가압가열 및 microwave에 의한 중력분 반죽 gliadin의 항원성 변화

곽지희(Ji-Hee Kwak),김꽃봉우리(Koth-Bong-Woo-Ri Kim),이청조(Chung-Jo Lee),김민지(Min-Ji Kim),김동현(Dong-Hyun Kim),선우찬(Chan Sunwoo),정슬아(Seul-A Jung),김현지(Hyun-Jee Kim),최정수(Jung-Su Choi),김성원(Seong-Won Kim),안동현(Dong-Hyun 한국식품과학회 2012 한국식품과학회지 Vol.44 No.1

본 연구에서는 가압가열 및 microwave 처리에 의한 중력분 반죽 추출물 내의 gliadin 단백질의 항원성 변화에 대해 살펴보았다. 중력분 반죽에 가압가열과 microwave를 단독 또는 병행으로 처리하여 ci-ELISA, SDS-PAGE 및 immunoblotting을 실시하였으며, 가압가열 처리에 의해서 anti-gliadin IgG 항체와 gliadin과의 결합력이 다소 감소한 것을 확인하였다. 특히 30 min 이상 처리시 더욱 감소한 것으로 나타났으며, SDS-PAGE와 immunoblotting 결과에서도 gliadin band의 강도가 약해지고 항체와의 반응도 나타나지 않았다. Microwave 처리의 경우, 5 min 이상 처리시 일부 gliadin 단백질의 소실이 관찰되었으나, 항원성에는 큰 변화가 없었다. 또한 가압가열 및 microwave 병행 처리에 의해 항원-항체 결합력이 더욱 감소되었으며, 특히 가압가열 50 min, microwave 10 min 처리시 약 35.0%로 감소되었다. 이상의 결과를 통해 가압가열 처리에 의해 중력분 반죽 추출물 내 gliadin의 항원성이 감소되는 것을 확인하였으며, microwave와 병행 처리하는 경우, 더욱 감소하는 것을 확인하였다. The aim of this study was to determine the optimal physical treatment to reduce the antigenicity of gliadin in wheat dough. Medium wheat dough was treated with an autoclave (5, 10, 30, and 50 min at 121℃, 1 atm), a microwave (1, 5, and 10 min) or both (10, 30, and 50 min/5, 10 min). The proteins in the dough extracts were analyzed by SDS-PAGE and the binding ability of anti-gliadin IgG to gliadin was examined by ci-ELISA and immunoblotting. Results showed that the ability of anti-gliadin IgG to bind to gliadin in wheat dough treated with an autoclave alone or in combination with a microwave was decreased. Especially, it declined to ~77% after autoclaving for 30 min and 35% after both autoclaving for 50 min and microwaving for 5 min. In addition, the intensity of gliadin bands in SDS-PAGE were weakened and anti-gliadin IgG did not recognize gliadin in immunoblotting. However, microwaving alone did not affect the antigenicity of gliadin in wheat dough. These results indicate that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat dough. Moreover, autoclaving in combination with microwaving is more effective for reducing the antigenicity of wheat dough.

Genomics Approach to Identify the Cause of the Missing Omega-5 Gliadin Protein in O-Free Wheat

( Yun Gyeong Lee ),( Sang Chul Choi ),( Yuna Kang ),( Chon-sik Kang ),( Changsoo Kim ) 한국육종학회 2018 Plant Breeding and Biotechnology Vol.6 No.4

A previous work developed and identified a new omega-5 gliadin deficient wheat line named O-free by crossing Keumkang and Olgeuru, which is nutritionally quite meaningful in that omega-5 gliadin is one of the known wheat allergens. To verify the characteristics of the O-free, we performed RNA sequencing (RNAseq) analysis of the O-free and the two parent lines (Keumkang and Olgeuru). The results of the similarity analysis with the ESTs for gliadins and glutenins showed that the O-free ESTs had no similarity with the omega-5 gliadin sequences but had similarity to other gliadins and glutenins. Furthermore, mapping results between the raw RNAseq data from the O-free and the omega-5 gliadin sequence showed a clear deletion of the N-terminal sequences which are an important signature of omega-5 gliadin. We also designed specific PCR primers that could identify omega-5 gliadin in the genomic DNA. The results showed that no omega-5 gliadin fragments were detected in the O-free. According to these results, we confirmed that the deficiency of omega-5 gliadin in the O-free is not caused by post-transcriptional or post-translational regulations such as epigenetic phenomena but by a simple deletion in the chromosome. Furthermore, we showed that the low-molecular weight glutenin subunit (LMW-GS) gene in the O-free had a single nucleotide polymorphism (SNP) causing a premature stop codon, resulting in a truncated polypeptide. We expect that the O-free line may serve as an excellent source of wheat that could prevail in the hypo-allergen wheat market, which has recently gained interest world-wide.

Gliadin intake induces oxidative-stress responses in <i>Caenorhabditis elegans</i>

Lim, So Dug,Min, Hyemin,Youn, Esther,Kawasaki, Ichiro,Shim, Yhong-Hee Elsevier 2018 Biochemical and biophysical research communication Vol.503 No.3

<P><B>Abstract</B></P> <P>Clinical attention to gluten-related disorders, such as celiac disease and nonceliac gluten sensitivity, is on the rise. However, identifying the pathophysiological mechanisms of gluten-related disorders remains elusive. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. <I>Caenorhabditis elegans</I> has been widely used as the predominant experimental animal model to study toxicity and stress response in biomedical research. We investigated the stress response induced by gliadin intake in <I>C. elegans</I> to evaluate its toxicity and found brood size, body bending, and pumping rates to be significantly altered in response to gliadin. Notably, reactive oxygen species (ROS) production and <I>Pgst-4::GFP</I> transgene expression, an indicator of the oxidative-stress response, were significantly increased after gliadin intake. Reduced pumping rates were most likely caused by gliadin-induced oxidative stress, since pumping rates in oxidative stress-sensitive <I>mev-1</I> mutants were more severely reduced than in oxidative stress-resistant <I>daf-2</I> mutants following gliadin intake. Our results indicated that gluten/gliadin intake in <I>C. elegans</I> triggered ROS production and induced an oxidative stress response that reduced pumping rates and decreased brood size. We suggest <I>C. elegans</I> to be a useful model system for studying gluten/gliadin toxicity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Locomotion behavior and brood size were reduced in <I>C. elegans</I> fed with gliadin. </LI> <LI> ROS production and oxidative-stress responses were increased with gliadin intake. </LI> <LI> Reduction in pumping rate was caused by the gliadin-induced oxidative stress. </LI> </UL> </P>

가압가열 및 Microwave 처리에 의한 중력분 Gliadin의 항원성 변화

곽지희(Ji-Hee Kwak),김꽃봉우리(Koth-Bong-Woo-Ri Kim),이청조(Chung-Jo Lee),김민지(Min-Ji Kim),김동현(Dong-Hyun Kim),선우찬(Chan Sunwoo),정슬아(Seul-A Jung),강주연(Ju-Youn Kang),김현지(Hyun-Jee Kim),최정수(Jung-Su Choi),김성원(Seong-Won 한국식품영양과학회 2011 한국식품영양과학회지 Vol.40 No.10

본 연구에서는 가압가열 및 microwave 처리가 gliadin의 항원성에 미치는 영향을 살펴보기 위해 중력분에 가압가열과 microwave를 단독 또는 병행으로 처리하여 Ci-ELISA, SDS-PAGE 및 immunoblotting을 실시하였다. 가압가열 처리의 경우, 처리 시간이 길어질수록 IgG와의 결합력이 감소하였으며, 특히 50분 처리구에서 약 69%로 가장 낮은 결합력을 보였다. 또한 SDS-PAGE와 immunoblotting 결과에서도 무처리구에서 강하게 보였던 gliadin band가 처리에 의해 거의 소실되고 항체와 반응하지 않았다. 가압가열 및 microwave를 병행 처리한 경우도 마찬가지로 gliadin의 결합력이 다소 감소하였으며, 처리구 중에서는 가압가열 50분, microwave 5분 처리구에서 약 73%로 가장 낮은 결합력을 보였다. 반면 microwave를 단독으로 처리하였을 때에는 일부 단백질의 변화는 관찰되었으나 항원성 감소에는 큰 영향을 미치지 않았다. 이상의 결과를 통해 가압가열을 단독 처리에 의해 gliadin의 항원성이 다소 감소되었으며, microwave 병행 처리에 의한 차이는 크게 나타나지 않은 것을 확인하였다. This study was conducted to evaluate the effect of physical treatments on the antigenicity of gliadin in medium wheat flour. The wheat flour was treated with an autoclave (5, 10, 30, 50 min), a microwave (1, 5, 10 min), and both (10, 30, 50 min/ 5, 10 min), and investigated by SDS-PAGE, immunoblotting and Ci-ELISA using anti-gliadin IgG. The results showed that the binding ability of anti-gliadin IgG to gliadin in wheat flour was slightly decreased when autoclaved or when autoclaved and microwaved. Especially, it was reduced to about 69% after autoclaving for 50 min and 73% after autoclaving for 50 min and microwaving for 5 min. In addition, gliadin bands in the 50 min autoclaved group disappeared in both SDS-PAGE and immunoblotting. On the other hand, the antigenicity of gliadin was unaffected by microwaving alone. Consequently, there were no considerable changes in using an autoclave alone or in combination with a microwave. These results suggest that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat flour.

Evaluation of Allergenicity on a ω-5 Gliadin-Deficient Cultivar in Wheat-Dependent Exercise-Induced Anaphylaxis

Lee Jongsun,Kim Sung-Ryeol,Park Jong Han,박경희,Jeong Kyoung Yong,이재현,Kang Chon-Sik,Kim Kyeong-Hoon,박중원 대한천식알레르기학회 2022 Allergy, Asthma & Immunology Research Vol.14 No.4

Purpose: ω-5 gliadin is the major allergen that causes wheat-dependent exercise-induced anaphylaxis (WDEIA). Recently, a missing mutant wheat cultivar at 1B chromosome Glu-B3 and closely linked Gli-B1 loci was bred. This cultivar (ω5D) has a deficiency in ω-5 and γ-gliadins as well as some low-molecular-weight glutenins. We evaluated specific immunoglobulin E (sIgE) reactivity of the ω5D in WDEIA patients compared to wild-type cultivar. Methods: Serum samples from 14 WDEIA and 7 classic wheat allergy patients were used to compare the allergenicity of ω5D and wild-type cultivars using immunoglobulin E immunoblotting, enzyme-linked immunosorbent assay (ELISA), and ImmunoCAP inhibition assays. Results: Immunoblotting revealed that ω5D extracts had less sIgE binding to gliadins and glutenins in WDEIA sera than wild-type extracts. Immunoblot inhibition assay for gliadin sIgE reactivity also showed that ω5D gliadins had less allergenicity than wild-type gliadins. ELISA inhibition assay showed stronger allergenicity of gliadins than glutenins, although they had cross-reactivity. This assay also showed that the 50% inhibitory concentrations (IC50) of ω5D extracts against gliadin- or glutenin-sIgE reactivity were approximately 4-fold higher in WDEIA patients than those of wild-type extracts. The inhibition capacity of ω5D gliadins against recombinant ω-5 gliadin-sIgE reactivity was also lower in WDEIA patients than that of wild-type. Conclusions: The allergenicity of the ω5D cultivar is markedly lower for WDEIA patients in the sIgE inhibition tests. These results suggest that the ω5D cultivar may be a safe alternative for WDEIA patients.

물리적 처리에 의한 강력분 밀가루 Gliadin의 항원성 변화

강보경(Bo-Kyeong Kang),김꽃봉우리(Koth-Bong-Woo-Ri Kim),김민지(Min-Ji Kim),박시우(Si-Woo Bark),박원민(Won-Min Pak),김보람(Bo-Ram Kim),안나경(Na-Kyung Ahn),최연욱(Yeon-Uk Choi),최정수(Jung-Su Choi),최호덕(Ho-Duk Choi),안동현(Dong-Hyun A 한국식품영양과학회 2014 한국식품영양과학회지 Vol.43 No.4

본 연구에서는 가압가열 및 microwave 처리가 gliadin의 항원성에 미치는 영향을 살펴보기 위해 강력분에 가압가열과 microwave를 단독 또는 병행으로 처리하여 Ci-ELISA, SDS-PAGE 및 immunoblotting을 실시하였다. 가압가열 처리의 경우 시간이 길어질수록 IgG와의 결합력이 감소하였으며, 특히 50분 처리구에서 약 87%로 가장 낮은 결합력을 보였다. 또한 SDS-PAGE와 immunoblotting 결과에서도 무처리구에서 강하게 보였던 gliadin band가 가압가열처리에 의해 거의 소실되고 항체와 반응하지 않았다. 가압가열 및 microwave를 병행 처리 시도 마찬가지로 gliadin의 결합력이 감소하였으며, 처리구 중 가압가열 50분, microwave 5분 처리구에서 약 93%로 가장 낮은 결합력을 보였다. 반면 microwave를 단독으로 처리한 경우에는 일부 단백질의 변화는 관찰되었으나, 항원성 감소에는 효과가 없음을 확인하여 단백질 변화가 항원성에는 큰 효과를 준 것 같지 않다. 이상의 결과를 통해 가압가열 단독 처리 및 가압가열 및 microwave의 병행처리 시 gliadin의 항원성이 감소함을 확인하였다. This study was conducted to evaluate the effects of physical treatments on the antigenicity of gliadin in strong wheat flour. Strong wheat flour was treated with an autoclave (5, 10, 30, 50 min), a microwave (1, 5, 10 min), or both (10, 30, 50 min/ 5, 10 min), followed by SDS-PAGE, immunoblotting, and Ci-ELISA using anti-gliadin IgG. The results indicated that the binding ability of IgG to gliadin in strong wheat flour slightly decreased after autoclaving or autoclaving/microwaving. In particular, the binding ability was reduced to about 87% after autoclaving for 50 min and to 89% after autoclaving/microwaving (50/5 min). In addition, gliadin bands in the 50 min autoclaved group disappeared in both SDS-PAGE and immunoblotting. On the other hand, the antigenicity of gliadin was unaffected by microwaving alone. In conclusion, the results of this study suggest that autoclaving may reduce the antigenicity of gliadin in strong wheat flour.

Production of omega-5 gliadin monoclonal antibodies for allergenic evaluation of WDEIA-causing wheat varieties

Sim Jae-Ryeong,Lee Jong-Yeol,Kim Sewon 한국응용생명화학회 2023 Applied Biological Chemistry (Appl Biol Chem) Vol.66 No.-

In allergic individuals, ingestion of wheat can lead to wheat-dependent exercise-induced anaphylaxis (WDEIA). Many studies have been conducted to find WDEIA allergen–deficient wheat, including by generating omega-5 gliadin antibodies. However, the reported antibodies have not been specific enough to detect omega-5 gliadins encoded on the 1B chromosome. In this study, we generated monoclonal antibodies against the major allergens causing WDEIA, omega-5 gliadins. Using these antibodies (mono-O5B-1C10), we assessed accumulation of omega-5 gliadins in wild-type and nullisomic-tetrasomic (NT) lines of the wheat (Triticum aestivum) varieties Chinese Spring (CS) by one- and two-dimensional gel electrophoresis, followed by Coomassie blue staining or immunoblotting with mono-O5B-1C10. We also tested mono-O5B-1C10 for major omega-5 gliadins in various wheat germplasms. Our results thus demonstrate the specificity of mono-O5B-1C10 for major omega-5 gliadins and potentially useful for identifying of omega-5 gliadin–deficient wheat varieties that should not cause WDEIA.

글루텐분해유산균 GLU70 섭취에 따른 글리아딘 투여 마우스의 장내 염증성 사이토카인 및 유전자 발현의 변화

윤슬기,장준호,손은심,윤주성,변지영,윤복근 한국식품과학회 2024 한국식품과학회지 Vol.56 No.2

Excessive intake of wheat flour can lead to digestive disorders and intestinal diseases due to gliadin in gluten, an indigestible protein that is a causative agent of inflammation. This study aimed to confirm the effects of glutendegrading Lactobacillus strain GLU70 intake on the intestines of gliadin-administered mice. The results showed that GLU70 intake increased the activity of pancreatic proteases and significantly alleviated gliadin-induced damage in the jejunum tissue. While reducing the expression of TMF-α in the intestine, GLU70 intake increased the expression of the anti-inflammatory cytokine IL-10. It also reduced the mRNA expression of cytokines IL-4 and IL-5, which are related to allergic diseases, and decreased the expression of TGM2, zonulin, and p65NF-κB, which are proteins related to celiac disease, leaky gut syndrome, and inflammatory response respectively. Therefore, gluten-degrading Lactobacillus strain GLU70 may be useful in the food industry for its effects of alleviating gliadin-induced complications.

천연 글루텐 필름의 기계적 물성 향상 연구

김학선(Hak Seon Kim),강호종(Ho-Jong Kang) 한국고분자학회 2016 폴리머 Vol.40 No.2

글루텐 단백질 구성 성분이 천연 글루텐 필름 물성에 미치는 영향을 확인하여 보았다. 글루텐/에틸알코올 수용액 원심분리에 의하여 글라이딘/글루테닌 조성비가 다른 글루텐을 얻어 천연 글루텐 필름을 제조하였다. 일반 글루텐 필름에 비하여 고분자량 글루테닌이 제거된 글루텐 필름은 신율이 증가되며 아울러 광투과 특성이 현저히 증가함을 알 수 있었다. 글루텐 필름의 가공 특성 증가를 위해 사용된 글리세린은 필름 신율을 증가시키는 반면 인장강도는 감소시킴을 확인하였다. 이러한 감소는 L-cysteine을 가교제로 사용하여 이황화 결합에 의한 글루텐 가교를 촉진시켜 최소화될 수 있으며 고분자량 글루테닌을 다소 함유하고 있는 글루텐 필름에서 그 효과가 두드러짐을 알 수 있었다. The effect of chemical composition of gluten protein on physical properties of natural gluten film was studied. The various glutens having different gliadin/glutenin compositions were obtained by the centrifugation of gluten/ethyl alcohol aqueous solution and then natural gluten films were produced from obtained glutens. It was found that both elongation at break and optical transmittance were enhanced in the film made by glutens without high molecular weight glutenin. The applied glycerin for processing aid resulted in increasing of elongation at break but the lowering of tensile strength was found. This tensile strength decrease might be minimized using L-cysteine as a crosslinking agent which caused the disulfide bonding between gluten molecules, specially for gluten having high molecular weight glutenin.

Characterization of two novel γ-gliadin genes encoded by K genome of Crithopsis delileana and evolution analysis with those from Triticeae

Zhi-Fu Guo,Li-Jun Zhang,Ming Zhong,Yu-Ming Wei,Li Zhang,Hui Ma,Hao-Ge Li,Li-Jing Chen,Jing-Wei Lin,You-Liang Zheng 한국유전학회 2010 Genes & Genomics Vol.32 No.3

By acid polyacrylamide gel electrophoresis (A-PAGE) analysis,it was indicated that the electrophoresis mobility of gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14,KK) had obvious difference with those from common wheat in α, γ and ω region. Using homologous primers, two γ-gliadin genes (gli-Kr1 and gli-Kr2) were isolated from C. delileana,which had been deposited in the GenBank under accession numbers EU283818 and EU283821, respectively. Two γ-gliadin genes of C. delileana had the similar primary structures to the corresponding gene sequences from other wheat related species. The differences were mainly resulted from substitutions,insertions and deletions involving single amino acid residues or motifs of γ-gliadins. The repetitive domains of gli-Kr1 and gli-Kr2 from C. delileana are shorter than most of other sequences. By the alignment of γ-gliadin genes from A, B, D, Am, Au, S, Sl, Ssh, Ss and Sb genomes of Triticum and Aegilops, R genome of Secale (γ-secalin), Ee genome of Lophopyrum and K genome of Crithopsis in Triticeae, phylogenetic analysis indicated that two γ-gliadin genes of C. delileana could be clustered together with a γ-gliadin genefrom Ssh genome of Aegilops by an interior paralleled branch. It was the first time that the γ-gliadin genes encoded by K genome of C. delileana were characterized. These could offer precious information for better understanding the qualities associated with gliadins, the response in coeliac disease and studying the evolutionary relationship of gliadins in Triticeae.