초록

○ 연구결과
1. 저급 식육으로 부터의 기능성 펩타이드 생산 연구
2. 노폐계를 이용한 식육 펩타이드 생산기술 개발
3. 식육 부산물에서의 콘드로이친의 추출기술 연구

Abstract ▼

IV. reslts of the Project and Their Application
1. Results of the project
Section 1. The production of functional peptides from low quality meat and offal
1. The ACE inhibiton activity of peptides, hydrolized by enzyme thermolysin+Protease A for 4hours at 37℃ was 53.36% and IC50 value was 7

IV. reslts of the Project and Their Application
1. Results of the project
Section 1. The production of functional peptides from low quality meat and offal
1. The ACE inhibiton activity of peptides, hydrolized by enzyme thermolysin+Protease A for 4hours at 37℃ was 53.36% and IC50 value was 7.50ug/100ul. Peptide sequnce of this hydrolysates was V-L-A-Q-Y-K.
2. Separated peptides(VLAQYK) was orally supplemented to the SHR for 8weeks to verify ACE inhibition activity. Concentrations of the peptide was 200mg/kg B.W, 500mg/kg B.W, 1000mg/kg B.W. The lowest blood pressure was showed on the 3rd week at 100mg/kg B.W treated group. Content of LDL-cholesterol of the SHR was dose dependently decreased.(p<0.001). In fact, its content of control group was 61.3mg/100ml but decreased to 36.4mg/100ml at 1000mg/100ml treatment group.
3. Seven commercial enzymes, Thermolysin+Prtease A, Trypsin, Protease K, Tyrosinase, Pepsin, Papain, Protease were used to hydrolize beef rump protein and separate peptides.
4. Separated peptides synthesized and used to determine antimicrobial activity, cytotxic activity and machrophage stimulation activity. Peptide GFHI was shown low antimicrobial on E.Coli and P. aeruginosa.
However, peptide FHG showed low antimicrobial activity on P. aeruginosa. In terms of cytotoxicity of synthesized peptide, 400 ug/ml of peptide GFHI showed 20% cytotoxicity on breast cancer cell and ca. 30% cytotoxicity on stomach cancer cell. Also, 400ug/ml of peptide GLSDGEWQ showed 80% cytotoxicity on stomach cancer cell.
However, there was no peptide showed cytotoxicity activity on lung cancer cell. eptide GFHI, which showed the highest cytotoxicity on stomach cancer, didin't change nitric oxide content. It means that peptide GFHI has not immune stimulation activity but cytotoxicity of breast cancer and stomach cancer cell.
5. ACE nhibition activity of separated peptide(0.1mg/ml) stored for 2months at 4℃ was evaluated on various pH level(pH 6.0,6.5,7.0,7.5,8.0) and heat treatment.(60,70,80,90,100℃) ACE inhibition activity of peptide 714,725 during storage was not changed compare to control group.
However, ACE inhibition activity of peptide 1155,1152,1134 changed at 60℃ heat treatment and showed different retention time on HPLC chromatogram from control's.
Section 2. Study of meat peptide using spent hen.
1. Sarcoplasmic protein(water soluble) and myofibrillar protein(salt soluble) of breast meat were extreacted from spent layer aged more than 80 weeks. The extreacts were treated with a 10 commercial enzymes under diiferent pH, temperature, and time. Optimum conditions of enzymes for carnosine, anserine and homocarnosine production were determined depending on yield. Acute toxicity of peptides produced by selected enzymes, such as neutrase, papain, alcalase were conducted to evaluate safety with mouse and rabbits.
The concentrations of carnosine, anserine and homocarnosine extraced from sarcoplasmic protein at optimum conditions of ten enzymes were ranged from 11.2 to 84.8, 18.0 to 365.2 and 10.1 to 29.9mg/100g, respectively. The concentrations of the dipeptides extracted from myofibrillar protein were higher, which were ranged from 15.8 to 222.0, 24.1 to 287.5, and 13.5 to 29.7 mg/100g respectively.
Neutrase was the best in terms of yields of three peptides following papain, alcalase, flavourzyme, protease, trypsin, carboxypeptidase A, protamex, aminopeptidase. takadiasetase.
2. There were no observed acute toxicity, side effect induction, specific induced symptoms in Sigle Dose General Toxicity Test, Primary Skin Irritation Test, and Eye Irritation Test.
The peroxide value, TBARS(thio barbituric acid reactive substances), selectron donating activity, nitrite-scavenging effect and SOD enzyme like activity were conducted to evaluate antioxidant activity of carnosine, anserine and homocarnosine extracted from spent hen. All peptide treatment samples were more effective than the blank sample. Peptides were indicated different response depend on the analysis method and metal type.
Carnosine was the best effect in terms of electron donating activity, nitrite-scavenging effect and SOD enzyme like activity.
3. The clinical assessment was conducted to evaluate wrinkle care effects of peptide using test sample cream containing 0.1% carnosine. The eye winkle area of 20 subjects were spread by test sample and placebo for 4 weeks. The analysis method, skin replicas of representative area of winkle were evaluated by using image-analysis computer software that reflects wrinkle and depth.
The rate of winkle decrease on test area were higher than placebo area but not significantly. In the survey that ask winkle improvement effect, 7 subjects responded that it is effect test area than placebo, and 13 subjects were similar to placebo. There was no observed side effect in clinical assessment.
Section 3. Development of chondroitin sulfate materials from the meat by-products.
1. The screening of meat by-products for chondroitin sulfate materials.
For the screening chondroitin sulfate materials, yields and chondroitin sulfate contents were estimated in various meat by-products such as liver, trachea, intestine, heart etc. of bovine, pork and chick. Liver, trachea, intestine of bovine and pork had higher levels of yields and chondroitin sulfates than others. They were good sources for preparation of chondroitin sulfate materials.
2. Development of liquefaction techniques for preparation of chondroitin sulfate materials.
Effective liquefaction conditions for chondroitin sulfate materials were studied from selected samples. Extraction methods included physical method using heat and pressure, and enzymatic method using various proteases. When compared those methods, 2% alcalase treatment in parallel with 1hrs autoclave was best conditions in every respects of economical, efficiency and convenience, resulted in higher levels of yields and chondroitin sulfate contents. In case of bovine liver, chondroitin sulfate contents was the highest in among selected samples.
3. Development of techniques for preparation and estimation of functional properties of crude chondroitin sulfate.
Hydrolysates of selected samples were filtered by centrifuge, concentrated under vacuum, freeze dried and ground to a fine powder for preparation of crude chondroitin sulfates. From results of general composition analysis, protein and ash contents in crude chondroitin sulfates of all selected samples were < 40%, <5% respectiviely, which is suggest that crude chondroitin sulfates would to be good source for food materials. Crude chondroitin sulfates of 1mM inhibited ACE activity above 60%, especially bovine liver and pork liver 70%. Furthermore, extracted chondroitin sulfates had strong cytotoxic effect on tumor cells such as HepG2 and SNU-16 cell. Above results suggest that extracted chondroitin sulfates would be used to materials for health promoting and/or medical foods.
2. The Proposed Applications for Results
Extraction and separation method of physiologically active(ACE inhibition and anti-oxidation-like) peptide from low quality meat and meat by-products was established. Moreover, analysis methods making higher yield of chondroitin from liver and esophagus of cows and swine were developed and their safety was determined. According to this results, production of functional meat products which possess these peptides and chondroitin will be possible. This established technology may be applied for industry, which can make bulk production inducing added-valued to producer and contribution to human health.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 10
• CONTENTS ... 19
• 목차 ... 23
• 제1장 연구 개발 과제의 개요 ... 28
• 제1절 연구 개발의 목적 ... 28
• 제2절 연구 개발의 필요성 ... 28
• 제3절 연구 개발의 범위 ... 31
• 제2장 국내외 기술 개발 현황 ... 32
• 제1절 국내외 관련 기술의 현황과 문제점 ... 32
• 제2절 앞으로의 전망 ... 34
• 제3장 연구개발 수행내용 및 결과 ... 35
• 제1세부 과제: 저급식육에서의 기능성 펩타이드 생산연구 ... 35
• 제1절 효소에 의한 식육부산물의 최적 분해조건 확립 및 생산 펩타이드 성질규명 ... 35
• 1. 재료 ... 35
• 2. 실험 방법 ... 35
• 가. 일반성분의 분석 ... 35
• 나. 식육내 단백질의 추출방법 ... 35
• 다. 선택된 효소의 종류 ... 36
• 라. 효소와 단백질 추출물의 배양조건 ... 37
• 마. 가수분해도의 측정 (Degree of hydrolysis) ... 37
• 바. 혈압 상승억제 활성 측정(ACE inhibition activity) ... 37
• 사. 한외여과(Ultra filtration)분리 ... 38
• 아. Gel filtration 분리 ... 38
• 자. RP-HPLC (Reverse phase HPLC) 분리 ... 38
• 차. 아미노산 조성 ... 39
• 카. SDS-PAGE를 이용한 단백질 분해 정도 판별 ... 40
• 타. 펩타이드 서열분석 ... 40
• 3. 결과 및 고찰 ... 41
• 가. 일반성분 분석 ... 41
• 나. 효소처리에 따른 우둔 단백질의 분해도 ... 42
• 다. ACE 억제효과(%) ... 44
• 라. SDS-PAGE electrophoregrams를 이용한 단백질 효소 분해도 결과 ... 46
• 마. 한외여과 분리된 펩타이드의 ACE 억제 효과 ... 48
• 바. Gel filtration을 이용해 분리한 분획의 크로마토그램과 각 분획의 분자량 예측 ... 49
• 사. Gel filtration을 통해 분리된 분획의 ACE 억제 효과 ... 54
• 아. Reversed-Phase HPLC를 이용하여 분리한 분획 ... 57
• 자. 순차적 분리단계를 통해 분리한 분획의 IC50 값 ... 61
• 차. 소 염통과 지라의 단백질 분해물의 ACE 억제 효과 ... 63
• 제2절 생산된 펩타이드의 동물 실험을(SHR, Spontaneously Hypertensive Rat) 통한 ACE 억제 효과의 검정 ... 69
• 1. 실험동물의 준비 ... 69
• 가. 실험동물과 처리물(펩타이드) 준비 ... 69
• 2. 실험 방법 ... 69
• 가. 혈압측정 ... 69
• 나. 혈청내 지질성분 분석 및 장기중량 측정 ... 70
• 다. 통계분석 ... 70
• 3. 결과 및 고찰 ... 70
• 가. 실험동물의 생리적 변화 ... 70
• 나. 실험동물의 혈압변화와 혈내 지질수준의 변화 ... 72
• 제3절 일곱 가지 효소를 이용하여 식육에서 분리한 ACE억제 활성 펩타이드 ... 74
• 1. 재료 및 방법 ... 74
• 가. 재료 및 추출조건 ... 74
• 나. Reversed-Phase Liquid Chromatography를 이용한 펩타이드의 분리 ... 75
• 2. 결과 및 고찰 ... 76
• 가. Ultrafiltraion 과 Gel filtration을 이용한 단백질 효소분해물의 DH(Degree of Hydrolysis)와 분해 패턴 ... 76
• 나. Gel filtraion을 이용하여 분리한 분획의 ACE 억제 효과 ... 81
• 다. RP-HPLC를 이용하여 ACE 억제 활성을 지닌 분획의 분리 ... 83
• 라. RP-HPLC를 통해 분리한 분획의 ACE 억제 활성 결과 ... 88
• 제4절 분리된 ACE억제 펩타이드의 그 외 다른 생리 활성기능의 스크리닝 ... 90
• 1. 실험재료 및 방법 ... 90
• 가. 펩타이드 ... 90
• 나. 분리된 펩타이드의 항균활성 측정(디스크 법) ... 90
• 다. 펩타이드의 암세포에 대한 cytotoxicity 측정 ... 90
• 라. 면역 활성 측정 ... 91
• 2. 결과 및 고찰 ... 92
• 가. 분리된 펩타이드의 항균 실험 결과 ... 92
• 나. 분리한 펩타이드의 세포독성 효과 ... 95
• 다. 분리한 펩타이드의 면역 활성 조사 ... 103
• 제5절 분리한 펩타이드의 저장 기간과 pH 변화에 따른 ACE 억제 활성 기능의 변화 ... 105
• 1. 재료 및 방법 ... 105
• 2. 결과 및 토론 ... 105
• 가. 다양한 온도, pH 조건에서 저장기간동안(℃) ACE 억제 활성펩타이드의 활성 변화 ... 105
• 나. 다양한 온도에서의 peptide의 RP-HPLC 크로마토그램 ... 106
• 제6절 결론 ... 110
• 제2세부 (협동과제) 노폐계를 이용한 식육펩타이드의 연구 ... 112
• 제1절 노폐계를 이용한 기능성 식육펩타이드 생산 및 기능 확인 ... 112
• 1. 실험재료 ... 112
• 2. 실험 방법 ... 112
• 가. 단백질의 추출 ... 112
• 나. 펩타이드의 분리 최적조건 탐색 ... 113
• 다. 효소가수분해 최적조건의 탐색 ... 113
• 라. 펩타이드의 정제 최적조건 탐색 ... 116
• 마. 펩타이드의 항산화능 평가 ... 119
• 바. 펩타이드의 안전성 평가 ... 122
• 사. 펩타이드(carnosine)를 주성분으로 하는 기능성(주름완화)화장품의 임상평가 ... 133
• 3. 연구수행 결과 ... 137
• 가. 펩타이드 분리정제 조건 ... 137
• 나. 펩타이드 생산함량 ... 139
• 다. 펩타이드의 항산화능 평가 ... 146
• 라. 아질산염 소거능(Nitrite-scavenging effect) ... 154
• 마. SOD 유사활성(SOD like activity) ... 157
• 바. 펩타이드의 안전성 평가 ... 158
• 사. 펩타이드(carnosine)를 주성분으로 하는 기능성(주름완화)화장품의 임상평가 ... 161
• 4. 결론 ... 162
• 제3세부 (위탁과제) : 식육부산물에서의 콘드로이친 생산기술연구 ... 164
• 제1절 식육 부산물에서 콘드로이친 생산기술 개발과 기능성 연구 ... 164
• 1. 실험재료 ... 164
• 2. 실험 방법 ... 164
• 가. 일반성분분석 ... 164
• 나. 콘드로이친황산의 정량 ... 166
• 다. 콘드로이친황산 추출 방법 ... 167
• 라. 콘드로이친의 기능성 검증 실험 ... 169
• 3. 실험결과 및 토론 ... 172
• 가. 일반성분 함량 ... 172
• 나. 콘드로이친황산 함량 ... 172
• 다. 효소에 따른 콘드로이친 수율 ... 179
• 라. 급속동결건조물의 일반성분 분석 ... 194
• 마. ACE(angiotensin I converting enzyme) 저해활성 ... 195
• 바. MTT assay 결과 ... 197
• 4. 결론 ... 198
• 제4장 목표 달성도 및 관련 분야에의 기여도 ... 200
• 제5장 연구 개발 결과의 활용 계획 ... 203
• 제6장 연구 개발 과정에서 수집한 해외 과학 기술 정보 ... 204
• 제7장 참고문헌 ... 206
• 끝페이지 ... 213