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1 pissn eissn J. Milk Sci. Biotechnol. 2019;37(1): ARTICLE 유아분변에서분리한비피도박테리아의프로바이오틱스기능성연구 강창호 * 김용경 한설화 정율아 박혜민 백남수 ( 주 ) 메디오젠 Probiotic Properties of Bifidobacteria Isolated from Feces of Infants Chang-Ho Kang *, YongGyeong Kim, Seul Hwa Han, Yulah Jeong, HyeMin Park, and Nam-Soo Paek MEDIOGEN, Co., Ltd., Jecheon, Korea Received: February 7, 2019 Revised: March 12, 2019 Accepted: March 12, 2019 *Corresponding author : Chang-Ho Kang MEDIOGEN, Co., Ltd., Jecheon, Korea Tel : Fax : changho-kang@naver.com Copyright 2019 Korean Society of Milk Science and Biotechnology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ORCID Chang-Ho Kang Yonggyeong Kim Seul Hwa Han Yulah Jeong HyeMin Park Nam-Soo Paek Abstract Bifidobacteria are a prototype probiotic, which normally inhabit the intestinal tract of humans. In the present study, four species of Bifidobacterium isolated from the feces of infants were characterized. The tolerance for acid or bile salt, autoaggregation, and antibiotic resistance of the bacteria were examined. The four species were resistant to low ph, bile salts, and up to 3% bile acid. Autoaggregation rates were as high as 90%. The bacteria were consistently resistant to gentamicin, kanamycin, streptomycin, ciprofloxacin, and nalidixic acid. Due to their tolerance to environmental factors like acid and bile salts, B. longum MG723, B. breve MG729, B. bifidum MG731, and B. animalis subsp. lactis MG741 are potentially valuable as probiotics and may be useful for industrial application. Keywords Bifidobacterium, probiotics, acid, bile salt, autoaggregation 서론 프로바이오틱스는숙주의장내미생물균형을유지시킴으로써유익한작용을하는살아있는미생물을 의미한다 (FAO/WHO, 2011). 일반적으로프로바이오틱스는장내균총의안정화, 유해세균의억제, 면역활성화작용, 항암작용, 유당불내증의경감등의기능을갖고있다고알려져있다 (Alander et al., 1999; Vrese et al., 2001; Seo et al., 2010). 일반적으로유산균은 GRAS(generally regarded as safe) 미생물로분류되고있어, 프로바이오틱스로서의이용가치가매우높은것으로알려 져있다. 프로바이오틱스로가장많이이용되고있는균주는 Lactobacillus 와 Bifidobacterium 이며, 발효유, 건강식품, 유산균정장제등에사용되고있다 (Vrese et al., 2001). 미생물이프로바이 오틱스로사용되기위해서는섭취에대한안전성, 소화기관내에서의안정성, 세포정착능력과항균활 성에대한기능성및배양에대한기술적인부분에대한검증이되어야하며, 제조공정이나완제품 유통과정중에생존성이충분히유지되어야한다 (Fukushima et al., 1998; Isolauri et al., 2001). 비피더스균 (Bifidobaterium spp.) 은인체장내에상재균으로다양한대사와면역조절기능으로인 체에유익한균으로잘알려져있다 (Poupard et al., 1973). Bifidobacterium 속은유아에서는 Bi. bifidum, Bi. longum, Bi. infantis, Bi. breve 등이검출되고, 성인에서는 Bi. bifidum, Bi. longum, Bi. adolescentis 등이주로검출된다고알려져있다 (Holt, 1997). 비피더스균은일반젖 산균과는달리담즙에비교적높은저항력을가지고있으며, 유기산과지방산을생성시켜항염증효 40 J Milk Sci Biotechnol Vol. 37, No. 1

2 유아분변에서분리한비피도박테리아의프로바이오틱스기능성연구 과 (Maslowski et al., 2009) 등숙주에게이로운작용을하며, 과민성대장증후군을완화시키고 (Guglielmetti et al., 2011), 유해균억제및혈중콜레스테롤감소, 면역조절작용 (Savard et al., 2011) 을한다. Bifidobacteria 의 probiotic 효능이밝혀지면서인체에서분리한 Bi. longum, Bi. lactis, Bi. breve, Bi. adolescentis, Bi. infantis 가주로이용되고있다 (Sanders, 1995; Kurmann, 1998). 이에본연구에서는한국의건강한어린이분변에서분리한비피더스균의내산성, 내담즙성과 autoaggregation 능력등을통해프로바이오틱스로적합한균주인지확인하고자한다. 재료및방법 1. 실험재료및배지본연구에사용된비피더스균주는강원도지역에서출생한건강상태가양호한유아 (2 15 개월 ) 의분변으로부터분리하여실험에사용하였다 (Table 1). 증식용배양배지는 0.05% L-cystein 을첨가한 MRS broth(mmrs, Difco, MI, USA) 를사용하였다. 균주의보관은 20% glycerol stock으로 70 에서보관하였다. 2. 비피더스균주의동정분리균주의동정을위하여 16S rrna gene sequencing 을수행하였으며, 유전자의증폭은 the universal rrna gene primers(27f and 1492R) 를사용하였고, 각과정은 SolGent Co.(Daejeon, Korea) 을통하여진행하였다. 이후분석된 16S rrna sequencing 결과는 National Center for Biotechnology Institute(NCBI) 의 Basic Local Alignment Search Tool(BLAST) analysis ( 를통해 Genbank database 와비교하여동정하였다. Sequence 는 PHYDIT 3.1을통하여 alignment 를진행하였고, 이후 MEGA 5.1 software 를이용하여 neighborjoining method로계통수를작성하였다 (Tamura et al., 2011). 3. 비피더스균주의산성및담즙염내성확인분리된비피더스균주의장내환경과같은산성및담즙염에대한생균력을확인하기위해 Guo의방법 (2010) 으로실험을진행하였다. 비피더스균주를 mmrs 배지에 24시간배양한후, OD 으로조정하여새로운 mmrs 배지에 1% 접종하여 18시간배양하였다. 배양액은원심분리 (4,000 g, 4, 5분 ) 한후, 0.85% saline 에 2번세척하였고, OD ( CFU/mL) 으로조정하여각각산성과담즙염에대한내성실험에사용하였다. 산성에대한내성을확인하기위하여비피더스균주희석액 1 ml를 9 ml PBS(pH 2와 7) 에첨가후진탕한뒤 37 에서 3시간배양한후생균수를확인하였다. 담즙염내성은 0 1% oxgall(difco, MI, USA) 을포함한 mmrs 배지에비피더스균주희석액을 1% 접종하고, 37 에 24시간배양한후생균수를확인하였다. 4. 비피더스균주의 autoaggregation Table 1. Bifidobacterial strains tested in this study Species/ strains Bifidobacterium longum MG723 Bifidobacterium breve MG729 Bifidobacterium bifidum MG731 Bifidobacterium animalis subsp. lactis MG741 Sources Korean infants feces Korean infants feces Korean infants feces Korean infants feces J Milk Sci Biotechnol Vol. 37, No. 1 41

3 Kang et al. 장내세포내부착능력을간접적으로확인하기위하여 Kassa 의연구 (2014) 를변형하여 autoaggregation 실험을진행하였다. mmrs 배지에유산균을전배양한배양액을 OD 으로조정하여새로운 5 ml mmrs 배지에 1% 접종한뒤, 18시간배양하여실험에사용하였다. 배양한유산균은원심분리 (4,000 g, 4, 5분 ) 한후, PBS에 2번세척하였고, OD 으로조정하여균주현탁액 5 ml를 10초간진탕한뒤 5시간동안방치하면서 autoaggregation 을확인하였다. 실험시작직후 (A0) 와 5시간후 (A), 각각 0.1 ml의상등액을취해 0.9 ml PBS와혼합한뒤 600 nm에서흡광도를측정 (A0, A) 하였고, 다음계산식에따라 autoaggregation 을계산하였다. Autoaggregation(%) = (A0 A)/A 비피더스균주의항생제감수성시험항생제감수성시험은 Clinical and Laboratory Standards Institute(CLSI) guidelines(2006) 를따라 MHA 배지를이용 disc method 에의해수행하였다. 분리된비피더스균주를 mmrs 액체배지에서배양한후, 10 4 CFU/mL 정도의균체를 MHA 배지에도말한후, 항생제디스크 (BBL, Becton Dickinson, USA) 를올려놓았다. 37 에서 24시간배양한후억제환의크기를 mm 단위로측정하여표준지표에따라판정하였다. 사용한항생제디스크는모두 BD BBL(Becton Dickinson and Company, USA) 사제품으로 ampicillin(am, 10 μg), cefotaxime(ctx, 30 μg), cefotetan (CTT, 30 μg), cephalothin(cf, 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. 6. 비피더스균주의효소활성확인본연구에사용된비피더스균주중프로바이오틱스성능이우수한비피더스균주를선별하여효소활성을확인하였다. mmrs 배지에평판도말한후 37 에서 18시간배양후균체를취하여 API ZYM enzyme system(bio Merieux, France) 의 API Suspension medium(2 ml, Bio Meriux, France) 에 McFarland standard(bio Meriux, France) 로탁도를맞추었다. API ZYM strip 각각의큐플에 Suspension medium 을 65 μl씩분주하고 37 에서 4시간동안배양한후, 결과를확인하였다. 결과및고찰 1. 분리된비피더스균주의동정현재국내에서는프로바이오틱스로사용가능한균주의확보가미흡한수준으로프로바이오틱스균주의확보가절실히요구되고있다 (Bang et al., 2012). 이에본연구에서분변으로부터분리된균주는 16S rrna 동정을통해 Bifidobacterium longum MG723, Bi. breve MG729, Bi. bifidum MG731, Bi. animali subsp. lactis MG741 로각각동정되었다 (Fig. 1). 분리균주에대한세포의형태는주사전자현미경을통하여관찰하였으며, 모두간균의형태인것을확인할수있었다 (Fig. 2). 2. 분리된비피더스균주의내산성및내담즙성 유산균이프로바이오틱스로기능을발휘하기위해서는소화관내의조건에서생존해야한다. 구강을 통해섭취된유산균은위액과각종효소가존재하는위를통과하고, 담즙이존재하는십이지장을거 42 J Milk Sci Biotechnol Vol. 37, No. 1

4 유아 분변에서 분리한 비피도박테리아의 프로바이오틱스 기능성 연구 Fig. 1. The neighbor-joining tree that is based on bacterial 16S rrna gene sequences from different bacterial isolates in the present study, along with sequences available in the GenBank database. Numerical values indicate the bootstrap percentile from 1,000 replicates. Fig. 2. SEM images of selected strains. (A) Bifidobacterium longum MG723; (B) Bifidobacterium breve MG729; (C) Bifidobacterium bifidum MG731; (D) Bifidobacterium animalis subsp. lactis MG741. 쳐 최종 목적 부위인 장에 도달해야 기능적인 효과를 나타낼 수 있다. 유산균이 위에서 위산과 같은 낮은 ph에서 생존하기 위해서 위산에 대한 내산성을 나타내야 하며(Sim et al., 1995; Saarela et al., 2000), 장내 극한 환경에서 생존하기 위해서는 쓸개에서 분비되는 담즙에 대한 내성을 지녀야 한다(Dunne et al., 2001). 산성에 대한 내성실험의 결과, ph 2에서 모든 균주가 102 CFU/mL이상 의 생균수를 나타냈으며, MG741(4.01 log CFU/mL)이 가장 높은 생균수를 보이는 것을 확인할 수 있었다(Table 2). 체내 위액의 ph는 대략 3.0으로 유지되며, 일반적으로 ph 3에서 3시간 이상 생균수를 유지하면 산성에 높은 내성을 보이는 것으로 알려져 있다(Guo et al., 2010). Mishra의 연구(2005)에 따르면 프로바이오틱스로 사용되는 여러 균주들이 ph 2에서 일반적으로 104 CFU/mL J Milk Sci Biotechnol Vol. 37, No. 1 43

5 Kang et al. Table 2. Effect of ph and bile salt on the survival of selected strains 1) 2) Selected strains ph tolerance 1) Bile salts tolerance 2) ph 7 ph 2 0% 0.3% 0.5% 1.0% MG ± ± ± ± ± ±0.03 MG ± ± ± ± ± ±0.02 MG ± ± ± ± ± ±0.05 MG ± ± ± ± ± ±0.15 The ph tolerance results are shown the viable counts (log CFU/mL) of each isolate at ph 7 and ph 2 for 3 h. Bile-salts tolerance results are shown as the viable counts (log CFU/mL) of each isolate at MRS media with 0% and 1% Oxgall at 37 for 24 h. 이하의생균수를보이는것을확인할수있으며, Lankaputhra and Shah의연구 (1995) 에따르면 9종의비피더스균주의 ph 에서의내산성에서 Bi. bifidum, Bi. breve, Bi. adolescentis 는내산성이매우약하다고보고하고있다. 이에본연구에서사용된비피더스균주의경우산성에대해우수한내성능력을가지고있는것을확인할수있다. 분리된비피더스균주의담즙염에대한내성을확인하기위해서일반적으로 0.3% 의담즙염농도를통해내성을확인하는데 (Gilliland et al., 1984), Maslowski 등 (2009) 의연구에의하면비피더스균주들은일반유산균과달리담즙염에비교적높은내성을가지고있는것으로알려져있다. 본연구에서사용된비피더스균주들은 0.3% 담즙염농도에서평균 7.67 log CFU/mL 의매우높은생균수를보여, 담즙염에대해매우우수한내성을보이는것을확인할수있다 (Table 2). MG 741균주의경우 1% 담즘염농도에서도 6.67 log CFU/mL 로다른비피더스균주들과비교하여매우높은내성을나타내는것을확인할수있다. Clark and Martind 의연구 (1994) 에따르면, 1% oxgall 농도에서생존율을비교한결과, Bi. bifidum, Bi. infantis, Bi. adolescentis 는 12시간후생존하지못하였으며, Bi. longum 은 12시간후에도높은생균수를보였다고보고하고있다. 3. 분리된비피더스균주의 autoaggregation 프로바이오틱스로사용되는유산균의경우, 체내에서지속적으로작용하기위해서장세포에부착할수있는세포부착능이요구된다 (Lin et al., 2006). Autoaggregation 능력은세포부착능을간접적으로확인할수있는방법으로, Kos(2003) 의연구결과에따르면, autoaggregation 능력이높은균주가실제세포부착능력또한높다고알려져있다. 본연구에서사용된비피더스균주에대한장내부착성을확인하기위해 autoaggregation 실험을진행하였다 (Fig. 3). 비피더스균주에대한 autoaggregation 능력은평균 96.2% 로매우높은 autoaggregation 능력을보였으며, MG723 균주가 99.5±0.03% 로가장높은 autoaggregation 능력을나타내었다. Malik의연구결과 (2013) 에따르면, 유산균주의 in vitro 에서 autoaggregation 이약 70% 로나타났으며, in vivo 상의상피세포에서의부착능도 50% 로 in vitro 상의결과와 in vivo 상의결과가연관되어있는것으로알려져있다. 4. 분리된비피더스균주의항생제내성유산균은프로바이오틱스로서유익한측면에서의장점과더불어기회감염균으로의가능성에대해확인해보아야한다. 최근 Streptococcus, Lactobacillus 등이심내막염수술후에감염된것으로보고 (Oakety et al., 1995) 되었는데, 질병예방및치료의목적으로사용되고있는항생제의빈도와농도가증가되면서장내미생물은유산균의항생제내성과함께유전자전이에대한연구가진행되고있다. 분리된비피더스균 4종에대해항생제내성특성은 Table 3과같다. RA, E, VA, AM, CEP 에서는대부분저해되는것을확인할수있었다. GM, K, S, CIP, NA에는내성을나타내는것을확인할수있는데, Ann의연구결과 (2011) 에따르면, 비피더스균주들은세포벽합성저해제인페니실린계항생제에대해서는감수성이높고, cephalosporin 계에도감수성이높은것으로알려져있다. 44 J Milk Sci Biotechnol Vol. 37, No. 1

유아분변에서분리한비피도박테리아의프로바이오틱스기능성연구 Fig. 3. Percentages of autoaggregation (%) of selected strains at 5 h. The results represent the means±sd of three independent experiments. Table 3.

6 유아분변에서분리한비피도박테리아의프로바이오틱스기능성연구 Fig. 3. Percentages of autoaggregation (%) of selected strains at 5 h. The results represent the means±sd of three independent experiments. Table 3. Antibiotic sensitivity and resistant activity of selected strains Antibiotics (µg/disc) Selected strains MG723 MG729 MG731 MG741 Sulphamethoxazole-trimethoprim (1.25/23.75) R R R S Tetracyclin (30) S R S I Cephalothin (30) I R S S Gentamicin (10) R R R R Erythromycin (15) S S S S Vancomycin (30) S S S S Ampicillin (10) S S S S Rifampicin (5) S S S S Ciprofloxacin (5) R R R R Cefotaxime (30) S R S S Cefepime (30) S I S S Cefotetan (30) R R S S Nalidixic acid (30) R R R R Kanamycin (30) R R R R Streptomycin (10) R R R R S: sensitive, I: intermediate, R: resistant. 단백질합성억제제인 aminoglycoside 계에는대부분내성을나타내며, DNA 합성저해제인퀴놀린계 에대해서도내성이강한것으로보고되고있다. 5. Bifidobacterium animalis subsp. lactis MG741의효소활성분리된비피더스균주중산성과담즙염에내성이우수한 MG741 균주의효소활성을측정하였으며, Table 4와같이 19종류의효소중 3종류 (Leucine arylamidase, Naphtol-AS-BI-phosphohydrolase, β glucuronidase) 의효소활성이확인되었다. Leucine arylamidase 는펩타이드가수분해효소이며, 효소, 에스트로젠대사및세포분열에관여하는 β-glucuronidase 에도활성이있는것을확인하였다. J Milk Sci Biotechnol Vol. 37, No. 1 45

7 Kang et al. Table 4. Enzyme activities of Bifidobacterium animalis subsp. lactis strain MG741 by API ZYM analysis Enzyme Result Alkaline phosphatase Esterase [C4] Esterase Lipase [C8] Lipase [C14] Leucine arylamidase + Valine arylamidase Crystinearylamidase Trypsin α Chymotrypsin Acid phosphatase Naphtol-AS-BI-phosphohydrolase + α Galactosidase β Glucuronidase + β Glucosidase α Glucosidase Bet Glucosidase N-Acetyl-beta-glucosaminidase α Mannosidase α Fucosidase 결론 본연구는한국인유아에서분리한비피더스균주를통해프로바이오틱스로사용가능성을확인하기위해산, 담즙염, 자가부착성및항생제내성에대한연구를진행하였다. 분리된비피더스균주는 16S rrna sequencing 결과, Bifidobacterium longum, Bi. breve, Bi. bifidum, Bi. animalis subsp. lactis 로동정되었으며, 모두건강기능식품의기준및규격에등재되어안정성이입증되어있는균주로확인되었다. 분리된비피더스균주는산성과담즙염에대한내성이우수하였으며, autoaggregation 능력이평균 96.2% 로장내부착성이우수할것이라사료된다. 본연구에사용된비피더스균주들은현재시판되는상업용종균과비교해서도동등한프로바이오틱스기능성을보이고있으며, 향후프로바이오틱스개발분야에주요한소재자원으로활용될수있을것이라기대된다. References Alander, M., Satokari, R., Korpela, R., Saxelin, M., Vilpponen-Salmela, T. and von Wright, A Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl. Environ. Microbiol. 65: Ann, Y. G Probiotic lactic acid bacteria. Korean J. Food. & Nutr. 24: Bang, J. H., Shin, H. J., Choi, H. J., Kim, D. W., Ahn, C. S., Jeong, Y. K. and Joo, W. H Probiotic potential of Lactobacillus isolates. J. Life Sci. 22: Clark, P. A. and Martin, J. H Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods. III. Tolerance to simulated bile concentrations of human small intestines. Cultured Dairy Products Journal. 46 J Milk Sci Biotechnol Vol. 37, No. 1

8 유아분변에서분리한비피도박테리아의프로바이오틱스기능성연구 CLSI Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline M45-A. Clinical and Laboratory Standards Institute, Waune, PA. USA. Dunne, C., O Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O Halloran, S., Feeney, M., Flynn, S., Fitzgerald, G., Daly, C., Kiely, B., O Sullivan, G. C., Shanahan, F. and Collins, J. K In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am. J. Clin. Nutr. 73: Food and Agricultural Organization of the United Nations and World Health Organization Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. World Health Organization. Fukushima, Y., Kawata, Y., Hara, H., Terada, A. and Mitsuoka, T Effect of a probiotic formula on intestinal immunoglobulin a production in healthy children. Int. J. Food Microbiol. 30: Gilliland, S. E., Staley, T. E. and Bush, L. J Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J. Dairy Sci. 67: Guglielmetti, S., Mora, D., Gschwender, M. and Popp, K Randomised clinical trial: Bifidobacterium bifidum MIMBb75 significantly alleviates irritable bowel syndrome and improves quality of life a double-blind, placebo-controlled study. Aliment. Pharmacol. Ther. 33: Guo, X. H., Kim, J. M., Nam, H. M., Park, S. Y. and Kim, J. M Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties. Anaerobe 16: Holt, J. G Bergey's manual of determinative bacteriology 8th ed. Williams and Wilkins, Baltimore, MD. pp Isolauri, E., Sutas, Y., Kankaanpaa, P., Arvilommi, H. and Salminen, S Probiotics: Effects on immunity. Am. J. Clin. Nutr. 73: Kassa, I. A., Hamze, M., Hober, D., Chihib, N. E. and Drider, D Identification of vaginal lactobacilli with potential probiotic propertied isolated from women in North Lebanon. Microb. Ecol. 67: Kos, B., Šuškovia, J., Vukovia, S., Šimpraga, M., Frece, J. and Matošia, S Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J. Appl. Microbiol. 94: Kurmann, J. A Starters for fermented milks: starters with selected intestinal bacteria. Bull. IDF. 227: Lankaputhra, W. E. V. and Shah, N. P Survival of Lactobacillus acidophilus and Bifidobacterium spp. In the presence of acid and bile salts. Cult. Dairy. Products J. 30:2 7. Lin, W. H., Hwangb, C. F., Chenc, L. W. and Tsen, H. Y Viable counts, characteristic evaluation for commercial lactic acid bacteria products. Food Microbiol. 23: Malik, S., Petrova, M. I., Claes, I. J. J., Verhoeven, T. L. A., Busschaert, P., Vaneechoutte, M., Lievens, B., Lambrichts, I., Siezen, R. J., Balzarini, J., Vanderleyden, J. and Lebeer, J Milk Sci Biotechnol Vol. 37, No. 1 47

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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