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dc.description.abstractThe use of soybean has consistently increased, and it is very difficult to find the food which contains absolutely no soybean. However, soybean is one of most allergenic foods not only in Korea but also in Western countries. Among soybean allergenic fractions such as 2S, 7S and 11S, the soybean 2S protein has not been studied as extensively as others. This study was performed to examine the effects of enzymatic hydrolysis and high pressure treatment of soybean 2S protein and its major allergen, soybean Kunitz trypsin inhibitor (SKTI), related to antigenicity and allergenicity. Soybean 2S protein was extracted by repetitive salting out and centrifugation method, and enzymatic hydrolysis was performed by pepsin, chymotrypsin, papain, bromelain, Flavourzyme, proteinase K and alkaline protease. Antigenicity and allergenicity were observed using rabbit polyclonal antibody to soybean trypsin inhibitor and soybean-sensitive patients’ sera, respectively. Only 13.3% (6/45) of soybean-sensitive patients reacted to SKTI. The distinct band of 20 kDa on SDS-PAGE was identified as SKTI by MALDI-TOF-MS, sequencing of five residues in N-terminal and immunological analysis with polyclonal antibody against SKTI. After peptic hydrolysis for 90 min at pH 1.2, the intensity of SKTI decreased to 25% although it was still visible on SDS-PAGE. Chymotryptic hydrolysis following peptic hydrolysis at pH 8 for 60 min showed a limited hydrolytic effect on soybean 2S protein. Peptic hydrolysis of soybean 2S protein partially reduced the allergenicity, while chymotryptic hydrolysis following peptic hydrolysis increased slightly the allergenicity. High pressure treatment seemed not to affect soybean 2S protein in terms of antigenicity and allergenicity. Among seven proteolytic enzymes, alkaline protease from B. licheniformis was most effective to decrease antigenicity and allergenicity. After hydrolysis with alkaline protease, all proteins completely disappeared from SDS-PAGE, and both the antigenicity and allergenicity decreased over 90%. Papain and bromelain somewhat reduced the antigenicity of soybean 2S protein, but the remained allergenicity was over 70% after papain hydrolysis. Flavourzyme could hardly hydrolyzed soybean 2S protein, and the antigenicity rather increased. In case of proteinase K, it seemed to reduce not the antigenicity of soybean 2S protein but that of SKTI. It is suggested that the alkaline protease treatment might be feasible to be used for less allergenic ingredients for soybean products.;대두는 단백질이 풍부하며 아미노산 균형이 잘 이루어져 있고 우리나라에서는 전통적으로 단백질 급원으로 사용되어 왔던 식품이며, 전세계적으로 가공식품의 원료로 널리 사용되고 있다. 하지만 대두는 국내 5대 알레르기 유발식품 중 하나이며, 전세계적으로는 8대 주요 알레르겐에 포함된다. 대두 알레르기를 유발하는 단백질 중 대두 2S 분획은 현재까지 연구가 많이 이루어져 있지 않다. 이 연구에서는 대두 2S 단백질을 반복적인 염석과 원심분리방법으로 분리하였으며, pepsin, chymotrypsin, papain, bromelain, Flavourzyme, proteinase K, alkaline protease를 이용하여 가수분해 특성을 확인하고 가수분해물의 알레르기성에 미치는 영향을 분석하였다. 알레르기성을 분석하기 위하여 대두에 감작된 환아 45명의 혈청을 실험에 사용하였으며, 대두 알레르기 환자 중 13.3% (6/45) 만이 대두 2S 단백질의 주요 알레르겐인 soybean Kunitz trypsin inhibitor (SKTI)에 반응하였다. 항원성 측정에는 토끼에 SKTI를 주입하여 얻은polyclonal 항체를 사용하였다. 추출한 대두 2S 단백질 중 SDS-PAGE에서 가장 뚜렷하게 나타나는 20 kDa단백질은 MALDI-TOF-MS, N-terminal sequencing, 항체를 이용한 면역반응(immunoblotting)으로 SKTI임을 확인하였다. SKTI는 pH 1.2에서 90분 동안 pepsin 가수분해를 진행하였을 때 일부가 분해되어 약 25% 정도가 남아있었다. 한편, 90분 간의pepsin 가수분해 후 연속적으로 수행된 60분간의 chymotrypsin 가수분해는 SKTI를 분해하지 못하였다. 대두 2S 단백질의 pepsin가수분해물은 알레르기성이 부분적으로 감소되었지만 6명 중 3명의 환아에서 여전히 알레르기성을 보였으며, chymotrypsin 처리 후에는 오히려 알레르기성이 증가하였다. 대두 2S 단백질은 alkaline protease처리 후 SDS-PAGE에서 모든 단백질 밴드가 사라졌으며, 항원성 및 알레르기성이 90%이상 감소하였으며, 특히 알레르기성은 negative control 수준으로 감소하였다. Papain 및 bromelain은 약간의 항원성 감소를 보였지만, papain 가수분해 후에도 알레르기성은 70% 이상 잔류하였다. Flavourzyme은 대두 2S 단백질을 분해하지 못하였으며, 오히려 항원성을 약간 증가시켰다. Proteinase K의 경우 SKTI의 항원성은 감소시켰지만 대두 2S 단백질의 항원성은 감소시키지 못하였다. 가수분해가 SKTI를 감싸고 있던 다른 단백질들을 분해함으로써 SKTI의 감춰진epitope이 노출되었기 때문으로 사료된다. 10%의 Alkaline protease를 이용한 대두 2S 단백질의 가수분해는 알레르기성을 유의적으로 감소시켰다. 이러한 결과를 토대로, alkaline protease를 이용하여 알레르기성이 감소된 대두 2S단백질 또는 대두 단백질 가수분해물을 이용한 새로운 식품소재의 가능성을 제시할 수 있을 것으로 사료된다.-
dc.description.tableofcontentsI. INTRODUCTION 1 1.1. Food allergy 4 1.2. Soybean allergy 11 1.3. Soybean allergens 13 1.4. Soybean Kunitz trypsin inhibitor 18 1.5. Soybean allergen mitigation 21 II. Characterization of soybean 2S protein and its allergenic properties 24 2.1. Materials and Methods 26 2.1.1. Patients sera 26 2.1.2. Preparation of soybean 2S protein 26 2.1.3. Heat treatment on soybean 2S protein 32 2.1.4. Hydrolysis of soybean 2S protein 32 2.1.5. SDS-PAGE 33 2.1.6. Enzyme linked immunosorbent assay (ELISA) 34 2.1.7. Immunoblotting 35 2.1.8. Fractionation of sequential peptic and chymotryptic hydrolysates of the soybean 2S protein by gel filtration 36 2.2. Results and discussions 37 2.2.1. Purification of soybean 2S protein 37 2.2.2. Heat treatment on soybean 2S protein 40 2.2.3. Peptic and chymotryptic hydrolysis of soybean 2S protein 40 2.2.4. Allergenicity of soybean 2S protein and its hydrolysates 48 2.2.5. Fractions from sequential peptic and chymotryptic hydrolyates of soybean 2S protein 55 III. The mitigation of allergenicity using proteolytic hydrolysis of soybean 2S protein 58 3.1. Materials and Methods 61 3.1.1. Patients sera 61 3.1.2. Preparation of soybean proteins 61 3.1.3. High Pressure treatment 63 3.1.4. SDS-PAGE 63 3.1.5. Hydrolysis of soybean proteins 64 3.1.6. Enzyme linked immunosorbent assay (ELISA) 65 3.2. Results and discussions 69 3.2.1. High Pressure treatment on soybean 2S protein 69 3.2.2. Enzymatic hydrolysis of soybean 2S protein 75 3.2.3. Effects of enzymatic hydrolysis on the allergenicity of soybean 2S protein 104 3.2.4. The optimal condition for reducing soybean 2S protein allergenicity 108 CONCLUSIONS 123 REFERENCES 126 국문초록 147-
dc.format.extent3712366 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleEffect of the soybean 2S protein proteolysis on its allergenicity-
dc.typeDoctoral Thesis-
dc.format.pagexiii, 149 p.-
dc.identifier.major대학원 식품공학과- 2-
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