From grape berries to wine: population dynamics of cultivable yeasts associated to “Nero di Troia” autochthonous grape cultivar

The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called “Uva di Troia”, monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations (AFs) were performed. Different restriction profiles of ITS–5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of AF revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of ‘cultivable’ microbiota diversity of “Uva di Troia” cultivar from the grape to the corresponding wine (“Nero di Troia”), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations.     

<Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis> yeasts: impact on wine and winemaking

Yeasts belonging to the Brettanomyces/Dekkera genus are non-conventional yeasts, which affect winemaking by causing wine spoilage all over the world. This mini-review focuses on recent results concerning the presence of Brettanomyces bruxellensis throughout the wine processing chain. Here, culture-dependent and independent methods to detect this yeast on grapes and at the very early stage of wine production are encompassed. Chemical, physical and biological tools, devised for the prevention and control of such a detrimental species during winemaking are also presented. Finally, the mini-review identifies future research areas relevant to the improvement of wine safety and sensory profiles.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Sequential inoculum of <Emphasis Type="Italic">Hanseniaspora guilliermondii</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for winemaking Campanino on an industrial scale

In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine.

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Discrimination of wine lactic acid bacteria by Raman spectroscopy

Species of Lactobacillus, Pediococcus, Oenococcus, and Leuconostoc play an important role in winemaking, as either inoculants or contaminants. The metabolic products of these lactic acid bacteria have considerable effects on the flavor, aroma, and texture of a wine. However, analysis of a wine’s microflora, especially the bacteria, is rarely done unless spoilage becomes evident, and identification at the species or strain level is uncommon as the methods required are technically difficult and expensive. In this work, we used Raman spectral fingerprints to discriminate 19 strains of Lactobacillus, Pediococcus, and Oenococcus. Species of Lactobacillus and Pediococcus and strains of O. oeni and P. damnosus were classified with high sensitivity: 86–90 and 84–85%, respectively. Our results demonstrate that a simple, inexpensive method utilizing Raman spectroscopy can be used to accurately identify lactic acid bacteria isolated from wine.     

Interaction between <Emphasis Type="Italic">Oenococcus oeni</Emphasis> and <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> isolated from wine. Modification of available nitrogen and biogenic amine production

During the mixed culture of Lactobacillus hilgardii 5w, a common spoilage wine bacteria and Oenococcus oeni X₂L, an amensalistic growth response of the malolactic bacteria was produced due to a competition for nitrogenous nutrients, mainly peptides. Arginine was fully consumed and peptide concentration diminished 60% with respect to both pure cultures at the end of exponential growth. Histamine release increased 34% with respect to L. hilgardii single culture. Under the poor nutritional conditions present during winemaking, L. hilgardii could increase histamine production and adversely affect malolactic fermentation conducted by O. oeni and hence the quality of the final product.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Improved cider fermentation performance and quality with newly generated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> × <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> hybrids

Yeast cryotolerance may be advantageous for cider making, where low temperatures are usually employed. Here, we crossed the cryotolerant S. eubayanus with a S. cerevisiae wine strain and assessed the suitability of the hybrids for low-temperature cider fermentation. All strains fermented the juice to 5% ABV, but at different rates; hybrid strains outperformed S. cerevisiae, which was sensitive to low temperatures. The best hybrid fermented similarly to S. eubayanus. S. eubayanus produced sulphurous off flavours which masked a high concentration of fruity ester notes. This phenotype was absent in the hybrid strains, resulting in distinctly fruitier ciders. Aroma was assessed by an independent consumer panel, which rated the hybrid ciders as identical to the wine strain cider. Both were significantly more pleasant than the S. eubayanus cider. Interspecific hybridization can apparently be used effectively to improve low-temperature fermentation performance without compromising product quality.     

Characterization of the genome of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from red berry wines

Using restriction analysis of noncoding rDNA regions, multiplex PCR, and molecular karyotyping, we have examined Saccharomyces strains isolated from red berry wine materials in Russia, Belarus, and Ukraine. According to the molecular analysis, all strains belong to the species S. cerevisiae. A correlation was revealed between microsatellite fingerprints of the strains and the source of their isolation. The strains isolated from juices and from the surface of different berries showed distinct PCR profiles. The genome compositions of interspecific Saccharomyces hybrids of natural and laboratory origin were studied.     

Genetic Differentiation of the Sherry Yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Molecular and genetic studies of the yeast Saccharomyces cerevisiae isolated at distinct stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from primary winemaking yeasts according to several physiological and molecular markers. All sherry strains, regardless of the place and time of their isolation, carry a 24-bp deletion in the ITS1 region of ribosomal DNA, whereas the yeasts of primary winemaking lack this deletion. Molecular karyotypes of sherry yeasts from different populations were found to be very similar.     

Employing oxygen pulses to modulate <Emphasis Type="Italic">Lachancea thermotolerans</Emphasis>–<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Chardonnay fermentations

Oxygen is sometimes deliberately introduced in winemaking at various stages to enhance yeast biomass formation and prevent stuck fermentation. However, there is limited information on how such interventions affect the dynamics of yeast populations. Our previous study in synthetic grape juice showed that oxygen supply enhances the persistence of Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia pulcherrima. The three non-Saccharomyces yeasts showed differences in growth as a function of oxygen. The present study focused on evaluating the influence of short oxygen pulses on population dynamics and the aroma profile of Chardonnay wine inoculated with L. thermotolerans and Saccharomyces cerevisiae. The results confirmed a positive effect of oxygen on the relative performance of L. thermotolerans. The mixed culture fermentation with L. thermotolerans with S. cerevisiae developed a distinct aroma profile when compared to monoculture S. cerevisiae. Specifically, a high concentration of esters, medium chain fatty acids and higher alcohols was detected in the mixed culture fermentation. The data also showed that the longer persistence of L. thermotolerans due to addition of oxygen pulses influenced the formation of major volatile compounds such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, propanol, isobutanol, butanol, isoamyl alcohol, hexanol, isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, octanoic acid, and decanoic acid.     

A novel killer protein from <Emphasis Type="Italic">Pichia kluyveri</Emphasis> isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts

A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index ? Σ FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose–response effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts.     

Identification of a second <Emphasis Type="Italic">PAD1</Emphasis> in <Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis> LAMAP2480

Volatile phenols are aromatic compounds produced by some yeasts of the genus Brettanomyces as defense against the toxicity of hydroxycinnamic acids (p-coumaric acid, ferulic acid and caffeic acid). The origin of these compounds in winemaking involves the sequential action of two enzymes: coumarate decarboxylase and vinylphenol reductase. The first one converts hydroxycinnamic acids into hydroxystyrenes, which are then reduced to ethyl derivatives by vinylphenol reductase. Volatile phenols derived from p-coumaric acid (4-vinylphenol and 4-ethylphenol) have been described as the major contributors to self-defeating aromas associated with stable, gouache, wet mouse, etc., which generates large economic losses in the wine industry. The gene responsible for the production of 4-vinylphenol from p-coumaric acid has been identified as PAD1, which encodes a phenylacrylic acid decarboxylase. PAD1 has been described for many species, among them Candida albicans, Candida dubliniensis, Debaryomyces hansenii and Pichia anomala. In Brettanomyces bruxellensis LAMAP2480, a 666 bp reading frame (DbPAD) encodes a coumarate decarboxylase. Recent studies have reported the existence of a new reading frame belonging to DbPAD called DbPAD2 of 531 bp, which could encode a protein with similar enzymatic activity to PAD1. The present study confirmed that the transformation of Saccharomyces cerevisiae strain BY4722 with reading frame DbPAD2 under the control of the B. bruxellensis ACT1 promoter, encodes an enzyme with coumarate decarboxylase activity. This work has provided deeper insight into the origin of aroma defects in wine due to contamination by Brettanomyces spp.     

Enhancing the tolerance of the <Emphasis Type="Italic">Starmerella bacillaris</Emphasis> wine strain to dehydration stress

A protocol was developed to obtain Starmerella bacillaris as an active dry wine yeast (ADWY), which will facilitate winemakers to realize sequential inoculations of grape must to improve wine complexity. In the present study, several compound solutions were analyzed during the dehydration–rehydration process for Starmerella bacillaris strains isolated from related environments of alcoholic beverages. The ADWY obtained from Starmerella bacillaris were evaluated in fermentations at laboratory scale, obtained by sequential- and co-inoculation with Saccharomyces cerevisiae; the fermentative and aromatic parameters were evaluated and discussed. Our results for one Starmerella bacillaris strain show that the enhancement of viability might lead to a 4-fold higher survival rate when cells are dried in the presence of 10% trehalose, followed by rehydration in 0.5% galactose solution. In co- and sequentially inoculated grape must fermentations with Saccharomyces cerevisiae, the laboratory-scale wines obtained with Starmerella bacillaris ADWY did not show major changes in terms of the main volatile compounds, but there was an improvement in the fermentation performance behavior. The present study paves the way to develop a protocol for developing Starmerella bacillaris as an ADWY.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and metabolic activators: HXT3 gene expression and fructose/glucose discrepancy in sluggish fermentation conditions

When exposed to mixtures of glucose and fructose, as occurs during the fermentation of grape juice into wine, Saccharomyces cerevisiae uses these sugars at different rates. Moreover, glucose and fructose are transported by the same hexose transporters (HXT), which present a greater affinity for glucose, so that late in fermentation, fructose becomes the predominant sugar. Only a few commercial fermentation activators are available to optimally solve the problems this entails. The aim of this study was to investigate the relation between HXT3 gene expression and fructose/glucose discrepancy in two different media inoculated with a commercial wine strain of S. cerevisiae in the presence of three metabolic activators. Fermentation kinetics, vitality and major metabolites were also measured. Rehydration with ergosterol improved the area under the curve and the growth rate (µ _max ) in both studied media. Also, the fructose/glucose discrepancy values were improved with all activator treatments, highlighting rehydration in the presence of ascorbic acid. The yeast rehydration process was demonstrated to influence HXT3 expression under the studied conditions. Tetrahydrofolic acid treatment greatly influenced HXT3 gene expression, especially on the 12th day of the fermentation process. To a lesser extent, ergosterol and ascorbic acid also improved this parameter.     

Heavy metal accumulation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.     

Bacteriocinogenic Potential of <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated from Wine

A total of 145 lactic acid bacteria isolated from a variety of Turkish red wines during malolactic fermentation were screened to find bacteriocin-producing strains. Among them, 14 isolates of Enterococcus faecium were identified to produce bacteriocins. PCR screening revealed that some isolates harbored entA and entB genes while some harbored entA, entB and entP genes. An isolate designated as Ent. faecium H46 was selected to characterize its bacteriocins. The bacteriocins were purified to homogeneity from culture supernatant by Amberlite XAD-16, cation-exchange and reverse-phase chromatography. MALDI-TOF mass spectrometry analysis identified the bacteriocins as enterocin A and enterocin B. The presence of Ent. faecium is noteworthy since it is not associated with wine fermentation. However, it has been reported as an important wine spoilage organism due to its potential to produce tyramine. Although species of Enterococcus is not known as wine bacteria, contamination by Ent. faecium may arise from grapes or wineries equipments used for wine production.