Workshop 2nd

Dear Colleagues, 

It is a great pleasure to invite you to participate in the 2nd FoodEnTwin Workshop “Experimental animal models for food and environment” that will be held in Vienna, Austria on 3th to 4th of February 2020. 

The venue of the 2nd FoodEnTwin Workshop is at the Medical University of Vienna (HörsaalZentrum, Kursraum 12).  

2nd FoodEnTwin Workshop “Experimental animal models for food and environment” program can be downloaded here.

Book of abstracts can be downloaded here.

The main topics 

  • Animal models for studying food and environmental allergy
  • Assessing allergenicity with proteomics 
  • Health effects of food components modifications
  • Novel in vitro and in vivo animal models for predicting effects of environmental stress
  • Food allergy: animal models used for risk assessment 

The organizers are looking forward to your participation. 

On behalf of scientific committee,

Michelle Epstein

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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 810752

logo-FoodEnTwin

2nd FoodEnTwin Workshop Programme

Experimental animal models for food and environment

Day 1 February 3, 2020
Time Title Speaker Name Institute
9:30-9:40 Opening of the Workshop Michelle Epstein Medical University of Vienna, Austria
9:40-10:10 Introduction to FoodEnTwin Tanja Cirkovic Velickovic University of Belgrade, Serbia
Session 1:Mouse and rat models for food allergy Chairpersons Michelle Epstein Marianne van Hage Medical University of ViennaKarolinska Institutet, Sweden
10:10-10:40 Rat models of food allergy Katrine Lindholm Bøgh Danish Food Institute, Denmark
10:40-11:10 Mouse models of food allergy Karine Adel-Patient CEA, France
11:10 Coffee break
Session 2: Risk Assessment in food allergy Chairpersons Tanja Cirkovic Velickovic Aleksandra Inic-Kanada University of Belgrade, Serbia Medical University of Vienna
11:40-12:10 Experimental in vitro and in vivo models for food allergy risk assessment Michelle Epstein Medical University of Vienna
12:10-12:40 Animal models for risk assessment of food allergy Antonio Dumont Fernandez European Food Safety Agency, EFSA, Parma, Italy
12:40 Lunch
Session 3: Protein allergens Chairperson Sanja Grguric-Sipka University of Belgrade
14:10-14:40 Protein structure of food allergens Karin Hoffmann-Sommergruber Medical University of Vienna
14.40-15:10 Allergy to red meat Marianne van Hage Karolinska Institutet, Sweden
15:10-15:50 Tomato allergens – multiomics identification and control in farming Wolfram Weckwerth University of Vienna, Austria
15:10 Coffee break
15:40-16:10 Oral immunotherapy (OIT) and peanut allergy Stefan Zeitler Aimmune, Germany
16:10-17:10 Round table discussion Chairpersons Michelle Epstein Tanja Cirkovic Velickovic Medical University of ViennaUniversity of Belgrade, Serbia
19:30 Gala dinner at the Melkerstiftskeller Schottengasse 3, 1010 Vienna
Day 2 February 4, 2020
Time Title Speaker Name Institute
Session 4: FoodEnTwin Seminars Chairpersons  Marianne van Hage Sanja Grguric-Sipka Karolinska Institutet, SwedenUniversity of Belgrade, Serbia
9:30-9:45 FoodEnTwin highlights Tanja Cirkovic Velickovic University of Belgrade, Serbia STSE BU/KULs
9:45-10:00 Allergenomics of ticks Danijela Apostolovic Karolinska Institutet, Sweden Training STSE KI/BU
10:00-10:15 Bovine γ-Globulin and Lactoperoxidase as major milk allergens among a mammalian meat allergic population Marija Perusko University of Belgrade, Serbia EAACI fellowship BU/KI
10:15-10:30 Development of an immune-polymerase chain reaction for detection and  quantification of shellfish tropomyosin Mirjana Radomirovic University of Belgrade, Serbia EAACI fellowship BU/KI
10:30-10:45 Discrete Hf18 metal-oxo cluster as a nanozyme for site-specific protein hydrolysis Jens Mons Katholieke University Leuven STSE KUL/BU
11:00 Coffee break
11:30-11:45 Proteomics and PTMs profiling of raw and thermally treated shellfish samples collected in Korea: focus on tropomyosin Urmila Khulal Ghent University STSE GU/BU
11:45-12:00 Characterisation of peanut allergens and possible post-translational modifications (PTMs) Shu Liu Medical University of Vienna, Austria STSE MUW/BU
12:00-12:15 Resolving Dactylis glomerata pollen proteins by 2D SDS PAGE and subsequent western transfer and immunoblots by antibodies against specific posttranslational modifications Jessika Obi & Sonja Schröfl Medical University of Vienna, Austria Training STSE MUW/BU
Session 5: Oral Session Chairpersons  Danijela Apostolovic Marija Perusko Karolinska InstitutetUniversity of Belgrade
12:15-12:30 Thermal treatment effect on the antioxidant activity of ethanolic extracts of Myrtus communis L. Ahmed Snoussi Ecole Supérieure des Industries Alimentaires de Tunis
12:30-12:45 Immunoproteomic study of raw and roasted peanut major allergen post-translational modifications (PTMs) Teodora Djukic University of Belgrade, Serbia
12:45-13:00 Higher degree of Mayar reaction induced by spray drying at high temperatures increases antioxidant activity of camel milk proteins. Ana Simovic University of Belgrade, Serbia
13:00-13:15 Microplastics determination in Korean clams Maria Krishna de Guzman Ghent University Global Campus
13:15 Lunch
Workshop ends
flag_yellow_high

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 810752

2nd FoodEnTwin Workshop

Experimental animal models for food and environment

February 3-4, 2020, Vienna, Austria

 

Book of Abstracts

 

Scientific Committee

Tanja Cirkovic Velickovic (President)

Paola Roncada

Andrea Urbani

Irena Vovk

Bruno De Meulenaer

Karin Hoffmann-Sommergruber

Marianne van Hage

Local Organizing Committee

Michelle Epstein

Karin Hoffmann-Sommergruber

Aleksandra Iniç-Kanada

Shu Liu

Sahar Kazemi

Jessika Obi

Sonja Schröfl

 

Session 1: Mouse and rat models for food allergy

EoodEnTwin highlights

Tanja Cirkovic Velickovic

University of Belgrade

The objective of FoodEnTwin (http://horizon2020foodentwin.rs/) is to create a networking collaboration among the University of Belgrade – Faculty of Chemistry (UBFC) and its Center of Research Excellence for Molecular Food Sciences (CoE MFS) and four high renowned institutes from Sweden (Karolinska InstitutetI), Austria (Medical University of Vienna) and Belgium (KULeuven and Ghent University) providing a unique opportunity for UBFC and its partners to increase their scientific excellence and visibility, technology innovation capacity and enable frontier research at the crossroad of food, agriculture, chemistry, nutrition and environmental sciences by the infusion of –Omics technologies and experimental animal models. The project focuses on the key target actions of twinning of research activities through networking, training and a lecturing program resulting in a roadmap for a future collaboration, organization of four public Summers Schools, internal and external expert-driven Academia-Industry meetings, two workshops, and finally, bringing the European Food Chemistry conference (EuroFoodChem) in 2021 to the UBFC in Serbia to increase the UBFC, the Serbian and the European visibility in the fields of food sciences. The scientific topic addresses the major challenge of how environmental pollution affects the food we eat at the molecular level. The project will have a significant societal impact. Our dissemination approach will present our networking ideas to a broad public, from experts, the science community and industry stakeholder organization, to interested, non-professionals, making society more aware of the impact that environment has on food and the importance of new approaches in food, nutrition and environmental sciences. The aim of this 3-year project is to use cutting-edge -omics technologies (proteomics, transcriptomics, digestomics, allergomics, metalomics and lipidomics) and experimental animal models to address the challenges in food, nutrition and environmental sciences in a way that enables the creation of a pan-European research network through the twinning research activities in this project..To achieve the objectives of the FoodEnTwin project, the consortium partners have implemented a comprehensive set of measures within the project’s key work packages (WPs): Short term staff exchanges; (WP1), Training workshops, and summer schools; (WP2), Dissemination and outreach. (WP3)

Rat models of food allergy

Katrine Lindholm Bøgh

Danish Food Institute

In this lecture, I will present an overview of the different parameters that you will need to consider when designing animal experiments. I will give examples of the impact of factors related i) to the proteins, such as dose-response relationship, protein preparation and processing, ii) to the host such as stain, gender and disease status, iii) to the environment such as diet, microbiome and housing condition, and iv) to the experimental design, such route of administration, use of adjuvant and end-point analyses. Allergenicity assessment of novel foods is a difficult task, and not animal models have been validation for such allergenicity assessment. This lecture will provide an example of how animal models can be used in the evaluation of the de novo sensitising as well as in cross-reaction capacity. Finally, as brief overview of how animal models can be used for preclinical assessment of new prevention and treatment strategies within food allergy.

Mouse models of food allergy

Karine Adel-Patient

CEA

In this lecture, we will have a view of mouse models of food allergy. which are not restricted to gastrointestinal sensitization, then mimicking the real life conditions. I'll present some of our own models and experiences, placing them in other published preclinical and clinical data.

Session 2: Risk Assessment in Food Allergy

Animal models for risk assessment of food allergy

Antonio Dumont Fernandez

EFSA

The allergenicity assessment is one of the most important and difficult aspects in the safety assessment of foods derived from biotechnology and novel sources. A weight-of-evidence approach is currently in place for the allergenicity assessment, which takes into account information of different nature and provides the risk assessor the appropriate elements to conclude on safety. Among this information, animal models are currently in used for the toxicological assessment but for different reasons their usefulness for the allergenicity assessment is not fully implemented in the regulatory frame. The development of novel strategies for the allergenicity assessment based on state-the-art in science on the field and considering novel in silico, in vitro and in vivo models are of major relevance and in urgent needs.

Experimental in vitro and in vivo models for food allergy risk assessment

Michelle Epstein

Medical University of Vienna

In this lecture, I will present an overview of in vitro and in vivo animal models used in food allergy aimed at predicting allergenicity of food proteins. This lecture will link to the lecture on risk assessment by Dr. Antonio Dumont Fernandez. I will focus on some of the reviews from this Special issue DOI: 10.1016/j.ddmod.2016.11.002; and DOI:10.1186/s13601-016-0110-2; DOI:10.1016/j.fct.2019.04.052; and DOI: 10.1111/all.13943.

Session 3: Protein allergens

Protein structure of food allergens

Karin Hoffmann-Sommergruber

Medical University of Vienna

Karin Hoffmann-Sommergruber, Dept. of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria. Only a restricted number of protein families account for IgE mediated food allergic reactions. For some protein families the rigid protein structure and / or enzymatic activities may account for their allergenic activity. However, allergens usually do not come alone. They are embedded in a mix of proteins, carbohydrates, small molecules and lipids. In the recent past increasing evidence has been provided on the interaction of lipids and the innate immune system contributing to the onset of an allergic immune response in predisposed individuals. For example, lipids are sensed by dendritic cells (DCs) via toll like receptors, such as TLR2. As a consequence activated DCs will present lipids via CD1d molecules to other immune cells such as invariant natural killer T-lymphocytes (iNKTs). It has been shown that a number of allergens carry pollen- or microbial derived lipids and activate via DCs Th1, Th2 and Th17 mediated allergic inflammation. Another example is the allergenic activity due to proteins’ ability to cross epithelial barriers and subsequently interact with the immune cells, thus triggering a Th2 prone response. An update of the recent underlying hypotheses will be presented and the features of the respective allergen protein families summarized.

Allergy to red meat

Marianne van Hage

Karolinska Institutet

Over the past years a new type of food allergy, red meat allergy or the galactose-α-1,3-galactose syndrome (AGS), has been recognized, wherein mostly middle-aged patients experience delayed severe allergic reactions after consumption of mammalian meat and mammalian products. The reactions are caused by IgE antibodies against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal), which is not only present on glycolipids and glycoproteins from mammalian sources, but also in some pharmaceuticals (cetuximab, antivenom, gelatin containing vaccines). We provide the first clinical and serological characterization of a large cohort of mammalian meat allergic patients from Northern Europe, in which the patients were well characterized and thoroughly investigated by the same physician with many years of experience with AGS. We noted an equal gender distribution of the disease. Furthermore, nearly half of the patients had experienced anaphylaxis and they had significantly higher IgE levels to α-Gal compared to non-anaphylactic patients. Atopy was found to be significantly more frequent in mammalian meat allergic patients compared to the general Swedish population. In addition, atopy increased the risk of anaphylaxis with respiratory symptoms. We also observed that patients belonging to blood group B were less than 2%, which is in line with our previous data showing that self-tolerance to the B-antigen results in a reduced risk of sensitization to α-Gal and development of AGS. As red meat allergy is a relatively new and upcoming disease, knowledge of the patients’ detailed clinical and immunological characteristics is warranted to increase the awareness and management of the disease.

Tomato allergens – multiomics identification and control in farming

Wolfram Weckwerth

University of Vienna

In the lecture, I will present multiomics technologies for the detection of known and novel tomato allergens. IgE reactivity and skin prick tests are used for the characterisation of these allergens. A remarkable result is how different individual responses of patients to the same tomate extract are with respect to these allergenicity tests. Furthermore, I will discuss the consequences of organic and conventional farmung as well genotype and environmental factors on the expression of potential allergens in tomato. 1. Welter S, et al. (2013) Identification of putative new tomato allergens and differential interaction with IgEs of tomato allergic subjects. Clin Exp Allergy 43(12):1419-1427; 2. Welter S, et al. (2013) Pepino mosaic virus infection of tomato affects allergen expression, but not the allergenic potential of fruits. Plos One 8(6):e65116; 3. Dolle S, et al. (2011) Tomato allergy: impact of genotype and environmental factors on the biological response. J Sci Food Agric 91(12):2234-2240; 4. Schwarz D, et al. (2011) Impact of arbuscular mycorrhizal fungi on the allergenic potential of tomato. Mycorrhiza 21(5):341-349; 5. Bassler OY, et al. (2009) Evidence for novel tomato seed allergens: IgE-reactive legumin and vicilin proteins identified by multidimensional protein fractionation-mass spectrometry and in silico epitope modeling. J Proteome Res 8(3):1111-1122.

Session 4: FoodEnTwin Seminars

FoodEnTwin highlights

Tanja Cirkovic Velickovic

University of Belgrade

Overview of FoodEnTwin STSE WP1 entitled Short Term Staff Exchanges (STSEs) aimed to increase research excellence of the coordinating institution in the field of –Omics technologies in food, nutrition and environmental sciences. STSE were conducted in cooperation with EU partners and resulted in a set of standard operating procedures (SOPs) or reports on the collaborative research performed during the collaborative STSE. Three tasks were foreseen to take place during the project implementation: training exchanges, already planned collaboration exchanges and exchanges in support of new collaborations between coordinator and partner institutions (not foreseen at the time of the application). During the first reporting period, the training missions objective was to create a collection of standard operating procedures (SOPs). Firstly, the application, evaluation and reporting procedures for the exchanges was established. The application procedure for STSEs was agreed upon during the kick-off meeting (September 26, 2018), and described and available for download at the project web portal: http://horizon2020foodentwin.rs/stse/ . SOP formats were agreed upon for each of the training STSEs. Continued discussions on the training missions were held during the kick off and first annual meeting of the FoodEnTwin. All exchanges originally planned within the GA were immediately approved. Additional training exchange objectives were fine-tuned by direct communication between home and host supervisors. Seven exchanges from coordinator (University of Belgrade – Faculty of Chemistry, UBFC) to partner institutions were planned. These STSEs included trainings targeting lipidomics, metallomics, allergomics, and transcriptomic-functionomic applications in both food and environmental sciences. In addition, four STSEs from partner institutions to coordinator were conducted. Training visits resulted in a collection of SOPs. Each visit of UBFC applicants was followed with a seminar open for public and also announced on the web site of the project. First part of seminars were given by UBFC researchers to interested audience within separate session of FoodEnTwin Workshop “Food and Environmental –Omics” held on June 20-21, 2019 in Belgrade. Several visits also resulted in seminar given both at the home and host institutions. The WP1 objectives set up to twinning through exchange of know-how and experience by staff exchanges towards achieving project’s S&T goals – enabling frontier research and infusion of –omics technologies into the fields of food, nutrition, agriculture and environmental sciences have been accomplished in a great deal within the first period of the project duration.

Allergenomics of ticks

Danijela Apostolovic

Karolinska Institutet

Background: The mammalian carbohydrate galactose-α-1,3-galactose (α-Gal) has shown to be the cause of a novel form of food allergy, red meat allergy. There is evidence for tick bites as a cause of IgE to α-Gal. Objective: By an allergenomic approach reveal α-Gal carrying proteins in the European tick Ixodes (I.) ricinus recognized by red meat allergic patients. Methods: IgE-reactivity, IgE-binding, and allergenicity of protein extracts from adults, and larvae as well as of saliva of adults I. ricinus ticks were assessed among 32 Swedish and 18 US red meat allergic patients by ImmunoCAP, ELISA and immunoblot. Basophil activation test was used for assessment of allergenic activity. The presence of the α-Gal epitope was verified using an anti-α-Gal antibody and comparative as well as shotgun proteomics for identifying the α-Gal carrying proteins. Results: Swedish and US patients had IgE reactivity against I. ricinus proteins. The presence of α-Gal was supported by IgE inhibition. IgE binding α-Gal-carrying proteins were recognized in saliva. All tested patients showed a higher allergenic activity to adult I. ricinus than to larvae and α-Gal. Allergenomics revealed vitellogenin and α-2-macroglobulin are IgE-binding α-Gal carriers. Vitellogenin was also identified in tick saliva from adult I. ricinus ticks. Conclusions: Red meat allergic patients have allergenic reactivity against I. ricinus adult and larvae and have IgE to α-Gal carrying proteins in tick saliva. Vitellogenin and α-2-macroglobulin were identified as IgE binding and α-Gal carrying proteins in I. ricinus. The results support the strong relationship between tick bites and the production of α-Gal-IgE, and development of red meat allergy. Acknowledgments: Swedish Research Council, the Stockholm County Council (ALF project), the Swedish Asthma and Allergy Association's Research Foundation, the King Gustaf V 80th Birthday Foundation, the Swedish HeartLung Foundation, the Hesselman Foundation, the Konsul Th C Bergh Foundation, the Swedish Cancer and Allergy Foundation, the Magnus Bergvall Foundation, the Swedish Association for Allergology, the European Union's Horizon 2020 Research and Innovation Program-IDLYME GA No. 720480, the European Union's Horizon 2020 FoodEnTwin project, GA No. 810752, and Ministry of Education Science and Technological Development Republic of Serbia GA No. 172024. The EC does not share responsibility for the content of the article.

Bovine γ-Globulin and Lactoperoxidase as major milk allergens among a mammalian meat allergic population

Marija Perusko

University of Belgrade

Objective. Mammalian meat allergy (MMA) is a severe form of food allergy with delayed symptoms where the IgE antibodies are directed against a carbohydrate epitope, galactose-α-1,3-galactose (α-Gal). Many MMA patients report allergic symptoms upon consumption of milk or dairy products. The aim of the project was to investigate the allergenicity of bovine milk proteins in a MMA population. Material and Methods. Adults with diagnosed MMA (n = 34) were recruited and their sIgE levels to α-Gal, beef and milk were analyzed by ImmunoCAP. Milk proteins were assayed by immunoblot and inhibition ELISA for the presence of the α-Gal epitope and for the binding to mammalian meat allergic patients’ IgE. Additionally, capacity of whole milk and milk proteins to activate basophils of MMA patients was tested. Results. Thirty-three out of 34 MMA patients were IgE positive to milk, and their IgE levels to milk were lower than those to α-Gal or beef. Significant correlations between IgE levels to milk and α-Gal (rs=0.55, P < 0.001), as well as between milk and beef (rs=0.77, P < 0.001) were observed. Immunoblot analysis of milk proteins revealed bovine γ-globulin (BGG) as α-Gal carrier. Other tested milk proteins, α-lactalbumin, β-lactoglobulin, α-casein, β-casein and κ-casein were negative for the presence of α-Gal epitope. BGG was also shown to bind IgE of MMA patients in immunoblot analysis. ELISA experiments showed that whey proteins, BGG and also lactoperoxidase (LPO) exerted a dose-dependent inhibition of MMA patients’ IgE binding to α-Gal indicating presence of the α-Gal epitope in these proteins. Importantly, activation of MMA patient’s basophils by milk, BGG and LPO was demonstrated. Conclusions. BGG and LPO were identified as α-Gal carrying proteins in milk that bound IgE antibodies and furthermore activated basophils of mammalian meat allergic patients. The study highlights the importance of milk as an allergenic food source among the MMA population. Acknowledgments: This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 172024 and by EAACI (MP was awarded a Research Fellowship). The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 810752.

Development of an immune-polymerase chain reaction for detection and quantification of shellfish tropomyosin

Mirjana Radomirovic

University of Belgrade

Food allergies represent important health problem in industrialized countries, with seafood being recognized as one of the 8 most common sources of allergens. While there are several proteins that have been linked to shellfish allergy, tropomyosin accounts for majority of diagnozed ingestion-related shellfish allergies. Presence of even traces of allergens in food can be a serious health hazard to consumers, which is why proper labeling of food products by food manufacturers is of critical importance for sensitized persons. On the other hand, development of reliable, specific and sensitive methods for detection and quantification of allergens in food products is of the high importance as well. The objective of this study was to develop highly sensitive immuno-polymerase chain reaction (immuno PCR) method for the detection and quantification of shellfish tropomyosin in food samples. Immuno PCR method couples standard sandwich enzyme-linked immunosorbent assay (ELISA) format with real time PCR. Monoclonal antibody was used as capture antibody, while biotinylated polyclonal antibody served as detection antibody. Reporter biotinylated DNA was coupled to detection antibody via streptavidin and subsequently amplified and quantified by real time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The results were compared to standard sandwich ELISA. This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 172024. The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 810752.

Discrete Hf18 metal-oxo cluster as a nanozyme for site-specific protein hydrolysis

Jens Mons

Katholieke University Leuven

Effective heterogeneous catalysts for the controlled transformation of large and complex biomolecules are rare and challenging to develop. In particular, selective hydrolysis of proteins by non-enzymatic catalysis is difficult to achieve, yet it is crucial for many modern applications in biotechnology and proteomics. Herein we report that discrete hafnium metal-oxo cluster (Hf18O10(OH)26(SO4)13 (H2O)33) (Hf18), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the rapid hydrolysis of horse heart myoglobin (HHM) protein in low buffer concentrations. Remarkably, among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues, Asp5, Asp21, Asp45, Asp110, Asp123 and Asp142 was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the synergistic actuation of Hf4+ Lewis acidic sites and the Brønsted acidic surface of Hf18. A combination of X-ray scattering and ESI-MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of solid Hf18 cluster, and not from smaller, soluble Hf species that could leach in solution. This study highlights the great potential of discrete metal-oxo cluster materials as inorganic proteases.

Proteomics and PTMs profiling of raw and thermally treated shellfish samples collected in Korea: focus on tropomyosin

Urmila Khulal

Ghent University

Shellfish, is a highly nutritive food resource in the world, but also among the eight allergic food groups accounting for approximately 90% of all immunoglobulin E food allergies worldwide [1]. The well-recognized major allergen muscle protein tropomyosin(TM) is responsible for cross reactivity between shellfish and other invertebrates [2]. while TM of vertebrates such as chicken, pig, and cow is not a prominent allergen. Therefore, further characterization is needed to shed more light of allergenic potential of TM in different shellfish species, including its expression pattern and post-translational (PTM) and chemical modifications upon 1 thermal treatment and 2 simulated in vitro gastro-intestinal digestion. Most in vitro digestibility studies are based on the protein extract rather than whole food matrix thus overlooking its effect on TM stability [3]. Our objectives are to primarily test the digestibility of shellfish Tropomyosin (raw and thermally treated based on their real life consumption modes) mimicking the gastro-intestinal digestion under standardized conditions. Secondarily and importantly to characterize shellfish TM protein focusing on its expression pattern and PTMs. Methods: Thermal treatment of selected shellfish samples from Korea to compare TM heat stability, Standardized static in vitro methods of simulated gastric digestion[4, 5] for the evaluation and comparison of TM resistance to pepsin and trypsin, Sodium Dodecyl Sulfate-Polyacryl amide Gel Electrophoresis (SDS-PAGE) of digesta supernatant under reducing and non-reducing conditions to quantify proteins and compare raw and thermally treated shellfish samples. Proteome extraction followed by in gel and in solution trypsin digestion [6], high resolution bottom-up tandem mass spectrometry (Nano liquid chromatography coupled to electrospray ionization tandem mass spectrometry on Orbitrap LTQ system), PEAKS Suite platform to assess expression levels of TM via label free quantification (LFQ) among different shellfish samples. Results and discussion: SDS-PAGE analysis shellfish samples showed a range of proteins in varied amounts between 10-250 kDa. Depending upon samples, varied numbers of prominent protein bands were observed including the distinct bands corresponding with the molecular weights of TM(37-39kDa). In agreement with publications, TM was, indeed, resistant against pepsin digestion as well as thermal treatment prominently in case of invertebrates than in vertebrates. This was confirmed upon Ab based Western blot analysis. Our results show that, upon thermal treatment but importantly with pepsin digestion, TM (allergen) is completely degraded in vertebrates in contrast to the invertebrates’ TM (which is pepsin resistant and heat stable). This result provides an insight on the differences in digestibility of allergenic versus non-allergenic TM in real food matrix and upon thermal treatments of solid food samples. Protein PTM modifications and expressions results will follow during our short term staff exchange (STSE) training.

Characterisation of peanut allergens and possible post-translational smodifications (PTMs)

Shu Liu

Medical University of Vienna

Peanut allergy is the most common type of food allergy causing severe reactions or even fatal anaphylaxis in sensitised individuals. The major peanut allergens are Ara h 1, Ara h 2, Ara h 3, and Ara h 6 which cause the most severe responses. Their molecular properties have been characterised but possible post-translational modifications (PTMs) are not well understood. The goal of this study was to utilise a combination of nanoLC-Mass Spectrometry (MS)/MS methods and PEAKS Studio 8.0 (Bioinformatics Solutions Inc., Ontario, Canada) program to evaluate PTMs in epitope sequences of purified Ara h 2 (Conglutin-7) and Ara h 6 (Conglutin). We identified 33 peanut proteins and found 242 epitopes, 29 potential PTMs and 4 mutations for Ara h 2, and 8 epitopes, 9 likely PTMs and no mutations for Ara h 6. Analyses of peanut extracts from raw, boiled and roasted samples revealed different PTMs on epitopes depending on previous treatment. These data suggest that PTMs on certain epitopes could influence allergenicity and digestibility of peanut proteins.

Resolving Dactylis glomerata pollen proteins by 2D SDS PAGE and subsequent western transfer and immunoblots by antibodies against specific posttranslational modifications

Jessika Obi&Sonja Schröfl

Medical University of Vienna

The prevalence and severity of respiratory allergic diseases is increasing worldwide and especially people living in urban areas are affected. There could be a number of factors responsible for this association, but air pollution seems to be a likely one. Studies have shown that air pollution can directly affect pollen grains and their allergenicity, although the significance of post-translational modifications (PTMs) in this context has not yet been studied as extensively as other aspects related to pollen pollution. The aim of the work during this exchange was to study processes of PTMs in response to the external environment. We wanted to compare extracts of two pollen samples of Dactylis glomerata, one collected in a rural, one in an urban area, using proteomics technology. We determined protein concentrations using Bicinchoninic acid assay (BCA assay), performed two-dimensional gel electrophoresis along with in solution digestion with subsequent ZipTips purification as a preparatory step for mass spectrometry measurements.

Session 5: Oral Session

Thermal treatment effect on the antioxidant activity of ethanolic extracts of Myrtus communis L.

Ahmed Snoussi

Ecole Supérieure des Industries Alimentaires de Tunis

Heat treatments employed in the food industries, to improve the sensorial, nutritional and hygienic quality, might affect the phenolic content and the biological effects of formulated foods. Thus, the understanding of the chemistry and the biological outcome of this process on bioactive compounds is crucial for the development of food with beneficial effects on health. The objective of this research is to study the effect of heat processing at 70, 90 and 110°C for 120 min on the stability of the antioxidant activity of Myrtus communis L. ethanolic extracts. The obtained results showed that the degradation of phenolics compounds in myrtle extracts is influenced by the temperature. Heating at 110 °C led to the highest decrease in total phenols, flavonoids and proanthocyanidins amounts. The antioxidant activity of the extracts was tested by the DBBH and ABTS scavenging assays. Despite, the decrease of the phenolic compounds amounts of extracts, their antioxidant activities are not lost. Heating at 70 °C led to a decrease in the antioxidant activity of extracts. However, an increase was observed for extracts treated at 90 and 110°C. These results suggest that the products of degradation of polyphenols could have an antioxidant activity which sometimes superior to the native one.

Immunoproteomic study of raw and roasted peanut major allergen post-translational modifications (PTMs)

Teodora Djukic

University of Belgrade

Peanuts are widely used for the preparation of a variety of foods and are primarily consumed in roasted or boiled form. They have a high protein content of 24–29% from which these six are major peanut allergens: Ara h 1, Ara h 2, Ara h 3 and Ara h 6 because they cause an immune response in over 50 % of the patients allergic to peanut. Arah 1, 2, 6 are peanut storage proteins where Arah 2 is very similar to Arah 6, they share 58 % of homology in sequence. Both of these are heat stable and resistant to digestion. PTM profile may differ between raw and thermally treated peanut, which could affect its allergic potential depending on type, size and position of modifications. We focused our research on post – translational modifications (PTMs) of Arah 1, 2, and 6, due to roasting of the peanut and compared raw and roasted samples to see the difference in PTMs. The objective was to compare PTMs found using bioinformatic methods with results got using methods like 2D-SDS PAGE electrophoresis, Western Blot analysis and ELISA immunoassay. Protein extracts of raw and roasted peanuts were analyzed by Western Blot using antibodies on 7 PTMs. Same extracts were analyzed on 2D-SDS PAGE electrophoresis. After Western Blot analysis we focused on 4 modifications that showed the biggest difference between the two samples. We tested those 4 modifications using ELISA immunoassay with patient’s sera who are allergic to peanut. What we concluded was that peanut allergens are indeed carriers of PTMs that differ in pattern and quantity between treated and non-treated. Roasted peanut extract carries more modifications on Arah 1, 2 and 6 than raw peanut especially Anti-Hydroxyproline (HyP), Anti-Carbamil Lysine (CarbK) and Methionine Sulfoxide (OxoM). Supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia GA 172024, and FoodEnTwin (Horizon 2020) GA 810752.

Higher degree of Mayar reaction induced by spray drying at high temperatures increases antioxidant activity of camel milk proteins.

Ana Simovic

University of Belgrade

Camel milk is highly nutritious food with many health benefits proposed. Demand for camel milk has increased worldwide. Production of camel milk powders facilitate its transport, prolonge shelf-life, and also offer an attractive additive for various food products. In this study we examined the effect of freeze/spray drying treatment for camel milk powder production, on physiochemical and functional properties of camel milk proteins. Whole camel milk powders were prepared by spray drying treatment at six different inlet temperatures (190°C - 250°C) or by freeze drying. The soluble protein fractions upon the treatments were analysed by combination of electrophoretic and spectroscopic techniques. Structural and functional properties of camel milk proteins such as Maillard reaction products formation, antioxidant activity and protein solubility were assessed. SDS-PAGE revealed non-uniform increase in Mw of major protein bands, while native electrophoresis revealed non-uniform decrease in pI values with increased inlet temperature of spray drying. That indicated attachement of lactose moieties to NH2-group of proteins via non-enzymatic Maillard reaction. Spectrophotometric analysis showed formation of intermediate Maillard reaction products (increased absorbance at 294 nm) and no detectable late Maillard reaction products formation. Higher inlet temperatures (230°C - 250°C) resulted in higher protein carbonyls formation and lower content of free amino groups as a result of Maillard reaction. Far-UV circular dichroism spectra showed no differences in secondary structures between freeze and spray dried samples. Antioxidant activity and protein solubility were increased with increase in inlet temperature. Our results showed that spray drying treatment promotes non-enzymatic glycation of camel milk proteins and exert significant effects on the techno-functional properties of CM powder such as nutritional value and shelf life. Thus, optimization of spray drying parametars is essential for production of high quality camel milk powders. This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 172024. The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 810752.

Microplastics determination in Korean clams

Maria Krishna de Guzman

Ghent University Global Campus

Microplastics (MP) pollution has reached a global scale. In the marine environment, it accounts for 92.4% of total plastic debris. Their high bioavailability to marine biota creates a serious health risk to consumers of seafood. In 2016, FAO identified South Korea as the top seafood consumer. With a consumption of 58.4 kg per person per year, South Koreans are more disposed to the negative health effects of MP. In this study, the MP content of 50 small (estimated age of ≤3 years) and 50 big clams (estimated age of >3 years) from the southwestern seas of South Korea was determined. Dissolution of organic matter was achieved by digestion in 10% w/w KOH. The isolated MP were visualized and counted using fluorescence microscopy with the aid of Nile red dye. Preliminary results from 24 small clams show that 95% of total MP detected are particles, followed by fragments (4%) and fibers (1%). In terms of size, 75% of the MP are < 250 μm, 16% are 250-1000 μm and the remaining 9% are ≥1mm. So far, the smallest particle detected from the clams is 10 μm. Given a total count of 523 MP from 24 small clams, the initial MP abundance is 22 microplastics per clam. In terms of weight, the MP concentration is 3.4 microplastics per gram wet weight. These current experiments will be continued to analyze all clam samples and chemical characterization will be conducted to identify the polymer type of the isolated MP. Additional studies will also be carried out to identify the effects that ingested MP has on human health.