Solid phase microextraction as a reliable alternative to conventional extraction techniques to evaluate the pattern of hydrolytically released components in Vitis vinifera L. grapes

In present research, headspace solid-phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry (GC–qMS), was evaluated as a reliable and improved alternative to the commonly used liquid–liquid extraction (LLE) technique for the establishment of the pattern of hydrolytically released components of 7 Vitis vinifera L. grape varieties, commonly used to produce the world-famous Madeira wine. Since there is no data available on their glycosidic fractions, at a first step, two hydrolyse procedures, acid and enzymatic, were carried out using Boal grapes as matrix. Several parameters susceptible of influencing the hydrolytic process were studied. The best results, expressed as GC peak area, number of identified components and reproducibility, were obtained using ProZym M with b-glucosidase activity at 35 °C for 42 h. For the extraction of hydrolytically released components, HS-SPME technique was evaluated as a reliable and improved alternative to the conventional extraction technique, LLE (ethyl acetate). HS-SPME using DVB/CAR/PDMS as coating fiber displayed an extraction capacity two fold higher than LLE (ethyl acetate). The hydrolyzed fraction was mainly characterized by the occurrence of aliphatic and aromatic alcohols, followed by acids, esters, carbonyl compounds, terpenoids, and volatile phenols. Concerning to terpenoids its contribution to the total hydrolyzed fraction is highest for Malvasia Cândida (23%) and Malvasia Roxa (13%), and their presence according previous studies, even at low concentration, is important from a sensorial point of view (can impart floral notes to the wines), due to their low odor threshold (μg/L). According to the obtained data by principal component analysis (PCA), the sensorial properties of Madeira wines produced by Malvasia Cândida and Malvasia Roxa could be improved by hydrolysis procedure, since their hydrolyzed fraction is mainly characterized by terpenoids (e.g. linalool, geraniol) which are responsible for floral notes. Bual and Sercial grapes are characterized by aromatic alcohols (e.g. benzyl alcohol, 2-phenylethyl alcohol), so an improvement in sensorial characteristics (citrus, sweet and floral odors) of the corresponding wines, as result of hydrolytic process, is expected.

Nematicidal activity of Solanum nigrum and S. sisymbriifolium extracts against the root-lesion nematode Pratylenchus goodeyi and its effects on infection and gene expression

The control of Pratylenchus goodeyi a common nematode parasite of banana crop in Madeira Island can benefit from searching for natural nematicides through plants extracts. With this aim we submitted Solanum nigrum and S. sisymbriifolium dried plants to a sequential extraction in the solvent sequence of dichloromethane, acetone, ethanol and water, and to na aqueous extraction of the fresh and dried plants. Analyses with the extracts at several concentrations were used to assess mobility and mortality on P. goodeyi. Results showed that the water extract and aqueous extracts from both plants at a concentration of 10 mg/mL affected nematode mobility and caused mortality but the acetone extract from S. nigrum was the most efficient, causing 100% mortality whereas dichloromethane had no effect on P. goodeyi. Determination of the lipophilic and phenolic compounds present in the two most effective Solanum extracts (acetone and water) and in dichloromethane extract revealed that some of these compounds had nematicidal activity. S. nigrum acetone extract (10 mg/mL) was used to find out the nematicidal potential following the effect at gene expression level and nematode behaviour. Genes coding for calreticulin and beta-1,4- endoglucanase related to parasitism and translocon-associated protein putatively connected to stress were obtained and its relative expression assessed in nematodes exposed to the extract. Results revealed that expression of Pg-CRT decreased showing to influence the infection, Pg-ENG remained steady and Pg-TRAPδ was induced over time exposure. Biological assays showed that P. goodeyi mobility and ability to infect the banana roots were affected as a decrease in the number of nematodes that reached the roots was obtained with the increased exposure time to the extract being implicated in the infection success. The information obtained from this thesis showed that S. nigrum has potential to be used for the development of a new control strategy against plant-parasitic nematodes.

Effectiveness of high-throughput miniaturized sorbent- and solid phase microextraction techniques combined with gas chromatography–mass spectrometry analysis for a rapid screening of volatile and semi-volatile composition of wines: a comparative study

In this study the feasibility of different extraction procedures was evaluated in order to test their potential for the extraction of the volatile (VOCs) and semi-volatile constituents (SVOCs) from wines. In this sense, and before they could be analysed by gas chromatography–quadrupole first stage masss spectrometry (GC–qMS), three different high-throughput miniaturized (ad)sorptive extraction techniques, based on solid phase extraction (SPE), microextraction by packed sorbents (MEPS) and solid phase microextraction (SPME), were studied for the first time together, for the extraction step. To achieve the most complete volatile and semi-volatile signature, distinct SPE (LiChrolut EN, Poropak Q, Styrene-Divinylbenzene and Amberlite XAD-2) and MEPS (C2, C8, C18, Silica and M1 (mixed C8-SCX)) sorbent materials, and different SPME fibre coatings (PA, PDMS, PEG, DVB/CAR/PDMS, PDMS/DVB, and CAR/PDMS), were tested and compared. All the extraction techniques were followed by GC–qMS analysis, which allowed the identification of up to 103 VOCs and SVOCs, distributed by distinct chemical families: higher alcohols, esters, fatty acids, carbonyl compounds and furan compounds. Mass spectra, standard compounds and retention index were used for identification purposes. SPE technique, using LiChrolut EN as sorbent (SPELiChrolut EN), was the most efficient method allowing for the identification of 78 VOCs and SVOCs, 63 and 19 more than MEPS and SPME techniques, respectively. In MEPS technique the best results in terms of number of extractable/identified compounds and total peak areas of volatile and semi-volatile fraction, were obtained by using C8 resin whereas DVB/CAR/PDMS was revealed the most efficient SPME coating to extract VOCs and SVOCs from Bual wine. Diethyl malate (18.8 ± 3.2%) was the main component found in wine SPELiChrolut EN extracts followed by ethyl succinate (13.5 ± 5.3%), 3-methyl-1-butanol (13.2 ± 1.7%), and 2-phenylethanol (11.2 ± 9.9%), while in SPMEDVB/CAR/PDMS technique 3-methyl-1-butanol (43.3 ± 0.6%) followed by diethyl succinate (18.9 ± 1.6%), and 2-furfural (10.4 ± 0.4%), are the major compounds. The major VOCs and SVOCs isolated by MEPSC8 were 3-methyl-1-butanol (26.8 ± 0.6%, from wine total volatile fraction), diethyl succinate (24.9 ± 0.8%), and diethyl malate (16.3 ± 0.9%). Regardless of the extraction technique, the highest extraction efficiency corresponds to esters and higher alcohols and the lowest to fatty acids. Despite some drawbacks associated with the SPE procedure such as the use of organic solvents, the time-consuming and tedious sampling procedure, it was observed that SPELiChrolut EN, revealed to be the most effective technique allowing the extraction of a higher number of compounds (78) rather than the other extraction techniques studied.