초록

○ 연구결과
• 물, 알콜, n-hexane으로 난황중 IgY, 인지질, 중성지질, 난황단백질을 계통적으로 분리하는 공정을 개발하고 중성지질과 난황단백질을 활용한 소세지제조
• phosvitin(인단백질) 분리 후 효소분해에 의한 칼슘흡착특이성 조사
• ovotransferrin의 분리, 정제공정개발과 항균성을 이용한 항균필름을 제조하여 신선육포장에 적용
• 난황고형분 기준 200톤 처리 가능한 모델시스템 수립하여 경제성분석

Abstract ▼

pH adjustment of diluted egg yolk to 5.0 increased the amounts of proteins and immunoglobulins extracted, but the profile of proteins extracted was not affected.
Lipid content and the turbidity of the supernatant decreased dramatically by lowering pH of the 10-fold diluted egg yolk, which is very

pH adjustment of diluted egg yolk to 5.0 increased the amounts of proteins and immunoglobulins extracted, but the profile of proteins extracted was not affected.
Lipid content and the turbidity of the supernatant decreased dramatically by lowering pH of the 10-fold diluted egg yolk, which is very important for the subsequent ultrafiltration step to concentrate the supernatant. Holding of diluted egg yolk in cold room up to 24 hr and the duration of centrifugation (30 min vs. 60 min at 2,800 x g) had practically no effect on protein and IgY extraction. As both the holding time and centrifugation time are expensive and rate limiting factors for a continuous, large-scale separation of IgY from egg yolk, holding time before centrifugation should be eliminated and centrifugation time should be limited to 30 min at 2,800 x g.
Comparison of water dilution method with polyethylene glycol, dextran sulfate, and xanthan gum precipitation methods found that water dilution method was superior to the other three precipitation methods(data not shown) in terms of IgY yield, purity, ease of use, potential scaling up, and immunoactivity of IgY. Increasing dilution ratio up to 10 times facilitated the precipitation of lipoproteins in egg yolk because lower ionic strength helped the aggregation of lipoproteins in egg yolk.
However, volume increase makes it difficult to handle for the subsequent preparation steps of IgY purification. Attempts were made to extract immunoglobulin directly from egg yolk by precipitating lipids with cold propane-2-ol and/or using two times dilution of yolk with distilled water and then treated them with a -70 °C freeze-thaw cycling (data not shown). These methods eliminated concentration step for immunoglobulin extract, but the yield of immunoglobulin was lower than the higher dilution method. To reduce volume and concentrate supernatant, ultrafiltration and precipitation of proteins with ammonium sulfate were suggested . Between the two methods, however, ultrafiltration was
more practical than ammonium sulfate precipitation for scaling up, continuous processing for IgY purification, and further separation of other components. In using ultrafiltration, a pre-treatment that removes lipids and lipoproteins is essential because they clog filters and reduce the productivity of ultrafiltration process. Various methods such as adding polyethylene glycol, dextran sulfate, γ -carrageenan gum, xanthan gum, hydroxypropylmethyl cellulose phthalate, caprylic acid or pectin, adjusting pH, or freeze-thaw cycling had been suggested to remove lipids and lipoproteins from water-diluted egg yolk or egg yolk extract. Among these methods, pH adjustment was the most practical for scaling up and continuous processing, and the yield of IgY was the highest. As shown in results, adjusting pH to 5.0 eliminated the lipids from the supernatant almost completely. However, lowering pH below 5.0 is not recommended because at pH< 5.0, the precipitants became loose and watery due to over-hydration of proteins. The pH adjustment of diluted egg yolk to pH 5.0 significantly increased moisture content in the precipitant (water-insoluble fraction of yolk) after centrifugation. This happened because the adjusted pH 5.0 is lower than the PIs of all egg yolk proteins and water molecules tend to bind proteins when the pH is lower or higher than the PI of a protein. About 15% of IgY was lost at ultrafiltration step.
Significant amount of concentrated IgY was lost in membrane of filter, tubing and empty spaces of filter. This problem, however, can be minimized in large-scale or continuous processing of ultrafiltration, instead of batch-type processing. Since yolk contains almost all the antibody in egg, egg yolk can be mixed in animal feed directly as an antibody source. Direct use of egg yolk as an antibody source is simple and costs less, but its application and efficiency is limited because the concentration of antibody is low. Additional process such as defatting step can be applied to concentrate antibodies from egg yolk. Supercritical fluid extraction and organic solvent extraction methods are currently used as defatting methods for egg yolk. Most of the current utilization of immunoglobulin (IgY) from egg yolk is a direct use of egg yolk obtained from hens immunized for specific diseases.
Spray-drying of the supernatant will produce a water-soluble egg yolk product with high IgY, which can be used as a dietary supplement. However, if antibodies from egg yolk are isolated and purified, their efficiency and application can be expanded significantly.
Because neutral lipids and phospholipids separated from the water-insoluble fraction of diluted egg yolk are aimed for human consumption, solvents of choices are limited to ethanol, hexane and acetone. Although acetone is not allowed as a lipid extracting solvent for human consumption in Korea yet, it's allowance increased gradually as a solvent for food usage worldwide. Both ethanol and acetone had strong extracting capabilities for phospholipids, but hexane was able to extract very small amount of lipids from the water-insoluble fraction of egg yolk. The major component of both ethanol and acetone extract was phospholipids, but some neutral lipids and cholesterol were also extracted as the volume of those solvents increased, especially with acetone. Using 4 volumes of ethanol or acetone was better than 9 volumes of ethanol or acetone because very high-purity(> 85%) phospholipids with little neutral lipids could be obtained. Repeated extraction of the precipitant of ethanol extraction with 4 volumes of ethanol increased the amounts of neutral lipids and cholesterol in the extract. It is also clear that decrease in ethanol concentration less than 4 volumes decreased its extractability. However, using 4 volumes of ethanol was better than that of acetone because ethanol had higher extractability for phospholipids from the water-insoluble fraction of egg yolk than acetone. It is very important point because almost all neutral lipids are eliminated from the ethanol extract without further purification steps. The remaining lipid (mainly neutral lipids) after ethanol extraction was extracted using hexane. Hexane is the most widely used solvent in extracting lipids from plant seeds and also has a strong extracting power for neutral lipids. Thus, hexane was selected as a second solvent to extract neutral lipids from the solids of ethanol extraction. As expected, the second (hexane) extract was mainly composed of neutral lipids but contained some phospholipids (PC and PE), indicating that there are still significant amount of phospholipids left in the solids after the first ethanol extraction. Four volumes of hexane were used to remove remaining lipids from the solids of ethanol extraction and the extraction was repeated 3 times.
Chromatography also has been proposed to further purify PLs by using alumina, silica gel, and ion exchange cellulose as media. The precipitants obtained after the second solvent extraction with hexane contained mainly proteins. After drying, the final egg yolk residue contained more than 90% of proteins with small amounts of lipids and carbohydrates.
Sequential separation of IgY, phospholipids, neutral lipids and yolk protein was successfully undertaken starting from egg yolk. Dilution of egg yolk with 9 volumes of water adjusted at pH 5 was selected as the first step for the consecutive separation of the components. The diluted egg yolk was centrifuged at 2800 x g and 4℃ for 30 minutes. Ultrafiltration of the supernatant with a molecular weight cut-off size of 30 kd concentrated IgY. The residues of centrifugation, water insoluble fraction of the diluted egg yolk, was treated with 4 volumes of 100% ethanol and centrifuged at 2800 x g for 10 minutes. Evaporation of the ethanol under vacuum recovered phospholipids. Further extraction of the precipitant with 4 volumes of hexane three times and centrifugation at 2800 x g for 5minutes isolated proteins. Hexane was removed from the supernatant to obtain neutral lipids. Removal of phospholipids using 2 volumes of 100% ethanol improved the purity of the neutral lipids. Overall mass balance and component mass balance during consecutive extraction of egg yolk with water, ethanol and hexane are made. Forty seven grams out of 51 g of total egg yolk solids (from 100 g egg yolk) remained in the precipitant fraction and 4 g (3.5 g proteins, 0.5 g lipids) went to the water-soluble fraction after water extraction. Considering 100 g egg yolk contain 17 g of proteins, more than 20% of the whole egg yolk proteins went to water-soluble fraction. Thus, a plan to recycle water-soluble proteins from the supernatant after recovering IgY may be needed in the future. Ethanol extract contained 11.5 g of solids most of which was phospholipids (9.4 g) and some cholesterol (1.1 g). The amounts of proteins and neutral lipids were small(0.4 g and 0.3 g, respectively). Because ethanol extract contains more than 85% phospholipids, it may be used in food products without further purification.
However, due to somewhat high cholesterol content in the ethanol extract, further purification to remove cholesterol may be necessary. The subsequent hexane extraction of the precipitant from ethanol extraction removed 21.5 g of lipids, which was composed of 20.2 g neutral lipids and 1.2 g phospholipids.
Phospholipids in hexane extract can be removed by repeated extraction with ethanol. The degree of purity should be decided by the specific applications or needs for phospholipids and neutral lipids. The final precipitant after hexane extraction was 14 g and was composed of 96% proteins, 3% ash and <1% of lipids after drying(dry weight basis. moisture <7%). This product can be an excellent source for protein supplementation of other food products or can be used as a starting material for other value-added components, such as phosvitin. This process is significant because multiple value-added components can be sequentially separated and used for various food and non-food purposes. With this sequential separation process of value-added components, natural resources can be maximally utilized with minimal wastes. Costs for separating value-added components from egg yolk can be decreased.
Ovotransferrin was separated from hen egg white by gel chromatography and heparin affinity chromatography. Protein characteristics under the pH and temperature and antimicrobial activity of ovotransferrin isolate was evaluated. The antimicrobial activity of κ-carrageenan based biodegradable packaging film incorporated with ovotransferrin, and effects on microorganisms of fresh chicken filet packaged with the antimicrobial biodegradable film during storage were determined. Heparin affinity chromatography with ferric ion saturated column resulted in the best separation of ovotransferrin compared with gel chromatography and heparin affinity chromatography with ferrous ion saturated column. Foaming ability and stability of ovotransferrin isolate were highest in neutral condition (pH 7.0), and temperature effect on foam stability of ovotransferrin isolate showed that the highest foam was produced at 60℃. Ovotransferrin isolate alone showed weak antimicrobial activity against E. Coli, S. typhi, P. aeruginosa. and C. albicans at dose of 12.5㎎/㎖ and 25㎎/㎖. Effect of ovotransferrin isolate with either lysozyme or albumin on antimicrobial activity shows that the effective dose was 25㎎/㎖ especially on S. typhi and C. albicans. Combined with κ-carrageenan, ovotransferrin and EDTA film showed antimicrobial activity on E. coli. In all treatment, total microorganisms and E. coli of chicken filet was inhibited during storage.
Generally demand for high quality food increases in accordance with economic growth. In case of developed countries, per capita consumption of egg is higher than less developed countries. Korea has achieved very high economic growth, so egg consumption rose gradually. This tendency will continue, but increasing rate will decrease. So we need to create new consumption of egg in order to grow egg industry. Purpose of the technology development is to improve the process of ingredients separation of egg. Feasibility of the developed technology was made in terms of cost-benefit analysis. There are several methods of technology assessment. In this research, modified cost approach method was applied to evaluate cost and benefit of the sequential separation of egg components.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 8
• CONTENTS ... 14
• 목차 ... 16
• 제 1 장 연구개발과제의 개요 ... 18
• 제 1 절 연구과제의 필요성 ... 18
• 제 2 절 연구개발의 목적 및 범위 ... 21
• 제 2 장 국내외 기술개발 현황 ... 24
• 제 3 장 난황주요성분의 계통적 분리공정개발 ... 26
• 제 1 절 연구내용에 따른 실험방법 ... 26
• 1. 성분별 분리 ... 26
• 2. 분석 방법 ... 29
• 3. 주요성분의 이용성 조사 ... 30
• 제 2 절 연구 내용과 결과 ... 38
• 1. 계란난황성분 분리공정총괄 ... 38
• 2. 성분별 분리와 정제 ... 50
• 3. 주요성분들의 이용성과 가공적성 ... 74
• 참고문헌 ... 104
• 제 4 장 기능성 난단백질의 분리와 효능특성연구 ... 111
• 제 1 절 연구내용에 따른 실험방법 ... 111
• 1. 난백 단백질의 항균성 단백질인 ovotransferrin의 분리 및 정제방법 연구 ... 111
• 2. 분리된 ovotransferrin의 기능성 연구 ... 113
• 3. Ovotransferrin 단백질의 용도 개발 ... 114
• 제 2 절 연구 내용과 결과 ... 119
• 1. 난백 단백질의 항균성 단백질인 ovotransferrin의 분리 및 정제방법 연구 ... 119
• 2. 분리된 ovotransferrin의 기능성 연구 ... 127
• 3. Ovotransferrin 단백질의 용도 개발 ... 138
• 참고문헌 ... 147
• 제 5 장 기술개발의 경제성 분석 ... 150
• 제 1 절 계란의 소비 및 가격 추세 ... 150
• 1. 계란의 소비 추세 ... 150
• 2. 계란의 가격변화 추세 ... 154
• 3. 계란의 수요함수 추정 ... 154
• 제 2 절 기술의 개요 및 관련제품의 시장현황 ... 157
• 1. 기술의 개요 ... 157
• 2. IgY ... 160
• 3. 인지질 ... 162
• 4. 중성지질 ... 164
• 5. 단백질 ... 164
• 6. 난백 ... 167
• 제 3 절 난황의 계통적 분리 방법의 경제성 분석 ... 171
• 1. 기술평가의 방법 ... 171
• 2. 단일성분 분리와 계통적 분리에 의한 경제성 비교 ... 173
• 3. 계란 성분분리 기술과 국민경제 ... 175
• 참고문헌 ... 180
• 제 6 장 목표달성도 및 관련분야에의 기여도 ... 181
• 제 7 장 연구개발결과의 활용계획 ... 186
• 제 8 장 연구개발과정에서 수집한 해외과학기술정보 ... 190
• 끝페이지 ... 190