Genetic and transcriptomic evidences suggest ARO10 genes are involved in benzenoid biosynthesis by yeast.

Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.

Melatonin and glycolytic protein interactions are related to yeast fermentative capacity.

Melatonin is an indole amine that interacts with some proteins in mammals, such as calreticulin, calmodulin or sirtuins. In yeast, melatonin is synthetized and interacts with glycolytic proteins during alcoholic fermentation in Saccharomyces cerevisiae. Due to its importance as an antioxidant molecule in both Saccharomyces and non-Saccharomyces yeasts, the aim of this study was to determine the intracellular and extracellular synthesis profiles of melatonin in four non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora uvarum, Starmeralla bacillaris and Metschnikowia pulcherrima) and to confirm whether glycolytic enzymes can also interact with this molecule in non-conventional yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced in a similar pattern in all non-Saccharomyces yeast, with M. pulcherrima and S. bacillaris being the highest producers. However, melatonin only bound to proteins in two non-conventional yeasts, S. bacillaris and T. delbrueckii, which specifically had higher fermentative capacities. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes, but an RNA-binding protein, the elongation alpha factor, which is related to mitochondria, was also identified. This study reports for the first time the interaction of melatonin with proteins inside non-Saccharomyces yeast cells. These results reinforce the possible role of melatonin as a signal molecule, likely related to fermentation metabolism and provide a new perspective for understanding its role in yeast.

Harnessing yeast metabolism of aromatic amino acids for fermented beverage bioflavouring and bioproduction.

Aromatic amino acid metabolism in yeast is an important source of secondary compounds that influence the aroma and flavour of alcoholic beverages and foods. Examples are the higher alcohol 2-phenylethanol, and its acetate ester, 2-phenylethyl acetate, which impart desirable floral aromas in wine, beer and baker's products. Beyond this well-known influence on the organoleptic properties of alcoholic beverages and foods, there is a growing interest in understanding and modulating yeast aromatic amino acid metabolism. The tryptophan derivatives melatonin and serotonin have bioactive properties and exert positive effects on human health, and aromatic amino acids are also the precursors of products of industrial interest, such as nutraceuticals, fragrances, and opium-derived drugs. This mini-review presents current knowledge on the formation of compounds from aromatic amino acids by Saccharomyces cerevisiae, from genetic and environmental influences on their flavour impacts in alcoholic beverages to their potential as bioactive compounds, and the use of yeast as microbial factories for the production of commercially relevant aromatic compounds.

The role of the membrane lipid composition in the oxidative stress tolerance of different wine yeasts.

Oxidative stress is a common stress in yeasts during the stages of the winemaking process in which aerobic growth occurs, and it can modify the cellular lipid composition. The aim of this study was to evaluate the oxidative stress tolerance of two non-conventional yeasts (Torulaspora delbrueckii and Metschnikowia pulcherrima) compared to Saccharomyces cerevisiae. Therefore, their resistance against H2O2, the ROS production and the cellular lipid composition were assessed. The results showed that the non-Saccharomyces yeasts used in this study exhibited higher resistance to H2O2 stress and lower ROS accumulation than Saccharomyces. Regarding the cellular lipid composition, the two non-Saccharomyces species studied here displayed a high percentage of polyunsaturated fatty acids, which resulted in more fluid membranes. This result could indicate that these yeasts have been evolutionarily adapted to have better resistance against the oxidative stress. Furthermore, under external oxidative stress, non-Saccharomyces yeasts were better able to adapt their lipid composition as a defense mechanism by decreasing their percentage of polyunsaturated fatty acids and squalene and increasing their monounsaturated fatty acids.

The production of aromatic alcohols in non-Saccharomyces wine yeast is modulated by nutrient availability.

Aromatic alcohols (tryptophol, phenylethanol, tyrosol) positively contribute to organoleptic characteristics of wines, and are also described as bioactive compounds and quorum sensing molecules. These alcohols are produced by yeast during alcoholic fermentation via the Erhlich pathway, although in non-Saccharomyces this production has been poorly studied. We studied how different wine yeast species modulate the synthesis patterns of aromatic alcohol production depending on glucose, nitrogen and aromatic amino acid availability. Nitrogen limitation strongly promoted the production of aromatic alcohols in all strains, whereas low glucose generally inhibited it. Increased aromatic amino acid concentrations stimulated the production of aromatic alcohols in all of the strains and conditions tested. Thus, there was a clear association between the nutrient conditions and production of aromatic alcohols in most of the wine yeast species analysed. Additionally, the synthesis pattern of these alcohols has been evaluated for the first time in Torulaspora delbrueckii, Metschnikowia pulcherrima and Starmellera bacillaris.

Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

Acetic acid bacteria from biofilm of strawberry vinegar visualized by microscopy and detected by complementing culture-dependent and culture-independent techniques.

Acetic acid bacteria (AAB) usually develop biofilm on the air-liquid interface of the vinegar elaborated by traditional method. This is the first study in which the AAB microbiota present in a biofilm of vinegar obtained by traditional method was detected by pyrosequencing. Direct genomic DNA extraction from biofilm was set up to obtain suitable quality of DNA to apply in culture-independent molecular techniques. The set of primers and TaqMan--MGB probe designed in this study to enumerate the total AAB population by Real Time--PCR detected between 8 × 10(5) and 1.2 × 10(6) cells/g in the biofilm. Pyrosequencing approach reached up to 10 AAB genera identification. The combination of culture-dependent and culture-independent molecular techniques provided a broader view of AAB microbiota from the strawberry biofilm, which was dominated by Ameyamaea, Gluconacetobacter, and Komagataeibacter genera. Culture-dependent techniques allowed isolating only one genotype, which was assigned into the Ameyamaea genus and which required more analysis for a correct species identification. Furthermore, biofilm visualization by laser confocal microscope and scanning electronic microscope showed different dispositions and cell morphologies in the strawberry vinegar biofilm compared with a grape vinegar biofilm.

Acetic acid bacteria and the production and quality of wine vinegar.

The production of vinegar depends on an oxidation process that is mainly performed by acetic acid bacteria. Despite the different methods of vinegar production (more or less designated as either "fast" or "traditional"), the use of pure starter cultures remains far from being a reality. Uncontrolled mixed cultures are normally used, but this review proposes the use of controlled mixed cultures. The acetic acid bacteria species determine the quality of vinegar, although the final quality is a combined result of technological process, wood contact, and aging. This discussion centers on wine vinegar and evaluates the effects of these different processes on its chemical and sensory properties.

Acetobacter malorum and Acetobacter cerevisiae identification and quantification by Real-Time PCR with TaqMan-MGB probes.

The identification and quantification of Acetobacter malorum and Acetobacter cerevisiae in wine and vinegar were performed using the Real-Time PCR (RT-PCR) with two TaqMan-MGB probes designed to amplify the internal transcribed spacer (ITS) region between the 16S-23S rRNA genes. The primers and probes were highly specific, with a detection limit of 10² cells/ml for both species, and the efficiency of the technique was >80%. The RT-PCR technique with these two new TaqMan-MGB probes, together with the five (Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter hansenii and Gluconacetobacter europaeus) that are already available (Torija et al., 2010), were validated on known concentrations of Acetic Acid Bacteria (AAB) grown in glucose medium (GY) and in inoculated matrices of wine and vinegar. Furthermore, this technique was applied to evaluate the AAB population in real wine samples collected in the Canary Islands. PCR enrichment performed prior to RT-PCR increased the accuracy of quantification and produced results similar to those detected with SYBR-Green. In real wine samples, the total AAB enumeration ranged from 9 × 10² to 106 cells/ml, and the seven AAB species tested were detected in more than one sample. However, AAB recovery on plates was poor; the isolates obtained on plates were A. malorum, G. oxydans, A. cerevisiae and A. pasteurianus species. RT-PCR with TaqMan-MGB probes is an accurate, specific and fast method for the identification and quantification of AAB species commonly found in wine and vinegar.

The Potential of Wild Yeasts as Promising Biocontrol Agents against Pine Canker Diseases.

Native wild yeasts from forest ecosystems represent an interesting potential source of biocontrol organisms in synergy with disease-tolerant forest materials. Yeasts have a combination of competitive mechanisms and low requirements for their biotechnological application as biocontrol agents. The current study aimed to increase the number of biocontrol candidates against Fusarium circinatum and Diplodia sapinea. The enzymatic and antagonistic activities of the biocontrol candidates were evaluated using different screening methods, in which the direct impact on the growth of the pathogen was measured as well as some properties such as cellulose and lignin degradation, tolerance to biocides, volatile compound production, or iron effect, which may be of interest in biotechnological processes related to the management of forest diseases. A total of 58 yeast strains belonging to 21 different species were obtained from oak forest and vineyard ecosystems and evaluated. The application of yeast treatment behaved differently depending on the pathogen and the plant clone. The 2g isolate (Torulaspora delbrueckii) showed the highest inhibitory activity for D. sapinea and 25q and 90q (Saccharomyces paradoxus) for F. circinatum. Clones IN416 and IN216 were the most susceptible and the most tolerant to D. sapinea, respectively, while the opposite was observed for F. circinatum.

Profiling potential brewing yeast from forest and vineyard ecosystems.

The brewing ability of wild yeast strains obtained from forest and vineyards ecosystems was analysed and compared with commercial yeast strains. The selection of new yeast strains as a way to create new beer aromas and flavours and to use local strains to promote the proximity ingredients in brewing is a topic of interest in the craft beer sector. Seventy-six wild Saccharomyces and non-Saccharomyces isolates and eighteen control strains were evaluated for their enzymatic activity and brewing capacity. The early screening system was set up to profile their enzymatic activity, utilisation of wort sugars and the effect of hop acids and ethanol on yeast growth. The microvolume screening method allows a large number of samples to be studied at the same time, permitting an affordable and rapid characterization in a relatively short period of time. Twenty-eight strains were selected using this method and tested in small-scale fermentations. Finally, three of these strains, all belonging to the species Lachancea thermotolerans, showed great potential and adaptability to ferment different wort styles, although further studies will be necessary to test their possibilities as beer starters. Understanding yeast enzymatic profiles and the influence of beer ingredients on their fermentation activity provides a platform to select strains for further consideration in brewing research.

Production of melatonin by Saccharomyces strains under growth and fermentation conditions.

Melatonin is a bioactive compound that is present in wine because it is contained in vinification grapes and synthesized by yeast during alcoholic fermentation. The purpose of this study was to determine the capacity of various Saccharomyces strains to form melatonin during its growth and alcoholic fermentation. A selection of yeasts including six S. cerevisiae (Lalvin CLOS, Lalvin ICV-D254, Enoferm QA23 Viniferm ARM, Viniferm RVA, and Viniferm TTA), one S. uvarum (Lalvin S6U) and one S. cerevisiae var. bayanus (Uvaferm BC) were tested to determine whether they produce melatonin in yeast extract peptose dextrose and synthetic must media in a variety of conditions. Two S. cerevisiae strains (ARM, and QA23), the S. uvarum and the S. cerevisiae var. bayanus, synthesized melatonin. The conditions in which they did so, however, were different: the QA23 strain produced melatonin best in a medium with a low concentration of reducing sugars and Lalvin S6U and Uvaferm BC required a synthetic must under fermentation conditions. Melatonin synthesis largely depended on the growth phase of the yeasts and the concentration of tryptophan, reducing sugars and the growth medium. These results indicate that melatonin may have a role as a yeast growth signal molecule.

Importance of micronutrients and organic nitrogen in fermentations with Torulaspora delbrueckii and Saccharomyces cerevisiae.

The current use of non-Saccharomyces yeasts in mixed fermentations increases the relevance of the interactions between yeast species. In this work, the interactions between Saccharomyces cerevisiae and Torulaspora delbrueckii were analyzed. For this purpose, fermentations with and without contact between strains of those yeast species were performed in synthetic must. Fermentation kinetics, yeast growth and dynamics were measured over time. Additionally, the effects of nitrogen and other nutrient supplementations on the mixed fermentations were determined. Our results showed that S. cerevisiae did not always dominate the sequential fermentations, and experiments without yeast contact (in which T. delbrueckii cells were removed from the medium before inoculating S. cerevisiae at 48 h) resulted in stuck fermentations except when the inoculum size was increased (from 2 × 106 to 108 cells/mL) or there was a supplementation of thiamine, zinc and amino acids at the same concentration as initially found in the synthetic must. Our findings highlight the importance of inoculum size and ensuring the availability of enough micronutrients for all yeast species, especially in sequential fermentations.

Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars.

Direct genomic DNA extraction from vinegars was set up and suitability for PCR assays performed by PCR/DGGE and sequencing of 16S rRNA gene. The method was tested on 12 intermediary products of special vinegars, fruit vinegars and condiments produced from different raw materials and procedures. DNAs extraction was performed on pellets by chemical, enzymatic, resin mediated methods and their modifications. Suitable yield and DNA purity were obtained by modification of a method based on the use of PVP/CTAB to remove polyphenolic components and esopolysaccharides. By sequencing of bands from DGGE gel, Gluconacetobacter europaeus, Acetobacter malorum/cerevisiae and Acetobacter orleanensis were detected as main species in samples having more than 4% of acetic acid content. From samples having no acetic acid content, sequences retrieved from excised bands revealed high similarity with prokaryotes with no function on vinegar fermentation: Burkholderia spp., Cupriavidus spp., Lactococcus lactis and Leuconostoc mesenteroides. The method was suitable to be applied for no-culture study of vinegars containing polyphenols and esopolysaccharides allowing a more complete assessment of vinegar bacteria.

Protective Effects of Melatonin on Saccharomyces cerevisiae under Ethanol Stress.

During alcoholic fermentation, Saccharomyces cerevisiae is subjected to several stresses, among which ethanol is of capital importance. Melatonin, a bioactive molecule synthesized by yeast during alcoholic fermentation, has an antioxidant role and is proposed to contribute to counteracting fermentation-associated stresses. The aim of this study was to unravel the protective effect of melatonin on yeast cells subjected to ethanol stress. For that purpose, the effect of ethanol concentrations (6 to 12%) on a wine strain and a lab strain of S. cerevisiae was evaluated, monitoring the viability, growth capacity, mortality, and several indicators of oxidative stress over time, such as reactive oxygen species (ROS) accumulation, lipid peroxidation, and the activity of catalase and superoxide dismutase enzymes. In general, ethanol exposure reduced the cell growth of S. cerevisiae and increased mortality, ROS accumulation, lipid peroxidation and antioxidant enzyme activity. Melatonin supplementation softened the effect of ethanol, enhancing cell growth and decreasing oxidative damage by lowering ROS accumulation, lipid peroxidation, and antioxidant enzyme activities. However, the effects of melatonin were dependent on strain, melatonin concentration, and growth phase. The results of this study indicate that melatonin has a protective role against mild ethanol stress, mainly by reducing the oxidative stress triggered by this alcohol.

Effect of a Multistarter Yeast Inoculum on Ethanol Reduction and Population Dynamics in Wine Fermentation.

Microbiological strategies are currently being considered as methods for reducing the ethanol content of wine. Fermentations started with a multistarter of three non-Saccharomyces yeasts (Metschnikowia pulcherrima (Mp), Torulaspora delbrueckii (Td) and Zygosaccharomyces bailii (Zb)) at different inoculum concentrations. S. cerevisiae (Sc) was inoculated into fermentations at 0 h (coinoculation), 48 h or 72 h (sequential fermentations). The microbial populations were analyzed by a culture-dependent approach (Wallerstein Laboratory Nutrient (WLN) culture medium) and a culture-independent method (PMA-qPCR). The results showed that among these three non-Saccharomyces yeasts, Td became the dominant non-Saccharomyces yeast in all fermentations, and Mp was the minority yeast. Sc was able to grow in all fermentations where it was involved, being the dominant yeast at the end of fermentation. We obtained a significant ethanol reduction of 0.48 to 0.77% (v/v) in sequential fermentations, with increased concentrations of lactic and acetic acids. The highest reduction was achieved when the inoculum concentration of non-Saccharomyces yeast was 10 times higher (107 cells/mL) than that of S. cerevisiae. However, this reduction was lower than that obtained when these strains were used as single non-Saccharomyces species in the starter, indicating that interactions between them affected their performance. Therefore, more combinations of yeast species should be tested to achieve greater ethanol reductions.

Pectinatus spp. - Unpleasant and recurrent brewing spoilage bacteria.

Traditionally, beer has been recognised as a beverage with high microbiological stability because of the hostile growth environment posed by beer and increasing attention being paid to brewery hygiene. However, the microbiological risk has increased in recent years because of technological advances toward reducing oxygen in beers, besides the increase in novel beer styles production, such as non-pasteurised, flash pasteurised, cold sterilised, mid-strength, and alcoholic-free beer, that are more prone to spoilage bacteria. Moreover, using innovative beer ingredients like fruits and vegetables is an added cause of microbial spoilage. To maintain quality and good brand image, beer spoilage microorganisms are a critical concern for breweries worldwide. Pectinatus and Megasphaera are Gram-negative bacteria mostly found in improper brewing environments, leading to consumer complaints and financial losses. Because of the lack of compiled scientific knowledge on Pectinatus spoilage ability, this review provides a comprehensive overview of the occurrence, survival mechanisms, and the factors affecting beer spoilage Pectinatus species in the brewing process.

Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts.

The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.

Transcriptomic Insights into the Effect of Melatonin in Saccharomyces cerevisiae in the Presence and Absence of Oxidative Stress.

Melatonin is a ubiquitous indolamine that plays important roles in various aspects of biological processes in mammals. In Saccharomyces cerevisiae, melatonin has been reported to exhibit antioxidant properties and to modulate the expression of some genes involved in endogenous defense systems. The aim of this study was to elucidate the role of supplemented melatonin at the transcriptional level in S. cerevisiae in the presence and absence of oxidative stress. This was achieved by exposing yeast cells pretreated with different melatonin concentrations to hydrogen peroxide and assessing the entry of melatonin into the cell and the yeast response at the transcriptional level (by microarray and qPCR analyses) and the physiological level (by analyzing changes in the lipid composition and mitochondrial activity). We found that exogenous melatonin crossed cellular membranes at nanomolar concentrations and modulated the expression of many genes, mainly downregulating the expression of mitochondrial genes in the absence of oxidative stress, triggering a hypoxia-like response, and upregulating them under stress, mainly the cytochrome complex and electron transport chain. Other categories that were enriched by the effect of melatonin were related to transport, antioxidant activity, signaling, and carbohydrate and lipid metabolism. The overall results suggest that melatonin is able to reprogram the cellular machinery to achieve tolerance to oxidative stress.

Effect of Several Nutrients and Environmental Conditions on Intracellular Melatonin Synthesis in Saccharomyces cerevisiae.

Melatonin is a bioactive compound that is present in fermented beverages and has been described to be synthesized by yeast during alcoholic fermentation. The aim of this study was to assess the capacity of intracellular and extracellular melatonin production by different Saccharomyces strains from diverse food origin and to study the effects of different fermentation parameters, such as sugar and nitrogen concentration, temperature or initial population, on melatonin production using a synthetic grape must medium. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry. Intracellular melatonin synthesis profile did not present differences between yeast strains. However, extracellular melatonin production depended on the yeast origin. Thus, we suggest that melatonin production and secretion during the different yeast growth phases follows a species-specific pattern. Other parameters that affected the fermentation process such as sugar content and low temperature had an impact on intracellular melatonin production profile, as well as the melatonin content within the cell. This study reports the effect of several conditions on the melatonin synthesis profile, highlighting its possible role as a signal molecule.