Signal pattern plot: a simple tool for time-dependent metabolomics studies by 1H NMR spectroscopy.

We show an alternative way to visualize time course NMR data without the application of multivariate data analysis, based on the temporal change of the metabolome of hazelnuts after mold infestation. Fresh hazelnuts were inoculated with eight different natural mold species and the growth was studied over a period of 14 days. The data were plotted in a color-coded scheme showing metabolic changes as a function of chemical shift, which we named signal pattern plot. This plot graphically displays alteration (trend) of a respected signal over time and allows visual interpretation in a simple manner. Changes are compared with a reference sample stored under identical conditions as the infected nuts. The plot allows, at a glance, the recognition of individual landmarks specific to a sample group as well as common features of the spectra. Each sample reveals an individual signal pattern. The plot facilitates the recognition of signals that belong to biological relevant metabolites. Betaine and five signals were identified that specifically changed upon mold infestation. Graphical abstract.

Cobalamin is produced by Acetobacter pasteurianus DSM 3509.

Only a few cobalamin-producing bacterial species are known which are suitable for food fermentations. The strain of Acetobacter pasteurianus DSM 3509 was found to have the capability to synthesize cobalamin. A survival test and a preliminary genetic study of the gene of uroporphyrinogen-III synthase indicated the ability to synthesize cobalamin. By a modified microbiological assay based on Lactobacillus delbrueckii ssp. lactis DSM 20355, 4.57 ng/mL of cyanocorrinoids and 0.75 ng/mL of noncorrinoid growth factors were detected. The product extracted and isolated by immunoaffinity chromatography in its cyanide form had the similar UV spectrum as standard cyanocobalamin and Coα-[α-(7-adenyl)]-(Coß-cyano) cobamide also known as pseudovitamin B12 produced by Lactobacillus reuteri DSM 20016. The chromatographically separated product of A. pasteurianus was subjected to mass spectrometrical analysis. There, its fragmentation pattern turned out to be equivalent to that of cyanocobalamin also produced by Propionibacterium freudenreichii ssp. freudenreichii DSM 20271 and clearly differs from pseudovitamin B12. Due to the presence of this species in several food applications, there might be cobalamin residues in food fermented with these bacteria.

New Taste-Active 3-( O-ß-d-Glucosyl)-2-oxoindole-3-acetic Acids and Diarylheptanoids in Cimiciato-Infected Hazelnuts.

Activity-guided fractionation in combination with sensory analytics, LC-TOF-MS, and 1D/2D-NMR spectroscopy enabled the identification of the bitter tasting diarylheptanoids asadanin, giffonin P, and the previously not reported ( E)-7,9,10,13-tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-en-11-one and 4,12,16-trihydroxy-2-oxatricyclo[13.3.1.13,7]-nonadeca-1(18),3,5,7(20),8,15,17-heptaen as well as the yet unknown astringent compounds 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3- O-6'-galactopyranosyl-2″-(2″oxoindolin-3″yl) acetate and 3-( O-ß-d-glycosyl) dioxindole-3-acetic acid in Cimiciato-infected hazelnuts exhibiting a bitter off-taste. Quantitative LC-MS/MS studies, followed by dose/activity considerations confirmed for the first time asadanin to be the key contributor to the bitter taste of Cimiciato-infected hazelnuts. Furthermore, quantitative studies demonstrated that neither the physical damage alone nor a general microbial infection is able to initiate a stress-induced asadanin generation, but most likely either specific Cimiciato-specific microorganisms associated with the bugs or specific chemical stimulants in the bugs' saliva is the cause triggering asadanin biosynthesis. Finally, also germination was found for the first time to activate diarylheptanoid biosynthesis, resulting in higher contents of bitter tasting phytochemicals and development of the bitter off-taste.

Genetic improvement of Bacillus licheniformis strains for efficient deproteinization of shrimp shells and production of high-molecular-mass chitin and chitosan.

By targeted deletion of the polyglutamate operon (pga) in Bacillus licheniformis F11, a derivative form, F11.1 (Δpga), was obtained that, along with lacking polyglutamate (PGA) formation, displayed enhanced proteolytic activities. The phenotypic properties were maintained in a strain in which the chiBA operon was additionally deleted: F11.4 (ΔchiBA Δpga). These genetically modified strains, carrying the Δpga deletion either alone (F11.1) or together with the ΔchiBA (F11.4) deletion, were used in fermentations (20-liter scale) aiming at the deproteinization of shrimp shells in order to obtain long-chain chitin. After chemical deacetylation, the resulting chitosan samples were analyzed by nuclear magnetic resonance spectroscopy, size exclusion chromatography, and viscometry and compared to a chitosan preparation that was produced in parallel by chemical methods by a commercial chitosan supplier (GSRmbH). Though faint lipid impurities were present in the fermented polysaccharides, the viscosity of the material produced with the double-deletion mutant F11.4 (Δpga ΔchiBA) was higher than that of the chemically produced and commercially available samples (Cognis GmbH). Thus, enhanced proteolytic activities and a lack of chitinase activity render the double mutant F11.4 a powerful tool for the production of long-chain chitosan.

Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin.

Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.