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1 농업생명과학연구 44(5) pp Journal of Agriculture & Life Science 44(5) pp 초음파처리가녹차침출액의추출수율및화학적특성에미치는영향 김병철 1 강성원 3 정창호 3 허호진 3 이승철 2 조성환 3 최성길 3* 1 SPC group 식품생명공학연구소, 2 경남대학교식품생명학과, 3 경상대학교응용생명과학부 ( 농업생명과학연구원 ) 접수일 (2010 년 08 월 25 일 ), 수정일 (2010 년 10 월 13 일 ), 게재확정일 (2010 년 10 월 19 일 ) Influence of Ultrasonification on Yield and Chemical Property of Green Tea Infusion Byung-Chul Kim 1 Sung-Won Kang 3 Chang-Ho Chung 3 Ho-Jin Heo 3 Seung-Cheol Lee Sung-Hwan Cho 3 Sung-Gil Choi 3* 1 R&D center, SPC group, Seoul , Korea, 2 Department of Food Science and Biotechnology, Kyungnam Univ., Masan , Korea 3 Division of Applied Life Science(Insti. of Agric. & Life Sci.) Gyeongsang Nat`l Univ., Jinju , Korea Received: AUG , Revised: OCT , Accepted: OCT 초록본연구에서는추출보조공정으로서초음파처리에따른녹차의성분특성변화를분석하기위하여, 에서 1, 5, 30, 60분간열수추출하고, 동일온도에서초음파추출하여각각가용성고형분, vitamin C, 총페놀성화합물, flavonols, catechins, caffeine, free amino acids 함량을분석하였다. 가용성고형분함량은초음파처리에의해약 4~5% 의추출수율이증가하였다. Viamin C 함량은 1, 5, 30분에서는각각약 0.21, 0.16, 0.31 mg/g 증가하였지만, 60분처리구에서는대조구와비교해 2.47 mg/g 에서 2.22 mg/g으로감소하였고, 총페놀성화합물함량이약 10~13 mg/g 증가하였다. Flavonols는 myricetin이 4.77 mg/100g에서 9.94 mg/100g, quercetin은 mg/100g에서 mg/100g, kaempferol은 7.95 mg/100g에서 mg/100g으로초음파처리에의해약 2배이상증가함을알수있었다. 초음파처리에의해녹차침출액의 EGCG, EGC, ECG, EC, (+)-C 함량이증가하는경향을보였다. 이러한경향과는달리, 유리아미노산의경우는초음파처리에따른영향이거의없었고, 추출시간이길어짐에따라증가하는경향을보였다. 결론적으로초음파처리에의해녹차의 vitamin C, 총페놀화합물, flavonol 류, catechins 의추출이증가함을알수있었고, 따라서녹차가공에있어초음파처리가매우효율적인추출보조공정으로사용될수있으리라사료된다. 검색어 - 녹차, 초음파, 카테킨, 플라보놀, 비타민 C ABSTRACT The objective of this work was to investigate the influence of ultrasonification on extraction yield and chemical properties of green tea infusion. Changes in total soluble matter(tsm), vitamin C, total phenolic compounds, flavonols, catechins, caffeine, free amino acids contents in green tea infusion(gti) influenced by ultrasonification at of extraction temperature for 1, 5, 30, and 60 min were investigated. The amount of infused TSM increased about 5.3% by ultrasonification for 60min. Vitamin C contents also increased 0.21, 0.16, 0.31 mg/g from 1 to 30 min by ultrasonification. However, vitamin C decreased from 2.47 to 2.22 mg/g at 60min. Total phenol compounds contents increased about 10~13 mg/g on all extraction times by ultrasonification. Flavonols such as, myricetin, quercetin, kaempferol were increased to doubled contents as an influence of ultrasonification. Catechins such as, EGCG, EGC, ECG, EC, (+)-C and caffeine contents showed same tendency as the results of vitamin C. On the other hand, *Corresponding author: Sung-Gil Choi Tel: Fax: sgchoi@gnu.ac.kr

2 92 Journal of Agriculture & Life Science 44(5) result of free amino acids showed different tendency. All amounts of free amino acids did not increase by ultrasonification. Consequently, content of bioactive compounds such as, vitamin C, total phenolic, flavonols and catechins in green tea infusion were influenced by ultrasonification. Key words - Green tea, Ultrasonification, Catechins, Flavonols, Vitamin C Ⅰ. 서론차나무 (Camellia sinensis L.) 는다년생상록관목수로북위 35 이하지방에서주로재배되고있으며, 차는전세계의음료중에서가장오랜역사를가지고있는기호음료로최근건강기능적우수성이입증됨에따라차음료산업매출신장과녹차함유가공품의소비가급증하는추세이다 (Tijburg et al., 1997; Drosti et al., 1997; Mukbtar & Ahmad, 1999). 차가중요한기호음료로서발전해온가장큰이유는차가여러민족의구미에맞는대중적인기호성을가지고있을뿐만아니라생체의복잡한생명활동을조절하는식품의 3차기능으로서생체리듬의조절, 면역력의증진, 질병의예방이나회복, 노화억제등신체조절기능을갖는기능성식품으로서중요성이새삼강조되고있기때문이다 (Kim et al., 1999). 차는차나무의잎을이용하여제조방법에따라채엽후바로덖음이나증제공정을거치는녹차와반발효차인우롱차, 발효차인홍차로구분 (Choi & Choi, 2003) 되며, 차의구성성분으로는섬유소, 단백질, Vit-E, β-carotene, 클로로필, catechin 류, flavonol 류, caffeine, Vit-C, 아미노산등이고, 대표적인가용성성분으로는주로 catechin 류, 아미노산, caffeine 등이있다. 그리고녹차침출액의주성분인 catechin 류는 (-)-epicatechin (EC), (-)-epicatechin 3-gallate (ECG), (-)-epigallocatechin (EGC) 및 (-)-epigallocatechin 3-gallate (EGCG) 등이들어있으며, 총카테킨류중 EGCG 가가장함량이높고, 생리할성능으로는항산화, 항암, 항당뇨병, 항균효과등이높다고보고 (Trevisanato & Kim, 2000; Shibata et al., 2000; Chung et al., 2003) 되고 있다. 유리아미노산은대표적으로 theanine 과 GABA 에관한연구가많이되고있으며, 이는주로혈압강하작용을하는것으로보고 (Park et al., 2001) 되고있다. 그리고 caffeine 은 alkaloid류의일종이며중추신경흥분, 강심, 이뇨, 항천식및대사항진 (Buchanan & Fletcher, 1978) 등의효과가밝혀지고있다. 초음파는주파수가약 20 khz 이상인음파를지칭하며 low-intensity (<1 W/cm 2 ) 와 high-intensity (10~1,000 W/cm 2 ) 초음파로대별되며, 전자는반사되는성질을이용하여식품의성분과구조등의비파괴검사에적용되고, 후자는물체를전단및압축시켜조직의물리적파괴, 유화형성, 화학반응촉진등의성질을가지는것으로알려져있다 (Earnshaw, 1998). 또한, 용매내에서초음파의공동 (cavitation) 효과에의해유발된분자운동이활성화에너지로작용하여세척이나반응성향상, 특정성분의추출공정에도입되어공정의효율을높이는효과가있다 (Namgoonga & John, 1984; Sehgel et al., 1980). 본연구에서는초음파처리에따른녹차침출액의 vitamin C, total phenolic compounds, flavanols, catechins, caffeine, free amino acids의함량변화를분석하여녹차추출가공에서보조공정으로서초음파의이용가능성을알아보고자하였다. Ⅱ. 재료및방법 2.1 실험재료경남하동군악양면에재배되고있는야생차잎을채취하여선별한후 300 ±10 의볶음솥에서 10 min 동안덖음처리하여제조한녹차를본실험에사용하였다.

3 Kim et al. : Influence of ultrasonification on extraction yield and chemical property of green tea infusion 침출액제조녹차침출액은차엽 1.5g을증류수 100 ml의비율로침출온도, ( 초음파처리 ), 침출시간 1 min, 5 min, 30 min, 60 min으로추출하여여과지 (Whatman No. 2) 로여과하여제조하였다. 2.3 침출조건에따른총가용성고형분함량 Aluminum weighing dish에침출액 30ml를취해 105 에서상압가열법에의해항량에이를때까지건조한후건조전과건조후의중량차이를이용해총가용성고형분함량을구하였다. 2.4 Vitamin C 비타민 C의분석은시료를물로추출하여 hexane 으로유지성분을제거하고, 0.45 μm membrane filter로여과하여 Sep-pak C 18 로색소및단백질성분을제거한후 HPLC (Hewlett packed 1100 series U.S.A) 로분석 (Nishi, 1989) 하였으며, 그분석조건에서 column은 u-bondapak C18 ( cm I.D) 을사용하였고, Sovent와 flow rate는각각 70% methanol(ph 4.0) 과 0.5 ml/min로하였으며, chat speed는 0.5 cm/min 이고, UV와 injection volumn 은각각 214nm와 50 μl이다. 동안환류냉각시킨다. 이추출물을 50 ml volumeteric flask를이용하여 60% ethanol로 volume을 50 ml가되게정용한다음 0.45 μm filter 로여과하여 HPLC (Hewlett packard 1100 series, U.S.A) 로분석하였다 (Kim et al., 2002). Column은 HP hypersil ODS ( mm), 30% acetonitrile in M KH 2PO 4 를이동상으로하여 40 에서유속 1.0 ml/min으로 UV detector 를이용해 280 nm에서검출하였다. 이때 myricetin, quercetin, kaempferol을표준물질 (Sigma Chemical Co., USA) 로사용하였다. 2.7 Catechin류및 caffeine Bae et al. (2002) 의방법을약간변형하여 HPLC (Agilent 1100 series, Hewlett packard, U.S.A) 로측정하였다. Column 은 CAPCELL PAK C18 UG 120 (5 μm, mm, Shiseido, Tokyo, Japan), methaol: water: formic acid = 19.5: 82.5: 0.3 (v/v/v) 을이동상으로하여 40 에서유속 1 ml/min 으로 280 nm 에서검출하였다. 이때 (-)-epigallocatechin (EGC), (+)-catechin (C), epicatechin (EC), (-)-epigallocatechin gallate (EGCG), epicatechin gallate (ECG) 와 caffeine을표준물질 (Sigma Chemical Co., USA) 로사용하였다. 2.5 총 phenol 성화합물녹차의총 phenol성화합물을분석하기위하여 50% Methyl alcohol로추출한후추출액 0.1 ml 에증류수 3 ml, M 포타슘페리시아나이드 (K 3Fe(CN) 6) 1mL, 0.01 M 삼염화철 (FeCl 3/0.1N HCl) 용액 1 ml를넣고혼합한후 15분간방치하고, 안정제 (H 2O : 1% gum arabic : 85% phosphoric acid = 3 : 1 : 1, v/v/v) 5 ml 첨가한후 700 nm 에서흡광도를측정하였으며, 몰식자산 (gallic acid) 으로작성한검량곡선으로함량을환산하였다 (Graham, 1992). 2.6 Flavonol류녹차에함유되어있는 flavonol 류를분석하기위하여침출액 16 ml에 99.99% ethanol 24 ml과 6 M-HCl 5 ml를첨가한후 95 수욕상에서 2시간 2.8 유리아미노산유리아미노산분석은 Oh et al.(2002) 의방법을약간변형하여침출액을일정량취하여 rotary vacuum evaporator 를이용하여수분을완전제거하고 Lithium citirate buffer (ph 2.2) 를 10 ml 로용해한후 sulfosallicylic acid 0.5 g을첨가하여상온에서 2시간동안방치한후 0.22 μm membrane filter로여과한여액을아미노산자동분석기 (Biochrom 30, Biochrom Ltd, Cambrige, UK) 를이용하여 Table 1의조건으로분석하였다. 2.9 통계처리각측정치의결과는 SAS (Statistics Analytical System, USA) program을이용하여 p<0.05 수준에서분산분석 (ANOVA) 를실시한후, Duncan's Multiple Range Test에의하여시료간의유의성을

4 94 Journal of Agriculture & Life Science 44(5) 검증하였다. Table 1. The operating condition for amino acids autoanalysis Items Conditions Instrument Column Biochrom 30, Biochrom Ltd, Cambrige, UK High resolution sodium column Flow rate Buffer 25 ml/hr, ninhydrin 20 ml/hr Column temp. 48 Injection volume 40 μl Ⅲ. 결과및고찰 3.1 침출조건에따른녹차침출액의총가용성고형분함량추출에의한처리후식품의음용시용매에용해되는가용성성분만이체내에흡수되기때문에추출조건에따른가용성고형분함량결과는식품의가공시매우중요한기초데이터로이용될수있다. 초음파처리에따른추출효과의차이를보기위해추출시간별녹차침출액의총가용성고형분함량을분석하여 Fig. 1에나타내었다. 추출시간이길어짐에따라가용성고형분함량이증가하는경향을보였고, 60분간추출한경우 와 초음파처리구각각 31.1, 36.4% 를보여, 초음파처리에의해약 5.3% 가용성고형분의추출이증가하였다. 추출보조공정으로초음파처리를할경우, 녹차침출액의추출효율을증가시킬수있으리라판단된다. 3.2 Vitamin C 초음파처리에따른침출시간별녹차침출액의 vitamin C 함량을분석한결과는 Table 2 과같다. 초음파처리에의해같은온도인 에비해시간 별로 1, 5, 30 분에서각각약 0.21, 0.16, 0.31 mg/g 증가하였다. 하지만, 침출시간 30 min 와 60 min 을비교하면 초음파처리구에의해는 2.47 mg/g 에서 2.22 mg/g 으로감소하였다. 이는 초음파처리시침출시간이 30 분을초과하여길어질 수록 vitamin C 산화의진행이높아지기때문으로 사료된다. 녹차추출가공품의제조시추출, 농축 등가열과정이길어질수록 vitamin C 의손실증가 하기때문에 5 분이내의추출시간에초음파추출하 는방법이 vitamin C 추출에는가장효율적인추출 방법이라사료된다. Total soluble matter (%) time (min) _Ultra Fig. 1. Changes in total soluble matter of green tea infused at with/without ultrasonification treatment. Table 2. Influence of ultrasonification on ascorbic acid contents of green tea infusion at different times (Unit : mg/g) time(min) ±0.006 dd 1.89±0.009 ca ±0.005 cb 2.21±0.030 ba ±0.006 bd 2.47±0.043 aa ±0.031 ab 2.22±0.002 bd Results are expressed as means ± SD. Values in the same column with different superscript small letters are significantly different (P< 0.05). a> b >c >d. Values in the same row of each infusion time with different superscript capital letter are significantly different (P< 0.05). A> B> C >D>E

5 Kim et al. : Influence of ultrasonification on extraction yield and chemical property of green tea infusion 총 phenol 성화합물 페놀성화합물은식물계에널리분포되어있는물질로, 다양한구조와분자량을가지며, 이것들의 phenolic hydroxyl기가단백질처럼거대분자와결합을하여항산화, 항균, 항암등의생리기능을가지는것으로보고되고있으며특히녹차에다량으로함유되어있다 (Choi et al., 1992; Kim et al., 2004) 는수확시기, 당해연도기상조건, 환경조건, 실험의분석방법에따라차이가나는것으로보고하였다. 초음파처리에따른침출시간별녹차침출액의총페놀성화합물함량은 Table 3에나타내었고 1, 5, 30, 60분추출한시료에서각각초음파처리에의해약 12.8, 13.2, 10.2, 11.5 mg/g 증가하는것으로나타났고, 추출시간이길어짐에따라증가하는경향을보였다. 또한, 60분간초음파처리하였을때 mg/g 으로가장높은값을보였고, 초음파처리에의한증가치는 5분처리한시료에서약 13.2 mg/g 으로가장높았다. Table 3. Influence of ultrasonification on total phenolic compounds contents of green tea infusion at different times (Unit : mg/g) time(min) ±9.76 cc 74.14±1.85 db ±3.58 bb ±4.92 ca ±4.61 ac ±4.08 bb ±3.29 ac ±4.04 ab Results are expressed as means ± SD. Values in the same column with different superscript small letters are significantly different (P< 0.05). a> b >c >d. Values in the same row of each infusion time with different superscript capital letter are significantly different (P< 0.05). A> B> C >D 3.4 Flavonol류 Flovonoid 계성분은항산화, 항암등의기능성을가진특유성분으로알려져있다. 초음파처리에따른침출시간별녹차침출액의 flavonol 류의함량변화는 Table 4와같다. Flavonol 류함량이추출시간 이길어짐에따라증가하는경향을보였고, 30분까지처리했을때는초음파처리한경우와대조구의값이거의차이가나타나지않았지만, 60분추출한경우, myricetin은 4.77 mg/100g에서 9.94 mg/100g, quercetin은 mg/100g에서 mg/100g, kaempferol은 7.95 mg/100g에서 mg/100g으로초음파처리에의해약 2배이상함량이증가하는것으로나타났다. Table 4. Influence of ultrasonification on flavonols contents of green tea infusion at different times (Unit : mg/100g) Myricetin Quercetin Keampferol time (min) ±0.30 c 2.13±0.18 d ±0.09 b 3.16±0.05 c ±0.68 a 4.87±0.68 b ±0.22 a 9.94±0.49 a ±0.57 c 5.98±0.56 d ±0.30 b 7.61±0.16 c ±1.13 a 11.01±0.84 b ±0.25 a 20.84±0.32 a ±0.51 c 4.11±0.70 c ±0.61 b 5.21±0.21 c ±0.67 a 7.76±0.69 b ±0.29 a 17.33±0.92 a Results are expressed as means±sd a-d Means in the same column with different superscripts are significantly different (P<0.05). a> b >c >d. 3.5 Catechin류및 caffeine Catechin 류는 C6-C3-C6 형탄소골격을가진물질로서 catechin의탄소 2, 3번위치에존재하는수소의결합방향에따라 trans 형이면 catechin 이라하고 cis형이면 epi-catechin 이라부르는데본실험에서는 catechin 및 epi-catechin 등총 5종을대상으로분석하였다. 초음파처리에따른침출시간별녹차침출액의 catechin 류함량의변화는 Table 5와같다. 초음파처리에의해녹차침출액의 EGCG, EGC, ECG, EC, (+)-C 함량이증가하는경향을보

6 96 Journal of Agriculture & Life Science 44(5) 였다. 한편, 추출시간에따라서는 에서는대부분증가하는경향을보였지만, 초음파처리구의경우는 30분까지는증가하였지만, 이후 60분에서는감소하였다. 이는초음파처리시간이 30분이상지속될경우에카테킨의화학구조가영향을받기때문인것으로사료된다. 또한, 추출시간 1분에서는 와 초음파처리군의결과가거의동일하게나타났는데, 이는추출시간이짧아초음파처리가녹차내성분의유리에영향을미치지못했기때문으로사료된다. Ya et al. (2003) 은 catechin 류의화학구조가열에불안정한구조를가지고있다고보고하였으며, 본연구에서는초음파처리시간의가중으로함량이감소한것으로사료된다. Caffeine 은 alkaloid의일종으로녹차의정미성분중에서약한쓴맛을나타내며카페인함량이높을수록녹차의맛에대한기호도가증가한다고알려져있으며, 녹차에함유된카페인은커피나홍차에함유되지않은 catechin 류나 theanine 등과분자화합물을이루고있기때문에흡수작용이서서히일어나순수한카페인을과잉섭취했을때나타나는정신불안, 불쾌감등의부작용을일으키지않고그작용이훨씬부드럽게나타나는것으로보고되고있다 (Kirishnamurthi, 1993). 녹차침출액의 caffeine 함량은 Table 6과같다. caffeine함량은초음파처리, 추출시간에따른차이가거의나타나지않았으며, 1.83 mg/g 에서 1/62 mg/g 으로감소하여, 카테킨류와유사한경향을보였다. 3.6 유리아미노산차엽의총질소화합물중약 20% 는카페인이며그외의질소화합물로는아미노산, 단백질, 핵산등이차지하고있다. 단백질은제조과정중탄닌과결합하거나가열에의해응고되어녹차침출액중에는거의용출되지않으나, 아미노산은수용성이기때문에침출액에용출되어차의맛에크게관여한다. 양질의녹차일수록녹차의특유의 amide가다량함유되어있고 theanine, 글루탐산, 아리기닌등유리아미노산총함량이매우중요하며상급의녹차맛은 theanine 함량및기타아미노산함량이많을수록맛이좋아진다고보고가있다 (Lee et al., 1998). 초음파처리에따른침출시간별녹차침출액의유리아미노산함량의변화는 Table 7과 같다. 유리아미노산의경우는초음파처리의영 Table 5. Influence of ultrasonification on catechins contents of green tea infusion at different times time (min) (Unit : mg/g) EGCG ±0.39 dc 8.94±0.39 dc ±0.19 cc 16.29±0.13 cb ±0.61 bd 22.23±0.10 ab ±0.45 aa 18.93±0.46 bb EGC ±0.18 cc 3.76±0.15 cc ±0.09 bc 6.31±0.60 aa ±0.28 ab 6.22±0.03 aab ±0.06 aa 4.88±0.42 bb ECG ±0.08 dc 2.10±0.05 cc ±0.06 cd 3.85±0.34 bc ±0.40 bb 5.98±0.03 aa ±0.18 aab 4.49±0.75 bb EC ±0.03 dc 0.99±0.05 cb ±0.02 cc 1.52±0.13 bab ±0.04 bb 1.90±0.01 aa ±0.08 abc 1.58±0.22 babc (+)-C ±0.02 ca 0.48±0.03 ca ±0.01 cc 0.68±0.06 bb ±0.05 bc 0.97±0.03 ab ±0.02 ac 0.77±0.08 bc Results are expressed as means ± SD. Values in the same column with different superscript small letters are significantly different (P< 0.05). a> b >c >d. Values in the same row of each infusion time with different superscript capital letter are significantly different (P< 0.05). A> B> C >D

7 Kim et al. : Influence of ultrasonification on extraction yield and chemical property of green tea infusion 97 향에따른함량의차이가거의거의나타나지않았고, 추출시간이길어짐에따라다소증가하는경향을보였다. 60분추출했을때, arginine이 에서열수추출했을때, 3.36 mg/g, 같은온도에서초음파처리했을때, 3.22 mg/g 으로가장높게나타났으며, 다음으로 asparagine, glutamic acid 순으로나타났다. 이러한결과는 Lee et al. (1998) 의 theanine이 , 0.31% 라는보고와 Oh et al. (2002) 의글루탐산이 mg/g 그리고아르기닌이 mg/g라는보고와는차이가있었다. 이것은유리아미노산함량이생육환경, 품종또는차나무의연령, 생육도등여러가지요인에따른차이때문인것으로사료된다. Table 6. Influence of ultrasonification on caffeine contents of green tea infusion at different times (Unit : mg/g) time (min) ±0.01 db 1.52±0.07 cab ±0.01 cc 1.69±0.03 abbc ±0.08 bb 1.81±0.00 aa ±0.01 aa 1.62±0.08 cc Results are expressed as means ± SD. Values in the same column with different superscript small letters are significantly different (P< 0.05). a> b >c >d. Values in the same row of each infusion time with different superscript capital letter are significantly different (P< 0.05). A> B> C >D Table 7. Influence of ultrasonification on free amino acids contents contents of green tea infusion at different times Content (mg/g) Free amino acids temperature/time 1min 5min 30min 60min 1min 5min 30min 60min Threonine Serine Theanine Asparagine Glutamic acid Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine GABA Lysine Histidine Arginine Total

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