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1 Korean Society for Biotechnology and Bioengineering Journal 32(4): (2017) ISSN / eissn Research Paper 유산균의생존율향상을위한동결건조보호제로서호박분말의효과 강창호 *, 김용경, 한설화, 김진성, 정율아, 백남수 Effect of Pumpkin Powder as Cryoprotectant to Improve the Viability of Freeze Dried Lactic Acid Bacteria Chang-Ho Kang*, YongGyeong Kim, Seul Hwa Han, Jin-Seong Kim, Yulah Jeong, and Nam-Soo Paek Received: 11 September 2017 / Revised: 13 October 2017 / Accepted: 23 October The Korean Society for Biotechnology and Bioengineering Abstract: Food-grade protective agents such as sucrose, skim milk, and pumpkin powder were studied for their ability to improve the viability of freeze-dried lactic acid bacteria, including Lactobacillus plantarum MG989, L. fermentum MG901, Streptococcus thermophilus MG5139, Lactococcus lactis MG 534, Enterococcus faecium MG89-2, and Bifidobacterium animalis ssp. lactis MG741. The best results were obtained with 5% sucrose contained 10% skim milk and 2% pumpkin powder; approximately 43.1 to 86.6% cell viability was observed during the freeze-drying process. During accelerated storage of freeze-dried lactic acid bacteria, those survival rate decrease rapidly, reaching 9.8~35.2% in 1 month. As a result, the group (5% sucrose, 10% skim milk, and 2% pumpkin powder) showed the protective effect much higher by 639.5% than the control group (5% sucrose and 10% skim milk). These results suggested that pumpkin powder was good candidate as a potential cryoprotectant agent. Keywords: pumpkin powder, cryoprotective agent, lactic acid bacteria, freeze drying 1. INTRODUCTION 프로바이오틱스는숙주의장내미생물균형을유지시킴으로써유익한작용을하는살아있는미생물을의미한다 [1]. 현재 ( 주 ) 메디오젠 MEDIOGEN, Co., Ltd., Seoul 04157, Korea Tel: , Fax: 프로바이오틱스로사용되고있는미생물은 Lactobacillus, Bifidobacterium, Lactococcus 와같은유산균이많이사용되어지고있다 [2]. 특히프로바이오틱스유산균은장내균총의안정화, 유해세균의정착억제에따른부패산물생성감소및질병예방, 면역활성화작용, 항암작용, 콜레스트롤저하, 유당불내증의경감, 변비억제등의다양한효과를가진다고알려져있다 [3-6]. 유산균이프로바이오틱스로서유용한기능을나타내기위해서는섭취한많은수의균이사람의소화기관을통과하여야함으로, 유산균이프로바이오틱스로사용되기위해서는사람장관의위산과담즙산에대한안정성을기본적으로보유해야한다. 프로바이오틱스는일반적으로발효, 정제, 보관, 섭취단계를거쳐인체내에들어오게되는데, 산도, 산소, 열등주변환경에매우민감하기때문에안정성이보장된유산균제제기술개발은프로바이오틱스제품개발에있어매우중요한요소이다 [7,8]. 유산균의지속적인안정성유지를위한장기보존방법으로동결건조방법을주로사용하고있으며, 동결건조는유산균균체를냉동건조시켜저장하는방법 [9] 으로대부분의유산균을효과적으로장기보존할수있는방법이다. 동결건조방법은오염방지, 저장, 간편성의장점을가지고있으나, 동결건조과정에서물리적, 생화학적인스트레스로세포의활성과생존율에많은영향을미치게되므로유산균의생존율을최대한높일수있는방법이요구되고있다 [10]. 현재유산균의동결건조기술로기능성고분자및다당류로캡슐화시키는방법과세포보호제를첨가하여동결건조하는방법이주로이용되고있다 [11,12]. 동결건조시에는온도와수분의손실을최소화하기위해탈지유 (skim milk), 전분 (starch) 와같은고분자물질과 glucose, lactose 와같은저분자물질등이이용된다 [12]. 보호제의역할은미생물의종류, 동결조건,

2 252 Korean Society for Biotechnology and Bioengineering Journal 32(4): (2017) 건조시간등에따라다르며, 미생물의종류에따라서는보호제가생존율에많은영향을미칠수있다 [13]. 이에본연구에서는다양한유산균주의동결건조과정에서생존율증대를위해식품첨가등급의동결건조보호제를탐색하고, 최적의동결건조보호제조건을통해가능성을확인하고자한다. 2. MATERIALS AND METHODS 생존율 (%) = 가혹조건에서의시료의 CFU 값 / 동결건조된시료의 CFU 값 통계처리모든실험은 3반복으로측정하여측정치를평균값 ± 표준편차로나타내었으며, 실험결과의통계적유의성은 Minitab program (Minitab 16, Minitab Inc., State College, PA, USA) one-way 분산분석 (ANOVA) 의 Turky HSD test에의해시료간의유의적차이 (p<0.05) 를검정하였다 사용균주및보존사용된균주는본연구팀에서보유하고있는 Lactobacillus plantarum MG989, Lactobacillus fermentum MG901, Streptococcus thermophilus MG5139, Lactococcus lactis MG534, Enterococcus faecium MG89-2, Bifidobacterium animalis ssp. lactis MG741 6종을사용하였다 [14,15]. 배양배지로 Bifidobacterium속은 BL배지 (Nissui, Japan) 를사용하였으며, 그외 5 종은 MRS배지 (Difco, MI, USA) 를사용하였다. 배양온도는 S. thermophilus 균주는 42 o C에서, 그외균주는 37 o C에서배양하였다. 실험균주의장기보관방법으로 25% 글리세롤로제조하여 -80 o C에서보존하여유전적변이를최소화하였다 동결건조보호제종류별유산균의생존율확인유산균의생존율향상을위한동결건조보호제로식품첨가물등급의 10% 탈지유 (Skim milk), 5% 자당 (Sucrose), 2% 호박분말 (Pumpkin powder) 을사용하였으며, 대조군으로인산완충용액 (PBS; phosphate buffered saline) 을사용하였다. 균주를 7L 발효조 (KoBioTech Co., Ltd. Korea) 에서배양한후원심분리 (Supra 22K, Hanil Co. Ltd., Korea) 를 5,000 rpm, 4 o C 로 30 분간하여균체를회수한다. 회수된균체는각각의동결건조보호제와혼합하여균질화한후, -40 o C 로동결시킨다음동결건조기 (Il Shin Co. Ltd., Korea) 에서건조하였다 [16]. 동결건조후건조된균체는분쇄하여분말화시킨다음멸균생리식염수 (0.85% NaCl) 에단계희석하여 MRS 및 BL 배지에도말한후, 48~72 시간동안배양하여생존율을비교하였다. 각각의동결건조보호제실험을통해, 5% 자당을기본구성으로각동결건조보호제를혼합하여동결건조보호제조합에의한유산균생존율의효과를검증하였다. 생존율 (%) = 동결건조후시료의 CFU 값 / 동결건조전시료의 CFU 값 가혹조건에서동결건조균원료의경시확인대조군으로 5% 자당을포함하여, 동결건조보호제로 10% 탈지유와 2% 호박분말을사용하였다. 동결건조한균원료를 10 g 씩소분및밀봉하여 40 o C 에 1 개월간보관하면서 1 주간격으로시료를채취하여멸균생리식염수에단계희석하여 MRS 및 BL 배지에도말한후, 48~72 시간배양한다음생균수를측정하여가혹조건에서의생존율을확인하였다. 3. RESULTS AND DISCUSSION 3.1. 동결건조보호제별생존율확인식품첨가등급의 3 가지동결건조보호제를첨가하여유산균의동결보호효과를확인한결과, 대조군과비교하여모든동결건조보호제에서동결건조후생존율이증가한것을확인할수있었다 (Fig. 1). 모든실험균주에서호박분말 (pumpkin) 을보호제로사용하였을경우보호효과가가장우수한것으로확인하였으며, B. lactis MG741 이동결건조직후 79.0±1.7% 의가장높은생존율을나타내었으며, L. plantarum MG989 와 Lac. lactis MG534 가각각 78.0±1.6% 와 69.4±2.1% 의높은생존율을나타내었다. 실험에사용된모든균주에서호박분말을동결건조보호제로사용하였을때생존율이 40% 이상으로매우높은생존율을나타내는것을확인할수있었다. 호박분말의주원료로사용되는호박 (Cucurbita maxima) 은박과에속하는 1 년생덩굴성초본으로남아메리카페루가원산지인서양계박으로기호성작물이며, 비타민 A, B1, B2, C 의함량이높으며그외에도칼슘, 나트륨, 인등의무기질함량이풍부한것으로알려져있다 [17]. 일반적으로건강식품으로다양한식품에기능성부재료로사용되고있으나, 현재까지유산균의동결건조보호제로적용된연구는보고된바없다. 또한호박분말은섬유질, 무기질, 각종비타민등과 β-carotene 을비롯한다양한아미노산류가풍부한식품소재로서 [18], 본연구에서는동결건조보호제로호박분말이식품첨가등급의경제적인소재로동결건조과정중에서유산균을보호해주는역할을수행하는것으로사료된다. 동결건조보호제는유산균의생존율이영향을미치는가장중요한인자이지만종류에따라부작용을야기할수도있다 [19]. 일반적으로동결건조보호제로사용되고있는탈지유의경우에는식품에첨가할경우풍미에영향을미칠수있으며섭취시유당불내증등소화에어려움이있으나, 호박분말의경우풍부한섬유질에식욕을개선해주는효능이있으며 [20], 다양한식품에기능성부재료 [21-23] 로사용하고있어부작용이없는소재가될수있다 동결건조보호제조합에따른유산균생존율확인기본동결건조보호제로결정된 5% 자당에첨가하여사용할수있는동결건조보호제중탈지유와호박분말에대한생존

3 유산균의생존율향상을위한동결건조보호제로서호박분말의효과 253 Fig. 1. Viability of lactic acid bacteria during freeze drying process depending on different protective agents. Data were expressed as mean±sd (n=3). Different characters were significantly different (p<0.05). Fig. 2. Viability of lactic acid bacteria during freeze drying process depending on the combination of different protective agents. C, 5% sucrose; SM, skim milk; PP, pumpkin powder. Data were expressed as mean± SD (n=3). Different characters were significantly different (p<0.05). 율은 Fig. 2 와같다. E. faecium MG89-2 균주의경우, 기본보호제에 10% 탈지유와 2% 호박분말을각각혼합한경우와 3 가지모두혼합하였을때 62.8±2.4%, 72.2±2.3%, 86.6±2.7% 로실험균주중가장높은생존율을나타내었다. 실험에사용된모든균주에서기본보호제에 10% 탈지유를혼합한조건보다 2% 호박분말을혼합한조건에서생존율이증가한것을

4 254 Korean Society for Biotechnology and Bioengineering Journal 32(4): (2017) Table 1. Survival rate of freeze-dried cell powder using the pumpkin powder as cryoprotectant after 1 month storage period at the 40 o C Survival rate(%) after 1 month storage period Strains 1 week 2 week 3 week 4 week C + 10%SM 36.0 ± ± ± ± 0.2 L. plantarum C + 2%PP 52.7 ± ± ± ± 1.2 MG989 C+10%SM + 2%PP 58.8 ± ± ± ± 0.7 C + 10%SM 20.8 ± ± ± ± 2.1 L. fermentum C + 2%PP 48.3 ± ± ± ± 1.9 MG901 C+10%SM + 2%PP 59.1 ± ± ± ± 2.0 C + 10%SM 16.2 ± ± ± ± 0.1 S. thermophilus C + 2%PP 24.9 ± ± ± ± 0.3 MG5139 C+10%SM + 2%PP 33.5 ± ± ± ± 0.2 C + 10%SM 32.8 ± ± ± ± 0.1 Lac. lactis C + 2%PP 44.5 ± ± ± ± 1.3 MG534 C+10%SM + 2%PP 70.2 ± ± ± ± 0.7 C + 10%SM 40.0 ± ± ± ± 0.4 E. faecium C + 2%PP 62.2 ± ± ± ± 2.4 MG89-2 C+10%SM + 2%PP 76.2 ± ± ± ± 1.4 C + 10%SM 44.0 ± ± ± ± 0.2 B. lactis C + 2%PP 53.0 ± ± ± ± 0.5 MG741 C+10%SM + 2%PP 68.5 ± ± ± ± 0.4 C: sucrose 5%, SM: skim milk, PP: pumpkin powder. 확인할수있었으나, 기본보호제에 10% 탈지유, 2% 호박분말을모두혼합한조건에서는실험균주에따른유의적인차이가없었다. 기존의연구결과 [13] 에따르면, 동결건조보호제는미생물의종류에따라서도생존율에영향을미친다고알려져있다. 본연구에서도실험균주에따라동결건조보호제조성에따른생존율이다르게영향을미치는것을확인할수있다 가혹조건에서의경시변화기본보호제에 10% 탈지유를첨가한조건, 2% 호박분말을첨가한조건, 10% 탈지유와 2% 호박분말을첨가한총 3 가지조건에서동결건조된균원료에대하여가혹조건 (40 o C) 에서 1 개월간보관하면서 1 주간격으로확인한생존율은 Table 1 과같다. 모든실험균주에서가속경시 1 주차에서기본보호제와 10% 탈지유만을혼합한조건에비해호박분말을동결건조보호제로첨가한경우 2 배이상의높은생존율을확인할수있었으며, 가속경시 4 주후에도매우높은생존율을유지하는것을확인할수있었다. 호박분말은단백질, 당, 무기질, 비타민을포함하고있으며, 단백질및당류가균체를포집및포괄하는작용을하며동결건조후에는건조균체의세포벽과결합하여미생물을코팅하여균체를보호하는특성을갖는것으로알려져있다 [24]. 이를통해호박분말이다양한유산균주의동결건조보호제및장기보존시보호제로사용가능함을확인할수있었다. 4. CONCLUSION 본연구는다양한유산균주의동결건조시생존율을높이기위한방안으로기존의동결건조보호제를대체하기위해식품첨가물등급의다양한동결건조보호제를통한생존율을비교분석하였다. 호박분말은단백질및당, 풍부한무기질과비타민으로구성되어있으며, 최근기능성식품소재로많이사용되고있는소재이다. 호박분말을유산균의동결건조보호제로사용한것은본연구가최초로시도되는부분이며, 자당과탈지유와비교하였을때동결건조후실험에사용된모든균주에서모두높은생존율을확인할수있었다. 또한 5% 자당과 2% 호박분말을혼합한조건에서실험에사용된모든균주에서생존율이증가한것을확인할수있었다. 가혹조건에서의경시변화에서도호박분말을사용한혼합동결건조분산매조건에서호박분말을사용하지않은동결건조분산매조건에비해높은생존율을지속적으로유지한것을통해호박분말이라는새로운동결건조보호제를선별한것에큰의의가있다고사료된다. REFERENCES 1. Food and Agricultural Organization of the United Nations and World Health Organization. (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. World Health Organization. 2. Isolauri, E., S. Salminen, and A. C. Ouwehand (2004) Probiotics. Best Prac. Res. Cl. Em. 18: Alander, M., R. Satokari, R. Korpela, M. Saxelin, T. Vilpponen- Salmela, and A. von Wright (1999) Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamno-

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