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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 43(9), 80~87(2014) 고체발효한약재추출물을함유한구강세척제개발 조병제 1 홍준영 2 김미정 2 송영옥 2 1 체담한방병원 2 부산대학교식품영양학과및김치연구소 Development of Mouthwash Products with Solid Fermented Oriental Medicinal Herb ByungJe Cho 1, Jun Young Hong 2, Mijeong Kim 2, and Yeong Ok Song 2 1 Chedam Hospital of Korean Medicine 2 Department of Food Science and Nutrition, and Kimchi Research Institute, Pusan National University ABSTRACT The purpose of this study was to develop a mouthwash product with solid fermented oriental medicinal herb (OMH). Solid fermentation of magnolia, liquorice, and cnidium by Phellinus linteus mycelium was carried out successfully when 30% water was added to the medium, whereas 10% brown rice powder was required as an extra nutrient for solid fermentation of mint besides water. The amount of total phenol compounds and DPPH radical scavenging activity of OMH increased significantly (P<0.0) upon solid fermentation. Antimicrobial activities of fermented OMH also increased and were approximately 100fold greater than those of unfermented samples. Oral pathogens such as Staphylococcus epidermis, Streptococcus pyogenes, Candida albicans, or Streptococcus mutans were used for determination of antimicrobial effects of OMH. Formulation of the mouthwash was developed based on the results of the sensory evaluation. Among seven formulas, the best formula chosen by the sensory evaluation was as follows: mouthwash prepared with 0.07% ethanol extract of solid fermented OMH as a main ingredient, 83.64% hot water extract of mint and clove (100:1, v/v) as a mouthwash base component, and other miscellaneous ingredients, including sodium fluoride, menthol, and surfactants. Data from a consumer's preference test with 30 participants, overall acceptance, and willingness to buy the product developed in this study were all significantly higher for the tested mouthwash compared to mouthwash on the market manufactured with OMH but with a different formula. Duration of freshness of the mouthwash after usage as determined by Breath Checker was not significantly different between the two samples, although the duration of our product was slightly longer than that of the commercial product mentioned above. Key words: mouthwash, solid fermentation, oriental medicinal herb, antimicrobial activity, Phellinus linteus mycelium 서 생활수준이향상되고대인활동의기회가증대됨에따라구강세척제는현대인의필수품으로그사용량이증가하고있다. 대부분의구강세척제는구강내항균기능및충치균생성억제효능이있는 chlorhexidine, triclosan 및 CPC cetylpyridinium chloride(cpc) 등의화학합성제재를함유하고있으며, 일부제품에는항생제를첨가한제품들도시판되고있어장기간사용시항생제내성부작용을일으킬수있는문제점이지적되고있다 (1). 뿐만아니라 chlorhexidine은항균효과는높으나장기사용시치아및혀의변색, 미각이상, 점막자극과민증을유발하고, 관능적으로 Received 11 August 2014; Accepted 27 August 2014 Corresponding author: Yeong Ok Song, Department of Food Science and Nutrition, and Kimchi Research Institute, Pusan National University, Busan 60973, Korea yosong@pusan.ac.kr, Phone: 론 소비자의기호를떨어뜨리는점이지적되고있다 (2). 이에화학성분의부작용을줄이고장기간사용해도안전한천연물을원료로하는구강세척제에대한요구가증가하고있고세신, 정향, 후박, 감초, 박하, 길경천궁및은행잎등과같은구강질환균에대한항균효과가높은한약재 (3) 가한방구강세척제제조에주로사용되고있다. 현대인은사회활동이증가함에따라타인과의만남의기회가많아지는데호기를통해발산되는나쁜입냄새인구취는자칫사회생활을방해하는요인으로작용할수있다. 구취의발생원인으로는노인에서흔히나타나는타액분비감소와같은생리적요인 (4), 구강내위생관리소홀로인한염증및흡연등과같은구강내요인 (,6) 또는간, 위, 식도질환에의한구강외적인요인 (7) 등이있다. 한의학에서는비장또는위장에열이있거나음식이소화되지않은채위에남아있거나호르몬교란에의해서도구취가발생된다고한다 (7). 뿐만아니라구강은외부와바로연결되어있는기관으로미생물들이쉽게들어올수있으며, 음식을섭취하는기관으로영양성분이

2 고체발효한방구강세척제개발 81 풍부하여균이서식하기에가장좋은조건을지니고있다. 일반적으로 30종이상의균이상주하고있으며이들의부패작용에의해구취가발생하는비율이가장높다고보고되고있다 (3). 구취를제거하기위해근본적인원인을제거하기도하지만일반적으로이를닦거나구강세척제로입안을헹구는간단한작업등으로구취를일시적으로제거한다. 최근한약재의약효를강화시키고자체의독성은약화시키기위해생물전환기법중발효기법을널리사용하고있다. 건조한약재를발효시키기위해고체발효가시도되고있는데고체발효는액체발효와달리미생물접종및생장이어려워건조약재를연화시키거나수분활성도를조절하거나, 영양배지를만드는등미생물이성장할수있는다양한조건이갖추어져야성공할수있다. 고체발효에주로사용하는균주는진균류인버섯균사체 (8) 와곰팡이 (9) 가있고유산균 (10) 또는고초균 (11) 등의세균류도사용하고있다. 버섯은항암, 항염증, 면역증진효과를지니고있고이러한건강기능성은균사체가자실체보다 4배이상높다고한다 (). 뿐만아니라균사체는자실체보다대량배양이가능하며효능이더우수한것으로알려져있다 () 상황버섯은항암등과같은다양한건강기능성이보고되고있으며 (14, 1) 상황버섯균사체역시항혈전능 (16), 항산화 (17), 항암및항염증 (18), 면역증진 (19) 등의약리작용이보고되어있다. 상황버섯, 영지버섯, 노루궁뎅이버섯의균사체로수삼을고체발효하였을때수삼의조지방함량은증가하고단백질함량은감소하였다고보고하여균사체에의한생물전환이일어났음을확인하였다. 뿐만아니라고체발효한약재수삼의면역증강활성은수삼및균사체보다높았으며, 특히상황버섯균사체로발효시킨수삼에서가장높았고이는버섯균사체로고체발효시킨수삼은수삼과는다른화학적조성을포함하고있기때문이라고설명하였다 (20). 상황버섯균사체를쌀에접종시켜술을제조하였을때일반주정을사용하여제조한술보다간의염증관련단백질발현을억제하고활성산소생성을저해하였다고보고하였다 (21). 따라서생리활성작용이탁월한버섯균사체가한약재를영양원으로하여생육할수있다면균사체가지닌생물학적변환능력에의해한약재의약리성분이변화하여그기능이증가될수있을것으로기대한다. 본연구에서는발효한약재를유효성분으로함유하는한방구강세척제를개발하고자후박, 감초, 천궁, 박하및정향을상황버섯균사체로고체발효하였다. 이를위하여각한약재의고체발효조건을확립하고, 발효전후의항산화및구강질환균에대한항균활성을확인한후구강세척제조성비를확립하며, 개발한발효한방구강세척제의효과를시판중인한방구강세척제와비교하였다. 재료및방법균주및한약재고체발효에사용한상황버섯균사체 (Phellinus linteus mycelium, KACC 43899) 는농업유전자원정보센터 (Korean Agricultural Culture Collection, KACC, Suwon, Korea) 에서분양받았다. 한방구강세척제에주로사용되고있는한약재중항균활성이있는것으로알려진후박, 감초, 천궁, 박하및정향은건조된국산한약재를사용하였다. 항균활성실험에사용한구강질환균인 Staphylococcus epidermis KACC 234, Streptococcus pyogenes KACC 1188, Candida albicans KACC 30004는농업유전자원정보센터에서, Streptococcus mutans KCTC 306는미생물자원센터 (Korean Collection for Type Cultures, KCTC, Daejeon, Korea) 에서분양받았다. 계대배양및액체종균액제조분양받은상황버섯균사체는 potato dextrose agar 고체배지에도말한후저온배양기 (27±1 C, J100M, Jisico, Seoul, Korea) 에서 14일간격으로계대배양하였다. 고체배지에서계대배양한 P. linteus를 8 cork borer로조각을내고 potato dextrose broth로제조한액체배지 10 ml 에 6조각을접종하여 shaking water bath(27±1 C, speed 0 rpm, HB20SW, Hanbaek Sci., Bucheon, Korea) 에서 14일간배양하였다. 배양후덩어리형태로자란균사체를한약재에접종시키기위해멸균된 glass beads 를넣고균사체를깨어균질화한것을액체종균액으로사용하였다. 한약재의고체발효후박, 감초, 천궁, 박하및정향을잘게파쇄하여 1 C에서 30분간고온가압멸균한후상온에서 30분간식혔다. 이를 00 ml 삼각플라스크에 100 g씩넣고 20%(w/v) 의상황버섯액체종균액을접종한후즉시실리스토퍼로입구를막아멸균환경을유지하였다. 균사체를접종한한약재는배양기에서 27±1 C, 21일간발효를진행하였다. 한약재의최적발효조건을확립하기위해수분첨가량, 영양배지농도를달리하여발효를진행하였다. 1차로수분을 0 30% 첨가하여고체발효를시도하였으며, 수분첨가에의해발효일어나지않은한약재의경우에는수분첨가와영양원으로현미가루 10%(w/w) 를함께첨가하였다. 고체발효중발생할수있는오염여부및균사의생장정도는육안으로확인하였다. 에탄올추출고체발효가종료된한약재에 10배의에탄올 (w/v, 99.% ethanol, Duksan, Korea) 을첨가한후추출기 (KMC 0sh; Vision Scientific Co., Ltd., Daegu, Korea; speed

3 82 조병제 홍준영 김미정 송영옥 level: max, temp. level: 7) 에서 90분간추출하였다. 동일시료를 2회반복추출한후얻어진추출액을여과 (No. 2 filter paper, Whatman, Clifton, NJ, USA) 하였다. 얻어진여액을진공회전농축기 (rotavator R200, Bϋchi, Flawil, Switzerland) 에서 1차농축하고동결건조 (SFDSM06, Samwon, Busan, Korea) 하여실험에사용하였다. 총페놀함량및 DPPH radical 소거능측정총페놀함량은 FolinCiocalteu 비색정량법 (22) 으로측정하였다. 즉농도별로제조한시료에증류수와 Folin Ciocalteu reagent를혼합하여상온에서 7분간반응시키고, 7% Na 2CO 3 을첨가하여상온에서 90분간반응시킨후 70 nm에서흡광도를측정하였다. 시료중의페놀함량은 tannic acid 표준곡선을이용하여 tannic acid에대한당량으로환산하여나타내었다. DPPH radical 소거능측정은시료와 60 μm 1,1diphenyl2picrylhydrazyl(DPPH) 을 96 well plate에넣고잘혼합하여암소에서 30분간방치시킨후 ELISA reader(model680, BioRad, Hercules, CA, USA) 40 nm 에서흡광도를측정하였다. DPPH radical 소거능은대조군에대한저해율 (%) 로나타내었다. DPPH radical 소거능은 IC 0 으로나타내었다. 항균활성측정구강세척제개발을위하여 4종의구강질환균을분양받아배양하여사용하였다. 즉구강인두와피부에상주하는화농성염증균인 Staphylococcus epidermis, 치주피부에침투하여연쇄구균성화농을유발하는 Streptococcus pyogenes, 아구창과의치성구내염및구각염의원인균인기회감염진균 Candida albicans, 사람의치아우식및충치원인균 Streptococcus mutans이다 (23). 항균활성은 Paper Disk법을이용하여발효전후의효과를상대적으로비교하였다 (24). 균주별배지 (Table 1) 를 petri dish에분주하여굳힌다음해당구강질환균을멸균증류수에 10 1 의농도로희석하여배지에도말하고멸균한 paper disk( 지름 8 mm, Advantec, Tokyo, Japan) 를배지위에올린후한약재에탄올추출물을 40 μl씩점적하여 24시간배양하였다. 한방 Table 1. Oral pathogens used in this study for the determination of antimicrobial activity of oriental medicinal herbs Bacteria Strain number Temp. Media 1) Staphylococcus epidermidis Streptococcus pyogenes Candida albicans Streptococcus mutans KACC 234 KACC 1188 KACC KCTC C 37 C 2 C 37 C TSA BHIA YMA BHIA 1) TSA: Tryptic Soy Agar (Difco, Detroit, MI, USA), BHIA: Brain Heart Infusion Agar (Difco), YMA: Yeast Malt extract Agar (Difco). 구강세척제의유효성분으로사용할발효한약재혼합물의최적배합비를찾기위하여각발효한약재에탄올추출물을다양한비율로혼합하여항균활성을측정하였다. 한방구강세척제기본베이스제조및조성비확립한방구강세척제의기본베이스로사용하기위하여박하와정향을각각중량대비 20배 (w/v) 의물로 2시간 30분, 2회추출하여열수추출물을제조한후이를다양한비율로혼합하여관능평가를실시한결과를바탕으로최적배합비를선정하였다. 한방구강세척제조성비를확립하기위하여한방베이스, 발효한약재혼합추출물, Lmenthol, sodium fluoride, poloxamer 407, glycerin, propylene glycol, 수소화 40 피마자유및 ethanol을조성물로하는구강세척제를제조하여관능평가에의해최적조성비를확립하였다. 최적의조성비로발효한방구강세척제시제품을제작하여시판중인한방구강세척제와효과를비교하였다. 관능검사및구강세척효과확인구강세척제의조성비를확립하기위해관능검사를실시하였다. 흰종이컵에 10 ml의발효한방구강세척제를담아제시하였다. 시료에대한편견을없애기위해종이컵에난수표에서추출한세자리숫자를표기하였으며, 시료는무작위순으로제시하였다. 구강세척제 10 ml를입안에머금은시간은총 30초이며이시간내에 3회가글하도록하였다. 관능평가항목은전반적인기호도, 쓴맛, 구강세척제사용후상쾌감 ( 박하맛 ), 구강세척제사용후불쾌감, 시료구매의사에대해 9점척도로평가하였고, 구강세척제사용후주관적으로느끼는상쾌감지속시간에대해질문하였다. 관능평가원은총 10명이었다. 개발한발효한방구강세척제시제품의소비자선호도조사에서구강세척제사용후상쾌감이지속되는시간을휴대용구취측정기 (Breath Checker, HC2M, Tanita, Tokyo, Japan) 로측정하였다. Breath Checker는입냄새의강도를 6단계로측정하여 0 점으로나타내는기기로냄새의강도가높을수록점수가높게나타난다. 본연구를위해각개인의입냄새를먼저측정하여기준치로설정하고, 구강세척제사용한후 30분까지 ( 예비검사결과 ) 분간격으로입냄새를측정하여측정수치가기준수치로되돌아오는데소요된시간을지속시간으로하였다. 각개인별로시간을측정한후 30명의산술적평균을구하였다. 측정시개인적인오차를줄이기위해참여자 30명은예비실험을통해측정기사용에익숙해지도록훈련하였다. 정확한측정을위하여입과측정기센서까지의간격을 1 1. cm로유지하고기기에수치가나타날때까지숨을불어넣었다. 구강세척제사용은상기의관능검사시와동일하게실시하였다. 시판중인한방구강세척제 ( 예닥터후레쉬파인, Fresh Fine Co., Seoul, Korea) 를대조군으로사용하였다. 실험은 2일에거쳐실시하였다.

4 고체발효한방구강세척제개발 83 통계처리모든실험결과는평균 ± 표준편차로나타내었고고체발효전후한약재의효과검증은 Student's ttest(spss 20.0 version, SPSS Inc., Chicago, IL, USA) 로실시하였다. 발효한방구강세척제개발및소비자선호도조사를위해실시한관능검사결과는 oneway analysis of variance (ANOVA) 를실시하고, 사후검증으로 Duncan's multiple range test를실시하였다. 유의수준 0.0에서유의성을검증하였다. 결과및고찰고체발효조건확립상황버섯균사체액체종균액을한약재의 20%(w/w) 로첨가하고물을첨가함으로써배지의수분함량을높여고체발효조건을조절하였다. 첨가한물의양이한약재중량의 1%(w/w) 이하였을때는모든한약재에서발효가일어나지않았으나물을 30% 첨가하였을때후박, 감초그리고천궁에서는발효가일어나고체발효에성공하였다. 그러나박하와정향에서는발효가진행되지않아영양원으로현미 가루를 10%(w/w) 첨가하였을때박하에서만발효가진행되었고정향에서는발효가일어나지않았다. 이는정향이진균류에대한항균력이높아상황버섯균사체의생장을억제하였기때문으로생각된다. 고체발효에소요된시간은한약재에따라다소다르나 3주정도소요되었으며 Fig. 1에서보는바와같이한약재표면적의 90% 이상덮을정도로상황버섯균사체가성장한것을육안으로확인할수있었다. 고체발효에의한한약재의총페놀함량및 DPPH radical 소거능증가상황버섯균사체로고체발효에성공한후박, 감초, 천궁그리고박하의총페놀함량은발효전에비해유의적으로증가하였다 (Table 2, P<0.001). 고체발효한약재의 DPPH radical 소거능을 IC 0 값으로나타내었을때후박과감초의 DPPH IC 0 은고체발효에의해유의적으로낮아져 (Table 2, P<0.01) DPPH radical 소거활성이증가하였음을확인하였다. 그러나천궁및박하는고체발효에의해 DPPH radical 소거활성은증가하였으나발효전과유의적인차이는보이지않았다. 고체발효에의한한약재의페놀함량증가정도와 DPPH 소거능 IC 0 은비례적으로나타나지않았 Fig. 1. Solid fermentation of dried oriental medicinal herb by Phellinus linteus mycelium. Solid fermentation of magnolia (A), liquorice (B), and cnidium (C) were carried out successfully when 30% water was added to the medium while mint (D) needed additional 10% brown rice powder as a nutrient source besides water. Table 2. Increase in total phenol compounds and DPPH radical scavenging activity of oriental medicinal herbs after solid fermentation by Phellinus linteus mycelium Herbs Magnolia Liquorice Cnidium Mint Values are mean±sd. NS: not significantly different. ** P<0.01, *** P<0.001 by Student's ttest. Phenol compounds (μg tannic acid/mg) DPPH radical scavenging activity (IC 0, μg/ml) Unfermented Fermented Unfermented Fermented 249.7± ±0.36 *** 111.3± ±1.97 ** 171.6± ±0. *** 98.6± ±1.3 *** 39.3± ±0.21 *** 788.± ±0.98 * 33.1± ±0.17 *** 762.4± ±40. NS

5 84 조병제 홍준영 김미정 송영옥 으나발효한약재의유리기소거능이증가한것은발효에의해페놀화합물이증가되었기때문으로생각된다. 한방약재추출물에송이버섯균사체와동충하초균사체를혼합하여발효시켰을때한방배지의총페놀함량과 DPPH 유리기소거능이증가하였다고보고하였다 (). 본연구에사용한후박, 감초, 천궁및박하는다양한약리작용에따른생리활성을조절하는효과가있으며이들한약재는항균및항산화효과를지니고있다. 후박은 quercetin, afzelin 및 rutin의 flavonoid와 magnolol, honokinol, ovovatol과같은 lignan계화합물및 sesquiterpene 등을함유하고있고 (23), 감초는뿌리에 saponin인 glycyrrhizin을함유하고있어항산화, 항바이러스효과가있다. 천궁은항산화 (2) 와소염및진통작용 (26) 이있고, 박하 (Menthae Herba) 는항산화 (27), 항염증 (28) 및방사선에대한보호효과 (29) 가있음이알려져있다. 상황버섯균사체도항산화활성을지니고있는데, 상황버섯균사체의생리활성성분인히스피딘은 BHA나 αtocopherol과유사한항산화활성을지니고있다 (17) 고보고되고있다. 본연구에서고체발효에의해한약재의페놀함량및유리기소거능이증가한것은발효중상황버섯균사체에의한한약재의항산화관련성분이증가하였을뿐만아니라상황버섯균사체증식에따른항산화활성증가도함께나타났기때문으로생각된다. 고체발효에의한항균효과증대후박, 감초, 천궁및박하를상황버섯균사체로고체발효시켜구강질환균에대한항균활성을증가시키고자하였다. 연구에사용된균은구강, 인후화농성염증균 Sta. epidermidis, 치주염을일으키는연쇄구균성화농균 Str. pyogenes, 아구창과의치성구내염및구각염의원인균인 Can. albicans, 치아우식및충치원인균인 Str. mutans 4종이었다. 감초는 4종의구강질환균에대해항균활성을나타내었고, 후박은 Can. albicans에대한항균활성은나타나지않은반면천궁은이균에대해서만항균활성을나타내었다. 그리고박하는고체발효에성공하였으나모든구강질환균에대해항균활성을나타내지않았다. 발효전한약재의항균활성은 1,000 mg/ml에서나타나미미하였으나고체발효에의해 10 mg/ml에서유사한항균효과가확인되어고체발효 에의해한약재의항균효과가유의적으로증가되었음을확인하였다 (Table 3). 구취는구강내염증반응이있는경우특히치주질환이있는사람에서흔히발견된다. 후박추출물은치주질환균의생육을억제할뿐만아니라치주인대세포내콜라겐분해효소의활성을저해하는효과가알려져있다 (30). 이러한후박의항균효과는 tetracycline 또는 chlorhexidine보다약하나 listerine보다높으며 (31) 세포독성도다른생약제에비해현저히낮다고보고되고있다 (32). 감초는혓바늘이라고부르는아프타성궤양에효과가있는데궤양의크기를줄여통증을감소시킨다고보고되고있으며 (33), 감초성분중 licoricidin과 licorisoflavan A는치아우식의원인균인 Str. mutans과 Str. sobrinus와치주균인 Porphyromonas gingivalis와 Prevotella intermedia의생장을억제한다고보고되고있다 (34). 박하정유성분은미생물의생장을억제하는효과가있는데주로대장균과살모넬라균에대해항균활성이높은것으로보고되고있다 (3). 박하의정유성분중 Lmenthol은진통제, 흥분제, 건위제로사용되기도하나신선하고상쾌한향때문에박하는청량제나향료로치약, 잼, 사탕, 화장품및담배등의제품에주로사용되고있다 (36). 상황버섯균사체는한약재에함유되어있는항균성분을발효에의해생물전환시킴으로써활성을더욱증가시킨것으로생각된다. Park 등 (20) 의연구에서수삼을버섯균사체로고체발효시켰을때발효하지않은수삼과는달리조지방함량은증가하고단백질함량은감소하는결과로보아발효에의해화학조성이달라졌다고보고하였다. 고체발효수삼은 lipopolysaccharide(lps) 로처리한대식세포에서수삼보다 nitric oxide(no) 의생성을촉진함으로써감염미생물의생육을억제하는효과를나타내었다. 고체발효한수삼의면역증진효과는상황버섯균사체로고체발효한수삼에서가장효과가높았다고보고하였다 (20). 발효한약재조성비확립및한방구강세척제의관능평가발효한한약재를유효성분으로하는구강세척제개발을위하여발효에의해항균활성이증가한후박, 감초, 천궁을선정하고, 이들의에탄올추출물을일정비율로혼합한후구강질환균의생육억제정도를살펴보았을때후박 : 감 Table 3. Increase in antimicrobial activity of oriental medicinal herbs after solid fermentation by Phellinus linteus mycelium (Clear zone, mm) Microorganism Sta. epidermidis Str. pyogenes Can. albicans Str. mutans 1, , , , Concentration (mg/ml) R 1) F 2) R F R F R F Magnolia Liquorice Cnidium Mint ) R: raw material, dried oriental medicinal herb. 2) F: fermented oriental medicinal herb. : antimicrobial activity was not detected.

6 고체발효한방구강세척제개발 8 Table 4. Antimicrobial activity of mixture of oriental medicinal herb prepared with different combination of each extract (10 mg/ml, clear zone, mm) Magnolia : Liquorice : Cnidium :3:2 3::2 1:1:1 Sta. epidermidis 11 Microorganism Str. pyogenes Can. albicans Str. mutans 1 초3 : 천궁2의배합비율에서가장높은항균활성을보였다 (Table 4). 본연구에서는한방구강세척제를개발하기위하여구강세척제에가장많은부분을차지하는베이스성분을박하와정향열수추출물로하였다. 박하와정향열수추출물을제조한후다양한비율로혼합하였을때, 박하열수추출물 100과정향열수추출물 1를부피비로혼합하였을때의관능검사결과가가장높은점수를받았다 (data not shown). 한방베이스에발효한약재에탄올추출물과 Lmenthol, sodium fluoride, poloxamer 407, glycerin, propylene glycol, 수소화 40 피마자유, 그리고 ethanol을참기하여조성비를개발하였다. 사전조사에의하면구강세척제에함유되어있는유효성분이 % 정도이고대부분 1% 미만임 ( 제품조사결과 ) 을감안하여본연구에서는발효한약재혼합물 0.1% 를기본으로하고, Lmenthol과 sodium fluoride의함량을달리하여 A D 한방구강세척제조성비를확립하고 (Table ) 관능검사를실시하였다. 모든한방구강세척제에서한약특유의쓴맛이강하여전체적인기호도가 9점척도에서 4점정도로낮았다. 쓴맛을보강하기위해 Lmenthol의함량을증가하였음에도불구하고큰차이가없었다 (Table 6). 이에발효한약재혼합유효성분을다양한농도로기본베이스에첨가하여쓴맛에대한평가를실시하였을때 0.07% 함유량이적합한것으로나타났다 (data not shown). 구강세척제의혼탁도를낮추기위해용해보조제또는계면활성제를첨가하고다른첨가제의농도를달리하여 E G 조성비를개발하고 (Table ) 관능검사를실시하였다. 발효한약재의함량을낮추고 glycerin을첨가하였을때구강세척제의쓴맛이감소함으로써기호도가증가하였다. LMenthol 사용량에따른관능평가결과를살펴 Table. Various formulation of mouthwash developed in this study (%) Mouthwash A B C D E F G Ethanol extracts of fermented oriental medicinal herbs LMenthol Sodium fluoride Poloxamer 407 Glycerin Propylene glycol 40 Hydrogenated castor oil Ethanol Base for the mouthwash 1) Total ) Dried mint and licorice were extracted with 20 volume of water for 10 minutes, respectively, for 2 times and then 100 portion of mint extracts and 1 portion of licorice extracts were mixed. Table 6. Sensory evaluation data of mouthwash developed with ethanol extracts of solid fermented oriental medicinal herbs as a main components Mouth wash samples 1) A B C D E F G Overall acceptance Bitterness Mint flavor Taste after use Willness to purchase Duration for sensing the refreshment after mouthwash use (min) 4.29±1.60 NS 3.86±1.07 ab2) 4.14±1.68 NS 4.86±2.34 ab 3.7±1.81 NS 9.29±4.0 bcd.00±1.63.7±1.27 a 4.29±1.0.43±1.40 ab 3.86± ±2.67 d 4.14± ±2.31 ab 4.43± ±1.1 b 3.86±2..14±2.67 b 3.71± ±1.70 b 4.43± ±2.14 ab 3.29± ±3.4 a.2± ±1.69 ab.38± ±1.77 ab 4.2± ±3.78 bcd.63± ±1.89 ab.38± ±2.03 a 4.38± ±3.4 bc.63± ±1.1 ab.00± ±2.07 ab 4.63± ±2.67 cd Values are mean±sd (n=10). 1) Refer Table 6 for the composition of mouthwash, A~G. 2) Data with different letters (ad) in the row are significantly different with oneway ANOVA followed by Duncan's multiple range test at P<0.0. NS: not significantly different.

7 86 조병제 홍준영 김미정 송영옥 Table 7. Results of consumer's preference test Overall acceptance Bitterness Mint flavor Taste after use Willness to purchase Duration of sensing the refreshment after mouthwash use (min) Time laps to the base line 3) after mouthwash use (min) Mouthwash Product from current study 1) Commercial product 2) 4.89±1. * 4.96±1.86 NS.43±1. NS 4.96±1.86 NS 4.86±1.67 ** 1.42±.82 NS 14.82±.69 NS 4.11±1.26.7± ±1.2.7± ± ±6.90.7±.20 Values are mean±sd (n=30). 1) Refer 'F' in the Table 6. 2) Ye Doctor Fresh Fine (Fresh Fine Co.). 3) Oral smell of individual participant was detected just before mouthwash use by Breath Checker (HC2M, Tanita). * P<0.0, ** P<0.01 by Student's ttest. NS: not significantly different. 보았을때민트향은차이가없었으나구강세척제사용후의느낌과사용후입안에서지속되는시간에대한본인의느낌에대한항목에서 F 시료는 G 시료에비해유의적으로높은점수를받았다 (P<0.0, Table 6). 이에 7종의시료중에서 F 시료를최종으로선택하고이조성비로구강세척제시제품을제작하여시판되고있는한방구강세척제 ( 예닥터후레쉬파인, Fresh Fine Co.) 와소비자기호도조사를실시하였다. 관능요원 30명을대상으로제품의관능평가및구강세척제사용후의효과를구취측정기로측정하였다 (Table 7). 시제품에대한종합적인평가점수는시판제품보다유의적으로높았으며 (P<0.0) 구매의사역시유의적으로높게나타났다 (P<0.01). Breath Checker를사용한지속성검사결과, 개발한시제품과시판한방제품사이에유의적인차이는없었다. 박하와정향열수추출물 (100:1, v/v) 83.64% 를베이스로하는한방구강세척제조성을관능검사에의거결정하고시제품을제조하였다. 본연구에서개발한발효한방구강세척제와시판하고있는한방구강세척제에대한소비자선호도조사 (n=30) 를실시하였을때본연구에서개발한제품이종합적인평가점수에서유의적으로높았으며구강세척제사용후지속효과를구취측정계로측정하여시간을산출하였을때유의적인차이는없었으나발효한방구강세척제의지속효과가다소길었다. 감사의글본과제 ( 결과물 ) 는교육부의재원으로지원을받아수행된산학협력선도대학 (LINC) 육성사업의연구결과입니다. 요 약 REFERENCES 본연구에서는발효한약재를사용하여한방구강세척제를개발하고자후박, 감초, 천궁, 박하및정향을상황버섯균사체로고체발효하였다. 후박, 감초천궁은수분을 30% 첨가한조건에서고체발효에성공하였으며, 박하는 30% 수분과현미가루 10% 를영양성분으로제공하였을때고체발효가진행되었다. 그러나정향은진균류에대한항균활성이강하여상황버섯균사체를이용한고체발효에성공하지못하였다. 고체발효한약재에탄올추출물의총페놀함량및 DPPH 소거능은발효후유의적으로상승하였으며항균효과역시상승하였다. 구강질환균인 Staphylococcus epidermis, Streptococcus pyogenes, Candida albicans 또는 Streptococcus mutans에대해후박, 감초및천궁이항균활성을보였으며, 항균활성은고체발효에의해약 100배정도증가하였다. 한방구강세척제조성비를확립하기위하여발효한후박, 감초및천궁의에탄올추출물을 :3:2로혼합하였을때가장높은항균활성을나타내었다. 이들발효한약재에탄올추출물혼합액 0.07% 를유효성분으로하고 1. Kim DH, Yoo YS, Joo KB, Lee KJ The evaluation of efficacy on oral cavity cleaner (dental cleaner). Korea Journal of Waters 2: Gagari E, Kabani S Adverse effects of mouthwash use: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 80: Yu YE, Park EY, Jung DH, Byun SH, Kim SC, Park SM Antibacterial effect of oriental medicinal herbs on dental pathogens. The Korean Journal of Microbiology 46: Kim YJ, Park KM. 20. Effects on salivation, xerostomia and halitosis in elders after oral function improvement exercises. J Korean Acad Nurs 42: Tonzetich J Production and origin of oral malodor: a review of mechanisms and methods of analysis. J Periodontol 48: Cho JW, Shin SC, Seo HS Tongue plaque removal effect according to tongue cleaner types. J Korean Acad Dent Health 27: Lee JH, Kim ME, Kim KS, Kim SB Study on the development of gargle solution containing medicinal herb extract for oral malodor. J Oral Med Pain 28: Liang CH, Syu JL, Mau JL Antioxidant properties

8 고체발효한방구강세척제개발 87 of solidstate fermented adlay and rice by Phellinus linteus. Food Chem 116: Dinis MJ, Bezerra RM, Nunes F, Dias AA, Guedes CV, Ferreira LM, Cone JW, Marques GS, Barros AR, Rodrigues MA Modification of wheat straw lignin by solid state fermentation with whiterot fungi. Bioresource Technol 100: Seesuriyachan P, Kuntiya A, Hanmoungjai P, Techapun C, Chaiyaso T, Leksawasdi N. 20. Optimization of exopolysaccharide overproduction by Lactobacillus confusus in solid state fermentation under high salinity stress. Biosci Biotechnol Biochem 76: Khan AW, Zohora US, Rahman MS, Okanami M, Ano T. 20. Production of iturin A through glass column reactor (GCR) from soybean curd residue (okara) by Bacillus subtilis RB14CS under solid state fermentation (SSF). Adv Biosci Biotechnol 3: Kim HS, You JH, Jo YC, Lee YJ, Park IB, Park JW, Jung MA, Kim YS, Kim SO. 20. Inhibitory effects of Phellinus linteus and rice with Phellinus linteus mycelium on obesity and diabetes. 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Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,