Continuous malolactic fermentation in red wine using free Oenococcus oeni

Malolactic fermentation (MLF) of wine in continuous culture was obtained by using Oenococcus oeni (formerly Leuconostoc oenos). The maximum malic acid degradation in our bioreactor system was reached at a dilution rate of 0.016h^–1, and 92–95% of the malic acid (3.9–4.0g/l) was converted to lactic acid and CO₂.     

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni

Aims:  Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine.
Methods and Results:  Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni .
Conclusion:  This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni . Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed.
Significance and impact of the study:  This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.     

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. Significance and Impact of the Study: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.     

Typification of Oenococcus oeni strains by multiplex RAPD-PCR and study of population dynamics during malolactic fermentation

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.     

Influence of phenolic acids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii

Aims: To determine the effect of several wine-associated, phenolic acids on the growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Methods and Results: Growth was monitored in ethanol-containing medium supplemented with varying concentrations of hydroxybenzoic acids (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids). Progressive inactivation was monitored in ethanol-containing phosphate buffer supplemented in a similar manner to the growth experiments. Hydroxycinnamic acids proved to be more inhibitory to the growth of O. oeni than hydroxybenzoic acids. On the other hand, some acids showed a beneficial effect on growth of Lact. hilgardii. p-Coumaric acid showed the strongest inhibitory effect on growth and survival of both bacteria. Conclusions: Most phenolic acids had a negative effect on growth of O. oeni, for Lact. hilgardii this effect was only noted for p-coumaric acid. Generally, O. oeni was more sensitive to phenolic acid inactivation than Lact. hilgardii. Significance and Impact of the Study: Eight wine-derived, phenolic acids were compared for their effects on wine lactic acid bacteria. Results indicate that phenolic acids have the capacity to influence growth and survival parameters. The differences found between phenolic compounds could be related to their different chemical structures.     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation

Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Deltapep4, which is deleted for the proteinase A gene. Fermentation with the mutant Deltapep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation, each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Deltapep4 strain took 10 days longer than the control. This difference may have been due to a difference in the nitrogen composition of the two media. Free amino acids and amino acids in peptides were poorly consumed by O. oeni. Thus, the qualitative aspects of nitrogen composition, which depend in part on yeast metabolism, may be a determinant for the optimal growth of O. oeni in wine.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

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.     

A new vector, pGID052, for genetic transfer in Oenococcus oeni

Despite the large number of techniques available for the transformation of bacteria, several species are still resistant to the introduction of foreign DNA. Oenococcus oeni are among the organisms that are particularly refractory to transformation. However, conjugal experiments from Lactococcus lactis to O. oeni with a new plasmid, pGID052, were performed via mobilization with success. This plasmid, a derivative of pORI19, encompasses: (i) the oriT of pIP501, which permitted the transfer to O. oeni, (ii) the replication genes of a native Leuconostoc citreum plasmid. Frequencies of 10(-7) conjugants per recipient were found. The transfer did not affect the structure of this low-copy-number plasmid. Moreover, pGID052 seems segregationally stable and could be used in the future as an expression vector.     

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

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.     

Effect of phenolic compounds on the co-metabolism of citric acid and sugars by Oenococcus oeni from wine

AIMS: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. METHODS AND RESULTS: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. CONCLUSIONS: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.     

Rapid detection of Oenococcus oeni in wine by real-time quantitative PCR

AIMS: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification of Oenococcus oeni in wine samples for monitoring malolactic fermentation. METHODS AND RESULTS: Specific primers and fluorogenic probe targeted to the gene encoding the malolactic enzyme of O. oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either from bacterial pure cultures or wine samples. Conventional CFU countings were also performed. The PCR assay confirmed to be specific for O. oeni species and significantly correlated to the conventional plating method both in pure cultures and wine samples (r = 0.902 and 0.96, respectively). CONCLUSIONS: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca 6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and direct detection of O. oeni in wine without the necessity for sample plating. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid quantification of O. oeni by a real-time PCR assay can improve the control of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.     

Influence of fatty acids on the growth of wine microorganisms Saccharomyces cerevisiae and Oenococcus oeni

The effects of fatty acids, extracted during prefermentation grape skin-contact on Saccharomyces cerevisiae and Oenococcus oeni, were studied. The influence of skin-contact on total fatty acid content was evaluated both in Chardonnay must and in synthetic medium. Prior to alcoholic fermentation, the skin-contact contributes to a large enrichment of long-chain fatty acids (C₁₆ to C_18:3). These results induced a positive effect on yeast growth and particularly on cell viability. In the skin-contact fermented media, levels of C₁₂ and especially C₁₀ are lower and macromolecules content higher than in controls. This production of extracellular mannoproteins and the reduction of medium-chain fatty acids in media by S. cerevisiae increased growth of O. oeni. The influence of fatty acids (C₁₀ to C_18:3), in their free and esterified forms, on bacterial growth and on malolactic activity was also examined. Only C₁₀ and C₁₂, especially in their esterified forms, always appeared to be toxic to O. oeni. Received 15 May 1997/ Accepted in revised form 02 December 1997     

Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking

Oenococcus oeni is an acidophilic member of the Leuconostoc branch of lactic acid bacteria indigenous to wine and similar environments. O. oeni is commonly responsible for the malolactic fermentation in wine and due to its positive contribution is frequently used as a starter culture to promote malolactic fermentation. In collaboration with the Lactic Acid Bacteria Genome Consortium the genome sequence of O. oeni PSU-1 has been determined. The complete genome is 1,780,517 nt with a GC content of 38%. 1701 ORFs could be predicted from the sequence of which 75% were functionally classified. Consistent with its classification as an obligately heterofermentative lactic acid bacterium the PSU-1 genome encodes all the enzymes for the phosphoketolase pathway. Moreover, genes related to flavor modification in wine, such as malolactic fermentation capacity and citrate utilization were readily identified. The completion of the O. oeni genome marks a significant new phase for wine-related research on lactic acid bacteria in which the physiology, genetic diversity and performance of O. oeni starter cultures can be more rigorously examined.