Biocontrol potential of wine yeasts against four grape phytopathogenic fungi disclosed by time-course monitoring of inhibitory activities.

Grapes' infection by phytopathogenic fungi may often lead to rot and impair the quality and safety of the final product. Due to the concerns associated with the extensive use of chemicals to control these fungi, including their toxicity for environment and human health, bio-based products are being highly preferred, as eco-friendlier and safer alternatives. Specifically, yeasts have shown to possess antagonistic activity against fungi, being promising for the formulation of new biocontrol products.In this work 397 wine yeasts, isolated from Portuguese wine regions, were studied for their biocontrol potential against common grapes phytopathogenic fungal genera: Aspergillus, Botrytis, Mucor and Penicillium. This set comprised strains affiliated to 32 species distributed among 20 genera. Time-course monitoring of mold growth was performed to assess the inhibitory activity resulting from either diffusible or volatile compounds produced by each yeast strain. All yeasts displayed antagonistic activity against at least one of the mold targets. Mucor was the most affected being strongly inhibited by 68% of the tested strains, followed by Botrytis (20%), Aspergillus (19%) and Penicillium (7%). More notably, the approach used allowed the detection of a wide array of yeast-induced mold response profiles encompassing, besides the decrease of mold growth, the inhibition or delay of spore germination and the complete arrest of mycelial extension, and even its stimulation at different phases. Each factor considered (taxonomic affiliation, mode of action and fungal target) as well as their interactions significantly affected the antagonistic activity of the yeast isolates. The highest inhibitions were mediated by volatile compounds. Total inhibition of Penicillium was achieved by a strain of Metschnikowia pulcherrima, while the best performing yeasts against Mucor, Aspergillus and Botrytis, belong to Lachancea thermotolerans, Hanseniaspora uvarum and Starmerella bacillaris, respectively. Notwithstanding the wide diversity of yeasts tested, only three strains were found to possess a broad spectrum of antagonistic activity, displaying strong or very strong inhibition against the four fungal targets tested. Our results confirm the potential of wine yeasts as biocontrol agents, while highlighting the need for the establishment of fit-for-purpose selection programs depending on the mold target, the timing, and the mode of application.

Insights into the transcriptional regulation of poorly characterized alcohol acetyltransferase-encoding genes (HgAATs) shed light into the production of acetate esters in the wine yeast Hanseniaspora guilliermondii.

Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.

Machine Learning Techniques Disclose the Combined Effect of Fermentation Conditions on Yeast Mixed-Culture Dynamics and Wine Quality.

The use of yeast starter cultures consisting of a blend of Saccharomyces cerevisiae and non-Saccharomyces yeasts has increased in recent years as a mean to address consumers' demands for diversified wines. However, this strategy is currently limited by the lack of a comprehensive knowledge regarding the factors that determine the balance between the yeast-yeast interactions and their responses triggered in complex environments. Our previous studies demonstrated that the strain Hanseniaspora guilliermondii UTAD222 has potential to be used as an adjunct of S. cerevisiae in the wine industry due to its positive impact on the fruity and floral character of wines. To rationalize the use of this yeast consortium, this study aims to understand the influence of production factors such as sugar and nitrogen levels, fermentation temperature, and the level of co-inoculation of H. guilliermondii UTAD222 in shaping fermentation and wine composition. For that purpose, a Central Composite experimental Design was applied to investigate the combined effects of the four factors on fermentation parameters and metabolites produced. The patterns of variation of the response variables were analyzed using machine learning methods, to describe their clustered behavior and model the evolution of each cluster depending on the experimental conditions. The innovative data analysis methodology adopted goes beyond the traditional univariate approach, being able to incorporate the modularity, heterogeneity, and hierarchy inherent to metabolic systems. In this line, this study provides preliminary data and insights, enabling the development of innovative strategies to increase the aromatic and fermentative potential of H. guilliermondii UTAD222 by modulating temperature and the availability of nitrogen and/or sugars in the medium. Furthermore, the strategy followed gathered knowledge to guide the rational development of mixed blends that can be used to obtain a particular wine style, as a function of fermentation conditions.

Application of Hyperspectral Imaging and Deep Learning for Robust Prediction of Sugar and pH Levels in Wine Grape Berries.

Remote sensing technology, such as hyperspectral imaging, in combination with machine learning algorithms, has emerged as a viable tool for rapid and nondestructive assessment of wine grape ripeness. However, the differences in terroir, together with the climatic variations and the variability exhibited by different grape varieties, have a considerable impact on the grape ripening stages within a vintage and between vintages and, consequently, on the robustness of the predictive models. To address this challenge, we present a novel one-dimensional convolutional neural network architecture-based model for the prediction of sugar content and pH, using reflectance hyperspectral data from different vintages. We aimed to evaluate the model's generalization capacity for different varieties and for a different vintage not employed in the training process, using independent test sets. A transfer learning mechanism, based on the proposed convolutional neural network, was also used to evaluate improvements in the model's generalization. Overall, the results for generalization ability showed a very good performance with RMSEP values of 1.118 °Brix and 1.085 °Brix for sugar content and 0.199 and 0.183 for pH, for test sets using different varieties and a different vintage, respectively, improving and updating the current state of the art.

Genome sequencing, annotation and exploration of the SO2-tolerant non-conventional yeast Saccharomycodes ludwigii.

BACKGROUND: Saccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO2). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome. RESULTS: Resequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall ß-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO2-tolerance genes, were identified in S. ludwigii genome. CONCLUSIONS: This work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming.

Phenotypic and transcriptional analysis of Saccharomyces cerevisiae during wine fermentation in response to nitrogen nutrition and co-inoculation with Torulaspora delbrueckii.

Nitrogen content of grape musts strongly impacts on fermentation performance and wine metabolite production. As nitrogen is a limiting nutrient in most grape musts, nitrogen supplementation is a common practice that ensures yeast growth during fermentation. However, preferred nitrogen sources -as ammonium- repress the genes related to alternative nitrogen sources consumption, usually involved in aromatic compounds production. Here, we describe the effect of high ammonium doses in Saccharomyces cerevisiae fermentation performance and wine properties, and how it is affected by yeast co-inoculation in mixed (S. cerevisiae + Torulaspora delbrueckii) fermentations. In addition, an RNA-seq analysis allowed us to study the S. cerevisiae transcriptional response to ammonium nutrition and yeast interaction, demonstrating that T. delbrueckii presence affects the global S. cerevisiae transcriptional response, reducing ammonium effects at both phenotypic -fermentation kinetics and metabolite production- and transcriptional levels, under experimental conditions.

The Role of Yeasts and Lactic Acid Bacteria on the Metabolism of Organic Acids during Winemaking.

The main role of acidity and pH is to confer microbial stability to wines. No less relevant, they also preserve the color and sensory properties of wines. Tartaric and malic acids are generally the most prominent acids in wines, while others such as succinic, citric, lactic, and pyruvic can exist in minor concentrations. Multiple reactions occur during winemaking and processing, resulting in changes in the concentration of these acids in wines. Two major groups of microorganisms are involved in such modifications: the wine yeasts, particularly strains of Saccharomyces cerevisiae, which carry out alcoholic fermentation; and lactic acid bacteria, which commonly conduct malolactic fermentation. This review examines various such modifications that occur in the pre-existing acids of grape berries and in others that result from this microbial activity as a means to elucidate the link between microbial diversity and wine composition.

Transcriptomic and chemogenomic analyses unveil the essential role of Com2-regulon in response and tolerance of Saccharomyces cerevisiae to stress induced by sulfur dioxide.

During vinification Saccharomyces cerevisiae cells are frequently exposed to high concentrations of sulfur dioxide (SO2) that is used to avoid overgrowth of unwanted bacteria or fungi present in the must. Up to now the characterization of the molecular mechanisms by which S. cerevisiae responds and tolerates SO2 was focused on the role of the sulfite efflux pump Ssu1 and investigation on the involvement of other players has been scarce, especially at a genome-wide level. In this work, we uncovered the essential role of the poorly characterized transcription factor Com2 in tolerance and response of S. cerevisiae to stress induced by SO2 at the enologically relevant pH of 3.5. Transcriptomic analysis revealed that Com2 controls, directly or indirectly, the expression of more than 80% of the genes activated by SO2, a percentage much higher than the one that could be attributed to any other stress-responsive transcription factor. Large-scale phenotyping of the yeast haploid mutant collection led to the identification of 50 Com2-targets contributing to the protection against SO2 including all the genes that compose the sulfate reduction pathway (MET3, MET14, MET16, MET5, MET10) and the majority of the genes required for biosynthesis of lysine (LYS2, LYS21, LYS20, LYS14, LYS4, LYS5, LYS1 and LYS9) or arginine (ARG5,6, ARG4, ARG2, ARG3, ARG7, ARG8, ORT1 and CPA1). Other uncovered determinants of resistance to SO2 (not under the control of Com2) included genes required for function and assembly of the vacuolar proton pump and enzymes of the antioxidant defense, consistent with the observed cytosolic and mitochondrial accumulation of reactive oxygen species in SO2-stressed yeast cells.

Effect of Controlled Microbial Fermentation on Nutritional and Functional Characteristics of Cowpea Bean Flours.

This study aimed to optimize bean flours fermentation through the use of appropriate technological procedure and, thereby, to obtain a high quality and safe product. In this line, cowpea bean flours with different moisture conditions (10, 20 and 30%) were incubated with (1) a single culture of Lactobacillus plantarum, or (2) a consortium of lactic acid and acetic acid bacteria, together with one strain of Saccharomyces cerevisiae. Effects of inoculation of cowpea beans flours on stability (i.e., evaluated by the decrease in pH), and variations in nutritional characteristics (i.e., protein, starch, water soluble carbohydrates, total dietary fibre) were investigated. In both fermented flours, the effect of fermentation was more noticeable in total water-soluble carbohydrate (WSC) concentration during the fermentation process (P < 0.001), accounted for by metabolic activity of the microorganisms. The pH values progressively decreased (P < 0.001) through the fermentation process, particularly in flours fermented with a single culture of L. plantarum. By contrast, titratable acidity increased (P < 0.001) throughout the fermentation process in F2 and F3, although more noticeable in F3. Total dietary fibre (TDF) was not variable over the time. In relation to the protein content, the fermentations behaved very similarly; F2 had a variation over the time, but the effect was not significant (P = 0.0690). Results revealed small changes in chemical composition except in the case of pH and sugar contents with the values lower in the fermented products (i.e., single- or mixed-culture fermentation), leading to a more stable and safety product. These results indicate that fermented dry beans flours have the potential as functional ingredients for new food formulations.

Characterizing the Potential of the Non-Conventional Yeast Saccharomycodes ludwigii UTAD17 in Winemaking.

Non-Saccharomyces yeasts have received increased attention by researchers and winemakers, due to their particular contributions to the characteristics of wine. In this group, Saccharomycodes ludwigii is one of the less studied species. In the present study, a native S. ludwigii strain, UTAD17 isolated from the Douro wine region was characterized for relevant oenological traits. The genome of UTAD17 was recently sequenced. Its potential use in winemaking was further evaluated by conducting grape-juice fermentations, either in single or in mixed-cultures, with Saccharomyces cerevisiae, following two inoculation strategies (simultaneous and sequential). In a pure culture, S. ludwigii UTAD17 was able to ferment all sugars in a reasonable time without impairing the wine quality, producing low levels of acetic acid and ethyl acetate. The overall effects of S. ludwigii UTAD17 in a mixed-culture fermentation were highly dependent on the inoculation strategy which dictated the dominance of each yeast strain. Wines whose fermentation was governed by S. ludwigii UTAD17 presented low levels of secondary aroma compounds and were chemically distinct from those fermented by S. cerevisiae. Based on these results, a future use of this non-Saccharomyces yeast either in monoculture fermentations or as a co-starter culture with S. cerevisiae for the production of wines with greater expression of the grape varietal character and with flavor diversity could be foreseen.

Volatile Composition and Sensory Properties of Mead.

Mead is a traditional beverage that results from the alcoholic fermentation of diluted honey performed by yeasts. Although the process of mead production has been optimized in recent years, studies focused on its sensory properties are still scarce. Therefore, the aim of this work was to analyse the sensory attributes of mead produced with free or immobilized cells of the Saccharomyces cerevisiae strains QA23 and ICV D47, and to establish potential correlations with its volatile composition. In the volatile composition of mead, the effect of yeast condition was more important than the strain. In respect to sensory analysis, the most pleasant aroma descriptors were correlated with mead obtained with free yeast cells, independently of the strain. Both sensory analysis and volatile composition indicates that the most pleasant mead was produced by free yeast cells. Although this study has provided a significant contribution, further research on the sensory quality of mead is still needed.

Production of blueberry wine and volatile characterization of young and bottle-aging beverages.

The aim of this study was the production of blueberry wine and the characterization of the volatile compounds of fermented and aging in bottle products. Multivariate data analysis indicated similarity of volatile compounds released when fermentations were conducted at laboratory-scale and midscale, with the exception of one replicate creating a distinctive group characterized by low concentrations of acetaldehyde, methanol, 1-hexanol, and ethyl hexanoate, and the production of polyalcohols such as 2,3-butanediols. This experiment was the only one where no adjustments of YAN were performed. Some of the major volatile compounds (acetaldehyde, ethyl acetate, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol) were found above their perception thresholds. Esters and terpenic compounds were the groups of volatiles expressed the most in blueberry wines, followed by volatile fatty acids, alcohols, and norisoprenoids (3-hydroxy-7,8-dihydro-ß-ionone, 3-oxo-α-ionol, and 3-hydroxy-7,8-dihydro-ß-ionol). The wines that experienced bottle-aging are characterized by high concentrations of ethyl esters, diethyl succinate, ethyl lactate, and diethyl malonate. The results contribute for deeper knowledge of the technological procedure, analytical characteristics, and volatile compounds of blueberry wines, reinforcing the interest in this beverage and opening perspectives for further studies on the production of new blueberry-based products with differential characteristics that value its nutraceutical and functional properties.

Genome sequence of the non-conventional wine yeast Hanseniaspora guilliermondii UTAD222 unveils relevant traits of this species and of the Hanseniaspora genus in the context of wine fermentation.

Hanseanispora species, including H. guilliermondii, are long known to be abundant in wine grape-musts and to play a critical role in vinification by modulating, among other aspects, the wine sensory profile. Despite this, the genetics and physiology of Hanseniaspora species remains poorly understood. The first genomic sequence of a H. guilliermondii strain (UTAD222) and the discussion of its potential significance are presented in this work. Metabolic reconstruction revealed that H. guilliermondii is not equipped with a functional gluconeogenesis or glyoxylate cycle, nor does it harbours key enzymes for glycerol or galactose catabolism or for biosynthesis of biotin and thiamine. Also, no fructose-specific transporter could also be predicted from the analysis of H. guilliermondii genome leaving open the mechanisms underlying the fructophilic character of this yeast. Comparative analysis involving H. guilliermondii, H. uvarum, H. opuntiae and S. cerevisiae revealed 14 H. guilliermondii-specific genes (including five viral proteins and one ß-glucosidase). Furthermore, 870 proteins were only found within the Hanseniaspora proteomes including several ß-glucosidases and decarboxylases required for catabolism of biogenic amines. The release of H. guilliermondii genomic sequence and the comparative genomics/proteomics analyses performed, is expected to accelerate research focused on Hanseniaspora species and to broaden their application in the wine industry and in other bio-industries in which they could be explored as cell factories.

Genome Sequence of the Wine Yeast Saccharomycodes ludwigii UTAD17.

This work describes, for the first time, the genome sequence of a Saccharomycodes ludwigii strain. Although usually seen as a wine spoilage yeast, S. ludwigii has been of interest for the production of fermented beverages because it harbors several interesting properties, including the production of beneficial aroma compounds.

A novel feedstuff: ensiling of cowpea (Vigna unguiculata L.) stover and apple (Malus domestica Borkh.) mixtures. Evaluation of the nutritive value, fermentation quality and aerobic stability.

BACKGROUND: Agro-industrial by-products are of low economic value as foods for human consumption but may have potential value as animal feedstuffs. This study evaluated a novel feedstuff, ensiled discarded apple (85%) and cowpea stover (15%) mixtures with two different ensiling periods (45 and 60 days), regarding the nutritive value, fermentation quality and aerobic stability. RESULTS: Generally, no differences (P > 0.05) were observed between ensiling periods for nutritive value and fermentation characteristics. Silages were stable after ensiling, presenting high lactic acid (77.3 g kg-1 dry matter (DM)) and acetic acid (54.7 g kg-1 DM) and low ethanol (15.7 g kg-1 DM) and NH3 -N (105.6 g kg-1 total N) concentrations. No butyric acid was detected in silages, and they were aerobically stable for up to 216 h. Lactic acid bacteria numbers were high at silo opening (7.14 log colony-forming units (CFU) g-1 ), while Enterobacteriaceae were not detected and yeasts/moulds were low (2.44 log CFU g-1 ). Yeast/mould and Enterobacteriaceae numbers grew considerably during 12 days of air exposure. CONCLUSION: A mixture of low calibre discarded apples with cowpea stover can be used as animal feed after the ensiling process owing to its nutritive value and long aerobic stability. © 2017 Society of Chemical Industry.

Genome Sequence of the Nonconventional Wine Yeast Hanseniaspora guilliermondii UTAD222.

In this work, we disclose the genome sequence and a corresponding manually curated annotation of the non-Saccharomyces yeast Hanseniaspora guilliermondii UTAD222, a strain shown to have interesting oenological traits for the production of wines with improved aromatic properties.

Genomic expression program of Saccharomyces cerevisiae along a mixed-culture wine fermentation with Hanseniaspora guilliermondii.

BACKGROUND: The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S. cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniaspora guilliermondii, this being the first time that this relevant yeast-yeast interaction is examined at a genomic scale. RESULTS: Co-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds. CONCLUSIONS: By integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation.

Comparative transcriptomic analysis reveals similarities and dissimilarities in Saccharomyces cerevisiae wine strains response to nitrogen availability.

Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23), under low (67 mg/L) and high nitrogen (670 mg/L) regimes, at three time points during fermentation (12 h, 24 h and 96 h). Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12 h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this nutrient in the grape-musts and the development of strategies to optimize yeast performance in industrial fermentations.

Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts.

Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase.

H. guilliermondii impacts growth kinetics and metabolic activity of S. cerevisiae: the role of initial nitrogen concentration.

Non-Saccharomyces yeasts include different species which comprise an ecologically and biochemically diverse group capable of altering fermentation dynamics and wine composition and flavour. In this study, single- and mixed-culture of Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used to ferment natural grape-juice, under two nitrogen regimes. In single-culture the strain H. guilliermondii failed to complete total sugar breakdown even though the nitrogen available has not been a limiting factor of its growth or fermentative activity. In mixed-culture, that strain negatively interfered with the growth and fermentative performance of S. cerevisiae, resulting in lower fermentation rate and longer fermentation length, irrespective of the initial nitrogen concentration. The impact of co-inoculation on the volatile compounds profile was more evident in the wines obtained from DAP-supplemented musts, characterised by increased levels of ethyl and acetate esters, associated with fruity and floral character of wines. Moreover, the levels of fatty acids and sulphur compounds which are responsible for unpleasant odours that depreciate wine sensory quality were significantly lower. Accordingly, data obtained suggests that the strain H. guilliermondii has potential to be used as adjunct of S. cerevisiae in wine industry, although possible interactions with S. cerevisiae still need to be elucidated.