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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 SO2 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.

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.

Fermentation processes have proceeded without microbiological control until starter cultures came up in 19th Century. However, in some processes, such as wine making a widespread use of starter cultures did not come up until the end of the 20th Century, when the cellar-friendly active dry wine yeast (ADWY) were available. However, this practice was challenged for the uniformity that brought a limited number of commercially available presentations and new tendencies in wine making have withdrawn the use of ADWY. The return to non-controlled wine fermentations may have considerable set backs especially in terms of economical losses, as these wines have much higher risks of presenting different levels of spoilage (presence of unwanted compounds that will be organoleptically detectable) that will not be acceptable for the consumer. The WILDWINE Project (EU contract 315065) proposed a system to overcome the criticism on the uniformity after use of ADWY by bringing “wild” microorganisms to the starter culture practice. The Project proposed mixed cultures as an alternative to single strain cultures that is the usual practice for starter cultures. This multi-strain and multi-species starter cultures aimed at reproducing the vineyard natural microbiota with the advantage of a selection of those strains and species that could add complexity to the final wines. The WILDWINE Project has focused the selection of this “wild” microorganism in 5 worldwide-recognised wine regions: Nemea and Crete (Greece), Piedmont (Italy), Bordeaux (France) and Priorat (Spain). The basic protocol was the same for all the regions: (i) to establish the natural biodiversity of the regions and determine the “microbial fingerprint” of the region, (ii) oenological screening of all the strains and species isolates to determine their quality for the wine making process and (iii) to use the microbial cocktails at pilot plants and in commercial cellars to determine the “microbial footprint” that the “wild” microorganisms leave on the final wines. In the Priorat region, a limited number of different strains from Saccharomyces cerevisiae were detected after sampling cellars and vineyards in two different harvests (2012 and 2013). These low number of S. cerevisiae strains is in sharp contrast with the high biodiversity observed in the same area 18 years before when a selection of strains of this species was preformed to provide a commercial “autochthonous” strain for the Priorat region. Furthermore, the main species found in the vineyards were the yeast-like fungus Aerobasidium pullulans and Hanseniaspora uvarum. A. pullulans disappeared when grapes were turned into must and then H. uvarum and Candida zemplinina were the main species. Minority species found in musts and later in wines were Metschnokowia pulcherrima and Torulaspora delbrueckii.

Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83 % of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.

Filiació URV: SIInclòs a la memòria: SI Although Oenococcus oeni is the main species that is responsible for malolactic fermentation (MLF), harsh wine conditions can limit its performance. Although several mechanisms underlying the response to stress have been studied in this species, little is known regarding the cellular systems that protect against oxidative stress in other bacteria, such as glutathione (GSH). O. oeni cannot synthesize GSH but contains several genes related to its utilization. In this study, the relative expression (RE) of the seven genes involved in the GSH redox system found in O. oeni PSU-1 (gshR, gpo, three glutaredoxin-like genes and two subunits of an hypothetical transporter) has been measured. The study was performed using three strains, with each exhibiting a different GSH uptake capacity. The strains were grown in a stress-adaptation medium supplemented with 5 mM GSH and under different adaptation stress conditions (pH 4 and 6% ethanol). The RE showed that only some of these genes, including one for a possible glutaredoxin (OEOE_RS04215) and cydC for a subunit of a putative GSH transporter (OEOE_RS1995), responded to the addition of GSH. The presence of ethanol had a relevant effect on gene expression. Among the studied genes, the one for a NrdH-redoxin (OEOE_RS00645) showed a common response to ethanol in the strains, being over-expressed when grown with GSH. In most cases, the transcriptional changes were more evident for the strain with a higher capacity of GSH uptake. Malolactic performance of the three strains after pre-adaptation was evaluated in wine-like media (12% ethanol and pH 3.4). It was observed that the addition of GSH during pre-adaptation growth had a protective role in the cells exposed to low pH and ethanol, resulting in a

Selected starter cultures of Oenococcus oeni are widely used to initiate malolactic fermentation (MLF) in wine. Nevertheless, the inoculated culture does not always develop as expected and undesired strains can grow causing wine spoilage. Therefore, methods that can reliably differentiate O. oeni strains are essential to monitor the population dynamics of MLF. This work presents a new multiplex PCR method that allows the simultaneous species identification and strain typification of O. oeni, based on the combined use of species-specific PCR primers and a Random Polymorphic DNA (RAPD)-PCR primer. This method represents an useful tool for the control of wine MLF.

Epiphytic bacteria on grape berries play a critical role in grape health and quality, which decisively influence the winemaking process. Despite their importance, the bacteria related with grape berry surface remain understudied and most previous work has been based on culture-dependent methods, which offer a limited view of the actual diversity. Herein, we used high-throughput sequencing to investigate the bacterial diversity on the surface from two grape varieties, Grenache and Carignan, and compared them across five vineyards included within the Priorat region (Spain). We could detect up to 14 bacterial phyla with Firmicutes (37.6% Bacillales and 14% Lactobacillales), Proteobacteria (16.8% Pseudomonadales and 11.6% Enterobacteriales) and Actinobacteria (3.4% Actinomycetales) being the most abundant. Bacterial community was different at each vineyard being grape varietal, geographical situation and orientation related with changes in bacterial populations. The most abundant bacterial taxa and those driving differences between the vineyards and grape varietals were identified. This study indicates that bacterial community heterogeneities can be influenced by geographic factors like orientation.

The goal of this study was to analyze how the profiles of membrane proteins of Oenococcus oeni change under particular stress conditions of wine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis protein profiles of membrane fraction showed that a 40-kDa protein was overexpressed in the presence of SO2. The sequence of its N-terminal fragment showed a significant identity with glyceraldehyde-3-phosphate dehydrogenases (GAPDHs), but the protein showed no GAPDH activity. This sequence was compared with those of other GAPDHs with ClustalW alignment, and it was found to be somewhat similar to that of the cell-wall and membrane proteins of other lactic acid bacteria.

A large number of strains of Oenococcus oeni (formerly Leuconostoc oenos) that had been isolated from wines were checked for lysogeny with mitomycin C as inducer. As a result of this test, 45% of the strains proved to be lysogenic, suggesting that lysogeny is widespread among bacteria isolated from wines during malolactic fermentation. The sensitivity of bacteria to phages was very different, depending on the strain. All the lysogenic strains were resistant to infection by the temperate phage they released. Some phages infected none of the strains. Phages of Oenoc. oeni had a classical morphology, an isometric head, and a long striated tail. With the broadest host strain as an indicator, phages were detected in wines after malolactic fermentation.

Four out of 187 strains, from enrichment cultures of dibenzothiophene (DBT), grew on DBT or thiophene 2-carboxylate as S sources. The four isolates, presumptively identified as Agrobacterium sp., Xanthomonas sp. and Corynebacterium spp., individually and together desulphurized DBT, producing 2-hydroxybiphenyl and sulphate.

The correct performance of wine malolactic fermentation (MLF) depends on the metabolic characteristics of the Oenococcus oeni strain/s responsible for this process. This study characterizes four O. oeni strains, which behave differently in terms of malolactic performance, the citric acid use related to acetic acid production, and stress adaptation. Metabolic evolution and its associated enzymatic activities were studied and the transcriptional response of the genes related to MLF, citrate metabolism and stress response was compared among strains. A higher initial expression of both the malolactic enzyme and the encoding gene mleA may be related to faster MLF. The initial transcriptional levels of citrate lyase (citE) proved indicative of early citrate consumption. Moreover, the strains that performed best in wine-like conditions presented a much higher relative expression of several stress responsive genes, particularly hsp18, clpP, ctsR and rmlB. Finally, an inter-strain comparison of the transcriptional levels of selected genes at different times during MLF proved a useful tool in characterizing strains based on their metabolic behaviour.

The growth and oxygen consumption of a variety of thermophilic, acidophilic bacteria in the presence of thiophene-2-carboxylate (T2C) and dibenzothiophene (DBT) have been determined. T2C was extremely toxic to the acidophiles in comparison with neutrophiles, but appeared to be degraded by a heterotrophicSulfolobus- like thermophile. DBT proved to be unstable at high temperatures, even in the absence of bacteria, and was not a substrate for the thermophiles.

Aims: The goal of this study was to develop a reproducible method for molecular typing strains of Oenococcus oeni, and also to apply it in the study of population dynamics of these strains during malolactic fermentation of wine. Methods and Results: A new method of multiplex randomly amplified polymorphic DNA (RAPD)-PCR has been developed, based on the combination of one random 10-mer and one specific 23-mer oligonucleotide in a single PCR. This method generates unique and discriminant DNA profiles for strains of O. oeni. The strains of this species were also clearly distinguished from other species of lactic acid bacteria. The method was applied to study the dynamics of O. oeni strains during malolactic fermentation, in three vintages in the same cellar. Conclusions: A fast and reliable method for typing strains of O. oeni has been designed and optimized. It improves the reproducibility and rapidity of conventional RAPD-PCR, and it has been validated monitoring the population dynamics during malolactic fermentation. Significance and Impact of the Study: This method will be a good tool to study the population dynamics of bacteria during malolactic fermentation and to evaluate the performance of new malolactic starter cultures and their dominance over the native microbiota.

This study shows that the growth of Oenococcus oeni CECT 4100 in a synthetic medium is affected by phenolic compounds in different ways, depending on their type and concentration. Generally they have no effects at low concentrations, but hydroxycinnamic acids are inhibitory at high concentrations. Malolactic fermentation was stimulated in the presence of catechin and quercetin, but increasingly delayed with increasing amounts of p-coumaric acid. Gallic acid appeared to delay or inhibit the formation of acetic acid from citric acid. This could lead to a better control of malolactic fermentation and suppress the increase in volatile acidity, which is undesirable in the wine-making process.

Phenol-degrading strain QT 31 ofPseudomonas sp. was selected among other phenol-resistant bacteria from activated sludges of wastewater treatment plant of an oil refinery. Its capacity of degradation was studied at different periods of adaptation, reaching a phenol biodegradation rate of 28 mg/l phenol per hour, from minimal, medium with 1000 mg/l phenol, after adaptation for 20 days.

A Technicon AutoAnalyzer has been adapted to analyse lysine of fermentation broths from lysine-producer mutants, with a photometric method with ninhydrin. Linearity and accuracy have been tested, with errors lower than 5.5%. The great advantadge is its speed, analysing one sample every 5 minutes, automatically.

Genetic diversity of 60 Oenococcus oeni strains from different wines was evaluated by numerical analysis of (i) pulsed-field gel electrophoresis (PFGE) patterns with endonuclease ApaI and (ii) randomly amplified polymorphic DNA (RAPD)-PCR fingerprints with four oligonucleotide primers. Sixty-two percent of the strains could be distinguished by PFGE, whereas most strains were identified by distinct RAPD-PCR profiles and associated according to the geographical origin. Because of its rapidity and reliability, RAPD-PCR appeared to be a suitable method for typing and monitoring O. oeni strains in winemaking.