Marzipan is a confectionary which is mostly offered in form of filled chocolate, pralines, or pure. According to the German guidelines for oil seeds only almonds, sugar and water are admitted ingredients of marzipan. A product very similar in taste is persipan which is used in the confectionary industry because of its stronger flavor. For persipan production almonds are replaced by debittered apricot or peach kernels. To guarantee high quality products for consumers, German raw paste producers have agreed a limit of apricot kernels in marzipan raw paste of 0.5%. Different DNA-based methods for quantitation of persipan contaminations in marzipan are already published. To increase the detection specificity compared to published intercalation dye-based assays, the present work demonstrate the utilization of a multiplex real-time PCR based on the Plexor technology. Thus, the present work enables the detection of at least 0.1% apricot DNA in almond DNA or less. By analyzing DNA mixtures, the theoretical limit of quantification of the duplex PCR for the quantitation of persipan raw paste DNA in marzipan raw paste DNA was determined as 0.05%.Keywords: marzipan; multiplex real-time PCR; persipan; Plexor technology; Prunus;

The present work demonstrates the first automated enrichment approach for antibiotics in milk using specific DNA aptamers. First, aptamers toward the antibiotic sulfanilamide were selected and characterized regarding their dissociation constants and specificity toward relevant antibiotics via fluorescence assay and LC-MS/MS detection. The performed enrichment was automated using the KingFisherDuo and compared to a manual approach. Verifying the functionality, trapping was realized in different milk matrices: (i) 0.3% fat milk, (ii) 1.5% fat milk, (iii) 3.5% fat milk, and (iv) 0.3% fat cocoa milk drink. Enrichment factors up to 8-fold could be achieved. Furthermore, it could be shown that novel implementation of a magnetic separator increases the reproducibility and reduces the hands-on time from approximately half a day to 30 min.Keywords: antibiotics; aptamer; automation; enrichment; milk; sulfanilamide;

Lebensmittel sind ein essentieller Bestandteil unseres täglichen Lebens. Sie werden meist als selbstverständlich angesehen und machen in der Presse eher mit Negativschlagzeilen auf sich aufmerksam. Dabei rücken nicht nur Lebensmittelskandale mit dem Potential der Gesundheitsgefährdung in den Fokus der Öffentlichkeit, wie zum Beispiel der „Melaminskandal“, sondern auch Verstöße gegen das Lebensmittelrecht, welche im Bereich der Verbrauchertäuschung („Food Fraud“, „Food Fakery“) anzusiedeln sind. Als Beispiel hierfür ist u. a. der „Pferdefleischskandal“ zu nennen, welcher mit großem Interesse von der Öffentlichkeit und den Medien verfolgt wurde (Johnson in Food Fraud and “Economically Motivated Adulteration” of food and food ingredients. Congressional Research Service Report. https://www.fas.org/sgp/crs/misc/R43358.pdf. Zugriff am 08. Oktober 2015, 2014). So ist es nicht verwunderlich, dass in den letzten Jahren ein Trend hin zum bewussteren Konsum von Lebensmitteln zu verzeichnen ist. Dabei zeigt sich, dass sowohl die Qualität als auch die Herkunft der Lebensmittel immer häufiger eine zentrale Rolle für den Verbraucher spielen. Die Bereitschaft, bei Edel- oder Premiumprodukten bzw. Produkten mit geschützten geografischen Angaben (g. g. A.) oder mit geschützter Ursprungsbezeichnung (g. U.) einen höheren Preis zu zahlen, wächst stetig. Deshalb ist es umso wichtiger, die Authentizität von Lebensmitteln sicherzustellen, da bei Aufdeckungen solcher Verbrauchertäuschungen, auch ein nicht unerheblicher Image-Schaden für die gesamte Lebensmittelbranche entstehen kann. Nicht immer geschehen solche Verfälschungen aus krimineller Absicht. Vermischungen und Beimengungen minderwertiger Ware können bereits bei der Ernte oder Produktion auftreten, wenn beispielsweise unterschiedliche Qualitätsstufen pflanzlicher Rohstoffe nahe beieinander angebaut werden oder auf derselben Produktionslinie unterschiedliche Produkte hergestellt werden (Herrmann et al. in J Agric Food Chem 62(50):12118–12127, 2014; Brüning et al. in J Agric Food Chem 59:11910–11917, 2011). Im Augenblick ist die Überprüfung der Lebensmittelrohstoffe meist nur anhand von beiliegenden Dokumenten (Frachtpapiere) möglich. Deswegen hat es sich die HAMBURG SCHOOL OF FOOD SCIENCE (HSFS) zum Ziel gesetzt, robuste analytische Methoden zu entwickeln, welche eine zeitnahe Überprüfung der Echtheit vor allem bei Rohware ermöglichen sollen.

We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans DXS as a model enzyme, targeting the thiamine diphosphate (TDP) pocket of DXS whilst also exploring the putative substrate-binding pocket, where selectivity over other human TDP-dependent enzymes could be gained. The STI methodology – suitable for weak binders – was essential to determine the binding mode in solution of one of the fragments, circumventing the requirement for an X-ray co-crystal structure, which is known to be particularly challenging for this specific enzyme and in general for weak binders. Based on this finding, we carried out fragment growing and optimisation, which led to a three-fold more potent fragment, about as potent as the well-established thiamine analogue deazathiamine. The STI methodology proved therefore its strong potential as a tool to support medicinal-chemistry projects in their early stages, especially when dealing with weak binders.

The intensely fluorescent lumazine protein is believed to be involved in the bioluminescence of certain marine bacteria. The sequence of the catalytically inactive protein resembles that of the enzyme riboflavin synthase. Its non-covalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine, is the substrate of this enzyme and also the committed precursor of vitamin B2. An extensive crystallization screen was performed using numerous single-site mutants of the lumazine protein from Photobacterium leiognathi in complex with its fluorophore and with riboflavin, respectively. Only the L49N mutant in complex with riboflavin yielded suitable crystals, allowing X-ray structure determination to a resolution of 2.5 Å. The monomeric protein folds into two closely similar domains that are structurally related by pseudo-C2 symmetry, whereby the entire domain topology resembles that of riboflavin synthase. Riboflavin is bound to a shallow cavity in the N-terminal domain of lumazine protein, whereas the C-terminal domain lacks a ligand.

•Aptamer-based trapping of phytosphingosine in real matrices.•Adaption of just in time-Selection for aptamer selection towards small molecules.•Determination of dissociation constants via fluorescence assay.•Developed application is adaptable with little effort to further targets.Usually, small molecules like single metabolites used in clinical diagnostic can be quantified by instrumental approaches like LC–MS or bioanalytical techniques using antibodies or aptamers as selective receptors. The present work comprises the generation of aptamers with an affinity towards the medically relevant metabolite phytosphingosine via the previously reported just in time-Selection approach (Hünniger et al., 2014). The whole approach could be seen as a proof of concept to extend the existing just in time-Selection protocol for selection towards small molecules with dissociation constants in the low nanomolar range. Moreover it is conceivable that the shown methods could be quickly adapted to further scopes. Aptamers could be applied for clean-up or concentration processes prior to further analysis. As an example, we used the selected aptamers towards phytosphingosine bound to magnetic particles for affinity enrichment in both selection buffer and urine samples. As an outcome, enrichment factors of up to 9-fold (selection buffer)/4-fold (urine samples) were achieved by this approach.

We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans DXS as a model enzyme, targeting the thiamine diphosphate (TDP) pocket of DXS whilst also exploring the putative substrate-binding pocket, where selectivity over other human TDP-dependent enzymes could be gained. The STI methodology – suitable for weak binders – was essential to determine the binding mode in solution of one of the fragments, circumventing the requirement for an X-ray co-crystal structure, which is known to be particularly challenging for this specific enzyme and in general for weak binders. Based on this finding, we carried out fragment growing and optimisation, which led to a three-fold more potent fragment, about as potent as the well-established thiamine analogue deazathiamine. The STI methodology proved therefore its strong potential as a tool to support medicinal-chemistry projects in their early stages, especially when dealing with weak binders.