TY - JOUR A1 - Uhr, Linda A1 - Wieland, Phillis A1 - Homann, Thomas A1 - Huschek, Gerd A1 - Rawel, Harshadrai Manilal T1 - Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products JF - European food research and technology : official organ of the EuCheMS, Division of Food Chemistry N2 - The use of technical enzymes in bakery industry is necessary for a consistent and good quality of baked products. Since the cultivation of cereals leads to low amounts of endogenous enzymes being present, a need of their commercial alternatives is becoming a routine process in order to meet the consumer quality demands. Targeted quantification proteomics-based methods are necessary for their detection to meet the regulatory criteria. Here, we initially report on the identification of Lipase FE-01, a lipase from fungus Thermomyces lanuginosus, as analyzed by SDS-PAGE, in-Gel digestion, and MALDI-TOF-MS. In further experiments, the focus of the study was directed toward an extensive use and optimization of in-solution enzymatic digestion in combination with LC-MS/MS techniques in identification of specific peptide markers and finally in utilization of the latter in delivering reproducible quantification data for several different technical enzymes (alpha-amylases, xylanase, and lipases from microbial origin) in complex matrices such as baked bread and wheat flour. Two digestion protocols (a fast option using thermocycler program and the well-established overnight method) were tested, and both of these can be successfully applied. The application of isotopically labeled analogs of the MRM targeted peptides as internal standards and the addition of an internal protein standard during the extraction/digestion experiment were compared to determine the optimal quantification algorithm of the recovered enzyme concentrations. Thus, a standardized sensitive LC-MS/MS method could be developed to determine technical enzymes as forthcoming ingredients in the prefabricated food formulations in concentrations lower than 10 ppm. KW - Technical enzymes KW - Amylase KW - Xylanase KW - Lipase KW - Baked products KW - Mass spectrometry Y1 - 2016 U6 - https://doi.org/10.1007/s00217-015-2536-5 SN - 1438-2377 SN - 1438-2385 VL - 242 SP - 247 EP - 257 PB - Springer CY - New York ER - TY - JOUR A1 - Reinkensmeier, Annika A1 - Steinbrenner, Katrin A1 - Homann, Thomas A1 - Bussler, Sara A1 - Rohn, Sascha A1 - Rawel, Harshadrai Manilal T1 - Monitoring the apple polyphenol oxidase-modulated adduct formation of phenolic and amino compounds JF - Food chemistry N2 - Minimally processed fruit products such as smoothies are increasingly coming into demand. However, they are often combined with dairy ingredients. In this combination, phenolic compounds, polyphenoloxidases, and amino compounds could interact. In this work, a model approach is presented where apple serves as a source for a high polyphenoloxidase activity for modulating the reactions. The polyphenoloxidase activity ranged from 128 to 333 nakt/mL in different apple varieties. From these, ‘Braeburn’ was found to provide the highest enzymatic activity. The formation and stability of resulting chromogenic conjugates was investigated. The results show that such adducts are not stable and possible degradation mechanisms leading to follow-up products formed are proposed. Finally, apple extracts were used to modify proteins and their functional properties characterized. There were retaining antioxidant properties inherent to phenolic compounds after adduct formation. Consequently, such interactions may also be utilized to improve the textural quality of food products. KW - Apple polyphenoloxidase KW - Phenol-amino-adducts KW - Post-translational protein modification KW - Functionality Y1 - 2016 U6 - https://doi.org/10.1016/j.foodchem.2015.07.145 SN - 0308-8146 SN - 1873-7072 VL - 194 SP - 76 EP - 85 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Sievers, Steven A1 - Rawel, Harshadrai Manilal A1 - Ringel, Karl Peter A1 - Niggemann, Bodo A1 - Beyer, Kirsten T1 - Wheat protein recognition pattern in tolerant and allergic children JF - Pediatric Allergy and Immunology N2 - BackgroundWheat is one of the most common food allergens in early childhood. In contrast to other food allergies, wheat-specific IgE correlates badly with clinical symptoms and relevant components have been identified mostly for wheat-depended exercise-induced anaphylaxis. Moreover, a high percentage of patients present with immediate type symptoms but wheat-specific IgE cannot be detected with commercial available systems. ObjectiveWe addressed the question whether the IgE recognition pattern between wheat allergic (WA) and clinically tolerant (WT) children differs in order to identify individual proteins useful for component-resolved diagnostics. MethodsSera of 106 children with suspected wheat allergy, of whom 44 children had clinical relevant wheat allergy and 62 were tolerant upon oral food challenge, were analyzed for wheat-specific IgE using the ImmunoCap system as well as immunoblots against water and salt soluble, and water-insoluble protein fractions. 40 randomly selected sera were analyzed for specific IgE to 5-gliadin. ResultsSixty-three percent of the WT and 86% of the WA children were sensitized to wheat with >0.35 kU(A)/l in ImmunoCAP analysis. We could confirm the role of -, ss-, -, and -gliadins, and LMW glutenin subunits as major allergens and found also IgE binding to a broad spectrum of water- and salt-soluble protein bands. It is of great importance that wheat allergic and tolerant patients showed IgE binding to the same protein bands. WT and WA did not significantly differ in levels of 5-gliadin-specific IgE. Conclusions & Clinical RelevanceChildren with challenge proven clinical relevant food allergy and tolerant ones had a similar spectrum of IgE binding to the same protein bands. These findings imply that component-resolved diagnostics might not be helpful in the diagnostic work-up of wheat allergy. KW - wheat KW - IgE KW - 5-gliadin KW - protein pattern KW - immunoblot Y1 - 2016 U6 - https://doi.org/10.1111/pai.12502 SN - 0905-6157 SN - 1399-3038 VL - 27 SP - 147 EP - 155 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Wilde, Sandra Catharina A1 - Treitz, Christian A1 - Keppler, Julia Katharina A1 - Koudelka, Tomas A1 - Palani, Kalpana A1 - Tholey, Andreas A1 - Rawel, Harshadrai Manilal A1 - Schwarz, Karin T1 - beta-Lactoglobulin as nanotransporter - Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide JF - Food chemistry N2 - The whey protein beta-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified beta-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified beta-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to beta-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein. (C) 2015 Elsevier Ltd. All rights reserved. KW - Beta-lactoglobulin KW - Covalent modification KW - LC-MS KW - CD, DLS KW - Thiol KW - Allicin KW - Garlic KW - Diallyl disulfide Y1 - 2016 U6 - https://doi.org/10.1016/j.foodchem.2015.11.011 SN - 0308-8146 SN - 1873-7072 VL - 197 SP - 1022 EP - 1029 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Bußler, Sara A1 - Rumpold, Birgit A. A1 - Fröhling, Antje A1 - Jander, Elisabeth A1 - Rawel, Harshadrai Manilal A1 - Schlüter, Oliver K. T1 - Cold atmospheric pressure plasma processing of insect flour from Tenebrio molitor: Impact on microbial load and quality attributes in comparison to dry heat treatment JF - Meteoritics & planetary science : journal of the Meteoritical Society N2 - In this study, the applicability of semi-direct cold atmospheric pressure plasma (CAPP) during postharvest processing of Tenebrio molitor flour is investigated. Besides analyzing the decontamination efficacy, plasma induced impact on techno-functionality, protein solubility, composition and structure was determined and compared to heat induced effects. Following CAPP treatment, the total microbial load of the Tenebrio flour of 7.72 log(10) cfu/g was reduced to 7.10 (1 min), 6.72 (2.5 min), 5.79 (5 min), 5.19 (7.5 min), 521 (10 min) and 4.73 (15 min) log(10) cfu/g. With increasing exposure to CAPP, protein solubility at pH 4 almost linearly decreased to a minimum of 54%. Water binding capacity decreased from 0.79 to 0.64 gwatedg whereas oil binding capacity increased from 0.59 to 0.66 g(oil)/g. Gel electrophoresis revealed a decrease of all protein fractions at pH 4 whereas at pH 10 the band pattern significantly shifted to protein fractions with higher molecular weights. Industrial relevance: Edible insects are rich in valuable protein, fat, fibre, minerals and micronutrients. Although a wide range of species represent a valuable alternative protein source that could contribute to food and feed security, they are industrially hardly exploited. The tailored application of proper processing technologies could lead to novel insect-based high-protein food and feed products with unique functional properties supporting the increase in acceptability among potential consumers. Current research concentrates on developing processing chains including innovative nonthermal approaches. Cold atmospheric pressure plasma (CAPP) has gained attention as an effective technology for the decontamination and modification of fresh and dry agricultural products. In the postharvest chain of edible insects, the application of CAPP could contribute to the development of safe and high-quality insect-based products in the food and feed sector. (C) 2016 Published by Elsevier Ltd. KW - Edible insects KW - Postharvest processing KW - Thermal and nonthermal treatment KW - Inactivation KW - Decontamination KW - Protein functionality and modification Y1 - 2016 U6 - https://doi.org/10.1016/j.ifset.2016.07.002 SN - 1466-8564 SN - 1878-5522 VL - 36 SP - 277 EP - 286 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Islam, Khan Shaiful A1 - Khalil, Mahmoud A1 - Männer, K. A1 - Raila, Jens A1 - Rawel, Harshadrai Manilal A1 - Zentek, J. A1 - Schweigert, Florian J. T1 - Effect of dietary alpha-tocopherol on the bioavailability of lutein in laying hen JF - Journal of animal physiology and animal nutrition N2 - Lutein and its isomer zeaxanthin have gained considerable interest as possible nutritional ingredient in the prevention of age-related macular degeneration (AMD) in humans. Egg yolk is a rich source of these carotenoids. As an oxidative sensitive component, antioxidants such as -tocopherol (T) might contribute to an improved accumulation in egg yolk. To test this, chickens were fed lutein esters (LE) with and without -tocopherol as an antioxidant. After depletion on a wheat-soya bean-based lutein-poor diet for 21days, laying hens (n=42) were equally divided into three groups and fed the following diets for 21days: control (basal diet), a LE group (40mg LE/kg feed) and LE+T group (40mg LE plus 100mg T/kg feed). Eggs and blood were collected periodically. Carotenoids and -tocopherol in yolk and blood plasma were determined by HPLC. Egg yolk was also analysed for total carotenoids using a one-step spectrophotometric method (iCheck(())). Lutein, zeaxanthin, -tocopherol and total carotenoids in egg yolk were highest after 14days of feeding and decreased slightly afterwards. At the end of the trial, eggs of LE+T group contained higher amount of lutein (13.72), zeaxanthin (0.65), -tocopherol (297.40) and total carotenoids (21.6) compared to the LE group (10.96, 0.55, 205.20 and 18.0mg/kg, respectively, p<0.05). Blood plasma values of LE+T group contain higher lutein (1.3), zeaxanthin (0.06) and tocopherol (20.1) compared to LE group (1.02, 0.04 and 14.90mg/l, respectively, p<0.05). In conclusion, dietary -tocopherol enhances bioavailability of lutein reflecting higher content in egg yolk and blood plasma. Improved bioavailability might be due to increased absorption of lutein in the presence of tocopherol and/or a greater stability of lutein/zeaxanthin due to the presence of -tocopherol as an antioxidant. KW - carotenoids KW - tocopherol KW - egg yolk KW - bioavailability KW - HPLC KW - iCheck Y1 - 2016 U6 - https://doi.org/10.1111/jpn.12464 SN - 0931-2439 SN - 1439-0396 VL - 100 SP - 868 EP - 875 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Huschek, Gerd A1 - Boenick, Josephine A1 - Loewenstein, Yvonne A1 - Sievers, Steven A1 - Rawel, Harshadrai Manilal T1 - Quantification of allergenic plant traces in baked products by targeted proteomics using isotope marked peptides JF - LWT - food science and technology : an official journal of the Swiss Society of Food Science and Technology (SGLWT/SOSSTA) and the International Union of Food Science and Technology (IUFoST) N2 - The right choice of analytical methods for plant allergen quantification is a deciding factor for the correct assessment and labeling of allergens in processed food in view of consumer protection. The aim of the present study was to develop a validated target peptide multi-method by LC/MS/MS providing high specificity and sensitivity for plant allergen protein detection, plant identification in vegan or vegetarian products using peptide markers for quantification. The methodical concept considers the selection of target peptides of thermostable allergenic plant proteins (Gly m6 soy, Ses i6 sesame and (beta-conglutin from white lupine) by data base research, BLAST and in silico digestion using Skyline software. Different allergenic concentration levels of these proteins were integrated into our own reference bakery products and quantified with. synthesized isotopically labeled peptides after in-solution digestion using LC/MS/MS. Recovery rates within the range of 70-113% and LOQ of 10 ppm-50 ppm (mg allergenic food/kg) could be determined. The results are independent of thermal processing applied during baking and of epitope binding site for the tested allergens. (C) 2016 Elsevier Ltd. All rights reserved. KW - Allergenic food KW - Plant allergen (soy, sesame, lupine) KW - LC/MS/MS; Quantification of allergenic plant traces KW - Food labeling Y1 - 2016 U6 - https://doi.org/10.1016/j.lwt.2016.07.057 SN - 0023-6438 SN - 1096-1127 VL - 74 SP - 286 EP - 293 PB - Elsevier CY - Amsterdam ER -