실제 식품에서 글리아딘 검출을 위한 금 나노입자 응집 기반 라벨프리 비색 압타센서 개발

학위논문(석사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2022. 8. 최영진.Gluten, composed of glutenin and gliadin, is an important raw material that determines the quality of wheat foods such as bread and pasta by giving dough viscoelasticity. However, gliadin in the gluten protein causes celiac disease and wheat allergens, which causes fatal damage to the small intestine, so many methods have been developed for the detection of gluten. The studied detection method has disadvantages that are difficult to use practically in the field due to complicated equipment, long time, and cost. In this paper, a label-free colorimetric aptasensor detection method that can detect gliadin in gluten, a cause of celiac disease, was developed in the field. Based on the salt-induced aggregation of gold nanoparticles, it was detected using a gli4 aptamer that specifically recognizes gliadin. The pre-treatment process of gliadin was simplified to be suitable for the field by extracting it by dissolving it in 60% ethanol. For induction of salt aggregation of gold nanoparticles, gli4 aptamer, gliadin, and NaCl were each optimized in concentration and volume, as confirmed by UV-Vis spectrophotometer and electron transmission microscope (TEM), NaCl and gli4 aptamer concentrations were optimized to 120 mM and 125 nM, respectively. When detecting gliadin based on optimization, the linear curve range (0-120 mg/L) that meets the gluten-free standard (20 mg/L) within 70 min, and the detection limit is 10.5 mg/L (UV-Vis spectrophotometer) and 12 mg/L (colorimetric). In addition, as a selectivity test, the aptasensor was observed for other grain proteins (barley, oats, corn, and rice), and it was confirmed that it responded only to gliadin. To confirm the detection in real foods, ELISA and aptasensor were compared by spiking gliadin at any concentration into gluten-free foods (pasta, bread, and cookie), and it was shown that it could be detected below the gluten-free standard. Therefore, the results of this study are expected to be applied in the food industry to gluten-sensitive allergen patients and environments that require verification of gluten-free food.글루테닌과 글리아딘으로 구성된 글루텐은 반죽에 점탄성을 부여하여 빵, 파스타 등 밀 식품의 품질을 결정짓는 중요한 원료이다. 그러나 글루텐 단백질의 글리아딘은 소장에 치명적인 손상을 주는 셀리악 병 및 밀 알러젠을 유발되며, 이를 검출하기 위해 많은 방법들이 개발되었다. 하지만 연구된 검출 방법들은 복잡한 장비, 긴 시간, 비용으로 인해 현장에서 실용적으로 사용되기 어려움이 있다. 본 논문에서는 셀리악 병의 원인이 되는 글루텐에 존재하는 글리아딘을 현장에서 검출이 간편하고 장비가 필요 없이 육안으로 식별이 가능한 라벨 없는 압타센서 검출법을 개발하였다. 금나노입자의 염 유도 응집을 기반으로 글리아딘을 특이적으로 인식하는 gli4 압타머를 활용하였다. 글리아딘의 전처리 과정은 추출하는 과정으로 에탄올 60%에 용해시켜 추출함으로써 현장에 적합하게 간소화했다. 금 나노입자의 염 응집 유도를 위해 gli4 압타머, 글리아딘 및 NaCl은 각각의 농도, 부피를 최적화하여, 분광광도계 및 전자 투과현미경(TEM)을 통해 NaCl 및 gli4 압타머 농도는 각각 120 mM, 125 nM로 선정했다. 최적화를 기반으로 글리아딘 검출시, 70분 이내로 글루텐 프리 기준(20mg/L)에서 선형 구간(0-120 mg/L)과 검출 한계는 각각 10.5 mg/L (분광광도계), 12mg/L(비색)로 검출되었다. 선택성 실험을 통하여 다른 곡물 단백질(귀리, 옥수수, 쌀, 보리)에 대해 관찰하였고 압타센서는 글리아딘에만 반응하는 것을 확인했다. 실제 식품에서의 검출을 확인하기 위해, 글루텐 프리 식품(파스타, 빵, 쿠키)에 글리아딘을 스파이킹하여 ELISA와 압타센서를 비교하였고 글루텐 프리 기준 이하로 정량화할 수 있음을 확인했다. 따라서 본 연구의 결과로 식품산업에서 글루텐에 민감한 알러젠 환자 및 글루텐 프리 식품의 검증이 필요한 환경에 적용이 될 수 있을 것으로 기대된다.Ⅰ. INTRODUCTION 6 Ⅱ. MATERIALS AND METHODS 9 2.1. Materials 9 2.2. Instrumentation 9 2.3. Synthesis of gold nanoparticles (AuNP) 10 2.4. Optimization of detection condition 10 2.4.1. Measurement of the absorbance 10 2.4.2. Optimization of NaCl concentrations 11 2.4.3. Optimization of gli4 aptamer concentrations 12 2.5. Detection of gliadin 12 2.6. Selectivity of the colorimetric aptasensor of gliadin 13 2.7. Validation of gliadin detection in real foods 14 2.8. Statistical analysis 15 Ⅲ. RESULTS AND DISCUSSION 16 3.1. Overall detection procedures 16 3.2. Optimization of detection condition 21 3.2.1. Optimization of NaCl concentrations 21 3.2.2. Optimization of gli4 aptamer concentrations 26 3.3. Detection of gliadin 31 3.4. Selectivity of the colorimetric aptasensor of gliadin 37 3.5. Validation of gliadin detection in real foods 39 CONCLUSION 42 REFERENCES 44 국문초록 49석

Reducing the Production of Acrylamide During the Roasting of Balloon Flower Roots in Consumer Appliances and Industrial Equipment

This study aimed to determine what conditions were needed to reduce the production of acrylamide when balloon flower roots [Platycodon grandiflorum (Jacq. A. DC.)] were heated. The conditions of temperature, time, and the type of equipment (i.e., consumer appliance or industrial equipment) were the important variables in the experiment. The official criterion for a recommended standard of acrylamide in tea product is less than 1000 µg/kg as determined by the Ministry of Food and Drug Safety in South Korea. A response surface methodology was used to determine whether the heated samples met the safety requirements. The most significant condition for consumer appliances was time and for industrial equipment was temperature. The optimal roasting time was 3.01 min with a consumer appliance and 4.18 min with industrial equipment at 110 ℃, a typical temperature in the field. The acrylamide content of the tested sample was significantly in agreement with the predicted amount (R2 \u3e 0.950)

A Novel Defined TLR3 Agonist as an Effective Vaccine Adjuvant

Synthetic double-stranded RNA analogs recognized by Toll-like receptor 3 (TLR3) are an attractive adjuvant candidate for vaccines, especially against intracellular pathogens or tumors, because of their ability to enhance T cell and antibody responses. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been limited due to heterogeneous molecular size, inconsistent activity, poor stability, and toxicity. To overcome these limitations, we developed a novel dsRNA-based TLR3 agonist named NexaVant (NVT) by using PCR-coupled bidirectional in vitro transcription. Agarose gel electrophoresis and reverse phase-HPLC analysis demonstrated that NVT is a single 275-kDa homogeneous molecule. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without any signs of serious toxicity. NVT stimulated TLR3 and increased the expression of viral nucleic acid sensors TLR3, MDA-5, and RIG-1. When intramuscularly injected into C57BL/6 mice, ovalbumin (OVA) plus NVT highly increased the migration of dendritic cells (DCs), macrophages, and neutrophils into inguinal lymph node (iLN) compared with OVA alone. In addition, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-β production. Furthermore, NVT exhibited an appropriate adjuvanticity because it elevated OVA-specific IgG, in particular, higher levels of IgG2c (Th1-type) but lower IgG1 (Th2-type). Concomitantly, NVT increased the levels of Th1-type T cells such as IFN-γ+CD4+ and IFN-γ+CD8+ cells in response to OVA stimulation. Collectively, we suggest that NVT with appropriate safety and effectiveness is a novel and promising adjuvant for vaccines, especially those requiring T cell mediated immunity such as viral and cancer vaccines

Effect of the Physical Fibrillated Sweet Potato (Ipomoea batatas) Stem on the Plant-Based Patty Analogues

In this study, a dietary fiber extracted from sweet potato stems (Ipomoea batatas, PS) was evaluated for its ability to improve the quality of vegetable patty analogues. A patty analogues containing 0–50 wt% dietary fiber were prepared to analyze the utilized dietary fiber\u27s performance. To evaluate the manufactured patty analogues, texture profile analysis, color analysis, emulsion stability, and microstructural analysis were conducted. As the PS increased, the hardness decreased, while the total expressible fluids tended to increase. The color analysis revealed that the a* value, which represents red, declined as the PS content increased, and heterogeneous colors showed at least 40 wt% of PS. According to the microstructural analysis, PS is a structure in which massive fiber bundles are integrated between textured vegetable protein networks, which is believed to have given the patty analogue soft characteristics. The findings of this study can serve as a foundation for future research into the application of carbohydrates to plant-based meat analogues

Dependence of reaction center-type energy-dependent quenching on photosystem II antenna size

AbstractThe effects of photosystem II antenna size on reaction center-type energy-dependent quenching (qE) were examined in rice plants grown under two different light intensities using both wild type and qE-less (OsPsbS knockout) mutant plants. Reaction center-type qE was detected by measuring non-photochemical quenching at 50 μmol photons m−2 s−1 white light intensity. We observed that in low light-grown rice plants, reaction center-type qE was higher than in high light-grown plants, and the amount of reaction center-type qE did not depend on zeaxanthin accumulation. This was confirmed in Arabidopsis npq1–2 mutant plants that lack zeaxanthin due to a mutation in the violaxanthin de-epoxidase enzyme. Although the electron transport rate measured at a light intensity of 50 μmol photons m−2 s−1 was the same in high light- and low light-grown wild type and mutant plants lacking PsbS protein, the generation of energy-dependent quenching was completely impaired only in mutant plants. Analyses of the pigment content, Lhcb proteins and D1 protein of PSII showed that the antenna size was larger in low light-grown plants, and this correlated with the amount of reaction center-type qE. Our results mark the first time that the reaction center-type qE has been shown to depend on photosystem II antenna size and, although it depends on the existence of PsbS protein, the extent of reaction center-type qE does not correlate with the transcript levels of PsbS protein. The presence of reaction center-type energy-dependent quenching, in addition to antenna-type quenching, in higher plants for dissipation of excess light energy demonstrates the complexity and flexibility of the photosynthetic apparatus of higher plants to respond to different environmental conditions

Impact of Fatty Liver on Acute Pancreatitis Severity

Aim. Acute pancreatitis is typically a mild disease, but some patients develop severe courses. Fatty liver changes are seen in patients with acute pancreatitis, but its clinical significance has not been well-studied. We aimed to investigate the relationship between fatty liver and the severity of acute pancreatitis. Methods. Unenhanced CT images of patients with acute pancreatitis were retrospectively reviewed by a radiologist, and mean hepatic and splenic attenuation was measured in Hounsfield units (HU). Fatty liver was defined as mean hepatic/splenic HU<1. Results. Among 200 patients, fatty liver was found in 67 (33.5%) and nonfatty liver in 133 (66.5%). Compared with patients without fatty liver, the severity of pancreatitis and levels of serum C-reactive protein were higher in fatty liver patients. The prevalence of local complications, persistent organ failure, and mortality were also higher in patients with fatty liver. Even after adjusting for age, sex, body mass index, and cause of pancreatitis, fatty liver was significantly associated with moderately severe or severe acute pancreatitis. Conclusions. Fatty liver may play a prognostic role in acute pancreatitis. Fatty liver could be incorporated into future predictive scoring models

Evapotranspiration and crop coefficients of rice (Oryza sativa L.) under sprinkler irrigation in a semiarid climate determined by the surface renewal method

The evapotranspiration (ETc) of sprinkler-irrigated rice was determined for the semiarid conditions of NE Spain during 2001, 2002 and 2003. The surface renewal method, after calibration against the eddy covariance method, was used to obtain values of sensible heat flux (H) from high-frequency temperature readings. Latent heat flux values were obtained by solving the energy balance equation. Finally, lysimeter measurements were used to validate the evapotranspiration values obtained with the surface renewal method. Seasonal rice evapotranspiration was about 750–800 mm. Average daily ETc for mid-season (from 90 to 130 days after sowing) was 5.1, 4.5 and 6.1 mm day−1 for 2001, 2002 and 2003, respectively. The experimental weekly crop coefficients fluctuated in the range of 0.83–1.20 for 2001, 0.81–1.03 for 2002 and 0.84–1.15 for 2003. The total growing season was about 150–160 days. In average, the crop coefficients for the initial (Kcini), mid-season (Kcmid) and late-season stages (Kcend) were 0.92, 1.06 and 1.03, respectively, the length of these stages being about 55, 45 and 25 days, respectively

Teachers’ Epistemological Assumptions about Educational Inequality in Four Societies: A Holistic Reading Strategy for Examining Sociocultural Epistemologies

Conventional approaches to analyzing cross-national data on teacher knowledge have often failed to recognize qualitative variations across and within different countries. A dilemma confronted by researchers is how to avoid the essentialization of cultures while benefiting from cultural intuition by attending to general national patterns. If researchers focus on exploring the diverse subjectivities of respondents, they are not likely to observe general national patterns because subtle nuances in meaning make it challenging to deal with data with broad categories. There may be too many subtle meanings. However, if researchers focus on general national patterns, they may lose the hidden scripts of the data, as little attention is paid to nuanced meanings. Our data suggest that a holistic reading approach examining different types of semantic foci can be an alternative method for dealing with such a methodological dilemma. This study provides an illustrative example analysis based on this alternative analytic approach