Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines

Aims: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. Methods and Results: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain‐specific primers. Strains were further grouped using a multiplex RAPD‐PCR analysis. Then, six strains were inoculated in two wine‐like media with two different ethanol concentrations (11 and 13% vol/vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT‐PCR) approach was adapted to monitor the physiological state of the strains selected. Conclusion: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. Significance and Impact of the Study: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.     

A multiplex PCR method for simultaneous species identification and strain typification of Oenococcus oeni

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.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

Effect of nitrogen limitation and nature of the feed upon Oenococcus oeni metabolism and extracellular protein production

AIMS: The aim of the study was to characterize the effect of various nitrogen sources on Oenococcus oeni growth, carbon source utilization, extracellular protease activity and extracellular proteins. More generally, the goal is to understand how nitrogen-based additives might act to enhance malolactic fermentation in wine. METHODS AND RESULTS: Five yeast extracts were used. As the amino acid and nitrogen analyses revealed, they were similar in global amino acid composition, except for arginine level. Nevertheless the ratio of amino acids between free/bound, and low/high molecular weight fractions were highly different. One of the yeast extracts led to a significant protease activity in the supernatant and to a poor final biomass of the IOB84.13 strain compared to the other ones. For the IOB84.13 strain specifically, arginine addition to the arginine poor yeast extract did not restore growth. 35S-methionine-labelled extracellular proteins were separated by SDS-PAGE. Signals were detected in all media early in the growth phase and were maintained during 48 h of culture. CONCLUSIONS: A significant protease activity was detected for O. oeni supernatants during growth under nitrogen limitation but only for certain nitrogen sources. Moreover, the activity was strain dependent. Peptides (0.5-10 kDa) seemed to be more favourable for growth of wine bacteria than <0.5 kDa nitrogen sources. The extracellular protein signal patterns differed more greatly between the bacterial strains tested than between the nitrogen molecules in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study extensively considering the role of the nitrogen source composition and level upon O. oeni growth and metabolism.     

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non‐ropy wines

Aims: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine . Methods and Results: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with β‐glucan formation and ropiness were grown on grape juice medium, dialysed MRS‐derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol‐sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf?). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. Conclusion: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than β‐glucan. Significance and Impact of the Study: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.     

Malolactic fermentation in wine with high densities of non-proliferating Oenococcus oeni

Five strains of Oenococcus oeni (syn. Leuconostoc oenos) under non-proliferating conditions were assessed for the performance of the malolactic fermentation in wine at various initial pH values, malic acid concentration and densities of cells. We succeeded in inducing the malolactic fermentation after inoculation of high densities of O. oeni G6 even in recalcitrant wines where the traditional malolactic fermentation was inhibited by adverse environmental conditions (low pH and high concentration of malic acid). Optimal degrading conditions in wine, under different physico-chemical environments, were determined in order to achieve rapid depletion of malic acid in red wine. Off-odour compounds were not formed under these conditions, suggesting an attractive alternative for wine production. This revised version was published online in November 2006 with corrections to the Cover Date.     

Isolation and characterization of Oenococcus oeni from Aglianico wines

A technological characterization of Oenococcus oeni strains isolated from Aglianico wines was performed to select starter cultures for malolactic fermentation (MLF). One hundred and fifty six O. oeni isolates were extracted from Aglianico wines, and identified by using species-specific PCR. Malolactic activity (MLA), sulphur dioxide (SO₂) resistance, acetaldehyde metabolism and other technological characteristics were tested. Differences in the technologically relevant characteristics were observed. All O. oeni strains were able to grow at low temperature and none in presence of 14% of ethanol. About 80% of O. oeni degraded more than 80% of acetaldehyde, producing ethanol and acetic acid as final products. Among nine O. oeni chosen, four isolates were sensitive to 60 mg of SO₂ l⁻¹, while the other five had high resistance. Considering their technological characteristics, five O. oeni strains could be selected starter cultures for MLF in Aglianico.     

In Vivo PCR-DGGE Analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.     

Comparative metabolic profiling to investigate the contribution of <Emphasis Type="Italic">O. oeni</Emphasis> MLF starter cultures to red wine composition

In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2-methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine.     

Correlation between indigenous <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strain resistance and the presence of genetic markers

This study reports on monitoring Oenococcus oeni intraspecific diversity evolution during winemaking. Three different wines were monitored. The proportion of O. oeni species was determined by species-specific PCR and O. oeni strains were distinguished by multiplex PCR-RAPD. Each strain was tested by PCR for 16 significant markers revealed by a previous genetic comparison between a strong oenological potential strain and one with poor oenological potential. Population levels and diversity changed according to winemaking stages, oenological practices and the chemical properties of the wine. In all situations, O. oeni was the best-adapted species. Within the O. oeni group, intraspecific strain diversity decreased and the malolactic fermentation was the result of the most resistant strains with the highest number of markers.     

Dynamics of Indigenous Lactic Acid Bacteria Populations in Wine Fermentations from La Rioja (Spain) During Three Vintages

Diversity of lactic acid bacteria (LAB) species has been analyzed for three consecutive years (2006, 2007, and 2008) during alcoholic and malolactic fermentations of Tempranillo wine in a winery at La Rioja. The results showed differences in malolactic fermentation duration, and in both diversity of LAB species and diversity of Oenococcus oeni genotypes. O. oeni was shown to be the predominant species (73% of total isolates). Monitoring the different strains of O. oeni using pulsed-field gel electrophoresis of chromosomal DNA digested with SfiI and ApaI allowed detection of a total of 37 distinct genotypes, most of them comprised at least two isolates. Six appeared in more than one vintage, one of them being present in the three studied years. Moreover, four genotypes were indistinct of the strains isolated from the air of this same winery in 2007 vintage. The frequency of participation of each genotype varied from year to year, thus dominant genotypes at one year were minority or not present at another year. This suggests that distinct indigenous O. oeni strains are better adapted to the different winery conditions every year. Predominant genotypes that appeared in more than one vintage and lead to quality wines with low histamine contents could be considered as interesting for selecting of new malolactic starter cultures.     

乙醇胁迫处理对酒酒球菌SD-2a生理特性的影响

为制备高效的葡萄酒乳酸菌发酵剂,以酒酒球菌SD-2a为试验材料,研究了5%、10%不同浓度的乙醇胁迫处理对菌体生长、细胞内MLE活性、冻干存活率及细胞膜脂肪酸组分的影响。试验结果表明乙醇胁迫处理明显降低了菌体的生长速率和生物产量。与对照相比,5%乙醇胁迫处理降低了菌体的冻干存活率,而10%乙醇处理却显著增加了菌体的冻干存活率。细胞膜脂肪酸组分的测定结果表明,前者细胞膜U/S比值为1.12,比对照降低了26.3%,而后者细胞膜U/S比值为2.29,比对照增加了50.6%。故认为酒酒球菌SD-2a为适应不同浓度的乙醇胁迫环境,在细胞膜脂肪酸水平上所采用的机制不同,且菌体的冻干存活率与其细胞膜脂肪酸组分密切相关。     

Identification of pOENI-1 and Related Plasmids in Oenococcus oeni Strains Performing the Malolactic Fermentation in Wine

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

Biochemical characterisation of the esterase activities of wine lactic acid bacteria

Esters are an important group of volatile compounds that can contribute to wine flavour. Wine lactic acid bacteria (LAB) have been shown to produce esterases capable of hydrolysing ester substrates. This study aims to characterise the esterase activities of nine LAB strains under important wine conditions, namely, acidic conditions, low temperature (to 10°C) and in the presence of ethanol (2–18% v/v). Esterase substrate specificity was also examined using seven different ester substrates. The bacteria were generally found to have a broad pH activity range, with the majority of strains showing maximum activity close to pH 6.0. Exceptions included an Oenococcus oeni strain that retained most activity even down to a pH of 4.0. Most strains exhibited highest activity across the range 30–40°C. Increasing ethanol concentration stimulated activity in some of the strains. In particular, O. oeni showed an increase in activity up to a maximum ethanol concentration of around 16%. Generally, strains were found to have greater activity towards short-chained esters (C2–C8) compared to long-chained esters (C10–C18). Even though the optimal physicochemical conditions for enzyme activity differed from those found in wine, these findings are of potential importance to oenology because significant activities remained under wine-like conditions.     

Growth Response and Modifications of Organic Nitrogen Compounds in Pure and Mixed Cultures of Lactic Acid Bacteria from Wine

The interactions between the proteolytic X₂L strain of Oenococcus oeni and the non-proteolytic 12p strain of Pediococcus pentosaceus were assayed. The characteristics of cell growth, protein degradation, and amino acid production of both strains were determined in pure and mixed cultures. O. oeni showed poor cell growth and greater ability in the release of amino acids to the extracellular medium, whereas P. pentosaceus showed a higher yield in cell production with a decrease in the amino acid concentration in the medium. P. pentosaceus especially consumed essential amino acids for growth, and O. oeni released several of the essential amino acids important for growth of P. pentosaceus. In the mixed culture, mutualism was observed. The higher activity of the proteolytic system of O. oeni in mixed culture produced an increase in cell growth and in the amount of essential amino acids released. These findings provide new knowledge about the metabolic interactions between lactic acid bacteria isolated from wine when proteins are degraded in mixed bacterial populations.     

Cloning and characterisation of a cystathionine β/γ-lyase from two <Emphasis Type="Italic">Oenococcus oeni</Emphasis> oenological strains

Sulphur-containing compounds in wine have been extensively studied because of their effect on wine flavour and quality. In this study, an enzyme that degrades sulphur-containing amino acids was cloned and characterised from two Oenococcus oeni strains of oenological origins. The enzyme has features of a cystathionine-γ-lyase (EC 4.4.1.1), a pyridoxal-5-phosphate-dependent enzyme catalysing an α,γ-elimination reaction of l-cystathionine to produce l-cysteine, α-ketobutyrate and ammonia. Moreover, it was able to catalyse an α,β-elimination reaction producing homocysteine, pyruvate and ammonia from l-cystathionine. An elimination reaction of l-cysteine and dl-homocysteine was also efficiently catalysed by the enzyme, resulting in the formation of hydrogen sulphide. Furthermore, the ability to demethiolate methionine into methanethiol, an unfavourable volatile sulphur compound in terms of wine aroma, was observed. The findings of this work suggest that O. oeni seems to play a minor role in the production of volatile sulphur compounds during the vinification process as the optimal conditions were far from the harsh wine environment.     

High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype

Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 10⁷ CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 10³ CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 10³ per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.     

Utilization of Ammonia Nitrogen by Intestinal Bacteria Isolated from Pigs

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of ¹⁵N from [¹⁵N]ammonia into bacterial amino acids was studied. ¹⁵N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.     

Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE)

The bacterial population during malolactic fermentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electrophoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable.