제출문 농림축산식품부장관귀하 이보고서를 전통발효유타락의산업화연구 과제 ( 세부과제 타락발효유의제품화기 술개발에관한연구 ) 의보고서로제출합니다 년 01 월 17 일 주관연구기관명 : 한국야쿠르트주관연구책임자 : 장성식세부연구책임자 : 장성식연구원 : 명길선연구

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1 발간등록번호

2 제출문 농림축산식품부장관귀하 이보고서를 전통발효유타락의산업화연구 과제 ( 세부과제 타락발효유의제품화기 술개발에관한연구 ) 의보고서로제출합니다 년 01 월 17 일 주관연구기관명 : 한국야쿠르트주관연구책임자 : 장성식세부연구책임자 : 장성식연구원 : 명길선연구원 : 김수아 협동연구기관명 : 성신여자대학교협동연구책임자 : 한영숙협동연구기관명 : 성신여자대학교협동연구책임자 : 윤현근협동연구기관명 : 성신여자대학교협동연구책임자 : 고성희

3 요약문 Ⅰ. 제목 전통발효유타락의산업화연구 Ⅱ. 연구개발의목적및필요성 연구개발의목적막걸리를발효원으로한전통발효유의표준화를위해발효미생물과발효산물을분석하고기능성성분을규명하며응용제품을개발함으로써산업화하고자함. (1) 타락발효유의제품화기술개발 (2) 전통발효유타락의제조표준화및영양적우수성규명 (3) 타락의기능성분석방법확립및 in vitro 기능성규명 (4) 타락의응용제품개발 연구개발의필요성 (1) 문화적측면 : - 세계적으로건강식에대한관심이높아지면서한식의건강기능성이부각되고있음. - 한식의대표적인발효음식인김치, 젓갈, 된장, 간장, 고추장은이미그우수성을과학적으로인정받고있음. - 현재까지발견된가장오래된한문필사본조리서인 수운잡방 ( 需雲雜方,1540년) 은고려말기에서조선전기에걸친한시대의음식법을추정할수있는귀중한자료임. - 수운잡방에는우유에탁주를첨가하여발효시킨발효유, 타락의제법을기록함으로써그당시부터발효유가섭취되었음을알수있음. - 우리나라에도전통발효유가존재했음에도불구하고그제법이나품질특성이거의알려지지않았으며체계적인연구는시작단계에불과한실정임. - 발효유는발효에사용된미생물의발효특성이나제조방법에따라그종류가매우다양하며, 염증억제, 혈압조절, 콜레스테롤저하, 비만억제등다양한기능성이확인됨으로써세계인들의관심이높은식품임. - 타락은우리나라전통발효식품이나타발효식품과는달리동물성원료인우유를발효시킨독특한식품임.

4 (2) 산업적측면 : - 현재, 국내발효유시장의연간매출규모는약 1조4000억원으로이가운데장과위, 간발효유등기능성발효유가전체발효유시장의 40% 를차지하고있으며차별화된프리미엄급발효유제품이출시되고있음. - 전세계적으로발효유제품은비만, 고혈압, 당뇨, 심장질환, 암등과같이건강과웰빙에문제가되는질환을예방해주는제품으로자리를확고하게구축하였으며, 유기농을비롯하여저지방 / 무지방, 비타민강화, 저콜레스테롤, 저나트륨, 무카페인, 무설탕등다양한분야에서의신제품개발도동시에이루어지고있음. - 유럽발효유의특징은각나라별로건강관심요인에차이를나타냄. 따라서, 주요관심요인이되는특정질환에초점을맞춘제품이주류를이루고있으며 L. casei, L. acidophilus, Bifidobacteria 등의 probiotic 젖산균을제품에활용하고있음 ( 한국산업보건진흥원, 2004). - 발효유의종류는젖산균발효유와젖산균-효모발효유가있으며특히젖산균-효모발효유의건강기능성이발표됨으로써새롭게주목을받고있음. - 본연구과제에서개발될새로운전통타락발효유의상품화를통하여기업매출증대및수출촉진에도크게이바지할것으로예상됨. (3) 사회적측면 : - 유산균을이용한전통기능성발효에관한기술축적및타락균발효유개발을위한균주의배양방법과발효조건및제조공정을습득한전문인력을양성할수있음. - 한식의디저트화를통하여타락발효유의브랜드화가능함. - 유제품의맛과기능성을향상시킴으로써유제품의소비를향상시킬수있음. - 우유의수요증가를통한낙농농가와유가공업체의수익증대에기여할수있음. (4) 기술적측면 : - 타락균을접종하여원유단독배양후그배양액을균질, 살균, 냉각하여제조하는타락균표준발효유대량제조공정기술확보가가능함. - 타락균의기능성물질과특유의맛, 영양성분을증가시킴으로서우유의영양과타락균이발효하면서생성되는생리활성물질성분을동시에제공할수있는전통건강기능성발효유제품개발기술을확보할수있음. Ⅲ. 연구개발내용및범위 타락발효유의제품화기술개발 - 유산균주의선발및동정 - 선발균주의표준배합비선정 - 발효공정최적화 - 타락발효유시제품개발및관련품질확보

5 전통발효유타락의제조표준화및영양적우수성규명 - 막걸리를발효원으로한타락의표준화제법개발 - 타락의발효산물및영양성분분석 타락의기능성분석방법확립및 in vitro 기능성규명 - 타락의추출물제조법확립과분획 - 타락의기능성분석 : 면역증진, 멜라닌생성억제, 항비만, 암세포생장억제기능성 타락의응용제품개발 - 타락응용젤리류개발 ( 젤리, 푸딩 ) - 타락응용프로즌요구르트개발 ( 아이스크림 ) Ⅳ. 연구개발결과 타락발효유의제품화기술개발 가. 유산균주의선발및동정 생막걸리내의효모의농도는 cfu/ml 수준이었음. 유산균이분리된생막걸리는약 32종으로수집된막걸리의 36.8% 였으며, 유산균이있는경우농도는 cfu/ml 수준이었음. 이것으로보아막걸리의주요발효균주는효모와유산균임을알수있었음. 대량생산에용이한것은유산균이므로유산균주를우선검토하였음. 현재까지분리된균주는효모가약 130종, 유산균이약 200종정도임. 분리균주중 milk 배지에서커드형성유산균을동정한결과, Lactobacillus plantarum 7 종과 Lactobacillus paracasei ssp. paracasei 5 종이각각규명되었음. 몽골유제품균주 89종중 10% skim milk에서커드를형성하는균주는 8종이었으며 Enterococcus 균주가 7종, Lactobacillus 균주 1종이었음. 그중대량생산에적용하기용이한것은 Lactobacillus 속유산간균으로 Lactobacillus helveticus를배양테스트에이용하였음. 타락균주 47종에서는 10% skim milk에서커드를형성하는균주가없었음. 나. 선발균주의표준배합비선정 선별 10종균주중파일럿테스트배양물의 curd 형성균은총 5종으로 MKRL 5-8, M , M , M , M 이었으며, 이중총유산균수가가장높은균주는 MKRL 5-8이었음. 이균주를적용하여배양액을제조할수있도록배양조건을설정하였음. 막걸리유래유산균의성장이유성분만으로는안정적이지않아당류를첨가하여발효가잘되도록유도하였음. 또한전통적인타락과동일하게유성분에막걸리를넣은효과를내기위해쌀즙을넣고막걸리유래유산균으로발효하였음. 다. 발효공정최적화

6 배합비와같이원료를섞고 130~135 로 2~3초간가열 (UHT, ultra-high temperature sterilization) 하여살균하였음. 살균후 35~40 로냉각하여막걸리유래유산균 (MKRL 5-8, M , M , 모두 Lactobacillus paracasei subsp. paracasei) 을 0.02% 농도로접종하였음. 37 water bath에서 24시간동안배양하여발효시키고발효가끝난샘플은 50/150 bar로균질하여드링크타입의발효유로제조하였음. 라. 타락발효유시제품개발및관련품질확보 선발균주중 L. paracasei M 이 24시간배양후, 산도가가장낮았음. L. paracasei M 이신맛이가장적었으며, 당도와균수는선발균주 3종의유의적차이가없었으며, 농후발효유법적기준에적합하였음. L. paracasei M 으로발효시킨타락발효유의 100 g 당영양성분은일반적인드링크발효유에비해지방함량은낮고당함량은높은편임. L. paracasei M 으로발효시킨타락발효유로관능검사수행결과전체적인맛점수는 4.0점 /7점척도로중간수준이었음 (Fig. 8). 원인으로는향에대한기호도가낮고신맛이강하기때문이라고판단이됨. MKRL5-8의배양액및균체는여드름원인균인 P. acnes ATCC11828 과 P. acnes ATCC6919 의생장을저해하는효능이있음을확인하였음. 전통발효유타락의제조표준화및영양적우수성규명 가. 막걸리를발효원으로한타락의발효산물및영양성분분석 막걸리에서추출한 5종의 L. paracasei를이용해 24시간 37 에서타락을발효하며, 타락의 ph, 산도, 점도, 유기산, 유리당함량, 지방산, 무기질함량및미세구조를측정하였음. 발효시간이길어짐에따라타락의 ph는감소하였으며, 특히발효 12시간에는 ph가급감, 우유의등전점인 ph 4.6 부근으로감소, 발효 12시간부터커드를형성하였음을알수있었음. ph, 산도와점도는발효시간에따라서현저하게달라지나, 균종간의차이는크지않았음 생균수역시발효시간이경과함에따라증가하는경향을보이나, 24시간후에는균종간의차이가없었음. 유기산중젖산 (lactic acid) 함량은발효 12시간에급증한후발효시간에따라증가하는경향을보였음. 균종간에는균 5번샘플이다른균종에비해높은수치의 lactic acid를형성하였음을알수있었음. M 로발효한샘플이가장낮은유당함량을보였음. 발효 24시간샘플들을동결건조하여균종간의무기질과지방산을비교한결과, 무기질은샘플들간에큰차이가없었지만, M 로발효한샘플이다른샘플들에비해높은미네랄함량을보였음. 지방산결과에서는약 30% 의불포화지방산을함유하는것으로나타났음. 대조군샘플과발효타락샘플의미세구조를전자현미경으로관찰한결과발효타락샘플에서는균의몸체와함께 EPS(exo-polysaccharides, 균이생성하는다당체 ) 로추정되는점

7 질물이함께발견되었음. L. paracasei 가발효가진행되는동안생체기능성물질로알려진다당체의형성가능성을보여, 발효유타락이항암, 항궤양, 면역증강작용등인체내에 bioactive agent 로서의충분한역할을할수있을것이라예상됨. 특히 M 는다른균종들에비해다당류를더많이생성시킴을알수있었음. 타락의기능성분석방법확립및 in vitro 기능성규명 가. 타락의추출물제조법확립과분획 제1세부제조타락동결건조시료제공 (MKRL-5-8, M , M , M , M ) 타락동결건조시료를 n-hexane, chloroform, ethyl acetate, n-butanol, phosphate buffer로분획하여고형분회수 나. 면역증진기능성분석및기능성강화조건탐색 세포증식도측정을위한 MC116 cell, Jurkat cell 의배양조건확립 M 시료는 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml 농도에서 jurkat cell 의세포증식도를유의하게증가시킴 (p<0.05). 다. 타락의멜라닌생성억제기능분석 MKRL-5-8, M , M , M , M 타락시료는 100 μg/ml 농도에서 tyrosinase 역가의약 20% 를저해함. MKRL-5-8 시료, M 시료는 100 μg/ml 농도에서 α-msh에의하여촉진된 B16 F10 cell의멜라닌생성을유의적으로억제함. (p<0.05) 라. 타락의항비만기능성규명 지방전구세포 (3T3-L1) 배양조건, 지방세포분화조건, 지방생성유도조건수립 타락추출물 (5-8, 71-2) 은지방세포의지방생성도를유의하게억제함. 타락추출물 (5-8, 71-2) 은지방세포의 PPARγ, C/EBPα 발현량감소기능이없음. 타락추출물 (5-8, 71-2) 은쥐를이용한동물실험에서체중감소와혈중지질개선효능이있음. 마. 타락의암세포생장억제기능성규명 타락추출물 (5-8, 23-3, 71-2) 에서는 A549 세포 ( 폐암세포 ), HepG2 세포 ( 간암세포 ) 의 생장억제기능이발견되지않았음.

8 타락의응용제품개발 가. 타락응용젤리류개발 ( 젤리, 푸딩 ) 본실험에사용된타락은수운잡방 (Sueunjabbang Research Institute 2011) 과 Lim 등 (2013) 의타락제조법을참고한자연발효타락을이용하였음. 타락을이용한젤리류인과편, 양갱, 젤리, 푸딩의실험조리결과, 타락의사용량이비교적많고, 관능상특성이우수하다고생각되는젤리와푸딩에대한제조조건을확립하였음. 타락을이용한젤리와푸딩의일반성분분석결과, 젤리와푸딩모두타락의첨가량이많아질수록수분의함량은감소하였고, 조단백질, 조회분, 조지방의함량은유의적으로증가하였음. 젤리와푸딩모두타락의첨가량이많아질수록 ph는유의적으로감소하고산도는유의적으로증가하였으며, 당도는증가하였음. 타락 75% 의타락젤리의관능품질이가장우수한것으로판단됨. 푸딩제조시타락특유의신맛과향이긍정적인영향을미치는것으로판단되며, 이상의결과타락 75% 와 100% 의첨가가바람직한것으로생각됨. 나. 타락응용프로즌요구르트개발 ( 아이스크림 ) 본연구에서는 1세부과제 ( 한국야쿠르트 ) 를통해제조된상업용타락을이용하여디저트식품으로선호도가높은아이스크림제품류중유지방분 6 % 이상, 유고형분 16 % 이상에해당하는아이스크림규격에맞춰타락응용프로즌요구르트 ( 아이스크림 ) 의제조조건을확립하고자하였음. 타락첨가아이스크림의젖산균수는 (log CFU/g) 의수준으로나타났으며, 타락의첨가에따라유의적으로증가하였음. 전반적인기호도에서타락을가장많이첨가한 TI-48( 타락 48%) 이가장높게평가되었음. 본연구결과를토대로향후연구에서는타락을각 50% 첨가하여아이스크림류의식품공전상의기준규격에맞춰샤베트, 저지방아이스크림, 아이스크림등제품군별제조를통해타락을이용한아이스크림의최적제품을제안하고자함. Ⅴ. 연구성과및성과활용계획 1. 연구성과 가. 특허출원 본연구결과의산업재산권확보를위해막걸리분리유산균에대한특허 여드름원인균프 로피오니박테리움아크네스의생장억제효능을가지는락토바실러스파라카제이 HY7301

9 및이를유효성분으로함유하는제품 과응용제품에대한특허 타락을이용한젤리의제조방법및타락을이용한젤리, 타락을이용한푸딩의제조방법및타락을이용한푸딩, 타락을이용한아이스크림의제조방법및타락을이용한아이스크림 을특허출원하였음. 향후막걸리균주에대한특허 피부미백활성을가지는락토바실러스파라카제이 HY7301 의우유발효물 을추가출원할예정임. 여드름원인균프로피오니박테리움아크네스의생장억제효능을가지는락토바실러스파라카제이 HY7301 및이를유효성분으로함유하는제품, 피부미백활성을가지는락토바실러스파라카제이 HY7301의우유발효물 특허가등록될경우, 피부개선관련원료로서상기균주를적용한고부가가치 피부기능성발효유제품 개발이가능할것으로기대됨. 나. 학술대회발표 타락발효유의제품화기술개발관련다수의연구결과를국내학술대회에지속적으로발표하였음. - The effects of L. paracasei subsp. paracasei HY7301 isolated from Makgeolli on cell viability and synthesis of procollagen in human fibroblast. 다산컨퍼런스 ~ 명길선외 3인 - The Probiotic Characteristics of Lactobacillus paracasei isolated from Makgeolli. 한국식품과학회. P ~ 김수아외 3인 - Antagonistic Activity of Potent Probiotic Lactobacillus Strain against Acne Pathogens. 한국미생물학회 ~ 명길선외 3인 전통발효유타락의제조표준화및영양적우수성규명관련다수의연구결과를국내학술대회에지속적으로발표하였음. - Historical and Cultural Study on Korean Traditional Fermented Milk, Tarak. 한국식품과학회. P ~ 오사다사치코외 3인 - Effects of three Lactobacillus paracasei strains found in Korean traditional rice wine on nutritional and sensory characteristics of fermented milk. 한국식품과학회. P ~ 김상숙외 7인 - Study on exo-polysaccharides of cultured milk inoculated with Lactobacillus paracasei strains isolated form Korean traditional rice wine, Makgeolli. 한국식품과학회. P ~ 김은정외 6인 - Studies on Fermentation Characteristics of Tarak, Korean Traditional Fermented Milk, through the Analysis of Microbial Succession and Metabolites. 한국식품과학회. P ~ 정상은외 8인 타락의응용제품개발관련다수의연구결과를국내학술대회에지속적으로발표하였음. - 타락을이용한아이스크림류의품질특성. 한국식품조리과학회. 식품가공 FPP 고성희외 4 인

10 - 전통발효유타락을이용한디저트상품개발. 한국식품조리과학회. 식품조리 FCO 이경연외 4 인 다. 논문발표및개제 타락의응용제품개발관련된논문을 3건개제하였음 ( 비SCI). - 전통발효유타락을이용한젤리의품질특성. 한국식품조리과학회지. 29(5); (2013) - 전통발효유타락을이용한푸딩의품질특성. 한국조리학회지. 20(3); (2014) - 전통발효유타락을이용한아이스크림의품질특성. 한국조리학회지. 20(6) (2014) 향후비SCI급 2건, SCIE급 4건의논문을추가개제할예정임. 2. 활용계획 전통발효유타락의제조공정및기술개발 유산균을이용한전통기능성발효에관한기술축적및타락균발효유개발을위한균주의배양방법과발효조건및제조공정을습득한전문인력양성 기능성발효유로서의타락제품개발 외식산업을통한한식의디저트로서타락발효유의브랜드화 전통발효유타락의제품개발을통해전통발효식품에다양한콘텐츠제공 타락의응용제품개발을통해타락의수요에대한다각화및홍보자료구축 항암및항비만, 항동맥경화, 항당뇨, 항산화기능성검증실험기술을이용하여타락발효산물의생리활성물질연구에응용

11 SUMMARY ( 영문요약문 ) Ⅰ. Title Industrialization of Tarak, Korean Traditional Fermented Milk Product Ⅱ. Objectives and background We analyzed microbial fermentation of Tarak and its product to standardize traditional fermented milk using rice wine products, and also to identify the functional components. This allows the industrialization of Tarak through the development of applications. (1) Development of technologies for commercialization of Tarak fermented milk (2) Establishment of manufacturing standards and elucidation of nutritional excellence of traditional fermented milk - Tarak (3) Establishment of functional analysis methods for Tarak and elucidation of in vitro functionality (4) Developments of commercial products using Tarak The need for research (1) Cultural aspects - As the interest in health is being raised worldwide, the health promoting functionality of Korean foods has emerged. - As representatives of Korean fermented foods, kimchi, fermented fish, miso, and red pepper paste were already recognized for scientific excellence. - Suunjapbang, the oldest cooking book written in Chinese letter, is a valuable resource to find the food recipes from late Goryeo Dynasty to early Joesun Dynasty. - In Suunjapbang, the recipe of Tarak using milk and Takju exists and it suggested that the fermented milk was consumed at that time. - Though the traditional fermented milk existed in Korea, the manufacturing process

12 and the quality characteristics were not revealed and systematic research has started recently. - Fermented milk products are categorized according to their fermentation characteristics and manufacturing process. Functions such as lowering cholesterol concentration and obesity were already confirmed. - Tarak is a unique traditional fermented food of Korea. However, unlike other traditional fermented foods, milk of animal origin was utilized as the ingredient. (2) Industry Aspects - Annual sales of yogurt products in domestic market is about 1.4 billion USD and functional yogurt have 40% market share of the total market. - Fermented milk is considered to improve symptoms such as obesity, hypertension, diabetes, and heart disease. New product development trends are low-fat, non-fat, vitamin enhanced, low cholesterol, low sodium, no caffeine, and no sugar in various ways. - European fermented milk products are mainly focused on the particular disease. Probiotic lactic acid bacteria such as L. casei, L. acidophilus, and bifidobacteria are used for commercial products (Korea Health Industry Development Institute, 2004). - Either combination of lactic acid bacteria and yeast or only lactic acid bacteria is utilized to ferment milk. Recently, the use of combination of lactic acid bacteria and yeast culture draws attention to the public. - In this research, the commercialization of new traditional fermented milk, Tarak, is expected to contribute to the company revenue. (3) Social aspects - Knowledge accumulation on traditional functional fermentation using the lactic acid bacteria is expected. - Tarak can be branded as a desert. - The consumption of dairy products can be increased by improving taste and functionality of dairy products. - Increase in milk demand will contribute to revenue growth of dairy farmers and dairy companies. (4) Technical aspects - Standardized manufacturing process for Tarak in large scale will be developed. - By improving the flavor and functionality of the milk, it is possible to achieve traditional functional dairy product technology with production of bio-active molecules.

13 III. Contents and scope of this study Development of commercial production technology for Tarak fermented milk - Selection and identification of lactic acid bacteria - Development of standard recipe with starter microorganism - Fermentation process optimization - Development of Tarak fermented milk test product and quality assurance Manufacturing standards and nutritional excellence of traditional fermented milk Tarak - Development of standardized method for tarak by the fermentation of rice wine - Analysis of fermentation products and nutrient Establishment of functional analysis methods for Tarak and examination of in vitro functionality - Establishment of manufacturing Tarak extracts and fractions - Functional analysis of Tarak: immune stimulation, inhibition of melanin, antiobesity, cancer cell growth inhibition Applications development of Tarak - Development of Tarak applications (jelly, pudding) - Development of frozen yogurt using Tarak (ice cream) IV. Results of research and development Development of commercialization technologies for Tarak fermented milk 1. Selection and identification of lactic acid bacteria The concentration of yeast was cfu/ml in raw rice wine. The lactic acid bacteria were isolated from 32 kinds of raw rice wine products. The concentration of lactic acid bacteria was cfu/ml. Yeast and lactic acid bacteria are the major strains for fermentation of rice wine. Lactic acid bacteria are suitable for production in large scale. The isolated strains from rice wine products were about 130 strains of yeast and about 200 strains of lactic acid bacteria with curd formation. Results of identification of lactic acid bacteria were 7 strains of Lactobacillus plantarum and 5 strains of Lactobacillus paracasei ssp. paracasei. Five kinds of enterococcus and one kind of lactobacillus strains from 89 Mongolia dairy products showed curd formation in 10% skim milk. From isolated strains,

14 Lactobacillus helveticus was used for fermentation test. Tarak strains (n=47) did not show curd formation in 10% skim milk. 2. Standard recipe formulation with selected strains MKRL 5-8, M13-2-3, M , M , M showed curd formation in pilot tests. From tested strains, MKRL 5-8 obtained the highest viable count results. The fermentation condition was optimized for the strain. Lactic acid bacteria derived from rice wine were not suitable for growing with milk ingredient only. Addition of sugar induced better fermentation. Also, rice soup was used for fermentation. 3. Fermentation Process Optimization Ingredients were mixed and sterilized for 2-3 seconds at degree (UHT, Ultra High Temperature sterilization). After that, it was cooled to degree and inoculated in the concentration of 0.02% with Lactobacillus paracasei subsp. paracasei strains (MKRL 5-8, M , M ) from Korean rice wine. It was fermented for 24 hours in 37 degree using a water bath and homogenized at 50/150 bar for the production of drink type yogurt. 4. Development of Tarak fermented milk test products and establishment of quality control The acidity of L. paracasei M after 24 hours of incubation showed the lowest results in comparison to other strains. L. paracasei M was the lowest in the level of sour taste. Sugar contents and the viable counts of bacteria showed no significant difference between three selected strains. Nutrients per 100 g of Tarak fermented milk with L. paracasei M were lower in fat contents and higher in sugar contents compared to the typical fermented milk products. The sensory evaluation of Tarak using L. paracasei M was around the normal level with 4.0 in 7.0 point scale(fig. 8). It was attributed to the low preference for flavor and the strong acidity. Incubated MKRL 5-8 and its fermentation products were effective to inhibit the growth of P. acnes ATCC11828 and P. acnes ATCC6919. Establishment of manufacturing standards and elucidation of nutritional excellence of traditional fermented milk Tarak 1. Nutritional analysis of the Tarak and fermentation products Five L. paracasei strains were used for fermentation for 24 hour at 37 degree. Acidity, ph, viscosity, organic acid concentration, free sugar content, fatty acid,

15 minerals were assayed and microstructure of Tarak was also observed. The ph of Tarak was decreased according to the incubation period and was dramatically decreased at 12hr to ph 4.6 (isoelectric point of milk). Curd was produced in the 12 hours of fermentation. Acidity, ph, and viscosity can vary significantly depending on the fermentation time. However, it was not great differences between the species. The number of live cells also show a tendency to increase according to the length of fermentation time. But it did not show significant differences between the species after 24 hours of incubation. Lactic acid contents was sharply increased in 12 hours, after that, slowly increased according to the fermentation time. M showed the highest level of lactic acid. Tarak incubated for 24 hours was freeze-dried. Minerals and fatty acids in the samples were analyzed. There was no difference between mineral contents of samples. The sample fermented with M showed a slightly higher mineral content than the other samples. And the samples contained an unsaturated fatty acid of about 30%. Microstructure of control and Tarak were observed by electron microscopy and EPS (exo-polysaccharide) was investigated. L. paracasei produced polysaccharide recognized as biological functional materials in the culture. So, Tarak might be used as a bioactive agent for anti-cancer, anti-inflammation, and immune stimulation. In particular, M produced more polysaccharide than other strains. Establishment of functional analysis methods in the Tarak and elucidation of in vitro functionality 1. Development of manufacturing method for Tarak extracts and fractions Freeze-dried samples manufactured in R&D center of Korea Yakult Co., Ltd. (MKRL-5-8, M , M , M , M ) were used. Freeze-dried samples were fractionated using n-hexane, chloroform, ethyl acetate, n-butanol, and phosphate buffer. 2. Analysis of immune stimulation and investigation for the condition of functionality improvement The establishment of culture conditions of MC116 cell, Jurkat cell for measurement of the degree of cell proliferation. Tarak sample(m ) significantly increased the cell proliferation of Jurkat cells in 10, 50, 100, and 200 μm/ml concentration (p<0.05).

16 3. Analysis for inhibition of melanin production by Tarak Tarak samples (MKRL-5-8, M , M , M , M ) reduced tyrosinase activity in 20% at 100 μg/ml concentration. Tarak samples(mkrl-5-8, M ) significantly inhibited the generation of melanin by B16F10 cell accelerated by α-msh in 100 μg/ml concentration (p<0.05). 4. Analysis of Tarak for antiobesity function Establishment of Preadipocytes(3T3-L1) culture conditions, adipocyte differentiation conditions, and lipogenesis induced conditions Tarak extract(5-8, 71-2) significantly inhibited the degree of adipocyte lipogenesis Tarak extract(5-8, 71-2) did not reduce the expression level of PPARγ, C/EBPα in the adipocytes. Tarak extract(5-8, 71-2) reduced body weight and improved the blood lipids profile in vivo test. 5. Cancer cell growth inhibition analysis of Tarak Tarak extract(5-8, 23-3, 71-2) showed no growth inhibitory function for A549 cell (Lung cancer cells), and HepG2 cell (Hepatoma cells). Development of commercial products using Tarak 1. Applications of Tarak for jelly type product(jelly, pudding) Tarak, which were traditionally produced according to Suunjapbang (Suunjapbang Research Institute,2011) and Lim (2013), were used for this research. We tested the possible application of Tarak for Gwapyeon, sweet jelly of beans, jelly, and pudding. As a result, manufacturing procedures with excellent sensory properties were established. We analyzed the general composition of the pudding and jelly with Tarak. As more Tarak was used, the less moisture contents were found. However, crude protein, ash, and crude fat were significantly increased. As the Tarak quantity increases, the ph is decreased significantly, Conversely acidity and sugar contents were significantly increased. The combination of 75% Tarak and jelly resulted in the best acceptance. A sour taste and flavor of Tarak goes well with pudding of Tarak applications. The 100% and 75% of Tarak in pudding were suitable. 2. Application of Tarak for frozen yogurt development (ice cream) The legal standard for ice cream defines the contents of milk fat as more than 6% and milk solids as 16% or more. Frozen yogurt manufacturing procedures had been

17 established according to the above specification. The number of lactic acid bacteria in Tarak ice cream was (logCFU/g) level. Bacterial counts were significantly increased according to the amount of Tarak. In the case that contents of Tarak was 48%, the taste of Tarak application showed the highest acceptance. Based on the present findings, we propose to include 50% of Tarak to manufacture ice cream products. V. The application of research and development results 1. Research results A. Patent To secure a industrial property right on this research result for lactic acid bacteria from makgeolli, we have applied patents of antimicrobial property of Lactobacillus paracasei HY7301 and comprising product against Propionibacterium acnes, Method of manufacturing jelly using Tarak, Method of manufacturing pudding using Tarak and pudding using Tarak, and Method of manufacturing ice cream using Tarak and ice cream using Tarak. In addition, we will apply for the patent for skin whitening effect of fermented milk using Lactobacillus paracasei HY7301. We anticipate that development of functional fermented milk for skin improvement with HY7301 will be possible when the patents of antimicrobial property of Lactobacillus paracasei HY7301 against Propionibacterium acnes and fermented milk of L. paracasei HY7301 with skin whitening effect are registered. B. Conference presentation We continuously published research results of commercialization of Tarak in domestic conferences. - The effects of L. paracasei subsp. paracasei HY7301 isolated from Makgeolli on cell viability and synthesis of procollagen in human fibroblast. Dasan conference ~ Gil Sun Myung et al. - The probiotic characteristics of Lactobacillus paracasei isolated from Makgeolli. Korean society of food science and technology. P ~ Su a Kim et al. - Antagonistic activity of potent probiotic Lactobacillus strain against acne pathogens. The microbiological society of Korea ~ Gil Sun Myung et al. We also continuously published results for standardization of manufacturing method and nutritional value of traditional fermented milk Tarak in domestic conferences.

18 - Historical and cultural study on Korean traditional fermented Milk, Tarak. Korean society of food science and technology. P ~ Sachiko Osada et al. - Effects of three Lactobacillus paracasei strains found in Korean traditional rice wine on nutritional and sensory characteristics of fermented milk. Korean society of food science and technology. P ~ Sang Suk Kim et al. - Study on exo-polysaccharides of cultured milk inoculated with Lactobacillus paracasei strains isolated form Korean traditional rice wine, Makgeolli. Korean society of food science and technology. P ~ Eun Jung Kim et al. - Studies on fermentation characteristics of Tarak, Korean traditional fermented milk, through the analysis of microbial succession and metabolites. Korean society of food science and technology. P ~ Sang Eun Jung et al. Application products of Tarak were reported in domestic conferences. - Quality characteristics of ice cream using Tarak. The Korean society of food and cookery science. food processing. FPP Sung Hee Go et al. - Development of dessert products using traditional fermented milk Tarak. Food and cookery science. Food cooking. FCO Kyung Yeon Lee et al. C. Published papers We published three papers for application products of Tarak. - Quality characteristics of jelly using traditional fermented milk Tarak. The Korean society of food and cookery science. 39(5); (2013) - Quality characteristics of pudding using traditional fermented milk Tarak. The culinary society of Korea. 20(3); (2014) - Quality characteristics of ice cream using traditional fermented milk Tarak. The culinary society of Korea. 20(6) (2014) We will publish two non-sci and four SCIE papers as research achievements. 2. Application plan Development of manufacturing process of traditional fermented milk Tarak Knowledge accumulation of traditional fermentation using lactic acid bacteria and training of specialist. Development of Tarak products as functional fermented milk products. Branding of the Tarak Korean desserts. Development of various fermented foods through development of Tarak products. Production of promotional literature through application products of Tarak Studies on biological activity of Tarak using results from assessment for prevention of anticancer, antiobesity, antiatherogenic, antidiabetic, and antioxidant.

19 CONTENTS ( 영문목차 ) SUMMARY 2 Chapter 1. Overview of the study 20 Chapter 2. Current status of related technologies in domestic and foreign countries 23 Chapter 3. Results 26 Section 1. Development of commercialization technologies for Tarak 27 Section 2. Establishment of manufacturing standards and elucidation of nutritional excellence of traditional fermented milk Tarak 55 Section 3. Establishment of functional analysis methods and in vitro examination of Tarak 101 Section 4. Development of commercial Tarak products and determination of the value in food culture 117 Chapter 4. Achievement and contribution to relevant fields 139 Chapter 5. Result and application plan of the study 142 Chapter 6. Overseas technical information and knowledge acquired from the study 144 Chapter 7. References 146

20 목 차 요약문 2 제 1 장연구개발과제의개요 20 제 2 장국내외기술개발현황 23 제 3 장연구개발수행내용및결과 26 제 1 절타락발효유의제품화기술개발 27 제 2 절전통발효유타락의제조표준화및영양적우수성규명 55 제 3 절타락의기능성분석방법확립및 in vitro 기능성규명 101 제 4 절타락의응용제품개발및식문화적가치규명 117 제 4 장목표달성도및관련분야에의기여도 139 제 5 장연구개발성과및성과활용계획 142 제 6 장연구개발과정에서수집한해외과학기술정보 144 제 7 장참고문헌 146

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51 μ μ μ μ μ μ μ μ μ μ

52 μ μ μ μ μ μ

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56 Sample-01 Sample-02 Sample-03 Sample ph Time(Hr)

57 Titratable acidity(%) Sample-01 Sample-02 Sample-03 Sample Time(Hr)

58 Sample-01 Sample-02 Sample-03 Sample Brix(%) Time(Hr)

59 Sample-01 Sample-02 Sample-03 Sample cp Time(Hr)

60 Log(CFU)/ml 8 7 Sample-01 Sample-02 Sample-03 Sample Time(Hr) Sample-01 Sample-02 Sample-03 Sample-04 Log(CFU)/ml Time(Hr)

61 Amount(mg/L) citric acid lactic acid succinic acid acetic acid EtOH Time(Hr) Amount(mg/L) citric acid lactic acid succinic acid acetic acid EtOH Time(Hr)

62 citric acid lactic acid succinic acid acetic acid EtOH Amount(mg/L) Time(Hr) Amount(mg/L) citric acid lactic acid succinic acid acetic acid EtOH Time(Hr)

63 60 50 Free sugar(mg/ml) lactose glucose fructose Time(Hr) 60 lactose 50 Free sugar(mg/ml) Time(Hr)

64 60 50 lactose glucose Free sugar(mg/ml) Time(Hr) 60 lactose 50 Free sugar(mg/ml) Time(Hr)

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69 μ μ

70 μ μ μ

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75 a b c d

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86 ω ω ω

87 m m m

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89 Band No. length closest type strain similarity Pseudomonas congelans DSM 14939(T) Lactobacillus paracasei subsp. paracasei ATCC 25302(T) Serratia proteamaculans DSM 4543(T) 99.35

90 Band No. Length Closest type strain Similarity Clostridium saccharoperbutylacetonicum N1-4(T) Lactobacillus paracasei subsp. paracasei ATCC 25302(T) Pseudomonas graminis DSM 11363(T) Band No. Length Closest type strain Similarity Lactobacillus graminis DSM 20719(T) 98.71

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98 Similarity Relative 시료 Closest type strain (%) abundance Pseudomonas psychrotolerans C36(T) /11 A 경장주 ( 원 ) Enterococcus durans CECT 411(T) /11 Lactobacillus paracasei subsp. tolerans JCM 1171(T) /11 Leuconostoc citreum ATCC 49370(T) /11 Lactobacillus pentosus JCM 1558(T) /11 B 오정주 ( 원 ) Enterococcus durans CECT 411(T) /8 Lactobacillus pentosus JCM 1558(T) /8 Leuconostoc citreum ATCC 49370(T) /11 Weissella cibaria KACC 11862(T) /11 C 술쩨게 ( 원 ) D 끓인 것 ( 부원 ) E 데운것 ( 부원 ) F 경장주 Pediococcus pentosaceus DSM 20336(T) /11 Streptococcus salivarius subsp. salivarius ATCC 7073(T) /11 Lactobacillus coryniformis subsp. torquens KCTC 3535(T) /11 Lactococcus lactis subsp. lactis NCDO 604(T) /11 Lactococcus lactis subsp. lactis NCDO604(T) /12 Leuconostoc citreum ATCC 49370(T) /12 Lactococcus lactis subsp. lactis NCDO 604(T) /12 Lactobacillus paracasei subsp. tolerans JCM 1171(T) /11 Lactobacillus fermentum NBRC 3956(T) /11 G 오정주 H 고리초 Lactococcus lactis subsp. lactis NCDO 604(T) /6 Lactobacillus fermentum NBRC 3956(T) /6 Pediococcus pentosaceus DSM 20336(T) /6 Lactobacillus fermentum NBRC 3956(T) /12 Lactococcus lactis subsp. lactis NCDO604(T) /12 Lactobacillus paracasei subsp. paracasei ATCC 25302(T) I /12 안동막걸리 Lactobacillus paracasei subsp. tolerans JCM 1171(T) /12 J Lactobacillu spentosus JCM 1558(T) /11

99 우유 Lactobacillus paracasei subsp. tolerans JCM 1171(T) /11 Pediococcus pentosaceus DSM 20336(T) /11 Enterococcus durans CECT 411(T) /11

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104 μ μ μ μ

105 μ μ μ μ μ μ μ μ

106 μ μ μ μ α μ μ μ μ μ μ μ

107 α μ α α

108 μ μ μ

109 μ μ

110 μ

111 γ α γ α γ α μ

112 γ α β γ α μ α μ α γ α γ

113 α 4 혈중 AST 역가, ALT 역가

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129 성분규격 (%) 유지방 조지방 MSNF ( 무지유고형분 ) 유고형분 아이스크림 6% 이상 16% 이상 아이스밀크 2% 이상 7% 이상 샤베트 2% 이상 저지방아이스크림 2% 이하 10% 이상 비유지방아이스크림 5% 이상 5% 이상 성상 아이스크림, 저지방아이스크림고유의향미를가지고이미 이취가없어야한다. 아이스밀크, 샤베트, 비유지방아이스크림고유의향미를가지고이미 이취가없어야한다. 유지방 (%) 세균수 6.0 이상 ( 단저지방아이스크림의경우 조지방 2.0 이하 ) 검사시료를녹인액체 1 ml당 100,000 이하 ( 단, 유산균함유제품, 발 효유함유제품의경우유산균수는제 외한다 ) 2.0 이상 ( 아이스밀크에한한다 ) 검사시료를녹인액체 1 ml당 50,000 이하 ( 단, 유산균함유제품, 발효유함 유제품의경우유산균수는제외한다 ) 대장균군 n=5, c=2, m=10, M=100 n=5, c=2, m=10, M=100 유산균수 표시량이상 ( 단, 유산균함유제품에 한한다 ) 표시량이상 ( 단, 유산균함유제품에 한한다 )

130 탄수화물 18g 당류 16g 단백질 3g 지방 1.8g 포화지방 1.2g 트랜스지방 0g

131 재료 시료 1) Control TI- 16 TI-32 TI-48 타락 우유 생크림 탈지분유 설탕 물엿 안정제 물

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146 국가기업명대표제품특성특이사항 한국야쿠르트 쿠퍼스 헛개나무열매에서추출한분말함유, 공격적인마케팅 국내발효유의대표적인기업 한국 뉴팜 셀바이오텍 온가족혼합유산균뉴팜혼합유산균베베락혼합유산균 듀오락 유산균을포함한다양한웰빙관련제품 한국과일본에서특허및출원, 유산균관련자체기술력확보 한미약품메디락 S 장용캡슐장질환의원인개선 대장암을치료하는유산균개발을목표로함 ( 주 ) 바이오리듬 코팅김치유산균 유산균의생존률과기능및활성유지 씨티바이오 김치로부터유산균분리, 과일로배양 AI 바이러스억제유산균관련특허획득 해피패밀리 해피멜츠 면역력향상을위한유아용제품, 약 20 억마리유산균함유 미국 스토니필드팜 뉴트리카 여러균주혼합, 다양한효능갖춤 Coromega 오메가 3 지방산 제품낱개포장하여소비자편리성확보및취향고려 일본캐나다유럽이탈리아 기린맥주그룹 KW 유산균 가열처리균체, 알레르기개선효과가짐 랄푸드코팅유산균초콜릿에사용 Sitia-Yomo SPA 프로바이오틱제품 DIY 형태로제작하여소비자취향고려 기린맥주와고이와이유업이공동개발한유산균에총력을기울임

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