초록 ▼

제 1 장 기능성 효소 생산 기술 개발
제 1 절 멸치액젓 유래 저분자 peptide의 생리활성
천연 숙성 멸치액젓의 숙성 기간별 생성되는 다양한 형태의 기능성 Peptide을 분리·정제하여 항암, 항산화 및 ACE 저해효과를 분석하였다.
제 2 절 멸치액젓 유래 저분자 물질의 항산화활성
천연 숙성 멸치액젓의 숙성 기간별 생성되는 다양한 형태의 항산화물질을 분리·정제하여 항산화활성 및 구조분석을 하였다.
제 3 절 멸치액젓 유래 Bacillus subtilis JM3 단백분해효소의 정제 및 특성
멸치

제 1 장 기능성 효소 생산 기술 개발
제 1 절 멸치액젓 유래 저분자 peptide의 생리활성
천연 숙성 멸치액젓의 숙성 기간별 생성되는 다양한 형태의 기능성 Peptide을 분리·정제하여 항암, 항산화 및 ACE 저해효과를 분석하였다.
제 2 절 멸치액젓 유래 저분자 물질의 항산화활성
천연 숙성 멸치액젓의 숙성 기간별 생성되는 다양한 형태의 항산화물질을 분리·정제하여 항산화활성 및 구조분석을 하였다.
제 3 절 멸치액젓 유래 Bacillus subtilis JM3 단백분해효소의 정제 및 특성
멸치액젓 유래 내염성 미생물 (Bacillus subtilis)이 생산하는 단백분해효소를 정제하여, 여러 가지 특성 (내염성, 내산성, 최적 pH 및 온도, 안정성, 기질 및 저해제 특이성, 동력학적 연구)을 분석하였다.
제 4 절 오징어 젓갈 유래 Bacillus megaterium KLP-98 단백분해 효소의 정제 및 특성
오징어 젓갈 유래 내염성 미생물 (Bacillus megaterium)이 생산하는 단백분해 효소를 정제하여, 여러 가지 특성 (내염성, 내산성, 최적 pH 및 온도, 안정성, 기질 및 저해제 특이성, 동력학적 연구)을 분석하였다.
제 5 절 된장으로부터 분리한 Asp- sp- 101 단백분해효소의 정제 및 특성
된장으로부터 분리한 곰팡이인 Asp. sp.가 생산하는 단백분해효소를 정제하여, 여러 가지 특성 (내염성, 내산성, 최적 pH 및 온도, 안정성, 기질 및 저해제 특이성, 동력학적 연구)을 분석하였다.
제 6 절 된장으로부터 분리한 효모(Yeast) 단백분해효소의 정제 및 특성
효모로부터 단백분해효소를 정제하였으며, 여러 가지 특성 (내염성, 내산성, 최적 pH 및 온도, 안정성, 기질 및 저해제 특이성, 동력학적 연구)을 분석하였다.
제 7 절 기능성 효소 (B. subtilis JM3 pretense)를 첨가한 멸치액젓의 품질특성
발효 및 숙성기간을 단축하기 위하여 멸치액젓에서 단백질분해 활성이 가장 큰 단백분해세균 (B. subtilis JM3)이 생산해내는 단백분해효소를 숙성 중의 멸치액젓에 첨가하여 여러 가지 품질 특성을 분석하였다.
제 8 절 멸치액젓에서 기능성 peptide의 정제 및 생리활성
단백분해세균 (B. subtilis JM3)이 생산해내는 단백분해효소를 숙성 중의 멸치 액젓에 첨가하여 생성되는 peptide을 분리·정제하였으며, 정제된 peptide의 생리활성을 분석하였다.
제 9 절 B- subtilis JM3 단백분해효소에 의한 멸치 근원섬유단백질 분해 특성
다양한 시판 단백분해효소 및 B. subtilis JM3 단백분해효소의 멸치 actomyosin에 대한 가수분해 특성을 분석하였으며, 액젓 및 자건품으로 널리 이용하고 있는 멸치육 단백질을 B. subtilis JM3 단백분해효소로 가수분해시, 반응표면분석법 (Response Surface Methodology, RSM)에 의한 최적의 가수분해 조건을 검토하였다.
제 2 장 기능성 미생물 (Starter) 개발
제 1 절 멸치액젖 유래 단백분해세균의 분리 및 동정
액젓의 속성발효 및 항혈전, 항고혈압, 항산화 그리고 나아가서는 항암과 같은 생리활성 기능을 부여한 신개념의 액젓을 제조할 목적으로 상업적으로 이용 가능한 기능성 액젓 제조용 starter를 분리하고자 국내 시판 액젓 중 기호도가 매우 높은 멸치액젓으로부터 단백질 분해활성이 강하고, 동시에 혈전용해활성이 우수한 균주를 분리하여, 최적 성장 조건 및 최적 효소 생산조건 등을 조사하였으며, 분리균주들의 동정은 형태학적, 배양학적 특징 및 PCR을 이용한 165 rRNA의 염기서열을 분석하여 동정하였다. 또한 분리한 균주를 대상으로 각종 생리활성기능의 유무를 알아보기 위하여 항산화 활성, 항균 활성, ACE 활성 저해 효과, 암세포 증식 억제 효과 등을 조사하였다.
제 2 절 멸치액젖 유래 Bacillus subtilis JM-3의 생리활성기능
앞으로 본 균주 (E. subtilis JM-3)의 멸치액젓 starter로서의 이용을 극대화하기 위하여서는 돌연변이에 의한 호염성 변이주의 유전적 육종이나 형질 전환 방법 등을 응용한 호염성 미생물과의 접목을 통한 내염성 균주의 개발도 필요할 것으로 여겨지며, 멸치액젓에 분리된 미생물의 돌연변이원성 실험 (Mutagenicity test), 항돌연변이원성 실험 (Antimutagenicity test), 변이원물질 및 항변이원활성을 측정하였다.
제 3 절 분리균주를 Starter로 이용한 가자미 식해 제조
발효 및 숙성기간을 단축하기 위하여 가자미 식해 제조시 단백질분해 활성이 가장 큰 단백분해세균 (B. subtilis JM3)을 starter로 첨가하여 숙성기간에 따른 품질특성을 분석하였다.
제 4 절 분리균주를 Starter로 이용한 멸치액젓 제조
발효 및 숙성기간을 단축하기 위하여 멸치액젓 제조시 단백질분해 활성이 가장 큰 단백분해세균 (B. subtilis JM3)을 starter로 첨가하여 숙성기간에 따른 품질 특성을 분석하였다.

Abstract ▼

The nutrition of food is traditionally emphasized to human beings so far. In accordance with the development of improvement of living levels, the functionality of food is recently highlighted and studied in the field of food science. A peptide hydrolyzed from protein has biofunctional activities suc

The nutrition of food is traditionally emphasized to human beings so far. In accordance with the development of improvement of living levels, the functionality of food is recently highlighted and studied in the field of food science. A peptide hydrolyzed from protein has biofunctional activities such as antitumor, antioxidant, opioid, antithrombic, antihypertention, anticholesterol activities, etc. Fermented seafood has been used as a very important role in Korean dietary life and may contain various biofunctional substances. Fish sauce, one of the fermented seafood, contains a lot of amino acid and peptide hydrolyzed from fish protein during fermentation. There are many disadvantages In the production of fish sauce; long fermentation period, use of high salt content, food safety, and marketing.
The objectives of this study are to develop the enzyme and microbial starter to shorten the fermentation period of fish sauce and to enforce the functionality of fish sauce. In addition, the functionalities of peptide and microorganism in fish sauce are also investigated.
Chapter I - Development of Functional Enzyme
1. Biofunctional activities of low molecular weight peptides from anchovy sauce
Biofunctional activities of peptides purified from anchovy sauce fermented at 15±3℃ for 1, 3, and 5 years, respectively, were investigated. 1 year-fermented anchovy sauce showed 3 peaks on gel permeation chromatography pattern, while 3 and 5 year fermented anchovy sauce showed 4 and 5 peaks, respectively. Longer fermentation period, lower molecular weight of peptides on gel permeation chromatography pattern. Antioxidant, antitumor, and ACE inhibitory activities of low molecular weight peptides were increased with increasing fermentation periods. Antioxidant and antitumor activities of peptide peak 3 from 3 year fermented anchovy sauce were the highest with 34 and 44 ㎍/mL of $IC_{50}$ values, respectively, while ACE inhibitory activity ($IC_{50}$, 32 ㎍/mL) of peak 3 from 1 year fermented was the highest.
2. Antioxidant activity of low molecular weight substances from anchovy sauce
Antioxidant activities of low molecular weight biocompounds purified from anchovy sauce fermented at 15±3℃ for 5 years were investigated. 5 year fermented anchovy sauce showed 5 peaks on gel chromatography pattern. Antioxidant activities of peak 2 was 82.7% followed by peak 1 (42.6%). Main antioxidant compounds of peak 1 were glutamic acid and lysine, but peak 2 were not confirmed by amino acid sequencer.
3. Purification and Characterization of Bacillus Subtilis JM-3 Protease from Anchovy sauce
Salt and acid tolerant bacteria, Bacillus subtilis JM3 was isolated from naturally fermented anchovy sauce at 15±3℃ for 3 years. B. subtilis JM3 was cultured in MRS media with 10% NaCl concentration at 37℃ for 8 days in a shaking incubator at 150 rpm. Protease activity was the highest in 40∼60% ammonium sulfate fraction. Protease showed three peaks on ion exchange chromatography patterns and purified further through gel filtration chromatography. Yield of purified protease was 5.33% and its purification ratio was 35.56 folds. Peak I protease showed a single band on SDS-PAGE and its molecular weight was 20,000 Da. Km and Vmax values were of B. subtilis JM3 were 1.75 mg/mL and 318 unit/min, respectively. Optimal temperature for protease activity was 60℃ but optimal stability temperature was 30℃. Optimal pH for protease activity was 5.5. B. subtilis JM3 protease was stable at pH 5. 0∼5.5. Therefore, B. subtilis JM3 protease was a acid pretense. Relative activity of B. subtilis JM3 protease was 69, 21, and 1.3% at 10, 20, and 30% NaCl concentrations, respectively. The best substrate for B. subtilis JM3 protease was azocasein followed by casein. TLCK was the strongest inhibitor followed by DTT, but NEM did not inhibit this enzyme. B. subtilis JM3 pretense was, therefore, presumed serine protease like trypsin type.
4. Purification and Characteristic of Bacillus megaterium KLP-98 Salt-tolerant Protease Derived from Squid Jeot-gal
Salt and acid tolerant bacteria, Bacillus megaterium KLP-98, was isolated from naturally fermented squid Jeot-gal purchased in a local market. B. megaterium KLP-98 was cultured in MRS media with 10% NaCl concentration at 37℃ for 5 days in a shaking incubator of 150 rpm. Pretense activity was the highest in 40∼60% ammonium sulfate fraction. Protease showed two peaks on ion exchange chromatography patterns. Yield of purified protease was 15.3% and its purification ratio was 18.83 folds. Peak I protease showed a single band on SDS-PAGE and its molecular weight was 64,000 Da. Km and Vmax values B. megaterium KLP-98 protease were 2 mg/mL and 283 unit/min, respectively. Optimal temperature for B. megaterium KLP-98 protease was 60℃ but optimal stability temperature was 30℃. Optimal pH of B. megaterium KLP-98 protease was 5.5 and this enzyme was very stable at pH 5.0∼6.0. Therefore, B. megaterium KLP-98 pretense was a acid protease. Relative activity of B. megaterium KLP-98 protease was 73, 33, and 4% at 10, 20, and 30% NaCl concentrations, respectively. The best substrate for B. megaterium KLP-98 protease was azocasein followed by casein. NEM was the strongest inhibitor followed by TPCK, but 2-Mercaptoethanol and DTT did not inhibit this enzyme. B. megaterium KLP-98, therefore, protease was presumed cysteine like protease.
5. Purification and Characterization of Salt-tolerant Acid Protease from Fungus, Asp. sp.
The microorganism producing salt-tolerant acid protease, isolated from the Korean traditional soybean paste, was identified as Asp. sp. 101. The enzyme was purified to homogeneity from the culture supernatant of Asp. sp. 101 by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, and SDS-polyacrylamide electrophoresis. The specific activity and the purification fold of the purified enzyme were 2,301 unit/mg and 11.6, respectively. The molecular weight of the purified enzyme was 25,000 Da on SDS-PAGE. The optimal pH and temperature for the enzyme activity were pH 6.5 and 50℃, respectively. The enzyme activity was relatively stable at pH 4.5-7.5 and temperature below 40℃. The protease was relatively stable at 5 and 10% salt concentrations. This protease was inhibited by $Ag^{2+}$ and $Zn^{2+}$, but, activated by $Cu^{2+}$ and $Mn^{2+}$. In addition, enzyme was potently inhibited by EDTA and PMSF. It was conclusively considered that the purified enzyme was a serine protease influenced by metal ions. Km and Vmaz values for hammastein milk casein were 1.04 mg/mL and 124.84 unit/min, respectively.
6. Purification and Characterization of a Novel Pretense from Yeast, Saccharomyces sp.
The microorganism producing salt-tolerant protease, isolated from the bakers' yeast dough, was identified as Saccharomyces sp. B101. The enzyme was purified to homogeneity from the culture supernatant of Saccharomycessp. B101. by ammonium sulfate fractionation, ultrafiltration, ion-exchange chromatography on DEAE-Sephadex A-50, and gel chromatography on Sephadex G-100. The specific activity and the purification fold of the purified enzyme were 4688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33,000 Da on SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 8.5 and 30℃, respectively. The enzyme activity was relatively stable at pH 6.5-8.5 and temperature below 35℃. This protease was relatively stable at 10 and 15% salt concentrations. This protease was Inhibited by $Ag^{2+}$ and $Fe^{2+}$, and on the contrary, activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by EDTA and PMSF. It was conclusively considered that the purified enzyme was a serine protease influenced by metal ions. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/min, respectively.
7. Quality Characteristics of Anchovy Sauce Manufactured with B. subtilis JM3 Protease
Crude B. subtilis JM3 pretense from naturally fermented anchovy sauce was purified in 40-60% ammonium sulfate fraction. In order to accelerate the fermentation of anchovy sauce, each 2 and 4% Crude B. subtilis JM3 protease was added to 6 month-ripened anchovy sauce, and it's quality was analyzed. pH was at constant during hydrolysis in all samples, while lactic acid contents of 2 and 4% protease added anchovy sauces were higher than control. The amino-nitrogen and volatile basic nitrogen contents of anchovy sauce with 2 and 4% proteases were twice higher than those of control. Anchovy sauces with 2 and 4% protease increased the hydrolysis rate by 27 and 32%, respectively. Browning degree of anchovy sauce with 4% was the higher than 2% and control. Anchovy sauce with 2 and 4% proteases was good in sensory evaluation of color, aroma, and taste.
8. Functionality of Anchovy Sauce Peptide hydrolyzed with B. subtilis JM3 Protease
Peptide was purified by ultrafiltration and gel permeation chromatography from anchovy sauce hydrolyzed with B. subtilis JM3 pretense. Control, 2 and 4% pretenses added anchovy sauces showed 5, 6, and 7 peaks, respectively on gel permeation chromatography patterns. Total ACE inhibitory activity was the highest on peak 6 (43.75%) of anchovy sauce with 2% pretense, followed by peak 5 (34.82%) of control. Total DPPH radical scavenging effects was more than 50% in all samples. Total cytotoxicity was the highest on peak 3 (44.12%) of control, followed by peak 5 (42.04%) of anchovy sauce with 4% pretense.
9. Hydrolysis Characteristics of Anchovy Muscle Actomyosin with B. subtilis MJ3 pretense
The optimal pHs for Alcalase, Protamex, Flavourzyme, and B. subtilis JM3 pretense for the hydrolysis of anchovy actomyosin were 7.5-8.5, 7.0-8.0, 5.0-6.0, and 5.5-6.0, respectively, while the optimal temperatures were 45-55, 50-60, 45-55, and 50-60℃, respectively. Relative activities of Alcalase, Protamex, Flavourzyme, and B. subtilis JM3 pretenses were 30.3, 65.1, 35.2 and 55.2% at 20% NaCl concentration, respectively. Protamex showed the highest proteolytic activities, followed by in order of Flavourzyme, Alcalase, and B. subtilis JM3 protease.
Chapter II. Development of Functional Microbial Starter
We isolated three bacterial strains, JM-1, JM-2 and JM-3 with proteolytic and fibrinolytic activity from anchovy sauce. Among the 3 bacterial strains, JM-3 showed the strongest proteolytic and fibrinolytic activity. Bacterial strain JM-3 was gram-positive rod, motile and formed endospore. The 16S rRNA of bacterial strain JM-3 was amplified by PCR and then its sequence was determined by ABI 310 genetic analyzer. The 16S rRNA sequence of bacterial strain JM-3 was compared to BLAST DNA database and identified to Bacillus subtilis with 99% of homology. The optimum temperature, pH and NaCl concentration for growth of B. subtilis JM-3 were 40℃, 5.0 and 0%, respectively. The optimum temperature, pH and NaCl concentration for proteolytic and fibrinolytic enzyme production of B. subtilis JM-3 were same as optimum conditions for growth. At 20% of NaCl concentration which is common NaCl concentration of fish sauce, B. subtilis JM-3 showed about 60% of proteolytic and fibrinolytic activity of 0% NaCl concentration. From above results, we found that B. subtilis JM-3 will be able to used for starter of functional fish sauce. The cell free culture of B. subtilis JM-3 showed antioxidative activity with 87%, strong antibacterial activity against L. monocytogenes and 88.9% of growth inhibition rate on the stomach cancer cell of human.
The amount of Amino-N was continuously increased during fermentation period of Gajami-Sikhae added B. subtilis JM-3 . When the 20% of B. subtilis JM-3 was added to Gajami-sikhae, the amount of Amino-N was dramatically increased on third day of fermentation.
The amount of Amino-N was continuously increased during fermentation period of Anchovy sauce added B. subtilis JM-3. Especially, When the 10% of B. subtilis JM-3 was added to Anchovy sauce, the amount of Amino-N of it was higher (above 100mg/100g ) than that of control.

목차 Contents

• 표지 ...1
• 제출문 ...2
• 요약문 ...3
• SUMMARY ...15
• CONTENTS ...21
• 목차 ...25
• 제 1 장 서 론...29
• 제 1 절 연구목적...29
• 제 2 절 연구개발 필요성...30
• 제 3 절 연구개발 범위...31
• 제 2 장 국내외 기술개발 현황...33
• 제 1 절 국내 기술현황...33
• 제 2 절 국외 기술현황...34
• 제 3 절 참고문헌...37
• 제 3 장 기능성 효소 생산 기술 개발...42
• 제 1 절 멸치액젓 유래 저분자 peptide의 생리활성...42
• 1. 서 론...42
• 2. 재료 및 방법...43
• 3. 결과 및 고찰...46
• 4. 결 론...52
• 5. 참고문헌...52
• 제 2 절 멸치액젓 유래 저분자 물질의 항산화활성...57
• 1. 서 론...57
• 2. 재료 및 방법...58
• 3. 결과 및 고찰...61
• 4. 결 론...65
• 5. 참고문헌...65
• 제 3 절 멸치액젓 유래 Bacillus subtilis JM3 단백분해효소의 정제 및 특성...68
• 1. 서 론...68
• 2. 재료 및 방법...70
• 3. 결과 및 고찰...74
• 4. 결 론...86
• 5. 참고문헌...87
• 제 4 절 오징어 젓갈 유래 Eacillus megaterium KLP-98 단백분해 효소의 정제 및 특성...91
• 1. 서 론...91
• 2. 재료 및 방법...93
• 3. 결과 및 고찰...96
• 4. 결 론...108
• 5. 참고문헌...108
• 제 5 절 된장으로부터 분리한 Asp. sp. 101 단백분해효소의 정제 및 특성...113
• 1. 서 론...113
• 2. 재료 및 방법...114
• 3. 결과 및 고찰...117
• 4. 결 론...126
• 5. 참고문헌...126
• 제 6 절 된장으로부터 분리한 효모(Yeast) 단백분해효소의 정제 및 특성...128
• 1. 서 론...128
• 2. 재료 및 방법...129
• 3. 결과 및 고찰...132
• 4. 결 론...142
• 5. 참고문헌...144
• 제 7 절 기능성 효소(B. subtilis JM3 단백분해효소)를 이용한 어간장의 속성 제조 및 특성...148
• 1. 서 론...148
• 2. 재료 및 방법...150
• 3. 결과 및 고찰...152
• 4. 결 론...158
• 5. 참고문헌...160
• 제 8 절 멸치액젓에서 기능성 peptide의 정제 및 생리활성...163
• 1. 서 론...163
• 2. 재료 및 방법...166
• 3. 결과 및 고찰...168
• 4. 결 론...173
• 5. 참고문헌...173
• 제 9 절 B. subtilis JM3 단백분해효소의 멸치 근원섬유단백질 분해 특성...177
• 1. 서 론...177
• 2. 재료 및 방법...178
• 3. 결과 및 고찰...180
• 4. 결 론...188
• 5. 참고문헌...194
• 제 4 장 기능성 미생물 (Starter) 개발...196
• 제 1 절 멸치액젖 유래 단백분해세균의 분리 및 동정...196
• 1. 서 론...196
• 2. 재료 및 방법...197
• 3. 결과 및 고찰...201
• 4. 결 론...208
• 5. 참고문헌...212
• 제 2 절 멸치액젖 유래 Bacillus subtilis JM-3의 생리활성기능...215
• 1. 서 론...215
• 2. 재료 및 방법...215
• 3. 결과 및 고찰...220
• 4. 결 론...228
• 5. 참고문헌...228
• 제 3 절 분리균주를 Starter로 이용한 가자미 식해 제조...229
• 1. 서 론...229
• 2. 재료 및 방법...229
• 3. 결과 및 고찰...230
• 4. 결 론...236
• 5. 참고문헌...236
• 제 4 절 분리균주를 Starter로 이용한 멸치액젓 제조...237
• 1. 서 론...237
• 2. 재료 및 방법...237
• 3. 결과 및 고찰...238
• 4. 결 론...242
• 5. 참고문헌...244
• 제 5 장 목표 달성도 및 관련분야에의 기여도...246
• 제 1 절 연구목표 및 내용...246
• 제 2 절 평가 착안점...248
• 제 3 절 목표 달성도 및 관련분야 기여도...248
• 제 6 장 연구개발결과의 활용계획...250
• 제 7 장 해외 과학 기술 정보...251