Enhancement of pyranoanthocyanin formation in blueberry wine with non-Saccharomyces yeasts.

The development of blueberry wine provides an alternative method for maintaining the nutritional value and extending the shelf life of blueberries. However, anthocyanin loss and off-flavor compound generation during fermentation impair blueberry wine color and quality. Hydroxycinnamate decarboxylase from yeast can catalyze the conversion of hydroxycinnamic acids to vinylphenols, which later may condense with anthocyanins to form more stable vinylphenolic pyranoanthocyanins. In this study, 10 non-Saccharomyces yeasts from Daqu that showed hydroxycinnamate decarboxylase activity were screened. Among the 10 strains, Wickerhamomyces anomalus Y5 showed the highest consumption (34.59%) of the total tested phenolic acids and almost no H2S production. Furthermore, Y5 seemed to produce four vinylphenol pyranoanthocyanins (cyanidin-3-O-galactoside/glucoside-4-vinylcatechol, cyanidin-3-O-galactoside/glucoside-4-vinylsyringol, malvidin-4-vinylguaiacol, and malvidin-4-vinylcatechol) during blueberry wine fermentation, which may improve the color stability of blueberry wine. These findings provide new insights for improving the quality of blueberry wine using non-Saccharomyces yeasts.

Non-Saccharomyces yeasts for beer production: Insights into safety aspects and considerations.

The application of non-Saccharomyces yeasts in beer as a natural tool for innovation, to create different aroma profiles and flavoured non-alcoholic beers, has attracted great interest from both researchers and commercial brewers. As a result, a higher diversity of non-Saccharomyces yeasts for beer production is expected on the market in the coming years. However, the safe use of non-Saccharomyces yeasts has not been broadly investigated and no guidance for the safety assessment of yeasts is published. The fundamentals of a safety assessment include an accurate taxonomic species identification using up-to date methods, along with a literature study regarding the yeast species in question. The strain-specific safety concerns that should be assessed involve pathogenic potential, antifungal resistance, production of biogenic amines and possible allergic reactions. However, yeast safety assessment is in its infancy compared to bacterial safety assessment and research is needed to set cut-off values for antifungal resistance, identify potential virulence genes and validate screening tools to assess yeast strains. Finally, the individual breweries are responsible for the safety related to the process in which yeasts are applied and throughout the shelf life of the beer. The application of non-Saccharomyces yeasts for industrial beer production is promising in terms of defining new prototypes and developing healthier and safer beers, but only if good food safety measures, i.e., both for the strain and the production process, are in place throughout the food value chain. In this way, the ancient role of yeasts in making beverages safer and thereby improving food safety is emphasized.

Enzymatic modification and adsorption of hydrophobic zein proteins on lactic acid bacteria stabilize Pickering emulsions.

The effect of enzymatic and physical modifications of the surface of two different strains from lactic acid bacteria, Lactobacillus rhamnosus (LGG) and Lactobacillus delbruekii subs. lactis ATCC 4797 (LBD), to stabilize medium-chain triglyceride (MCT) oil based Pickering emulsions were investigated. A section of cell wall degrading enzymes, lysozyme from chicken egg white and human, lysostaphin, mutanolysin from Streptomyces globisporus and proteinase k and the hydrophobic protein zein were used for enzymatic and physical surface modifications. Cell surface modifications were characterized by optical microscopy, scanning electron cryo-microscopy (Cryo-SEM), transmission electron microscopy (TEM), microbial adhesion to hexadecane (MATH) test and zeta potential measurements. The modified cell hydrophobicity in terms of MATH values were increased (around four times) by the enzymatic and physical modifications for LBD and LGG compared to the control. Emulsions stabilized by modified bacterial cells showed higher stability in comparison with unmodified samples, especially for the samples modified with chicken egg lysozyme. Confocal microscopy revealed that the modified bacterial cells were absorbed at the interface between oil and water and preventing the oil particles from coalescence. Thus, modified bacterial cells can be used to formulate food-grade stable Pickering emulsions. Such Pickering emulsions can potentially be clean label alternatives to replace the conventional emulsion preparations.

Multi-species colloidosomes by surface-modified lactic acid bacteria with enhanced aggregation properties.

HYPOTHESIS: Surface modification of lactic acid bacteria enhances their adsorption and aggregation at air-water interface and enables stabilization of microbubbles that spontaneously transform into water-filled colloidosomes, which can be further modified using LBL formulations. EXPERIMENTS: The bacterial physicochemical properties were characterized using water contact angle (WCA) measurement, bacterial aggregation assay and zeta potential measurement. Cell viability was enumerated using plate-counting method. The LBL reinforcement of colloidosomes was examined by zeta potential measurement and the formed microstructure was investigated using bright-field microscopy, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Shell permeability of colloidosomes was evaluated using a dye release study. FINDINGS: Bacteria surface-modified using octenyl succinic anhydride (OSA) expressed strong adsorption and aggregation at air-water interface when producing microbubbles. Bacteria with enhanced aggregation ability formed stable shells, enabling complete removal of air and air-water interface without shell disintegration. The formed colloidosomes were studied as they were, or were further reinforced by LBL deposition using polymer or hybrid formulations. Hybrid coating involved assembly of two bacterial species producing colloidosomes with low shell porosity. The findings can be exploited to organize different living bacteria into structured materials and to encapsulate and release substances of diverse sizes and surface properties.

Saccharomyces cerevisiae does not undergo a quorum sensing-dependent switch of budding pattern.

Saccharomyces cerevisiae can alter its morphology to a filamentous form associated with unipolar budding in response to environmental stressors. Induction of filamentous growth is suggested under nitrogen deficiency in response to alcoholic signalling molecules through quorum sensing. To investigate this further, we analysed the budding pattern of S. cerevisiae cells over time under low nitrogen conditions while concurrently measuring cell density and extracellular metabolite concentration. We found that the proportion of cells displaying unipolar budding increased between local cell densities of 4.8 × 106 and 5.3 × 107 cells/ml. This increase in unipolar budding was not reproduced with cells growing at the critical cell density and in conditioned media. Growth under high nitrogen conditions also resulted in increased unipolar budding between local cell densities of 5.2 × 106 and 8.2 × 107 cells/ml, but with differences in metabolite concentration compared to low nitrogen conditions. Neither cell density, metabolite concentration, nor nitrogen deficiency were therefore sufficient to increase unipolar budding. Therefore, by using the budding pattern as an early indicator of filamentous growth, our results suggest that quorum sensing may not control the switch of budding behaviour in S. cerevisiae. Only a high concentration of the putative signalling molecule, 2-phenylethanol, resulted in an increase in unipolar budding. However, this concentration was not physiologically relevant, suggesting toxicity rather than a known quorum sensing mechanism.

Reliable budding pattern classification of yeast cells with time-resolved measurement of metabolite production.

Filamentous growth in Saccharomyces cerevisiae is a stress response commonly induced under nutrient deprivation and by certain alcohols. It is a compound phenotype characterized by pseudohyphal growth, invasion and a shift to more polarized budding. Previous methods have not allowed the time-resolved determination of filamentous growth. Here we present a new method for budding pattern characterization that enables the measurement of filamentous growth and metabolite concentration during yeast cell growth at precise time intervals. By combining chemical cell immobilization and single-cell imaging using an oCelloScope™, this method provides more accurate budding pattern classification compared with previous methods. The applications of the method include, for example, investigation of quorum sensing-controlled yeast filamentous growth and metabolism under stress and identification of toxic metabolites.

The effects of cell-cell contact between Pichia kluyveri and Saccharomyces cerevisiae on amino acids and volatiles in mixed culture alcoholic fermentations.

This study used a double-compartment fermenter to assess yeast growth, fermentation activity, and aroma production in response to cell-cell contact during mixed culture fermentation of Pinot noir grape must with Pichia kluyveri and Saccharomyces cerevisiae. Furthermore, amino acids were analyzed in order to study yeast interactions and possible reasons for aroma modulation as a response to cell-cell contact. Our results show that cell-cell contact between the two yeasts decreased cell viability of each yeast during mixed culture fermentation, and that it increased acetate and ethyl ester production and decreased varietal volatile levels. Moreover, it increased the consumption of glutamic acid and the biosynthesis of some specific amino acids related to cell growth, mainly histidine, glycine and proline, while suppressing the production of higher alcohols through the Ehrlich pathway. These results may contribute to an improved understanding, and thus control, of aroma production in mixed culture wine fermentations.

Efficient chemical hydrophobization of lactic acid bacteria - One-step formation of double emulsion.

A novel concept of stabilizing multiple-phase food structure such as emulsion using solely the constitutional bacteria enables an all-natural food grade formulation and thus a clean label declaration. In this paper, we propose an efficient approach to hydrophobically modifying the surface of lactic acid bacteria Lactobacillus rhamnosus (LGG) using lauroyl ahloride (LC) in non-aqueous media. Compared to the unmodified bacteria, cell hydrophobicity was dramatically altered upon modification, according to the higher percentages of microbial adhesion to hexadecane (MATH) and water contact angles (WCA) of LC-modified bacteria. No evident changes were found in bacterial surface charge before and after LC modification. By using one-step homogenization, all the modified bacteria were able to generate stabile water-in-oil-in-water (W/O/W) double emulsions where bacteria were observed on oil-water interfaces of the primary and secondary droplets. Modification using high LC concentrations (10 and 20 w/w%) led to rapid autoaggregation of bacteria in aqueous solution. A long-term lethal effect of modification primarily came from lyophilization and no apparent impact was detected on the instantaneous culturability of modified bacteria.

Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds.

The antagonistic activities of native Debaryomyces hansenii strains isolated from Danish cheese brines were evaluated against contaminating molds in the dairy industry. Determination of chromosome polymorphism by use of pulsed-field gel electrophoresis (PFGE) revealed a huge genetic heterogeneity among the D. hansenii strains, which was reflected in intra-species variation at the phenotypic level. 11 D. hansenii strains were tested for their ability to inhibit germination and growth of contaminating molds, frequently occurring at Danish dairies, i.e., Cladosporium inversicolor, Cladosporium sinuosum, Fusarium avenaceum, Mucor racemosus, and Penicillium roqueforti. Especially the germination of C. inversicolor and P. roqueforti was significantly inhibited by cell-free supernatants of all D. hansenii strains. The underlying factors behind the inhibitory effects of the D. hansenii cell-free supernatants were investigated. Based on dynamic headspace sampling followed by gas chromatography-mass spectrometry (DHS-GC-MS), 71 volatile compounds (VOCs) produced by the D. hansenii strains were identified, including 6 acids, 22 alcohols, 15 aldehydes, 3 benzene derivatives, 8 esters, 3 heterocyclic compounds, 12 ketones, and 2 phenols. Among the 71 identified VOCs, inhibition of germination of C. inversicolor correlated strongly with three VOCs, i.e., 3-methylbutanoic acid, 2-pentanone as well as acetic acid. For P. roqueforti, two VOCs correlated with inhibition of germination, i.e., acetone and 2-phenylethanol, of which the latter also correlated strongly with inhibition of mycelium growth. Low half-maximal inhibitory concentrations (IC50) were especially observed for 3-methylbutanoic acid, i.e., 6.32-9.53 × 10-5 and 2.00-2.67 × 10-4 mol/L for C. inversicolor and P. roqueforti, respectively. For 2-phenylethanol, a well-known quorum sensing molecule, the IC50 was 1.99-7.49 × 10-3 and 1.73-3.45 × 10-3 mol/L for C. inversicolor and P. roqueforti, respectively. For acetic acid, the IC50 was 1.35-2.47 × 10-3 and 1.19-2.80 × 10-3 mol/L for C. inversicolor and P. roqueforti, respectively. Finally, relative weak inhibition was observed for 2-pentanone and acetone. The current study shows that native strains of D. hansenii isolated from Danish brines have antagonistic effects against specific contaminating molds and points to the development of D. hansenii strains as bioprotective cultures, targeting cheese brines and cheese surfaces.

Analysis of the response of the cell membrane of Saccharomyces cerevisiae during the detoxification of common lignocellulosic inhibitors.

Gaining an in-depth understanding of the response of Saccharomyces cerevisiae to the different inhibitors generated during the pretreatment of lignocellulosic material is driving the development of new strains with higher inhibitor tolerances. The objective of this study is to assess, using flow cytometry, how three common inhibitors (vanillin, furfural, and acetic acid) affect the membrane potential, the membrane permeability and the concentration of reactive oxygen species (ROS) during the different fermentations. The membrane potential decreased during the detoxification phase and reflected on the different mechanisms of the toxicity of the inhibitors. While vanillin and furfural caused a metabolic inhibition and a gradual depolarization, acetic acid toxicity was related to fast acidification of the cytosol, causing an immediate depolarization. In the absence of acetic acid, ethanol increased membrane permeability, indicating a possible acquired tolerance to ethanol due to an adaptive response to acetic acid. The intracellular ROS concentration also increased in the presence of the inhibitors, indicating oxidative stress. Measuring these features with flow cytometry allows a real-time assessment of the stress of a cell culture, which can be used in the development of new yeast strains and to design new propagation strategies to pre-adapt the cell cultures to the inhibitors.

The Effects of NaCl and Temperature on Growth and Survival of Yeast Strains Isolated from Danish Cheese Brines.

Yeasts play an important role in cheese making, by contributing to microbial community establishment and improving flavor. This study aimed at investigating the impact of NaCl and temperature on growth and survival of 20 strains belonging to the yeast species Candida intermedia (2 strains), Debaryomyces hansenii (11), Kluyveromyces lactis (1), Papiliotrema flavescens (1), Rhodotorula glutinis (1), Sterigmatomyces halophilus (2) and Yamadazyma triangularis (2) isolated from Danish cheese brines. All yeasts could grow in Malt Yeast Glucose Peptone (MYGP) medium with low NaCl (≤ 4%, w/v) concentrations at 25 °C and 16 °C. Further, none of the strains, except for one strain of D. hansenii (KU-9), were able to grow under a condition mimicking cheese brine (MYGP with 23% (w/v) NaCl and 6.3 g/L lactate) at 25 °C, while all yeasts could grow at 16 °C, except for the two strains of C. intermedia. In the survival experiment, D. hansenii, S. halophilus and Y. triangularis survived in MYGP with 23% (w/v) NaCl throughout 13.5 days at 25 °C, with Y. triangularis and S. halophilus being the most NaCl tolerant, while the remaining yeasts survived for less than 7 days. These results enable the selection of relevant yeasts from cheese brines for potential use in the cheese industry.

Fast measurement of phosphates and ammonium in fermentation-like media: A feasibility study.

Real-time monitoring of bioprocesses plays a key-role in modern industries, providing new information on full-scale production, thus enabling control of the process and allowing it to run at optimal conditions while minimizing waste. Monitoring of phosphates and ammonium in fermentation processes has a twofold interest: they are important nutrients for living organisms while at the same time constituting environmental nutrient pollutants, for which unnecessary use and disposal must be avoided. In this report, the possibility of simultaneous analysis of phosphates and ammonium in fermentations was verified using spectroscopy-based methods combined with chemometrics to construct calibration models. To achieve this, the models were based on synthetic samples mimicking real fermentation media, providing a dataset where the analytes were completely uncorrelated. Different at-line techniques (mid- and near- infrared spectroscopy, MIR and NIR) were evaluated for their ability to monitor quickly both analytes, in a wide range of concentrations (10-100 mM), in three media of different complexities. Partial Least Squares (PLS) models on MIR spectroscopy gave very good results, with prediction errors lower than 5 % for both analytes in all datasets. In contrast, the results for PLS models on NIR spectroscopy were inferior (prediction errors between 3 and 26 %) for both analytes, as, in the case of phosphate, it could be demonstrated that the model was based on based on indirect predictions.

Proteomics insights into the responses of Saccharomyces cerevisiae during mixed-culture alcoholic fermentation with Lachancea thermotolerans.

The response of Saccharomyces cerevisiae to cocultivation with Lachancea thermotolerans during alcoholic fermentations has been investigated using tandem mass tag (TMT)-based proteomics. At two key time-points, S. cerevisiae was sorted from single S. cerevisiae fermentations and from mixed fermentations using flow cytometry sorting. Results showed that the purity of sorted S. cerevisiae was above 96% throughout the whole mixed-culture fermentation, thereby validating our sorting methodology. By comparing protein expression of S. cerevisiae with and without L. thermotolerans, 26 proteins were identified as significantly regulated proteins at the early death phase (T1), and 32 significantly regulated proteins were identified at the late death phase (T2) of L. thermotolerans in mixed cultures. At T1, proteins involved in endocytosis, increasing nutrient availability, cell rescue and resistance to stresses were upregulated, and proteins involved in proline synthesis and apoptosis were downregulated. At T2, proteins involved in protein synthesis and stress responses were up- and downregulated, respectively. These data indicate that S. cerevisiae was stressed by the presence of L. thermotolerans at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from stress, perhaps increasing its enzymatic machinery to ensure better survival.

Can community-based signalling behaviour in Saccharomyces cerevisiae be called quorum sensing? A critical review of the literature.

Quorum sensing is a well-described mechanism of intercellular signalling among bacteria, which involves cell-density-dependent chemical signal molecules. The concentration of these quorum-sensing molecules increases in proportion to cell density until a threshold value is exceeded, which triggers a community-wide response. In this review, we propose that intercellular signalling mechanisms can be associated with a corresponding ecological interaction type based on similarities between how the interaction affects the signal receiver and producer. Thus, we do not confine quorum sensing, a specific form of intercellular signalling, to only cooperative behaviours. Instead, we define it as cell-density-dependent responses that occur at a critical concentration of signal molecules and through a specific signalling pathway. For fungal species, the medically important yeast Candida albicans has a well-described quorum sensing system, while this system is not well described in Saccharomyces cerevisiae, which is involved in food and beverage fermentations. The more precise definition for quorum sensing proposed in this review is based on the studies suggesting that S. cerevisiae may undergo intercellular signalling through quorum sensing. Through this lens, we conclude that there is a lack of evidence to support a specific signalling mechanism and a critical signal concentration of these behaviours in S. cerevisiae, and, thus, these features require further investigation.

Occurrence of lactic acid bacteria and yeasts at species and strain level during spontaneous fermentation of mawè, a cereal dough produced in West Africa.

Mawè is a West African spontaneous fermented cereal-based dough. Different types of mawè exist varying in type of cereal and/or production condition, with fermentations lasting 24-48 h. With the aim of obtaining a comprehensive understanding of the microbial ecology of mawè processing, a microbiological characterisation was performed for four mawè types, produced at eight sites in Benin. At the onset of the fermentations lactic acid bacteria (LAB) and yeast counts were on average 7.5 ±â€¯1.03 and 4.8 ±â€¯0.79 Log10 cfu/g, which increased to 9.2 ±â€¯0.38 and 7.4 ±â€¯0.42 Log10 cfu/g, respectively, at the end of the fermentations. LAB (n = 321) and yeasts (n = 298), isolated during the fermentations, were identified. The predominant LAB and yeast species were Lactobacillus fermentum and Pichia kudriavzevii, respectively, followed by Kluyveromyces marxianus, all present throughout the mawè fermentations. Further, microbial successions took place with Weissella confusa occurring mostly at the onset, while Pediococcus acidilactici and Saccharomyces cerevisiae were mainly associated with the end of the fermentations. Species diversity was influenced both by type of cereal and production condition. The dominating strain clusters of L. fermentum and P. kudriavzevii were ubiquitous and strain diversities were influenced by type of cereal and production site.

Cheese brines from Danish dairies reveal a complex microbiota comprising several halotolerant bacteria and yeasts.

The Danish Danbo cheese is a surface ripened semi-hard cheese, which before ripening is submerged in brine for up to 24 h. The brining is required in order to obtain the structural and organoleptic properties of the cheeses. Likewise, the content of NaCl in the cheese will influence especially the surface microbiota being of significant importance for flavour development and prevention of microbial spoilage. Even though the microbiota on cheese surfaces have been studied extensively, limited knowledge is available on the occurrence of microorganisms in cheese brine. The aim of the present study was to investigate by both culture-dependent and -independent techniques the brine microbiota in four Danish dairies producing Danbo cheese. The pH of the brines varied from 5.1 to 5.6 with a dry matter content from 20 to 27% (w/w). The content of lactate varied from 4.1 to 10.8 g/L and free amino acids from 65 to 224 mg/L. Bacteria were isolated on five different media with NaCl contents of 0.85-23.0% (w/v) NaCl. The highest count of 6.3 log CFU/mL was obtained on TSA added 4% (w/v) NaCl. For yeasts, the highest count was 3.7 log CFU/mL on MYGP added 8% (w/v) NaCl. A total of 31 bacterial and eight eukaryotic species were isolated including several halotolerant and/or halophilic species. Among bacteria, counts of ≥6.0 log CFU/mL were obtained for Tetragenococcus muriaticus and Psychrobacter celer, while counts between ≥4.5 and < 6.0 log CFU/mL were obtained for Lactococcus lactis, Staphylococcus equorum, Staphylococcus hominis, Chromohalobacter beijerinckii, Chromohalobacter japonicus and Microbacterium maritypicum. Among yeasts, counts of ≥3.5 log CFU/mL were only obtained for Debaryomyces hansenii. By amplicon-based high-throughput sequencing of 16S rRNA gene and ITS2 regions for bacteria and eukaryotes respectively, brines from the same dairy clustered together indicating the uniqueness of the dairy brine microbiota. To a great extent the results obtained by amplicon sequencing fitted with the culture-dependent technique though each of the two methodologies identified unique genera/species. Dairy brine handling procedures as e.g. microfiltration were found to influence the brine microbiota. The current study proves the occurrence of a specific dairy brine microbiota including several halotolerant and/or halophilic species most likely of sea salt origin. The importance of these species during especially the initial stages of cheese ripening and their influence on cheese quality and safety need to be investigated. Likewise, optimised brine handling procedures and microbial cultures are required to ensure an optimal brine microbiota.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Metabolic footprint analysis of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations.

INTRODUCTION: There has been a growing interest towards creating defined mixed starter cultures for alcoholic fermentations. Previously, metabolite differences between single and mixed cultures have been explored at the endpoint of fermentations rather than during fermentations. OBJECTIVES: To create metabolic footprints of metabolites that discriminate single and mixed yeast cultures at two key time-points during mixed culture alcoholic fermentations. METHODS: 1H NMR- and GC-MS-based metabolomics was used to identify metabolites that discriminate single and mixed cultures of Lachancea thermotolerans (LT) and Saccharomyces cerevisiae (SC) during alcoholic fermentations. RESULTS: Twenty-two metabolites were found when comparing single LT and mixed cultures, including both non-volatiles (carbohydrate, amino acid and acids) and volatiles (higher alcohols, esters, ketones and aldehydes). Fifteen of these compounds were discriminatory only at the death phase initiation (T1) and fifteen were discriminatory only at the death phase termination (T2) of LT in mixed cultures. Eight metabolites were discriminatory at both T1 and T2. These results indicate that specific metabolic changes may be descriptive of different LT growth behaviors. Fifteen discriminatory metabolites were found when comparing single SC and mixed cultures. These metabolites were all volatiles, and twelve metabolites were discriminatory only at T2, indicating that LT-induced changes in volatiles occur during the death phase of LT in mixed cultures and not during their initial growth stage. CONCLUSIONS: This work provides a detailed insight into yeast metabolites that differ between single and mixed cultures, and these data may be used for understanding and eventually predicting yeast metabolic changes in wine fermentations.

Saccharomyces cerevisiae accumulates GAPDH-derived peptides on its cell surface that induce death of non-Saccharomyces yeasts by cell-to-cell contact.

During wine fermentations, Saccharomyces cerevisiae starts to excrete antimicrobial peptides (AMPs) into the growth medium that induce death of non-Saccharomyces yeasts at the end of exponential growth phase (24-48 h). Those AMPs were found to derive from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). On the other hand, the early death of non-Saccharomyces yeasts during wine fermentations was also found to be mediated by a cell-to-cell contact mechanism. Since GAPDH is a cell-wall-associated protein in S. cerevisiae, we put forward the hypothesis that the GAPDH-derived AMPs could accumulate on the cell surface of S. cerevisiae, thus inducing death of non-Saccharomyces yeasts by cell-to-cell contact. Here we show that 48-h grown (stationary phase) cells of S. cerevisiae induce death of Hanseniaspora guilliermondii and Lachancea thermotolerans by direct cell-to-cell contact, while 12-h grown cells (mid-exponential phase) do not. Immunological tests performed with a specific polyclonal antibody against the GAPDH-derived AMPs revealed their presence in the cell wall of S. cerevisiae cells grown for 48 h, but not for 12 h. Taken together, our data show that accumulation of GAPDH-derived AMPs on the cell surface of S. cerevisiae is one of the factors underlying death of non-Saccharomyces yeasts by cell-to-cell contact.

Effect of sequential fermentations and grape cultivars on volatile compounds and sensory profiles of Danish wines.

BACKGROUND: There has been an increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. In this work, three non-Saccharomyces yeast strains (Metschnikowia viticola, Metschnikowia fructicola and Hanseniaspora uvarum) indigenously isolated in Denmark were used in sequential fermentations with S. cerevisiae on three cool-climate grape cultivars, Bolero, Rondo and Regent. During the fermentations, the yeast growth was determined as well as key oenological parameters, volatile compounds and sensory properties of finished rosé wines. RESULTS: The different non-Saccharomyces strains and cool-climate grape cultivars produced wines with a distinctive aromatic profile. A total of 67 volatile compounds were identified, including 43 esters, 14 alcohols, five acids, two ketones, a C13-norisoprenoid, a lactone and a sulfur compound. The use of M. viticola in sequential fermentation with S. cerevisiae resulted in richer berry and fruity flavours in wines. The sensory plot showed a more clear separation among wine samples by grape cultivars compared with yeast strains. CONCLUSION: Knowledge on the influence of indigenous non-Saccharomyces strains and grape cultivars on the flavour generation contributed to producing diverse wines in cool-climate wine regions. © 2017 Society of Chemical Industry.