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1 Korean J. Food Sci. Ani. Resour. Vol. 30, No. 2, pp. 286~290(2010) ARTICLE Ovotransferrin 가수분해물의 Angiotensin-converting Enzyme 활성억제효과및생산최적화 이나경 안동욱 1 박근규 2 백현동 * 건국대학교동물생명과학부및생명분자정보학센터 1 아이오와주립대학교동물과학과및서울대학교 Biomodulation 전공, 2 건국대학교동물자원연구센터 Inhibitory Effect on Angiotensin-converting Enzyme (ACE) and Optimization for Production of Ovotransferrin Hydrolysates Na-Kyoung Lee, Dong Uk Ahn 1, Keun-Kyu Park 2, and Hyun-Dong Paik* Division of Animal Life Science and Bio/Molecular Informatics Center, Konkuk University, Seoul , Korea 1 Department of Animal Science, Iowa State University, Iowa 50011, USA and Major in Biomodulation, Seoul National University, Seoul , Korea 2 Animal Resources Research Center, Konkuk University, Seoul , Korea Abstract Angiotensin-converting enzyme (ACE) inhibitory activity and production optimization of ovotransferrin hydrolysates were studied. Ovotransferrin was hydrolyzed by several enzymes (protamex, alcalase, trypsin, pepsin, neutrase, and flavorzyme) and acid (0.03 N HCl). Ovotransferrin hydrolysate reduced ACE activity by 60.2%, 55.8%, and 42.6% when treated with trypsin, acid, and pepsin, respectively. Trypsin was selected for production of peptide having maximum ACE inhibitory effect, which was greatest with 7 h hydrolysis. Central composite design determined that optimum composition of ACE inhibitory substances using substrate concentration of 20-35%, temperature of o C, and ph of The optimum composition was 1% trypsin, substrate concentration of 26.32%, o C, and ph Under this conditions, a maximum ACE inhibitory effect of 69.1% was evident, similar to the predicted value. Key words: ovotranferrin, hydrolysate, angiotensin-converting enzyme, trypsin, response surface methodology 서 인간의평균수명이연장되고점차노령화사회가되어가면서, 각종성인병인구가늘어가고있는추세이다. 성인병중에서도순환기계통의고혈압, 심장병과당뇨병등의질환이증가되고있는추세이며, 특히고혈압은많은성인병의원인이되고있다 (Shinogle and Limon, 1989). Angiotensin-converting enzyme(ace) 은 renin-angiotensin aldosteron 계의 angiotensin I을생리적혈압상승물질인 angiotensin II로전환시키거나혈관이완작용이있는 bradykinin을분해시키는효소로동물체의각조직에널리분포되어있으며, ACE 저해제는이 ACE를특이적으로 *Corresponding author: Hyun-Dong Paik, Division of Animal Life Science and Bio/Molecular Informatics Center, Konkuk University, Seoul , Korea. Tel: , Fax: , hdpaik@konkuk.ac.kr 론 저해한다. Angiotensin II가감소되면혈중 catecholamine 값이감소되고혈관이확장되며동시에항이뇨호르몬인 aldosteron의분비가억제된다. 이에따라나트륨이온 (Na + ) 및수분배설이촉진되어순환혈액량이감소되고혈압강하가일어난다. 그러므로 ACE 작용억제는결국혈관수축을막고체내수분저류를막아혈압을낮추는효과를나타낸다 (Petrillo and Ondetti, 1983). 기존의화합물질에의한 ACE 저해치료제의경우, 마른기침, 식욕부진, 미각이상, 발진등과같은부작용사례가있으며, 이를대체하기위해계란 (Ibrahim et al., 2000), 우유 (Do et al., 2007), 자색고구마 (Shin et al., 2008), 뽕잎추출물 (Cho et al., 2006)), 전통된장 (Kim et al., 1999), 미생물 (Papadimitrio et al., 2007; Lim et al., 2008; Yu et al., 2009), 소고기 (Jang et al., 2003) 로부터 ACE 저해천연소재펩타이드를분리하기도하고, 펩타이드의합성 (Jang et al., 2008) 에관한연구가진행된바있다. 286

2 ACE Inhibitory Effect and Optimized Production of Ovotransferrin Hydrolysate 287 천연의완전식품으로잘알려진계란은각성분들의생리적기능과상호작용에의한역할이밝혀지고있고, 기능성건강식품의주요한소재로사용될수있다는점에서그중요성이더욱강조되고있다 (Jeon et al., 2002). Ovotransferrin은난백중 ovalbumin 다음으로차지하는비중이많은단백질이며, 이단백질의기능성으로는항균, 항바이러스, 항산화능 (Ibrahim et al., 2003; Ibrahim et al., 2007) 등이알려져있다. 하지만, ovotransferrin에대한국내연구는 ovotransferrin의생산 (Jang et al., 2005), 항균효과 (Jang et al., 2005) 에대한사례밖에없다. 또한, 특허를통해서도국내의 ovotranferrin과관련한연구는아주미흡한실정이다. 하지만, 북미, 일본및유럽각국에서는 ovotransferrin의추출, 분리에관한연구가지속되고있으며, 점차시장이확대되고있다. 또한, ACE 저해효과의경우, 단백질인 ovalbumin에대해서보고된사례는있으나, ovotransferrin의경우는확인되지않았다 (Fujita et al., 2000; Huopalahti et al., 2007; Miguel et al., 2004). 따라서, 본연구에서는산업적이용가능성이우수한 ovotransferrin을여러가수분해효소를이용하여얻은가수분해물의 ACE 저해효과를확인하고, 그중효과가우수한 trypsin에의한 ovotransferrin 가수분해물의반응시간, 기질의농도, ph, 반응온도를조건으로하여가수분해물의최적화연구를수행하였다. 재료및방법 실험재료본연구를위해 ovotransferrin( 순도 : 98%) 은미국의아이오와주립대학교 (Iowa State University, Ames, IA, USA) 에서제조된시료를사용하였다. 가수분해를위해서 protamex(novozymes Co., Bagsvaerd, Denmark), alcalase (Novozymes), trypsin(sigma Chemical Co., St Louis, MO, USA), pepsin(sigma), neutrase(novozymes), flavorzyme (Novozymes) 을사용하였다. ACE 저해효과를확인하기위해 rabbit lung acetone powder, hippuryl-l-histidyl-l-leucine (HHL, Sigma), sodium borate(duksan Pure Chemical Co., Gyunggi-do, Korea) 를사용하였다. ACE 억제활성측정 ACE 억제활성측정은 Cushman and Cheung(1971) 에 의해보고된실험방법으로실험하였다. Rabbit lung acetone powder(sigma Chemical Co., USA) 를 10배의 0.1 M sodium borate buffer(ph 8.3) 로현탁하고 4 o C에서 24시간동안효소를추출하였다 (Cushman and Cheung, 1971). 추출된효소액을 1,900 g에서 40분간원심분리하여그상등액을 ACE 조효소로사용하였다. 0.3 M NaCl을함유한 0.1 M sodium borate buffer(ph 8.3) 에기질 Hippuryl-histidyl-leucine(HHL, Sigma Chemical Co., USA) 2.5 mm을녹인용액 50 µl, ACE 조효소 50 µl와시료 50 µl( 시료농도 : 30 mg/ml) 를혼합하였으며, 대조구는시료대신증류수 50 µl를첨가하여 37 o C에서 30분간반응시키고 1 N HCl 250 µl 첨가하여반응을중지시켰다. 여기에 ethyl acetate 1.5 ml를가하여 15초간교반한후, 1,900 g에서 20분간원심분리하여상등액 1mL를취하였다. 이상등액을 121 o C에서가열하여완전히건조시킨뒤증류수 3mL를가하여용해시킨후 228 nm에서흡광도를측정하여 ACE 저해율을구하였다. ACE 저해율 (%) = [(SC S)/SC] 100 SC, 시료대조구의흡광도 ; S, 시료의흡광도 가수분해물의제조 Ovotranferrin의가수분해물은산가수분해와 protamex, alcalase, trypsin, pepsin, neutrase, flavorzyme에의한효소로가수분해되었다 (Shinha et al., 2007). 산가수분해는 0.03 N HCl에녹여, 100 o C, 150분간반응시킨후, ammonium water로 ph 7.0으로중화시켰다. 효소가수분해는효소의농도는 1%(w/v) 로첨가되었으며, 기질 (ovotransferrin) 의농도는 25% 로하여 pepsin은 ph 2.5에서, pepsin외다른효소는 ph 6.5로 45 o C, 3시간반응시킨후, pepsin의경우 ph 7.0으로조절한후 100 o C에서 10분동안가열을통해반응을정지시켰으며, 나머지는그대로반응정지를위해 100 o C에서 10분동안가열하였다. 이들은각각원심분리 (Hanil Co., Korea, 1,900 g, 20 min) 하여상등액을동결건조하여본실험에사용하였다. 반응시간에따른 ACE 저해효과검증기질 (ovotransferrin) 의농도를 25%, trypsin 1%, ph 6.5, 반응온도 45 o C로하여 9시간동안반응시키고, 1시간단위로시료를채취하여 ACE 저해효과를검증하였다. Table 1. Levels of independent variables for experimental design X n Independent variables Levels Concentration of substrate (%) ph Temperature ( o C)

3 288 Korean J. Food Sci. Ani. Resour., Vol. 30, No. 2 (2010) Response surface methodology를이용한기질의농도, ph, 반응온도의최적화기질의농도 (20-40%), ph( ), 반응온도 (35-55 o C) 로하여중심합성계획에의해총 16개의조건으로결정하여최적화하였다. 이들요인변수 (X n ) 에의해영향을받는반응변수 (Y) 를회귀분석에사용하였다. 회귀분석에의한모델식의예측에는 SAS program이사용되었다. 결과및고찰 Ovotransferrin의산분해및효소가수분해에따른 ACE 억제효과혈압상승의중요한기전인 rennin-angiotensin system에서주요효소인 angiotensin-converting enzyme(ace) 은 angiotensin I을 angiotensin II로전환하는효소로서, 전환된 angiotensin II가 angiotensin II-type 1 receptor에작용하여혈관을수축시키고 aldosteron의분비를증가시켜혈압을상승하는것으로알려져있다 (McFarlane et al., 2003). Ovotransferrin과산가수분해및효소별가수분해하여얻은가수분해물의 ACE 저해효과를확인하였다 (Table 2). ACE 저해효과는 trypsin>pepsin> 산분해의순으로확인되었다. Trypsin에의해가수분해되어생산된가수분해물이가장높은 ACE 저해효과 (60.2%) 를보이는것으로확인되었다. 이수치는대조군인 ovotransferrin(7.3%) 의 ACE 저해효과에비해월등히높은수치이며, 산가수분해물과비교해서도 5% 정도높은수치이다. 산가수분해물보다효소가수분해물의소비자들의선호및안전성과다른장점을생각한다면이결과는매우긍정적인결과라고판단된다. 반면에, flavorzyme, alcalase, neutrase에의한가수분해물들은오히려 ACE 활성을상승시키는것으로확인되었다. 반응시간에따른 ovotansferrin 가수분해물의 ACE 저해펩타이드의생산 Ovotransferrin의가장높은 ACE 저해효과를나타낸 trypsin을처리하여 0-9시간동안 1시간간격으로확인하 Table 2. ACE inhibitory effect of ovotransferrin hydrolysate ACE inhibitory effect (%) Control (ovotransferrin) 07.3 Acid hydrolysate 55.8 Enzyme hydrolysate Pepsin 42.6 Flavorzyme - a Alcalase - Neutrase - Trypsin 60.2 a Negative effect. 였다 (Fig. 1). 반응시간이 7시간일때 ACE 저해효과가 64% 로가장높게나타났으며, 그이후에는비교적일정하게유지되는것을확인할수있었다. 따라서 7시간을기준으로최적화실험을계속하였다. 중심합성계획에의한생산최적화기질과효소의비, ph, 반응온도를변수로사용하여중심합성계획에의해총 16개의조건으로하여최적화하였다 (Table 3). 각조건의결과를이용해 SAS를통한회귀분석결과는 Table 4에나타내었다. 세가지조건인기질의농도 ( ), ph( ), 반응온도 ( ) 가변할때 ACE 저해효과에대한반응표면회귀식은다음과같았다. Y(%) = Fig. 1. ACE inhibitory effect of an ovotransferrin hydrolysate using trypsin. Table 3. Experimental design and ACE inhibitory effect of the central composite design No. Substrate Conc. (%) ph Temperature ( o C) ACE inhibitory effect (%)

4 ACE Inhibitory Effect and Optimized Production of Ovotransferrin Hydrolysate 289 Table 4. Least square fit and parameters for ACE inhibitory effect Model term a Parameter estimate t-value p-value Intercept < < < a X1, substrate concentration;, ph;, temperature. 회귀식의전체 R 2 는 였고, 0.01% 유의수준을나타내었다. Fig. 2를통해두성분간의상관관계를확인하였다. 기질의농도 ( ) 와 ph( ) 의상관관계, 기질의농도 ( ) 와반응온도 ( ) 의상관관계는확인이되었으나, ph( ) 와반응온도 ( ) 의상관관계는확인할수없었다. 중심합성계획에따른 maximum response는기질농도 26.32%, ph 6.32, o C에서 ACE 저해효과 70.67% 로예측되어졌으며, 실제로실험한결과도이와유사하게 69.1% 로나타내어본최적화실험이성공적으로이루어졌음이확인되었다. 요 약 해물의항고혈압효과를얻을수있는 ACE 저해효과가최초로확인되었다. Ovotransferrin과산분해및효소가수분해물의 ACE 저해효과순서는 trypsin>pepsin> 산분해순인것으로나타났다. 따라서, trypsin에의해가수분해되어생산된가수분해물이가장큰 ACE 저해효과 (60.2%) 를나타내는것으로확인되었으며, 이결과를토대로 trypsin 에의한가수분해물의최적화연구를수행하였다. ACE 저해효과를효소반응시간에따른생산을검토한결과, 7시간일때 64% 로가장높게나타났으며, 7시간을반응시간으로최적화실험을계속진행하였다. 기질과효소의비, ph, 반응온도를변수로사용하여중심합성계획에의해총 16개의조건으로하여최적화하였다. 회귀식의전체 R 2 는 이었고, 0.01% 유의수준을나타내었다. 중심합성계획에따른 maximum response는기질농도 26.32%, ph 6.32, o C에서 ACE 저해효과 70.67% 로예측되어졌으며, 실제실험으로검증한결과도이와유사하게 69.1% 로확인됨으로써, 본실험을통해 ACE 저해활성을나타내는가수분해물의생산이최적화되었다. 감사의글본연구는한국연구재단의중점연구소지원사업 ( ) 및농수산식품기술기획평가원지원과제 ( SB240) 의지원에의해이루어진것으로이에감사드립니다. 또한, 아이오와주립대와교육과학기술부 WCU 프로그램 (R ) 의지원에도감사드립니다. 본연구를통해난백단백질인 ovotransferrin 및가수분 Fig. 2. Contour graph of substrate concentration ( ), ph ( ), and temperature ( ). Table 5. Analysis of variance (ANOVA) for ACE inhibitory effect Sources of variations DF Mean square F Value Pr>F Model < Root MSE = 2.10; CV = 4.64%; R 2 =0.9908

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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,