Korean J Food Preserv, 29(2), (2022) pissn: , eissn: Korean Journal of Food Pre

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1 Korean J Food Preserv, 29(2), (2022) pissn: , eissn: Korean Journal of Food Preservation 한국식품저장유통학회지 Research Article Preparation of β-aminoisobutyric acid and branched chain amino acid-enhanced hydrolysates from chicken breast: Effect of protease types and hydrolysis conditions 베타 - 아미노이소부티르산과분지쇄아미노산함량이증강된닭가슴살가수분해물의제조 : 프로테아제의종류및가수분해조건의영향 Eun-Bee Kim 1, Dae-Woon Kim 1, Han-Suk Choi 2, Young-Hoi Kim 1, Myung-Kon Kim 1 * 김은비 1 김대운 1 최한석 2 김영회 1 김명곤 1 * 1 Department of Food Science and Technology, Jeonbuk National University, Jeonju 54896, Korea 2 Department of Agriculture and Fisheries Processing, Korea National College of Agriculture and Fisheries, Jeonju 54874, Korea 1 전북대학교식품공학과, 2 국립농수산대학농수산가공학과 OPEN ACCESS Citation: Kim EB, Kim DW, Choi HS, Kim YH, Kim MK. Preparation of β-aminoisobutyric acid and branched chain amino acidenhanced hydrolysates from chicken breast: Effect of protease types and hydrolysis conditions. Korean J Food Preserv, 29(2), (2022) Received: October 20, 2021 Revised: November 23, 2021 Accepted: November 24, 2021 These authors contributed equally to this study. *Corresponding author Myung-Kon Kim Tel: kmyuko@jbnu.ac.kr Copyright 2022 The Korean Society of Food Preservation. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( es/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Beta-aminoisobutyric acid (BAIBA) and branched-chain amino acids (BCAA) have recently received considerable attention because of their various health benefits. In this study, the hydrolysis characteristics of chicken breast for 11 commercially proteolytic enzymes were studied to evaluate the effect of protease type, reaction ph (5-8), leaction time (2-24 h) and enzyme concentration (0.25-8%), and the effect of two combined enzymes for the preparation of BAIBAand BCAA- enriched chicken breast hydrolysates. The content of BAIBA was higher in hydrolysate by prozyme 2000P (38.72±3.01 mg/g, dry weight base), foodpro alkaline protease (30.24±2.54 mg/g) and alcalase (26.24±1.01 mg/g) while the BCAA content was higher in prozyme 2000P (108.96±2.63 mg/g), flavourzyme (81.12±4.78 mg/g) and sumizyme LP (62.88±3.83 mg/g). As the effects of reaction ph, time, and amount of added enzymes, the optimum condition for the preparation of BAIBA- and BCAA-enriched hydrolysates were as follows; reaction ph 6-7, reaction time 8-16 h and the amount of enzyme 4% (w/w) based on the dried chicken breast. However, no significant increase of BAIBA and BCAA content was observed when hydrolyzed with two combined enzymes. These results suggest that commercial proteolytic enzymes can be applied to prepare BAIBA- and BCAA-enriched chicken breast hydrolysates. Keywords chicken breast, proteolytic enzymes, BAIBA, BCAA, hydrolysis conditions 1. 서론 아미노산류는단백질을합성하기위한구성요소로서중요한역할을하지만, 유리형의아미 노산자체도종류에따라각기다른다양한생리적기능성을지니고있다는것이밝혀져있다 (Pasini 등, 2018; Wu, 2010; Wu, 2013). 최근까지자연계에서는 300 종이상의아미노산류 276

2 Korean J Food Preserv, 29(2) (2022) 가발견되어있으나이들중오직 20종의아미노산이단백질구성에참여하며, 이들은모두 α-형의아미노산류이다 (Wu, 2009). 자연에존재하는아미노산류중에는 citrulline, ornithine 과같은비단백질성아미노산류나 taurine 및 β- alanine과같은분자구조상 α-형이아닌아미노산일지라도인간의성장과세포의재생및건강유지에중요한역할을한다 (Curis 등, 2007; Hu 등 ; 2008). 근래에는의약분야뿐만아니라, 식품산업분야에서도기능성식품소재로서아미노산및관련제품들이시판되고있다 (Kim 등, 2015). 예로서 arginine과 citrulline은혈관을이완하는물질인산화질소합성의전구물질로작용하여이완에도움을주며, 고혈압등혈관질환의예방및치료를위한주요기능성물질로서사용되고있으며, 이외에도골밀도감소지연, 성기능개선, 체지방감소등다양한생리활성에관한연구결과들이보고되어있다 (Bahadoran 등, 2021). 또한, γ- aminobutyric acid(gaba) 는뇌에서신경전달물질인아세틸콜린의분비를증가시켜뇌기능을촉진하는물질로서, 혈압강하, 불안, 불면, 우울증, 통증완화와지방대사및간기능을부분적으로개선시키는것으로알려져있다 (Diana 등, 2014; Ngo와 Vo, 2019). 비필수아미노산류에속하는 β-aminoisobutyric acid (BAIBA) 는포유동물에서염기성분인 thymine과필수아미노산인 valine이체내에서대사되는과정에서생성되는것으로알려진물질로서 (Begriche 등, 2010; Tanianskii 등, 2019) 백색지방조직에서갈색지방세포유전자의발현을증가시키고, 간에서지방산 β-산화를증가시킴으로써비만및관련대사장애개선기능이있는것으로밝혀져있다 (Barlow 등, 2020; Roberts 등, 2014). 이외에도 BAIBA 는근섬유에의해생성및방출되어운동량을증가시키고, 심혈관질환예방효과가있는것으로밝혀져있고 (Roberts 등, 2014; Stautemas 등, 2019), 생쥐를이용한동물실험결과에서 BAIBA는포도당대사를개선함으로써비만과만성대사질환예방에도움을줄뿐만아니라, 노화로인한뼈손실과근육기능손실을방지하는효과도있는것으로보고되어있다 (Begriche 등, 2010; Kitase 등, 2018; Shi 등, 2016). 또한, 필수아미노산이면서분지쇄아미노산 (branched chain amino acid, BCAA) 인류신 (leucine), 이소류신 (isoleucine) 및발린 (valine) 은단백질구성아미노산류의 30-40% 를차지하며, 다른아미노산류는주로간에서대사되지만 BCAA의대부분은체순환계로들어가골격근, 지방조직및뇌에도달하여대사된다 (Chung 등, 2010; Zhang 등, 2018). 따라서 BCAA의보충은운동중에피로를감소시킬수있으며, BCAA가풍부한식단은근육단백질대사, 신체유지및유산소운동을개선하는효과가있음이밝혀져있어현재는건강기능성소재, 의약품, 동물사료첨가제및화장품원료등으로널리사용되고있다 (Liu 등, 2021). 한편, 프로테아제에의한단백질가수분해물들은오래전부터의료용, 식품용, 동물사료용및기타산업용으로사용되어왔다 (Neklyudov 등, 2000). 단백질가수분해물은주로유리된아미노산류와저분자의펩타이드류가주성분으로서이러한가수분해생성물들은단백질에비해장내흡수가용이하고각종생리활성을지닌저분자량의펩타이드들이함유되어있어영양공급소재또는치료용소재로이용되고있다 (Korhonen 과 Pihlanto, 2006; Sánchez 와 Vázquez, 2017). 특히저분자의펩타이드류는체내에서쉽게흡수되기때문에스포츠영양에있어최적의질소공급원이될뿐아니라, 단백질보조제로서이용성이높으며, 연구결과에따르면 di- 및 tripeptide와분자량이작은일부의펩타이드들은유리아미노산보다더빨리흡수되는것으로알려져있다 (Manninen, 2009). 그동안영양기능증진및생리활성강화를목적으로단백질함량이높은동물성식품 (Ahhmed 와 Muguruma, 2010; Rezaharsamto 와 Subroto, 2019; Ryan 등, 2011; Vercruysse 등, 2005), 식물성식품 (Daliri 등, 2019; Kong 등, 2008; Leo 등, 2009; Sun, 2011; Wang 등, 2019; Wouters 등, 2016) 및해산물 (In 등, 2016; Lee 등, 2010; Nikoo 등, 2016; Vijaykrishnaraj 와 Prabhasankar, 2015) 을대상으로효소에의한단백질가수분해연구가많이수행되었다. 특히닭가슴살은지방과탄수화물함량은매우낮고단백질함량은높아웰빙다이어트식품으로각광을받고있다. 닭가슴살은저렴한가격에구할수있는고단백저지방식재료로꾸준하게그수요가유지되고있기는하나, 육질이부드럽지못하고퍽퍽한식감때문에소비자들에게각광을받지못하고있다. 따라서닭가슴살의소비를증대시키기위해서는새로운가공기술적용을통한고부가가치제품의개발이요구되고있으며, 닭가슴살에함유된단백질가수분 277

3 Preparation of β-aminoisobutyric acid and branched chain amino acid-enhanced hydrolysates from chicken breast 해에관한연구도다수수행되어있다 (Kurozawa 등, 2009; Lindberg 등, 2021; Nchienzia 등, 2010; Saiga 등, 2003; Sangsawad 등, 2017; Sun 등, 2012; Terashima 등, 2010; Wu 등, 2020). 닭가슴살이나닭의가공과정에서산출되는부산물들의프로테아제에의한가수분해연구는주로단백질을정상적으로소화하기곤란한고연령자나병약자들을위한영양공급원또는생리활성을지닌펩타이드류의함량을증대시킬목적으로되었으나프로테아제에의한닭가슴살의가수분해조건이 BAIBA 및 BCAA의생성에미치는영향에대해서는거의알려진바없다. 따라서본연구에서는상업적으로시판되는 11종의프로테아제를사용하여닭가슴살을가수분해시킨다음생성된아미노산의조성을분석하고, 가수분해조건 ( 프로테아제종류, 반응 ph, 반응시간, 효소첨가량 ) 이 BAIBA 및 BCAA 생성에미치는영향을조사하고자하였다. 2. 재료및방법 2.1. 재료닭가슴살은 ( 주 ) 하림 (Iksan, Korea) 에서가공된것을제 공받아 ( 주 ) 코아 (Wanju, Jeonbuk, Korea) 에서열풍건조방법으로건조후분말형태로분쇄하여사용하였다. 분쇄한시료는밀폐된용기에넣은다음 -20 에서보관하면서이후의실험에사용하였다 효소및시약단백질가수분해효소인 alcalase 2.4 L FG(from Bacillus licheniformis), nutrase 0.8 L(from B. amyloliiensquefaciens), flavourzyme 1,000 L(from Aspergillus. oryzae), protamex (Bacillus sp.), papain T100 MG, bromelain DR 1200 은 대종상사 (Seoul, Korea) 에서입수하였고, prozyme 2000P와 foodpro alkaline proteases는 비젼바이오켐 (Seongnam, Korea) 으로부터입수하여사용하였다. Sumizyme LP(from Asp. oryzae) 는 옥전바이오텍 (Seoul, Korea), ficin(from fig tree latex), Asp. saitoi protease 및 thermolysin (from Geobacillus stearothermophilus) 은 Sigma-Aldrich(St. Louis, MO, USA) 에서구입하여사용하였으며, 본실험에서사용한 protease의특성 ( 가수분해 type, 반응최적 ph 및최적온도 ) 은 Table 1과같다. Casein(sodium salt from bovine milk), Folin- Table 1. List of proteases used in this study Enzyme Type Activity (U/mg) Optimal condition 1 Alcalase 2.4 L FG Endo 0.54±0.05 ph , Flavourzyme 1,000 L Exo 0.76±0.09 ph 6, Neutrase 0.8 L Endo 2.10±0.06 ph 8.0, 50 4 Protamex Endo 3.02±0.31 ph , Papain T100 MG Endo 0.24±0.01 ph 5-8, 65 6 Bromelain BR 1200 Endo+exo 0.30±0.10 ph 7.0, 50 7 Prozyme 2000P Exo 3.60±0.10 ph 5-5.5, Foodpro alkaline protease Endo 1.09±0.06 ph 6.5-9, Protease (Asp. saitoi) ±0.04 ph , Ficin (fig tree latex) Endo 0.20±0.05 ph 6.5, Sumizyme LP Endo 0.69±0.03 ph 6.0, 50 Protease activity was assayed using casein sodium salt as the substrate, according to the He et al. (2004) and Joung et al. (2018) with some modfication. The reaction mixture (2 ml) containing each 1 ml of diluted enzyme, 2% casein and 0.9 ml of 50 mm Tris-HCl (ph 7.0) was incubated at 40 for 20 min. The reaction was terminated by adding 2 ml of 0.4 M trichloroacetic acid. After centrifugation, 1 ml of supernatant was mixed with 5 ml of 0.4 M sodium carbonate and 1 ml of 1 N Folin-Ciocalteu reagent, and incubated at 40 for 20 min. The absorption value of the mixture was measured at 660 nm. One unit of protease activity was defined as the amount of the enzyme required to release 1 μm of tyrosine from casein per minute under the above conditions

4 Korean J Food Preserv, 29(2) (2022) Ciocalteu 시약, L-BAIBA, L-leucine, tyrosine 및 sodium dodecyl sulfate(sds) 는 Sigma-Aldrich Co. Ltd(St. Louis, MO, USA) 에서구입하여사용하였고, TNBS (2,4,6-trinitrobenzenesulfonic acid, 5% w/v methanol solution) 는 Thermo Scientific Co.(Rockford, IL, USA) 에서구입하여사용하였다. Amino acid standard mixture(1.0 μm/ml) 는 Sykam GmbH(Gewerbering, Eresing, Germany) 에서구입하였고, 기타일반시약은 analytical grade를대한과학 (Wonju, Korea) 에서구입하여사용하였다 포로테아제활성측정프로테아제활성은 He 등 (2004) 과 Joung 등 (2018) 의방법을약간변형하여측정하였다. 즉, 증류수를사용하여적정한농도로희석한효소액 1 ml와 50 mm Tris-HCl buffer(ph 7.0) 0.9 ml 및 2% casein 용액 1 ml를혼합하고 50 에서 10분간반응시킨다음 0.4 M trichloroacetic acid 용액 2 ml를가하여반응을종료시켰다. 이어서반응액은원심분리 (12,000 g, 20분 ) 하여얻어진상등액 1 ml를취하여 0.4 M sodium carbonate 용액 5 ml와 1 N Folin-Ciocalteu 시약 1.0 ml를가한다음 40 에서 20분간반응시킨후 UV-vis. spectrophotometer (Shimadzu UV-1601, Kyoto, Japan) 를사용하여 660 nm에서흡광도를측정하였다. 별도로 tyrosine( μg/ml) 을사용하여작성한검량선을사용하여프로테아제활성을계산하였다. 효소활성의 1 unit(u) 는 casein에서분당 1 μm의 tyrosine을유리시키는데필요한효소의양으로하였다 닭가슴살의가수분해에대한 ph의영향측정시판의프로테아제에의한건조닭가슴살의가수분해에대한반응액 ph의영향은 Fig. 1과같은순서로수행하였으며, 반응액의 ph는 0.1 M sodium acetate 완충용액 (ph 5) 과 0.1 M sodium phosphate 완충용액 (ph 6-8) 을사용하여조절하였다. 즉, 건조닭가슴살 1 g에 ph를달리한완충용액 8 ml씩을가하고예비실험결과에서 BAIBA 및 BCAA 생성량이높았던효소인 alcalase, prozyme 2000P 또는 foodpro alkaline protease를건 Fig. 1. Flow diagram for the preparation of protein hydrolysates from chicken breast by commercial proteases. 조닭가슴살에대하여각각 4% 씩첨가한다음 50 의수욕 조에서진탕하면서 12 시간동안반응시켰다. 각반응액은 85 에서 20 분간가열하여효소를불활성화시킨후 3,400 g (gravity) 로 10 분동안원심분리얻어진상등액의일부 를단백질가수분해도측정용시료로사용하였고, 나머지 시료는동결건조하였다 닭가슴살의가수분해에대한반응시간측정 건조닭가슴살 1 g 에 0.1 M sodium phosphate 완충 용액 (ph 7) 8 ml 를가하여혼합한다음건조닭가슴살에 대하여 4% 가되도록 alcalase 를첨가하고, 50 의수욕조 에서 24 시간진탕반응을시키면서주기적으로시료를채취 하였다. 각각의반응액은 ph 의 85 에서 20 분간가열하여 효소를불활성화시킨후 3,400 g (gravity) 로 10 분동안 원심분리하여얻어진상등액은동결건조하였다

5 Preparation of β-aminoisobutyric acid and branched chain amino acid-enhanced hydrolysates from chicken breast 2.6. 닭가슴살의가수분해에대한효소첨가량측정건조닭가슴살 1 g에 0.1 M sodium phosphate 완충용액 (ph 7) 8 ml를가하여혼합한다음 alcalase, prozyme 2000P 또는 foodpro alkaline protease를건조닭가슴살에대하여각각 % 씩첨가하고, 50 의수욕조에서 12시간동안반응시켰다. 각각의반응액은 ph 의 85 에서 20분간가열하여효소를불활성화시킨후 3,400 g (gravity) 로 10분동안원심분리하여얻어진상등액은동결건조하여실험에사용하였다 단백질가수분해도측정시료의가수분해는건조닭가슴살 1 g에 0.1 M sodium phosphate 완충용액 (ph 7) 8 ml를가하여혼합한다음, 각각의효소를건조닭가슴살에대하여 4% 씩첨가하고 50 의수욕조에서 50 에서 12시간진탕반응시켰다. 또한 2종의프로테아제를혼합처리를하였을때단백질가수분해도에미치는영향을알아보기위하여, 건조닭가슴살에대하여 alcalase(endo-type) 와 foodpro alkaline protease (endo-type) 를각각 2% 또는 alcalase(endo-type) 와 prozyme 2000P(exo-type) 를각각 2% 씩을첨가한다음 50 의수욕조에서 12시간진탕반응시켰다. 각각의반응액은 ph의 85 에서 20분간가열하여효소를불활성화시킨후, 원심분리 (4,500 rpm, 10분 ) 하여얻어진상등액을단백질가수분해도측정용시료로사용하였다. TNBS법에의한단백질가수분해도 (degree of hydrolysis, DH %) 는 Adler-Nissen(1979) 의방법에따라측정하였다. 즉, 효소가수분해후얻어진원심분리상등액 1 ml를 1% SDS를함유하는증류수로 200배희석한용액 0.25 ml에 0.2 M sodium phosphate buffer(ph 8.2) 2.0 ml 및 0.1% TNBS 용액 2.0 ml를가하고 50 에서 1시간동안반응시켰다. 이어서반응액에 0.1 M HCl 용액 4.0 ml를가하고상온에서 30분간유지한다음 UV-Vis spectrophotometer 를사용하여 340 nm에서흡광도를측정하였으며, 1% SDS를함유하는증류수로를대조구로하였다. TNBS법에의한단백질가수분해도는 SDS를 1% 의농도로첨가한증류수에용해시킨 L-leucine을사용하여작성한검량선을이용하여계산하였다 (Adler-Nissen, 1979; Spellman 등, 2003). Formol법에의한가수분해도측정을위하여아미노태질소 (α-amino nitrogen) 함량은닭가슴살을효소가수분해한다음 3,400 g (gravity) 로 10분동안원심분리하여얻어진상등액을증류수로 200배희석한다음, 그중 2 ml 를취하여 Sorensen법 (AOAC, 1995) 에따라 35% formaldehyde 용액으로적정하였고, 건조닭가슴살중의총질소함량은 animal food analyzer(kjeltec 8400, FOSS, Hillerod, Denmark) 로정량한다음아래와같은식에따라가수분해를계산하였다. α-amino nitrogen (AN) DH (%) = 100 Total nitrogen (TN) 2.8. 아미노산분석닭가슴살중의총아미노산은분말시료 0.5 g에 6 N HCl 용액 10 ml를가하고질소가스로치환한다음 110 에서 24시간분해하였다. 분해액은감압농축한다음농축액에증류수를 10 ml씩을가하여다시감압농축하는과정을 2 회반복하여 HCl을완전히제거하였다. 농축액은 sample dilution buffer(ph 2.2) 로희석한다음 membrane filter(0.45 μm) 로여과한후아미노산분석기로분석하였다. 프로테아제에의한가수분해물을원심분리하여얻어진상등액의동결건조분말은시료 0.2 g을 sample dilution buffer 25 ml에용해시킨다음분석에사용하였으며, 가수분해물중의 BAIBA 및 BCAA 분석결과는 mg/g(dry weight base) 으로표시하였다. 또한프로테아제에의한가수분해물에서분석된총유리아미노산 (total amount of free amino acid, TFAA) 중에서 BAIBA 또는 BCAA가차지하는비율을백분율 (%) 로표시하였다. 아미노산분석기는 Sykam GmbH 사 (Gewerbering, Eresing, Germany) 의 S7130 amino acid reagent organizer, S5200 sample injector 와 S2100 solvent delivery system 을사용하였으며, column은 cation separation column LCA K06/NA(4.6 mm 250 mm) 를사용하였다. 이동상의유속은 0.45 ml/min, ninhydrin은 0.4 ml/min으로하였으며, 각성분의함량은 Sykam사에제공하는유리아 280

6 Korean J Food Preserv, 29(2) (2022) 미노산 (45 개성분 ) 표준용액을분석한다음아미노산분석 기에내장된프로그램을이용하여정량하였다 통계분석각실험은 3회반복하여얻은결과를평균 ± 표준편차로나타내었다. 또한, 실험값에대한통계분석에는 Statistical Package for the Social Sciences SPSS, IBM 20, NYC, USA) 프로그램을이용하였고, one-way analysis of variance(anova) 로분석한후 Duncan s multiple range test 방법을이용하여평균값간에유의수준 p<0.05 에서유의성을검정하였다. 3. 결과및고찰 3.1. 단백질가수분해도효소에의한단백질의가수분해도측정방법으로는 phstat법, TNBS법, formol 적정법, o-phthaldialdehyde (OPA) 법, trichloroacetic acid-soluble nitrogen(sn- TCA) 법, osmometric법등다양한방법들이사용된다. 가수분해도는측정에사용되는반응시약이단백질의가수분해에의해생성된유리아미노산및펩타이드에반응하는기전이나감도가달라단일방법으로전반적인가수분해도를나타낼수는없는것으로알려져있다 (Rutherfurd, 2010; Spellman 등, 2003). 따라서단백질가수분해도를측정에있어서는 2-3가지방법을함께적용하여측정하는것이바람직하며, 그중에서도 ph-stat법, TNBS법, OPA법, formol법이가장널리이용되는방법들이다 (Rutherfurd, 2010; Spellman 등, 2003). 본실험에서는 11종의프로테아제를건조닭가슴살에대하여각각 4% 씩첨가후 50 에서 12시간반응시킨다음 TNBS법과 formol법으로가수분해도를측정한결과는 Fig. 2와같다. TNBS법에의한가수분해도측정결과, prozyme 2000P(58.90±3.42%), flavourzyme(50.08±4.28%), alcalase(45.48±3.60%) 및 sumizyme LP(44.55±0.97%) 로가수분해하였을때가수분해도가높았으며, formol법에의한측정결과에서도역시 prozyme 2000P(24.62±0.20%), flavourzyme(22.04± 0.57%) 및 alcalase(16.50±0.14%) 에의한가수분해물에서높았다. 이와같이사용한효소에따라닭가슴살에함유 Fig. 2. Comparison of degree of hydrolysis of chicken breast by proteases. A, TNBS method; B, formol method; 1, alcalase; 2, nutrase; 3, flavourzyme; 4, protamex; 5, papain; 6, bromelain; 7, prozyme 2000P; 8, foodpro alkaline protease; 9, Asp. saitoi protease; 10, ficin; 11, sumizyme LP; 12, mixture alcalase (2%)+foodpro alkaline protease (2%); 13, mixture of alcalase (2%)+prozyme 2000P (2%). Values are means±standard deviation (SD) (n=3). Different superscripts in the same row indicate significant differences (p<0.05). 된단백질에대한가수분해도가다른이유는, 효소별단위무 게당활성 (specific acivity), 효소별기질특이성 (substrate specificity), 펩타이드결합에의해연결된아미노산사슬 에대한작용방식의차이에기인한다고할수있다 (Tavano, 2013). 측정방법별로비교하였을때에는 TNBS 법에의한가수 분해도측정결과가 formol 법에의한측정결과보다전반적 으로높게나타났다. 프로테아제에의한단백질의가수분해 도는가수분해에사용된효소의종류, 반응온도, 반응액의 ph, 반응시간, 사용된기질, 기질과효소의비율및측정방 법에따라달라진다 (Beaubier 등, 2019; Nielsen 등, 2001; Spellman 등, 2003). 예로서 Rutherfurd(2010) 는 hemoglobin 을 esperase 와 flavourzyme 을연속적으로 처리한후 ph-stat 법, SN-TCA 법, formol 법으로가수분 해도를측정하여비교한결과, formol 법에의한측정값이 가장낮았고, SN-TCA 법에의한측정값은 formol 법에의 281

7 Preparation of β-aminoisobutyric acid and branched chain amino acid-enhanced hydrolysates from chicken breast 한측정값보다 2배이상높게나타났다고보고하였다. 또한, Kurozawa 등 (2009) 은닭가슴살을 ph 8.0의완충용액에현탁시킨다음, alcalase를 4.2% 첨가하고반응시킨다음 ph-stat법으로가수분해도를측정하였을때최적조건에서의가수분해도는약 30% 였다고보고하였다. Fallah- Delavar와 Farmani(2018) 는 5종의프로테아제로닭껍질을 ph 7-8.5, 온도 50 에서 2시간가수분해한다음가수분해도를비교하였을때, alcalase 처리구는 49.19%, protamex 31.65%, nutrase 25.05%, 그리고 flavourzyme 처리구는 8.92% 였다고보고하였고, Jung(2019) 은중-초고압조건에서닭가슴살을 alcalase와 flavourzyme으로 12시간동안가수분해하였을때, 가수분해도는 alcalase 처리구에서는효소첨가량에따라 50-52%, flavourzyme 처리구에서는 46-48% 범위이었다고보고하였다. 반면에 Nchienzia 등 (2010) 은닭고기를 alcalase, flavourzyme 및 protamex로가수분해 (ph 7.0, 50, 6시간, 효소첨가량 1-3%) 하였을때가수분해도는 10-11% 수준이었다고보고하였다. 또한, 효소의혼합처리효과를알아보기위하여, alcalase (endo-type) 와 foodpro alkaline protease(endo-type) 를각각 2% 또는 alcalase와 prozyme 2000P(exo-type) 를각각 2% 씩혼합처리하여얻어진시료의가수분해도측정결과, alcalase와 foodpro alkaline protease의혼합처리구는 2종의효소를단독으로처리하였을때보다가수분해도가뚜렷하게증가하였으나, alcalase와 prozyme 2000P의혼합처리구는 prozyme 2000P의단독처리구에비해가수분해도가거의증가하지않았다 가수분해물의아미노산조성본실험에서프로테아제에의한닭가슴살가수분해물에서는 22종의아미노산이검출되었다. 이중에서가수분해도가낮았던 Asp. saitoi 프로테아제에의한가수분해물을제외하고, 나머지프로테아제에의한가수분해물중의아미노산조성을분석한결과는 Table 2와같다. 효소가수분해에의해서생성된총유리아미노산함량은사용한프로테아제에따라많은차이를보였는데 prozyme 2000P (492.71±35.03 mg/g), flavourzyme(379.84±27.44 mg/g), sumizyme LP(307.36±23.81 mg/g) 에의한가수분해물에서높았고, nutrase(121.07±11.42 mg/g), protamex(164.32±19.24 mg/g), papain(172.48±17.41 mg/g) 및 ficin(187.68±19.57 mg/g) 에의한가수분해물에서낮았다. 개별아미노산의함량도처리한프로테아제의종류에따라차이가있었는데, 모든처리구에서대체적으로 glutamic acid, leucine 및 lysine의함량이높은편이었다. 개별아미노산의함량을보면 alcalase 처리구에서는 BAIBA(26.24±1.01 mg/g), glutamic acid(25.76±2.27 mg/g), leucine(23.84±1.43 mg/g), β-alanine(23.04± 0.89 mg/g), flavourzyme 처리구에서는 glutamic acid (42.88±2.34 mg/g), leucine(40.32±2.32 mg/g), lysine (35.84±0.89 mg/g) 및 arginine(30.08±1.14 mg/g) 의함량이높았다. 또한, prozyme 2000P 효소처리구에서는 leucine(52.80±1.58 mg/g), glutamic acid(50.72±3.22 mg/g), lysine(42.88±1.23 mg/g), arginine(39.5±3.14 mg/g) 및 BAIBA(38.72±3.01 mg/g) 의함량이높았고, 식물성프로테아제인 bromelain, papain 및 ficin에의한가수분해물은, 미생물에서유래하는프로테아제에비해닭가슴살단백질의가수분해도도낮고, 유리된총유리아미노산의함량도낮은편이었다. 이와같이사용한효소에따라닭가슴살가수분해물의아미노산조성이다른것은단백질에대한효소별기질특이성 (substrate specificity), 펩타이드결합에의해서연결된사슬에대한각효소의작용방식의차이에기인한다고할수있다 (Tavano, 2013). Kurozawa 등 (2009) 은 alcalase에의한닭가슴살가수분해물에서, 양적으로많이검출된아미노산은 glutamic acid(14.80 g/100 g), tryptophan(9.01 g/100 g), lysine(8.79 g/100 g), aspartic acid(8.71 g/100 g) 및 leucine(7.91 g/100 g) 이라고보고한바있다. 또한, 육계와산란계의닭가슴살을 alcalase와 flavourzyme을혼합처리한가수분해물에서는 aspartic acid의함량이가장높았고, 그다음으로 leucine, glutamic acid, BAIBA 및 arginine의함량이높은것으로보고되었다 (Jo 등, 2021). 프로테아제에의한닭가슴살의가수분해물의아미노산조성에서본실험과문헌보고된결과와다소차이를보이는것은, 가수분해에사용된효소의종류와첨가량, 반응액의 282

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9 Preparation of β-aminoisobutyric acid and branched chain amino acid-enhanced hydrolysates from chicken breast ph, 반응온도와시간등의차이에기인할것으로판단된다 가수분해물중 BAIBA 및 BCAA 함량비교 BAIBA 는인체내에서섭취된단백질이대사되는과정에서 thymine 으로부터 D-BAIBA, valine 으로부터는 L-BAIBA 가 생성되는것으로보고되어있다 (Begriche 등, 2010; Roberts 등, 2014; Tanianskii 등, 2019). BAIBA 는근육량을증가 시키고, 글리코겐절약작용, 단백질합성과분해속도조절 작용, 운동에의한피로감소, 유산소운동을개선하는효과 가있는것으로밝혀져있다 (Begriche 등, 2010; Liu 등, 2021; Roberts 등, 2014). 닭가슴살을가수분해도가낮았 던 Asp. saitoi 프로테아제를제외한 10 종의프로테아제로 가수분해한다음, 원심분리하여얻어진상등액의동결건조 분말에서 BAIBA 의함량을분석비교한결과는 Fig. 3(A) 와 같다. Prozyme 2000P 에의한닭가슴살의가수분해물에 서 BAIBA 의함량은 38.72±3.01 mg/g 으로본실험에서 사용한 10 종의프로테아제중에서가장높았고, 그다음은 foodpro alkaline protease(30.24±2.54 mg/g), alcalase Fig. 3. Comparison of BAIBA (A) and BCAA (B) content in chicken breast hydrolysates by proteases. 1, alcalase; 2, nutrase; 3, flavourzyme; 4, protamex; 5, papain; 6, bromelain; 7, prozyme 2000P; 8, foodpro alkaline protease; 9, ficin; 10, sumizyme LP; TFAA, total amount of free amino acid in hydrolysate. Values are means±sd (n=3). Different superscripts in the same row indicate significant differences (p<0.05). (26.24±1.01 mg/g) 및 sumizyme AP(25.76±1.76 mg/g) 에의한가수분해물에서많이검출되어가수분해에사용된프로테아제의종류에따라 BAIBA 생성량에많은차이가있음을알수있다. 또한, 닭가슴살가수분해물에서검출된총유리아미노산중에서 BAIBA 가차지하는비율을백분율 (%) 로환산하였을때에는 foodpro alkaline protease (14.96%) 와 alcalase(12.76%) 에의한가수분해물에서높았다. Jo 등 (2021) 은 alcalase와 flavourzyme에의한육계가슴살의가수분해물에서 BAIBA 함량은 0.98±0.9%, 산란계에서는 1.10±0.22% 로서 aspartic acid, glutamic acid 및 leucine 다음으로많이함유되어있었다고보고하였다. 한편, 본실험에서프로테아제에의한닭가슴살분수분해물에서 BCAA의함량을분석하여비교한결과는 Fig. 3(B) 와같다. BCAA의함량은 prozyme 2000P의가수분해물에서가장높았고, 다음으로 flavourzyme과 sumizyme LP에의한가수분해물에서높은편이었다. 각가수분해물에서검출된총유리아미노산중에서, BCAA가차지하는비율은 nutrase에의한가수분해물에서약 31% 로서가장높았고기타프로테아제에의한가수분해물에서는 20% 수준이었다. Jo(2021) 는육계와산란계의닭가슴살을 alcalase와 flavourzyme을혼합처리하여얻어진가수분해물에함유된총유리아미노산중에서 BCAA가차지하는비율은각각 15.3% 및 16.8% 로보고한바있으며, Sun 등 (2012) 은 papain에의한닭가슴살가수분해물중의 BCAA 함량은 54.2 mg/g이라고보고하였다. 또한, 옥수수단백질인글루텐을 alcalase와 flavourzyme으로혼합처리하여얻어진가수분해물에함유된총유리아미노산에서 BCAA가차지하는비율은약 25.1% 정도인것으로보고되어있다 (Chung 등, 2010). Kurozawa 등 (2008) 은닭가슴살을 alcalase에의해가수분해하여생성된아미노산을 phenylisothiocyanate(pitc) 로유도체화하여분석하는 Pico-Tag method로분석하였을때, BCAA 함량은 mg/g으로보고하여본실험에서분석결과보다높았는데, 이러한차이는분석방법의차이로인한것으로판단된다. 따라서시판되는프로테아제로닭가슴살을가수분해하여 BAIBA와 BCAA의함량이높은가수분해물을얻기 284

10 Korean J Food Preserv, 29(2) (2022) 위해서는 prozyme 2000P, foodpro alkaline protease, alcalase 또는 flavourzyme으로가수분해하는것이바람직할것으로판단된다 BAIBA 및 BCAA 생성에대한 ph의영향본실험에서특히 BAIBA 의생성량이높았던효소를중심으로대표적으로 3종의효소 (alcalase, prozyme 2000P, foodpro alkaline protease) 를선정하여반응액의 ph가 BAIBA의생성량에미치는영향을조사한결과는 Fig. 4와같다. Alcalase 에의한가수분해물의경우, ph 6-8 범위에서 BAIBA의생성량은 32.3± ±1.09 mg/g으로서특히 ph 6-7 사이에서높았다. 반면에 prozyme 2000P에의한가수분해물의경우반응액 ph 5 부근에서 BAIBA 생성량이가장높았고, ph가증가할수록생성량이 유의적으로감소하는경향을보였다. Foodpro alkaline protease의경우, BAIBA의생성량은 ph 6-7 사이에서높았으며, 이러한결과는각효소별단백질가수분해를위한최적 ph 범위와관련이있을것으로판단된다. 한편, 3 종의프로테아제에의한가수분해물에서 BCAA의함량을비교하였을때 alcalase와 foodpro alkaline protease의경우 ph 7.5 부근의용액에서가수분해하였을때가장많이생성되었으며, 이때가수분해물중의 BCAA의함량은 alcalase에의한가수분해물에서 64.8±0.74 mg/g, foodpro alkaline protease에의한가수분해물에서는 70.4±0.85 mg/g이었다. Prozyme 2000P에의한가수분해물에서 BCAA의함량은 ph 5-6 사이에서유의적으로높았고, 그중에서도특히 ph 6에서가수분해하였을때 119.0±0.92 mg/g으로서가장높았다 (Fig. 5). Fig. 4. Effect of ph on BAIBA content in chicken breast hydrolysates by three proteases. A, alcalase; B, prozyme 2000P; C, foodpro alkaline protease; TFAA, total amount of free amino acid in hydrolysate. The ph of the reaction solution was adjusted using 0.1 M sodium acetate buffer (ph 5) and sodium phosphate buffer (ph 6-8). Values are means±sd (n=3). Different superscripts in the same row indicate significant differences (p<0.05). Fig. 5. Effect of ph on BCAA content in chicken breast hydrolysates by three proteases. A, alcalase; B, prozyme 2000P; C, foodpro alkaline protease; TFAA, total amount of free amino acid in hydrolysate. The ph of the reaction solution was adjusted using 0.1 M sodium acetate buffer (ph 5) and sodium phosphate buffer (ph 6-8). Values are means±sd (n=3). Different superscripts in the same row indicate significant differences (p<0.05)

11 Preparation of β-aminoisobutyric acid and branched chain amino acid-enhanced hydrolysates from chicken breast 3.5. 반응시간의영향예비실험결과에서 alcalase, prozyme 2000P 및 foodpro alkaline protease를사용하여반응시간의영향을조사하였을때반응시간이연장됨에따라 BAIBA와 BCAA도그에비례하여증가하는경향을보였다. 따라서대표적으로 alcalase를사용하여 24시간동안반응시키면서 BAIBA 및 BCAA의생성량을분석한결과는 Fig. 6과같다. BAIBA와 BCAA의생성량은반응시작후 2시간이내에급격히증가한다음그이후 8시간까지는완만하지만지속적으로증가하였고, 8시간이후 16시간에도달해도별차이가없었다. 이러한결과를바탕으로판단할때프로테아제에의한닭가슴살에함유된단백질의가수분해는, 반응초기단계인 2시간이내에급속하게일어나기때문에 BAIBA와 BCAA도반응초기단계에급격히생성될것으로판단된다 효소첨가량의영향프로테아제에의한단백질의가수분해나가수분해물의조성은반응 ph, 반응시간이외에효소첨가량에따라많은영향을받는다 (Kurozawa 등, 2008; Kurozawa 등, 2009; Jung 2019). 프로테아제로서 alcalase, prozyme 2000P 및 foodpro alkaline protease를닭가슴살에대해서 % 를첨가한다음, 50 에서 12시간반응시켜얻어진가수분해물에서 BIABA 함량을분석한결과는 Fig. 7과같다. Alcalase에의한가수분해물의경우 % 까지는첨가농도에비례하여 BAIBA 생성량도유의적으로증가하였으나, 그이후 8% 까지효소첨가량을증가시켰을때에는 BAIBA 생성량이감소하였다. Prozyme 2000P와 foodpro alkaline protease 첨가구에서도역시 % 까지는효소첨가량과비례하여 BAIBA 생성량도유의적으로증가하였으나, 효소첨가량을 8% 까지증가시켜도유의적인 BAIBA 생성량의증가는관찰되지않았다. 이러한결과는본실험에서사용한 3종의효소모두 BAIBA 생성량을극대화하기위한적정첨가량은약 4% 라는것을나타내주는결과이다. 효소첨가량 4% 를기준으로비교하였을때 alcalase 에의한가수분해물에서 BAIBA의생성량 31.44± 1.36 mg/g이었으나 prozyme 2000P와 foodpro alkaline protease에의한가수분해물에서는각각 39.84±1.31 mg/g 및 33.60±2.00 mg/g이었다. 한편, BCAA 생성량의경우 alcalase 에의한가수분해물에서는효소첨가량이증가함에따라 BCAA의생성량도비 Fig. 6. Effect of hydrolysis time on BAIBA and BCAA content in chicken breast hydrolysates by alcalase. Values are means±sd (n=3). Different superscripts in the same row indicate significant differences (p<0.05). Fig. 7. Effect of enzyme concentration on BAIBA content in chicken breast hydrolysates by three proteases. A, alcalase; B, prozyme 2000P; C, foodpro alkaline protease; TFAA, total amount of free amino acid in hydrolysate. Values are means±sd (n=3). Different superscripts in the same row indicate significant differences (p<0.05)

12 Korean J Food Preserv, 29(2) (2022) 례적으로증가하여, 효소첨가량이 4% 였을때최대값에도달하였다 (Fig. 8). 또한, prozyme 2000P와 foodpro alkaline protease의경우도 % 까지는효소첨가량에비례하여 BCAA 생성량도유의적으로증가하였으며, 효소첨가량을 4% 이상으로증대시켰을때 BCAA 생성량은그이상증가하지않았다. 이러한결과는 3종의프로테아제를이용하여 BAIBA와 BCAA 함량이증대된닭가슴살의가수분해물을제조하기위해서는닭가슴살을기준으로효소를 4% 정도첨가하는것이바람직하다는것을시사해주는결과이다. 4. 요약 BAIBA와 BCAA의함량이높은닭가슴살의가수분해물을제조하기위하여, 시판되는 11종의프로테아제를닭가슴살에대하여 4% 씩첨가후 50 에서 12시간반응시켰을때단백질가수분해도는 alcalase, flavourzyme, prozyme 2000P에의한가수분해물에서높았다. 총유리아미노산함량은 prozyme 2000P(492.71±35.03 mg/g), flavourzyme (379.84±27.44 mg/g) 및 sumizyme AP(307.36±23.81 mg/g) 에의한가수분해물에서높았고, papain, bromelain, ficin과같은식물성프로테아제는미생물유래프로테아제에비해가수분해도와유리아미노산의생성량이낮은편이었다. 가수분해물중의 BAIBA 함량은 prozyme 2000P foodpro alkaline pretease alcalase flavourzyme 의순으로높았으나 BCAA의함량은 prozyme 2000P, flavourzyme, sumizyme LP에의한가수분해물에서높았고, 기타효소처리구에서는 45 mg/g 이하로낮은편이었다. 이효소들중 BAIBA와 BCAA의성량이높은 3종의프로테아제 (alcalase, prozyme 2000P, foodpro alkaline protease) 를선정하여반응액의 ph, 반응시간및효소첨가량의효과를조사한결과, BAIBA 생성량의경우 alcalase와 foodpro alkaline protease에의한가수분해물에서는 ph 6-7 범위에서높은반면 prozyme 2000P는 ph 5에서높았다. BCAA 생성량의경우, alcalase와 foodpro alkaline protease에의한가수분해물에서는 ph 7.5에서높은반면에 prozyme 2000P는 ph 사이에서높았다. 효소첨가량의영향을조사한결과, 3종의효소모두첨가량이 4% 였을때 BAIBA와 BCAA 생성량이최대값에도달하였으며, 대표적으로 alcalase를이용하여반응시간의영향을조사한결과, 두성분의최대생성을위한적정반응시간은 8시간이었다. 본연구결과는시판프로테아제를이용하여 BAIBA와 BCAA의함량이높은닭가슴살가수분해물을제조하는데유용한자료로활용할수있을것으로판단된다. 감사의글 본연구는중소벤처기업부 ( 과제번호 S ) 지원에 의한연구과제의일부로이에감사드립니다. Fig. 8. Effect of enzyme concentration on BCAA content in chicken breast hydrolysates by three proteases. A, alcalase; B, prozyme 2000P; C, foodpro alkaline protease; TFAA, total amount of free amino acid in hydrolysate. Values are means±sd (n=3). Different superscripts in the same row indicate significant differences (p<0.05). Conflict of interests The authors declare no potential conflicts of interest. Author contributions Conceptualization: Kim MK. Data curation: Kim EB

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