG501, L. reuteri MG505, and L. gasseri MG570. Results show that resistance to low ph and bile salts. Also, the selected strains were resistant to bile acid up to 3% and their autoaggregation rates were as high as 60%. All strains tested were resistance to nalidixic acid and kanamycin. Keywords: Probiotic, Lactic acid bacteria, Human feces, Antimicrobial activity ( 주 ) 메디오젠 MEDIOGEN, Co., Ltd., Seoul 04157, Korea Tel: +82-43-644-4216, Fax: +82-43-644-4215 e-mail: changho-kang@naver.com 1. INTRODUCTION 프로바이오틱스는숙주의장내미생물균형을유지시킴으로써유익한작용을하는살아있는미생물을의미한다 [1]. 이와같은프로바이오틱스는장내균총의안정화, 유해세균의정착억제에따른부패산물생성감소및질병예방, 면역활성화작용, 항암작용, 콜레스트롤저하, 유당불내증의경감, 변비억제등의기능을갖고있다고알려져있다 [2-5]. 미생물이프로바이오틱스로이용되기위해서는섭취에대한안전성, 소화기관내에서의안정성, 세포정착능력과항균활성에대한기능성및배양에대한기술적인부분에대한검증이되어야하며, 최근에는프로바이오틱스에대한규제및검증이강화되고있어기초연구결과에대한중요성이대두되고있다 [6,7]. 일반적으로유산균은장내의낮은 ph 및담즙염에대한내성을지니며안정성이확보된 GRAS (generally regarded as safe) 미생물로분류되고있어, 프로바이오틱스로서의이용가치가매우높은것으로알려져있다 [8]. 프로바이오틱스로사용되고있는미생물은 Lactobacillus, Bifidobacterium, Enterococcus 와같은유산균이주류를이루고있으며, Leuconostoc, Pediococcus, Bacillus 그리고효모등은주로동물에사용되고있다. 현재, 프로바이오틱스로가장많이이용되고있는균주는 Lactobacillus 와 Bifidobacterium 이며, 발효유, 건강식품, 유산균정장제, 동물약품등에사용되고있다 [9]. Lactobacillus 는통성혐기성간균이며, 숙주동물의장내균총균형을유지하기위하여발효유형태로가장먼저이용되어왔으며, 사람의장내에서식하는균주로서다양한종류의제품에사용되고있다.

154 Korean Society for Biotechnology and Bioengineering Journal 32(2): 153-159 (2017) 이에본연구에서는다양한유산균을분리하기위해, 한국인의분변으로부터유산균을순수분리하여선별하였으며, 분리유산균에대한프로바이오틱스로서의기능을확인하기위해항균활성, 내산성, 내담즙성, 세포표면소수성및항생제내성등의기능을확인하였다. 2. MATERIALS AND METHODS 2.1. 유산균분리실험에사용된시료는유아 (2 명 ), 성인 (5 명 ) 등총 7 명의남녀로부터분변을혐기적으로채취하였다. 분변시료 1 g 에 0.85% saline 용액 9 ml 를넣고 Stomacher (3M, Korea) 를이용하여혼합하였다. 혼합용액을 Whatman No.2 paper 를통해여과한후, 1 ml 을취하여십진희석법으로희석한후, Rogosa agar (Difco, MI, USA) 배지에도말하여 37 o C, 48 시간배양하였다. 각시료별배지에서형성된단일콜로니는 bromocresol purple 이첨부된 MRS (Difco, MI, USA) 배지에도말하여노란색을보이는콜로니를최종선별하였다. 선별된균주의보관은 25% glycerol stock 으로 -70 o C 에서보관하도록한다. 2.2. 분리된유산균주의항균활성확인분리된유산균주의항균활성은액체배지희석법을이용해확인하였으며, 5종의병원균 (Staphylococcus aureus KCCM 12214, Escherichia coli KCCM41300, E. coli O157:H7 KCCM 40406, Vibrio parahaemolyticus KCTC2729, V. alginolyticus KCTC2472) 에대한항균력을확인하였다. 분리된유산균주를 MRS 액체배지에 24시간정치배양한후, OD 600 1.0 (10 8 CFU/mL) 으로조정하여새로운 MRS 배지에 1% 접종하였다. 18시간동안본배양한후, 원심분리 (4,000 g, 4 o C, 5분 ) 하여 0.22 µm 필터를이용해여과한상등액을본실험에사용하였다. 항균활성측정은 Vibrio 속을제외한병원균 3종은 Müller- Hinton Agar (MHA, BBL, Spark, USA), Vibrio속 2종은 2% NaCl이포함된 Brain-Heart Infusion (BHI, Bacto, USA) agar 배지에각각도말한후, 멸균된 paper disc에각각분리된유산균주의상등액을 50 µl씩흡수시켜균이도말된배지에올리고 24시간동안 37 o C에서배양하였다. 배양후 paper disc 주변에생성되는저해환의크기를측정하여항균활성을측정하였다. 2.3. 선별된유산균주의특성확인및동정선별된유산균주의생화학적특성확인을위하여 MRS 배지, 37 o C에서 24시간동안배양하였다. 배양된균은그람염색법을실시하였으며, 균의 oxidase 활성과 catalase 활성은각각 1% N,N,N,N -Tetramethyl-p-phenylenediamine dihydro-chloride (BDH Limited, Poole, England) 시약의색변화와 3% H 2 O 2 시약의기포생성여부를통하여확인하였다. 균의생화학적특성은 API 50CHL kit (BioMerirux, France) 로확인하였다. 선별된유산균주의동정을위하여 16S rrna gene sequencing 을수행하였으며, 유전자의증폭은 the universal rrna gene primers (27F and 1492R) 를사용하였고, 각과정은 Sol- Gent Co. (Daejeon, Korea) 을통하여진행하였다. 이후분석된 16S rrna sequencing 결과는 National Center for biotechnology Institute (NCBI) 의 Basic Local Alignment Search Tool (BLAST) analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi) 를통해 Genbank database 와비교하여동정하였다. Sequence 는 PHYDIT 3.1 을통하여 alignment 를진행하였고, 이후 MEGA 5.1 software 를이용하여 neighborjoining method 로계통수를작성하였다. 2.4. 선별된유산균주의산성및담즙염내성확인선별된유산균주의장내환경과같은산성및담즙염에대한내성을확인하기위해 Guo 의방법 [10] 으로실험하였다. 선별된유산균주를 18 시간동안배양한후, 원심분리 (4,000 g, 4 o C, 5 분 ) 하여 phosphate-buffer saline (PBS, ph 7) 에 2 번세척하였고, OD 600 1.0 으로조정하여각각산성과담즙염에대한내성실험에사용하였다. 산성에대한내성을확인하기위하여유산균희석액 1 ml 를 9 ml PBS (ph 2 와 7) 에첨가한후진탕한뒤 37 o C 에서 3 시간배양한후생균수를확인하였다. 담즙염내성은 1~3% oxgall (Difco, MI, USA) 을포함한 MRS 배지에유산균희석액을 1% 접종하고 37 o C 에 24 시간배양한후생균수를확인하였다. 2.5. 선별된유산균주의 Autoaggregation 장내세포내부착능력을간접적으로확인하기위하여 Kassa 의연구 [11] 를변형하여 autoaggregation 실험을진행하였다. MRS 배지에유산균을전배양한배양액을 OD 600 1.0 으로조정하여새로운 5 ml MRS 배지에 1% 접종한후 18 시간배양하였다. 배양한유산균은원심분리 (4,000 g, 4 o C, 5 분 ) 한후, PBS 에 2 번세척하였고, OD 600 1.0 으로조정하여균주현탁액 5 ml 을 10 초간진탕한뒤 5 시간동안방치하면서 autoaggregation 을확인하였다. 실험시작직후 (A0) 와 5 시간후 (A), 각각 0.1 ml 의상등액을취해 0.9 ml PBS 와혼합한뒤 600 nm 에서흡광도를측정 (A0, A) 하였고, 다음계산식에따라 autoaggregation 을계산하였다. Autoaggregation (%) = (A0 A) / A0 100 2.6. 선별된유산균주의항생제감수성시험항생제감수성시험은 Clinical and Laboratory Standards Institute (CLSI) guidelines [12] 를따라 MHA 배지를이용 disc method 에의해수행하였다. 선별된유산균주를 MRS 액체배지에서배양한후, 10 4 CFU/mL 정도의균체를 MHA 배지에도말한후, 항생제디스크 (BBL, Becton Dickinson, USA) 를올려놓았다. 37 o C 에서 24 시간배양한후억제환의크기를 mm 단위로측정하여표준지표에따라판정하였다. 사용한항생제디스크는모두 BD BBL (Becton Dickinson and Com-

한국인장관에서분리한유산균의항균활성및프로바이오틱스특성연구 155 pany, USA) 사제품으로 ampicillin (AM, 10 µg), cefotaxime (CTX, 30 µg), cefotetan (CTT, 30 µg), cephalothin (CF, 30 µg), chloramphenicol (C, 30 µg), ciprofloxacin (CIP, 5 µg), cefepime (CEP, 30 µg), erythromycin (E, 15 µg), gentamicin (GM, 10 µg), kanamycin (K, 30 µg), nalidixic acid (NA, 30 µg), rifampin (RA, 5 µg), streptomycin (S, 10 µg), tetracycline (TE, 30 µg), trimethoprim/sulfamethoxazole (SXT, 1.25 µg/23.75 µg), vancomycin (VA, 30 µg) 을사용하였다. 3. RESULTS AND DISCUSSION 3.1. 분리된유산균주의항균활성유산균의항균활성은유산균이생성하는유기산, hydrogen peroxide, diacetyl, bacteriocin 및일부지방산등과같은대사산물과장내상피세포에대한경쟁적부착성에의해병원성미생물의생육을억제한다고알려져있다 [13]. 본연구에서는한국인 7 명의분변으로부터잠재적인유산균주총 25 종을분리하였으며, 분리된유산균주의병원균에대한항균활성결과는 Table 1 과같다. 대조군으로는유산균주를배양하지않은 MRS 배지를사용하였으며, 저해환은나타나지않았다. 선별된유산균주의항균효과는최대 12.5 mm 까지저해환을 나타내었으며, E. coli, E. coli O157:H7, S. aureus 에서는 10 mm 이상의저해환을보이는유산균주를확인할수있었다. 비브리오속에대해서는대부분 10 mm 이하의저해환을확인할수있었으며, V. alginolyticus 에대해서는 MG316, MG 501 이 10 mm 이상의저해환을나타내었다. 본연구결과와유사하게기존의여러연구결과에서도다양한유산균주가 S. aureus, E. coli, Pseudomonas aeruginosa, Salmonella typhi 등에항균효과가있다고보고되고있다 [14-16]. 3.2. 선별된유산균주의동정현재국내에서는프로바이오틱스로사용가능한균주의확보가미흡한수준으로프로바이오틱스균주의확보가절실히요구되고있다 [17]. 이에본연구에서는분변으로부터분리된 25종의유산균주중항균활성효과가우수한 4종의유산균주를선별하였다. 선별된균주는 16S rrna 동정을통해 Lactobacillus rhamnosus MG316, L. acidophilus MG501, L. reuteri MG505, L. gasseri MG570으로각각동정되었다 (Fig. 1). 3.3. 선별된유산균주의내산성및내담즙성유산균이프로바이오틱스로제제이용하기위해서는안전성과안정성을갖추고있는균주의선별이요구된다. 구강을통해섭취된유산균은위액과각종효소가존재하는위를통 Table 1. Origin and direct antibacterial activity against various pathogens of selected strains isolated from fermented foods Origin Human feces Pathogen (mm) Selected Staphylococcus Vibrio strains Escherichia coli E. coli O157:H7 V. alginolyticus aureus parahaemolyticus MG316 11 12.5 11 9.5 10.5 MG501 10 11 10 9.5 10.5 MG5025 7 9 11 7 9 MG5026 11 10 9 6 9 MG5031 8 9 9 6 7.5 MG5034 7 9 9 12 7 MG5040 0 9 11 7 6.5 MG5041 11 9 10 7 6.5 MG5042 7 9.5 9 7 0 MG505 10 10 10 9 10 MG570 11 11 10.5 9 9 MG5055 8 7.5 8.5 9 9 MG5056 7 6 4 7 9 MG5063 7 7 8 9 8.5 MG5064 6.5 6 7 7.5 8 MG5065 7 6 8 6 8 MG5072 6.5 7 6.5 5 8 MG5073 7 5 7.5 9 7.5 MG5074 7.5 5 6 9 7.5 MG5075 7 8 6 7 7 MG5077 6 5 9 6.5 7 MG5078 6.5 9 8 8 6.5 MG5083 4 10 7 7 6 MG5085 10 7 6.5 5 0 MG5086 10 7 6.5 4 0 Zones of inhibition were defined as the diameter of the circle formed as a result of the inhibitory activity of isolates against indicator organisms, excluding the diameter of the cork borer used (6 mm).

156 Korean Society for Biotechnology and Bioengineering Journal 32(2): 153-159 (2017) Fig. 1. Phylogenetic analysis of selected Lactobacillus spp. compared to other lactobacilli. 과하고, 담즙이존재하는십이지장을거쳐최종목적부위인장에도달해야기능적인효과를나타낼수있다. 유산균이위에서위산과같은낮은 ph 에서생존하기위해서위산에대한내산성을나타내야하며 [18,19], 장내극한환경에서생존하기위해서는쓸개에서분비되는담즙에대한내성을지녀야한다 [20]. 산성에대한내성실험의결과, ph 2 에서모든균주가 10 4 CFU/mL 이상의생균수를나타냈으며, MG501 (6.46 log CFU/ ml), MG505 (5.86 log CFU/mL) 이가장높은생균수를보이는것을확인할수있었다. Hood 와 Zottola 의연구 [21] 에따르면 Lactobacillus 중 L. casei 와 L. acidophilus 등의내산성이높으며특히 L. acidophilus 의경우 ph 2 에서도 30~40 분정도생존하는것으로보고되고있다. 체내위액의 ph 는대략 3.0 으로유지되며, 일반적으로 ph 3 에서 3 시간이상생균수를유지하면산성에높은내성을보이는것으로알려져있다 [10]. Mishra 의연구 [22] 에따르면프로바이오틱스로사용되는여러균주들이 ph 2 에서일반적으로 10 4 CFU/mL 이하의생균 수를보이는것을확인할수있는데, 본연구에서선별된유산균주의경우산성에대해우수한내성능력을가지고있는것을확인할수있다. 선별된균주의담즙염에대한내성을확인하기위해서일반적으로 0.3% 의담즙염농도를통해내성을확인하는데 [23], Lin 의연구결과 [24] 에따르면 0.3% 담즙염에서유산균들의생균수가 4 log CFU/mL 이하로담즙염에민감하다고알려져있다. 본연구에서선별된 4 종의균주는 3% 담즙염농도에서평균 7.38 log CFU/mL 의매우높은생균수를보여, 담즙염에대해매우우수한내성을보이는것을확인할수있다. 콜레스테롤을분해하는균주는담즙염에대한저항성및담즙염의탈포합 (deconjugation) 능력이강한특징이있다고알려져있다 [25]. 본연구에서선별된 4 종의유산균주의경우 3% oxgall 이포함된배지에서도저해를받지않는것으로나타나, 콜레스테롤분해기능에대한잠재적인가능성을가지고있다고사료된다. Table 2. Effect of ph and bile salt on the survival of selected strains Selected strains ph tolerance 1) Bile salts tolerance 2) ph 7 ph 2 0% 1% 2% 3% MG316 8.44±0.03 4.62±0.44 9.31±0.08 8.27±0.06 8.19±0.04 7.96±0.03 MG501 8.57±0.05 6.46±0.04 9.23±0.10 8.00±0.02 7.58±0.11 7.45±0.04 MG505 8.98±0.06 5.86±0.04 9.18±0.10 8.11±0.04 7.71±0.08 7.08±0.01 MG570 8.47±0.01 5.61±0.04 8.46±0.11 7.38±0.08 7.02±0.11 7.01±0.61 1) The ph tolerance results are shown the viable counts (log CFU/mL) of each isolate at ph 7 and ph 2 for 3 h. 2) Bile-salts tolerance results are shown as the viable counts (log CFU/mL) of each isolate at MRS media with 0% and 3% Oxgall at 37 o C for 24 h.

한국인장관에서분리한유산균의항균활성및프로바이오틱스특성연구 157 Fig. 2. The autoaggregation ability for 5 h. 3.4. Autoaggregation 프로바이오틱스로사용되는유산균의경우, 체내에서지속적으로작용하기위해서장세포에부착할수있는세포부착능이요구된다 [26]. Autoaggregation 능력은세포부착능을간접적으로확인할수있는방법으로, Del [26] 과 Kos [27] 의연구결과에따르면, autoaggregation 능력이높은균주가실제세포부착능력또한높다고알려져있다. 본연구에서선별된 4 종의균주에대한장내부착성을확인하기위해 autoaggregation 실험을진행하였다 (Fig. 2). 4 종의균주에대한 autoaggregation 능력은평균 62.1% 로높은 autoaggregation 능력을보였으며, MG505 균주가 66.3% 로가장높은 autoaggregation 능력을나타내었다. Malik 의연구결과 [28] 에따르면, L. plantarum CMPG5300 의 in vitro 상의 autoaggregation 이약 70% 로나타났으며, in vivo 상인상피세포에서의부착능도 50% 로 in vitro 상의결과와 in vivo 상의결과가연관되어있는것으로알려져있다. 3.5. 선별된균주의항생제내성최근유산균이항생물질내성을유발할수있는 plasmid 와 transposon 을보유하고있다는연구가지속적으로발표되고있다. Enterococci 는발효유제품의숙성및풍미형성에관여하는프로바이오틱스로오랜기간이용되어왔으나, 높은항생물질내성유전자를가지고있음에따라기회감염성균으로도인식되고있다 [29,30]. 항생물질내성의문제는 enterococci 뿐만아니라 Lactococcus, Lactobacillus, Leuconostoc, Pediococcus 속유산균및 Bifidobacteria 에서도나타나고있 Table 3. Antibiotic sensitivity and resistant activity of selected strains Antibiotics (µg/disc) Selected strains MG316 MG501 MG505 MG570 Chloramphenicol (C, 30) S 1 S R 3 S Sulphamethoxazole-trimethoprim (SXT, 1.25/23.75) R S R R Tetracyclin (TE, 30) S S S S Cephalothin (CF, 30) S S S S Gentamicin (GM, 10) R I 2 R R Erythromycin (E, 15) S S S S Vancomycin (VA, 30) R S R S Ampicillin (AM, 10) S S S S Rifampicin (RA, 5) S S S S Ciprofloxacin (CIP, 5) S S R R Cefotaxime (CTX, 30) S S R S Cefepime (CEP, 30) S S I S Cefotetan (CTT, 30) R R R S Nalidixic acid (NA, 30) R R R R Kanamycin (K, 30) R R R R Streptomycin (10) R R R I 1 sensitive, 2 intermediate, 3 resistant.

158 Korean Society for Biotechnology and Bioengineering Journal 32(2): 153-159 (2017) 어, 프로바이오틱스균주는항생제내성양상을명확하게밝혀야한다 [31]. 본연구에서선별된유산균 4 종에대해항생제내성특성은 Table 3 과같다. TE, CF, E, AM, RA, CEP 에서는대부분저해되는것을확인할수있었다. CTT, K, S, CIP, NA 에는내성을나타내는것을확인할수있는데, 기존의연구결과 [31] 에따르면, 많은 Lactobacilli 가 aminoglycoside 계항생제인 K, S 에내성을가지고있으며, 본연구결과와같이유사한결과를통해유산균주가갖는특징중의하나라고사료된다. 4. CONCLUSION 본연구는한국인의분변으로부터분리한유산균을통해프로바이오틱스로사용가능성을확인하기위해항균활성효과와생리활성기능을확인하였다. 분리된 25 종의유산균주중항균활성효과가우수한 4 종을선별하였으며, 16S rrna sequencing 결과 Lactobacillus rhamnosus MG316, L. acidophilus MG501, L. reuteri MG505, L. gasseri MG570 으로동정되었다. 선별된유산균주는모두건강기능식품의기준및규격에등재되어안정성이입증되어있는균주로상용화가가능하다. 선별된유산균주는산성과담즙염에대한내성이우수하였으며, autoaggregation 능력이평균 62.1% 로장내부착성이우수할것이라사료된다. 본연구를통해선별된유산균주들은향후프로바이오틱스개발분야에주요한소재자원으로활용될수있을것이라기대된다. REFERENCES 1. Food and Agricultural Organization of the United Nations and World Health Organization. (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. World Health Organization. 2. Alander, M., R. Satokari, R. Korpela, M. Saxelin, T. Vilpponen- Salmela, and A. von Wright (1999) Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl. Environ. Microbiol. 65: 351-354. 3. Axelsson, L. T., T. C. Chung, W. G. Dobrogosz, and S. E. Lindgren (1989) Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri. Microb. Ecol. Health Dis. 2: 131-136. 4. Seo J. G., G. S. Lee, J. E. Kim, and M. J. Chung (2010) Development of probiotic products and challengers. KSBB J. 25: 303-310. 5. Vrese, M., A. Stegelmann, B. Ritcher, S. Fenselau, C. Laue, and J. Schrezenmeir (2001) Probiotics: Compensation for lactase insufficiency. Am. J. Clin. Nutr. 73: S421-S429. 6. Fukushima, Y., Y. Kawata, H. Hara, A. Terada, and T. Mitsuoka (1998) Effect of a probiotic formula on intestinal immunoglobulin a production in healthy children. Int. J. Food Microbiol. 30: 39-44. 7. Isolauri, E., Y. Sutas, P. Kankaanpaa, H. Arvilommi, and S. Salminen (2001) Probiotics: Effects on immunity. Am. J. Clin. Nutr. 73: 444-450. 8. Isolauri, E., S. Salminen, and A. C. Ouwehand (2004) Microbialgut interactions in health and disease. Probiotics. Best Prac. Res. Cl. Em. 18: 299-313. 9. Vrese, M., A. Stegelmann, B. Ritcher, S. Fenselau, C. Laue, and J. Schrezenmeir (2001) Probiotics: compensation for lactase insufficiency. Am. J. Clin. Nutr. 73: 421?429. 10. Guo, X. H., J. M. Kim, H. M. Nam, S. Y. Park and J. M. Kim (2010) Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties. Anaerobe 16: 321?326. 11. Kassa, I. A., M. Hamze, D. Hober, N. E. Chihib, and D. Drider (2014) Identification of vaginal lactobacilli with potential probiotic propertied isolated from women in North Lebanon. Microb. Ecol. 67: 722-734. 12. CLSI. (2006) Methods for Antimicrobial Dilution and Disk Susceptibility Testing ofinfrequently Isolated or Fastidious Bacteria; Approved Guideline M45-A.Clinical and Laboratory Standards Institute, Waune, PA. 13. Tagg, J. R., A. S. Dajani, and L. W. Wannamaker (1976) Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40: 722-756. 14. Adesokan, I. A., B. B. Odetoyinbo, and A. O. Olubamiwa (2008) Biopreservative activity of lactic acid bacteria on suya produced from poultry meat. Afr. J. Biotech. 7: 3799-3803. 15. Obadina, A. O., O. B. Oyewole, L. O. Sanni, and K. I. Tomlins (2006) Biopreservative activities of Lactobacillus plantarum strains in fermenting cassava fufu. Afr. J. Biotech. 5: 620-623. 16. Puttalingamma, V., K. Begum, and A. S. Bawa (2006) Antimicrobial peptides-new weapons against enteric pathogens. Pak. J. Nutr. 5: 432-435. 17. Bang, J. H., H. J. Shin, H. J. Choi, D. W. Kim, C. S. Ahn, Y. K. Jeong, and W. H. Joo (2012) Probiotic potential of Lactobacillus isolates. J. Life Sci. 22: 251-258. 18. Sim, J. H., S. J. Oh, S. K. Kim, and Y. J. Baek (1995) Comparative tests on the acid tolerance of some lactic acid bacteria species isolated from lactic fermented products. Korean J. Food Sci. Technol. 27: 101-104. 19. Saarela, M., G. Mogensen, R. Fondn, J. Mtt, and T. Mattila-Sandholm (2000) Probiotic bacteria: Safety, functional and technological properties. J. Biotechnol. 84: 197-215. 20. Dunne, C., L. O Mahony, L. Murphy, G. Thornton, D. Morrissey, S. O Halloran, M. Feeney, S. Flynn, G. Fitzgerald, C. Daly, B. Kiely, G. C. O Sullivan, F. Shanahan, and J. K. Collins (2001) In vitro selection criteria for probiotic bacteria of human origin: Correlation with in vivo findings. Am. J. Clin. Nutr. 73: 386-392. 21. Hood, S. K., E. A. Zottola (1988) Effect of low ph on the ability of Lactobacillus acidophilus to survive and adhere to human intestinal cells. J. Food Sci 53: 1514-1520 22. Mishra, V., and D. N. Prasad (2005) Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. Int. J. Food Microbiol. 103: 109-115. 23. Gilliland, S. E., T. E. Staley, and L. J. Bush (1984) Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct.

한국인장관에서분리한유산균의항균활성및프로바이오틱스특성연구 159 J. Dairy Sci. 67: 3045-5301. 24. Lin, W. H., C. F. Hwangb, L. W. Chenc, and H. Y. Tsen (2006) Viable counts, characteristic evaluation for commercial lactic acid bacteria products. Food Microbiol. 23: 74-81. 25. Gilliland, S. E. and D. K. Walker (1990) Factors to consider when selecting a culture to produce of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans. J. Dairy Sci. 73: 905-911. 26. Del Re, B., B. Sgorbati, M. Moglioli, and D. Palenzona (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of bafidobacterium longum. Lett. Appl. Microbiol. 31:438-442. 27. Kos, B., J. Šuškoviæ, S. Vukoviæ, M. Šimpraga, J. Frece, and S. Matošiæ (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J. Appl. Microbiol. 94: 981-987. 28. Malik, S., M. I. Petrova, I. J. J. Claes, T. L. A. Verhoeven, P. Busschaert, M. Vaneechoutte, B. Lievens, I. Lambrichts, R. J. Siezen, J. Balzarini, J. Vanderleyden, and S. Lebeer (2013) The highly autoaggregative and adhesive phenotype of the vaginal Lactobacillus plantarum strain CMPG5300 is sortase dependent. Appl. Environ. Microbiol. 79: 4576-4585. 29. Franz, C. M. A. P., M. Huch, H. Abriouel, W. Holzapfel, and A. Gálvez (2011) Enterococci as probiotics and their implications in food safety. Int. J. Food Microbiol. 151: 125-140. 30. Ogier, J. C. and P. Serror (2008) Safety assessment of dairy microorganisms: The Enterococcus genus. Int. J. Food Microbiol. 126: 291-301. 31. Mathur, S. and R. Singh (2005) Antibiotic resistance in food lactic acid bacteria - A review. Int. J. Food Microbiol. 105: 281-295.