Oenococcus Oeni Strains Research Articles

Increase in antioxidant and antihypertensive activity by Oenococcus oeni in a yeast autolysis wine model

Accelerated autolysis of Saccharomyces cerevisiae mc2 in synthetic wine medium enabled the release of 3.7 mg peptide nitrogen/l, concomitantly with an increase in antioxidant properties (243 micromol FeSO(4)/l in the case of ferric reducing antioxidant power and 0.5% in 2,2-diphenyl-1-picrylhydrazyl radical scavenging) and antihypertensive activity (22% in angiotensin I-converting enzyme inhibitory activity). Sequential inoculation of a proteolytic Oenococcus oeni strain in the synthetic medium after yeast autolysis produced an increase in peptide nitrogen concentration of 1.5 mg/l after 48 h of growth. After this incubation time an improvement in antihypertensive and antioxidant activities was detected. Oenococcus oeni X(2)L could give additional value to wine because of the bioactive peptides with multifunctional beneficial activity released as consequence of its proteolytic activity.

Cloning and characterisation of a cystathionine β/γ-lyase from two Oenococcus oeni 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.

Evolution of genome organization in Oenococcus oeni revealed by physical and genetic map comparison

The genomic organization of nine strains of Oenococcus oeni belonging to two previously suggested divergent groups was examined by a top-down approach, including analysis of isolated genes and construction of physical and genetic maps. Genomic sequence data from Oenococcus oeni strain PSU-1 was also examined by a bottom-up approach, using sequence data accessible from Joint Genome Institute, which enabled the confirmation of gene location and assessment of transcription direction. Comparison of genomic maps revealed O. oeni as a homogeneous species and supported the existence of two different genomic groups, although in a too much early phase of divergence to be considered as subspecies. O. oeni genomic organization is characterized by an unusual conserved distribution of the two rrn operons located at least 500 kb apart from the putative chromosome replication origin. Differential degrees of conservation may be observed in O. oeni chromosomes, being the neighbouring region of the replication terminus the most conserved one. Since most of the structural polymorphisms can be correlated to the presence of transposase genes and sites of prophage integration, occurrence of macrodiversity events, as insertions-deletions, duplications or inversions of larger genomic regions seems to be ruled out in O. oeni evolution.

Technological properties ofOenococcus oenistrains isolated from typical southern Italian wines

To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. 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 winelike 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. 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. 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.

Technological properties of Oenococcus oeni strains isolated from typical southern italian wines.

Technological properties of Oenococcus oeni strains isolated from typical southern italian wines.

Selection of autochthonous Oenococcus oeni strains according to their oenological properties and vinification results

The goal of this study is to carry out a characterization of 84 Oenococcus oeni strains isolated from Tempranillo wine samples taken at the cellars in Castilla-La Mancha, in order to select those showing the highest potential as oenological starter cultures. Various oenological properties were analyzed and the ability of some of these strains to grow and undergo MLF in simulated laboratory microvinifications was tested. Twenty-two strains were selected on the basis of fermentation assays and the eight that produced the best results in the chemical analysis of the wines were chosen for further assays. None of the eight strains was either able to produce biogenic amines or displayed tannase or anthocyanase activities. On the other hand all presented activity against p-NP-βGlucopyranoside, p-NP-αGlucopyranoside and p-NP-β xylopyranoside. Randomly Amplified Polymorphic DNA (RAPD)-PCR was used to determine the colonizing ability of the inoculated strains. C22L9 and D13L13 strains showed the highest implantation values. On the basis of this characterization, two strains have been selected which are suitable as starter cultures for MLF of Tempranillo wine. Use of these strains will ensure that MLF proceeds successfully and gives retention of the organoleptic characteristics of wines made in Castilla-La Mancha.

Metabolomics Reveals Alterations in Both Primary and Secondary Metabolites by Wine Bacteria

Lactic acid bacteria (LAB) were isolated from Korean Meoru (Vitis coigneties) wine and identified as Lactobacillus plantarum meoru0711 (KACC 91436C). The fermentative behavior and metabolic effects of L. plantarum during malolactic fermentation (MLF) were compared with those of the commercial Oenococcus oeni strain through 1H NMR- and GC-based metabolic profiling. Twenty-two primary metabolites of amino acids, carbohydrates, and organic acids, and 55 secondary metabolites of volatile compounds were identified in wines by 1H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography (GC), respectively. Principal component analysis (PCA) revealed that malolactic (ML)-fermented and non-ML-fermented wines, and wines ML-fermented with O. oeni and L. plantarum were clearly differentiated. Both the primary and secondary metabolites were responsible for these differentiations. Compared to non-MLF wines, MLF wines were characterized by increased levels of primary metabolites such as lactic acid, phenylalanine, uracil, ornithine, alanine, threonine, leucine, isoleucine, and valine with decreased levels of monosaccharides, glycerol, malic, and citric acids. In addition, higher levels of secondary metabolites such as butanal, ethyl isobutylate, isobutanol, isoamyl acetate, 2-butanoate ethyl ester, isoamyl alcohol, ethyl hexanoate, glycine, acetic acid, and benzaldehyde characterized the MLF wine. Higher levels of primary metabolites such as tyrosine, monosaccharides, glycerol, alanine, 2,3-butanediol, valine, and leucine, and of secondary metabolites such as propyl acetate, isobutanol, isoamyl acetate, 1-butanol, ethyl hexanoate, prenyl alcohol, glycine, 2-hexen-1-ol, ethyl octanoate, acetic acid, benzaldehyde, and butyric, together with lower levels of lactic acid, were observed in the wines fermented by L. plantarum compared with those by O. oeni. This present study demonstrates that different genera of LAB affect both the primary and second metabolites in wine. Moreover, metabolomics with multivariate statistical analysis provide insight into wine fermentation.

Survey on indigenous Oenococcus oeni strains isolated from red wines of Valtellina, a cold climate wine-growing Italian area

Spontaneous MLF in high acidity wines produced in cool-climate regions remains problematic though indispensable for the development of sensory characteristics. Genetic aspects and phenotypic traits of thirty-six Oenococcus oeni strains, most of them isolated from Valtellina wines over three consecutive years, were investigated. Molecular typing achieved by RAPD PCR and PFGE analyses allowed 27 different genotypes to be discriminated, whereas from the comparison of results arising by physiological tests (sugar fermentation, alcohol resistance, growth at low temperatures, biogenic ammines production) 28 different phenotypic profiles were obtained. Particularly, 69% of Valtellina isolates were able to develop at 5 °C in cultural broth. Micro-vinification experiments allowed the selection of strains with potential oenological performances and an interesting capability to grow in cold conditions was confirmed. Some O. oeni strains formed phenylethylamine (up to 47 mg/L) and tyramine (up to 36 mg/L) both in cultural broth and wine.

Evidence for exopolysaccharide production byOenococcus oenistrains isolated from non-ropy wines

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. Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with beta-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. Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than beta-glucan. These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.

Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures

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. 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. 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. 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.

Fingerprinting analysis ofOenococcus oenistrains under stress conditions

Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12-13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 degrees C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.

Characterization of Fermentative Behaviors of Lactic Acid Bacteria in Grape Wines through 1H NMR- and GC-Based Metabolic Profiling

The effects of five commercial Oenococcus oeni strains (MCW, Enoferm alpha, Wyeast, Vinibacti111, and Vinibacti222) on fermentative behaviors, and variations of metabolites in Meoru ( Vitis coigneties ) wines during malolactic fermentation (MLF) were investigated by metabolomic analysis of (1)H NMR and GC data sets. In the development of MLF with various O. oeni strains, the fastest conversions of malic acid to lactic acid occurred in wines fermented with Enoferm alpha and Vinibacti111 strains. Seventeen primary metabolites and 65 secondary metabolites of volatile compounds in the wines were identified by (1)H NMR spectroscopy and GC-MS, respectively. In pattern recognition models of principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA), significant differentiations between wines with O. oeni strains were identified by the secondary metabolites rather than by the primary metabolites, showing the effects of O. oeni strains only on the secondary metabolites. Twelve volatile compounds, 2-phenylethanol, isoamyl alcohol, 2-butanol, ethyl octanoate, ethyl hexanoate, hexadecanoic acid, diethyl succinate, butyl butyrate, octanoic acid, 9-hexadecanoic acid, isobutyric acid, and 2-ethyl-1-hexanol, contributed to the differentiation of wines according to O. oeni strain, including spontaneous MLF. This study demonstrates that O. oeni strains affect the secondary metabolites, which are easily identified through multivariate statistical analysis of GC-MS data set.

Genetic and phenotypic evidence for two groups of Oenococcus oeni strains and their prevalence during winemaking

Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis was the most relevant method to follow the diversity of lactic acid bacteria during winemaking. By targeting the rpoB gene, two types of Oenococcus oeni strains were distinguished resulting from a single mutation in the rpoB region targeted in PCR and generating two different electrophoresis profiles. The first one prevailed during fermentation and the second during ageing. Some strains of each type were isolated during winemaking and were studied using several genetic methods (real-time PCR, PCR-random amplified polymorphic DNA, multiple locus sequence typing and the presence of gene markers). Physiological characters related to environmental conditions were examined. The results confirmed the relevance of the rpoB mutation for characterising the two O. oeni subgroups. The relationship between the physiological response to stress and the rpoB genetic groups raised the question of O. oeni intraspecies grouping. A possible division within this species, of great technological interest to the wine industry, was also raised.

Potential formation of ethyl carbamate in simulated wine inoculated with Oenococcus oeni and Lactobacillus plantarum

SummaryThe production of ethyl carbamate (EC) and its precursor citrulline by two strains of Oenococcus oeni and one of Lactobacillus plantarum during malolactic fermentation (MLF) was studied in different conditions of pH, temperature, and ethanol and l‐malic acid concentrations. The potential EC, defined as the concentration of EC after wine is heated at 80 °C for 48 h, was also investigated. The O. oeni strains were able to degrade arginine in the conditions studied and to excrete some citrulline. In these strains, the conditions that led to a slight increase in EC were a high ethanol concentration, low pH and a high l‐malic acid concentration. However, the potential EC increased with higher pH. In the case of L. plantarum, arginine was not degraded and citrulline was not produced, although the potential EC was comparable with that of the O. oeni strains studied.

Determination of the essential nutrient requirements of wine-related bacteria from the genera Oenococcus and Lactobacillus

Wine lactic acid bacteria (LAB) are responsible for the malolactic fermentation (MLF) in wine production. Wine LAB have fastidious nutrient requirements but their auxotrophies remain little studied. The ability of specific wine nutrients to meet the nutritional requirements of wine LAB, and thus support MLF, remains unclear. This work investigated the essential growth requirements of four strains of wine LAB from the genera Oenococcus and Lactobacillus using the single omission technique with a suitable chemically defined medium. For the determination of auxotrophies, at least 3 (and up to 15) subcultures in deficient media were made, and intra- and extracellular nutrient carry over was reduced by small inoculation rates and washing cells 3 times between transfers. This careful methodology revealed more auxotrophies than those described for wine LAB in the literature. The essential bacterial nutrient requirements were found to be strain specific. 10 compounds were essential for all wine LAB tested, the carbon and phosphate source, manganese, as well as several amino acids (proline, arginine and the branched amino acids valine, leucine and isoleucine) and vitamins (nicotinic acid and pantothenic acids). Nucleotides were not essential for any of the bacteria studied. The two Oenococcus oeni strains revealed a larger number of auxotrophies (18 and 21) and had a higher degree of nutritional similarity (86%) defined as percentage of common requirements per maximum total requirements. The two Lactobacillus strains only had 11 and 14 auxotrophies and the similarity was 79%, but both were auxotroph for riboflavin, which was not needed by the O. oeni strains. Data on the common requirements may be used to further study the ability of wines or commercial nutrients to support MLF and to consider the microbiological stability of finished wines. The results indicate that absence of riboflavin in oenological nutrient preparations may allow to create a specific advantage for indigenous or inoculated O. oeni, which are generally desired for MLF.

Biogenic amine production by Oenococcus oeni isolates from malolactic fermentation of Tempranillo wine.

In this article, we examine the production of biogenic amines, histamine, putrescine, tyramine, and cadaverine by 90 strains of Oenococcus oeni isolated from different cellars of Castilla-La Mancha (Spain) during wine malolactic fermentation. Amino biogenic capacity of strains was qualitatively analyzed on agar. After that, production of amines on a synthetic medium and on wine, and presence in strains of histidine, ornithine, and tyrosine decarboxylase genes were determined. Only two strains were able to produce histamine or putrescine, both on synthetic medium and wine. The presence of the corresponding genes in these strains was also confirmed. These results suggest that O. oeni does not significantly contribute to the overall biogenic amine content of wines. The main contribution of this work is the isolation of a putrescine-producing O. oeni strain that harbors the ornithine gene, since this gene appears to be rarely present in the genome of O. oeni.

Oenococcus oeni genome plasticity is associated with fitness.

Oenococcus oeni strains are well-known for their considerable phenotypic variations in terms of tolerance to harsh wine conditions and malolactic activity. Genomic subtractive hybridization (SH) between two isolates with differing enological potentials was used to elucidate the genetic bases of this intraspecies diversity and identify novel genes involved in adaptation to wine. SH revealed 182 tester-specific fragments corresponding to 126 open reading frames (ORFs). A large proportion of the chromosome-related ORFs resembled genes involved in carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, and replication, recombination, and repair. Six regions of genomic plasticity were identified, and their analysis suggested that both limited recombination and insertion/deletion events contributed to the vast genomic diversity observed in O. oeni. The association of selected sequences with adaptation to wine was further assessed by screening a large collection of strains using PCR. No sequences were found to be specific to highly performing (HP) strains alone. However, there was a statistically significant positive association between HP strains and the presence of eight gene sequences located on regions 2, 4, and 5. Gene expression patterns were significantly modified in HP strains, following exposure to one or more of the common stresses in wines. Regions 2 and 5 showed no traces of mobile elements and had normal GC content. In contrast, region 4 had the typical hallmarks of horizontal transfer, suggesting that the strategy of acquiring genes from other bacteria enhances the fitness of O. oeni strains.

Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae

Abstract Twenty-six wild Oenococcus oeni strains were investigated for their ability to form biogenic amines during malolactic fermentation in synthetic medium and in wine. Eight strains produced histamine and tyramine in screening broth at concentrations of 2.6–5.6 mg/L and 1.2–5.3 mg/L, respectively. Based on their ability to form biogenic amines, five strains were selected to inoculate three wines obtained by the fermentation of three different Saccharomyces cerevisiae strains (A, B, and C). All bacterial strains could perform malolactic fermentation for short periods in wine C, whereas only one strain performed complete malolactic fermentation in wines A and B. Two O. oeni strains (261 and 351) produced histamine and tyramine in wine C. Time-course analysis of these compounds showed that for both strains, histamine and tyramine production began at day 10 and finished on day 25, after the end of malolactic fermentation. These results indicate that the ability of O. oeni to produce histamine and tyramine is dependent on the bacterial strain and on the wine composition, which in turn depends on the yeast strain used for fermentation, and on the length of bacteria–yeast contact time after the completion of malolactic fermentation.

Genotyping by Amplified Fragment Length Polymorphism and malate metabolism performances of indigenous Oenococcus oeni strains isolated from Primitivo wine

The Amplified Fragment Length Polymorphism (AFLP) technique was applied for the first time to investigate the genotyping of Oenococcus oeni, the most important species involved in malolactic fermentation (MLF) in wine. A total of 87 out of 220 lactic acid bacteria, isolates from “Primitivo” wine (Apulia, Italy) undergoing MLF, identified as O. oeni by species-specific PCR and 16S rRNA sequence analysis, were studied by AFLP analysis. Four main clusters were distinguished and three of them showed intraspecific homology higher than 60%. A total of 28 strains, representative of AFLP clusters, were tested for malate metabolism in order to gain information on their malolactic performances. Significant differences were observed among strains for malic acid consumed, biomass produced and specific malic acid consumption rate. These findings indicated that AFLP technique is reliable for typing O. oeni strains and that, together with metabolism studies it may be used to individuate possible candidates as industrial malolactic starters.

Characterization of gtf, a glucosyltransferase gene in the genomes of Pediococcus parvulus and Oenococcus oeni, two bacterial species commonly found in wine.

"Ropiness" is a bacterial alteration in wines, beers, and ciders, caused by beta-glucan-synthesizing pediococci. A single glucosyltransferase, Gtf, controls ropy polysaccharide synthesis. In this study, we show that the corresponding gtf gene is also present on the chromosomes of several strains of Oenococcus oeni isolated from nonropy wines. gtf is surrounded by mobile elements that may be implicated in its integration into the chromosome of O. oeni. gtf is expressed in all the gtf(+) strains, and beta-glucan is detected in the majority of these strains. Part of this beta-glucan accumulates around the cells forming a capsule, while the other part is liberated into the medium together with heteropolysaccharides. Most of the time, this polymer excretion does not lead to ropiness in a model medium. In addition, we show that wild or recombinant bacterial strains harboring a functional gtf gene (gtf(+)) are more resistant to several stresses occurring in wine (alcohol, pH, and SO(2)) and exhibit increased adhesion capacities compared to their gtf mutant variants.