Microbial community dynamics in phyto-thermotherapy baths viewed through next generation sequencing and metabolomics approach.

Phyto-thermotherapy is a treatment consisting in immersing oneself in baths of self-heating alpine grass, to benefit of the heat and rich aromatic components released by the process. The aim of this study was to characterize the bacterial and fungal diversity of three phyto-thermal baths (PTB) performed in three different months, and to compare the data with the profile of the volatile organic compounds (VOCs) of the process. All the data collected showed that PTBs were structured in two stages: the first three days were characterised by an exponential rise of the temperature, a fast bacterial development, higher microbial diversity and higher concentrations of plant aliphatic hydrocarbons. The second stage was characterised by a stable high temperature, shrinkage of the microbial diversity with a predominance of few bacterial and fungi species and higher concentrations of volatiles of microbial origin. Erwinia was the dominant microbial species during the first stage and probably responsible of the self-heating process. In conclusion, PTBs has shown both similarities with common self-heating processes and important peculiarities such as the absence of pathogenic bacteria and the dominance of plant terpenoids with health characteristics among the VOCs confirming the evidence of beneficial effects in particular in the first three days.

Methyl Salicylate Glycosides in Some Italian Varietal Wines.

Glycosides are ubiquitous plant secondary metabolites consisting of a non-sugar component called an aglycone, attached to one or more sugars. One of the most interesting aglycones in grapes and wine is methyl salicylate (MeSA), an organic ester naturally produced by many plants, particularly wintergreens. To date, nine different MeSA glycosides from plants have been reported, mainly spread over the genera Gaultheria, Camellia, Polygala, Filipendula, and Passiflora. From a sensorial point of view, MeSA has a balsamic-sweet odor, known as Wintergreen. MeSA was found in Vitis riparia grapes, in Vitis vinifera sp. and in the Frontenac interspecific hybrid. We found that the MeSA glycosides content in Verdicchio wines and in some genetically related varieties (Trebbiano di Soave and Trebbiano di Lugana) was very high. In order to understand which glycosides were present in wine, the methanolic extract of Verdicchio wine was injected into a UPLC-Q-TOF-HDMS and compared to the extracts of different plants rich in such glycosides. Using pure standards, we confirmed the existence of two glycosides in wine: MeSA 2-O--d-glucoside and MeSA 2-O--d-xylopyranosyl (1-6) -d-glucopyranoside (gaultherin). For the first time, we also tentatively identified other diglycosides in wine: MeSA 2-O--l-arabinopyranosyl (1-6)--d-glucopyranoside (violutoside) and MeSA 2-O--d-apiofuranosyl (1-6)--d-glucopyranoside (canthoside A), MeSA 2-O--d-glucopyranosyl (1-6)-O--d-glucopyranoside (gentiobioside) and MeSA 2-O--l-rhamnopyranosyl (1-6)--d-glucopyranoside (rutinoside). Some of these glycosides have been isolated from Gaultheria procumbens leaves by preparative liquid chromatography and structurally annotated by 1H- and 13C-NMR analysis. Two of the peaks isolated from Gaultheria procumbens leaves, namely MeSA sambubioside and MeSA sophoroside, were herein observed for the first time. Six MeSA glycosides were quantified in 64 Italian white wines, highlighting the peculiar content and pattern in Verdicchio wines and related cultivars. The total concentration in bound and free MeSA in Verdicchio wines varied in the range of 456-9796 g/L and 5.5-143 g/L, respectively, while in the other wines the bound and free MeSA was below 363 g/L and 12 g/L, respectively. As this compound's olfactory threshold is between 50 and 100 g/L, our data support the hypothesis that methyl salicylate can contribute to the balsamic scent, especially in old Verdicchio wines.

Applying novel approaches for GC × GC-TOF-MS data cleaning and trends clustering in VOCs time-series analysis: Following the volatiles fate in grass baths through passive diffusion sampling.

Phytothermotherapy ("grass baths") is a traditional phytotherapy for rheumatism consisting of taking baths in hot fermenting grass. Scientific studies have demonstrated its efficiency in treating several rheumatic diseases. However the efficiency and repeatability of the therapy is dependent on the wild fermentations, determining sometimes the appearance of unpleasant conditions leading to the early abandonment of the therapy. The metabolism undergoing in the grass baths is unknown and there is not an established method to evaluate and predict grass baths quality. The aim of this study is to establish a simple VOCs profiling method able to evaluate the grass baths, predicting their evolution, through the identification of marker volatiles related to the best conditions and/or the spoilage. After replicating in real scale the traditional grass baths, the volatile profiles were measured using passive diffusion samplers injected in a thermal desorption-comprehensive GC × GC-TOF-MS. The high dimensionality of the data coupled with the limited number of time points, required a rigorous method development for the analysis of the data, achieved through the development of a novel R package for variable selection in GC × GC data matrices. The further application of a fuzzy clustering approach demonstrated to be a useful tool dealing with short time series, allowing to discard un-trending volatiles and giving a clear snapshot of the main trends in the data. A broad coverage of the volatolome was provided, thus suitable to describe the main metabolic changes ongoing in the grass baths. Coupling this data with the temperature and pH, and comparing it to the data from similar processes, like silage and compost, we demonstrated that the established method can be helpful to evaluate short time series, allowing us to obtain a list of volatiles as candidate markers for the quality of the grass baths. The established method gave a list of markers applicable to real scale grass baths to predict spoilage; furthermore it provides a list of volatiles where to search for candidate markers with reported health-related effects and can be used to generate hypothesis on the mechanisms of action of the treatment.

Characterization of 14 raspberry cultivars by solid-phase microextraction and relationship with gray mold susceptibility.

Fourteen raspberry varieties were evaluated over two cropping seasons by solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry. Thirty-six compounds were fully identified, and 10 more compounds were tentatively identified. Despite interannual variability, raspberry varieties can be divided in two main groups on the basis of terpenes and C-13 norisoprenoids. Susceptibility toward Botrytis cinerea , one of the most relevant pathogenic fungi for soft fruits during storage, was also evaluated. On the basis of volatile profiles, it was possible to highlight the relationship between different volatile compounds and resistance to B. cinerea. Volatile profiles and Botrytis susceptibility of the different raspberry varieties evaluated should assist future breeding programs.