발간등록번호 11-1541000-000771-01 비만억제효과를갖는키토올리고당의건강기능식품개발 고지방식이쥐와비만마우스를이용한키토산올리고당의비만억제효능연구 키토올리고당의항비만효과
제출문 농림수산식품부장관귀하 이보고서를 비만억제효과를갖는키토올리고당의건강기능식품개발에관한연구 과 제 ( 세부과제 고지방식이쥐와비만마우스를이용한키토산올리고당의비만억제효능연구에 관한연구 / 키토올리고당의항비만효과에관한연구 ) 의보고서로제출합니다. 2010 년 11 월 30 일 주관연구기관명 : ( 주 ) 키토라이프주관연구책임자 : 정특래세부연구책임자 : 양현필연구원 : 허지연연구원 : 김원석협동연구기관명 : 대구대학교협동연구책임자 : 윤종원협동연구기관명 : 바이오푸드네트워크사업단협동연구책임자 : 권미란 - 1 -
연구개발보고서초록 - 2 -
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요약문 Ⅰ. 제목 비만억제효과를갖는키토올리고당건강기능식품개발 Ⅱ. 연구개발의목적및필요성최근비만인구의증가와더불어웰빙열풍에따라다이어트에관한관심이높아지면서시중에다양한다이어트식품이유통되고있으며전체건강기능식품시장에서다이어트제품시장이차지하는비율도매우높다. 그러나이들제품대부분이식약청에서요구하는건강기능식품의기준에들지않는제품이대부분이며이에따라다이어트제품에대한소비자의이미지는좋지않은상태이다. 따라서 in vivo 및임상실험을통하여제품의기능이증명되고또한개별인증의건강기능식품으로등록이된다이어트식품이개발된다면소비자들은믿고제품을복용가능하게될것이다. 실제로다이어트식품의부작용으로인해접수된한국소비자보호원의상담건을보면대부분이일반식품에체중감량등의표현을강조한제품이거나부작용을유발할수있는부적격의성분이포함된것이대부분이었다. 따라서다이어트식품은어느식품분야보다더과학적인근거가절실히필요한부분이라할수있으며이는정확한동물실험과임상실험에의해서만얻어질수있다고생각하며, 키토올리고당을특정분자량으로분취한다음동물실험과임상실험을통해비만억제효과를확인하여건강기능식품개별인정신청과제품화를모색하고자한다. Ⅲ. 연구개발내용및범위 - 각종분리막을이용하여분자량별키토산올리고당최적조건확립 - 분리된분자량별키토산올리고당의분자량분포확인 - 고지방식이쥐와비만마우스를이용한키토산올리고당의비만억제활성확인 - 키토올리고당의항비만효과의유효성평가를위한인체적용시험 - 최적조건하에서분자량별키토산올리고당의대량생산화모색 - 건강기능식품개별인정신청및제품화 Ⅳ. 연구개발결과 - 4종류 (1K이하, 1-3K, 3-5K 및 5K이상 ) 분자량별키토산올리고당의분리조건을확립 - 광산란검출기가장착된겔투과크로마토그래피 (SEC-MALS GPC) 를이용하여분자량분포도를확인 - 키토산올리고당처리군에서 4주간식이, 체중감소효과 12% 로지방세포의지질대사개선효과확인 - 지질대사개선효과관련바이오마커 8종발견 - 유효성평가분석결과 BMI의감소효과는부족하였으나복부지방 ( 내장지방 + 피하지방 ) 의감소효과를확인 - 4 -
- BMI 30kg/ m2이상인고도비만과허리둘레남녀각각 90cm, 85cm이상및내장지방형복부비만에서대조군과대비하여유의한지방감소효과를보임 - 분자량별 Millipore membrane을이용하여대량생산체계구축함 - 건강기능식품개별인정신청 Ⅴ. 연구성과및성과활용계획 - 복부지방 ( 내장지방 + 피하지방 ) 의감소효과를서울성모병원임상결과를통해확인하였으며, 국외논문 2편투고 (Plasma proteome analysis for anti-obestiy and anti-diabetic potentials of chitosan oligosaccharide in ob/ob mice, Proteomics, 9(8), pp2149~2162, 2009/ Proteomic analysis in adipose tissue of ob/ob mice in response to chitosan oligosaccharides treatment, Biotechnology and Bioprocess Engineering 15(4), pp559~571, 2010) 와특허출원 1편 ( 키토산올리고당을활성성분으로포함하는항비만용조성물, 출원번호 10-2009-0101785) 의성과를거두었으며, 키토올리고당의기능성표기확대차원에서개별인정을신청하여비만억제기능성식품과원료를상품화하여침체된키토올리고당시장을확대하고자하며, 중소바이오업체에기초연구에서제품개발, 출시및매출전단계에성공적인모델을제시코자한다. - 5 -
SUMMARY Ⅰ. Title Development of Health functional Food of chitooligosaccharides with obestiy inhibitory effect Ⅱ. Purpose and necessity of the development With the recent rise in obesity and well-being craze resulting in more concern on diet, a wide variety of diet foods are on the market and their share in the total health foods market is already high. Most of the diet foods, however, fall short of the standards for health foods required by Korean Food and Drug Administration (KFDA) and accordingly, consumers impression on diet foods are not so favorable. Hence, if the functions of diet foods are verified via in vivo and clinical tests, and individual registrations are obtained as functional health foods, consumers may take the foods with confidence. According to the complaints on diet foods received by The Korea Consumer Agency, most of the cases are about general foods which are used to deceive consumers as diet foods by exaggerating their weight loss effects, and foods containing inappropriate ingredients that may cause adverse effects on health. As a matter of fact, development of diet foods requires stricter scientific basis than that of any other foods, and it can be attained only through rigorous animal experiments and clinical tests. We want to manufacture diet foods containing chitosan oligosaccharides that have inhibiting effects against obesity confirmed by animal experiments and clinical tests, and then obtain individual registrations as functional health foods. Ⅲ. Contents and scope of research and development - Establishment of an optimal condition for chitosan oligosaccharides by using various separation membranes - Confirmation of molecular-weight distribution of chitosan oligosaccharides by separated molecular weight - Confirmation of inhibiting activities of chitosan oligosaccharides against obesity by using mice put on a fatty diet and obese mice. - In vivo tests in humans to evaluate the effectiveness of anti-obesity effects of chitosan oligosaccharides against obesity - Method of mass producing chitosan oligosaccharides by molecular weight under the optimal condition - Application for individual registrations as functional health foods and production of - 6 -
functional health foods Ⅳ. Results of research and development - Established 4 kinds (1K and less, 1-3K, 3-5K and 5K and more) of separation condition for chitosan oligosaccharides by molecular weight - Confirmed a distribution chart of molecular weight by using a gel-permeation chromatography installed with a light scattering detector - Obtained clinical test results of decrease in weight by 12% from the oligosaccharides-treated group after a 4-week diet program - Confirmation of improvement effect on lipid metabolism of the fat cell - Detection of Biomarker of the eight kind with improvement effect on lipid metabolism - Based on the effectiveness evaluation, confirmed fat reduction effects on the abdomen (visceral fat + subcutaneous fat), even though body mass index (BMI) reduction was insufficient - Confirmed significant fat reduction effects on the morbid obesity group whose BMI is 30 kg/m 2 or over, and the male and female groups whose waists are 90 cm or more and 85 cm or more, respectively - Set up a mass production system by molecular weight by using Milipore membranes - Application for individual registrations as functional health foods Ⅴ. Research performances and our future plan to make a use of them The fat reduction effects on the abdomen were confirmed by the clinical tests organized by Seoul Catholic Medical Center. We could come up with an English research paper (Plasma proteome analysis for anti-obesity and anti-diabetic potentials of chitosan oligosaccharide in ob/ob mice, Proteomics, 9(8), pp2149~2162, 2009/ Proteomic analysis in adipose tissue of ob/ob mice in response to chitosan oligosaccharides treatment, Biotechnology and Bioprocess Engineering 15(4), pp559~571, 2010) and a Korean patent application (Anti-obesity composition containing chitosan oligosaccharides as active ingredients, #10-2009-0101785). We plan to apply for individual registrations to expand the opportunity to mark the functionality of chitosan oligosaccharides and to reinvigorate the sluggish chitosan oligosaccharides market. In addition, we try to provide a successful model of full-scale diet foods business including preliminary research, development, launching and sale of products of a small and medium sized company. - 7 -
CONTENTS Chapter 1 An overview of research and development projects... 11 Chapter 2 The current status of research and development in Korea and abroad... 12 Chapter 3 Details and results of research and development... 15 3. 1. Details of research... 15 3. 1. 1. Optimal condition for chitosan oligosaccharides by molecular weight.. 15 3. 1. 2. Confirmation of the distribution of the molecular weight of chitosan oligosaccharides by molecular weight... 16 3. 1. 3. Confirmation of inhibiting activities of chitosan oligosaccharides against obesity by using mice put on a fatty diet and obese mice... 16 3. 1. 4. Analysis on proteome in major organs of test animals... 16 3. 1.5. Investigation on the inhibiting mechanism of chitosan oligosaccharides against obesity via analysis on proteome... 17 3. 1. 6. Evaluation on in vivo tests in humans to evaluate the effectiveness of anti-obesity effects of chitosan oligosaccharides against obesity... 17 3. 1. 7. Application for individual registrations as functional health foods and production of functional health foods... 19 3. 2. Results of research... 21 3. 2. 1. Separation condition for chitosan oligosaccharides by molecular weight... 21 3. 2. 2. Average molecular weight of chitosan oligosaccharides by molecular weight... 21 3. 2. 3. Confirmation of inhibiting effects of chitosan oligosaccharides against obesity... 29 3. 2. 4. Analysis on proteome in major organs of test animals... 29 3. 2. 5. Investigation on the inhibiting mechanism of chitosan oligosaccharides against obesity via analysis on proteome... 41 3. 2. 6. Evaluation on in vivo tests in humans to evaluate the effectiveness of anti-obesity effects of chitosan oligosaccharides against obesity... 49-8 -
3. 2. 7. Application for individual registrations as functional health foods and production of functional health foods... 50 Chapter 4 Ratios of accomplishment and contribution to related fields... 51 Chapter 5 The outcome of research and development... 53 Chapter 6 Overseas information on science and technology collected in the process of research and development... 54 Chapter 7 References... 55 Appendix. Clinical test Report Animal test Report - 9 -
목 차 제 1 장 연구개발과제의개요... 11 제 2 장 국내외기술개발현황... 12 제 3 장 연구개발수행내용및결과... 15 3. 1. 연구내용... 15 3. 1. 1. 분자량별키토산올리고당최적조건... 15 3. 1. 2. 분자량별키토산올리고당분자량분포확인... 16 3. 1. 3. 고지방식이쥐와비만마우스를이용한키토산올리고당의비만억제활성 확인... 16 3. 1. 4. 실험동물의주요조직별단백체분석... 16 3. 1. 5. 단백체분석을통한키토산올리고당의비만억제작용기전규명... 17 3. 1. 6. 키토올리고당의항비만효과의유효성평가를위한인체적용시험평가... 17 3. 1. 7. 건강기능식품개별인정신청및제품화... 19 3. 2. 연구결과... 21 3. 2. 1. 분자량별키토산올리고당분취조건... 21 3. 2. 2. 분자량별키토산올리고당의평균분자량... 21 3. 2. 3. 키토산올리고당의비만억제효능확인... 29 3. 2. 4. 실험동물의주요조직별단백체분석... 29 3. 2. 5. 단백체분석을통한키토산올리고당의비만억제작용기전규명... 41 3. 2. 6. 키토올리고당의항비만효과의유효성평가를위한인체적용시험평가... 49 3. 2. 7. 건강기능식품개별인정신청및제품화... 50 제 4 장 목표달성도및관련분야에의기여도... 51 제 5 장 연구개발성과및성과활용계획... 53 제 6 장 연구개발과정에서수집한해외과학기술정보... 54 제 7 장 참고문헌... 55 별첨. 임상시험보고서 동물실험보고서 ( 자사추가동물실험 ) - 10 -
제 1 장연구개발과제의개요 - 11 -
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Thermometer Pump Prefilter, centrification UF membrane Pump Pump Stirrer Enzyme reactor NaOH sol. tank Pump Reservoir tank NF membrane Water bath Feed in Storage Tank Storage Tank Storage Tank Storage Tank MWCO30,000 MWCO5,000 MWCO3,000 MWCO1,000 Pump Pump Pump Pump (MW5,000Da higher) (MW5,000Da~3,0 00Da) (MW3,000Da~1,0 00Da) (MW1,000D lower) 그림 1. 분자량별키토산올리고당제조모식도 - 18 -
건강기능식품기능성원료인정신청처리절차 구비서류 건강기능식품기능성원료인정신청서 전자문서로된신청서포함 제출자료 부 원본 부포함 제출자료를수록 개 제품또는시제품및기능성분 또는지표성분 표준품 - 19 -
국내건강기능식품검사기관이발행한시험성적서 제출자료 제출자료전체의총괄요약본 기원 개발경위 국내 외인정및사용현황등에관한자료 제조방법및그에관한자료 원료의특성에관한자료 기능성분 또는지표성분 에대한규격및시험방법에관한자료 유해물질에대한규격및시험방법에관한자료 안전성에관한자료 기능성내용및그에관한자료 섭취량 섭취시주의사항및그설정에관한자료 의약품과같거나유사하지않음을확인하는자료 주의사항 제출자료를생략하는경우에는그사유를구체적으로기재하여야함 모든제출자료는원문과요약본을함께제출하여야하며 영어이외의외국어자료는한글번역문을제출하여야함신청기관 식품의약품안전청소비자담당관실 처리부서 영양기능식품기준과 수수료 원 수입인지 처리기간 신규원료 일 기능성추가 일 - 20 -
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반응시간 키토산 키토산 키토산 키토산 - 22 -
표 분자량별키토올리고당수율표 - 23 -
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Control CO-treated Vimentin Aldehyde reductase IDO RABP GST SOD - 42 -
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WAT (8% PAGE) MW (kda) WAT (12% PAGE) MW (kda) 240 140 240 140 100 100 72 72 50 50 35 35 25 25 15 ph3 ph10 ph3 ph10 BAT (8% PAGE) MW (kda) 240 140 BAT (12% PAGE) MW (kda) 240 140 100 100 72 72 50 50 35 25 35 15 25 ph3 ph10 ph3 ph10-47 -
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주 의 1. 이보고서는농림수산식품부에서시행한농림수산식품기 술개발사업의연구보고서입니다. 2. 이보고서내용을발표할때에는반드시농림수산식품부에 서시행한농림수산식품기술개발사업의연구결과임을밝혀 야합니다. 3. 국가과학기술기밀유지에필요한내용은대외적으로발표 또는공개하여서는아니됩니다.