TY - THES A1 - Uhr, Linda T1 - Technische Enzyme in Backwaren T1 - Technical enzymes BT - Untersuchung der technologischen Wirkung und Entwicklung eines Multiparameterverfahrens zum quantitativen Nachweis mittels LC-MS/MS im Spurenbereich BT - investigation of technological impact, and development of a multi-parameter method for the quantitative detection by LC/MS/MS in trace concentration N2 - Die sensorisch einwandfreie, konstant gute Qualität von Backprodukten, die beim Verbraucher einen hohen Stellenwert hat, wird maßgeblich durch den Gehalt endogener Getreideenzyme beeinflusst. Seit dem Auftreten züchtungsbedingter Enzymdefizite ist der Einsatz technischer Enzyme zur Gewährleistung dieser geforderten Qualität eine feste Größe in der Backwarenindustrie. Lebensmittelrechtlich werden technische Enzyme nicht als Zutat betrachtet, da sie theoretisch während des Backprozesses umgesetzt werden und im Endprodukt keine technologische Wirkung mehr zeigen. Vor allem in gebackenen Produkten bedarf es der Prüfung, dass die eingesetzten technischen Enzyme nicht mehr als Zutat vorliegen und sich somit einer potentiellen Deklarationspflicht entziehen. Zur Gewährleistung der Wirtschaftlichkeit muss der quantitative Einsatz technischer Enzyme in der Backwarenindustrie gesteuert werden, um optimale Effekte zu erzielen und Kosten zu sparen. Ziel dieser Arbeit war daher die Entwicklung eines Analysenverfahrens, das den simultanen Nachweis verschiedener technischer Enzyme und deren Quantifizierung im Spurenbereich auch in gebackenen Produkten ermöglicht. Für die Einschätzung der Wirkung der technischen Enzyme Fungamyl (Novozymes), Amylase TXL (ASA Spezialenzyme GmbH) sowie Lipase FE-01 (ASA Spezialenzyme GmbH) wurden Backversuche durchgeführt, die zeigten, dass Fungamyl und Amylase TXL zu einer verbesserten Brotqualität (Volumenausbeute, Feuchtegehalt, Sensorik) beitrugen. Die Zugabe der Lipase FE-01 führte zu einer vermehrten Bildung freier Fettsäuren und wirkte sich negativ auf die sensorische Brotqualität aus. Dieser bisher nicht beschriebene Effekt konnte auf die Nutzung eines Spezialöls als Backzutat zurückgeführt werden, welches ausschließlich aus gesättigten Fettsäuren besteht. Dies bestätigt die Bedeutung der Auswahl eines geeigneten Fettes beim Zusatz technischer Lipase zum Backprozess. Um die in Fungamyl und Lipase FE-01 enthaltenen Enzyme zu identifizieren, wurden SDS-PAGE und anschließender In-Gel-Verdau angewendet um die Analyse proteolytisch gespaltener Proteine mit MALDI-TOF-MS zu ermöglichen. Es konnte gezeigt werden, dass Fungamyl ein Gemisch aus 9,8 % alpha-Amylase (Aspergillus oryzae) und 5,2 % Endo-1,4-Xylanase (Thermomyces lanuginosus) enthält. Lipase FE-01 besteht aus der Lipase (Thermomyces lanuginosus), Amylase TXL wurde als alpha-Amylase (Aspergillus oryzae) identifiziert. Zur Analyse der technischen Enzyme in Backwaren wurde aufgrund seiner Robustheit und Sensitivität das Verfahren der LC-MS/MS gewählt. Die Entwicklung einer solchen Methode zur Detektion spezifischer Peptide ermöglichte den qualitativen Nachweis der 3 Enzyme alpha-Amylase (Aspergillus oryzae), Endo-1,4-Xylanase (Thermomyces lanuginosus) und Lipase (Thermomyces lanuginosus). Durch eine lineare Kalibrierung aus synthetisch hergestellten Peptiden unter Einbeziehung eines Protein-Internen-Standards sowie isotopenmarkierter Peptidstandards erfolgte darüber hinaus die quantitative Bestimmung in selbst hergestellten Referenzmaterialien (Weizenmehl, Toastbrot und Biskuitkeks). In weniger als 20 Minuten Messzeit kann das Enzym alpha-Amylase ab einer Konzentration von 2,58 mg/kg (Mehl, Keks), bzw. 7,61 mg/kg (Brot) quantitativ nachgewiesen werden. Zeitgleich können die Enzyme Endo-1,4-Xylanase ab einer Konzentration von 7,75 mg/kg (Brot), 3,64 mg/kg (Keks) bzw. 15,60 mg/kg (Mehl) sowie Lipase ab einer Konzentration von 1,26 mg/kg (Mehl, Keks), bzw. 2,68 mg/kg (Brot) quantifiziert werden. Die Methode wurde nach allgemein verwendeten Richtlinien im Zuge einer Validierung statistisch geprüft und lieferte sehr robuste und reproduzierbare quantitative Werte mit Wiederfindungsraten zwischen 50 % und 122 %. Das primäre Ziel dieser Arbeit, die Entwicklung eines quantitativen Multiparameterverfahrens zum Nachweis technischer Enzyme in Backwaren, wurde somit erfolgreich umgesetzt. N2 - The consistent and good quality of baked products, which is of great importance for consumers, is significantly influenced by endogenous grain enzymes. Since cereal breeding techniques have caused deficits of endogenous enzymes, the use of technical enzyme alternatives became a routine process in the bakery industry to ensure the expected quality. According to food law, technical enzymes are not listed as ingredients, because theoretically, enzymes are destroyed during the baking process so that there is no more technological activity in the end product. To prevent a potentially required declaration, it has to be verified that technical enzymes are no ingredients in baking products. To secure economic viability, the quantitative application of technical enzymes should be regulated to gain optimal effects and save costs. Therefore, the aim of this thesis was the development of a quantitative analytical method with the potential to analyze several technical enzymes simultaneously and sensitively (ppm concentration) quantify them in baking products. The effects of the use of the technical enzymes Fungamyl (Novozymes), Amylase TXL (ASA Spezialenzyme GmbH) and Lipase FE-01 (ASA Spezialenzyme GmbH) were monitored during baking experiments. The application of Fungamyl and Amylase TXL resulted in a perfect bread quality (high volume, good bread humidity, good sensory grading). The addition of Lipase FE-01 led to an increased release of free fatty acids, resulting in bread with a bad sensory quality. Reason for that previously not described negative effect is the use of an oil consisting only of saturates fatty acids as a baking ingredient. Thereby the importance of the choice of fat as a baking ingredient was demonstrated and confirmed. To identify the enzymes contained in the technical enzymes Fungamyl and Lipase FE-01, SDS-PAGE and in-gel digestion were applied, enabling the analysis of proteolytically splitted proteins by MALDI-TOF-MS. It was shown that Fungamyl contains 9.8 % alpha-Amylase (Aspergillus oryzae) and 5.2 % Endo-1,4-Xylanase (Thermomyces lanuginosus). Lipase FE-01 consists of Lipase (Thermomyces lanuginosus) and Amylase TXL could be qualified as alpha-Amylase (Aspergillus oryzae). Based on its robustness and sensitivity LC/MS/MS was the analytical method of choice. The development of a specific LC/MS/MS method capable of detecting characteristic peptides, allowed for the qualitative determination of the enzymes alpha-Amylase (Aspergillus oryzae), Endo-1,4-Xylanase (Thermomyces lanuginosus) and Lipase (Thermomyces lanuginosus). Quantification of self-made reference material (wheat flour, bread and cookies) was performed by linear calibration with synthetically produced peptide standards and the use of a protein internal standard as well as isotopically labeled peptide standards. Less than 20 minutes are necessary to quantify alpha-Amylase at a minimum concentration of 2.58 mg/kg (flour, cookies) and 7.61 mg/kg (bread). Simultaneously the enzyme Endo-1,4-Xylanase can be quantified at concentrations as low as 3.64 mg/kg (cookies), 7.75 mg/kg (bread) and 15.60 mg/kg (flour) as well as the enzyme Lipase at a minimum concentration of 1.26 mg/kg (flour, cookies) and 2.68 mg/kg (bread). Finally the method was validated in accordance with generally accepted guidelines. It was shown that the method leads to very stable and reproducible quantitative results for the three tested enzymes with recovery rates between 50 % and 122 %. The primary aim of this thesis, developing a quantitative multi parameter method for analyzing technical enzymes in baking products, was successfully implemented. KW - Enzyme KW - LC-MS/MS KW - quantitativ KW - enzymes KW - LC/MS/MS KW - quantitative Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-96432 ER - TY - GEN A1 - Hocher, Berthold A1 - Reichetzeder, Christoph A1 - Dwi Putra, Sulistyo Emantoko A1 - Slowinski, Torsten A1 - Neuber, Corinna A1 - Kleuser, Burkhard A1 - Pfab, Thiemo T1 - Increased global placental DNA methylation levels are associated with gestational diabetes N2 - Background: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM. Methods: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study. Results: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1–4 (2.9–5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation. Conclusions: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 370 KW - Placenta KW - Gestational diabetes KW - Insulin resistance KW - LC-MS/MS KW - Global DNA methylation KW - Epigenetics KW - Hypermethylation Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-400914 ER - TY - GEN A1 - Zwickel, Theresa A1 - Kahl, Sandra M. A1 - Rychlik, Michael A1 - Müller, Marina E. H. T1 - Chemotaxonomy of mycotoxigenic small-spored Alternaria fungi BT - do multitoxin mixtures act as an indicator for species differentiation? T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - Necrotrophic as well as saprophytic small-spored Altemaria (A.) species are annually responsible for major losses of agricultural products, such as cereal crops, associated with the contamination of food and feedstuff with potential health-endangering Altemaria toxins. Knowledge of the metabolic capabilities of different species-groups to form mycotoxins is of importance for a reliable risk assessment. 93 Altemaria strains belonging to the four species groups Alternaria tenuissima, A. arborescens, A. altemata, and A. infectoria were isolated from winter wheat kernels harvested from fields in Germany and Russia and incubated under equal conditions. Chemical analysis by means of an HPLC-MS/MS multi-Alternaria-toxin-method showed that 95% of all strains were able to form at least one of the targeted 17 non-host specific Altemaria toxins. Simultaneous production of up to 15 (modified) Altemaria toxins by members of the A. tenuissima, A. arborescens, A. altemata species-groups and up to seven toxins by A. infectoria strains was demonstrated. Overall tenuazonic acid was the most extensively formed mycotoxin followed by alternariol and alternariol mono methylether, whereas altertoxin I was the most frequently detected toxin. Sulfoconjugated modifications of alternariol, alternariol mono methylether, altenuisol and altenuene were frequently determined. Unknown perylene quinone derivatives were additionally detected. Strains of the species-group A. infectoria could be segregated from strains of the other three species-groups due to significantly lower toxin levels and the specific production of infectopyrone. Apart from infectopyrone, alterperylenol was also frequently produced by 95% of the A. infectoria strains. Neither by the concentration nor by the composition of the targeted Altemaria toxins a differentiation between the species-groups A. altemata, A. tenuissima and A. arborescens was possible. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 696 KW - small-spored Alternaria fungi KW - Alternaria species-groups KW - Alternaria mycotoxins KW - chemotaxonomy KW - secondary metabolite profiling KW - LC-MS/MS KW - wheat KW - perylene quinone derivatives Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-426623 SN - 1866-8372 IS - 696 ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Bönick, Josephine A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett–Burman and Box–Behnken Designs T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 805 KW - wheat KW - α-amylase/trypsin inhibitors KW - extraction KW - Plackett–Burman design KW - Doehlert design KW - SDS-PAGE KW - MALDI-TOF/MS KW - LC-MS/MS Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-442229 SN - 1866-8372 IS - 805 ER - TY - GEN A1 - Tchewonpi Sagu, Sorel A1 - Landgräber, Eva A1 - Henkel, Ina M. A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Bußler, Sara A1 - Schlüter, Oliver K. A1 - Rawel, Harshadrai Manilal T1 - Effect of cereal α-amylase/trypsin inhibitors on developmental characteristics and abundance of digestive enzymes of mealworm larvae (Tenebrio molitor L.) T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1153 KW - growth behavior KW - Tenebrio molitor larvae KW - feeding KW - cereal meals KW - α-amylase/trypsin inhibitors KW - digestive enzymes quantification KW - LC-MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-520924 SN - 1866-8372 IS - 5 ER - TY - GEN A1 - Tchewonpi Sagu, Sorel A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Effect of sample preparation on the detection and quantification of selected nuts allergenic proteins by LC-MS/MS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1157 KW - nut allergenic proteins KW - sample preparation KW - protein extraction KW - tryptic digestion KW - microwave assisted digestion KW - SDS PAGE KW - LC-MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-521871 SN - 1866-8372 IS - 15 ER - TY - GEN A1 - Figueroa Campos, Gustavo A. A1 - G. K. T. Kruizenga, Johannes A1 - Sagu Tchewonpi, Sorel A1 - Schwarz, Steffen A1 - Homann, Thomas A1 - Taubert, Andreas A1 - Rawel, Harshadrai T1 - Effect of the Post-Harvest Processing on Protein Modification in Green Coffee Beans by Phenolic Compounds T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The protein fraction, important for coffee cup quality, is modified during post-harvest treatment prior to roasting. Proteins may interact with phenolic compounds, which constitute the major metabolites of coffee, where the processing affects these interactions. This allows the hypothesis that the proteins are denatured and modified via enzymatic and/or redox activation steps. The present study was initiated to encompass changes in the protein fraction. The investigations were limited to major storage protein of green coffee beans. Fourteen Coffea arabica samples from various processing methods and countries were used. Different extraction protocols were compared to maintain the status quo of the protein modification. The extracts contained about 4–8 µg of chlorogenic acid derivatives per mg of extracted protein. High-resolution chromatography with multiple reaction monitoring was used to detect lysine modifications in the coffee protein. Marker peptides were allocated for the storage protein of the coffee beans. Among these, the modified peptides K.FFLANGPQQGGK.E and R.LGGK.T of the α-chain and R.ITTVNSQK.I and K.VFDDEVK.Q of β-chain were detected. Results showed a significant increase (p < 0.05) of modified peptides from wet processed green beans as compared to the dry ones. The present study contributes to a better understanding of the influence of the different processing methods on protein quality and its role in the scope of coffee cup quality and aroma. View Full-Text T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1256 KW - Arabica coffee KW - coffee processing KW - protein modification KW - bound phenolic compounds KW - peptide biomarkers KW - LC-MS/MS Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-557643 SN - 1866-8372 VL - 11 SP - 1 EP - 19 PB - Universitätsverlag Potsdam CY - Potsdam ET - 2 ER -