Characterization of Bioactive Compounds Obtained from Antarctic Krill (E. superba) by Sub- and Supercritical Fluids
- Alternative Title
- 아임계 및 초임계유체공정를 용하여 남극 크릴새우(E. superba) 로부터 어진생리활성 물질의 특성
- Abstract
- 남극 크릴새우 (Euphausia superba)는 난바다곤쟁이목 난바다곤쟁이과에 속하는 종으로 남극해 생태계의 중심역할을 한다. 다른 수산자원들에 비하여 높은 생체량(60-155만 톤)을 가지며 수산업에 새로운 가능성이 대두되고 있는 자원이다. 현재, 남극 크릴새우는 낚시, 양식업 그리고 식용 등 대부분 상업적으로 사용되고 있다. 그러나 불포화지방산(PUFAs), 주로 오메가-3지방산이 풍부한 고품질의 저지방 동물성 단백질이 풍부한 자원이며, 항산화능이 다른 어류에 비하여 특이적으로 높다. 또한, 가공 후 부산물은 부가가치상품으로 사용이 가능하다. 본 연구에서는 아임계 및 초임계 유체 (SCF) 공정을 통하여 남극 크릴새우로부터 생리활성물질을 추출하고 그 특성을 조사하였다.
먼저, 친환경적 공정인 초임계 이산화탄소 (SC-CO2) 추출법과 hexane 을 이용한 유기용매 추출법을 통하여 남극 크릴새우 오일을 추출하였다. SC-CO2 추출은 온도 범위 35 - 45°C, 압력 15 –25 MPa 범위에서 수행하였으며 CO2 유량은 22g/min 로서 총 추출 시간인 2 시간 30 분 동안 지속적으로 흐르게 하였다. 오일의 최대 추출 수율은 온도와 압력이 높을수록 높았다. 오일의 지방산 분석을 위해 gas chromatography (GC)를 이용하였다. 그 결과, 고도불포화지방산(PUFAs)의 함량이 높았으며, 그 중 특히 eicosapentaenoic acid (EPA) 와 docosahexaenoic acid (DHA) 의 함량이 높았다. SC-CO2 를 이용하여 추출한 남극 크릴새우 오일와 hexane 으로 추출한 오일의 품질을 비교해 본 결과 SC-CO2 추출 오일이 hexane 추출 오일보다 높은 안정성을 보였다. 또한 high performance liquid chromatography (HPLC) 를 이용하여 각기 다른 추출조건에 따른 astaxanthin 함량을 비교 분석한 결과 25MPa, 45℃ 추출조건에서 astaxanthin 의 수율이 가장 높음을 확인하였다. 이는 SC-CO2 가 남극 크릴새우로부터 지질을 효과적으로 얻을 수 있음을 나타낸다. 그리고 남극 크릴새우의 fluorine 함량에 대한 SC-CO2 추출 효과를 조사하였다. 그 결과, SC-CO2 추출하는 동안 fluorine함량은 초기농도의 40%정도 줄어들었다. 이러한 결과를 확인하기 위하여 새로운 SC-CO2 추출과정 설계가 추가적으로 필요하다.
제2장에서는 남극 크릴새우의 SC-CO2 추출 후 부산물로부터 인지질을 분리, 특성을 파악하였다. 인지질 정제는 순도 93-97%범위로 HPLC –ELSD 로 정량 분석하였으며 정제된 인지질은 산, 과산화수소 값 및 산화안정성을 조사하였다, Thin layer chromatography (TLC)를 수행하여 각각의 인지질을 정제하였으며, 총 인지질, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI)의 조성은 GC로 분석하였다. 그 결과, 남극 크릴새우 인지질의 산화안정성은 불포화지방산 함량이 높음에도 불구하고 높게 나타났다.
제3장에서는 효소정제시스템에서 SC-CO2 의 적용가능성을 조사하였다. 남극 크릴새우로부터 SC-CO2, hexane, acetone 으로 오일을 추출한 후, 주요 3가지 소화효소에 대하여 조사하였다. 먼저 최적조건 (25 MPa, 45°C) 에서 추출된 남극 크릴새우의 추출 부산물 내의 소화효소 특성을 조사한 결과, 남극 크릴새우의 SC-CO2 추출 부산물은 hexane, acetone 의 유기용매 추출 부산물과 비교하여 프로테아제, 리파아제, 아밀라아제의 활성을 가장 감소시켰다. 또한 SC-CO2 추출 부산물의 모든 소화효소들이 높은 열안정성을 보였지만 SC-CO2 와 hexane, acetone 의 추출 부산물 모두 각각의 소화효소들에 대하여 거의 동일한 최적pH, pH안정성을 보였다. SDS-PAGE 를 이용한 결과, SC-CO2, hexane, acetone 을 처리하거나 처리하지 않은 남극 크릴새우의 조추출물에서 단백질 패턴의 큰 차이를 보이지 않았으며, 이는 단백질 변성이 일어나지 않았음을 나타낸다.
제4장에서는 남극 크릴새우의 SC-CO2 추출 부산물로부터 고부가가치 소재 생산을 위한 아임계 수 가수분해공정을 수행하였으며, 이를 미가공 크릴새우와 비교 분석하였다. 남극 크릴새우의 탈지시료와 원시료의 아임계 수 가수분해능을 알아보기 위하여 200°C 에서 280ºC 의 조건하에서 시료와 물의 비율을 1:50으로 하고 아미노산 분해를 줄이기 위하여 반응시간은 5분 동안 수행하였다. 또한 0.25MPa 의 질소와 공기가 사용되었다. 글라이신, 아르기닌, 류신의 가수분해능은 높은 온도에서 높은 용해도를 가지듯이 수온이 증가됨에 따라 증가되었다. 오일이 제거된 남극 크릴새우 가수분해물의 아미노산 수율은 280 °C에서 가장 높았다. 반면, 처리하지 않은 남극 크릴새우의 가수분해물의 아미노산 수율은 200 °C에서 가장 높았다. 또한, 환원당 수율도 오일이 제거된 남극 크릴새우 가수분해물에서 더 높게 나타났다.
Antarctic krill (Euphausia superba) is a species which belongs to the order of Euphausiacea within the crustacean superorder Eucarida, playing a central role in the Southern Ocean pelagic ecosystem. Due to its high biomass (60-155 million tons) compared with the commercial fish resources; it is a new potential source for the fishing industry. Currently, krill is mostly commercially exploited for the fishing industry and aquaculture and only a small percentage is used for human consumption. However, Krill is a rich source of high-quality protein, with the advantage over other animal proteins of being low in fat and a rich source of polyunsaturated fatty acids (PUFA), mainly omega-3 fatty acids. Antioxidant levels in krill are higher than in other fish, signifying benefits against oxidative damage. Finally, the by-product generated by the processing of krill into edible products can be used for the production of value-added materials. In this thesis, the application of sub- and supercritical fluids (SCF) processes for the recovering and the characterization of the bioactive compounds from Antarctic Krill was investigated. Initially, krill oil was extracted using an environmental friendly solvent, supercritical carbon dioxide (SC-CO2) and an organic solvent, hexane. The SC-CO2 extraction was carried out at temperatures range of 35 to 45ºC and pressures ranging from 15 to 25 MPa. The flow rate of CO2 (22 g min-1) was constant during the extraction period of 2.5 hrs. The maximum oil yield was found at higher extraction temperature and pressure. The extracted oil was analyzed using gas chromatography (GC) for fatty acid profile compositions. High percentage of PUFAs was found, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The quality of krill oil obtained by SC-CO2 extraction was compared with that of hexane extracted oil. The SC-CO2 extracted oil demonstrated higher stability than the oil obtained by hexane extraction. High performance liquid chromatography (HPLC) was used for astaxanthin analysis among the different extraction conditions. The highest yield of astaxanthin was found in krill oil extracted at 25 MPa and 45ºC. These results indicate that SC-CO2 is effective in obtaining nonpolar lipids from krill by only one-step extraction. On the other hand, the effect of SC-CO2 extraction on the fluorine content of krill was investigated. Almost 40 % of the fluorine content was removed from the initial concentrations during the SC-CO2 extraction. Further works with a special SC-CO2 extraction design are required to confirm these results.
In the second part of this study, Phospholipids was isolated and characterized from krill residues extracted by SC-CO2 extraction. The purity of phospholipids ranged between 93 and 97% and was evaluated by HPLC–ELSD. The purified phospholipids were characterized by their acid value, peroxide value, and the oxidative stability. Thin layer chromatography (TLC) was performed to purify the individual phospholipids. The fatty acid compositions of total phospholipids, Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were analyzed by GC. The oxidative stability of krill phospholipids was high in spite of its high content of PUFAs.
The third step of the thesis consists of the applicability of SC-CO2 in enzyme purification system. Three major classes of digestive enzymes of krill were investigated after oil extraction using SC-CO2, hexane and acetone. The residues of krill extracted at optimum conditions (25 MPa and 45ºC) were used to characterize the digestive enzymes. The digestive enzyme activities of protease, lipase and amylase of SC-CO2 treated krill residues were slightly decreased comparing to organic solvent, hexane and acetone treated residues. In SC-CO2 treated samples, all of the digestive enzymes showed slightly higher temperature stability. On the other hand the crude extracts of SC-CO2, hexane and acetone treated krill samples showed almost same optimum pH and pH stability for each of the digestive enzymes. It was also found in SDS-PAGE that there are no significant differences in protein patterns of the crude extracts of untreated and SC-CO2, hexane and acetone treated krill indicating no denaturation of proteins.
The last part of the thesis deals with the use of subcritical water hydrolysis to produce valued materials from krill residues extracted by SC-CO2 and to compare the results with those obtained from raw krill. Subcritical water hydrolysis efficiency from raw and deoiled krill was examined over the temperature range of 200 to 280ºC, ratio of material to water for hydrolysis was 1:50 and for water-sample contact equilibration times of 5 min to decrease the decomposition of amino acids. Nitrogen and air were used as atmosphere at pressure of 0.20MPa. The hydrolysis efficiencies of glycine, arginine, and leucine were found to be increased with increasing water temperature, consistent with higher solubility at higher temperatures. The highest yield of amino acids in deoiled krill hydrolysate was at 280ºC. While, the highest amino acid yield in raw krill hydrolysate was at low temperature 200ºC. Also, reducing sugar content was analyzed in both samples, and the results showed that the yield of reducing sugar in deoiled krill hydrolysate was higher than that of raw krill hydrolysate.
- Issued Date
- 2011
- Awarded Date
- 2011. 8
- Type
- Dissertation
- Keyword
- Marine Biotechnology Advanced Food Separation Processes Supercritical Fluids Technology and its Applicaion marine microbiology Fisheries Genetic resources and Analysis Biochemistry Biotelemetry
- Publisher
- Pukyong National University
- Affiliation
- Pukyong National University, Fisheries Scineces
- Department
- 대학원 수산해양학ㆍ연협동과정
- Advisor
- Chun Byung-Soo
- Table Of Contents
- Contents……………………………………………i
List of Figures……………………………………vi
List of Tables…………………………………………………ix
Abstract………………………………………………………x
Chapter 1: General Introduction
1.1. Background……………………………1
1.2. Krill (Euphausiids)…………………………………………2
1.2.1. Antarctic krill (Euphausia superba) ……………………3
1.2.2. Habitat and biology of Euphausia superba …………3
1.3. Krill oils………………………………………………………11
1.3.1. Nutritional value……………………………………………11
1.3.2. Cardiovascular health benefits…………………………12
1.3.3. Other possible health benefits…………………………12
1.4. Krill protein quality…………………………………………13
1.4.1. Health benefits of krill protein……………………………13
1.5. Value added products…………………………………14
1.5.1. Chitin………………………………………………………14
1.5.2. Krill enzymes………………………………………………14
1.6. Supercritical Fluids………………………………………14
1.6.1. History of supercritical fluids.……………………………15
1.6.2. Properties of supercritical fluids………………………16
1.6.3. Supercritical carbon dioxide (SC-CO2) extraction…20
1.6.4. Comparison between SC-CO2 and organic solvent extraction…20
1.7. Objectives of the thesis……………………………………23
1.8. References…………………………………………………24
Chapter 2: Characterization of oil including astaxanthin extracted from krill (E. superba) using SC-CO2 and organic solvent as a comparative method
Abstract…………………………………………………………30
2.1. Introduction…………………………………………………31
2.2. Materials and Methods…………………………………33
2.2.1. Materials…………………………………………………33
2.2.2. Sample preparation………………………………………33
2.2.3. SC-CO2 extraction………………………………………33
2.2.4. Soxhlet extraction by hexane…………………………34
2.2.5. Determination of extraction yield………………………36
2.2.6. Gas Chromatography analysis for fatty acid compositions…36
2.2.7. Measurement of oil stability……………………………36
2.2.7.1. Free fatty acid content of extracted oil………………36
2.2.7.2. Peroxide value ………………………………………38
2.2.7.3. Colour……………………………………………………38
2.2.8. Astaxanthin analysis by High Pressure Liquid Chromatography……39
2.2.9. Measurement of fluorine content of the raw and extracted residues……40
2.2.9.1. Procedure of bomb Combustion……………………41
2.2.9.2. The photometric method………………………………41
2.2.10. Statistical Analysis………………………………………42
2.3. Results and Discussion……………………………………43
2.3.1. SC-CO2 extraction………………………………………43
2.3.2. Comparison of oil yield obtained by SC-CO2 and hexane extraction… 46
2.3.3. Fatty acid compositions……………………………… 46
2.3.4. Oil stability……………………………………………… 49
2.3.5. Colour…………………………………………………… 49
2.3.6. Extraction yield of astaxanthin……………………… 50
2.3.7. Correlation of astaxanthin solubility using Chrastil model…………… 55
2.3.8. Fluorine content in krill residues extracted by SC-CO2 ……………57
2.4. Conclusions…………………………………………………59
2.5. References……………………………………………………60
Chapter 3: Purification and characterization of phospholipids from Krill (E. superba) residues deoiled by SC-CO2
Abstract……………………………………………………………65
3.1. Introduction…………………………………………………66
3.2. Materials and Methods……………………………………67
3.2.1. Materials……………………………………………………67
3.2.2. Isolation of Phospholipids……………………………67
3.2.3. Quantification of purity of isolated Phospholipids……68
3.2.4. Major phospholipids quantification by HPLC–ELSD…70
3.2.5. Characterization of purified phospholipids……………70
3.2.5.1. Free fatty acids…………………………………………70
3.2.5.2. Peroxide value…………………………………………71
3.2.5.3. Thin layer chromatography of the purified phospholipids ………71
3.2.5.4. Analysis of fatty acids composition by Gas chromatography…71
3.2.5.5. Oxidative stability……………………………………72
3.2.5.5.1. Thiocyanate method…………………………………72
3.2.5.5.2. TBA method……………………………………………72
3.3. Statistical analysis…………………………………………73
3.2. Results and Discussion……………………………………73
3.3.1Organic solvent extraction for comparison and solvent selection………73
3.4.2. Quantification of major phospholipids composition…73
3.4.3. Phospholipids characterization …………………………74
3.4.3.1. Free fatty acids, acid value and peroxide value …74
3.4.3.2. Fatty acid compositions of total phospholipids, PC, PE and PI……75
3.4.4. Oxidative stability…………………………………………79
3.5. Conclusions…………………………………………………81
3.6. References……………………………………………………82
Chapter 4: Comparative study of digestive enzymes of krill (E. superba) after SC-CO2 and organic solvent extraction
Abstract……………………………………………………………86
5.1. Introduction…………………………………………………87
5.2. Materials and method……………………………………88
5.2.1. Materials…………………………………………………88
5.2.2. Digestive enzyme assay………………………………89
5.2.2.1. Preparation of crude enzyme…………………………89
5.2.2.2. Protease assay…………………………………………89
5.2.2.3. Lipase assay……………………………………………89
5.2.2.4. Amylase assay…………………………………………90
5.2.3. Effect of pH and pH stability of protease, lipase and amylase………90
5.2.4. Effect of temperature and temperature stability of protease, lipase and amylase………………………………90
5.2.5. Electrophoresis……………………………………………91
5.3. Results and discussion……………………………………92
5.3.1. Digestive enzyme activities………………………………92
5.3.2. Optimum pH of protease, lipase and amylase ………92
5.3.3. pH stability…………………………………………………93
5.3.4. Optimum temperature of protease, lipase and amylase…100
5.3.5. Temperature stability ……………………………………100
5.3.6. Electrophoresis …………………………………………101
5.4. Conclusions…………………………………………………101
5.5. References…………………………………………………107
Chapter 5: Production of value added materials by subcritical water hydrolysis from krill residues extracted by SC-CO2
Abstract……………………………………………………………110
4.1. Introduction…………………………………………………111
4.2. Materials and Methods……………………………………112
4.2.1. Materials……………………………………………………112
4.2.2. Proximate Composition…………………………………112
4.2.3. SC-CO2 extraction………………………………………112
4.2.4. Subcritical water hydrolysis……………………………113
4.2.5. Protein content measurement of hydrolysates……113
4.2.6. Reducing sugar content measurement of hydrolysates……113
4.2.7. Amino acids analysis. …………………………………114
4.3. Results and Discussion……………………………………117
4.3.1. Proximate compositions of raw and SC-CO2 extracted krill residues…117
4.3.2. Protein yield in hydrolysates of raw and SC-CO2 extracted residues …117
4.3.3. The effect of atmosphere used on amino acid yield....118
4.3.4. Reducing sugar yields…………………………………118
4.3.5. Amino acid yields………………………………………121
4.4. Conclusions………………………………………………125
4.5. References…………………………………………………126
Summary…………………………………………………………129
Abstract (In Korean)……………………………………………130
Acknowledgement ……………………………………………133
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