Genomic and transcriptomic analysis of Saccharomyces cerevisiae isolates with focus in succinic acid production.

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10 selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.

Phenylacetaldehyde real-time release kinetics in wine like model solutions.

The present work shows key possibilities in modelling the kinetics of phenylacetaldehyde formation as a function of sugar, phenolic compounds, metals and sulphur dioxide. The release kinetics were measured online by proton transfer reaction-mass spectrometry (PTR-MS). Phenylacetaldehyde formation was fitted using Weibull models and an activation energy of 73 kJ/mol estimated. Also, a confirmation that glucose can inhibit the aldehyde formation was demonstrated, and the sequential additions in real time showed that the inhibition level was dependent on metal ions presence. Moreover, for the first time it was observed in real time the capacity of SO2 to bind with phenylacetaldehyde, and by trapping it, lowering its release. Finally, the impact of pH and temperature in the stability of the formed adducts and underling release mechanism is also elucidated.

Response surface methodology: A tool to minimize aldehydes formation and oxygen consumption in wine model system.

A response surface methodology was applied to study the effect of precursors on o-quinone and phenylacetaldehyde formation in wine model systems stored at 40 °C during 24 h. The results confirmed that glucose plays an important role in reducing aldehyde formation by inhibiting the formation of o-quinone. The regression equations showed that oxygen consumption followed a 2nd polynomial equation whereas phenylacetaldehyde and o-quinone were best fit with a polynomial function containing quadratic terms. These behaviors indicate that different pathways are involved in the respective aldehyde formation and oxygen consumption. RSM has been shown to be a powerful tool to better understand key chemical reactions. By considering a number of factors, individually and in combinations, the derived equations predicted that the best combination to minimize phenylacetaldehyde was achieved for high glucose levels and low amounts of gallic acid and metals. This is valuable information when trying to improve wines sensorial properties during shelf-life.

Study of major aromatic compounds in port wines from carotenoid degradation.

The carotenoids degradation and the formation of volatiles were examined by simulating Port wine aging. A two year old red Port wine was saturated with oxygen, supplemented with lutein and ß-carotene and kept at 60°C during 87h. A similar study was performed in a model wine solution. Results showed that the percentage decrease in lutein levels was, respectively, 79% and 95%, in the wine model solution and in the Port wine, and 55% and 10% for ß-carotene, indicating that lutein was more sensitive to degradation than ß-carotene. Two other unknown degradation carotenoid compounds were identified by HPLC/DAD (reverse phase λmax: 422; 445; 475 and 422; 445; 472) in the lutein supplemented wine. Levels of ß-ionone and ß-cyclocitral increased (2.5 times) in both, wine and wine model solution, supplemented with ß-carotene. Along with these compounds, the same behaviour was observed in ß-damascenone in the supplemented lutein wine and wine model solution. New insights were provided into the understanding of aroma modifications occurring during Port wine aging. The relationship between carotenoid molecules (ß-carotene and lutein) and some volatiles has also been provided.

Carotenoid, chlorophyll, and chlorophyll-derived compounds in grapes and port wines.

Carotenoids and chlorophyll-derived compounds in grapes and Port wines were investigated by HPLC-DAD and HPLC-DAD-MS (ESP+) analysis. A total of 13 carotenoid and chlorophyll-derived compounds are formally reported in grapes, 3 are identified for the first time, pheophytins a and b and (13Z)-beta-carotene, and 3 others remain unknown. In Port wines 19 compounds with carotenoid or chlorophyll-like structures are present, 8 still unidentified. The young wines showed higher total carotenoid content and chlorophyll-like compounds compared to aged Ports, with lutein and beta-carotene as major carotenoids. Among samples analyzed of monovarietal Vitis vinifera L. cultivar wines produced with the five most important Douro varieties, Tinta Roriz contained the highest levels of carotenoids and Touriga Franca the lowest. The forced-aging study indicated that lutein was more sensitive to temperature than beta-carotene. Additionally, aged wines showed higher ratios of beta-carotene/lutein concentrations compared to new Ports. Rates of degradation of chlorophyll derivative compounds were higher than those for carotene and lutein.

Influence of the temperature and oxygen exposure in red Port wine: A kinetic approach.

Although phenolics are recognized to be related with health benefits by limiting lipid oxidation, in wine, they are the primary substrates for oxidation resulting in the quinone by-products with the participation of transition metal ions. Nevertheless, high quality Port wines require a period of aging in either bottle or barrels. During this time, a modification of sensory properties of wines such as the decrease of astringency or the stabilization of color is recognized to phenolic compounds, mainly attributed to anthocyanins and derived pigments. The present work aims to illustrate the oxidation of red Port wine based on its phenolic composition by the effect of both thermal and oxygen exposures. A kinetic approach toanthocyanins degradation was also achieved. For this purpose a forced red Port wine aging protocol was performed at four different storage temperatures, respectively, 20, 30, 35 and 40°C, and two adjusted oxygen saturation levels, no oxygen addition (treatment I), and oxygen addition (treatment II). Three hydroxycinnamic esters, three hydroxycinnamic acids, three hydroxybenzoic acids, two flavan-3-ols, and six anthocyanins were quantitated weekly during 63days, along with oxygen consumption. The most relevant phenolic oxidation markers were anthocyanins and catechin-type flavonoids, which had the highest decreases during the thermal and oxidative red Port wine process. Both temperature and oxygen treatments affected the rate of phenolic degradation. In addition, temperature seems to influence mostly the phenolics kinetic degradation.

Identification of key odorants related to the typical aroma of oxidation-spoiled white wines.

The oxidative degradation of white wines rapidly leads to a loss of their sensorial qualities. The identification of the most important descriptors related with oxidation-spoiled wine was performed by a trained sensory panel. The terms selected were "honey-like", "farm-feed", "hay", and "woody-like". By gas chromatography-olfactometry analysis three aromatic zones related to these descriptors in the oxidation-spoiled white wines could be determined. Comparison of the aroma extract dilution analysis aromagrams of oxidation-spoiled white wines and a nonspoiled wine showed the highest values of dilution factors were attributed to 3-(methylthio)propionaldehyde, phenylacetaldehyde, 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), and 4,5-dimethyl-3-hydroxy-2(5H)-furanone (sotolon). A "forced aging" experiment was implemented to simulate the typical oxidation-spoiled aroma. Samples rated with the highest score in the ranking test were also those that presented the highest concentration of these four molecules. To test the sensory impact of these substances, a normal wine (unspoiled) was spiked with these molecules (with the exception of TDN) singly and in combination, and the similarity value (SV) between samples and the oxidation-spoiled white wines was then determined. The highest value from the similarity tests was 5.4 when the three compounds were added simultaneously; 3-(methylthio)propionaldehyde alone was found to be responsible for 3.6, suggesting that, among the molecules studied, it is the most important contributor to the typical aroma of an oxidation-spoiled white wine.

Heterocyclic acetals from glycerol and acetaldehyde in Port wines: evolution with aging.

In Port wine, isomers of glycerol and acetaldehyde acetals have been found at total contents ranging from 9.4 to 175.3 mg/L. During oxidative aging, the concentrations of the 5-hydroxy-2-methyl-1,3-dioxane and 4-hydroxymethyl-2-methyl-1,3-dioxolane isomers increased with time showing a linear correlation (r > 0.95). The flavor threshold for the mixture of the four isomers was evaluated in wine at 100 mg/L. Thus, it is expected that they contribute to "old Port wine" aroma in wines older than 30 years. Experiments with model solutions and wine clearly demonstrated that SO(2) combines with acetaldehyde and blocks the acetalization reaction.

Influence of some technological parameters on the formation of dimethyl sulfide, 2-mercaptoethanol, methionol, and dimethyl sulfone in port wines.

Volatile sulfur compounds of 15 young port wines and 12 old port wines were determined. As there is a great difference in the pool of sulfur compounds between the two groups of wines, an experimental protocol was performed to determine which technological parameter (dissolved O(2), free SO(2) levels, pH, and time/temperature) was related with the formation/consumption of these compounds. Four sulfur compounds were selected for this purpose: dimethyl sulfide, 2-mercaptoethanol, dimethyl sulfone, and methionol. The synergistic effects of increasing temperature and O(2) at lower pH had the largest impact. Dimethyl sulfide was formed during the experimental period in the presence of O(2). Dimethyl sulfone had the same behavior. Methionol decreased significantly in the presence of O(2), but no methional was formed. 2-Mercaptoethanol, considered to be an important "off-flavor" in dry wines, also decreased during the experimental period (54 days) in the presence of O(2), and the respective disulfide was formed. These results corroborate the fact that old port wine (barrel aged) never develops "off-flavors" associated with the presence of methionol (cauliflower), 2-mercaptoethanol (rubber/burnt), or methional (cooked potato). In fact, temperature and oxygen are the major factors in the consumption of these molecules. However, some notes of "quince" and "metallic" can appear during port wine aging, and these can be associated with the presence of dimethyl sulfide.

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).

Untangling the chemistry of port wine aging with the use of GC-FID, multivariate statistics, and network reconstruction.

Chromatography separates the different components of complex mixtures and generates a fingerprint representing the chemical composition of the sample. The resulting data structure depends on the characteristics of the detector used, univariate for devices such as a flame ionization detector (FID) or multivariate for mass spectroscopy (MS). This study addresses the potential use of a univariate signal for a nontargeted approach to (i) classify samples according to a given process or perturbation, (ii) evaluate the feasibility of developing a screening procedure to select candidates related to the process, and (iii) provide insight into the chemical mechanisms that are affected by the perturbation. To achieve this, it was necessary to use and develop methods for data preprocessing and visualization tools to assist an analytical chemist to view and interpret complex multidimensional data sets. Dichloromethane Port wine extracts were collected using GC-FID; the chromatograms were then aligned with correlation optimized warping (COW) and subsequently analyzed with multivariate statistics (MVA) by principal component analysis (PCA) and partial least-squares regression (PLS-R). Furthermore, wavelets were used for peak calling and alignment refinement, and the resulting matrix was used to perform kinetic network reconstruction via correlation networks and maximum spanning trees. Network-target correlation projections were used to screen for potential chromatographic regions/peaks related to aging mechanisms. Results from PLS between aligned chromatograms and target molecules showed high X to Y correlations of 0.91, 092, and 0.89 with 5-hydroxymethylfurfural (HMF) (Maillard), acetaldehyde (oxidation), and 4,5-dimethyl-(5H)-3-hydroxy-2-furanone, respectively. The context of the correlation (and therefore likely kinetic) relationships among compounds detected by GC-FID and the relationships between target compounds within different regions of the network can be clearly seen.

Simultaneous determination of ketoacids and dicarbonyl compounds, key Maillard intermediates on the generation of aged wine aroma.

The aim of this work was the simultaneous determination of both ketoacids and dicarbonyl compounds in wine. To detect ketoacid compounds in wine, a method based on the quinoxaline derivatives by the reaction with diaminobenzene, currently employed to detect alpha-dicarbonyl compounds, was developed. The quinoxaline derivatives were detected by RP-HPLC with UV detection, which allows the determination of the major dicarbonyl compounds in wine: glyoxal, methylglyoxal, diacetyl and pentane-2,6-dione, and the quinoxaline/quinoxalinol derivatives of alpha-keto-gamma-(methylthio)butyric acid and beta-phenylpyruvic acid (intermediate ketoacid compounds of methional and phenylacetaldehyde) were simultaneously detected by a fluorescence detector. The identification was performed by comparison with standards and also by using LC-MSMS. The levels found in 15 wines analyzed (white wines, Madeira wines, and Port wines) diverge according to the type and the age of the wine. The ketoacid compounds ranged from 0.2 to 5.7 mg/L for alpha-keto-gamma-(methylthio)butyric acid and 0.1 to 9.6 mg/L for beta-phenylpyruvic acid. The quantities observed for dicarbonyl compounds were similar to those already reported.