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[Display omitted] • Nonconventional yeasts displayed suitable fermentation profiles for pineapple wine. • Brettanomyces and Lachancea yeasts exhibited suitable fermentation metabolites. • Alcoholic fermentation enhanced the bioaccessibility of phenolic compounds. • Nonconventional yeasts improved the fruity esters of pineapple wines. Alcoholic fermentation is one of man's most efficient food preservation processes, and innovations in this area are a trend in food science and nutrition. In addition to the classic Saccharomyces yeasts, various other species may have desirable characteristics for obtaining fruit wines. This study investigated the profile of non- Saccharomyces commercial yeasts compared with S. cerevisiae regarding pineapple wine's chemical composition and bioaccessibility. The fermentation profile of the yeasts Lachancea thermotolerans , Brettanomyces bruxellensis , Brettanomyces lambicus , and S. cerevisiae was evaluated for sugar and alcohol content, and the pineapple wines obtained were analyzed for amino acids, phenolics, and organic acids by HPLC and volatile profile by GC/MS. All yeast strains were able to produce ethanol and glycerol at acceptable levels. L. thermotolerans produced higher levels of lactic acid (0.95 g/L) and higher consumption of free amino acids. B. bruxellensis produced higher levels of individual phenolics and ethanol 109 g/L. The alcoholic fermentation process improved the bioaccessibility of phenolics such as catechin (237 %), epigallocatechin gallate (81 %), procyanidin B1 (61 %) and procyanidin B2 (61 %). The yeasts differed in their volatile profiles, with Brettanomyces and Lachancea producing higher levels of compounds associated with pineapple aroma, such as ester ethyl butyrate (260–270 µg/L). These results demonstrate the importance of choosing the yeast strain for the conduction of alcoholic fermentation and that the yeasts Brettanomyces and Lachancea showed technological potential in obtaining pineapple wines. This study contributes to developing processes for obtaining fruit wines by highlighting two non- Saccharomyces yeast species with technological potential for alcoholic fermentations. [ABSTRACT FROM AUTHOR]

The aromatic profile of wine determines its overall final quality, and among the volatile molecules that define it, varietal thiols are responsible for shaping the distinctive character of certain wine varieties. In grape must, these thiols are conjugated to amino acids or small peptides in a non-volatile form. During wine fermentation, yeasts play a principal role in expressing these aromatic compounds as they internalise and cleavage these precursors, releasing the corresponding free and aroma-impacting fraction. Here, we investigate the impact of three wine yeasts (Saccharomyces cerevisiae , Torulaspora delbrueckii and Lachancea thermotolerans) on thiol releasing in synthetic grape must fermentations supplemented with different cysteinylated (Cys-4MSP and Cys-3SH) and glutathionylated (GSH-4MSP and GSH-3SH) precursors. We demonstrate higher consumption levels of cysteinylated precursors, and consequently, higher amounts of thiols are released from them compared to glutathionylated ones. We also report a significant impact of yeast inoculated on the final thiols released. Meanwhile T. delkbrueckii exhibits a great 3SHA releasing capacity, L. thermotolerans stands out because of its high 3SH release. We also highlight the synergic effect of the co-inoculation strategy, especially relevant in the case of S. cerevisiae and L. thermotolerans mixed fermentation, that has an outstanding release of 4MSP thiol. Although our results stem from a specific experimental approach that differs from real winemaking situations, these findings reveal the potential of unravelling the specific role of different yeast species, thiol precursors and their interaction, to improve wine production processes in the context of wine aroma enhancement. • Yeasts exhibit a high capacity for metabolising cysteinylated precursors. • Td greatly produces 3-SHA, while Lt is unable to do it. • Lt exhibit high 3SH release and great 4MSP production under mixed fermentation. [ABSTRACT FROM AUTHOR]

The genus Hanseniaspora is a non- Saccharomyces yeast frequently isolated from wine-associated environments and has been recognized to play a key role in winemaking. However, most of the research on Hanseniaspora focused on its role in fermentation or its coordination with Saccharomyces cerevisiae , but the genome data of Hanseniaspora is very scarce, which is not conducive to better utilization and development of this yeast. Therefore, in this study, the genome data of two Hanseniaspora vineae strains CC-P5 and CC-ZZ6 with flocculation and antimicrobial abilities were sequenced and bioinformatics analyses were performed. The results showed that the genomes of the two strains have a high degree of homology with an average nucleotide identity of 97.7 %. Both H. vineae CC-P5 and CC-ZZ6 have corresponding genes for adaptability to osmotic stress, sulfur dioxide stress, ethanol toxicity and other stresses, as well as various genes involved in the biosynthesis of ethyl esters, volatile organic acids, aromatic amino acid, benzyl alcohol/benzaldehyde and terpenoid, which are useful for modulating wine flavor and aroma. These results shed light on why H. vineae has the biotechnology to make wine. In addition, the number of publicly available H. vineae genome databases have increased. This has laid the foundation for further research on Hanseniaspora. • The genomes of Hanseniaspora vineae CC-P5 and CC-ZZ6 were sequenced. • Both CC-P5 and CC-ZZ6 exhibit bacteriostatic and flocculating properties. • The biotechnological properties of CC-P5 and CC-ZZ6 for winemaking were evaluated. • Genes encoding flocculation and superkiller proteins were found in CC-P5 and CC-ZZ6 genomes. [ABSTRACT FROM AUTHOR]

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography–mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives. • Phenylalanine addition enhances floral and fruity wine aromas produced with H. vineae. • High phenylalanine at natural levels did not cause negative effects to wine aroma. • Phenylalanine derivatives are not converted into tyrosine derived aromas in H. vineae. [ABSTRACT FROM AUTHOR]

In this study, yeasts were isolated from an indigenous starter culture for cachaça production (Brazilian spirit) and brewer's spent grain and characterized through a series of phenotypic assays: the killer profile, ascospore formation capacity, growth at high temperatures, resistance to natamycin and actidione, and exoenzyme production. One hundred thirty-four (n = 134) yeasts were isolated and identified as belonging to 6 genera and 10 different species. The ascospore formation in potassium acetate agar was observed in 86% of the isolates (73 Saccharomyces cerevisiae, 3 Torulaspora delbrueckii, and 1 Wickerhamomyces anomalus) highlighting their potential for microencapsulation. Only S. cerevisiae LMQA SNR 70 expressed an effective killer factor (ability to destroy sensitive strains), while only 3 S. cerevisiae, 1 Clavispora lusitaniae, and 1 Pichia kudriavzevii isolates were able to grow up at 45 'C. At 50 and 100 mg/L, actidione showed selectivity action for Candida parapsilosis, while natamycin inhibited the growth of all isolates. The non-Saccharomyces isolates stood out in the qualitative and quantitative tests for amylolytic, pectinolytic, cellulolytic, and xylanolytic activities. A predominance of the species W. anomalus and T. delbrueckii was observed. Besides, quantitative tests revealed that 23% of the isolates exhibited exoenzymatic activity in a nutrient-poor environment, and W. anomalus LMQA CSC 5 e LMQA CSC 43 stood out for the multi-enzymatic in agar and liquid medium response. The phenotypic profile showed that 16% of the isolates expressed two or more characteristics studied, with 2/3 represented by the non-Saccharomyces species. These results highlight the importance of studying yeast species for new biotechnological purposes. © 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. [ABSTRACT FROM AUTHOR]

The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50–200 mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels. Nitrogen nutrient supplementation impacts yeast competition and ester biosynthesis during mixed-culture fermentation of synthetic grape must. Unlabelled Image • The timings rather than levels of nitrogen addition impact mixed fermentation. • Nitrogen addition at the mid-stage eases yeast competition and enhances esters. • Nitrogen addition improves MCFA ethyl esters by activating the MCFA metabolism. • Enhanced MCFAs may derive from redistributed carbon fluxes in the central pathway. [ABSTRACT FROM AUTHOR]

To obtain beverages with reduced alcohol content, the use of unripe grapes, with low sugar and high malic acid concentration, was recently explored. Due to the low sugar, ethanol and glycerol production is limited during fermentation affecting important sensory aspects such as the palate fullness of these beverages. The high acidity influences their organoleptic quality, as well. So far, only S. cerevisiae starter, used in conventional fermentations, have been tested in this condition, and no selection has been performed to identify alternative yeasts suitable for unripe grape fermentation. S. bacillaris is known for the low ethanol tolerance, high glycerol and moderate volatile acidity production. Therefore, this non- Saccharomyces yeast have been investigated to improve the quality of low-alcohol beverages. Seven S. bacillaris strains were tested in synthetic musts with different sugar and malic acid levels, mimicking natural ripe and unripe grape musts. In all the tested conditions, S. bacillaris produced higher glycerol than S. cerevisiae. In single-strain fermentation at low sugar and high malic acid no S. bacillaris strains was able to transform all the sugars, although the produced ethanol was lower than that at high sugar condition. Therefore, sequential fermentations with S. cerevisiae were evaluated at low sugar and high malic acid. In this condition all the sugars were consumed and a significant glycerol increase was found. These results were confirmed when sequential fermentations were run in natural unripe grape must. Moreover, an increase in malic acid degradation, with respect to EC1118 single-strain fermentation, was observed. • At lab condition S. bacillaris showed the best growth when nitrogen was low. • During fermentations at high and low sugar S. bacillaris produced high glycerol. • At low sugar S. bacillaris did not complete the fermentation. • In sequential fermentation with S. cerevisiae all the sugar was consumed. • In unripe grape must fermentation malic acid degradation and high glycerol were found. [ABSTRACT FROM AUTHOR]

The use of Starmerella bacillaris in combination with Saccharomyces cerevisiae is considered as a state-of-the-art biological application to modulate wine composition. This application implies a detailed understanding of yeast-yeast interactions during mixed fermentations and their effect on the composition of the resulting wines. In this context, ten commercial S. cerevisiae strains were used as partners of an indigenous, previously characterized Starm. bacillaris strain in order to get a better insight into the impact of S. cerevisiae strain employed. The different combinations of strains tested influenced the growth dynamics, the fermentation behavior and, as a consequence, wine composition in a couple-dependent manner. In addition, wines produced from mixed fermentations had significantly lower levels of ethanol, acetic acid and ethyl acetate, and showed higher amounts of glycerol, higher alcohols and esters compared to pure S. cerevisiae control fermentations. This study reveals the importance of S. cerevisiae strain choice on the chemical composition of the wines produced from mixed culture fermentations with Starm. bacillaris. Unlabelled Image • Ten Saccharomyces cerevisiae strains were used as partners of Starmerella bacillaris. • Combinations of strains tested influenced growth dynamics and wines composition. • Mixed fermentation significantly decreased ethanol content. • Mixed fermented wines showed higher concentrations of higher alcohols and esters. [ABSTRACT FROM AUTHOR]

Highlights • Sun-dried grape musts were fermented by co-inoculation of indigenous strains. • Lachancea thermotolerans strain tolerates and ferments high-sugar must. • Multivariate Curve Resolution is a suitable tool for wine volatile profile processing. • Wines volatile profile depended on the ratio of each yeast strain in the inoculum. • Co-inoculated fermentations seem to increase aromatic complexity in wines. Abstract The aim of this work was to evaluate how the use of mixed cultures of Saccharomyces cerevisiae and Lachancea thermotolerans indigenous yeast strains influences the volatile composition of wine. Multivariate curve resolution (MCR) method has been applied to data analysis. Five fermentation trials were carried out: three co-inoculated with L. thermotolerans:S. cerevisiae , at the ratio of 50:1, 20:1 and 5:1 respectively, and two with a pure culture of each strain. A must from sun-dried Pedro Ximénez grapes was employed. Volatile composition was determined by dual sequential stir bar sorptive extraction, followed by GC–MS analysis. MCR provided 171 peaks. Results in co-inoculation fermentations revealed that the majority compounds obtained in each case followed the same trend as that observed for the pure culture of the strain that was present in a higher proportion. Finally, 50:1 and 20:1 seemed to be the best proportions to obtain a wine with a quality volatile profile. [ABSTRACT FROM AUTHOR]

Abstract A total of 512 yeasts, including 422 non- Saccharomyces yeasts, were isolated from various fruits including apple, aronia, Muscat Bailey A grapes, and persimmon. These were used to prepare persimmon wine and apple cider starters that produced high levels of aromatic compounds, which contribute to high-quality fermented products. Environmental tolerance testing with 20% glucose and 8% EtOH, alongside a sniffing test, led to the selection of Wickerhamomyces anomalus (Synonym Pichia anomala) SJ20, Meyerozyma caribbica (Synonym Pichia caribbica) YP1, Pichia kluyveri CD34, Hanseniaspora uvarum SJ69 (for persimmon wine), W. anomalus CS7-16 (for apple cider), and Starmerella bacillaris (Synonym Candida zemplinina) CD80 (for both wines) as wine starters. These strains had high environmental stress tolerance and the highest sniffing test scores. Persimmon wine and apple cider were fermented using these strains in single- or mixed-culture with S. cerevisiae W-3 to determine the improved effect on wine aroma. In accordance with the results of volatile ester compounds and sensory evaluation, W. anomalus SJ20, H. uvarum SJ69, and W. anomalus CS7-16 had an excellent potential as persimmon wine and apple cider starters. Moreover, other strains also showed a good potential for a distinctive persimmon wine and apple cider because of the different compositions of the various volatile ester compounds. Six types of sugars (fructose, glucose, maltose, sucrose, raffinose, sucrose, and trehalose), four types of rehydration solutions (distilled water, 1× phosphate buffered saline, 0.85% NaCl, and 1% peptone water), and two types of antioxidants (l -ascorbic acid and glutathione) were examined to improve the survival rate of air-blast dried non- Saccharomyces yeast cells. Optimal sugar and rehydration conditions for each strain were validated, and scanning electron microscopy showed that each cell was surrounded by protectants, including sugar, skim milk, and lactomil. Storability assessment of air-blast dried yeast cells maintained at 4 °C for two months indicated that at least one condition in each strain had a higher survival rate than the control, regardless of the concentration or type of antioxidant treatment, except for M. caribbica YP1. These results suggest that antioxidant treatment contributes to maintaining the viability of air-blast dried cells in hostile environments. Highlights • Selecting 6 non- Saccharomyces yeast strains as persimmon and apple wine starters • Characterization of persimmon wine and apple cider fermented using selected strains • Optimizing protectant, rehydration and antioxidant conditions for yeast starters • Antioxidant additives improved starters storability after 2-month of storage at 4 °C • Yeast starters maintained appropriate fermentation rate for wine fermentation [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to characterize the yeast consortium isolated from Grignolino grapes in a newly planted vineyard in Piedmont (Italy) via analysis of the intra-vineyard yeast distribution of grape samples from single rows. A two-phase approach allowed the identification of culturable yeasts present on grape skins and, through an enriching procedure via grape fermentation, the isolation of low frequency non- Saccharomyces and Saccharomyces spp. fermentative species, including S. paradoxus , which is highly unusual during grape fermentation, along with the intra-specific characterization of S. cerevisiae isolates. Culture-based molecular techniques revealed a grape yeast microbiota formed by (in order of abundance) Hanseniaspora uvarum , the yeast-like fungus Aerobasidium pullulans , Candida zemplinina , Pichia kluyveri , Candida californica , Curvibasidium cygneicollum , Meyerozima caribbica , Rhodotorula babjevae , Metschnikowia pulcherrima and Cryptococcus flavescens. Technological properties of isolated Saccharomyces spp. strains were analysed, identifying strains, including S. paradoxus , potentially suitable as an ecotypical starter for territorial wines. Highlights • The first study of yeast distribution on Grignolino grapes has been carried out • High intra-vineyard variability in terms of yeast abundance was found and depended upon row position • Differences in yeast species diversity and richness were evident in the vineyard within a few metres • S. cerevisiae strains potentially suitable as a starter for territorial wines were characterized • S. paradoxus strain with interesting technological features was found [ABSTRACT FROM AUTHOR]

Abstract The use of mixed culture fermentations with selected Starmerella bacillaris and Saccharomyces cerevisiae strains is gaining winemaking attention, mainly due to their ability to enhance particular characteristics in the resulting wines. In this context, yeast interspecies interactions during fermentation have a fundamental role to determine the desired product characteristics, since they may modulate yeast growth and as a consequence metabolite production. In order to get an insight into these interactions, the growth and death kinetics of the abovementioned species were investigated in pure and mixed culture fermentations, using cv. Nebbiolo grape must. Trials were conducted in flasks but also in a double-compartment fermentation system in which cells of the two species were kept separate by a filter membrane. Although the two species had similar growth pattern during the first days of fermentation, Starm. bacillaris died earlier when tested in the flask than in the double-compartment fermentor. The early death of Starm. bacillaris seemed to be not caused by nutrient limitation nor by accumulation of growth inhibitory compounds (which were not measured in the present study). Rather, cell-to-cell contact mechanism, dependent on the presence of viable S. cerevisiae cells, appears to be responsible for the observations made. These results contribute to better understand the factors that influence Starm. bacillaris death during wine fermentations. Highlights • Yeast interspecies interactions determine wine characteristics • The growth and death kinetics of S. cerevisiae and Starm. bacillaris were investigated in pure and mixed fermentations • The early death of Starm. bacillaris is not caused by nutrient limitation or by inhibitory compounds accumulation • Cell-to-cell contact mechanism appears to be responsible for S. cerevisiae dominance [ABSTRACT FROM AUTHOR]

Non- Saccharomyces wine yeasts are recognized for their ability to enhance the aroma complexity of wines. New trends in winemaking have highlighted the concept of microbiological terroir thus encouraging the use of indigenous non- Saccharomyces starter cultures in mixed fermentations with Saccharomyces cerevisiae to overcome the lack of authenticity and provide the wines with unique regional character and distinct aromatic profiles. In this study, 257 indigenous non- Saccharomyces isolates were obtained for the first time from 11 Maraština vineyards located within the winegrowing region of Dalmatia (Croatia). The isolates were identified at the molecular level and characterized for desirable oenological traits such as growth at different temperatures, high ethanol and SO 2 tolerance, and for negative features such as high hydrogen sulphide (H 2 S) and acetic acid production as well as hydroxycinnamic acid decarboxylase (HCDC) activity. Among 27 identified species, those from Aureobasidium genera prevailed, followed by Metschnikowia , Hanseniaspora and Lachancea. The representatives from different species showed high SO 2 (up to 250 mg/L) and ethanol (up to 12%) tolerance, low acetic acid, and medium-low H 2 S production. The HCDC activity was detected exclusively among Metschnikowia , Aureobasidium and Kabatiella species. Some regionally distinct characteristics within the isolates from the same species were observed. The encouraging insights into potential new starter cultures emerged after the evaluation of enzymatic activities of selected isolates. Most of them showed high leucine and valine arylamidase activity, whereas Hanseniaspora and Metschnikowia isolates were additionally characterized by high β -glucosidase and esterase activity, respectively. [Display omitted] • Indigenous non- Saccharomyces yeasts were isolated from Croatian Maraština grapes. • Among 257 isolates, 27 different species were identified and characterized. • Potential candidates for development as starter cultures were found. • Hanseniaspora isolates showed high β -glucosidase activity. • Some regionally distinct characteristics within the species were observed. [ABSTRACT FROM AUTHOR]

Abstract Strains of Schizosaccharomyces pombe are being increasingly investigated with regards to their grape winemaking potential either in combination with the typical production yeast, Saccharomyces cerevisiae, or in monoseptic fermentations. Their ethanol tolerance and ability to degrade L-malic acid is oenologically convenient but contrasts with the comparatively high acetic acid and acetaldehyde formation potential which is considered undesirable, especially in white winemaking. The purpose of this work was to investigate the performance of a selected S. pombe strain in monoseptic femerntations of white grape must. Traditional batch fermentations were compared with an innovative and automated fed-batch fermentation technique were sugar concentrations are kept low during fermentations to decrease sugar induced osmotic stress. Because of its known effect on growth and ethanol tolerance, the effect of Mg was also tested. While Mg supplementation was not shown to significantly influence residual values of sugars, ethanol, glycerol, organic acids and acetaldehyde, the application of the fed-batch technique led to a fundamental change in yeast physiology. While glycerol values were only slightly reduced, the fed-batch approach allowed obtaining wines devoid of acetic acid whose levels were considerable in wines produced by the traditional batch technique (0.6 g/L). The work demonstrates that the acetic acid metabolism of S. pombe is associated to sugar induced osmotic stress such as for S. cerevisiae , too, and may be controlled by application of suitable fermentation techniques for winemaking. Graphical abstract Unlabelled Image Highlights • Significant production of acetic acid in traditional batch fermentation • The fed-batch approach allos to prevent acetic acid formation by S. pombe. • Grape must supplementation with Mg did not affect metabolism. [ABSTRACT FROM AUTHOR]

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

Chitosan is an active highly charged polysaccharide that has initially been developed in oenology to eliminate the spoilage yeast B. bruxellensis. However, different forms of chitosan exist, some complying with EU regulation for their use in wines, others not. Moreover, with the trend in oenology of limiting SO 2 , more and more questions arise as to the impact of chitosan on other microorganisms of the grape and wine environment. We investigated the antimicrobial efficiency of chitosan on a large oenological microbial collection, englobing technological as well as spoilage microorganisms. Results show that most species are affected at least transiently. Furthermore, a high variability prevails within most species and sensitive, intermediate and tolerant strains can be observed. This study also highlights different efficiencies depending on the wine parameters or the winemaking stage, giving important indications on which winemaking issues can be solved using chitosan. Chitosan treatment does not seem to be appropriate to limit the musts microbial pressure and Saccharomyces cerevisiae cannot be stopped during alcoholic fermentation, especially in sweet wines. Likewise, acetic acid bacteria are poorly impacted by chitosan. After alcoholic fermentation, chitosan can efficiently limit non- Saccharomyces yeast and lactic acid bacteria but special care should be given as to whether malolactic fermentation is wanted or not. Indeed, O. oeni can be severely impacted by chitosan, even months after treatment. Finally, this study highlights the crucial importance of the chitosan type used in its efficiency towards microbial stabilization. While a high molecular weight chitosan has limited antimicrobial properties, a chitosan with a much lower one, complying with EU and OIV regulation and specifications for its use in wine is much more efficient. • Wine microbial species display variable sensitivity to fungal chitosan treatment. • Within a given species, different behaviours can be observed, depending on the strain considered. • The chitosan MW modulates its antiseptic efficiency. • The wine in which the treatment is performed also modulates the chitosan antiseptic efficiency. • Certain winemaking issues can be efficiently solved with chitosan, but not all. [ABSTRACT FROM AUTHOR]

[Display omitted] • L. thermotolerans/H. vineae inhibited acidification up to 0.41 g/L of lactic acid. • L. thermotolerans/H. opuntiae; synergistic effect achieved 2.44 g/L of lactic acid. • pH reduction of up to 0.16 was always greater with Nutrient Vit BlancTM. • 2-phenylethyl acetate was higher (day7) than the control with Nutrient Vit BlancTM. • 2-phenylethyl acetate was (day 7) at least 3 times higher than the control with Nutrient Vit BlancTM. • The sensory analysis of the co-inoculations with H. vineae obtained better scores. The use of non- Saccharomyces yeast in the winemaking industry and even more their co-inoculations to maximize their growth and to express phenotypic characteristic is gaining more and more relevance. This study aimed to shed light on the biocompatibilities between Lachancea thermotolerans and Hanseniaspora spp., using different types of nutrients and considering the effect on Yeast Assimilable Nitrogen (YAN), at low temperature (16 °C) and medium SO 2 (50 mg/L), in white must. L. thermotolerans has been used for its positive effect on pH reduction and Hanseniaspora spp. for improving the sensory profile. The behaviour of these yeasts was evaluated in co-inoculation, always finishing the fermentation with the sequential inoculation of S. cerevisiae. Significant results were obtained on the population count (CFU/mL) in CHROMagar™, with higher populations of Hanseniaspora spp. with respect to L. thermotolerans. Fermentations with L. thermotolerans/H. vineae , showed inhibition of acidification, generating up to 0.41 g/L of lactic acid. On the contrary, a synergistic effect when L. thermotolerans/H. opuntiae was used, achieved 2.44 g/L of lactic acid and a pH reduction of up to 0.16 and always more significant with Nutrient Vit BlancTM. At the same time ethanol concentration decreased by 3.4 % and volatile acidity never exceeded 0.5 g/L. Aromatic composition was analysed and it was found that all fermentations retained more aromatic esters and that on day 7 the amount of 2-phenylethyl acetate was at least 3 times higher in all fermentations compared to the control (Sc + Nutrient Vit BlancTM) which had 5.96 mg/L. Less yellow intensity (-17.3 %) typical of oxidation were observed in all fermentations in which Nutrient Vit BlancTM had been used and in the sensory analysis the co-inoculations with H. vineae generated better scores. [ABSTRACT FROM AUTHOR]

Abstract: Among non-Saccharomyces wine yeasts, Candida zemlpinina is one of the frequently isolated and oenologically important species. It is mostly known from European winemaking areas and it has become one of the key species of non-Saccharomyces wine yeasts to study. Investigating the diversity of C. zemplinina isolates is important for a deeper understanding of the non-Saccharomyces wine yeasts and for the yeast starter industry, as numerous researches have pointed to the potential use of this species in winemaking. For assessing the biodiversity of a larger number of strains, RAPD and micro/minisatellite PCR is often the method of choice, however, this technique is often unstandardized. Whereas some laboratories use these methods for species identifications, others apply RAPD primers for determining intraspecies diversity. In this study, we have tested 5 different RAPD and micro/minisatellite primers on strains of C. zemplinina isolated from different locations. We show that after a rigorous PCR-optimization aimed at reproducibility and comparability of band patterns with these PCR-reactions, diversity of different strains from a wide range of geographic locations is relatively low. The analysis of several oenologically important physiological traits of the strains showed a relatively low level of diversity as well. We also demonstrate that the intraspecific diversity of C. zemplinina observable with different techniques (RAPD, micro/minisatellite or physiological analysis) may be fairly different and not necessarily comparable. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The airborne yeast flora associated with four different areas in 3 Spanish wineries was analyzed over the course of a year (winter, spring, summer and fall) in order to distinguish the yeast species present in this ecosystem and to evaluate the risk of air as a means of wine contamination by spoilage yeasts. Sampling was conducted in the vinification, bottling, cask aging and bottle aging areas. 367 yeasts were identified from two different culture media: 258 in a generic medium for yeast species and 109 in a more selective medium for the Brettanomyces/Dekkera genus. Low levels of yeasts were found in the air with different values depending on the activity being performed at the time: the population in the bottling area increases when bottling is being carried out, and it also increases in the air present in the winemaking premises during vinification. Most of the isolated yeasts were members of the non-Saccharomyces group. Yeasts from the Saccharomyces cerevisiae species were only detected in the air present in the vinification area during the vinification period. Aureobasidium and Cryptococcus were the dominant genera in the air and they were found permanently in the 3 wineries in every area analyzed. The highest level of yeast cells was found in the bottling line, which makes it the area in the winery that requires the greatest vigilance. However, the types of yeast detected are not a real risk to the quality of the wine being bottled. Spoilage yeast Brettanomyces/Dekkera was detected in the air present in the cask area of one winery when wine containing these yeast species was being handled. This suggests that air can be a means for these yeast cells to spread in the wineries and that yeast dissemination is associated with the handling of the wine containing them. No significant differences were found between the wineries, neither in the count of yeasts in the air nor in the main genera present. Therefore, this data establishes an initial description of the yeast levels in the air, the genera and species usually present and the risk to the stability of the wine and its spoilage. [Copyright &y& Elsevier]

Highlights: [•] Application of yeast Hanseniaspora vineae in real wine co-fermentation. [•] H. vineae and spontaneous fermentations showed higher flavour intensity and diversity. [•] Genetic strains identification showed increased diversity compared to conventional treatment. [•] Increased formation of esters and decrease formation of alcohols may explain fruit intensity. [Copyright &y& Elsevier]

A complex consortium of yeasts is the principal responsible of wine fermentation, being Saccharomyces cerevisiae the main driver. The use of selected yeast, beginning with Saccharomyces strains, is one of the main achievements in microbiological control in the wine industry. However, the use of single strain starters of S. cerevisiae and the limited variability of strains has increased the objections to its use due to the production of wines with a homogeneous profile. New tendencies in winemaking have emphasized the microbiological terroir and challenged the use of selected starters from different areas, including Non- Saccharomyces yeast or multi-strain starters in order to add complexity to the biochemical composition of wines. Nevertheless, these strategies also harbor their own challenges. In the present mini-review, we focus on the alternatives to current commercial yeast starters mainly based on the local multispecies starters or controlled spontaneous fermentations, considering the advantages and limitations of each strategy. Also, we present an ancestral technique based on the use of pre-fermented must (Pied-de-Cuve) because it provides certain microbial control to the alcoholic fermentation while allows the development of autochthonous microorganisms that might confer microbial typicity to the produced wines. Nevertheless, the latest strategy needs further research to establish a scientific background for the selection of the best Pied-de-Cuve development. Finally, the tendencies in winemaking should find a commitment between microbial control to guarantee alcoholic fermentation fulfillment and the production of wines reflecting microbial typicity to respond to consumer demands, without forgetting the scaling up in the cellars. • Strategies to use the microbial terroir for wine production are reviewed. • Alternatives to single local strain starters with limited diversity • Multi-starters are difficult to reproduce at cellars. • The non-inoculated but controlled pied-de-cuve is a promising alternative. [ABSTRACT FROM AUTHOR]

Forty-three South African Torulaspora delbrueckii yeast isolates from the ARC Infruitec-Nietvoorbij yeast culture collection, the T. delbrueckii type strain (CBS 1146), one reference T. delbrueckii strain (CBS 4663), two T. delbrueckii strains isolated from commercial yeast blends (Viniflora® Harmony.nsac and Viniflora® Melody.nsac), and a commercial Saccharomyces cerevisiae yeast (VIN 13) had their identities confirmed and were characterised using conventional and molecular microbiological techniques. These included a selection of growth media as well as CHEF electrophoretic karyotyping and PCR-RFLP analyses. Based on the biochemical and physiological results the strains were divided into 13 groups. The performances of the yeasts were also monitored by means of laboratory-scale fermentations in grape must at 15°C and 22°C. The fermentation kinetic data showed that at 22°C, the yeasts were divided into two distinct groups, a faster and a slower fermenting group. The fermentation curves of the laboratory-scale study at 15°C showed that, at this lower temperature, the yeasts also fermented at different speeds, but the fermentation curves showed greater separation. The biochemical and physiological grouping did not coincide with the fermentation abilities and good fermenters could be found in more than one group. Chemical analyses of the resultant wines (alcohol, volatile acidity, glycerol, total SO2, residual sugar) were used in Principle Component Analyses. The yeasts that grouped close to the S. cerevisiae reference strain (VIN 13) showed more acceptable wine chemical profiles, while those further away displayed less acceptable profiles. Three locally isolated strains and one commercial T. delbrueckii yeast strain, Viniflora® Harmony.nsac. produced wines with acceptable chemical profiles at both temperatures. These strains also had comparable fermentation kinetics to the S. cerevisiae reference. Therefore, depending on the fermentation temperature, different T. delbrueckii strains will be suitable for specific wine styles and some may even be considered for single inoculations without S. cerevisiae in industrial fermentations. [ABSTRACT FROM AUTHOR]

Abstract: The airborne yeast flora associated with four different areas in a Spanish winery was analyzed monthly over the course of one year (February 2008–January 2009), in order to characterize the yeasts in this particular ecosystem, an aspect relatively unknown until now. The sampling areas were: vinification, bottling, cask aging and bottle cellar aging. 382 yeasts were identified from two different culture media: one generic for yeasts and the other selective for the Brettanomyces/Dekkera genus. The levels of yeasts present in the air throughout the year were low and the highest presence of yeasts was in the bottling area. Most of the yeasts isolated were members of the non-Saccharomyces groups. Yeasts of the Saccharomyces cerevisiae species were only detected in the air in the vinification area during the vinification period. Sporidiobolus and Cryptococcus were the dominant genera in the air and they were found continuously in every area analyzed. The spoilage yeast Brettanomyces/Dekkera was not detected in any sampling made. [Copyright &y& Elsevier]

Abstract: Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS–S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations. [Copyright &y& Elsevier]

Abstract: Grappa is an Italian alcoholic beverage obtained from distillation of grape marc, the raw material derived from separation of must during the winemaking process. Marc is stored for a period lasting from few days to several weeks, when fermentation of residual sugars occurs mainly by yeast activity. Many distilleries have adopted different solutions to manage this critical phase in order to avoid spoilage microorganisms: marc acidification is the most widely diffused. In this work, Prosecco grape pomace was acidified with sulphuric acid (to pH 2.9) and stored, whereas non-acidified grape marc was used as control (pH 3.9). Samples for microbiological analysis were collected at the beginning of the storage period, after 15 and 43days. At the beginning of the ensilage (time T0) the indigenous microflora was represented both by yeasts and bacteria at a concentration of about 106 cfu/g. During the first 15days, when the fermentation generally takes place, yeast population grew considerably (up to 107 cfu/g) in acidified grape marc, where bacterial population was maintained at low levels. Moreover, yeast populations recovered at the three sampling times in both treated and untreated marc were genetically characterised. This analysis showed that the species succession lead to non-Saccharomyces species dominance (in particular Issatchenkia and Pichia genera) in both conditions although acidified marc showed a lower percentage of Saccharomyces at any sampling time analysed, this meaning that non-Saccharomyces species were favoured in this environment. Gas chromatographic analysis showed a remarkable change in the aromatic profile of distilled grape marcs at the end of the storage, thus evidencing that concentration of monitored volatile compounds usually produced by microflora was generally lowered by the acidification treatment. This work demonstrates for the first time the strong effect of a persistent acidification treatment both on the microbiota of grape pomace and on the aromatic profile of the distillate. Indeed, the lowering of the pH caused significant changes in yeast–bacteria populations ratio and in yeast species turnover. These microbiological changes determine an improvement of the aromatic profile of the distillate, due to the reduction of the main volatile products associated with potential off-flavours. [Copyright &y& Elsevier]

Abstract: Several studies have reported the beneficial influence of non-Saccharomyces yeasts and their potential applications in the wine industry, mainly in mixed-culture fermentation with S. cerevisiae. The potential impact of 15 non-Saccharomyces strains from 7 species on 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) release in model medium and Sauvignon Blanc must was evaluated after partial fermentation. Whereas the impact of non-Saccharomyces on 4MSP release in both media was low, some M. pulcherrima, T. delbrueckii and K. thermotolerans strains had a high capacity to release 3SH, despite their minimal fermentation activity. As previously demonstrated for Saccharomyces yeast, this contribution is strain dependant. Taking into account their dynamic and quantitative presence during the whole process, the real impact of non-Saccharomyces yeast on 4MSP and 3SH release was evaluated using a recreated community simulating the yeast ecosystem. Our results revealed a positive impact on 3SH release in Sauvignon Blanc wines by promoting non-Saccharomyces yeast activity and delaying the growth of S. cerevisiae. Some non-Saccharomyces yeast strains are capable of making a positive contribution to volatile thiol release in wines, essentially during the pre-fermentation stage in winemaking, when this microbiological sub-population is dominant. [Copyright &y& Elsevier]

Abstract: The strains of two species of yeast, Saccharomyces cerevisiae and Pichia guillermondii, both with high hydroxycinnamate decarboxylase (HCDC) activity (56% and 90% respectively), were used in the fermentation of musts enriched with grape anthocyanins, to favour the formation of highly stable vinylphenolic pyranoanthocyanin pigments. The different strains were used to ferment the must separately, simultaneously, or sequentially, the latter involving an initial period using the yeast with the greatest HCDC activity (P. guillermondii). The must was made from concentrated grape juice diluted to 220g/l of sugar, and enriched with grape anthocyanins to 100mg/l and with p-coumaric acid to 120mg/l. The pH was fixed to 3.5. All 50ml micro-fermentations were done in triplicate. The development of anthocyanin-3-O-glucoside precursors, the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol and vinylphenolic pyranoanthocyanin derivatives were studied during the fermentation. The fermentation strategy used and the yeast HCDC activity significantly influenced the formation of vinylphenolic pyranoanthocyanins. The latter molecules were separated, identified, and quantified using high performance liquid chromatograph with diode array detection and electrospray-mass spectrometry (HPLC-DAD-ESI/MS). The volatile compounds profile was screened during fermentation using gas cromatogrphy-flame ionisation detection (GC-FID), in order to detect and quantify the main molecules. The best results were reached with the sequential fermentation (3.74mg/l of malvidin-3-O-glucoside-4-vinylphenol). This work shows that during mixed or sequential fermentations carried out with non-Saccharomyces or highly fermentative Saccharomyces strains, with high HCDC activity, the content of stable pigments can be increased without sensorial modifications. [ABSTRACT FROM AUTHOR]

Abstract: For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of “Serranía de Ronda” (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied. [ABSTRACT FROM AUTHOR]

Abstract: In this study, several strains of Torulaspora delbrueckii yeast species were evaluated in the laboratory for their enological properties. In a preliminary step, the ability of different molecular methods to discriminate among T. delbrueckii strains was compared. A combination of 7 PCR methods was able to separate 21 strains into 18 groups, while an REA-PFGE method allowed, in one experiment, the separation into 19 groups. The T. delbrueckii strains used presented a wide phenotypic variability in fermentation behaviour, e.g. Lag Phase (LP) duration, T50 parameter (time necessary to ferment half the sugar), and ethanol production. These 3 parameters have to be considered for industrial selection, particularly the LP duration. The majority of T. delbrueckii strains produced 8 to 11% and 7 to 10% ethanol vol. at 17°C and 24°C, respectively, with a maximum ethanol concentration of 12.35 at 17°C and 10.90% vol. at 24°C. The phenotypic variability of this species was also reflected in volatile acidity, glycerol, and aroma production. These experiments confirmed the low volatile acidity and glycerol production of this species and revealed a difference in osmotic stress response, compared to Saccharomyces cerevisiae. T. delbrueckii presented high fermentation purity and produced low levels of undesirable volatile compounds, such as hydrogen sulphide and volatile phenols. [Copyright &y& Elsevier]

Oenococcus oeni is the main agent responsible for malolactic fermentation (MLF) in wine. This usually takes place in red wines after alcoholic fermentation (AF) carried out by Saccharomyces cerevisiae. In recent years, there is an increasing interest in using non- Saccharomyces yeast, usually in combination with S. cerevisiae , to improve wine quality. Current studies report a stimulatory effect of non- Saccharomyces on MLF, generally related to a decrease in the inhibitor compounds found in wine. In this work, we followed a comparative multi-omics approach, including transcriptomic and proteomic analysis, to study the molecular adaptation of O. oeni in wines fermented with Torulaspora delbrueckii and Metschnikowia pulcherrima , two of the most frequently used non- Saccharomyces , in sequential inoculation with S. cerevisiae. We compared the results to the adaptation of O. oeni in S. cerevisiae wine to determine the main changes arising from the use of non- Saccharomyces. The duration of MLF was shortened when using non- Saccharomyces , to half the time with T. delbrueckii and to a quarter with M. pulcherrima. In this work, we observed for the first time how O. oeni responds at molecular level to the changes brought about by non- Saccharomyces. We showed a differential adaptation of O. oeni in the wines studied. In this regard, the main molecular functions affected were amino acid and carbohydrate transport and metabolism, from which peptide metabolism appeared as a key feature under wine-like conditions. We also showed that the abundance of Hsp20, a well-known stress protein, depended on the duration time. Thus, the use of non- Saccharomyces reduced the abundance of Hsp20, which could mean a less stressful wine-like condition for O. oeni. • Use of M. pulcherrima and T. delbrueckii brought about shorter MLF. • Changes in O. oeni transcriptome and proteome depended on the fermenting yeast. • O. oeni nitrogen metabolism is one of the most affected by yeast strain/s. • Specific use of peptides might explain the better O. oeni adaptation to wine. • Hsp20 was confirmed as a reliable O. oeni stress marker. [ABSTRACT FROM AUTHOR]

Abstract: Four pre-selected indigenous yeast strains belonging to Candida guilliermondii (V2 and V5), Candida pulcherrima (V6) and Kloeckera apiculata (V9), were used as β-glucosidase (βGL) and β-xylosidase (βXL) sources. The optimization of yeast culture conditions was carried out and the effects of oenological parameters on β-glycosidase activities were evaluated. C. guilliermondii V2 and C. pulcherrima V6 strains were selected. These strains showed intracellular (C. pulcherrima V6) and parietal (C. guilliermondii V2) constitutive βGL and βXL. The enzymatic activities were active at pH, glucose, ethanol and SO2 concentrations usually found in winemaking and they were able to release monoterpenols and alcohols from grape juice glycoside extracts. Additionally, these yeast strains were not able to produce volatile acidity and off flavour. Regional ecological relevance of these species was also discussed. Our results evidence that the selected C. guilliermondii V2 and C. pulcherrima V6 strains have interesting oenological characteristics and allow us to think in their potential application in winemaking. [Copyright &y& Elsevier]

Abstract: Although the use of starter cultures in winemaking has improved the reproducibility and predictability of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. In this study, we identified for the first time the yeast population occurring during spontaneous fermentation of the Catalanesca white grape, a variety from Campania (Italy). Yeasts were identified using molecular tools: PCR-DGGE and partial sequence analysis of the 26S rRNA gene from isolates. Eighteen different species belonging to 11 different genera were identified. Hanseniaspora spp., Issatchenkia spp. and Candida spp. were the dominant yeasts during the early stages of fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. Four species of Issatchenkia spp., rarely isolated from wine fermentation, were found in this study accounting for the 33.5% of the total isolates. The RAPD-PCR screening of the isolates followed by partial rRNA gene sequencing proved to be a very effective approach to first differentiate the isolates and then identify yeast species involved in a wine making procedure. The results show very high yeast diversity in this natural wine fermentation and also highlight the possibility of considering interesting autochthonous strains for starter selection. [Copyright &y& Elsevier]

Abstract: To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40–50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4–5% (v / v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine. [Copyright &y& Elsevier]

Abstract: An ecological study of spontaneous fermentation in red wine carried out in two wineries located in the same village of the D.O.Ca. Rioja appellation (Spain). These two wineries were very different: one was very old and the other commenced its operations with the 2000 vintage. The study was conducted over seven consecutive years (1997–2003); the first 3 years in the old winery; and the other 4 years in the new one. Another difference between the wineries was the use of commercial strains: no starters were ever used in the old winery and two known strains (A and B) were employed only in a white must vat in the new winery. The number of different Saccharomyces cerevisiae strains detected for each vintage and the frequency of their appearance varied between the old winery and the new winery. There was no one clearly dominant clone in the fermentations studied in the old winery, whereas in the vinifications monitored in the new winery, a clear majority strain was detected. The same S. cerevisiae clone took over the spontaneous fermentation of all the vintages studied in this new winery. Analysis of the mtDNA restriction pattern of this strain revealed that it was identical to one of the commercial strain used (starter A), despite the fact that the fermentation studied was spontaneous. Microbial analysis conducted on winery equipment and winery surfaces prior to the 2003 vintage revealed that starter A had become a resident of the winery with a percentage of 50% among all S. cerevisiae. The presence of non-Saccharomyces yeasts in the early stages of vinification also differed between the old and new wineries. [Copyright &y& Elsevier]

The isolation of autochthonous yeast species presents a good strategy to select new microorganisms for developing an adequate inoculum to carry out fermentations and generate representative products of the cider production zone. However, non- Saccharomyces yeasts have been considered to have low capacity to carry out a complete fermentation as Saccharomyces cerevisiae. In this work, five autochthonous yeasts from a cider fermentation process were isolated and identified as Saccharomyces cerevisiae , Kluyveromyces marxianus , Pichia membranaefaciens , P. kluyveri and Zygosaccharomyces rouxii. A series of fermentations were developed at laboratory level, using each species individually and it was observed that only S. cerevisiae was able to finish the process. K. marxianus consumed less than 50% of the sugars; P. kluyveri and Z. rouxii consumed less than 70% and P. membranaefaciens consumed more than 90% but the yield (ethanol produced for sugar consumed (Y P/S)) was 0.39. Nevertheless, the addition of magnesium, zinc and nitrogen increased the fermentative capacity of almost all species: K. marxianus , Z. rouxii and P. kluyveri , showed an increase in ethanol production when nutrients were added, obtaining more than 80 g/L of ethanol, and showing that those nutrients are necessary to complete the fermentation. This work describes the potential use of different non- Saccharomyces species to carry out fermentation of apple juice and highlights the importance of certain nutrients to enable an efficient alcoholic fermentation and the generation of desirable volatile compounds for cider production. • Isolation, identification and characterization of autochthonous cider yeasts • Non- Saccharomyces autochthonous yeasts did not finish the fermentation on apple juice. • Magnesium, zinc and nitrogen improved the fermentation of non- Saccharomyces yeast. • The non- Saccharomyces can be used as unique yeast to cider production using nutrients. • The volatile profile depends on the yeast species and of the nutrients concentration. [ABSTRACT FROM AUTHOR]

• Importance of related enzymes and precursors on ester production was evaluated. • Fatty acid was added in alcoholic fermentation to lift fruity ester production. • Fine-tuning fatty acids contents is a good way in winemaking for aroma enhancement. Pichia fermentans Z9Y-3 and its intracellular enzymes were inoculated along with S. cerevisiae in synthetic grape must to modulate fruity ester production. The levels of ester-related enzymes, ester precursors, and fruity esters were monitored every 24 h during fermentation. Results showed that the levels of ethyl acetate, acetate higher alcohol esters (AHEs), short chain fatty acid ethyl esters (SFEs), and medium chain fatty acid ethyl esters (MFEs) were significantly enhanced in mixed fermentation. Pearson correlation analysis further revealed that higher alcohols and fatty acids played a more important role in fruity ester production than enzymes; Particularly, the correlation coefficient between fatty acids and MFEs was 0.940. In addition, supplementation of medium chain fatty acids (7.2 mg/L) at the metaphase of single S. cerevisiae fermentation improved ethyl acetate, AHE, SFE, and MFE production by 42.56%, 21.00%, 61.33%, and 90.04%, respectively, although the high level of ethyl acetate might result in off-flavors. [ABSTRACT FROM AUTHOR]

Non- Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae , to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non- Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni , under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non- Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO 2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii , that seemed to promote the development of O. oeni and improve MLF performance. • Effect of two non- Saccharomyces on MLF in white and red winemaking was evaluated. • MLF was faster in most of the wines inoculated with non- Saccharomyces. • SO 2 and MCFA concentrations were lower when non- Saccharomyces were used. • The fastest MLF was observed in white wines inoculated with T. delbrueckii. • T. delbrueckii enhanced the diversity of O. oeni strains in red wines. [ABSTRACT FROM AUTHOR]

• The effect of non- Saccharomyces on malolactic fermentation depends on the yeast. • Torulaspora delbrueckii and Metschnikowia pulcherrima were the best MLF compatible. • Hanseniaspora uvarum and Starmerella bacillaris had negative impact on MLF. Interest in using non- Saccharomyces yeasts in winemaking has increased in recent years due to their ability to improve wine quality. However, little information has been published regarding the possible effect on malolactic fermentation (MLF), carried out mostly by Oenococcus oeni. The aim of this paper is therefore to evaluate the effect of the most representative non- Saccharomyces species on O. oeni and wine MLF. Different strains of Torulaspora delbrueckii , Metschnikowia pulcherrima , Hanseniaspora uvarum , Hanseniaspora vineae and Starmerella bacillaris (syn. Candida zemplinina) were used in sequential alcoholic fermentation with Saccharomyces cerevisiae. The resulting wines were inoculated with four O. oeni strains. The action of non- Saccharomyces affected the final wine composition and the later role of O. oeni. Some of its strains could not perform MLF in H. uvarum wine due to high SO 2 concentrations. In some cases, MLF was inhibited in wines inoculated with S. bacillaris. All the H. uvarum and H. vineae strains notably increased acetic acid concentrations, thus threatening wine quality. The best conditions for MLF were provided by some T. delbruecckii and M. pulcherrima strains , which showed increased concentrations of mannoproteins – compounds described as MLF activators –, no production of SO 2 , and low consumption of L-malic acid. In conclusion, non- Saccharomyces yeasts have diverse effects on O. oeni and MLF depending on the species, with T. delbrueckii and M. pulcherrima being those that showed the best compatibility with MLF development. [ABSTRACT FROM AUTHOR]

• Interactions among Starm. bacillaris and LAB were explored in wine mixed fermentations. • MLF was differently affected depending on the strains and yeast inoculation protocol. • Couple Starmerella /LAB stimulated, inhibited, or unaffected the outcome of the MLF in co-inoculum. • O. oeni always performed MLF successfully and minimally impacting on the wine aromatic profile. • Strain-depending interactions affected time of vinification and quality of the wine. This work aims to improve the management of the malolactic fermentation (MLF) in red wines by elucidating the interactions between Starmerella bacillaris and Saccharomyces cerevisiae in mixed fermentations and malolactic bacteria. Two Starm. bacillaris strains were individually used in mixed fermentations with a commercial S. cerevisiae. MLF was performed using two autochthonous Lactobacillus plantarum and one commercial Oenococcus oeni inoculated following a simultaneous (together with S. cerevisiae) or sequential (at the end of alcoholic fermentation) approach. The impact of yeast inoculation on the progress of MLF was investigated by monitoring the viable microbial populations and the evolution of the main oenological parameters, as well as the volatile organic composition of the wines obtained in mixed and pure micro-scale winemaking trials. Our results indicated that MLF was stimulated, inhibited, or unaffected in mixed fermentations depending on the strains and on the regime of inoculation. O. oeni was able to perform MLF under all experimental conditions, and it showed a minimal impact on the volatile organic compounds of the wine. L. plantarum was unable to perform MLF in sequential inoculation assays, and strain-depending interactions with Starm. bacillaris were indicated as factor affecting the outcome of MLF. Moreover, uncompleted MLF were related to a lower aromatic complexity of the wines. Our evidences indicate that tailored studies are needed to define the appropriate management of non- Saccharomyces and malolactic starter cultures in order to optimize some technological parameters (i.e. reduction of vinification time) and to improve qualitative features (i.e. primary and secondary metabolites production) of red wines. [ABSTRACT FROM AUTHOR]

The use of non- Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non- Saccharomyces , through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non- Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania , Aureobasidium , Meyerozyma , Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H 2 S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds. • Indigenous yeast from South Australian Shiraz must were identified by ITS-PCR. • Oenological attributes (aroma, enzymes, ethanol tolerance, etc.) were characterised. • Kazachstania , Wickerhamomyces and Torulaspora isolates were sequentially fermented. • Phenylethyl and isoamyl acetate/alcohol levels were elevated in Kazachstania spp. [ABSTRACT FROM AUTHOR]