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1 pissn eissn J. Milk Sci. Biotechnol. 2018;36(3): ARTICLE 백윤서 1 하호경 2 이지홍 1 이미령 3 이원재 1* 1 경상대학교동물생명과학과 ( 농업생명과학연구원 ), 2 순천대학교동물자원과학과, 3 대구대학교식품영양학과 Formation and Characterization of Casein Phosphopeptide/Chitosan Oligosaccharide NanoComplex Yun-Seo Baek 1, Ho-Kyung Ha 2, Ji-Hong Lee 1, Mee-Ryung Lee 3, and Won-Jae Lee 1* 1 Department of Animal Bioscience and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea 2 Department of Animal Science and Technology, Sunchon National University, Sunchon, Korea 3 Department of Food and Nutrition, Daegu University, Gyeongsan, Korea Received: September 17, 2018 Revised: September 18, 2018 Accepted: September 18, 2018 These authors contributed equally to this study. *Corresponding author : Won-Jae Lee Department of Animal Bioscience and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea Tel : Fax : wjleewisc@gnu.ac.kr Copyright 2018 Korean Society of Milk Science and Biotechnology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ORCID Yun-Seo Baek Ho-Kyung Ha Ji-Hong Lee Mee-Ryung Lee Won-Jae Lee Abstract The objectives of this study were to manufacture casein phosphopeptide (CPP)/chitosan oligosaccharide (CSO) nanocomplexes and to investigate the impacts of manufacturing variables, such as CPP concentration and ph, on their morphological and physicochemical characteristics. Transmission electron microscopy (TEM) and particle size analysis were used to assess the morphological and physicochemical properties of the CPP/CSO nanocomplexes, respectively. Based on the images obtained by TEM, the spherical shapes of the CPP/CSO nanocomplexes ranged from 50 to 150 nm. As the concentration of CPP was increased and the ph was decreased, the average particle size of the nanocomplexes significantly (p<0.05) increased. The CPP/CSO nanocomplexes had a highly uniform distribution with a polydispersity index value of less than 0.3. In addition, they had a negative surface charge with a zeta-potential value between 17 and 26 mv. The CPP/CSO nanocomplexes showed good stability during the freeze-drying process. In conclusion, CPP/CSO nanocomplexes were successfully manufactured, and the CPP concentration and ph were found to be key factors that affected their morphological and physicochemical properties. Keywords casein phosphopeptide, chitosan oligosaccharide, nanocomplex 서론 우리나라는출산율저하와평균수명증가로인해고령화가빠르게진행됨에따라시니어들의노년기 질환문제가크게대두되고있다. 당뇨병, 암, 우울증, 치매등다양한노인성질환중작은충격에 의해서도쉽게골절로이어질수있는골다공증과같은뼈질환문제는더욱더심각하게인식되고 있다. 사람의몸에존재하는가장많은무기질중하나인칼슘은뼈의주요구성요소로체중의약 2% 를차지하고있는데, 칼슘이체내에부족하게되면구루병이나골절, 골다공증과같은뼈질환 문제의발생위험이커지게된다 (Cilla et al., 2011; Cao et al., 2017). 국민건강영양조사의통계에 따르면우리나라만 65 세이상성인의골관절염유병률은 32.7% 이며, 연간전체뼈무게의약 0.3% 가소실되는것으로알려져있다 (KCDC, 2015). 또한칼슘을부족하게섭취하는사람의비율은 74% 인데, 특히 65 세이상성인 83% 가칼슘섭취가부족하다고보고되었다 (KCDC, 2016). 따라서뼈 질환문제를예방하고이를통한노년기삶의질향상을위해서는충분한칼슘섭취가반드시이루어 164 J Milk Sci Biotechnol Vol. 36, No. 3

2 져야한다. 현재칼슘섭취를위해이용되어지는칼슘보충제로는 calcium carbonate, calcium gluconate, calcium lactate 와같은이온화칼슘 (ionized calcium) 이있다. 칼슘이장내에서흡수되기위해서는용해된상태로존재해야하는데, 이온화칼슘은소장에서의용해도저하로인한침전생성으로인해칼슘흡수가저하되고, 결과적으로생체이용률이낮은문제점이있으며, 또한변비나복부팽창등과같은부작용이발생할수있다 (Sun et al., 2016). 본연구에서는기존의이온화칼슘이용시나타나는문제점들을개선하기위해새로운칼슘이용률향상을위한식품소재로케이신포스포펩티드, casein phosphopeptide(cpp) 와키토올리고당, chitosan oligosaccharide(cso) 가선택되었다. CPP는우유단백질인케이신이분해되어만들어진펩티드로음전하를띠는아미노산인 3개의인산화세린과 2개의글루탐산으로구성되어있어칼슘과효율적으로결합할수있다 (Ferraretto et al., 2001). 또한 CSO는분자량이커서물에잘녹지않는키토산을분해한 (<20 kda) 물질로수용해성이높아식품적용성이뛰어나며 (Bharathiraja et al., 2018), paracellular pathway 를통해이루어지는칼슘의장내흡수과정에서세포사이공간인 tight junction 을열어 paracellular uptake 를증진시켜칼슘의흡수를향상시킬수있다 (Ranaldi et al., 2002). 본연구에서 CPP와 CSO를이용하여제조할나노복합체는입자크기가 200 nm 이하로표면적이상대적으로넓어장상피세포와같은 target 세포와상호작용할수있는가능성이높으며, 이로인해칼슘을포함한여러생리활성물질들의흡수율및생체이용률을향상시킬수있을것으로기대된다 (Ha et al., 2015). 또한칼슘결합부위를지닌 CPP와 paracellular enhancer 인 CSO를이용하여제조한나노복합체가칼슘의용해도뿐만아니라, 칼슘의흡수를증진시킬수있을것으로가정하였다. 본연구의목적은칼슘흡수를향상시키기위한 CPP/CSO 나노복합체를제조하고, 나노복합체제조공정요인인 CPP 농도와 ph가나노복합체의형태학적, 물리화학적특성에미치는효과를연구하는것이다. 재료및방법 1. 시약케이신포스포펩티드는 FrieslandCampina 에서, 키토올리고당은 Amicogen Inc. 에서제공받아사용하였다. Hydrochloric acid(hcl) 는 Sigma-Aldrich 에서구입하였다. 2. 나노복합체제조 Casein phosphopeptide(cpp)/chitosan oligosaccharide(cso) 나노복합체는 modified ionic gelation 방법 (Ha et al., 2013) 을사용하여제조하였다. 우선케이신포스포펩티드와키토올리고당을증류수에용해시켜 0.4%, 0.6%, 0.8%(w/v) 농도의케이신포스포펩티드수용액과 0.006% (w/v) 농도의키토올리고당수용액을준비하였다. 케이신포스포펩티드수용액과키토올리고당수용액을혼합하여다양한농도의케이신포스포펩티드 ( 최종농도 : 0.2, 0.3, 0.4%(w/v))/ 키토올리고당 ( 최종농도 : 0.003%(w/v)) 혼합용액을준비한후, 0.2M HCl을이용하여 CPP/CSO 혼합액의 ph를조절하였다 ( 최종 ph: 5.5, 6.0, 6.5). 3. 나노복합체의물리화학적특성분석 CPP/CSO 나노복합체의입자크기 (particle size), 다분산지수 (polydispersity index), 제타전위 (zeta-potential) 는입도분석기 (particle size analyzer, Nano-ZS, Malvern, UK) 를이용하여측정하였다. 나노복합체를증류수를이용하여 20배희석한후 173 의 scattering angle에서입자 J Milk Sci Biotechnol Vol. 36, No

Baek et al. 크기와다분산지수를측정하였다. 또한제타전위는나노복합체를증류수를이용하여 10 배희석한 후측정하였다. 4. 나노전달체의형태학적특성평가나노복합체의형태학적특성은투과전자현미경 (transmission electron microscopy, TEM, Phillips Tecnai 12, Phillips, Netherlands) 을이용하여측정하였다.

3 Baek et al. 크기와다분산지수를측정하였다. 또한제타전위는나노복합체를증류수를이용하여 10 배희석한 후측정하였다. 4. 나노전달체의형태학적특성평가나노복합체의형태학적특성은투과전자현미경 (transmission electron microscopy, TEM, Phillips Tecnai 12, Phillips, Netherlands) 을이용하여측정하였다. 제조된나노전달체를탄소- 코팅된 200-mesh copper grid 위에올린다음 2% uranyl acetate 를이용하여 45초간염색하였다. 그런다음염색된그리드를투과전자현미경에삽입하여 120 kv에서나노복합체의형성유무와형태를관찰하였다. 5. 나노복합체의동결건조동결건조기 (FD-1000, Tokyo Rikakikai Co., Ltd, Japan) 를이용하여나노복합체의식품적용성향상을위한분말화를수행하였다. 제조된나노복합체현탁액을 80 C 에서 12시간동결시킨후동결건조기를이용하여분말화된나노복합체를 1 mg/ml 농도로증류수에분산시킨후, 투과전자현미경을이용하여입자의형태학적특성을측정하였다. 결과및고찰 1. Casein phosphopeptide(cpp) 농도에따른나노복합체의형태학적, 물리화학적특성 CPP/CSO 를이용한나노복합체의제조공정확립을위해제조공정요인 (CPP 농도, ph) 에따른나노복합체의물리화학적, 형태학적특성을평가하였다. 투과전자현미경을사용하여 CPP 농도에따른나노복합체의형성유무와형태학적특성을측정한결과, CPP 농도가 0.2% 에서 0.4%(w/v) 로증가함에따라좀더큰응집체가형성됨을확인하였고, 약 nm 크기의구형의입자가형성되었음을확인할수있었다 (Fig. 1). CPP 농도에따른나노복합체의물리화학적특성평가결과, 우선 CPP 농도가 0.2에서 0.4%(w/v) 로증가함에따라 CPP/CSO 나노복합체의크기가 187에서 280 nm로유의적 (p<0.05) 으로증가하였다 (Fig. 2A). 그러나 CPP 농도가 0.6%(w/v) 이상이었을경우침전이발생하였다 (data not shown). Hu 등 (2012) 의연구에따르면단백질과탄수화물을이용하여복합체를제조할경우, 단백질의농도가증가할수록형성되는복합체의입자크기가증가한다고보고되었다. 본실험조건인 ph 6.5에서는유단백질인 CPP가음의전하를지니며, CSO는양의전하를지녀정전기적상호작용 (electrostatic attraction) 에의해나노복합체가형성되었다. CPP Fig. 1. TEM images of CPP/CSO nano complexes. Nano complexes were prepared with 0.2% (A), 0.3% (B), and 0.4% (C) (w/v) CPP concentration levels at ph 6.5 (CSO concentration: 0.003%, w/v). Scale bar=100 nm. 166 J Milk Sci Biotechnol Vol. 36, No. 3

Fig. 2. Effects of casein phosphopeptide (CPP) concentration level on the particle size (A), polydispersity index (B), and zeta-potential (C) of CPP/CSO nano complex.

Different letters on a column indicate significant differences (p<0.05). 의농도가증가할수록더많은 CPP가 CSO와의정전기적상호작용에관여하여결과적으로더큰복합체를형성하였다고사료된다. 다분산지수 (PDI) 의경우, CPP 농도가 0.2에서 0.4%(w/v) 로증가함에따라유의적인차이는없었다 (Fig.

4 Fig. 2. Effects of casein phosphopeptide (CPP) concentration level on the particle size (A), polydispersity index (B), and zeta-potential (C) of CPP/CSO nano complex. CPP solutions with various concentration levels (0.2%, 0.3%, and 0.4%, w/v) was mixed with 0.003% (w/v) CSO solution and then adjusted to ph 6.5. Different letters on a column indicate significant differences (p<0.05). 의농도가증가할수록더많은 CPP가 CSO와의정전기적상호작용에관여하여결과적으로더큰복합체를형성하였다고사료된다. 다분산지수 (PDI) 의경우, CPP 농도가 0.2에서 0.4%(w/v) 로증가함에따라유의적인차이는없었다 (Fig. 2B). 그러나 0.2%, 0.3%(w/v) CPP 농도로제조된 CPP/CSO 나노복합체는 0.3 이하의 PDI 값을가진반면, 0.4%(w/v) CPP 농도로제조된나노복합체의 PDI 값은 0.3 이상이었다. 결과적으로 PDI 값이 0.3 이하인 0.2와 0.3%(w/v) CPP 농도조건에서는상대적으로균일한나노복합체가생성되었음을알수있었다 (Lippacher et al., 2001). CPP 농도에따른제타전위실험의경우, CPP 농도가 0.2에서 0.4%(w/v) 로증가함에따라나노복합체의제타전위가 19.0 에서 25.7 mv로유의적 (p<0.05) 으로감소함을알수있었다 (Fig. 2C). 본연구를위한예비실험에서 CPP의 pi( 등전위점 ) 는 ph 3.1이었고 (data not shown) 따라서 ph 6.5에서 CPP는음의전하를가지게된다. 즉, CPP의농도가증가할수록나노복합체는더많은음전하를띠게되어결과적으로나노복합체의표면전하역시감소된다고사료된다. 2. ph에따른나노복합체의형태학적, 물리화학적특성나노복합체제조공정요인중하나인 ph(5.5, 6.0, 6.5) 에따른나노복합체의형태학적특성평가결과, ph가 5.5에서 6.5로증가함에따라좀더작은응집체가형성됨을확인하였고, 약 nm 크기의구형의입자가형성되었음을확인할수있었다 (Fig. 3). 나노복합체의물리화학적특성에대한 ph의영향평가의경우, ph가 5.5에서 6.5로증가함에따라 CPP/CSO 나노복합체의입자 Fig. 3. TEM images of CPP/CSO nano complexes. Nano complexes were prepared with 0.3% (w/v) CPP concentration and 0.003% (w/v) CSO concentration at 5.5 (A), 6.0 (B), and 6.5 (C) ph levels, Scale bar=100 nm. J Milk Sci Biotechnol Vol. 36, No

3 이하값을지니고있었다 (Fig. 4B). 따라서본연구에서 5.5 6.5 의 ph 조건에서형성되는 CPP/CSO 나노복합체의입도분포는매우균일함을확인하였다. 또한 ph가 5.5에서 6.5로증가함에따라 CPP/CSO 나노복합체의제타전위값은 16.8 mv에서 24.5 mv로유의적 (p<0.05) 으로감소함을알수있었다 (Fig. 4C). ph가 5.5에서 6.5로증가함에따라 CPP가지니는음전하가증가하게되고, 결과적으로제조된 CPP/CSO 나노복합체의표면전하는감소했다고사료된다.

5 Baek et al. 크기가 246 nm에서 191 nm로유의적 (p<0.05) 으로감소함을확인하였다 (Fig. 4A). CPP의 pi가 3.1(data not shown) 이고, CSO의 pka는 6.3(Bhattarai et al., 2010) 이기때문에 ph가 5.5 에서 6.5로증가함에따라 CPP는더많은음전하를지니게되고, 이에따라양전하를지닌 CSO와의정전기적인력작용 (electrostatic attraction) 이증가되어결과적으로더작은크기의나노복합체를형성하였다고사료된다. ph에따른다분산지수값은 ph 5.5일때 0.200, ph 6.0일때 0.156, ph 6.5일때 0.189로유의적인차이없이세조건모두에서 0.3 이하값을지니고있었다 (Fig. 4B). 따라서본연구에서 의 ph 조건에서형성되는 CPP/CSO 나노복합체의입도분포는매우균일함을확인하였다. 또한 ph가 5.5에서 6.5로증가함에따라 CPP/CSO 나노복합체의제타전위값은 16.8 mv에서 24.5 mv로유의적 (p<0.05) 으로감소함을알수있었다 (Fig. 4C). ph가 5.5에서 6.5로증가함에따라 CPP가지니는음전하가증가하게되고, 결과적으로제조된 CPP/CSO 나노복합체의표면전하는감소했다고사료된다. 3. 동결건조과정중나노복합체의물리적안정성제조된 CPP/CSO 나노복합체를다양한종류의식품에적용하기위해동결건조기를이용하여분말화를수행하였다. 투과전자현미경을사용하여동결건조전후의나노복합체의물리적안정성을평가한결과, 동결건조후에도매우균일한분포의구형의나노복합체가형성되었음을확인하였다 (Fig. 5). 결과적으로동결건조과정을통해생성된나노복합체분말은향후다양한종류의식품에적용 Fig. 4. Effects of ph on the particle size (A), polydispersity index (B), and zeta-potential (C) of CPP/CSO nano complex. 0.3% (w/v) CPP solution was mixed with 0.003% (w/v) CSO solution and then adjusted to ph 5.5, 6.0, and 6.5. Different letters on a column indicate significant differences (p<0.05). Fig. 5. Morphological properties of CPP/CSO nano complex before (A) and after (B) freeze drying process. 0.3% (w/v) CPP solution was mixed with 0.003% (w/v) CSO solution and then adjusted to ph 6.5. Scale bar=100 nm. 168 J Milk Sci Biotechnol Vol. 36, No. 3

6 가능한잠재력이높음을알수있었다. 요약 본연구에서는 CPP와 CSO를사용하여약 nm 크기의균일한구형의나노복합체를성공적으로제조하였다. 나노복합체는제조공정요인인 CPP 농도와 ph 조절을통해입자크기, 다분산지수, 그리고표면전하와같은나노복합체의물리화학적특성을조절할수있음을확인하였다. 또한 CPP/CSO 나노복합체의분말화공정을통해분말화된나노복합체의식품적용성을증진시킬수있음을알수있었으며, food-grade 물질인 CPP와 CSO를사용하여제조한나노복합체는향후잠재적인칼슘전달체로써식품산업에이용될수있을것으로기대된다. 감사의글 This work was supported by Basic Science Research Program(NRF-2017R1D1A1B ) through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Republic of Korea. References Bharathiraja, S., Bui, N. Q., Manivasagan, P., Moorthy, M. S., Mondal, S., Seo, H., Phouc, N. T., Vy Phan, T. T., Kim, H., Lee, K. D. and Oh, J Multimodal tumor-homing chitosan oligosaccharide-coated biocompatible palladium nanoparticles for photobased imaging and therapy. Sci. Rep. 8:500. Bhattarai, N., Gunn, J. and Zhang, M Chitosan-based hydrogels for controlled, localized drug delivery. Adv. Drug Deliv. Rev. 62: Cao, Y., Miao, J., Liu, G., Luo, Z., Xia, Z., Liu, F., Yao, M., Cao, X., Sun, S., Lin, Y., Lan, Y. and Xiao, H Bioactive peptides isolated from casein phosphopeptides enhance calcium and magnesium uptake in caco-2 cell monolayers. J. Agric. Food Chem. 65: Cilla, A., Lagarda, M. J., Alegría, A., Ancos, B., Cano, M. P., Sánchez-Moreno, C., Plaza, L. and Barberá, R Effect of processing and food matrix on calcium and phosphorous bioavailability from milk-based beverages in caco-2 cells. Food Res. Int. 44: Ferraretto, A., Signorile, A., Gravaghi, C., Fiorilli, A. and Tettamanti, G Casein phosphopeptides influence calcium uptake by cultured human intestinal ht-29 tumor cells. J. Nutr. 131: Ha, H. K., Kim, J. W., Lee, M. R. and Lee, W. J Formation and characterization of quercetin-loaded chitosan oligosaccharide/β-lactoglobulin nanoparticle. Food Res. Int. 52: Ha, H. K., Kim, J. W., Lee, M. R., Jun, W. and Lee, W. J Cellular uptake and cytotoxicity of β-lactoglobulin nanoparticles: The effects of particle size and surface charge. Asian-Aust. J. Anim. Sci. 28: J Milk Sci Biotechnol Vol. 36, No

7 Baek et al. Hu, B., Ting, Y., Zeng, W. and Huang, Q Cellular uptake and cytotoxicity of chitosan-caseinophosphopeptides nanocomplexes loaded with epigallocatechin gallate. Carbohyd. Polym. 89: Korea Centers for Disease Control & Prevention(KCDC). 2015~2016. 질병관리본부 Lippacher, A., Müller, R. H., and Mäder, K Preparation of semisolid drug carriers for topical application based on solid lipid nanoparticles. Int. J. Pharm. 214:9-12. Ranaldi, G., Marigliano, I., Vespignani, I., Perozzi, G. and Sambuy, Y The effect of chitosan and other polycations on tight junction permeability in the human intestinal caco-2 cell line. J. Nutr. Biochem. 13: Sun, N., Wu, H., Du, M., Tang, Yue., Liu, H., Fu, Y. and Zhu, B Food protein-derived calcium chelating peptides: A review. Trends. Food Sci. Technol. 58: J Milk Sci Biotechnol Vol. 36, No. 3

갓 태어난 송아지는 초유의 이행 항체에 의한 수동면역의 획득에 크게 의존하고 있다

갓 태어난 송아지는 초유의 이행 항체에 의한 수동면역의 획득에 크게 의존하고 있다 pissn 2384-0269 eissn 2508-3635 J Milk Sci Biotechnol 2017;35(3):208-214 https://doi.org/10.22424/jmsb.2017.35.3.208 ARTICLE 하호경 1 김진욱 1 한경식 2 윤숭섭 3 이미령 4 이원재 1* 1 경상대학교동물생명과학과 ( 농업생명과학연구원 ), 2 삼육대학교동물생명자원학과,