J Korean Soc Food Sci utr 한국식품영양과학회지 (2), 327~333(11) DOI:.3746/jkfn.11..2.327 단백질분해효소처리가청국장의항산화활성에미치는영향 연구노트 박민경청운대학교식품영양학과 Effect of Enzymatic Hydrolysis by roteases on ntioxidant ctivity of hungkukjang Min-Kyung ark Dept. of Human utrition and Food Science, hungwoon University, hungnam 3-71, Korea bstract hungkukjang and soybean powder were enzymatically hydrolyzed with, and mu of 3 commercially available proteases (alcalase 2.4L, protamex and neutrase.8l) at o for 1 min. The degree of hydrolysis and antioxidant activities of hydrolysates were comparably evaluated. lcalase and protamex yielded higher content of peptide compared to neutrase in both hungkukjang and soybean powder hydrolyzed samples. Both hungkukjang and soybean hydrolysates showed also greater increases of antioxidant activities compared to those prepared with neutrase. The rates of increment of DH, BTS and hydroxyl radical scavenging activities were similar between hungkukjang and soybean powder hydrolyzates. These results show that alcalase and protamex are not specific for hungkukjang but enhance its antioxidant activity. Key words: hungkukjang, soybean powder, alcalase, protamex, neutrase, peptide, antioxidant activity 서론최근식품단백질유래펩타이드의다양한생리활성에관한연구가주목받고있으며유제품에서최초로언급된항고혈압물질인 angiotensin I 전환효소 (angiotensin converting enzyme, E) 저해펩타이드는가장많이연구된생리활성펩타이드중하나로 (1) 된장 (2-4), 청국장 (5,6), 간장 (7) 및 natto(8) 등에서도분리되어대두발효식품의건강기능성이주목받도록한요인이다. 또한다양한효소처리에의해생성된펩타이드가심혈관질환관련요인에미치는영향이많은연구자에의해발표되었다. Zhong 등은상업용단백질분해효소인 alcalase 처리대두가수분해물이소화효소인 pepsin 및 pancreatin에도안정하며콜레스테롤의용해도저해를통해장에서의흡수를방해하므로혈청 low density lipoprotein(ldl)-콜레스테롤과 very low density lipoprotein(vldl)-콜레스테롤을저하시킨다고보고하였으며 (9), 활성펩타이드를분리, 동정하였다 (). Wu와 Ding(11) 도 alcalase를이용하여대두로부터 E 저해펩타이드를분리, 동정하고소화효소에대한안정성을보고하였으며, hiang 등 (12) 은분리대두단백으로부터 E 저해펩타이드활성이높은가수분해물생산에가장효율적인효소종류및반응조건등을보고하였다. Ringseis 등 (13) 은 pepsin, trypsin, chymotrypsin 등의효소처리로얻은대두가수분해물이인체동맥내피세포의증식및 nitric oxide(o), prostaglandin I 2 등의분비에영향을미치는것으로보고하였으며, O 및 eicosanoids의비정상적인분비는혈관손상및동맥경화를유발하는것으로알려져있다 (14,15). 식품단백질유래펩타이드의항산화기능또한중요생리활성으로활발히연구되고있으며 casein(16), 유청 (17), 난백 (18), 난황 (19) 을비롯하여대두 (-22) 의효소분해를통해얻어진펩타이드의항산화활성이보고되었다. 펩타이드의항산화활성은특정아미노산서열과관련이있으며 (16), hen 등 () 은대두단백질가수분해물로부터분리한펩타이드를이용하여 proline-histidine-histidine이높은항산화활성을나타낸다고밝혔다. 지금까지대두펩타이드에관한연구는주로분리대두단백을통해이루어졌으며, Gibbs 등 (23) 이 natto 및 temphe를가수분해하여얻은펩타이드의생리활성을보고한바있으나발효대두를이용한연구는미미한상태이다. 대두단백의효소가수분해율은동일효소에의해서도전처리조건에의해달라질수있는데 (24) 발효로인한대두단백질의변화또한단백분해효소활성에영향을미칠것으로사료된다. 따라서본연구에서는청국장을 alcalase, protamex 및 E-mail: mkpark@chungwoon.ac.kr hone: 82-41-6-3241, Fax: 82-41-634-87
328 박민경 neutrase 등상업용단백분해효소로가수분해하고펩타이드생성및항산화활성을측정한후대두분말과비교하여차이가있는지알아보았다. 재료및방법실험재료청국장시료는발효후열풍건조하여분쇄한분말을맛가마식품 ( 충남논산 ) 에서구입하여사용하였다. 대두분말은대두 [Glycine max(l.) Merrill] 를분쇄기 (GFM-S1, LG, Seoul, Korea) 를이용하여분쇄한것으로하였으며두시료모두체 (1 mesh) 로친후실험에사용하였다. lcalase 2.4L(2.4 U/g), protamex(1.5 U/g), neutrase.8l(.8 U/g) 은 ovozymes /S(Bagsvaerd, Denmark), 2,4,6-trinitrobenzene sulfonic acid(tbs), L-leucine, luminol, 1,1-diphenyl-2-picryl hydrazyl(dh), 2,2'- azinobis-3-ethylbenzothiazoline-6-sulfonic acid(bts), microperoxidase-8(m-8), FeSO 4 7H 2O 및 ascorbic acid 는 Sigma(St. Louis, MO, US) 로부터구입하였으며기타시약은일반특급시약을사용하였다. 효소처리청국장및대두분말 5% 수용액을 95 o 에서 분간살균한후상온에서냉각하고반응액으로사용하였다. 반응액의 ph는 6.8~7. 사이로실험에사용한세효소의적정 ph 범위에해당되므로 ph 보정없이 alcalase, protamex 및 neutrase를각각 mu, mu 및 mu가되도록첨가하고진탕배양기 (SJ88H, Sejong, Seoul, Korea) 를이용하여온도 o, 회전속도 8 rpm의조건에서 분, 분, 분, 분및 1분간반응시켰다. 반응이끝나면끓는물에 분가열하여효소를불활성화하고원심분리기 (Union 32R, Hanil, Seoul, Korea) 를이용하여 2,322 g로 분원심분리한후상등액을취하여펩타이드생성정도및항산화활성측정의시료로사용하였다. 대조구는효소를첨가하지않고살균후 1분간동일한진탕과정을진행한시료로하였다. 펩타이드생성측정펩다이드의측정은 TBS 법으로측정하였다 (25). 즉, 효소처리가끝나면원심분리하고상등액 1 ml에 1%(w/v) SDS 용액 9 ml를혼합한후 125 μl를취하여 1 ml의 phosphate buffer(.2125 M, ph 8.2) 와 1 ml의.1% TBS(w/v) 수용액을첨가하였다. 교반한후 o 수욕조에서빛을차단하고 1시간반응시킨후 2 ml의.1 Hl을첨가하여반응을종료하였다. 상온에서 분냉각한후반응액 μl를취하고분광광도계 (Microplate reader, VERSmax, Molecular Devices, Sunnyvale,, US) 를이용하여 3 nm에서흡광도를측정하였다. L-Leucine을표준물질로사용하여펩타이드생성정도를계산하였다. 항산화활성 DH radical 소거활성은건조고형분함유량이.6~9 mg/ml이되도록희석한시료액.5 ml을.2 mm DH 에탄올용액 1 ml와혼합하고 분간상온에서방치한후분광광도계를이용하여 515 nm에서흡광도를측정하여알아보았다 (26). BTS radical 소거활성은 rnao 등 (27) 과 Obon 등 (28) 의방법을응용하여측정하였다. 즉, 2 mm BTS,.2 mm M-8 및.1 mm H 2O 2 를함유한 1 ml의 BS(potassium phosphate-buffered saline,.1 M, ph 7.4) 와건조고형분함유량이 1~5 mg/ml 되도록희석한시료액.1 ml을혼합하고상온에서 6분간방치한후 735 nm에서흡광도를측정하였다. Hydroxyl radical(oh ) 소거활성은 Fenton 반응에의해 OH 을생성하고 luminol을발광증폭제로사용하여화학발광법 (hemiluminescence) 으로측정하였다 (29,). 즉, 건조고형분함유량이.4~2 mg/ml 되도록희석한시료액 μl와 1 mm H 2O 2 μl를 96-well microplate에넣고화학발광기 (Microtiterplate Luminometer, EG&G Berthold LB96, Bad Wildbad, Germany) 에장착한후자동주입기를통해 mm luminol μl를자동주입하였다. 5초후 1 mm FeSO 4 μl를자동주입하고 초간화학발광정도를측정하였다. 시료의항산화활성즉 DH, BTS 및 hydroxyl radical 소거활성은 vitamin 를표준물질로이용하여환산하고 vit. eq μm/mg으로표시하였다. 통계처리결과는평균 ±SD로표시하였으며, 통계적유의성은 Student's t-test 및 OV 실시후 Tukey's test로검증하였다. 결과및고찰펩타이드생성효소첨가를제외한나머지실험조건을동일하게하여추출한청국장건조분말과대두분말의펩타이드평균함량은각각 7.2±.72 및 5.64±.68 mm로 ( min) 청국장이유의적으로높았다 (p<.5, Student's t-test). lcalase는 Bacillus licheniformis 유래의 endoproteinase 로 mu를처리한청국장의경우,,, 및 1분에서각각 15.32±1.34,.77±1.79, 25.69±2.37, 26.42±2.41, 27.18±2.76 mm의팹타이드생성을보였으며, mu에서는 26.55±2.17, 37.26±3.73, 46.89±4.48, 47.31±4.54, 47.46 ±4.65 mm 및 mu의처리에의해서는 33.77±2.56, 44.54±3.94,.78±4.83, 51.67±4.76, 51.34±4.91 mm의펩타이드생성을보였다 (Fig. 1). 대두분말은 alcalase 처리시 mu에서 13.1±1.38,
단백질분해효소처리가청국장의항산화활성에미치는영향 329 lcalase rotamex eutrase mm Leucine 8 1 8 1 8 1 Hydrolysis time (min) Fig. 1. eptide contents of hungkukjang () and soybean (S) hydrolysates prepared with different concentrations of alcalase, protamex and neutrase., mu-;, mu-s;, mu-;, mu-s;, mu-;, mu-s. 16.74±1.85, 21.76±2.9, 22.42±2.15, 21.59±2.22 mm, mu에서는.8±1.98, 28.98±2.28, 35.57±3.71, 36.1± 3.32, 37.88±3.61 mm 및 mu의처리에의해서는 27.44 ±2.26, 35.34±3.24, 39.85±3.73,.11±3.85,.85±4.1 mm의팹타이드생성을보였다. 즉, 청국장및대두분말두시료모두에서효소량이증가함에따라펩타이드생성량이증가하였으며 분및 분에급격히증가하여 분이후최대에이르렀으며이는 alcalase를사용하여분리대두단백 (24) 및탈지대두 (31) 를분해한연구보고와유사하였다. rotamex는 Bacillus protease complex로청국장의경우 mu 처리시,,, 및 1분에서각각 15.3± 1.26, 23.89±1.85, 25.31±2.7, 25.24±2.27, 25.72±2.31 mm, mu에서는 23.92±2.61, 37.±3.58, 39.98±3.25,.62 ±3.91,.75±3.85 mm 및 mu의처리에의해서는 29.36±2.26, 44.75±4.54, 45.81±4.35, 45.96±4.16 45.65± 4.24 mm로 alcalase와유사한결과를보였다 (Fig. 1). 대두분말을 protamex 처리시 mu에서 12.15±1.1, 19.67±1.54,.91±2.17,.78±1.73,.71±1.95 mm, mu에서는 18.88±1.63,.72±2.74, 33.±3.25, 34.47± 3.49, 34.51±3.61 mm 및 mu의처리에의해서는 24.24 ±2., 35.43±3.39, 36.79±3.18, 35.9±3.28, 36.65±3.45 mm의펩타이드생성을보였다. eutrase는 Bacillus amyloliquefaciens로부터얻어진 endoproteinase이며, mu 처리시,,, 및 1분에서청국장의경우각각 9.34±1.2 11.98±1.16, 13.66±1.47, 13.39±1.11, 13.57±1.23 mm, mu에서는 15.1± 1.29, 21.9±1.96, 23.74±2.9, 24.25±2.24, 25.61±2.26 mm 및 mu의처리에의해서는 13.5±1.17, 22.82±2.14, 27.23±2.59, 27.45±2.43, 28.1±2.56 mm의펩타이드생성을보였다. 대두분말은 mu에서 7.97±.66, 9.82±.79,.69±.89,.98±1.11, 11.25±.95 mm, mu에서는 9.61±.97, 15.33±1.23, 17.62±1.57, 17.73±1.79, 17.82±2.2 mm 및 mu의처리에의해서는.14±1.5, 18.11±2.7,.99±2.18, 21.49±2.5, 22.43±2.19 mm로효소첨가량이증가함에따라펩타이드의생성량이증가하였으나 alcalase 및 protamex 처리와비교하여청국장및대두분말모두에서펩타이드생성량이낮은것으로나타났다 (Fig. 1). 분리대두단백, β-conglycinin 및탈지대두등을효소분해한연구보고에의하면효소의종류에따라가수분해율에차이가있는것으로알려졌다 (21,24,31). 또한시료의가열전처리가효소가수분해율에영향을주어 alcalase의경우분해율이낮아지는반면 protamex는증가하는등동일효소의작용이전처리조건에의해달라질수있는것으로보고되었다 (24). 본연구의결과에서도 alcalase 및 protamex가 neutrase와비교하여펩타이드생성량이높아효소에의한차이가있음을보여주고있다. 반면청국장과대두분말두시료모두에서효소처리하지않은대조구 ( min) 대비펩타이드증가율이유사한것으로미루어본연구조건에서는가열및발효등청국장제조공정에의한시료의변화가효소분해에유의한영향을주지않는것으로나타났다. 항산화활성청국장및대두분말효소분해물의항산화활성은 DH, BTS 및 hydroxyl radical 소거활성정도를통해알아보았으며통계적유의성은효소처리를하지않은대조구와비교하여 95% 유의수준에서 Tukey's test로검정하였다. 청국장의 DH radical 소거활성은 Fig. 2에서보는바와같이고형분.6, 1.2, 3, 6 및 9 mg/ml에서효소처리를하지않은대조구는 123.7±7.12, 213.6±15.45, 479.77±39.9,
3 박민경 hungkukjang Soybean DH radical scavenging activity 8 DH radical scavenging activity 8 2 4 6 8 2 4 6 8 Fig. 2. DH radical scavenging activity of hungkukjang and soybean hydrolysates prepared with alcalase (), protamex () and neutrase (). : non-enzymatically hydrolyzed sample. The values are means±sd, which obtained from alcalase and protamex treatments are significantly different (p<.5) from the control () by Tukey's test. 6.53±54.8, 75.1±47.96, alcalase 가수분해물은 176.5 ±6.2, 3.88±31., 754.7±53.75, 863.33±59.65, 968.93 ±54.12 vit. eq μm로 alcalase 처리구가대조구와비교하여시료량에따라약 37~57% 증가하였다 (p<.5). rotamex 가수분해물도 175.±17.69, 6.26±.66, 721.33±64.3, 845.66±63.51, 954.±83.14 vit. eq μm로대조구와비교하여시료량에따라 35~% 증가하였다 (p<.5). 반면 neutrase 가수분해물은 132.25±11.45, 261.62±.13, 567.84 ±35.24, 729.±55., 815.±76.66 vit. eq μm로대조구와비교하여통계적으로유의한차이가없었다 (p>.5). 대두분말의 DH radical 소거활성은대조구가 68.24± 4.1, 119.72±8.88, 247.47±24.19, 381.71±25.93, 414.36± 36.19 vit. eq μm이며 alcalase 가수분해물은 3.29±7.51, 185.16±11.19, 376.42±26.35, 525.7±45.3, 57.19±.84 vit. eq μm로대조구와비교하여 37~55% 유의적으로높았다 (p<.5). rotamex 가수분해물도 87.66±3.5, 172.49 ±11.5, 356.92±31.95, 524.67±49.87, 567.44±47.9 vit. eq μm로대조구와비교하여시료량에따라 36~44% 유의적으로높았다 (p<.5). eutrase 가수분해물은 82.41± 6.14, 1.7±13.36, 5.±26., 433.33±37.52, 49.36± 45.94 vit. eq μm로대조구와비교하여통계적으로유의한차이가없었다 (p>.5). BTS radical 소거활성은 Fig. 3에서보는바와같이청국장가수분해물의경우시료건조고형분 1, 2, 3, 4 및 5 mg에서대조구가 44.±4.6, 2.5±., 133.38±9.7, 169.27±13.19, 185.33±15.56 vit. eq μm인반면 alcalase 가수분해물은 91.59±6.63, 169.49±13.94, 231.66±24.94, 279.66±24.11, 294.33±22. vit. eq μm로대조구와비교하여 59~6% 유의적으로증가하였다 (p<.5). rotamex 가수분해물도 77.44±.75, 165.8±16.86, 2.72±18.15, 268.69±25.9, 289.52±18.75 vit. eq μm로대조구와비교하여 56~74% 유의적으로증가하였으나 (p<.5), neutrase 가수분해물은 53.±5.56, 119.±12.77, 157.±.33, 191.1±13.18, 211.15±14.2 vit. eq μm로대조구와비교하여통계적으로유의한차이가없었다 (p>.5). 대두분말효소분해물의 BTS radical 소거활성은대조구가 24.29±2.6, 49.11±2.64, 65.±5.4, 85.12±6.14, 96.96±7.78 vit. eq μm이며 alcalase 가수분해물은 38.66 ±4.16, 84.16±6.41, 129.54±9.95, 135.38±.7, 146.44± 12.17 vit. eq μm로대조구와비교하여 51~99% 유의적으로높았다 (p<.5). rotamex 가수분해물도.42±3.3, 77.41±6.7, 1.74±7.69, 128.±.33, 1.43±.82 vit. eq μm로대조구와비교하여 44~66% 유의적으로높았으나 (p<.5), neutrase 가수분해물은 27.16±3.7, 58.87±5.22, 77.53±4.84, 98.8±.32, 114.53±12.36 vit. eq μm로대조구와비교하여통계적으로유의한차이가없었다 (p>.5). Hydroxyl radical 소거활성은 Fig. 4에서보는바와같이청국장가수분해물의경우시료건조고형분.4,.8, 1.2, 1.6 및 2 mg에서대조구는 9.53±1.27, 19.38±1.59, 28.84±2.31, 34.9±2.63, 37.±2.44 vit. eq μm이며 alcalase 가수분해물은 18.52±1.44, 36.37±3.,.2±4.97, 58.17±4.95, 62.7±6.4 vit. eq μm로대조구와비교하여 67~94% 유의적으로증가하였다 (p<.5). rotamex 가수분해물도 16.71 ±1.34,.84±2.51, 44.75±3.28, 53.35±4.21, 57.6±4.57 vit. eq μm로대조구와비교하여 52~75% 유의적으로높았으나 (p<.5), neutrase 가수분해물은 11.69±1.4, 23.19 ±1.92, 32.57±2.55,.41±4.6, 42.75±3.59 vit. eq μm로대조구와비교하여유의적차이가없었다 (p>.5). 대두분말가수분해물의 hydroxyl radical 소거활성은대조구가 5.11±.71, 11.19±2.71, 14.63±3.26, 17.53±4.6,
단백질분해효소처리가청국장의항산화활성에미치는영향 331 3 hungkukjang 3 Soybean BTS radical scavenging activity 2 1 BTS radical scavenging activity 2 1 1 2 3 4 5 6 1 2 3 4 5 6 Fig. 3. BTS radical scavenging activity of hungkukjang and soybean hydrolysates prepared with alcalase (), protamex () and neutrase (). : non-enzymatically hydrolyzed sample. The values are means±sd, which obtained from alcalase and protamex treatments are significantly different (p<.5) from the control () by Tukey's test. 7 hungkukjang 7 Soybean Hydroxyl radical scavenging activity Hydroxyl radical scavenging activity..5 1. 1.5 2...5 1. 1.5 2. Fig. 4. Hydroxyl radical scavenging activity of hungkukjang and soybean hydrolysates prepared with alcalase (), protamex () and neutrase (). : non-enzymatically hydrolyzed sample. The values are means±sd, which obtained from alcalase and protamex treatments are significantly different (p<.5) from the control () by Tukey's test..48±4.59 vit. eq μm, alcalase 가수분해물은 8.53±1.36, 19.42±1.51, 26.71±1.8, 31.7±2.65, 34.49±2.57 vit. eq μm로대조구와비교하여 67~83% 유의적으로높았다 (p<.5). rotamex 가수분해물도 8.42±.94, 16.85±1.67, 23.77 ±1., 28.84±1.61, 32.39±2.23 vit. eq μm로대조구와비교하여 51~65% 유의적으로높았으나 (p<.5), neutrase 가수분해물은 6.±.84, 12.59±1.96, 17.42±1.88, 21.8 ±1.99, 23.74±2.28 vit. eq μm로대조구와비교하여유의한차이가없었다 (p>.5). 이상의결과는펩타이드생성이상대적으로높은 alcalase 와 protamex 처리시료가 DH, BTS 및 hydroxyl radical 소거활성이대조구와비교하여유의적으로증가하는반면 펩타이드생성이적은 neutrase 처리시료는유의적효과가없으며항산화활성의증가정도 (%) 는대두분말과청국장두시료에서유사함을보여주고있다. 그러나청국장이대두분말에비해항산화활성이높은것은 (p<.5) 대조구비교를통해볼수있듯이청국장자체의상대적으로높은항산화활성에서기인한것으로보인다. 대두발효식품은발효하지않은콩과비교하여항산화활성이증가하는데 (32,33) 이는총폴리페놀함량증가 (33,34) 및 daidzein과 genestein의지용성 aglycone 분리등에의한것으로보고되었으며 (35) 발효중미생물효소에의한펩타이드함량증가도항산화활성의증가에기여한것으로사료된다.
332 박민경 본연구의결과는 alcalase와 protamex가대두분말과비교하여청국장에특이적이지는않으나펩타이드생성을높여청국장의항산화활성을더욱증대시킬수있음을보여주고있으며이는청국장을이용한새로운기능성가공식품의개발등에활용될수있을것으로기대된다. 요 lcalase, protamex 및 neutrase 효소처리가청국장의펩타이드및항산화활성증가에미치는영향을동일한조건으로효소처리한대두분말과비교하였다. 효소, 및 mu를 o 에서 1분간처리한결과청국장과대두분말모두에서 alcalase 및 protamex에의한펩타이드생성이 neutrase보다높게나타났다. 항산화활성은청국장의경우 alcalase 처리에의해 DH, BTS 및 hydroxyl radical 소거활성이효소처리를하지않은대조구와비교하여시료량에따라각각 37~57%, 59~6% 및 67~83%, protamex 처리에의해 35~%, 56~74% 및 52~75% 통계적으로유의하게증가하였다. 대두분말을효소처리한시료에서도유사한결과를보여 alcalase 처리에의해 DH, BTS 및 hydroxyl radical 소거활성이각각 37~55%, 51~99% 및 67 ~83%, protamex 처리에의해 36~44%, 44~66% 및 51~ 65% 증가하였다. 그러나두시료모두에서 neutrase 처리에의한항산화활성의유의적증가는없었다. 이상의결과는 alcalase와 protamex가청국장에특이적이지는않으나펩타이드생성및항산화활성증가에효율적임을보여주고있다. 문 1. Berthou J, Migliore-Samour D, Lifchitz, Delettre J, Floc'h F, Jolles. 1987. Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins. FEBS Lett 218: 55-58. 2. 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