N. Moreira

Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must

Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h(-1) for H. uvarum, 0.23 h(-1) for H. guilliermondii and 0.18 h(-1) for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3-mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.

Volatile sulphur compounds in wines related to yeast metabolism and nitrogen composition of grape musts

The influence of nitrogen compounds in grape musts on the content of sulphur compounds of wines was studied. Different vinifications were performed with the addition of methionine (20 mg l −1 ) and/or cysteine (40 mg l −1 ) to grape musts before alcoholic fermentation. Six grape musts, with different nitrogen composition, from cultivars of the ‘Vinhos Verdes’ Region, in Portugal, were used. Addition of methionine to grape musts enhanced the content of wines in 3-(methylthio)-1-propanol, acetic acid-3-(methylthio)propyl ester, 3-(methylthio)propionic acid and some unidentified sulphur compounds. Increase of cysteine concentration in musts led to the production of wines with high levels of hydrogen sulphide and cis-2-methyltetrahydrothiophene-3-OL and also unidentified sulphur compounds; however, the content of 3-(methylthio)propionic acid in the wines decreased considerately with the addition of cysteine to grape musts. This work showed that cultivars from the Vinho Verde Region show different sulphur compound contents. Avesso wines, elaborated from grape musts with low amino acids level, presented the highest total sulphur compounds content. Wines from Azal branco and Alvarinho were characterised by high contents of 4-(methylthio)-1-butanol and 3-(methylthio)propionic acid, respectively. A high content of N-(3-(methylthio) propyl)-acetamide and dimethylsulphone characterise the Loureiro wines. In contrast, Trajadura wines, produced from a must rich in amino acids, presented a low total sulphur compounds content; however, these wines were also characterised by high concentrations of 4-(methylthio)-1-butanol, acetic acid-3-(methylthio)propyl ester and hydrogen sulphide.

Development and validation of automatic HS-SPME with a gas chromatography-ion trap/mass spectrometry method for analysis of volatiles in wines.

An automated headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-ion trap/mass spectrometry (GC-IT/MS) was developed in order to quantify a large number of volatile compounds in wines such as alcohols, ester, norisoprenoids and terpenes. The procedures were optimized for SPME fiber selection, pre-incubation temperature and time, extraction temperature and time, and salt addition. A central composite experimental design was used in the optimization of the extraction conditions. The volatile compounds showed optimal extraction using a DVB/CAR/PDMS fiber, incubation of 5 ml of wine with 2g NaCl at 45 °C during 5 min, and subsequent extraction of 30 min at the same temperature. The method allowed the identification of 64 volatile compounds. Afterwards, the method was validated successfully for the most significant compounds and was applied to study the volatile composition of different white wines.

Optimization of the HS-SPME-GC-IT/MS method using a central composite design for volatile carbonyl compounds determination in beers

An automated headspace solid-phase microextraction (HS-SPME) combined with gas chromatography and ion trap mass spectrometry detection (GC-IT/MS) was developed in order to quantify a large number of carbonyl compounds in beers. Carbonyl compounds were previously derivatized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA). Volatile carbonyl compounds associated with staling beer aroma includes alkanals, alkenals, alkadienals, dicarbonyl compounds, Strecker aldehydes, ketones and furans. The HS-SPME was performed using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber. The procedures were optimized for HS-SPME pre-incubation temperature and time, extraction temperature and time, and PFBHA addition. A central composite design was used in the optimization of extraction conditions and PFBHA addition. The volatile compounds showed optimal extraction incubating 5 ml of beer with 700 mg l(-1) of PFBHA for 7 min and extracted for more 20 min at 45 °C. The method was validated with regard to the linearity, repeatability, inter and intra-day precision and accuracy. The method achieved detection limits ranging from 0.003 to 0.510 µg l(-1), except for furans (1.54-3.44 µg l(-1)). The quantification limits varied from 0.010 to 1.55 µg l(-1), except for 2-furfural (4.68 µg l(-1)), 5-methyl-2-furfural (5.82 µg l(-1)) and 5-hyfroxymethylfurfural (10.4 µg l(-1)). Repeatability values of all compounds were lower than 17%. The method accuracy was satisfactory with recoveries ranging from 88% to 114%. The validated method showed to be suitable for a fast and reliable determination of main carbonyl compounds in beers.

Relationship between nitrogen content in grapes and volatiles, namely heavy sulphur compounds, in wines

Ammonium salts were added to white grape musts, before alcoholic fermentation, in order to evaluate their influence on the heavy sulphur compound and aliphatic higher alcohol composition of resulting wines. Six grape musts were used (Trajadura, Pedernã, Loureiro, Azal Branco, Avesso and Alvarinho). Ammonium supplementation of Trajadura and Pedernã grape musts, with the highest nitrogen level, did not influence the content of heavy sulphur compounds and aliphatic higher alcohols in wines; however, the addition of ammonium salts to grape musts with low initial nitrogen content, such as Loureiro, Azal Branco and Avesso, led to a higher production of 1-propanol and a lower production of isoamyl alcohols and sulphur compounds, e.g. S-methyl thioacetate, 2-mercaptoethanol, acetic acid-3-(methylthio)propyl ester, 3-mercapto-1-propanol, 4-(methylthio)-1-butanol, 3-(ethylthio)-1-propanol, 3-methylthiopropionic acid and N-3-(methylthiopropyl)acetamide. For Alvarinho grape must, a decrease in sulphur compound concentrations in wines was only observed for 3-methylthiopropionic acid, acetic acid-3-(methylthio)propyl ester and 2-mercaptoethanol.

Non-targeted and targeted analysis of wild toxic and edible mushrooms using gas chromatography-ion trap mass spectrometry

Mushrooms are known all over the world both due to the remarkable gastronomic value of some species and for severe intoxications mediated by other species that are frequently difficult to distinguish from the edible ones, by the common user. Therefore, it is important to develop strategies to discover molecules that can identify mushroom species. In the present work, two GC-MS methodologies were applied in the chemical characterization of 22 mushroom species (12 edible, 3 toxic and 7 potentially toxic) - a multi-target procedure to simultaneously determine amino acids (AA), fatty acids (FA) and sterols by previous derivatization procedure with MSTFA, and a Head Space-Solid Phase Microextraction method to determine volatiles. For both methods, two approaches to data analysis were used: (I) targeted analysis, to identify and quantify AA, FA sterols and volatiles; (II) untargeted analysis, including Principal Component Analysis and Partial Least Square Discriminant Analysis, in order to identify metabolites/metabolite pattern with potential species identification and/or differentiation. Multi-target experiment allowed the identification and quantification of twenty one primary metabolites (9 AA, 11 FA and 1 sterol). Furthermore, through untargeted data analysis, it was possible to identify a 5-carbon sugar alcohol structure molecule, which was tentatively identified as xylitol or adonitol, with potential to be a species-marker of the edible Suillus bovinus mushrooms. Volatile profiling studies resulted in the identification of the main volatiles in mushrooms. Untargeted analysis allowed the identification of 6 molecules that can be species- or genus-specific: one secondary metabolite specific to the edible species Lycoperdon perlatum, an ester of hexanoic acid, tentatively identified as allyl or vinyl caproate; and five other secondary metabolites, whose identification was not achieved, which were only detected in Lactarius aurantiacus specimens (edibility/toxicity unknown).

Analysis of volatile human urinary metabolome by solid-phase microextraction in combination with gas chromatography–mass spectrometry for biomarker discovery: Application in a pilot study to discriminate patients with renal cell carcinoma

A new and simple analytical approach consisting of headspace-solid phase microextraction (HS-SPME) sampling coupled with gas chromatography-ion trap/mass spectrometry (GC-IT/MS) was developed to study the volatile human urinary metabolome. A central composite design (CCD) was used in the optimisation of extraction conditions. Fibre selection and evaluation of pH influence were performed using an univariate mode and the influence of other parameters, such as the time and temperature of extraction, time of incubation and salt addition, that affect the efficiency of the SPME sampling, was carried out using a CCD. With a sample volume of 2 mL, the optimal conditions in terms of total response values and reproducibility were achieved by performing analyses with a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre, in an acidic pH (pH 2) with the addition of 0.59 g of NaCl, allowing the sample to equilibrate for 9 min and extracting at 68 °C for 24 min. The applicability of the optimised method was then tested in a pilot non-target analysis of urine samples obtained from patients with renal cell carcinoma (RCC) and healthy individuals. Chemometric unsupervised analyses performed on the volatile pattern acquired for these samples clearly showed the potential of volatile urinary metabolome to discriminate between RCC and control patients.

Effect of storage time and heat processing on the volatile profile of Senegalese sole (Solea senegalensis Kaup, 1858) muscle

The effect of heat treatment and the presence or absence of fish skin on the volatile composition of Senegalese sole muscle was studied. The volatile profile of Senegalese sole at different storage periods was also evaluated. All samples were analysed by HS-SPME-GC-IT/MS and subjected to sensory evaluation. As expected, cooking enhanced the production/liberation of volatile compounds. Fish with the skin present, after cooking, had higher levels of sulphur compounds, 2-nonanone, ethyl octanoate and lower contents of hexanol and heptanol than skinned fish; moreover, the samples with the skin had a better overall sensory acceptability. During storage, changes on the volatile composition of Senegalese sole samples were found. The major differences were obtained after 2 weeks of storage. Compounds such as hexanal, heptanal, octanal, decanal, (E)-2-hexenal, (E)-2-decen-1-al, (E,Z)-2,6-nonadienal, benzaldehyde, 4-ethyl-benzaldehyde, 1-penten-3-ol, heptanol and (E)-2-octen-1-ol decreased after 2 weeks of storage, and other compounds, such as 3-methyl-1-butanal, 2-methyl-1-butanal, 2-heptanone, dimethyl trisulphide, dimethyl tetrasulphide and 2-methyltetrahydrothiophen-3-one increased. These differences were confirmed by sensory evaluation. Principal component analysis was applied to the chemical data.

Optimisation and validation of a HS-SPME-GC-IT/MS method for analysis of carbonyl volatile compounds as biomarkers in human urine: Application in a pilot study to discriminate individuals with smoking habits.

A new and simple analytical approach consisting of an automated headspace solid-phase microextraction (HS-SPME) sampler coupled to gas chromatography-ion trap/mass spectrometry detection (GC-IT/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to detect volatile carbonyl metabolites with low molecular weights in human urine. A central composite design (CCD) was used to optimise the PFBHA concentration and extraction conditions that affect the efficiency of the SPME procedure. With a sample volume of 1 mL, optimal conditions were achieved by adding 300 mg/L of PFBHA and allowing the sample to equilibrate for 6 min at 62°C and then extracting the samples for 51 min at the same temperature, using a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre. The method allowed the simultaneous identification and quantification of 44 carbonyl compounds consisting of aldehydes, dialdehydes, heterocyclic aldehydes and ketones. The method was validated with regards to the linearity, inter- and intra-day precision and accuracy. The detection limits ranged from 0.009 to 0.942 ng/mL, except for 4-hydroxy-2-nonenal (15 ng/mL), and the quantification limits varied from 0.029 to 1.66 ng/mL, except for butanal (2.78 ng/mL), 2-butanone (2.67 ng/mL), 4-heptanone (3.14 ng/mL) and 4-hydroxy-2-nonenal (50.0 ng/mL). The method accuracy was satisfactory, with recoveries ranging from 90 to 107%. The proof of applicability of the methodology was performed in a pilot target analysis of urine samples obtained from 18 healthy smokers and 18 healthy non-smokers (control group). Chemometric supervised analysis was performed using the volatile patterns acquired for these samples and clearly showed the potential of the volatile carbonyl profiles to discriminate urine from smoker and non-smoker subjects. 5-Methyl-2-furfural (p<0.0001), 2-methylpropanal, nonanal and 2-methylbutanal (p<0.05) were identified as potentially useful biomarkers to identify smoking habits.

Effect of two experimental diets (protein and lipid vegetable oil blends) on the volatile profile of Senegalese sole (Solea senegalensis Kaup, 1858) muscle

The aim of this study was to determine differences among volatile compounds composition of Senegalese sole muscle fed with extruded diets containing different plant protein (PP) and vegetable oil (VO) sources. Two set of experiments were performed on growing sole. One growth trial used a control diet containing fish meal (FM) as the main protein source and different PP-based diets. Another growth trial compared a control diet containing fish oil (FO) as the main lipid source and different VO-based diets; after a period, all sole were fed with the FO diet. Results showed that the incorporation of PP sources up to 75% allowed the production of a similar content of major volatile compounds to the control diet. In VO-based diets, some significant differences were found in the levels of some volatile compounds in sole muscle; however, no significant differences were obtained through sensory evaluation.