A modified aroma extract dilution approach (AEDA), followed by the determination of flavor dilution (FD) factors, a quantitative analysis and calculation of the relative flavor activity (RFA) and odor activity values (OAVs) as well as recombination experiments were conducted to evaluate the odor- and taste-relevant components of cold-pressed Citrus latifolia peel oil. A 2-fold concentration by distillation and reanalysis, compared with the original oil, revealed relevant components. Partition of the odor-active substances into four reconstitution groups according to their respective FD factors, followed by a recombination, allowed for a better understanding of the contribution of each FD-factor group to the overall aroma. Especially alpha-pinene, limonene, gamma-terpinene, and 7-methoxycoumarin contribute significantly to the distinct aroma profile of C. latifolia. Heptadecanal (CAS 629-90-3) was described for the first time as an odor-active substance in an enriched C. latifolia peel oil. Campherenyl acetate (CAS 18530-07-9) was identified in nature for the first time and described with a herbal, minty and citrus-like odor. The odor profile of the final recombinant mixture, containing 36 components, was similar to cold-pressed C. latifolia peel oil for most descriptors, whereas the taste profile was described as more aldehydic and citral-like.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar 'Yae-no-amacha' using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.

AbstractDie Chalconisomerase (CHI) ist ein Schlüsselenzym in der Biosynthese von Flavonoiden in Pflanzen. Die erste bakterielle CHI (CHIera) wurde in Eubacterium ramulus identifiziert, aber Häufigkeit, evolutionärer Ursprung, Substratspektrum und Stereoselektivität sind bislang unklar. Hier beschreiben wir die Identifizierung von 66 neuen bakteriellen CHIs in Genbank durch eine neuartige Sequenz‐Struktur‐Funktions‐Evolutions (SSFE)‐Strategie. Diese neuen bakteriellen CHIs weisen ein breites Substratspektrum gegenüber einer Reihe hydroxylierter und methoxylierter Chalcone auf. Die Mutagenese der CHIera, basierend auf einem Substratbindemodell dieser neuen bakteriellen CHIs, resultierte in mehreren Varianten mit deutlich verbesserter Aktivität gegenüber diesen Chalconen. Zusätzlich wurden präparative Reaktionen mit diesen bakteriellen CHIs für fünf Chalcone durchgeführt, die (S)‐Selektivität mit bis zu 96 % ee ergaben. Dies ermöglicht eine alternative biokatalytische Route zur Synthese von (S)‐Flavanonen in hohen Ausbeuten.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

So far, the occurrence of the flavor constituent 1-phenylethyl acetate in a natural source has not been unambiguously confirmed. The present work provides the detailed identification of 1-phenylethyl acetate from clove (Syzygium aromaticum (L.) Merr. & L.M. Perry) buds. In addition, headspace solid-phase microextraction-gas chromatography/mass spectrometry (GC/MS) analysis revealed further occurrence of 1-phenylethyl acetate in cocoa pulp and grape hyacinth flowers. A total of 15.2 g of essential oil was recovered from 7.2 kg of clove buds by simultaneous distillation-extraction followed by vacuum distillation. The distillate obtained was fractionated by silica column chromatography, whereby a significant enrichment of 1-phenylethyl acetate was achieved. The fraction containing the target analyte was further purified by preparative high-performance liquid chromatography, resulting in a final purity of ∼93.0%, yielding a total of 1 to 2 mg of 1-phenylethyl acetate. Identification of the isolated compound was achieved by GC/MS, infrared spectroscopy, enantioselective GC, isotope ratio MS, and nuclear magnetic resonance spectroscopy. Enantioselective GC/MS analysis revealed an enantiomeric excess of 60% (1S)-(-)-1-phenylethyl acetate in the isolate. The δ13CV-PDB value of -32.5 ± 0.5‰ was in accordance with that of C3-plants and other constituents found in genuine clove extracts.

Model studies have been performed with individual citrus related aroma compounds such as citral, α-terpineol, α-terpinene, and others, in order to de-couple the complexity of citrus flavors. Degradation pathways and transformation rates have been determined for the most relevant compounds leading to the most prominent off-note compound p-methyl acetophenone. Citral is one of the main precursors. Catechins (flavan-3-ols) are often used as antioxidants for stabilization of flavor mixtures. The reactivity of catechins towards aldehydes is underestimated. In our study, we demonstrate the formation of covalent catechin–citral adducts – a reaction that might influence the aroma stability of citrus flavors in the presence of catechins. As a result, the freshness of citrus flavors and stability of key aroma-active substances can be improved, on the result of this study.

Asafetida is an important condiment based on an oleogum resin with an onion-like aroma obtained from various plants of the genus Ferula (family Umbelliferae), which are cultivated in countries like India. Since the flavor of Asafetida is significantly dominated by sulfur compounds, a combination of specific analytical and sensorial techniques was selected to investigate the flavor profile. In particular the combination of high-temperature liquid chromatography with preparative GC isolation of the volatile compounds has been employed. Newly identified substances are reported and sensory attributes of isolated sulphur-containing aroma compounds are described.

Due to certain off-flavor problems and lacking bitter masking effects with Yerba Santa (Eriodictyon angustifolium and E. californicum) extracts, which are also described as bitter, herbal, medicinal, phenolic, or astringent, methanolic extracts were fractionated and evaluated for their taste properties using a high temperature liquid chromatography (HTLC)-based approach. The taste-guided fractionation led to the identification of a series of novel bisprenylated benzoic acids (erionic acids A (1), B (2), C (3), D (4), E (5), and F (6) and eriolic acids A (7), B (8), C (9), and D (10), respectively), along with the known flavonoids eriodictyol, homoeriodictyol, hesperetin, and chrysoeriol. The new compounds were isolated in larger amounts for characterization from Narrow Leaf Yerba Santa (E. angustifolium) and California Yerba Santa (E. californicum), respectively, using fast centrifugal partition chromatography (FCPC) and HTLC. The structures were elucidated using one and two-dimensional NMR spectroscopy and high resolution mass spectrometry (HR-MS). For E. californicum, data regarding seasonal and climatic variation of the eriolic acid contents and of the flavonoids were collected. The flavor properties of some of the isolated new compounds were evaluated; they showed strong off-flavor characteristics, such as bitter, astringent, phenolic, or woody, and may contribute to the sensory effects observed for crude Yerba Santa extracts. Erionic acid C (3) was not only able to increase the absolute bitterness but also to extinguish the bitter masking effect of homoeriodictyol in a caffeine solution.

An ethanolic extract of Chamomilla recutita [L.] Rauschert was analyzed by the hyphenated techniques HPLC/MS and HPLC/NMR, speeding up a reliable and comprehensive characterization of the main constituents in an extract from chamomile flowers. In addition to the cis - and trans -isomers of 2-(glucosyloxy)-4-methoxy-cinnamic acid and also herniarin and apigenin, apigenin-7-O- β -glucoside itself and further derivatives of the latter could unambiguously be identified as main constituents.

Liquid chromatography/mass spectrometry and liquid chromatography/nuclear magnetic resonance techniques with ultraviolet/diode array detection were used as complementary analytical tools for the reliable identification of polymethoxylated flavones in residues from molecular distillation of cold-pressed peel oils of Citrus sinensis. After development of a liquid chromatographic separation procedure, the presence of several polymethoxy flavones such as sinensetin, nobiletin, tangeretin, quercetogetin, heptamethoxyflavone, and other derivatives was unambiguously confirmed. In addition, proceranone, an acetylated tetranortriterpenoid with limonoid structure, was identified for the first time in citrus.

An oil of the piperitone type of Eucalyptus dives produced from trees cultivated in South Africa was analyzed by GC and GC/MS. Seventy-three constituents were characterized with piperitone ranging from 29.2% to 64.9%, depending on whether the oil was produced from dried or fresh leaves. Two novel constituents such as p-menth-5-en-2-one and p-menth-6-en-3-one were characterized as minor constituents of the oil; the former being found in nature for the first time.

Unlike gap genes in the trunk region of Drosophila embryos, gap genes in the head were presumed not to regulate each other's transcription. Here, we show that in tailless (tll) loss-of-function mutants the empty spiracles (ems) expression domain in the head expands, whereas it retracts in tll gain-of-function embryos. We have identified a 304bp element in the ems-enhancer which is sufficient to drive expression in the head and brain and which contains two TLL and two BCD binding sites. Transgenic reporter gene lines containing mutations of the TLL binding sites demonstrate that tll directly inhibits the expression of ems in the early embryonic head and the protocerebral brain anlage. These results are the first demonstration of direct transcriptional regulation between gap genes in the head.

Embryonic development in Drosophila is characterized by an early phase during which a cellular blastoderm is formed and gastrulation takes place, and by a later postgastrulation phase in which key morphogenetic processes such as segmentation and organogenesis occur. We have focused on this later phase in embryogenesis with the goal of obtaining a comprehensive analysis of the zygotic gene expression that occurs during development under normal and altered environmental conditions. For this, a functional genomic approach to embryogenesis has been developed that uses high-density oligonucleotide arrays for large-scale detection and quantification of gene expression. These oligonucleotide arrays were used for quantitative transcript imaging of embryonically expressed genes under standard conditions and in response to heat shock. In embryos raised under standard conditions, transcripts were detected for 37% of the 1,519 identified genes represented on the arrays, and highly reproducible quantification of gene expression was achieved in all cases. Analysis of differential gene expression after heat shock revealed substantial expression level changes for known heat-shock genes and identified numerous heat shock-inducible genes. These results demonstrate that high-density oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large scale gene expression in Drosophila embryos.

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.

The neostriatum caudolaterale (NCL) of birds is thought to be equivalent to the mammalian prefrontal cortex (PFC) due to its dense dopaminergic innervation, its associative structure, and its importance for cognitive tasks which are known to be affected in mammals with prefrontal lesions. The aim of the present study was to analyze the functional importance of the NCL and its main thalamic afferent structure, the n. dorsolateralis posterior thalami (DLP), in reversal and go/no-go tasks, two behavioral procedures which are often used to assess mammalian prefrontal functions. Using a multiple regression analysis in which structure-specific lesion extents are correlated with different postoperative behavioral measures, the specific contribution of the relevant structures were differentiated from the neighbouring areas CDL (area corticoidea dorsolateralis) and NC (neostriatum caudale). The analyses showed a highly significant contribution of the NCL to reversal but not to go/no-go or to visual discrimination performance, while all other structures under analysis had no impact on any behavioral measure. These results underline the specific contribution of the pigeons' NCL on a subset of cognitive tasks which are known to be affected by prefrontal lesions in mammals.

Studies in vertebrates show that homeotic genes are involved in axial patterning and in specifying segmental identity of the embryonic hindbrain and spinal cord. To gain further insights into homeotic gene action during CNS development, we here characterize the role of the homeotic genes in embryonic brain development of Drosophila. We first use neuroanatomical techniques to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS, and demonstrate that this order is virtually identical in the CNS of Drosophila and mammals. We then carry out a genetic analysis of the labial gene in embryonic brain development. Our analysis shows that loss-of-function mutation and ubiquitous overexpression of labial results in ectopic expression of neighboring regulatory genes. Furthermore, this analysis demonstrates that mutational inactivation of labial results in regionalized axonal patterning defects which are due to both cell-autonomous and cell-nonautonomous effects. Thus, in the absence of labial, mutant cells are generated and positioned correctly in the brain, but these cells do not extend axons. Additionally, extending axons of neighboring wild-type neurons stop at the mutant domains or project ectopically, and defective commissural and longitudinal pathways result. Immunocytochemical analysis demonstrates that cells in the mutant domains do not express neuronal markers, indicating a complete lack of neuronal identity. An alternative glial identity is not adopted by these mutant cells. Comparable effects are seen in Deformed mutants but not in other homeotic gene mutants. Our findings demonstrate that the action of the homeotic genes labial and Deformed are required for neuronal differentiation in the developing brain of Drosophila.