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Introduction Silver fir (Abies alba Mill.) is one of the most valuable conifer wood species in Europe. Among the main opportunistic pathogens that cause root and butt rot on silver fir are Armillaria ostoyae and Heterobasidion abietinum. Due to the different enzymatic pools of these wood-decay fungi, different strategies in metabolizing the phenols were available. Objective This work explores the changes in phenolic compounds during silver fir wood degradation. Methodology Phenols were analyzed before and after fungus inoculation in silver fir macerated wood after 2, 4 and 6 months. All samples were analyzed using high-performance liquid chromatography coupled to a hybrid quadrupole-orbitrap mass spectrometer. Results Thirteen compounds, including simple phenols, alkylphenyl alcohols, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxybenzaldehydes, hydroxyphenylacetic acids, hydroxycinnamic acids, hydroxybenzoic acids and hydroxycoumarins, were detected. Pyrocatechol, coniferyl alcohol, acetovanillone, vanillin, benzoic acid, 4-hydroxybenzoic acid and vanillic acid contents decreased during the degradation process. Methyl vanillate, ferulic acid and p-coumaric were initially produced and then degraded. Scopoletin was accumulated. Pyrocatechol, acetovanillone and methyl vanillate were found for the first time in both degrading and non-degrading wood of silver fir. Conclusions Despite differences in the enzymatic pool, both fungi caused a significant decrease in the amounts of phenolic compounds with the accumulation of the only scopoletin. Principal component analysis revealed an initial differentiation between the degradation activity of the two fungal species during degradation, but similar phenolic contents at the end of wood degradation.

Purpose γ-Aminobutyric acid (GABA) is recognised as a potential metabolic bioactive food ingredient with increasing evidence of its effects on the gut-brain axis and systemic metabolic health. Different lactic acid bacteria are capable of producing GABA, particularly strains of Lactobacillus brevis. In this study, we characterized a Lb. brevis isolated from traditional alpine cheese (Lb. brevis DSM 32386) for its ability to accumulate high levels of GABA in the culture medium and for other important probiotic phenotypic traits. Methods In vitro analysis were used to study the Lb. brevis DSM 32386 probiotic traits and the gene expression involved in GABA production Result Lactobacillus brevis DSM 32386 converted monosodium glutamate to GABA more efficiently than the type strain Lb. brevis DSM 20054, resulting in more than 200% of GABA produced. This ability seemed to be related to the higher transcriptional activation of the gene encoding for the glutamate (gad) decarboxylase antiporter (gadC) and regulator (gadR). Lactobacillus brevis DSM 32386 performed well in vitro under the stress conditions mimicking the gastro-intestinal tract, being resistant to acid pH (pH 2.5) and growing in simulated pancreatic fluid and 0.3% ox-bile. Conclusion These preliminary studies indicate that Lb. brevis DSM 32386 holds promise as a starter for GABA-rich dairy fermented foods and possibly a promising next-generation probiotic microorganism in the context of the gut (microbiota):brain axis.

Gum arabic (GA) is the oldest and best known of all natural gums. Thanks to the particular characteristics resulting from its complex mixture of polysaccharides and glycoproteins, its field of application is extensive. In the oenological sector, it is used to prevent colour depigmentation and protein precipitation and although there is no declared limit, the International Code of Oenological Practices suggests not exceeding a dosage of 300 mg L−1. In this study, the botanical origin (Acacia senegal vs A. seyal) and commercial description (Kordofan region vs other Sub-Saharan areas, based on the traditional geographic provenance) were defined studying 44 GA samples with a non-targeted screening approach using high resolution mass spectrometry. From an initial group of 88 identified masses, 15 were selected to create a bi-factor PLS-DA statistical model able to correctly reclassify samples with 94.4% accuracy. Moreover, the study addressed the question of identifying gum addition in wines, analysing 98 untreated wines and comparing them with 40 wines to which gum arabic had been added (200 mg L−1). Considering the database of 88 masses, 4 of them were established to be markers of gum arabic addition (m/z 152.1067, 166.1225, 585.1428 and 643.1403). Principal component analysis performed with the signal (area) of the 4 masses highlighted three groupings differentiating untreated wines, wines treated with A. senegal gum and wines treated with A. seyal gum. m/z 152.1067 was tentatively identified as N-methyltyramine, while m/z 166.1225 as N,O-dimethyltyramine.

In this work, the anthocyanins’ profile of two hybrid grape varieties—Cabernet Carbon and Prior—grown in two experimental plots in Trentino (Northern Italy) was investigated during 2016 and 2017 harvests. The focus was on the mono- and di-glycoside anthocyanins extraction during the fermentative maceration. It was optimized an HPLC-DAD method, which allows the separation and quantification of mono- and di-glucoside anthocyanin forms (non-esterified, acetylated, and p-coumaroylated) in a single chromatographic run. Furthermore, an HPLC-DAD/HQOMS approach was used to confirm the compound identification. The skin contact fermentation was conducted for 7 days at 24 °C, punching down manually the cap twice a day. Must samples were collected every 24 h. As expected, every anthocyanin showed an initial net extraction phase described by a first-order kinetic, followed by a decrease until the end of the maceration. The analysis of the accumulation rate showed a faster increase of the di-glucoside anthocyanins concentration compared to the corresponding mono-glucoside, due to their richness in the initial grapes. Moreover, the di-glucosides concentration at the end of the alcoholic fermentation underwent a three times minor percentage decrease with respect to the maximum reached. This paper intend to improve the knowledge of the kinetics of each anthocyanin form during the maceration step, providing information about the behavior of di-glucoside compounds, never investigated before in terms of accumulation and percentage decrease.

The biodiversity of Saccharomyces cerevisiae was studied in the Montalcino area (Italy). Two wineries were involved in the study, which compared the genotypic and oenological characteristics of the S. cerevisiae strains isolated in spontaneous fermentations. After isolation yeasts were identified by 26S rRNA gene sequence analysis, and S. cerevisiae strains were characterized through interdelta sequence analysis (ISA). Oenological tests were performed in synthetic grape must by varying the magnitude of the main wine-imiting factors. The evolution of alcoholic fermentation was monitored by measuring sugar consumption and flow cytometry. The results revealed the prevalence of S. cerevisiae from the third day of fermentation and the presence of a wide range of S. cerevisiae strains having ISA profiles characteristic of each winery. From an oenological point of view, the features of such strains, in terms of resistance to wine-limiting factors, seemed to be linked to the main oenological variables applied in the production process of each winery. Extreme fermentation temperatures and copper residues are the variables that mostly depress the yeast population, in terms of fermentation rate and cell viability. Flow cytometry revealed the different impact of limiting factors on the viability of yeast by the quantification of the ratio between live/dead yeast cells of each strain, suggesting different mechanisms of inhibition, for instance stuck of cell growth or cell killing, in response to the different stress factors.

Vitis vinifera is one of the most widespread grapevines around the world representing the raw material for high quality wine production. The availability of more resistant interspecific hybrid vine varieties, developed from crosses between Vitis vinifera and other Vitis species, has generated much interest, also due to the low environmental effect of production. However, hybrid grape wine composition and varietal differences between interspecific hybrids have not been well defined, particularly for the simple phenols profile. The dynamic of these phenols in wines, where the glycosylated forms can be transformed into the free ones during winemaking, also raises an increasing health interest by their role as antoxidants in wine consumers. In this work an on-line SPE clean-up device, to reduce matrix interference, was combined with ultra-high liquid chromatography-high resolution mass spectrometry in order to increase understanding of the phenolic composition of hybrid grape varieties. Specifically, the phenolic composition of 4 hybrid grape varieties (red, Cabernet Cantor and Prior; white, Muscaris and Solaris) and 2 European grape varieties (red, Merlot; white, Chardonnay) was investigated, focusing on free and glycosidically bound simple phenols and considering compound distribution in pulp, skin, seeds and wine. Using a targeted approach 53 free simple phenols and 7 glycosidic precursors were quantified with quantification limits ranging from 0.001 to 2mgKg-1 and calibration R2 of 0.99 for over 86% of compounds. The untargeted approach made it possible to tentatively identify 79 glycosylated precursors of selected free simple phenols in the form of -hexoside (N=30), -pentoside (21), -hexoside-hexoside (17), -hexoside-pentoside (4), -pentoside-hexoside (5) and -pentoside-pentoside (2) derivatives on the basis of accurate mass, isotopic pattern and MS/MS fragmentation.

Red-brown pigmentation can occasionally form in smeared-ripened cheese such as Fontina during the ripening process. This reaction is due to over-development of the typical microbiota present on the rind. Previous studies have demonstrated the relationship between red-brown pigmentation and the traditional utilization of wooden shelves during cheese ripening. The first part of the paper focuses on the characterisation of yeast and bacterial microbiota: plate counts and 454-pyrosequencing were performed in spoiled (n = 6) and non-spoiled cheeses (n = 6) and on the wooden shelves used during ripening. The second part shows different systems tested for cleaning the wooden shelves and avoiding the development of the red-brown defect in cheese: washing with hot water and ozone treatment. Actinobacteria, dominated on the wooden shelves, suggesting to be responsible for the red-brown pigmentation; they were also found in traces in the defected cheese samples. Galactomyces and Debaryomyces were the main species characterizing the yeast population, with Debaryomyces being the most dominant species on the shelves used during ripening of the red-brown defective cheese. Hot water treatment reduced the microbial contamination of shelves, whereas only the ozone treatment ensured complete elimination of both yeast and bacteria, resulting in the cheese rind not having the red-brown defect.

The effect of ultrasound (20 kHz, 153 &mu, m) on the prefermentation extraction mechanisms in Sauvignon Blanc grapes was studied, focusing on 3-mercaptohexan-1-ol (3MH) and 4-mercapto-4-methyl-pentan-2-one (4MMP) precursors linked to glutathione (GSH) and cysteine (Cys). The treatment determined a positive extraction trend between the duration (untreated, 3 and 5 min) and the conductivity or the concentration of catechins and total phenols, significantly differentiated after 5 min. Nevertheless, the concentration of the thiol precursors in grape juice not only remained undifferentiated, but that of 3-S-glutathionyl mercaptohexan-1-ol showed a negative trend with the treatment time applied (168 &plusmn, 43, 156 &plusmn, 36, and 149 &plusmn, 32 &mu, g/L, respectively, for control, 3 and 5 min). The divergence on the effect between families of compounds suggests an interaction between the sonication treatment and thiol precursor molecules. In order to evaluate the possible degradation properly, ultrasound was applied in a model solution spiked with 3MH and 4MMP precursors, reproducing the conditions of grapes. Except for Cys-3MH, the mean concentration (n = 5) for the rest of the precursors was significantly lower in treated samples, predominantly in those linked to glutathione (~&minus, 22% and ~18% for GSH-3MH and GSH-4MMP) rather than to cysteine (~&minus, 6%~&minus, 8% for Cys-3MH and Cys-4MMP). The degradation of precursors was associated with a significant increase of 3MH and 4MMP. The formation of volatile thiols following sonication is interesting from a technological point of view, as they are key aroma compounds of wine and potentially exploitable in the wine industry through specific vinification protocols.

Gum arabic (GA) is a natural, edible gum with particular characteristics resulting from its complex mixture of polysaccharides and glycoproteins. GA is used extensively in several fields and global demand is steadily increasing; for this reason, the determination of GA origin is fundamental to counteract commercial frauds and ensure food safety. The purpose of the study was to investigate whether elemental and isotopic profiles could discriminate, first, between GA produced from plants belonging to the same genus (Acacia) but to 2 different species (A. senegal, SN; A. seyal, SY), and, second, considering only GA of the same botanical origin (SN), between gums produced from different geographic areas (Kordofan region in Sudan vs other Sub-Saharan areas) and commercially resulting in products with different characteristics. The Principal Component Analysis (PCA), combining elemental and stable isotope profiles, could clearly separate the 45 gum samples in 2 clusters corresponding to the botanical species of origin. Results were screened using a partial least squares regression (PLS-R) model developed following the leave-one-out cross-validation approach and including a validation subset of 8 samples. All the calibration and validation samples showed a 100% correct classification, both for SN and SY. Furthermore, the PCA, using only the C, N, Cs, Mg and Na content, could adequately discriminate the SN samples according to their geographic origin, distinguishing the Kordofan samples, traditionally renowned as top-quality products, from the generic gum samples produced in other Sub-Saharan areas.

Purpose The role of fermentation temperature was studied for its impact on the evolution of malolactic fermentation performed by simultaneous inoculum of yeast and bacteria in grape must. Results were discussed considering the different fermentative kinetics and the composition of obtained wines. Methods Two strains of bacteria belonging to the O. oeni and L. plantarum species were inoculated 24 h after the beginning of the alcoholic fermentation in 2 grape musts having different acidic and sugar profiles. Fermentations were conducted at 3 different temperature profiles (16/22 °C in 3 days, 18/24 °C in 3 days, 22/32 °C in 5 days). Evolution of microbiota was followed by flow cytometry and plate count. Chemical analysis of grape musts and wines were performed by instrumental approaches (FT-IR, enzymatic quantification of malic acid, GC-MS). Results L. plantarum resulted more efficient in malic acid consumption in the entire set of tests. These results are unexpected because, generally, Lactobacillus has been reported to be more sensitive to an oenological environment than O. oeni. In our experiments, O. oeni resulted inhibited by the highest fermentation temperature profile, causing incomplete malic acid degradation. Similarly, S. cerevisiae showed a higher sensitivity to environmental limiting factors in respect to what is generally known. Differences in the chemical composition of wines were observed in relation to the bacteria strain and the temperature profile. However, the statistical treatment of data identified temperature as the main variable able to influence the features of wines. Conclusions Simultaneous inoculum of yeast and bacteria in grape must is an alternative approach in the management of malolactic fermentation which showed some interesting features. However, it is necessary to consider that the dynamics of the microbial population are different to that observed in traditional winemaking and the environmental variables act against the microorganisms in a peculiar, and in certain cases unexpected, way.

Background Over recent years, a new wave of disease-tolerant to mildew varieties has flooded the viticulture sector, for reasons of human safety and economic expediency. These hybrid grape cultivars are selected mainly on the basis of their intrinsic capability to counter the attack of the main fungal diseases that affect grape production, such as downy mildew and powdery mildew. However, their organoleptic and oenological characteristics have not yet been studied in depth for purposes of both juice and wine production, due to the high number of newly proposed germplasms and the lack of information about their adaptability to different environments. This work examines the thiol aroma precursors concentration in 64 red and white disease-tolerant hybrid varieties in the vine germplasm collections of Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria-Viticulture and Enology Research Center and Fondazione Edmund Mach, both from the north-east of Italy. Results All cultivars showed the presence of 3-mercaptohexan-1-ol (3MH) precursors, ranging from 4.4 to 1141 μg kg-1 for 3-S-glutathionyl hexan-1-ol (GSH-3MH), and from 0.3 to 136 μg kg-1 for 3-S-cysteinyl 3-hexan-1-ol. The concentration of GSH-3MH exceeded 600 μg kg-1 in eight varieties, with values comparable to those of the richest Vitis vinifera reported so far. On average, red grapes showed higher concentrations of 3MH precursors than white ones did. Only two hybrids had 4-mercapto-4-methylpentan-2-one (4MMP) precursors over the limit of quantification, albeit with a much higher concentration than those normally reported in Sauvignon Blanc. Conclusion This is the first detailed survey of 3MH and 4MMP precursors carried out taking into account a considerable number of hybrid grape varieties. The results show that some of these varieties could be interesting for the production of tropical juices or tropical-aromatic wines and soft drinks, through the enzymatic liberation of thiol aromas, as well as for the production of aromatizing tannins to be used in the food industry. © 2020 Society of Chemical Industry.

Volatile sulfur compounds (VSCs) greatly influence the sensory properties and quality of wine and arise via both biological and chemical mechanisms. VSCs formed can also act as precursors for further downstream VSCs, thus elucidating the pathways leading to their formation is paramount. Short-term additions of exogenous hydrogen sulfide (H2S), ethanethiol (EtSH), S-ethylthio acetate (ETA), methanethiol (MeSH) and S-methylthio acetate (MTA) were made to exponentially growing fermentations of synthetic grape medium. The VSC profiles produced from live yeast cells were compared with those from dead cells and no cells. Interestingly, this experiment allowed the identification of specific biochemical and/or chemical pathways; e.g. most of the conversion of H2S to EtSH, and the further step from EtSH to ETA, required the presence of live yeast cells, as did the conversion of MeSH to MTA. In contrast, the reaction from MTA to MeSH and ETA to EtSH was due primarily to chemical degradation. Ultimately, this research unravelled some of the complex interactions and interconversions between VSCs, pinpointing the key biochemical and chemical nodes. These pathways are highly interconnected and showcase the complexity of both the sulfur pathways in yeast and the reactive chemistry of sulfur-containing compounds.

According to EU law, the country of origin in which the honey was produced must be declared on the label and the botanical origin can be indicated. Conventional honey analyses are not always applicable and effective for determining the geographical and botanical origin of honey. In this study 265 honey samples of different botanical origin (polyfloral, citrus, rhododendron, eucalyptus, acacia, chestnut and honeydew) produced throughout Italy in different years were analysed to determine stable isotope ratios (SIRs) using Isotope Ratio Mass Spectrometry and mineral element content using Inductively Coupled Plasma Optical Emission Spectroscopy. The aim was to verify the relationship between these parameters and the geographical origin of honey and the botanical species, as has already successfully taken place for other commodities. The characteristic ranges of variability in terms of SIRs and mineral content in genuine Italian honey samples are presented, as well as their compliance with AOAC limit.

Free simple phenols have positive effects on health and influence the organoleptic profile of cocoa products, contributing towards defining their aroma and nutritional properties. Glycosidically bound simple phenols can be hydrolysed during the production phase to the corresponding free forms, and thus potentially contribute to the final sensory profile. In this work, 60 samples of Forastero cocoa beans from all over the world were analysed, combining on-line solid phase extraction (SPE) clean-up with ultra-high performance liquid chromatography (UHPLC), coupled with high resolution mass spectrometry. Operating in negative ion mode and with a heated electrospray, 62 simple phenols were measured, of which four were glucosidic precursors, with quantification limits ranging from 0.04 to 40mgkg-1, calibration R2 of 0.99 for over 93% of compounds, and precision (R.S.D.%) always lower than 12%. On the basis of accurate mass, isotope pattern and MS/MS spectrum, 32 monoglycosylated simple phenols such as hexoside and pentoside precursors, and 14 diglycosylated simple phenols such as hexoside-hexoside, hexoside-pentoside and pentoside-hexoside precursors, were tentatively identified. The untargeted approach was validated using 3 glucosidic precursors synthesized by an external supplier. Honestly Significant Difference Tukey's test (p

Commercial tannins from several botanical sources and with different chemical and technological characteristics are used in the food and winemaking industries. Different ways to check their botanical authenticity have been studied in the last few years, through investigation of different analytical parameters. This work proposes a new, effective approach based on the quantification of 6 carbohydrates, 7 polyalcohols, and 55 phenols. 87 tannins from 12 different botanical sources were analysed following a very simple sample preparation procedure. Using Forward Stepwise Discriminant Analysis, 3 statistical models were created based on sugars content, phenols concentration and combination of the two classes of compounds for the 8 most abundant categories (i.e. oak, grape seed, grape skin, gall, chestnut, quebracho, tea and acacia). The last approach provided good results in attributing tannins to the correct botanical origin. Validation, repeated 3 times on subsets of 10% of samples, confirmed the reliability of this model.

Background and Aims The safety of foods and beverages is a prerequisite for consumers. The aim of this study is to check whether a low concentration of pesticide residues can be further reduced with the addition of a low dose of charcoal during white fermentation. Methods and Results Thirteen fungicides, boscalid, cyprodinil, fludioxonil, fenhexamide, pyrimethanil, cyazofamid, dimethomorph, fluopicolide, iprovalicarb, metrafenone, penconazole, spiroxamine and trifloxystrobin, were added together to five well-clarified and stabilised juices to reach a final fungicide concentration in juice slightly higher than that observed in bottled wines. Juices were fermented with two types of charcoal, both added at 20 and 50 mg/L. The course of fermentation was monitored, and the concentration of residual fungicides and of fermentative aromas was analysed by ultra-high performance liquid chromatography-MS/MS and GC-flame ionisation detector, respectively. Conclusions Charcoal removed up to 130 µg/L of fungicides. Compared with the control wines, those fermented with charcoal had 30–80% less fungicides. Only iprovalicarb was not significantly reduced. Aroma composition was not affected. Significance of the Study Low doses of charcoal added during fermentation can positively contribute to wine safety without penalising aroma and colour.

The cheese-derived strains Streptococcus thermophilus 84C isolated from Nostrano cheese, and Lactobacillus brevis DSM 32386 isolated from Traditional Mountain Malga cheese have been previously reported as γ-aminobutyric acid (GABA)-producers in vitro. In the present study, the ability of these strains to produce GABA was studied in experimental raw milk cheeses, with the aim to investigate the effect of the culture and the ripening time on the GABA concentration. The cultures used consisted on S. thermophilus 84C alone (84C) or in combination with L. brevis DSM 32386 (84C-DSM). The control culture was a commercial S. thermophilus strain, which was tested alone (CTRL) or in combination with the L. brevis DSM 32386 (CTRL-DSM). The pH evolution, microbiological counts, MiSeq Illumina and UHPLC-HQOMS analysis on milk and cheese samples were performed after 2, 9, and 20 days ripening. During the whole ripening, the pH was always under 5.5 in all batches. The concentration of GABA increased during ripening, with the highest content in 84C after 9 days ripening (84 ± 37 mg/kg), in 84C-DSM and CTRL-DSM after 20 days ripening (91 ± 28 and 88 ± 24 mg/kg, respectively). The data obtained support the hypothesis that S. thermophilus 84C and L. brevis DSM 32386 could be exploited as functional cultures, improving the in situ bio-synthesis of GABA during cheese ripening.

This study aimed to evaluate the effects of fermentation with lactic acid bacteria (LAB) on organic acids, ascorbic acid, phenolic compounds, anthocyanins, and carotenoids of sweet orange (Citrus sinensis) juices. Juices from ‘Tarocco’ and ‘Washington navel’ oranges were fermented at 30 °C for 48 h with L. plantarum, L. rhamnosus, L. paracasei, and L. brevis without supplementation. The juice from ‘Washington navel’ resulted the best medium to grow LAB, especially L. brevis (1.98 ± 0.36 log CFU mL−1 at 48 h). Malic acid was thoroughly converted into lactic acid. Ascorbic acid increased in ‘Tarocco’ juice, peaking to 295 ± 25.3 g L−1 when fermented by L. brevis. The effects of LAB on phenolic compounds were matrix-specific, e.g., L. rhamnosus increased the levels of phenolics in ‘Washington navel’ juice (36.8 ± 1.51 mg L−1; p

Aims The work investigates the impact of grapes processing at the beginning of winemaking on the composition of microbiota during the oenological fermentations and on the composition of obtained wines. Methods and Results The experiments were conducted in a biodynamic winery to exclude interference due to microbial starters. Three winemaking protocols, with different pre-fermentative management of grapes, were followed by plate count and next generation sequencing on 16S for bacteria and internal transcribed spacer sequencing (ITS) for yeast. Chemical and sensory characterization of wine was performed. The grape processing influenced the evolution of microbiota (especially lactic and acetic acid bacteria) and the fermentation rate. The highest biodiversity was observed in the experiment carried out with whole grapes and carbonic maceration, with the presence of bacterial groups not usually found in winemaking (Bacteroidales, Clostridiales, Oscillospira). The different microbiotas influenced the organic acid profile of wines, the content of biogenic ammines and the perception of organoleptic descriptors linked to the vine cultivar (Syrah). Conclusions Carbonic maceration impacts on the evolution of the microbiota and the wine features. The absence of addition of starters and sulphur dioxide would seem to be correlated with the high microbial biodiversity. Significance and Impact of the Study Carbonic maceration is a traditional winemaking practice, today there are difficulties in its managing because the anaerobiosis stimulates spoilage micro-organisms. The work elucidates the reasons of these difficulties and identified some microbial groups rarely associated with winemaking. The ratio of ethanol accumulation along with physical management of grapes and the supply of oxygen during the early stages of winemaking are powerful instruments of oenological variability, able to offer new possibilities to winemakers in order to defining the quality of red wines.

Aroma plays an important role in food quality, and knowledge of volatile composition is essential to support technological choices during industrial transformation processes. In this study, an innovative GC-MS/MS method useful for fast analysis of the broad aroma profile of oenological products was developed. Fifty volatile compounds of oenological interest belonging to the main chemical classes (acetates, acids, alcohols, aldehydes, esters, ketones, phenols, terpenes, and thiols) were identified and quantified in a single chromatographic run taking only 15 minutes. The high sensitivity of the mass spectrometer, combined with accurate fast chromatography performed with a suitable capillary column, allowed us to avoid further concentration steps after solid phase extraction. Validation of the method was performed in relation to the limit of detection, limit of quantification, linearity range, repeatability, reproducibility, and recovery. Once validated, the method was successfully tested on commercial oenological samples, demonstrating its applicability to fast routine multiaroma analysis.

Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for their growth ability in the presence of ethanol, high sugar content, low pH, different temperatures and sulphur dioxide, using optical density and flow cytometry measurement. The ability of yeasts to produce ethyl phenols in red wines with different chemical compositions was evaluated by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD). The results highlighted wide variability in B. bruxellensis in response to wine limiting factors and in terms of the accumulation of ethyl phenols. As regards this last aspect, the differences found among strains were closely related to chemical composition of wine and strain resistance to environmental stress factors, making a priori evaluation of risk of wine alteration quite difficult. These results suggest that strategies for the control of Brettanomyces should be tailored on the basis of strain distribution and wine characteristics.

AIMS Straw wine is a highly valuable oenological production, affected by relevant economical losses due to proliferation of spoilage micro-organisms during drying grapes, after being harvested. In this work, ozone was evaluated as a tool to preserve grapes during drying, in terms of both qualitative and quantitative changes induced in the epiphytic microflora. In addition, the alteration exerted by ozone on grapes' chemical composition was analysed. METHODS AND RESULTS Grapes from four vine varieties were treated with ozone produced by a cold plasma generator during the entire drying period (6 weeks). The microflora was quantified weekly by plate counts and characterized by 454-pyrosequencing, and was compared with identical, untreated grape samples. At the end of drying, an extensive chemical characterization of the whole mass of grapes by FT-IR and GC-MS was performed. Ozone counteracted the growth of microflora by reducing the microbial population up to 3 log units. From the qualitative point of view, ozone reduced the incidence of spoilage micro-organisms, such as Acetobacter and Botrytis cinerea, among the microbiota of grapes. CONCLUSIONS The statistical analysis discriminates grape samples based on the residual epiphytic microflora at the end of drying and not on their chemical composition. These evidences confirmed that ozone did control spoilage micro-organisms without altering the volatile profile of grapes. Chemical analysis revealed that untreated grapes are less suitable for winemaking owing to the deprivation of some valuable compounds during the microbial proliferation. SIGNIFICANCE AND IMPACT OF THE STUDY Ozone is an emerging tool in winemaking, recently applied for controlling spoilage micro-organisms in winery. The present study describes a new application of ozone as an effective and safe alternative to chemical preservatives which are actually involved in the control of microbial alterations of grapes.

The recent finding that grape tannin may contain significant amount of S-glutathionylated (GSH-3MH) and S-cysteinylated (Cys-3MH) precursors of the varietal thiols 3-mercapto-1-hexanol and 3-mercaptohexyl acetate, characteristic of Sauvignon blanc wines, offers new opportunities for enhancing the tropical aroma in fermented beverages. In this study this new hypothesis was investigated: Muller Thurgau (17 samples) and Sauvignon blanc (15 samples) grapes were fermented with and without addition of a selected grape tannin. As expected, the tannin-added juices were higher in precursors, and they produced wines with increased free thiols. Preliminary informal sensory tests confirmed that in particular the Sauvignon wines produced with the tannin addition were often richer with increased “fruity/green” notes than the corresponding reference wines. This outcome confirms that grape tannin addition prior to fermentation can fortify the level of these compounds.

Many metabolites naturally occur as glycosides, since sugar moieties can be crucial for their biological activity and increase their water solubility. In the plant kingdom they may occur as glycosides or sugar esters, depending on precursor chemical structure, and in wine they have traditionally attracted attention due to their organoleptic properties, such as astringency and bitterness, and because they affect the colour and aroma of wines. A new approach directed at detailed description of glycosides in a large selection of monovarietal wines (8 samples each of Pinot Blanc, Muller Thurgau, Riesling, Traminer, Merlot, Pinot Noir and Cabernet Sauvignon) was developed by combining high performance liquid chromatography with high resolution tandem mass spectrometry. Analytical separation was performed on an Accucore™ Polar Premium LC column, while mass analysis was performed in negative ion mode with an non-targeted screening approach, using a Full MS/AIF/NL dd-MS2 experiment at a resolving power of 140,000 FWHM. Over 280 glycoside-like compounds were detected, of which 133 (including low-molecular weight phenols, flavonoids and monoterpenols) were tentatively identified in the form of pentose (6), deoxyhexose (17), hexose (73), hexose-pentose (16), hexose-deoxyhexose (7), dihexose (5) and hexose ester (9) derivatives. It was not possible to univocally define the corresponding chemical structure for the remaining 149 glycosides. Non-parametric statistical analysis showed it was possible to well characterise the glycosylated profile of all red and Traminer wines, while the identified glycosides were almost entirely lacking in Pinot Blanc, Riesling and Muller Thurgau wines. Also Tukey’s Honestly Significant Difference test (p

Commercial tannins are natural polyphenolic compounds extracted from different plant tissues such as gall, the wood of different species and fruit. In the food industry they are mainly used as flavourings and food ingredients, whereas in winemaking they are classified as clarification agents for wine protein stabilisation, although colour stabilisation, metal removal, unpleasant thiol removal and rheological correction are also well-known and desired effects. Due to their particular technical properties and very different costs, the possibility of correct identification of the real botanical origin of tannins can be considered a primary target in oenology research and in fulfilling the technical and economic requirements of the wine industry. For some categories of tannins encouraging results have already been achieved by considering sugar or polyphenolic composition. For the first time this work verifies the possibility of determining the botanical origin of tannins on the basis of the mineral element profile and analysis of the 13C/12C isotopic ratio. One hundred two commercial tannins originating from 10 different botanical sources (grapes, oak, gall, chestnut, fruit trees, quebracho, tea, acacia, officinal plants and tara) were analysed to determine 57 elements and the 13C/12C isotopic ratio, using inductively coupled plasma mass spectrometry and isotope-ratio mass spectrometry, respectively. Forward stepwise discriminant analysis provided good discrimination between the 8 most abundant groups, with 100% correct re-classification. The model was then validated five times on subsets of 10% of the overall samples, randomly extracted, achieving satisfactory results. With a similar approach it was also possible to distinguish toasted and untoasted oak tannins as well as tannins from grape skin and grape seeds. Copyright © 2014 John Wiley & Sons, Ltd.

The microflora of grapes involved in the production of a traditional Italian straw wine, “Vino Santo Trentino”, was evaluated as a biocontrol agent against Botrytis cinerea, one of the main diseases affecting fruit and grapes. The microbiota was described using plate counts and genotypic characterisation (sequencing of 16S rRNA for bacteria and 26s rRNA for yeast), allowing identification of yeasts belonging to the Hanseniaspora, Metschnikowia, Cryptococcus and Issatchenkia genera and bacteria (Bacillus, Microbacterium, Acetobacter and Gluconobacter spp.). The distribution of these species is related to the extent of B. cinerea infection. 7 isolates were able to halt the growth of B. cinerea in antagonistic cultures grown in Petri plates, using both synthetic growth and grape juice media. Technological characterisation of potential biocontrol agents, performed with the help of flow cytometry and HPLC-ECD, demonstrated that these microorganisms did not represent a risk for wine production due to their low resistance to ethanol, low pH and the absence of off-flavours. This ensures that the biocontrol agents disappear during winemaking and excludes a negative impact on the quality of wines. In conclusion, the microflora associated with dried grapes is a precious source of biocontrol agents against B. cinerea, both in terms of preventing disease in the vineyard and in control of the grape drying process for the production of straw wines.

Thiol compounds responsible for tropical fruit associated aroma have been extensively studied over the last 20 years. The occurrence of their non-aromatic precursors in grapes and musts is reported largely mainly for the cultivar Sauvignon Blanc. The presence of these thiols as precursors or free molecules in grape, juice, and wine has been reported in several different varieties, suggesting that they are more or less ubiquitous both for Vitis spp. and interspecific hybrids. The biosynthetic pathways resulting in these compounds are yet to be completely elucidated, but, in the meantime, industry needs to improve technological knowledge to better manage winemaking steps to enhance the variety-dependent aroma of wine. In this work, we studied the implications of the use of grape skin tannins—rich and poor in thiol precursors—on the final content of 3-sulfanylhexan-1-ol (3MH) and its acetate (3MHA) in wine and the effect in terms of sensory appreciability. The evaluation of 36 vinifications carried out in a semi-industrial scale permitted us to prove that only a tannin originally rich in precursors (High), when added to juice at the beginning of fermentation, enhanced both the concentration of precursors in the juice and the final concentration of aromatic thiols in the resultant wine. The 3MH and 3MHA developed as a consequence of the juice supplementation with tannin High and increased pleasantness and typicality of Gewurztraminer wines. A later supplementation with tannin High at the end of the alcoholic fermentation was sensorially not effective.

Careful control of spoilage microflora inside wine containers is a key issue during winemaking. To date, attention has been paid to the development of an effective protocol for the eradication of spoilage agents, especially Brettanomyces, from barrels. Few studies have taken into account the modifications caused by sanitation treatments in wine and wood barrels. In the present study the effects of two sanitation treatments (ozone and sodium hydroxide) on barrel spoilage microflora and the composition of the wine stored inside them were evaluated. The phenols of wine (38 compounds) were characterised using a UHPLC-MS during the first 3 months of wine ageing, to see possible alterations in composition due to the chemical exchange from wood to wine in presence of sanitising agents. With the same scope, a panel of 13 judges carried out sensorial analysis of wines. The results showed that the tested treatments had little effect on the organoleptic characteristics of wines, but underline the different performance of the sanitation treatments in terms of eradicating microorganisms.

A broad elemental profile incorporating 54 elements (Li, Be, B, Na, Mg, Al, P, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Y, Mo, Pd, Ag, Cd, Sn, Sb, Te, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Er, Tm, Yb, Re, Ir, Pt, Au, Hg, Tl, Pb, Bi and U) in combination with δ2H, δ13C, δ15N and δ18O was used to characterise the composition of 62 green arabica (Coffea arabica) and robusta (Coffea canephora) coffee beans grown in South and Central America, Africa and Asia, the four most internationally renowned areas of production. The δ2H, Mg, Fe, Co and Ni content made it possible to correctly assign 95% of green coffee beans to the appropriate variety. Canonical discriminant analysis, performed using δ13C, δ15N, δ18O, Li, Mg, P, K, Mn, Co, Cu, Se, Y, Mo, Cd, La and Ce correctly traced the origin of 98% of coffee beans. Copyright © 2012 John Wiley & Sons, Ltd.

Due to the large size (e.g. sections of tree trunks) and highly heterogeneous spatial distribution of deadwood, the timescales involved in the coarse woody debris (CWD) decay of Picea abies (L.) Karst. and Larix decidua Mill. in Alpine forests are largely unknown. We investigated the CWD decay dynamics in an Alpine valley in Italy using the chronosequence approach and the five-decay class system that is based on a macromorphological assessment. For the decay classes 1–3, most of the dendrochronological samples were cross-dated to assess the time that had elapsed since tree death, but for decay classes 4 and 5 (poorly preserved tree rings) radiocarbon dating was used. In addition, density, cellulose, and lignin data were measured for the dated CWD. The decay rate constants for spruce and larch were estimated on the basis of the density loss using a single negative exponential model, a regression approach, and the stage-based matrix model. In the decay classes 1–3, the ages of the CWD were similar and varied between 1 and 54 years for spruce and 3 and 40 years for larch, with no significant differences between the classes; classes 1–3 are therefore not indicative of deadwood age. This seems to be due to a time lag between the death of a standing tree and its contact with the soil. We found distinct tree-species-specific differences in decay classes 4 and 5, with larch CWD reaching an average age of 210 years in class 5 and spruce only 77 years. The mean CWD rate constants were estimated to be in the range 0.018 to 0.022 y−1 for spruce and to about 0.012 y−1 for larch. Snapshot sampling (chronosequences) may overestimate the age and mean residence time of CWD. No sampling bias was, however, detectable using the stage-based matrix model. Cellulose and lignin time trends could be derived on the basis of the ages of the CWD. The half-lives for cellulose were 21 years for spruce and 50 years for larch. The half-life of lignin is considerably higher and may be more than 100 years in larch CWD. Consequently, the decay of Picea abies and Larix decidua is very low. Several uncertainties, however, remain: 14C dating of CWD from decay classes 4 and 5 and having a pre-bomb age is often difficult (large age range due to methodological constraints) and fall rates of both European larch and Norway spruce are missing.

To describe the decay stage of coarse woody debris (CWD) a five decay-class system has been introduced and it is currently the most commonly applied. This system is based on visual, geometric and tactile features of the wood in the field; however, a detailed chemical characterization is often missing. Furthermore, the driving mechanisms (particularly substrate quality vs. environmental conditions) of deadwood decay are controversially discussed. Consequently, we investigated how typical major and minor chemical parameters of wood were correlated with the decay stage. The decomposition patterns of Norway spruce (Picea abies (L.) Karst) and European larch (Larix decidua Mill.) CWD of an Alpine setting were analyzed, and how the chemical and physical parameters were affected by the substrate and environmental conditions was checked. Two altitudinal sequences, having a different exposure (north- vs. south-facing sites), were sampled. We measured main biochemical compounds (lignin and cellulose), physical properties (density and water content), element concentrations (C, N, P, K, Ca, Mg, Fe, Mn), and the carbon isotopic signature (δ13C) of living trees and CWD at five decomposition stages (decay classes). Most investigated wood physico-chemical parameters such as wood density, water content, lignin and cellulose and even minor constituents (N, Ca, Mg, P, Fe, Mn) correlated well to the five decay-class system. Some important components, such as the carbon concentration and δ13C, did not vary with increasing decomposition. Our hypothesis that the different substrate should be traceable during CWD decay had to be rejected, although some statistically significant chemical differences between larch and spruce were measured in the living trees. The chosen tree species were probably not different enough to be chemically traceable in the CWD. Already in decay class 1, these differences were zeroed. The site conditions (expressed by the different altitudes and exposure) influenced only some of the investigated parameters, namely lignin, the δ13C isotopic ratio and nutrients such as P, Ca and K.

Malolactic fermentation (MLF) is a key feature in the production of high-quality wines. Its evolution is not always guaranteed, especially in white wine, due to certain limiting factors (low pH, sulphur dioxide, low temperature) acting against malolactic bacteria. The inoculation of grape must with bacteria is an alternative approach to the management of oenological fermentation, favouring the survival of bacteria due to the absence of ethanol or sulphur dioxide – toxic compounds made by yeasts in the first stages of winemaking. We compared the activity of two strains of Oenococcus oeni during MLF in wines made from an emerging white grape variety, native to north-eastern Italy, namely Incrocio Manzoni. Different winemaking protocols were assayed, comparing sequential or simultaneous inoculation of microbial starters. The monitoring of bacterial viability through fermentations and a comprehensive characterisation of the volatile profile of the wines were achieved by advanced analytical approaches, flow cytometry and GC-MS respectively. According to the preliminary hypothesis, the chemical composition of the grape must was characterised by high acidity, which represented a serious barrier to bacterial development. Simultaneous inoculation of the two O. oeni strains ensured a regular evolution of MLF. Some differences were highlighted, both in terms of fermentation kinetics and the aromatic profile of the wines obtained, in relation to the strain of lactic bacteria. The work provides an exhaustive overview of the opportunities and risks related to different wine fermentation approaches in order to enhance the quality of white wines made from “new” or “local” wine grapes.

In the field of winemaking, malolactic fermentation is a key aspect in obtaining high quality wines. Unfortunately, in some oenological contexts effective evolution does not take place because of the occurrence of certain limiting factors for malolactic bacteria in wine. Simultaneous alcoholic and malolactic fermentation in grape must is a promising alternative that promotes the survival of bacteria, due to the absence of certain limiting factors such as ethanol or other toxic substances produced by yeasts in the native grape must. The risk of wine depreciation due to the spoilage activity of malolactic bacteria can be reduced by using selected strains of Oenococcus oeni, with proven behaviour in terms of malolactic fermentation occurring in the presence of sugars. In this work we compared the activity of a strain of Oenococcus oeni in malolactic fermentation of a Chardonnay grape must, using different winemaking protocols characterised by sequential or simultaneous inoculums of microbial starters. The results are discussed both in terms of fermentative behaviour and the quality of the wines obtained, with careful analysis of the main chemical parameters of the wines and of 47 different volatile compounds, giving an exhaustive overview of the opportunities and the risks related to different wine fermentation strategies.

“Nostrano-cheeses” are traditional alpine cheeses made from raw cow’s milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produceγ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n=97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated wereLactobacillus paracasei,Streptococcus thermophilus, andLeuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers wereLactobacillus paracaseibut other GABA producing species includedLactococcus lactis,Lactobacillus plantarum,Lactobacillus rhamnosus,Pediococcus pentosaceus, andStreptococcus thermophilus. NoEnterococcus faecalisorSc. macedonicusisolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was aSc. thermophilus.

The Traditional Mountain Malga (TMM) cheese is made from raw cow's milk by spontaneously fermentation in small farms called “Malga” located in Trentino region. This study was designed to characterize the lactic acid bacteria (LAB) growing on MRS medium, of TMM-cheese at the end of the ripening. Ninety-five LAB were isolated and genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. The 95 LAB clustered in 70 biotypes. Pediococcus pentosaceus and Lactobacillus paracasei were the dominant species. Isolates were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase (AP) activity, biogenic amines production, bile salts hydrolysis, conjugated linoleic acid and γ-aminobutyric acid production. Lb. paracasei isolates resulted to be well adapted to Malga environment and to show the best AP activity and acetoin production. TMM-cheese related LAB showed also interesting health promoting properties and produced bioactive substances. In particular, one Lb. brevis biotype produced a GABA mean value of 129 mg/L that is considered a high concentration. The results confirmed that TMM-cheese resident LAB could be exploited for dairy production.

Grape flavonols are involved in the phenomenon of copigmentation in red wines. These compounds are characterised by nutraceutical properties, have antioxidant activity and are studied for chemotaxonomy of grapes. In general, hybrid grapes are characterised by presence of polyphenols often qualitatively and quantitatively different from Vitis vinifera varieties. In this work, flavonols of 34 hybrid grape varieties (22 red and 12 white) produced by crossing of V. vinifera, Vitis riparia, Vitis labrusca, Vitis lincecumii and Vitis rupestris species, were studied. Compounds were characterised by combining different liquid chromatography/mass spectrometry (LC/MS) methods: precursor-ion and neutral-loss multiple-reaction-monitoring (MRM), and high-resolution mass spectrometry. Twenty-four glycoside flavonols were identified, including 4 quercetin, 5 myricetin, 4 kaempferol, 3 isorhamnetin, 2 laricitrin, 3 syringetin and 3 dihydroflavonol derivatives; myricetin hexoside-glucuronide, myricetin O-di-hexoside, syringetin O-di-hexoside, isorhamnetin rutinoside and kaempferol rutinoside were found in grape for the first time. Statistical analysis (PCA and cluster analysis) divided the samples in four groups on the basis of their flavonol profiles.

Tannins are widely used in winemaking and food and beverage preparation for the many different contributions they can give to the overall characteristics of the product (e.g., colour stability, mouthfeel and aromatic composition). Varietal thiols and their precursors are one of the most interesting research areas in food science and a lot of effort has been put to further the current understanding on their formation and on the impact of different production strategies on their concentration in the final product. This paper reports the identification of two important thiol precursors (Cys-3MH and GSH-3MH) in commercial grape oenological tannins and, to the best of our knowledge, this information is reported here for the first time. This finding allows potential new perspectives in the winemaking and the food industry, offering the possibility of controlled additions of thiol precursors in pre-fermentative stages in order to tune the aroma profile of fermented products.

The anthocyanins of 21 hybrid red varieties produced by crossing V. vinifera, V. riparia, V. labrusca, V. lincecumii and V. rupestris species, the profiles for which have not yet been reported, were studied. Profiles were determined by LC/DAD, and identification of single anthocyanins was confirmed by LC/MS precursor-ion analysis. Anthocyanidin precursors (pelargonidin at m/z 271, dephinidin at m/z 303, cyanidin at m/z 287, petunidin at m/z 317, peonidin at m/z 301, and malvidin at m/z 331) and precursors of monoglucoside compounds allowed 24 different compounds to be identified. Analysis of precursor ions of monoglucoside anthocyanins at low capillary voltage revealed the signals of diglucosides only, providing a very selective method for analysis of diglucoside anthocyanins in grape. According to anthocyanin profile, the samples were subdivided into two groups: one characterized by the substantial presence of diglucoside compounds (particularly Seyve Villard 23-399 and Seyve Villard 23-369) and one by the scarce presence or practical absence of diglucosides (Seibel 10878, Burdin 4077, and Galibert 238-35). Particularly interesting for producing anthocyanin for the natural colorant industry were the varieties Siebel 8357, Baco 30-12 and Terzi 100-31.

This work was performed to evaluate the evolution of indigenous yeasts during wine productions carried out following the principles of biodynamic agriculture. Five trials were designed with different technological interventions consisting of the addition of nitrogen (in the form of ammonium salt), thiamine salt, oxygen, and pied de cuvee at varying concentrations. Yeasts were estimated by haemocytometer chamber and plate counts and identified by sequencing of the D1/D2 domain of the 26S rRNA gene. The isolates identified as Saccharomyces cerevisiae were found to dominate must fermentations and were genetically differentiated by interdelta sequence analysis (ISA). Several non-Saccharomyces species, in particular Hanseniaspora spp. and Candida spp., were found at subdominant levels during must fermentation. The trial added with both nitrogen and thiamine (NTV) showed the highest fermentation rate and microbial richness. The internal surfaces of the cellar equipment were characterised by a certain yeast biodiversity and hosted the species found during winemaking; the wooden surfaces represented the primary source of inoculation of a strain of S. cerevisiae found dominant in all winemaking trials.

Barrels play an important role in winemaking, giving wines a specific flavour and assisting the ageing process. However, the porosity and inertness of wood encourage the proliferation of spoilage yeasts which can severely affect wine quality. The yeast microbiota resident inside barriques located in a traditional Tuscan winery were identified by partial sequencing of the 26S rDNA gene. Most of them belonged to the Saccharomyces, Candida and Pichia genera, while Brettanomyces/Dekkera represented a limited part of the population. The ability of the isolated yeasts to spoil wine through the production of volatile phenols was evaluated. Brettanomyces showed significant production of ethylphenols, with a conversion rate of over 70%; many other species of yeast showed extensive production of vinylphenols, which can have a negative impact on wine quality. To improve the microbiological control of barrels, 4 treatments were tested for their efficacy in eradicating the yeast inside the barrels: aqueous steam (100 °C, 5–30-min treatment), UV irradiation (36-W lamp, 5–30-min treatment), gaseous O3 (40 mg/m3, 30-min treatment) and aqueous O3 (2 mg/L, 30-min treatment). Steam and O3 were the most effective treatments, eliminating about 70% of yeasts. UV appeared to be less effective, probably due to the porous nature of wood, which protects cells from direct irradiation.