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Aims To determine the effect of three different freezing temperatures on post-freeze-drying survival rates of wine yeasts and lactic acid bacteria (LAB). To know if a similar freeze-drying protocol can be used for both micro-organisms. Methods and Results Cells from liquid culture media were recovered and concentrated in appropriate lyoprotectants. Aliquots of each strain were frozen at −20, −80 and −196°C before vacuum drying. Viable cell counts were done before freezing and after freeze-drying. Survival rates were calculated. Freezing temperatures differently affected yeast and bacteria survival. The highest survival rates were obtained at −20 and −80°C for yeasts, but at −196°C for LAB. Major differences in survival rates were recorded among freeze-dried yeasts, but were less drastic for LAB. Yeasts Pichia membranifaciens, Starmerella bacillaris and Metschnikowia pulcherrima, and LAB Lactobacillus paracasei, Pediococcus parvulus and Lactobacillus mali, were the most tolerant species to freeze-drying, regardless of freezing temperature. Conclusions Yeast and LAB survival rates differed for each tested freezing temperature. For yeasts, −20°C ensured the highest post-freeze-drying viability and −196°C for LAB. Significance and Impact of the Study Freezing temperature to freeze-dry cells is a crucial factor for ensuring good wine yeast and LAB survival. These results are important for appropriately preserving micro-organisms and for improving starter production processes.

Aim A survey on indigenous malolactic bacteria populations isolated from wines produced in 13 different wineries of Aosta Valley, the highest vine-growing area in Europe, was carried out in order to characterize the dominant strains in spontaneous malolactic fermentation (MLF) and to reveal the appearance of psychrotrophic ones. Methods and Results Fifty-four isolates were identify by ITS rDNA region analysis and partial sequencing of 16S rDNA gene: 80% were ascribed to Oenococcus oeni while the remaining 20% were attributed to Pediococcus damnosus species. The genetic diversity of 43 O. oeni isolates was investigated through PFGE analysis after ApaI and SfiI restriction: 28 different pulso-types were discriminated at a level of similarity of 90%. In general, the MLF was led by more than one strain simultaneously. No connection between genotype and grape variety or vine-training system or geographical site of isolation was observed. The histidine decarboxylase gene was not found in any isolate. Pediococci proved to be more resistant than oenococci to lysozyme. Three O. oeni strains (2A1, 6A2 and 11A4) were able to develop at 10°C in Petit Rouge wine. Conclusions Psychrotrophy is a phenotypic trait present in O. oeni species and it may be possible to select strains for the management of MLF in cold climate territories where this biologic transformation is very difficult to control. Significance and Impact of the Study The natural emergence of strains able to perform the MLF at 10°C in wine is a new finding, interesting because it confirms the ecological ability of O. oeni species to adapt itself to environmental conditions by strain phenotype variations. It can be also a starting point for more sustainable oenological practices, since it would be alternative to the conditioning systems of the tanks or of the wineries where they are costly in terms of investment and energy consumption.

International audience; Aims To characterize Oenococcus oeni strains isolated from North-Apulian wines where malic acid degradation is usually achieved by spontaneous fermentations, and to determine the influence of bacterial inoculation time on the malolactic performances in ‘Nero di Troia’ wine using a complete autochthonous microbial regime. Methods and Results Oenococcus oeni strains from wines produced with the autochthonous (Apulia Region, southern Italy) grape variety ‘Uva di Troia’ were isolated, selected and characterized. Multilocus sequence typing and variable number tandem repeat analysis were used to investigate intraspecific diversity. Oenococcus oeni strains were tested in co-inoculation and in sequential inoculation, with two autochthonous yeast strains previously isolated from ‘Nero di Troia’ wine. After a preliminary screening using co-inoculation regime, the O. oeni strains were grouped in reason of the different behaviour in malic acid performances. Results suggested that the efficient degradation of malic acid in co-inoculation is a strain-dependent characteristic. Conclusions Autochthonous yeast/bacterium combinations were identified as starter culture, and used in a co-inoculation approach, for vinification of regional wines. Significance and Impact of the Study The ‘microbial terroir’ of typical fermented food and beverage production represents a dynamic sector of applied research in food microbiology. In this work, we propose the use of autochthonous bacteria and yeast for wine production from an indigenous grape variety.

[Aim]: This study was aimed to investigate the influence of malolactic fermentation (MLF) on sensory profile and organoleptic characteristics of Albariño and Caiño white wines. [Methods and Results]: Autochthonous bacteria were isolated from wines after alcoholic fermentation (AF) and further identified as Pediococcus damnosus by 16S rRNA gene sequence analysis. When a commercial Oenococcus oeni starter was inoculated into Albariño and Caiño white wines to perform MLF, which was checked by HPLC quantification of malic and lactic acids, it was shown that autochthonous Ped. damnosus strains were able to predominate over the commercial O. oeni starter and perform MLF in Caiño wine. By contrast, neither commercial strain nor indigenous Pediococcus carried out MLF in Albariño wine. However, MLF was achieved when autochthonous strains that predominated in Caiño were inoculated into Albariño. Sensory analysis showed that after the MLF Albariño increased its body and softness, while Caiño result a more mature wine. [Conclusions]: MLF can positively affect Albariño and Caiño wines giving them new attributes. Pediococci isolated and characterized in this work can successfully perform MLF without negative effects on the wine, because no production of biogenic amines or exopolysaccharides by the selected pediococcus strains was detected. [Significance and Impact of the Study]: The effect of MLF in the sensory profile of Albariño and Caiño wines has never been studied before. Results obtained in this work showed that Ped. damnosus strains can be considered as a new topic of investigation on malolactic starter. © 2013 The Society for Applied Microbiology., Funded by: Bodega Terras Gauda LTD; Xunta de Galicia. Grant Numbers: PGIDIT04TAL035E, 2004-7-OE-242, AGL2006-02558, A36108900, ALIBIRD-CM-S-0505/AGR-0153; CONSOLIDER INGENIO 2010. Grant Number: CSD2007-00063FUN-C-FOOD and BIODATI. Grant Number: DM 16101/7301/08.

Aims To assess the abilities of commercial wine lactic acid bacteria (LAB) to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production. Methods and Results Oenococcus oeni AWRI B551 produced significant levels of ethyl hexanoate and ethyl octanoate following growth in an ethanolic test medium, and ester formation generally increased with increasing pH (4·5 > 3·5), anaerobiosis and precursor supplementation. Cell-free extracts of commercial O. oeni strains and Lactobacillus plantarum AWRI B740 were also tested for ester-synthesizing capabilities in a phosphate buffer via: (i) acyl coenzyme A: alcohol acyltransferase (AcoAAAT) activity and (ii) reverse esterase activity. For both ester-synthesizing activities, strain-dependent variation was observed, with AcoAAAT activity generally greater than reverse esterase. Reverse esterase in O. oeni AWRI B551 also esterified 1-propanol to produce propyl octanoate, and deuterated substrates ([2H6]ethanol and [2H15]octanoic acid) to produce the fully deuterated ester, [2H5]ethyl [2H15]octanoate. Conclusions Wine LAB exhibit ethyl ester-synthesizing capability and possess two different ester-synthesizing activities, one of which is associated with an acyl coenzyme A: alcohol acyltransferase. Significance and Impact of the Study This study demonstrates that wine LAB exhibit enzyme activities that can augment the ethyl ester content of wine. This knowledge will facilitate greater control over the impacts of malolactic fermentation on the fruity sensory properties and quality of wine.

Aims: The objective of this study was to investigate the presence of genes coding for enzymes of oenological relevance in wine Lactobacillus strains isolated from South African grape and wine samples during the 2001 and 2002 harvest seasons. Methods and Results: A total of 120 wine lactobacilli isolates belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus sakei and Lactobacillus paraplantarum were genetically screened for enzyme-encoding genes using PCR with primers specific for β-glucosidase, protease, esterase, citrate lyase and phenolic acid decarboxylase. The results of PCR screening showed that the Lactobacillus strains possessed different combinations of enzymes and that some strains did not possess any of the enzymes tested. Confirmation analysis with gene sequencing also showed high similarity of genes with those available in GenBank database. Conclusion: In this study, we have demonstrated the existence of genes coding for wine-related enzymes in wine lactobacilli that could potentially hydrolyse wine precursors to positively influence wine aroma. Significance and Impact of the Study: An expansion of knowledge on the genetic diversity of wine-associated lactic acid bacteria will enable the selection of novel malolactic fermentation starter cultures with desired oenological traits for the improvement of the organoleptic quality of the wine, and hence wine aroma.

AIMS To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular beta-glucosidase from this strain. METHODS AND RESULTS Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for beta-glycosidase activities. A strain of Lact. brevis showing high intracellular beta-D-glucosidase, beta-D-xylosidase and alpha-L-arabinosidase activities was selected for purification and characterization of its beta-glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3.5. The K(m) for p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside is 0.22 and 1.14 mmol l(-1), respectively. The beta-glucosidase activity was strongly inhibited by gluconic acid delta-lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7.0 (t(1/2) = 50 day) but not at pH 4.0 (t(1/2) = 4 days). CONCLUSIONS The beta-glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei (Coulon et al. 1998) and Lactobacillus plantarum (Sestelo et al. 2004). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long-term stability could be of great interest for the release of aroma compounds during winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular beta-glucosidase isolated from a wine-related strain of Lact. brevis.

Aims: To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters. Methods and Results: One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3·5, 3·2 and 3·0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery. Conclusions: The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment. Significance and Impact of the Study: This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.

Aims: To study the effect of ethanol on Oenococcus oeni activity at the single cell level. Methods and Results: The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l-malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells. Conclusion: From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni. Significance and Impact of the Study: The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production

Aims: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. Methods and Results: Two yeast expression ⁄secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase ⁄xylanase gene cassette (pEXS) containing the endo-b-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-b-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d’Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase ⁄xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase ⁄xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase ⁄xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d’Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. Conclusions: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d’Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. Significance and Impact of the Study: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharidedegrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price : quality ratio of wine according to consumer expectations.

International audience; AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.

Aims: This study examined the effects of yeast strains in a novel winemaking process that had been designed to optimize phenolic extraction and improve production efficiency for Pinot noir winemaking. Methods and Results: Microwave maceration with early pressing and co-inoculation of yeast and malolactic bacteria for simultaneous alcoholic and malolactic fermentation was investigated. Yeast treatments ( Saccharomyces cerevisiae RC212 and EC1118, and Saccharomyces bayanus AWRI1176) were co-inoculated with Oenococcus oeni PN4 immediately after must microwave maceration. Alcoholic and malolactic fermentation were complete 17 days postinoculation for all three yeast treatments. At 16-month bottle age, the AWRI1176-treated wines had approximately twice the nonbleachable pigment and colour density of wines fermented by EC1118 and RC212. Conclusions: The novel winemaking process produced Pinot noir wine that was stable 37 days after fruit had been harvested and yeast strain choice significantly impacted the stability and phenolic character of wine. Significance and Impact of Study: Successful simultaneous alcoholic and malolactic fermentation in 17 days, and a demonstrated lack of inhibition between the yeast strains and malolactic strain applied in this study, provide proof of concept for very rapid red winemaking using the novel winemaking approach described herein. Further investigation would be required to assess strain effects on wine aroma, mouth feel and taste, however, this novel winemaking approach may offer significant industry efficiencies.

Aims: The purpose of this work was to study the effect of L-aspartic acid concentration on bacterial growth, D-glucose fermentation and L-malic acid consumption of Oenococcus œni NCFB 1707. Methods and Results: Bacterial cultures were performed in synthetic media. Bacterial growth, D-glucose fermentation and L-malic acid consumption were reduced when L-aspartic acid concentration became excessive. This inhibitory effect of high concentrations of L-aspartic acid on bacterial growth was also observed with several Oenococcus œni strains, except O. œni BL01. The L-aspartic acid inhibitory effect on bacterial growth could be reduced by increasing the concentration of L-glutamic acid. L-glutamic acid transport was found to be competitively inhibited by L-aspartic acid. In addition, an excessive amount of L-aspartic acid modified D-glucose metabolism, with an overproduction of acetic acid and reduced ethanol production. Conclusions: Since L-glutamic acid is an essential amio acid for the bacterial strain used, the L-aspartic acid inhibitory effect on bacterial growth could be linked to its involvement in an antagonistic interaction with L-glutamic acid. Significance and Impact of the Study: Such antagonistic interactions between amino acids in O. œni strains could be another explanation for the difficulties of inducing malolactic fermentation in wines.

Oenococcus oeni is the main lactic acid bacteria species which induces malolactic fermentation during wine-making. It is able to break down arginine via the arginine deiminase pathway, a potential source of energy already considered for many bacteria. The production of ATP by starved cells from arginine was quantified with a bioluminescence assay, and efficient coupling of amino acid catabolism and cell growth was monitored. Therefore, molecular growth yield was determined after glucose exhaustion. With colony plate counting and a direct epifluorescence technique, it was shown that addition of arginine to viable but non-culturable cells obtained after nutrient starvation restored their ability to grow during its degradation. Therefore, arginine produced more than maintenance energy. It is concluded that strains which are able to metabolize arginine might take advantage of this additional energy source for growth.

The excretion of citrulline, a precursor of carcinogenic ethyl carbamate, formed from arginine degradation by malolactic bacteria in wine is of toxicological concern. The arginine metabolism of resting cells of Lactobacillus buchneri CUC-3 and Oenococcus oeni Loll 1 was examined. The citrulline excretion rate was found to be linearly correlated to the arginine degradation rate. It was possible to calculate an arginine to citrulline conversion ratio which could be used to predict the amount of citrulline expected after the degradation of a known quantity of arginine. The conversion ratios determined in this study were similar to data calculated from other authors for fermentations in wine and ranged between 4.0% and 7.7%. Ribose, fructose and glucose inhibited the degradation of arginine in Lact. buchneri CUC-3, and inhibition of arginine degradation by glucose correlated with higher arginine to citrulline conversion ratios. The work presents new results of arginine metabolism in malolactic bacteria and gives starting points for investigations in wine.

Pilot scale fermentations with grape pomace from two different wineries were investigated during the 24 weeks of the ensiling period, along with laboratory scale experiments in which the environmental temperatures were held constant at 20, 25, 30 and 35 °C. During this period, yeast and lactic acid bacteria (LAB) counts were made, after which the identity of both groups of organisms was studied, as were the major microbial metabolites present. Major microbial and chemical alterations occurred during the first 3 weeks of ensilage, leaving a more stable product differing significantly from the initial substrate. The results obtained indicated that after initial growth, yeast and LAB populations undergo progressive inactivation at environmental temperatures above 20 °C, although LAB seem to adjust better to this specific, post-fermentation environment. Homofermentative species of Lactobacillus were the dominant LAB. The initial yeast flora of non- Saccharomyces species was replaced by a typical wine yeast flora, i.e. predominantly Saccharomyces cerevisiae. At the chemical level, major alterations were due to an alcoholic fermentation and a malolactic conversion within the first 3 weeks.

This article begins with an introduction to malolactic fermentation in wine, followed by a review of the occurrence of arginine degradation in wine lactic acid bacteria and the pathway of arginine catabolism, the distribution of enzymes responsible, and the formation of products. The bioenergetics of wine lactic acid bacteria and arginine degradation are then reviewed. This is followed by a review of the possible mechanisms of arginine transport, and regulation of arginine metabolism and synthesis of the enzymes for arginine catabolism. Finally, the practical significance of arginine metabolism in wine lactic acid bacteria is reviewed with respect to taxonomic utility, biological significance and oenological implications.

Aims The aim of this study was to investigate the effect of ethanol exposure on the expression level of geranylgeranyl pyrophosphate synthase gene involved in the metabolism of Oenococcus oeni to probe the mechanisms of ethanol tolerance correlated with adaptive changes. Methods and Results The evaluation of ten potential internal control genes and the comparative study of their stability were performed to select the most stable internal controls for the normalization of expression data. The expression level analysis by qPCR and changes after exposure to ethanol stresses highlighted a significant increase in the presence of higher ethanol concentrations. Conclusions The analysis of results suggest that O. oeni adjusts the expression of genes to adapt to stress conditions and the high expression level of ggpps would allow a flow of isoprenoid precursors towards the carotenoids and related pathways to stabilize bacterial cell membranes, improving the cell membrane disturbances and preventing cell death induced by ethanol. Significance and Impact of the Study The involvement of ggpps gene in physiological changes of bacterial behaviour confirmed the exposure to stress requires the activation of defence mechanism to be more tolerant to adverse conditions. Improving the knowledge of stress tolerance and adaptation mechanisms of O. oeni is essential to enhance the efficiency of the malolactic starter in wine and to obtain the development of starters able to survive to direct inoculation with a large benefit for wine technology.

Malolactic fermentation, a spontaneous step in vinification, is frequently controlled by use of malolactic bacterial starters of Leuconostoc œnos. Strains isolated from fermenting wines and then submitted to industrial processes to obtain freeze-dried preparations lose their natural adaptation to survive and to grow in wine. However, culture conditions influence their adaptability as well as their survival after exposure to stress. As the plasma membrane was considered as the primary target for the deleterious effects of the environment, the changes of its components after cell heating and incubation in hostile conditions were studied. The ratio of phospholipids to proteins of the membrane decreased significantly when cells were submitted to any kind of stress. Phospholipids decreased while the protein content increased up to fivefold. Only some membrane proteins were over-synthesized compared to control cells. Seventeen protein bands were separated by gel electrophoresis of which three in particular were always over-expressed after the cells were shocked. Some also appeared when the cells entered the stationary phase. These results show that Leuc. œnos responded to environmental stresses by changing its membrane composition. Stress proteins appeared whatever the nature of the shock. This phenomenon may be involved in the acquisition of resistance of Leuc. œnos to harsh conditions in wine.

Aim To clone and characterize two related intracellular esterases from Oenococcus oeni and Lactobacillus hilgardii under wine-like conditions. Methods and Results The published genome sequences for O. oeni and Lact. hilgardii were used to identify, clone and purify putative esterase genes from these species designated EstCOo8 and EstC34, respectively. Both esterases are members of family V of lipolytic enzymes. However, EstC34 contains an SGSLG nucleophilic elbow structural motif instead of the usual GGSLG motif which is conserved in other lactic acid bacteria. Both esterases exhibited greatest specificity for C2–C4 pNP-linked substrates and retained activity under wine-like conditions. EstCOo8 had an optimum temperature, pH, and ethanol concentration of 40°C, 5·5 and 6% (v/v), respectively. Whereas EstC34 had an optimum temperature, pH and ethanol concentration of 50°C, 5·0 and 10% (v/v), respectively. Conclusions Both esterases were stable and retained activity under conditions that would be encountered in wine. They have the potential to reduce short-chain ethyl esters such as ethyl acetate. Significance and Impact of the Study This study provides information that might help improve the performance of LAB during malolactic fermentation in wine in the future, either by strain selection, optimization or direct enzyme addition.

Aims To investigate the Oenococcus oeni population occurring during spontaneous malolactic fermentation (MLF) of Amarone wine, a peculiar and hostile environment for malolactic bacteria. Methods and Results Pulsed-field gel electrophoresis (PFGE) analysis showed a high level of genetic heterogeneity within the O. oeni population involved in MLF throughout an industrial vinification of Amarone wine. The 13 strains with distinct PFGE profile displayed different capability to hydrolyse esters and glycosides, as well as great variability to growth under stress parameters, such as high ethanol content (15% v/v), low pH (3·0) and temperature (15°C), and presence of SO2. Moreover, polymorphism in the gene sacB involved in exopolysaccharide production was observed among the strains. The strains showed differences to convert l-malic acid into l-lactic acid in wine. Conclusions The occurrence of spontaneous MLF in stressful ecosystems such as Amarone wine is related to the heterogeneity of O. oeni community; biodiversity indexes and strain evolution analyses suggested that its success depends on its initial strain evenness. Significance and Impact of the Study Remarkable intraspecies complexity within the O. oeni natural population could explain the great versatility of this species as key of successful adaptation to harsh winemaking conditions.

Aims: The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. Methods and Results: Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l-lactic acid, and sulphur dioxide (SO2) had no effect on enzyme synthesis and activity. Bacterial species differ in the relative enzymatic activity but all the factors affected similarly to L. hilgardii, P. parvulus and O. oeni. Conclusions: The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. The conditions that normally occur during malolactic fermentation and later on, could favour histamine production. SO2 could prevent bacterial growth, but does not diminish the HDC enzyme activity. Significance and Impact of the Study: Information on hdc expression and HDC activity can contribute to the prevention of histamine formation during wine production and storage.

Aims: Brettanomyces ⁄Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine. Methods and Results: Sterile red wines were inoculated with 5 · 10 3 viable cells ml )1 of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10 6 –10 7 colony forming units (CFU) ml )1 and volatile phenol concentrations ranged from 500 to 4000 l gl )1 . Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C2 to C10), short chain acid ethyl-esters and the ‘mousy off-flavour’ component 2-acetyl-tetrahydropyridine. Conclusions: Different kinds of ‘Brett character’ characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs. Significance and Impact of the Study: We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.

Aims: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO2. The current work investigated the degradation of acetaldehyde and SO2-bound acetaldehyde by two commercial Oenococcus oeni starters in white wine. Methods and Results: Wines were produced by alcoholic fermentation with commercial yeast and adjusted to pH 3.3 and 3.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both pH values, SO2-bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. Conclusions: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO2-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO2 should be considered next to free SO2 in order to evaluate malolactic fermentability. Significance and Impact of the Study: The current study provides new results regarding the metabolism of acetaldehyde and SO2-bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO2.

Aims: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). Methods and Results: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. Conclusions: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (

Aims: The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. Methods and Results: Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5·5 and 3·5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. Conclusions: The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. Significance and Impact of the Study: Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.

Aims: To assess glycosidase activities from a range of Lactobacillus and Pediococcus species and characterize these activities under conditions pertinent to the wine industry. Methods and Results: Lactic acid bacteria were cultured in MRS broth supplemented with apple juice before being harvested, washed and assayed for glycosidase activity using p-nitrophenol-linked substrates. All strains exhibited a detectable capacity for the hydrolysis of the β- and α-d-glucopyranosides. The magnitude of these activities and their response to the physico-chemical parameters investigated varied in a strain-dependent manner. The use of an assay buffer with a pH below 4 generally resulted in a reduced hydrolysis of both substrates while temperature optima ranged between 35 and 45°C. The effect of the inclusion of ethanol in the assay buffer (up to 12%, v/v) ranged from near complete inhibition to increases in activity approaching 80%. With the clear exception of a single strain, glucose and fructose (0·1–20 g l−1) acted as inhibitors. An assessment of glycosidase activity during simultaneous exposure to glucose and ethanol at a pH of 3·5 suggested that ethanol decreased loss of activity under these wine-like conditions. Conclusions: Lactobacillus spp. and Pediococcus spp. possess varying degrees of β- and α-d-glucopyranosidase activities, which in turn are influenced differently by exposure to ethanol and/or sugars, temperature and pH. Several strains appeared suited for further evaluation under winemaking conditions. Significance and Impact of the Study: This work highlights the fact that strains of Lactobacillus and Pediococcus have the potential to influence the glycoside composition of wine. Tailoring of wine may therefore be possible through selective application of strains or enzymatic extracts thereof.

Aims The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. Methods and results Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. Conclusions The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. Significance and impact of the study The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.

F. COMITINI, R. FERRETTI, F. CLEMENTI, I. MANNAZZU AND M. CIANI. 2005. Aims: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions.Methods and Results: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast–malolactic bacteria (MLB) coupling. Heatand protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that theinhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts eitherbacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grapejuice and wine.Conclusions: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth andmalic acid fermentation was characterized.Significance and Impact of the Study: The individuation, characterization and exploitation of yeastproteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management ofmalolactic fermentation.Keywords: antibacterial compounds, malolactic fermentation, Oenococcus oeni, Saccharomyces cerevisiae, yeast–bacteria interactions.

Aims: Little genetic information exists on the ability of wine lactic acid bacteria (LAB) to hydrolyse glycoconjugates during malolactic fermentation. We tried to fill this important gap by characterizing a gene codifying for a putative β-glucosidase enzyme from wine Lactobacillus plantarum and from a commercial strain of Oenococcus oeni. Methods and Results: The coding region of the putative β-glucosidase gene is 1400 nucleotides long and started with an ATG codon. The gene is widespread among LAB and the highest identity was observed between the nucleotide of L. plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum and O. oeniβ-glucosidase gene. The protein sequence deduced from the isolated genes has a calculated molecular mass of 61·19 kDa. Furthermore, the expression of the β-glucosidase gene in L. plantarum strain was analysed, under several stress, by reverse transcriptase (RT)-PCR and Northern-blot analysis. The gene was apparently regulated by abiotic stresses such as temperature, ethanol and pH. Conclusions: The β-glucosidase gene is widespread among LAB and its expression is probably regulated by a wide range of abiotic stresses. Significance and Impact of the Study: The inhibitory effect of temperature and ethanol on the L. plantarumβ-glucosidase gene may be useful to explain the differences found in β-glucosidase activity reported in wines by several authors.

Aims: The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway. Methods and Results: Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline. Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway. Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia. Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis. The strains were further classified as belonging to L. plantarum species. Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3·6 and 4·5). Conclusions: The results suggest that citrulline production in wine, could be performed by homofermentative LAB. Significance and Impact of the Study: Homofermentative malolactic bacteria (L. plantarum) may degrade arginine through the ADI pathway.

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.