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pissn 1229-1153 J. Fd Hyg. Safety Vol. 27, No. 4, pp. 449~455 (2012) Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 다양한유래에서분리된황색포도상구균에대한유기산과천연항균물질의저해효과 김보람 1 유진희 1 정규석 2 허성기 2 이선영 1 * 1 중앙대학교식품공학부, 2 농촌진흥청국립농업과학원농산물안전성부유해생물과 Inhibitory Effect of Organic Acids and Natural Occurring Antimicrobials Against Staphylococcus aureus Isolates from Various Origins Bo-Ram Kim 1, Jin-Hee Yoo 1, Kyu-Seok Jung 2, Sung gi Heu 2, and Sun-Young Lee 1 * 1 Department of Food Science and Technology, Chung-Ang University, 72-1, Nae-ri, Daedeok-myeon, Anseong-si, Gyeonggi-do 456-756, Korea 2 Microbial Safety Division, Department of Agro-food Safety, National Academy of Agricultural Science, Rural Development Administration, Gyeonggi-do 441-857, Korea (Received October 18, 2012/Revised October 30, 2012/Accepted November 14, 2012) ABSTRACT - The purpose of this study is to evaluate antimicrobial effects of organic acid and some natural occurring antimicrobials against Staphylococcus aureus isolated from various origins (vegetables, peanut, pea leaf, kim- bab, person, perilla leaf, and animal) and to calculate their MIC and MBC values. Five organic acids (acetic, lactic, citric, malic, and propionic acid), three essential oils (carvacrol, thymol, and eugenol), and two other natural antimicrobials (nisin and cinnamic acid) were evaluated for their antimicrobial effects against 113 strains of S. aureus using combination treatments. Propionic acid (7%), nisin (1%), thymol (1%), carvacrol (1%) showed antimicrobial activities against S. aureus strains in agar disc diffusion test. And, carvacrol, thymol, and nisin were found to be the most effective with the lowest MIC values of 0.0313%, 0.0625%, and 0.0625% against S.aureus, respectively. Propionic acid (0.2313%) and citric acid (0.6000%) were the most effective among organic acids tested. Therefore, these five antimicrobials were selected for next combination treatments. Combination of propionic acid and citric acid were showed the strongest inhibitory effectiveness against S. aureus among combination treatments. These results suggest that organic acid such as propionic and citric acid, and natural occurring antimicrobial such as nisin, carvacrol, and thymol might be possibly used as preservatives for inhibiting S. aureus in foods. Key wards: Stapylococcus aureus, Essential oil, Antimicrobial 서론 최근경제성장에따른소득수준의향상으로식생활패턴이바뀌고외식및즉석조리식품소비증가에따라식중독이증가하는추세이다 1). 실제로우리나라의식중독발생통계에따르면외식의생활화로인해 5년전보다발생건수가 2.5배증가하였고, 환자수는 20% 증가하였다 2). 특히, 유원지나터미널에서판매되는김밥의위생점검결과총 *Correspondence to: Sun-Young Lee, Department of Food Science and Technology, Chung-Ang University, 72-1 Nae-ri, Daedeokmyeon, Anseong-si, Gyeonggi-do 456-756, Korea Tel: 82-31-670-4587, Fax: 82-31-676-8741 E-mail: nina6026@cau.ac.kr 77개의김밥중 21개에서식중독균이검출되었으며, 원인균으로는황색포도상구균 (Stapylococcus aureus) 과 Bacillus cereus가검출되었다 3). 따라서최근식중독을야기할수있는병원성미생물을제어하는노력이중요시되고있다. 식중독주요원인균인황색포도상구균은사람과동물의장관및피부등에분포하며, 식중독이외에화농성염증, 표피박탈성피부염등의원인이된다. 이균은독소형으로단백독소인장관독소 (enterotoxin) 는열에강하여 100 o C 에서 30분정도가열처리로는거의무독화되지않고섭취한사람에게급성위장장해를일으킨다 4). 이에따라식품에서의황색포도상구균을제어하기위하여여러종류의합성보존료를사용하여식중독의발생빈도를줄이려는추세이다. 합성보존료는사용농도가높을수록효과적이나 449

450 Bo-Ram Kim, Jin-Hee Yoo, Kyu-Seok Jung, Sung gi Heu, and Sun-Young Lee 목적하는기능외에바람직하지못한부작용을나타내어안전성문제가대두되고있다. 또한장기간사용할경우돌연변이및만성독성을유발시킨다고보고되었다 5-6). 이러한안전성의문제로소비자들의불안이고조됨에따라합성보존료보다는천연보존료를선호하게되어천연항균성물질에대한연구및개발이진행되고있다 7). 천연항균물질로는전통적으로사용해온소금, 식초등일반식품소재뿐만이아니라동물이나식물에천연적으로존재하는특정단백질및효소류, 갑각류의키틴질에서추출한키토산, 유기산, 식물의정유 (essential oil) 및미생물에서유래된 nisin, ε-polylysine, natamysin 등이있다 8). 다양한천연항균물질중식물에서유래된항균물질은대부분이 alkaloid류, flavonoid류, terpenoid류, phenolic compound 류, quinone류및휘발성오일등의이차대사산물이거나그유도체들로알려져있다 9-10). 식물의휘발성이차대사산물인정유 (essential oil) 성분은항산화, 항균효과를가지고있고, 수세기동안천연의항균복합제나단일물질로서이용되어왔다 11). 항진균성과항균성을갖고있다고알려진유기산은초산, 젖산, 구연산, 사과산등여러종류가있으며식품의원료에천연적으로혹은발효산물로존재하거나인공적으로첨가되는경우가있다 12). 항균기작은비해리된분자가이온화되면서세포내 ph를변화시키거나세포막의투과성을변경시켜기질이동을방해하고, NADH 산화를막아전자전달체계에이상을준다고알려져있다 13). 유산균이생성해낸항균성펩타이드또는단백질인박테리오신 (bacteriocin) 은장내유익균에영향을주지않고, 잔류성이없어현재식품등의천연보존제로각광받고있다 14). 다양한박테리오신중유일하게그사 용이허가된 Lactococcus lactis의균주에의해생성되는 nisin은분자량이약 3,500 Da인펩타이드성항균물질 15-16) 로써펩티도글라이칸의생성을저지하여 Bacillus, Clostridium, Corynebacterium, Stapylococcus, Streptococcus 등의그람양성균의생육을효과적으로억제또는사멸시킨다고알려져있으며 17-20), 발효유, 치즈, 통조림식품, 알코올음료, 김치, 어패류, 과일및야채류, 냉동제품등의저장성향상을위해사용되고있다. 또한유기산, lysozyme, lauricidin, lactoperoxidase 등의다른항균제와의병합처리에대한연구도활발하게진행중이다 21-22). 그러나아직까지천연항균물질이맛, 냄새등관능적문제와중량기준으로볼때합성보존료에비해상대적으로낮은항균성으로인해실용화되는경우는많지않은실정이다. 이에본연구에서는황색포도상구균에대한정유, 유기산, nisin 및그밖의자연유래항균물질의항균성을 agar disc diffusion test와최소저해농도 (MIC) 로측정하고이물질들의혼합처리시항균성에대한상승효과를살펴보고자한다. 재료및방법 사용균주 Agar disc diffusion test에는 113개의서로다른식품및동물에서분리된 S. aureus를사용하였다 (Table 1). 각각의균주를 5 ml Tryptic Soy Broth (TSB: Difcolaboratory, Detroit, MI, USA) 를이용하여 37 o C에서 24시간동안배양한뒤실험에사용하였다. 자연유래항균물질인유기산과그밖의자연유래항균물질의최소저해농도와최소 Table 1. Source and origin of Staphylococcus aureus used in this study. Strains No. Origins Source R-1, R-2, R-4, R-5 1), R-9, R-10, R-11 7 Leaf vegetable Rural Development Administration R-12 1 Peanut Rural Development Administration H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-8, H-9, H-10, H-11, H-12, H-13, H-14 1), H-15, H-16, H-17, H-18, H-19, H-20, H-21, H-22, H-23 23 Pea leaf Rural Development Administration D-1, D-3, D-4, D-5, D-6, D-7, D-8, D-9, D-10 1), D-12, D-13, D-14, D-15, D-16, D-17, D-18, D-19, D-20, D-22, D-23, D-24, D-25, 26 Perilla leaf Rural Development Administration D-26, D-28, D-29, D-30, K-13, K-14, K-15, K-17, K18, K-19, K-20, K-21, K-23, K-24, K-25, K-26 12 Kim-bab Kyungwon univ. S-198, SS-25 2 Person Rural Development Administration K-1, K-3, K-4, K-5, K-7, K-8, K-9, K-10, K-11, K-12 10 Person Kyungwon C-1, C-2, C-5, C-6, C-7 5 Person Asan Medical Center C-30, C-32, C-34, C-35, C-36, C-37 6 Person Korea University Guro Hospital C-64, C-65, C-66, C-68, C-69, C-80, C-71 7 Person Kyeongbuk univ. C-76, C-78 1), C-79, C-80, C-88 5 Person Institute of health C-93, C-96, C-98, C-99, C-100 5 Person KNOAR C-50, C-51 1), C-52, C-53 4 Animal Chonbuk National univ. 1) Selected strains used MIC and combination experiment

Natural Antimicrobials' Activities Against S. aureus 451 사멸농도측정, 자연유래항균물질혼합처리시최소저해농도를측정하는실험에는 5개의유래별로선별한 S. aureus ( 쌈채소 : S. aureus R-5, 완두잎 : S. aureus H-14, 들깨잎 : S. aureus D-10, 동물 : S. aureus C-51, 사람 : S. aureus C- 78) 를사용하였다. 항균물질의준비유기산은 acetic acid (Duksan pure chemical, Seoul, Korea), lactic acid (Kanto Cemical, Tokyo, Japan), malic acid (Junsei chemical, Tokyo, Japan), citric acid (Sigma, Steinheim, Germany), propionic acid (Samchun chemical Co., Seoul, Korea) 를사용하였다. 다섯종류의유기산은 6 N NaOH로희석시켜 ph 4의용액으로사용하였다. 그밖의자연유래항균물질은 carvacrol (Sigma, Steinheim, German), thymol (Daejung chemicals & Metals, Incheon, Korea), nisin (Sigma, Steinheim, Germany), eugenol (Wako pure chemical, Osaka, Japan), cinnamic acid (Junsei chemical, Tokyo, Japan) 을실험에사용하였다. Carvacrol, thymol, eugenol, cinnamic acid는 95% ethanol로용해시켜 1% 용액으로사용하였고 nisin은 0.02 N HCl로용해시켜 1% 용액으로사용하였다. 각각자연유래항균물질의최소저해농도와최소사멸농도측정은항균효과가나타나지않은 lactic acid, cinnamic acid를제외한 8종의항균물질을사용하였으며, 자연유래항균물질혼합처리시최소저해농도측정은항균효과가높게나타난 propionic acid, citric acid와 carvacrol, nisin, thymol을 8MIC (MIC의 8배농도 ) 가되도록만든후두배씩희석하여 2 MIC (MIC의 2배농도 ) 까지만들었다. 다음과같은항균물질 carvacrol과 nisin, carvacrol과 thymol, carvacrol과 propionic acid, carvacrol과 citric acid, propionic acid와 citric acid를각각의농도로결합하여최소저해농도를측정하였다. Agar disc diffusion test 0.75% Bacto-agar (Difco) 를포함하고있는 TSB를멸균하여 45 o C로식힌뒤각각의배양된미생물을 0.1 ml 접종 하고멸균 petri-dish에분주하였다. 상온에서 agar가굳도록말린뒤멸균 paper disc ( 지름 : 6 mm, Toyo Roshi Kaisha, Ltd., Japan) 를 agar배지위에정렬하고각각의준비된항균물질 10 µl 를 paper disc에분주하여 37 o C에서 18시간동안배양하였으며배양후에생성된 clear zone의지름 (cm) 을측정하여항균성을비교하였다. 자연유래항균물질의최소저해농도 (MIC) 와최소사멸농도 (MBC) 조사멸균된 TSB가 100 µl씩있는 96-well plate에만들어진항균물질용액을 100 µl씩넣은후서로다른식품및동물에서분리된 S. aureus를각각의항균물질에 20 µl씩접종하고 37 o C, 20시간동안배양하였다. 배양한 96-well plate는 595 nm에서흡광도를측정하였고소수둘째자리에서반올림했을때의흡광도가 0.4와 0.1 이하인최소농도를각각결합용액의최소저해농도 (MIC) 와최소사멸농도 (MBC) 로설정하였다. Agar disc diffusion test 결과및고찰 S. aureus을자연에서유래된여러항균물질에대한항균력을평가하기위하여 agar disc diffusion test를시행하였다 (Table 2). 5종류의유기산중 propionic acid는 clear zone의크기가 2.5-3.0 cm로가장좋은항균력을나타냈으며 (Fig. 1) 그밖의자연유래항균물질중 carvacrol, nisin, thymol의 clear zone 크기가 1.0-1.5 cm로측정되어항균효과가높게관찰되었다. 이외에도유기산중 citric acid의 clear zone 크기가 1.5-2.0 cm로나타나 propionic acid 다음으로항균력이높게측정되었다. 이러한항균물질의항균작용은항균물질이세포막등의세포기관에손상을일으키거나세포의항상성을방해함으로써 ATP 에너지의소비를촉진시키고, 이에의한대사고갈을야기함으로써발생하는것으로보고되고있다 23). 기존에보고된타연구의결과에서도본연구와유사한연구결과를보고하고있다. Table 2. Grouping of 113 Staphylococcus aureus strains by clear zone (cm) as a result of agar diffusion tests of organic acids and other natural antimicrobials. Clear zone size (cm) Organic acids Other antimicrobials Acetic acid Propionic Citric acid Malic acid Lactic acid Thymol Carvacrol Eugenol Nisin acid Cinnamic acid 0.0-0.5 0 0 0 0 0 0 0 0 0 0 0.5-1.0 96 26 102 111 113 80 100 113 72 113 1.0-1.5 17 40 10 2 0 33 13 0 41 0 1.5-2.0 0 43 1 0 0 0 0 0 0 0 2.0-2.5 0 3 0 0 0 0 0 0 0 0 2.5-3.0 0 1 0 0 0 0 0 0 0 0

452 Bo-Ram Kim, Jin-Hee Yoo, Kyu-Seok Jung, Sung gi Heu, and Sun-Young Lee Fig. 1. Digital photographs of agar diffusion test results using organic acids (A: Acetic acid, B: Propionic acid, C: Citric acid, D: Malic acid, E: Lactic acid) against Staphylococcus aureus H-17, C-68, and SS-25. 안등 1) 의유기산항균활성비교연구에서유기산의 S. aureus 에대한증식저해효과를관찰하였으며, 이중 propionic acid의경우본실험의 agar disc diffusion test 결과와동일하게가장높은증식저해효과및항균력을보여주었다. 또한이등 24) 의연구에서매실과육에함유된유기산 (citric acid, malic acid, oxalic acid, succinic acid, tartaric acid, fumaric acid, maleic acid, α-ketoglutaric acid 등 ) 의농도수준에따른항균력을본결과유기산이함유된매실착즙액 3% 와 4% 를함유한배지상에서 S. aureus에대한성장은배양후 24시간이내모두저해되었다는연구결과를나타내었다. 이외에도 thyme과 oregano의정유에는 carvacrol과 thymol이주성분으로함유되어있으며이러한유기산성분들이다양한진균과 S. aureus 등과같은박테리아에효과가있음이보고된바있다고확인되었다 23). 따라서본실험의연구결과와여러연구의결과 propionic acid나항균효과가높았던자연항균물질을통해식중독의대표적균인 S. aureus의증식을저해할수있으며, 이를통해식품에사용할수있는천연항균물질개발에기초자료로활용할수있을것으로사료된다. 자연유래항균물질의최소저해농도 (MIC) 와최소사멸농도 (MBC) 쌈채소, 완두잎, 들깨잎, 사람, 동물등에서유래된 113 종의 S. aureus 중 origin별로선별한 5종의 S. aureus를최소저해농도측정실험에이용하였으며, agar disc diffusion test 결과항균효과가나타나지않은 cinnamic acid와 lactic acid를제외한총 8개의항균물질로최소저해농도를측정하였다. Table 3는 4종의유기산과 4종의그밖의자연유래항균물질에대한최소저해농도 (MIC) 와최소사멸농도 (MBC) 의결과이다. 유기산의최소저해농도측정결과, acetic acid는 0.38%, propionic acid는 S. aureus C-78를제외하면 0.23%, citric acid는 S. aureus C-51를제외한균주에서 0.60% 로나타났다. Malic acid는 S. aureus R-5, D- 10을제외한균주에서 1.68% 로나타났다. 이결과 4종의유기산항균물질중 propionic acid가 S. aureus C-78을제외한 4종의 S. aureus 균주에서최소저해농도가 0.23% 로항균효과가가장높음을알수있었으며, agar disc diffusion test를통한항균력측정결과와동일하게나타남을알수있었다. 그밖의자연항균물질의경우최소저해농도가 tymol은 5종의 S. aureus에대하여 0.06%, carvacrol은 S. Table 3. Minimum inhibitory concentration (MIC) 1) and minimum bactericidal concentration (MBC) 2) of organic acid and other antimicrobials against 5 strains of Staphylococcus aureus. Strain S.aureus R-5 S.aureus H-14 S.aureus D-10 S.aureus C-51 Acetic acid Organic acids Propionic acid Other antimicrobials Citric acid Malic acid Thymol Cavacrol Eugenol Nisin MIC 0.38 0.23 0.60 3.35 0.06 0.03 0.13 0.06 MBC 1.50 1.85 19.20 13.40 0.13 0.13 0.13 0.13 MIC 0.38 0.23 0.60 1.68 0.06 0.06 0.13 0.13 MBC 1.50 1.85 9.60 6.70 0.13 0.13 0.25 0.13 MIC 0.38 0.23 0.60 3.35 0.06 0.06 0.13 0.06 MBC 1.50 1.85 9.60 13.40 0.13 0.13 0.25 0.13 MIC 0.38 0.23 1.20 1.68 0.06 0.06 0.13 0.13 MBC 1.50 3.70 4.80 6.70 0.06 0.13 0.25 0.13 MIC 0.38 0.46 0.60 1.68 0.06 0.03 0.06 0.06 S.aureus C-78 MBC 1.50 1.85 4.80 6.70 0.06 0.13 0.13 0.13 1) MIC: Optical density at 595 nm was less than 0.4 2) MBC: Optical density at 595 nm was less than 0.1

Natural Antimicrobials' Activities Against S. aureus 453 aureus R-5, C-78에대하여 0.03%, S. aureus D-10, C-51 에대하여는 0.06% 로나타났다. Eugenol은 S. aureus C- 78을제외하고 4종의균주에서 0.13% 로나타났다. Nisin 은 S. aureus H-14, C-51에대하여 0.13%, S. aureus R-5, D-10, C-78에대하여 0.06% 로나타났다. 따라서본실험결과 carvacrol의최소저해농도가 0.03% 로유기산을제외한그밖의자연유래항균물질중항균효과가가장높음을알수있었다. 최소사멸농도 (MBC) 의결과에서유기산중 acetic acid는 1.50%, propionic acid 는 S. aureus C-51은 3.70%, 나머지 4종은 1.85% 로나타났다. Citric acid는 S. aureus C- 51, C-78에서 4.80% 로나타났으며 malic acid는 S. aureus H-14, C-51, C-78에서 6.70% 로나타났다. 따라서최소사멸농도측정결과유기산중 acetic acid가 1.50% 로가장높은사멸효과를나타내었다. 그밖의자연유래항균물질에서 thymol은 S. aureus C-51, C-78에서 0.06% 로나타났고, carvacrol은 5종모든균주에서 0.13% 로나타났다. Eugenol에서는 R-5, C-78 에서 0.13% 로나타났다. Nisin 은모든균주에서 0.13% 로나타났다. 따라서유기산을제외한그밖의자연유래항균물질중 thymol의최소사멸농도 (MBC) 가 0.06% 로가장높은사멸효과를나타내었다. 본실험결과유기산의 propionic aicd와 acetic acid는각각최소저해농도와최소사멸농도에서높은항균효과를보였으며안등 1) 의연구에서유기산에대한최소저해농도측정시 E. coli O157:H7의경우도 acetic acid와 propionic acid의증식저해효과및항균효과를나타내었고, Chung 등 25) Salmonella의항균효과에대해연구한결과증식저해에가장효과적인유기산은본실험에서항균력이높다고측정된유기산의 acetic acid와 propionic acid로동일한결과를확인할수있었다. 여러연구들과본실험에서유기산과그밖의자연유래항균물질들중 propionic acid가강한항균활성을나타내었다. 이는유기산이세포내부로들어갈수있는정도와해리되는효과의차이때문에발생하는것으로사료된다. 예를들어 citric acid와 lactic acid 는세포내부로들어갈수있는능력은낮지만, 세포내부에서해리되는능력이매우뛰어나며, 따라서 cytoplasm을산성화하여항균작용을일으키는것으로알려져있다 26). 기존의몇몇연구결과는 acetic acid와 propionic aicd 등의유기산이그람음성균에게높은항균효과를나타내는것을보고하고있다. 그러나본연구결과를통해서그람양성균인 S. aureus에서도 propionic acid와같은유기산이높은항균력을나타내어이같은유기산이나다른자연유래항균물질을이용하여식품에활용할수있는자연친화적항균제및보존제등을개발할수있을것으로사료된다. 항균물질결합처리시최소저해농도 Table 4 는총 5 종의자연항균물질인유기산과그밖의자연유래항균물질을각각의최소저해농도로부터 1/2 MIC, Table 4. Minimum inhibitory concentration (MIC) 1) of combination. Strain Antimicrobial Cavacrol Citric acid S. aureus R-5 S. aureus H-14 S. aureus D-10 S. aureus C-51 Nisin 1/2 MIC - Thymol MIC - Propionic acid MIC 1/2 MIC Thymol MIC - Propionic acid MIC 1/2 MIC Thymol 2 MIC - Propionic acid 1/4 MIC 1/8 MIC Thymol 2MIC - Propionic acid MIC 1/16 MIC Citric acid 2MIC - Thymol 2 MIC - S. aureus C-78 Propionic acid MIC 1/4 MIC 1) MIC: Optical density at 595 nm was less than 0.4 1/4 MIC, 1/8 MIC까지다양한농도로혼합처리시 S. aureus에대한최소저해농도를측정한결과이다. 본연구결과 S. aureus D-10의균은 propionic acid와 citric acid의 1/8 MIC로결합시최소저해농도로나타났으며, S. aureus C-51 균은 propionic acid와 citric acid의 1/16 MIC로결합시최소저해농도로나타났다. S. aureus C-78 균은 propionic acid와 citric acid의 1/4 MIC로결합시최소저해농도가나타났다. 따라서본연구결과를바탕으로 propionic acid와 citric acid의혼합처리시 S. aureus에대한최소저해농도가가장낮아항균효과가높음을알수있었다. Fernandes 등 26) 의연구에서 catfish fillet에서미생물의증식저해에가장효과적인유기산또한본실험의항균력이가장좋다고측정된 propionic acid이었으며그다음으로 lactic acid 와 acetic acid로나타났다. 이러한 propionic acid와같은유기산의항균활성은비해리된유기산의작용때문이며비해리된유기산은세포내부로수송되어세포의 ph를변화시켜세포내효소의변성을초래함으로써 27) 항균작용이일어난다. 몇몇유기산의결합처리에서의항균력이증가되는효과에대하여는보다많은항균기작에대한연구가이루어져야할것으로보인다. 또한일반적으로보존제는식품에첨가시식품성분과의상호작용에의하여단독으로처리시길항되는경향이있기때문에이러한단점을극복하기위하여다른물질과병용처리를하는데 27) 본연구에서도항균효과가높게측

454 Bo-Ram Kim, Jin-Hee Yoo, Kyu-Seok Jung, Sung gi Heu, and Sun-Young Lee 정된물질들을혼합처리를하였으며, propionic acid와 citric acid의혼합처리시최소저해농도에서도단독처리시보다항균효과가더높아져상승효과를가져오는결과를볼수있었다. 따라서이와같은혼합처리방법을통해최소저해농도에서의높은항균성을지닌항균물질을찾고, 혼합처리시항균력에대한상승효과를갖는천연물질들을이용하여합성보존료를대체할수있는천연보존제물질을개발할수있도록하는전문적인연구가필요할것으로사료된다. 요약 본연구에서는다양한유래에서분리한병원성세균 S. aureus에대한 5종의유기산 (acetic, propionic, citric, malic, lactic acid) 과 3종의정유성분 (carvacrol, thymol, eugenol) 및그밖의자연유래항균물질 (nisin, cinnamic acid) 의항균효과, 최소저해농도및각각의최소사멸농도를연구하였으며, 이를바탕으로항균물질간의혼합처리시항균효과를평가함으로써이와같은자연유래항균물질들의화학합성보존제대체가능성을연구하고자하였다. Agar disc diffusion실험을통해 propionic acid (1.38 cm) > nisin (1.00 cm) > thymol (0.98 cm) > carvacrol (0.90 cm) 순으로 S. aureus에대해서높은항균력을나타내었다. 최소저해농도는 carvacrol (0.06%), thymol (0.06%), nisin (0.06%) 이항균효과가가장좋은것으로나타났다. 그이외의항균물질의경우 eugenol (0.13%), propionic acid (0.23%), acetic acid (0.38%), citric acid (0.60%), malic acid (1.66%) 순으로효과가나타났다. 이를바탕으로항균물질간의혼합처리시 propionic acid와 citric acid의결합처리효과가가장높게나타났다. 자연유래항균물질은합성보존료에비해항균활성이미약해보존료의이용이어렵지만본연구결과자연유래항균물질들의혼합처리를통해시너지효과를알수있었다. 따라서자연유래항균물질의혼합처리를통한연구가계속적으로이루어진다면인공합성보존료를대신하는천연보존료의상업적이용이가능할것으로사료된다. 감사의글 본연구는농촌진흥청공동연구사업 ( 과제번호 : PJ008513-022012) 의지원에의해이루어진것이며이에감사드립니다. 참고문헌 1. Ahn, Y.S. and Shin, D.H.: Antimicrobial effects of organic acid and ethanol on several foodborne microorganisms. Kor. J. Food Sci. Technol., 31, 1315-1323 (1999). 2. KFDA: http://www.kfda.go.kr/index.html (2007). 3. KFDA: http://www.kfda.go.kr/index.html (2008). 4. Jang, J.S., Go, J.M. and Kim, Y.H.: Inhibitory effect of Staphylococcus aureus and Bacillus cereus by lactic acid and hydrogen peroxide. Kor. J. Env. Hlth., 31, 115-119 (2005). 5. Branen, A.L.: Toxicological and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. JAPCS, 52, 59-63 (1975). 6. Kim, D.H and Lee, Y.C.: Quality changes in minced ginger prepared with frozen ginger during storage. Kor. J. Food Sci. Technol., 36, 943-951 (2004). 7. Oh, D., Ham, S.S., Park, B.K., Ahn, C. and Yu, J.Y.: Antimicrobial activities of natural medicinal herbs on the food spoilage or foodborne disease microorganisms. Kor. J. Food Sci. Technol., 30, 957-963 (1998). 8. Kim, Y.S. and Shin, D.H.: Researches on the volatile antimicrobials compounds from edible plants and their food application. Kor. J. Food Sci. Technol., 35, 159-165 (2003). 9. Mitscher, L.A, Park, Y.H. and Clark, D.: Antimicrobial agents from higher plants, antimicrobials isoflavonoids and related substance from Glycyrrhiza glabra L.var Typica. J. Nat. Prod., 43, 259-269 (1980). 10. Lee, B.W. and Shin, D.H.: Screening of natural antimicrobial plant extract on food spoilage microorganism. Kor. J. Food Sci. Technol., 23, 200-204 (1991). 11. Misaghi, A. and Basti, A.A.: Effects of Zataria Multiflora Boiss. essential oil and nisin on Bacillus cereus ATCC 11778. Food Control., 18, 1043-1049 (2007). 12. Beuchat, L.R. and Golden, D.A.: Antimicrobials occurring naturally in foods. Food Technol., 43, 134-142 (1989). 13. Freese, E., Sheu, C.W. and Galliers, E.: Function of lipophilic acids as antimicrobials food additives. Nature., 241, 321-325 (1973). 14. Ha, S.D., Cho, M., Bae, E.K. and Park, J.: Application of natural antimicrobials to food industry. Food Sci. Ind., 38, 36-45 (2005). 15. Hurst, A.: Nisin. Adv. Appl. Microbiol., 27, 85-123 (1981). 16. Jarvis, B., Jeffcoat, J. and Cheeseman, G.C.: Molecular weight distribution of nisin. Biochem. Biophys. Acta., 168, 153-155 (1968) 17. Anderson, A.A., Michener, H.D. and Olcott, H.S.: Effect of some antibiotics on Clostridium botulinum. Antibiotics Chemother., 3, 521-526 (1953). 18. Hawley, H.B.: Nisin in food technology. Food Manuf., 32, 370-376 (1957). 19. Linnet, P.E. and Strominger, J.L.: Additional antibiotic inhibitors of peptidoglycan synthesis. Antimicrob. Agents Chemother., 4, 231-236 (1973). 20. O Brein, R.T., Titus, D.S., Delvin, K.A., Stumbo, C.R. and Lewis, J.C.: Antibiotics in food preservation II. Studies on the influence of subtilin and nisin on the thermal resistance of food spoilage bacteria. Food Technol., 10, 352-355 (1956). 21. Eswaranandam, S., Hettiarachchy, N.S. and Johnson, M.G.: Antimicrobial activity of citric, lactic, malic, or tartaric acids and nisin-incorporated soy protein film against Listeria mono-

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