초록 ▼

제 1 장 굴에 있는 Transglutaminase (TGase)의 정제와 특성
제 1 절 TGase의 정제와 특성
제 2 절 패각 단백질의 단백분해효소 저해효과
제 2 장 굴 부분 가수분해물의 제조와 기능성 확인
제 1 절 산 가수분해물의 제조와 특성
제 2 절 일단 효소 가수분해물의 제조와 특성
제 3 절 이단 효소 가수분해물의 제조와 특성
제 4 절 효소 가수분해물의 극성 기능성 물질의 정제와 구조
제 5 절 효소 가수분해물의 비극성 기능성 물질의 정제와 구조
제 3 장 새로운

제 1 장 굴에 있는 Transglutaminase (TGase)의 정제와 특성
제 1 절 TGase의 정제와 특성
제 2 절 패각 단백질의 단백분해효소 저해효과
제 2 장 굴 부분 가수분해물의 제조와 기능성 확인
제 1 절 산 가수분해물의 제조와 특성
제 2 절 일단 효소 가수분해물의 제조와 특성
제 3 절 이단 효소 가수분해물의 제조와 특성
제 4 절 효소 가수분해물의 극성 기능성 물질의 정제와 구조
제 5 절 효소 가수분해물의 비극성 기능성 물질의 정제와 구조
제 3 장 새로운 펩티드의 생리학적 기능성
제 1 절 새로운 펩티드의 세포 독성과 간 기능 보호 효과
제 2 절 굴 가수분해물과 굴 가수분해 조합물이 SD-렛트 혈청과 간 균질물에 미치는 영향
제 4 장 굴 부분 가수분해물의 응용
제 1 절 기능성 고형 제품의 제조와 생리적 기능성
제 2 절 혼합물 실험계획법에 의한 효소가수분해물과 한약추출물들의 최적 혼합비 분석
제 5 장 유용균주로부터 생리활성물질 대량 생산 및 활용
제 1 절 해양유래의 유용균주의 탐색
제 2 절 탐색 균주의 생물활성 및 생리활성물질 생산의 최적 조건 검토
제 3 절 생리활성 물질의 분리 정제 및 이화학적 특성
제 4 절 굴 가수분해물을 이용한 생리활성 물질의 대량생산 검토 및 해양 유래 미생물에 대한 적용
제 6 장 기능성 소재로서 활용을 위한 시장 조사

Abstract ▼

Chapter 1. Purification and Properties of Transglutaminase from Oyster
Section 1. Purification and Properties of Transglutaminase from Oyster
The specific activities of transglutaminase from water extract of 8 shellfishes were compared, and transglutaminase of oyster was purified. Transglutami

Chapter 1. Purification and Properties of Transglutaminase from Oyster
Section 1. Purification and Properties of Transglutaminase from Oyster
The specific activities of transglutaminase from water extract of 8 shellfishes were compared, and transglutaminase of oyster was purified. Transglutaminase was purified by 20-80% saturated ammonium sulfate fractionation, DEAE-Sepharose ion-exchange and Superdex 200 prep grade gel chromatography. The purity and yield of final purified transglutainase were 1895 fold and 1.9%, respectively. The optimum pH and temperature of transglutaminase were 9.0 and 45 $^{\circ}C$ against N,N`-dimethylated casein as substrate. The half activity was lost after heating at 55 $^{\circ}C$ for 30 min. The activity of purified transglutaminase depended on $Ca^{2+}$ ion, and showed the highest in 10 mM $CaCl_2$. The activity was decreased up to 10 mM NaCl, and not significant difference in the range of 50 -200 mM NaCl(p<0.05).
Section 2. Properties of Protease Inhibitor from Oyster Shell
The protease inhibitor was investigated from oyster shell. The crude protein was 0.8%, and calcium oxide was composed of 97.15% in calcium acetate from oyster shell. The fraction below 10 kDa had higher protease inhibitor compared with the fraction above 10 kDa in acetic acid soluble fraction. It was more effective against bromelain inhibition. The antimicrobial and hepatic toxicity were not observed in protease inhibitor fraction and the dried calcium acetate. The acetic acid soluble fraction did not protect ultra-violet ray. The results suggested that partial purified acetic acid soluble fraction had a potential ability as food ingredient for protease inhibition.
Chapter 2. Preparation and Functional Properties of Acidic and Enzymatic Hydrolysates from Oyster
Section 1. Preparation and Functional Properties of Acidic Hydrolysate from Oyster
The objective of this work was to prepare acid hydrolysate based on the functionality such as anti-oxidative effect and angiotensin I converting enzyme (ACE) inhibitor, and to investigate into properties. The optimum conditions for maximum yield were 10 N for HCl concentration and 22 hr for hydrolysis time. The main parameters for acidic hydrolysis were hydrolysis time, HCl concentration and hydrolysis temperature in order. Protein content of hydrolysate and desalted hydrolysate were 6.5% and 14.0%, respectively. The peptide size of hydrolysate was in the range of 400-2500 dalton. The amino acid of hydrolysate was high in order of lysine, leucine, tyrosine, histidine, glycine and valine. Threonine, serine and proline were not detected. Acidic hydrolysate did not showed toxicity on HepG2 cells up to 200 mg/mL. DPPH radical scavenging activity was showed in acidic hydrolysate, and $EC_{50}$ value was 0.28 mg/mL. The anti-oxidative effect for linoleic acid was not significant difference in the range of 0.1-0.0 mg/mL (p<0.05), and was strong with decreasing peptide size. The fraction of desalted hydrolysate with 500-1000 Da showed high ACE inhibition compared with other fraction. The results suggested that the desalted hydrolysate had a potential ability as functional ingredients. However, we need more the safety data of acidic hydrolysate such as 3-MCPD(3-monochloro propare-1,2-diol).
Section 2. Preparation and Functional Properties of Enzymatic Hydrolysates from Oyster by One-Step Processing
The study was carried out to prepare oyster hydrolysates by using Alcalase, Flavourzyme, Neutrase, Protamex, pepsin and trypsin, and to investigate its functional properties. The ACE inhibitory activity and antioxidant activity of enzymatic oyster hydrolysates did not increase with hydrolysis time. Among enzymatic oyster hydrolysates, hydrolysates incubated with Protamex for 1 hr (OHP) showed the most excellent ACE inhibitory activity and antioxidant activity, and their $IC_{50}$ and $EC_{50}$ values were 1.16 mg/mL and 1.49 mg/mL, respectively. However, Antimicrobial effect was not detected in all hydrolysates. Unhydrolysate from oyster had a higher molecular fraction(29-66 kDa) and lower molecular fraction below 6.5 kDa. The high and low molecular fractions decreased with increasing hydrolysis time. The free amino acid of hydrolysates after 1 hr and 3 hr hydrolysis increased up to 16% and 21% compared with that of raw oyster, respectively.
Section 3. Preparation and Functional Properties of Enzymatic Hydrolysates from Oyster by Two-Step Processing
The objectives of this study were to prepare hydrolysates from oyster by two-step processing and to investigate its physiological functionalities. The putrid odor in all hydrolysates were not observed. The degree of hydrolysis by two-step processing was a similar to that by one-step processing. ACE inhibitory activities were improved by two-step processing. Especially, the hydrolysate of Protamex followed by Neutrase showed the highest ACE inhibitor, and its $IC_{50}$ value was 0.40 mg/mL. Antimicrobial effect was not observed in all hydrolysates. Antioxidative effect of hydrolysate treated with Protamex and Neutrase in order was improved, compared with Protamex hydrolysate. The fractions of high molecular weight in range of 29-150 kDa shifted to the fraction of lower molecular weight of 6.5 kDa. The free amino acids were increased with two-step processing.
Section 4 Purification and Structure of Functional Polar Substance from Oyster Hydrolysate
The objective of this work was to purify and investigate the functional polar substances from oyster enzymatic hydrolysates. The hydrolysates from oyster were prepared by two step processing using Protamex, followed by Neutrase. The optimum conditions for preparing functional polar substances were 60 min for reaction time, 1% for protease concentration, and 40 $^{\circ}C$ for temperature. The protein yield was higher in 3% NaCl medium than distilled water. DPPH radical scavenging activities of hydrolysates were higher in small molcular size (<5 kDa). Low molecular peptides extended the induced time up to 168 hr in a linoleic acid oxidation, and had antioxidant effect above 85%. TGPN hydrolysate inhibited the angiotensin-I converting enzyme up to 80% compared to control. Most hydrolysates did not have the reducing effect.
Section 5 Purification and Structure of Bioactive Nonpolar Peptides from Oyster Hydrolysate
The objective of this work was to purify and investigate properties and structure of the functional nonpolar peptides from oyster enzymatic hydrolysates. The nonpolar peptides of TGPN hydrolysate were purified by ultrafiltration (1 KDa), size exclusion chromatography, reverse-phase and ion-exchange HPLC. TGPN hydrolysate had high angiotensin-I converting enzyme(ACE) inhibition below 500 Da molecular size. The purified peptide showed single peak in 8% $CH_{3}CN$ containing 0.1% TFA, and 38% ACE inhibition effect composed to control. The amino acid of purified peptide was composed of isoleucine, leucine, valine, alanine, aspartic acid, glycine, proline and asparagine by Edman sequence. The molecular weights of peptides were equal to the results of ESI-MS for peptide sequence.
Chapter 3. Physiological Functions of New Peptides from Oyster Hydrolysate
Section 1. Toxicity and Protective Effect of New Peptides on HepG-2 Cell
The objective of this work was to investigate the effect of optimum formulation product with two herbs and hydrolysate(HepaCare) on toxicity and cytoprotection of THA-induced toxicity for HepG-2 cell. The toxicity for HepG-2 cell did not find in O and I extracts, and enzymatic hydrolysates. TGPN and 3PN did not show the cytoprotoective effect. However, HepaCare revealed survival capacity of 116% for 50 ug/mL and 128% for 200 ug/mL, respectively, compared to control. HepaCare had significantly hepatoprotective effects lowering the activity of GOT, GPT and ${\gamma}$-GOT after oral administration. This results suggested that HepaCare had significant hepatoprotective effect for acute chronic, and alcoholic hepatitis. In addition, HepaCare played a protective role against lipid peroxidation by TGPN and two herbs.
Section 2. Effect of Oyster Hydrolysate and Herb Mixture on Serum and Liver Homogenate of SD-Rat
The effect of enzymatic hydrolysate from oyster and its formulated products with herb on SD-rat were investigated. The protein content of basic diet group was $45.16\pm2.25$ mg/mL for serum, and increased up to 7% for HepaCare-100 and up to 9.0% for Herb-200, significantly. compared with control group(p<0.05). However, the protein content of TGPN-200 and PN diet group was decreased, Total cholesterol was decreased in all diet group compared with control group. Especially, the total cholesterol was significantly decreased up to 29.9% for TGPN-200 and 24.1% for HapaCare-200, respectively(p<0.05). TGPN and HapaCare group decreased LDL-cholesterol of serum. The neutral lipid in serum was decreased in all enzymatic hydrolysate. Superoxide radicals of TGPN-100 and TGPN-200 groups was decreased up to 7.4% and 34.7%, respectively, compared with control groups. Hydroxy radicals in hydrolysate and HapaCare group were not significant difference compared with control group. TGPN-200 and HepaCare group decreased greatly carbonyl content in serum. The lipid peroxide in serum was decreased in TGPN and HepaCare groups. PN-100 and PN-200 groups increased superoxide dismutase activity. Especially, PN-200 group showed significant difference compared with control group, and increased superoxide dismutase activity up to 19.0%. The catalase activity was $10.26{\pm}0.10$ nmol/mg protein/min for control groups, $0.30{\pm}0.10$ nmol/mg protein/min for TGPN-100, and 0.11 nmol/mg protein/min for TGPN-200 group. PN group increased the catalase activity significantly (p<0.05).
Chapter 4. Application of Oyster hydrolysate
Section 1. Manufacture of the Functional Solid Tablet Using Oyster Hydrolysates and Herb Extracts
The objective of this work was to manufacture the solid tablet based on the functionality with oyster hydrolysate and extracts from plants such as O and I herb. The hydrolysate was obtained by polymerization with transglutaminase, followed by hydrolysis with Protamex and Neutrase, while O and I herb were extracted with water at 100 $^{\circ}C$ for 5 hr, and lyophilized. Wheat powder was used as basis of the tablet forming. The functional properties such as radical scavenging activity and angiotensin-I converting enzyme(ACE) inhibitory activity were determined before and after formulation of the tablet. I extract showed the highest radical scavenging activity. On the other hand, the oyster hydrolysate and O extract showed higher radical scavenging activity than positive control such as vitamin C and tocopherol. According to the ACE inhibitory activity and forming condition, the optimum levels of oyster hydrolysate, O extract, I extract and wheat powder were 2.74g, 0.75 g, 0.75 g and 3.5 g, respectively. Each functional property was verified before and after forming the tablet, and optimum mixing ratio for the solid tablet was provided based on the manufacturing process and functionality.
Section 2 Optimum Formation of the Functional Drink with Oyster Hydrolysate and Herb Extract by Using the Mixture Design
The objective of this work was to manufacture the functional drink with oyster hydrolysate and extracts from plants such as O and I herb by using mixture design and to provide the optimum ratio for the drink using the response surface methodology (RSM). The oyster hydrolysate was obtained by polymerization with transglutaminase, followed by hydrolysis with Protamex and Neutrase, while O and I herb were extracted with water at 100 $^{\circ}C$ for 5 hr. Interaction effects of these mixtures were investigated by modified distance based design and analyzed by regression canonical model, and trace plot. The optimization of mixture ratio was made by statistical modeling. Antiradical activity and sensory properties which are the important target constraints in drink showed linear canonical form, while color and viscosity of drink showed nonlinear canonical form indicating the highly interaction among mixtures. The reponse trace plot revealed that antiradical activity, sensory properties, color and viscosity were quite sensitive to the drink blending. According to the RSM, the optimum formulation of drink was 3% of oyster hydrolysate, 3.83% of I extract, and 8.17% O extract. This work shows the interaction effects of each mixture and provides the optimum ratio of the functional drinks for antiradical activity, ACE inhibition, color and viscosity.
Chapter 5. Mass Production and Application of Bioactive Materials from Marine Bacterium
A marine bacteria which produced the subtilisin-like proteinase inhibitor was screened from the sediment in Gwangyang bay of Korea. The cultural, morphological and physiological characteristics of an isolated strain were investigated for identification. Cultural characteristics based on ISP(International Streptomyces Project) were as follows: white and gray aerial mycelium and good growth on various medium. The strain did not produce the soluble pigment and this strain could be grown up to 9% salt concentration. Morphological and physiological characteristics showed cylindrical spore chain and smooth spore surface by SEM(Scanning Electron Microscope). A phylogenetic analysis of the 16S rDNA provided a clue that the isolated strain was actually a member of the genus Streptomyces, because DNA sequence exhibited a higher homology with Streptomyces thermocarboxydus.
A subtilisin-like proteinase inhibitor inhibited effectively the activity of subtilisin and proteinase K by complexing with the enzyme in the ratio of 1 : 1, while did not inhibit the trypsin and collagenase.
The optimum culture conditions for the production of subtilisin-like proteinase inhibitor were determined after cultivation for 3 days at 28 $^{\circ}C$. Glucose, galactose, starch and fructose were good carbon sources for the production of the inhibitors. On the other hand, maltose, lactose and mannose was only good for the cell growth, and potently inhibited the production of inhibitor. Natural organic nitrogen sources such as poly peptone and proteose peptone were good for the production of inhibitor, while beef extract, urea, casamino acid and inorganic nitrogen sources such as $(NH_{4})_{2}SO_{4}$ and $NH_{4}Cl$ were poor. Optimal medium conditions for inhibitor production were composed of 1.6% galactose, 0.5% proteose peptone, 1% NaCl and 1 mM $Li^{+}$, respectively. The optimal temperature and initial pH for production of the subtilisin-like proteinase inhibitor were 40 $^{\circ}C$ and pH 8.0, respectively. The subtilisin-like protease inhibitor from Streptomyces thermocarboxydus C12 showed a maximum inhibitor activity after the cultivation for 60 h under the optimized medium.
A subtilisin-like proteinase inhibitor produced by Streptomyces thermocarboxydus C12 was purified by ultrafiltration, ammonium sulfate fraction, DEAE Sepharose CL-6B and Superdex 200 column chromatography. The inhibitor was purified 119.9 fold, with a yield of 20.7%. The inhibitor has a monomer with a molecular weight of 33.1 kDa by SDS-PAGE. Also, the inhibitor showed one band in native-gel PAGE. The isoelectric point was 4.4. The inhibitor activity was stable in the range of $6.0{\sim}10.0$ for pH, and $30{\sim}60^{\circ}C$ for temperature respectively. The purified inhibitor was slowly activated by $Mg^{2+}$ and $K^{+}$. The N-terminal sequence of subtilisin-like proteinase inhibitor was determined as DAPSALYAPSALVLTVGKGVSAT. The sequence (residues from 1 to 23) was completely identified with the Streptomyces subtilisin inhibitor(SSI) produced in the Streptomyces albogriseolus. The $K_{m}$ and $V_{max}$ of the inhibitor were calculated to be 2.67 mg and 15.3 unit, respectively. The inhibitor was a noncompetitive inhibitor for subtilisin. As a result of a amino acid compositon inhibitor had a high level of serine and alanine. The inhibition effect of subtilisin-like proteinase inhibitor was much greater than that of food inhibitor such as bovine plasma, pork plasma and egg white protein.
Chapter 6. Market Survey for Application as Functional Ingredients

목차 Contents

• 제 1 장 굴에 있는 Transglutaminase (TGase)의 정제와 특성...99
• 제 1 절 TGase의 정제와 특성...99
• 1. 어종에 따른 transglutaminase 활성과 동결 저장 중 활성의 변화...106
• 2. Transglutaminase의 정제...108
• 3. Subunit의 조성...111
• 4. 최적 pH와 온도...112
• 5. 열안정성...113
• 6. $Ca^{2+}$ 이온 및 NaCl 농도의 영향...114
• 제 2 절 패각 단백질의 단백분해효소 저해효과...120
• 1. 일반성분과 무기성분...123
• 2. 단백질 분해효소 저해제의 정제와 저해활성...124
• 3. 항균효과와 세포독성...126
• 제 2 장 굴 부분 가수분해물의 제조와 기능성 확인...131
• 제 1 절 산 가수분해물의 제조와 특성...131
• 1. 산 가수분해물 제조를 위한 굴 가수분해물의 최적 반응 조건...140
• 2. 가수분해물의 분자량 분포...143
• 3. 아미노산 조성...144
• 4. 세포독성...147
• 5. 산 가수분해물의 기능성...149
• 제 2 절 일단 효소 가수분해물의 제조와 특성...159
• 1. 굴의 원료 특성...163
• 2. 휘발성 염기질소, pH 및 관능검사...163
• 3. 가수분해율...167
• 4. Angiotensin I converting enzyme(ACE) 저해특성...168
• 5. 항균특성...170
• 6. 항산화능...172
• 7. 분자량 분포...173
• 8. 가수분해물의 유리아미노산의 변화...175
• 제 3 절 이단 효소 가수분해물의 제조와 특성...182
• 1. 휘발성 염기질소 및 관능검사...187
• 2. 가수분해도...188
• 3. 2단 가수분해물의 Angiotensin I converting enzyme 저해효과...190
• 4. 이단가수분해물의 항균성...192
• 5. 이단가수분해물의 항산화 활성...193
• 6. 분자량 분포...194
• 7. 가수분해물의 유리 아미노산 조성...196
• 8. 가수분해물의 안전성...198
• 제 4 절 효소 가수분해물의 극성 기능성 물질의 정제와 구조...201
• 1. 효소 가수분해에 의한 굴 가수분해물의 제조 조건...208
• 2. 효소가수분해물의 부분 정제...217
• 3. 효소가수분해물의 아미노산 조성...221
• 4. 효소가수분해물의 기능적 특성...222
• 제 5 절 효소 가수분해물의 비극성 기능성 물질의 정제와 구조...237
• 1. ACE 저해 peptide 정제...242
• 2. 아미노산 서열분석...254
• 3. ESI-MS & MS/MS...255
• 제 3 장 새로운 펩티드의 생리학적 기능성...259
• 제 1 절 새로운 펩티드의 세포독성과 간 기능 보호 효과...259
• 1. 간세포에 미치는 영향...263
• 2. 간세포 손상의 회복 효과...264
• 3. HepaCare의 경구 투여 효과...265
• 제 2 절 굴 가수분해물과 굴 가수분해 조합물이 SD-렛트 혈청과 간 균질물에 미치는 영향...274
• 1. 단백질 함량 변화...283
• 2. 콜레스테롤 조성의 변화...285
• 3. 활성 라디칼에 미치는 영향...292
• 4. 산화적 스트레스에 미치는 영향...297
• 5. 항산화 효소의 활성의 변화...301
• 제 4 장 굴 부분 가수분해물의 응용...310
• 제 1 절 기능성 고형 제품의 제조와 생리적 기능성...310
• 1. 일반성분과 물리적 특성...314
• 2. 라디칼 소거능...315
• 3. 고형 환 제조...315
• 4. 고형 환 추출물의 항산화성...316
• 제 2 절 혼합물 실험계획법에 의한 효소 가수분해물과 한약추출물들의 최적 혼합비 분석...321
• 1. Design point and drink mixture...325
• 2. Statistical modeling and analysis...326
• 3. Optimization of mixture...331
• 제 5 장 유용균주로부터 생리활성물질 대량 생산 및 활용...336
• 제 1 절 해양유래의 유용균주의 탐색...336
• 1. 저해제 생산 균주의 분리...340
• 2. 분리균주의 일차동정...341
• 3. 16S rDNA 염기서열 분석 및 계통도 분류...344
• 제 2 절 탐색 균주의 생물활성 및 생리활성물질 생산의 최적조건 검토...349
• 1. 배양온도의 영향...352
• 2. 초기 pH의 영향...353
• 3. 탄소원의 영향...353
• 4. 질소원의 영향...356
• 5. NaCl 농도의 영향...358
• 6. Metal Salts의 영향...359
• 7. 최적배양조건에서 저해제의 생산...359
• 제 3 절 생리활성 물질의 분리.정제 및 이화학적 특성...364
• 1 Subtilisin-like protease inhibitor의 분리.정제...373
• 2. Subtilisin-like protease inhibitor의 이화학적 특성...380
• 제 4 절 굴 가수분해물을 이용한 생리활성물질의 대량생산 검토 및 해양 유래 미생물에 대한 적용...395
• 1. 저해제 대량생산을 위한 접종량의 영향...398
• 2. 저해제 대량생산을 위한 C/N비의 영향...399
• 3, 저해제 대량생산을 위한 교반속도의 영향...400
• 4. 저해제 대량생산을 위한 통기량의 영향...401
• 5. 미이용 굴가수분해물을 이용한 protinase inhibitor의 생산...402
• 6. 저해제 대량생산을 위한 회분배양...403
• 7. 굴 가수분해물 배지를 이용한 다른 해양유용미생물의 적용...405
• 제 6 장 기능성 소재로서 활용을 위한 시장 조사...410