Technologies for the Selection, Culture and Metabolic Profiling of Unique Rhizosphere Microorganisms for Natural Product Discovery.

Small molecule discovery has benefitted from the development of technologies that have aided in the culture and identification of soil microorganisms and the subsequent analysis of their respective metabolomes. We report herein on the use of both culture dependent and independent approaches for evaluation of soil microbial diversity in the rhizosphere of canola, a crop known to support a diverse microbiome, including plant growth promoting rhizobacteria. Initial screening of rhizosphere soils showed that microbial diversity, particularly bacterial, was greatest at crop maturity; therefore organismal recovery was attempted with soil collected at canola harvest. Two standard media (Mueller Hinton and gellan gum) were evaluated following inoculation with soil aqueous suspensions and compared with a novel "rhizochip" prototype buried in a living canola crop rhizosphere for microbial culture in situ. Following successful recovery and identification of 375 rhizosphere microbiota of interest from all culture methods, isolates were identified by Sanger sequencing and/or characterization using morphological and biochemical traits. Three bacterial isolates of interest were randomly selected as case studies for intensive metabolic profiling. After successful culture in liquid media and solvent extraction, individual extracts were subjected to evaluation by UHPLC-DAD-QToF-MS, resulting in the rapid characterization of metabolites of interest from cultures of two isolates. After evaluation of key molecular features, unique or unusual bacterial metabolites were annotated and are reported herein.

Metabolic fingerprinting of must obtained from sun-dried grapes of two indigenous Cypriot cultivars destined for the production of 'Commandaria': A protected destignation of origin product.

Grapes's sun-dried process is one of the most critical steps in the production of 'Commandaria', a dessert wine product that is exclusively produced in Cyprus and derived from must obtained from two indigenous grape cultivars, namely 'Mavro' and 'Xynisteri'. Despite its significant economic importance, no data regarding the primary and secondary metabolites of the aforementioned cultivars exist. Thus, in the current study, the effect of sun drying process on the qualitative attributes and phenolic profile of 'Mavro' and 'Xynisteri' musts was dissected. Musts were analyzed at harvest and at the end of the sun-drying process that corresponds to ca. 30-40% water loss. Results highlighted significant differences in chemical composition of the must before and after the sun-drying process. Except for the increase of soluble solids content, a significant increment in glucose, fructose, total acidity, total phenols and total flavonoids contents was monitored. Subsequently, forty-two phenolic compounds were identified by LC-DAD-qTOF-MS revealing the polyphenolic fingerprint of the two cultivars. Results also indicated that changes in the phenolic composition of the obtained must are not only correlated with the dehydration effect, but both synthesis or degradation reactions occurred. In particular, the increases in the concentration of hydroxybenzoic acids were higher than the concentration effect for both cultivars. Regarding to hydroxycinnamates, dehydration caused a six-fold increase of hydroxycinnamic acid content in both cultivars. Intriguingly, the concentration of some hydroxycinnamic acids such as caffeic acid dihexoside and fertaric acid isomer went descending. Although the degradation of the internal side of the skin facilitate improved extractability from the skins to the grape pulp and therefore to the grape must, the sun-drying process may also induced stilbene and lignans synthesis production. A significant effect of dehydration on the postharvest biosynthesis of three groups of flavonoids (flavonols, flavan-3-ols, flavanonols), was also observed. This study sheds some light in the substantial changes that occur in specific metabolites during the sun drying process; such metabolites can be considered as potential factors that may determine organoleptic characteristics and biological properties of the end-product.