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.     

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.     

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.     

Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analyses.

The intraspecific genetic diversity of Oenococcus oeni, the key organism in the malolactic fermentation of wine, has been evaluated by random amplified polymorphic DNA (RAPD), ribotyping, small-plasmid content, and sequencing of RAPD markers with widespread distribution among the strains. Collection strains representing the diversity of this species have been studied together with some new isolates, many of which were obtained from wines produced by spontaneous malolactic fermentation. The RAPD profiles were strain specific and discerned two main groups of strains coincident with clusters obtained by macrorestriction typing in a previous work. Ribotyping and the conservation of RAPD markers indicates that O. oeni is a relatively homogeneous species. Furthermore, identical DNA sequences of some RAPD markers among strains representative of the most divergent RAPD clusters indicates that O. oeni is indeed a phylogenetically tight group, probably corresponding to a single clone, or clonal line of descent, specialized to grow in the wine environment and universally spread.     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Comparative analysis of the Oenococcus oeni pan genome reveals genetic diversity in industrially-relevant pathways

ABSTRACT: BACKGROUND: Oenococcus oeni, a member of the lactic acid bacteria, is one of a limited number of microorganisms that not only survive, but actively proliferate in wine. It is is also unusual as, unlike the majority of bacteria present in wine, it is beneficial to wine quality rather than causing spoilage. These benefits are realised primarily through catalysing malolactic fermentation, but also through imparting other positive sensory properties. However, many of these industrially-important secondary attributes have been shown to be strain-dependent and their genetic basis it yet to be determined. RESULTS: In order to investigate the scale and scope of genetic variation in O. oeni, we have performed whole-genome sequencing on eleven strains of this bacterium, bringing the total number of strains for which genome sequences are available to fourteen. While any single strain of O. oeni was shown to contain around 1800 protein-coding genes, in-depth comparative annotation based on genomic synteny and protein orthology identified over 2800 orthologous open reading frames that comprise the pan genome of this species, and less than 1200 genes that make up the conserved genomic core present in all of the strains. The expansion of the pan genome relative to the coding potential of individual strains was shown to be due to the varied presence and location of multiple distinct bacteriophage sequences and also in various metabolic functions with potential impacts on the industrial performance of this species, including cell wall exopolysaccharide biosynthesis, sugar transport and utilisation and amino acid biosynthesis. CONCLUSIONS: By providing a large cohort of sequenced strains, this study provides a broad insight into the genetic variation present within O. oeni. This data is vital to understanding and harnessing the phenotypic variation present in this economically-important species.     

Molecular and biochemical diversity of Oenococcus oeni strains isolated during spontaneous malolactic fermentation of Malvasia Nera wine

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, was molecularly characterized by the amplified fragment length polymorphism (AFLP) technique. Three main groups resulted from cluster analysis and showed intraspecific homology higher than 50%, and a total of seven subgroups, with similarity values ranged from 80% to 98%, were obtained within these groups. Enzymatic activities, such as esterase, β-glucosidase, protease, and the consumption rate of l-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains, according to AFLP analysis. The results showed different enzymatic activities and consumption rates of the tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among Malvasia Nera strains demonstrated that the wine aroma can be modulated depending on the strains involved in MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes.     

酒酒球菌(Oenococcus oeni)胁迫适应性反应机制

苹果酸-乳酸发酵有利于提高葡萄酒品质,为了获得高活性的直投式酒酒球菌发酵制剂,从生理和分子生物学的角度理解该菌种胁迫耐受性增强的机制是必要的.本文就酒酒球菌利用苹果酸-乳酸发酵和膜结合的H -F0F1-ATP酶以维持细胞内环境的稳定和能量供给;胁迫适应过程中细胞膜组分的调整;小热休克蛋白Lo18等胁迫蛋白及其相应的基因的表达和调控等方面进行了综述.胁迫适应性反应机制的研究对发酵剂菌株的筛选、发酵剂的制备及其他工程菌株的构建具有重要意义.     

Intraspecific diversity of Oenococcus oeni isolated during red wine-making in Japan

Using molecular and chemotaxonomic techniques, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium isolated during red wine-making in Japan. The results confirmed high values of DNA-DNA relatedness and strong similarity among 16S rDNA sequences of the isolates with the O. oeni-type strain. Pulsed-field gel electrophoresis (PFGE) by NotI identified four patterns among the strains. Three different patterns of lactate dehydrogenase mobility were seen and there was a strong correlation between PFGE pattern and mobility. The present results suggest that the different strains of O. oeni comprise one species, and that variations in the genomic profiles of the different strains of O. oeni, including Japanese isolates are well correlated.     

Lactobacillic Acid Accumulation in the Plasma Membrane of Oenococcus oeni: A Response to Ethanol Stress?

It is known that ethanol strongly interferes with the development and activity of lactic acid bacteria in wine. In this work, it was observed that membrane composition was dependent of ethanol concentration and cell physiological state. The protein electrophoretic profile was modified in the membranes of Oenococcus oeni cultured in presence of 8 and 10% ethanol. Concerning the membrane lipid composition, it was observed that O. oeni maintained a high level of phospholipid biosynthesis via the relative increased biosynthesis of phosphoethanolamine and sphingomyelin in presence of ethanol. On the other hand, ethanol induced an increase in the membrane lactobacillic acid percentage at the expense of cis-vaccenic acid. This increased synthesis of lactobacillic acid appears as the more significant change induced by ethanol in O. oeni membrane. The increase of lactobacillic acid in the membrane of O. oeni clearly appears as a factor that provides protection against the toxic effect of ethanol, balancing the increase of membrane fluidity normally attributed to ethanol. The results presented in this paper constitute evidence that lactobacillic acid may have a part in the survival and or adaptive mechanisms developed by O. oeni under culture adverse conditions, allowing these bacteria to maintain their activity in the presence of ethanol, namely performing malolactic fermentation in wine.     

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.     

Evaluation of intra-specific diversities in Oenococcus oeni through analysis of genomic and expressed DNA

In winemaking Oenococcus (O.) oeni is the most frequent species of lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). Several studies have demonstrated that O. oeni is a quite homogeneous species and strains are difficult to differentiate especially when isolates from the same region are analyzed. In this study, the molecular biodiversity of O. oeni isolated from wines of the same region (Aglianico produced in Basilicata Region, Southern Italy) was evaluated with the aim of designing a molecular approach for discrimination and characterization of the isolates at the strain level. Three molecular techniques were applied: random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction endonucleases analysis-pulsed field gel electrophoresis (REA-PFGE) and differential display PCR (DD-PCR). The results obtained by RAPD-PCR confirmed the difficulty in differentiating isolates. By means of REA-PFGE a higher polymorphism, often related to the origin (winery) of strains, was revealed. However, on analyzing strains isolated from the same winery, only in some cases was more than one REA-PFGE pattern obtained. By analyzing dendrograms constructed on the basis of DD-PCR profiles differentiation of strains isolated from the same winery, in some cases, could be accomplished. The reliability of the DD-PCR in the differentiation of closely related strains suggests that this method could represent an alternative and/or additional tool to other molecular methods, such as REA-PFGE, for fine characterization of oenococcal strains.     

Differential real-time PCR assay for enumeration of lactic acid bacteria in wine

Oenococcus oeni is often employed to perform the malolactic fermentation in wine production, while nonoenococcal lactic acid bacteria often contribute to wine spoilage. Two real-time PCR assays were developed to enumerate the total, and nonoenococcal, lactic acid bacterial populations in wine. Used together, these assays can assess the spoilage risk of juice or wine from lactic acid bacteria.     

Microbiology of the malolactic fermentation: Molecular aspects

Abstract Malolactic fermentation conducted by lactic acid bacteria follows alcoholic fermentation during winemaking, and several positive effects make it indispensable for most wines. Research has focused on the growth and physiology of lactic acid bacteria in wine; resulting in the design of malolactic starter cultures. Future work on these starters will concentrate on aromatic changes as additional criteria for strain selection. Although the main features of the malolactic enzyme and its gene are known, the detailed mechanism of the malolactic reaction remains unclear. Cloning and expression of this activity in enological strains of Saccharomyces cereuisiae might be one of the next most important advances in the control of malic acid degradation in wine.     

Typical metabolic traits of two Oenococcus oeni strains isolated from Valpolicella wines

AIMS: Physiological comparison of two indigenous Oenococcus oeni strains, U1 and F3 isolated in the same area (Valpolicella, Italy) in order to select a performant starter for MLF in wine. METHODS AND RESULTS: Growth rate, sugar and malate metabolism in FT80 media at pH 5.3 and 3.5 were analysed. The amount of total protein synthesized and the level of expression of the small Hsp Lo18 were evaluated by radiolabelling and immunodetection experiments after heat (42 degrees C), acid (pH 3.5) and ethanol (12% v/v) stresses. Strain U1 showed significantly lower specific growth rate and growth yield in acid conditions than strain F3. However, strain U1 had a higher malate consumption capacity at pH 3.5 than strain F3, in relation with an higher malolactic activity determined on whole cells. Strain U1 exhibited about half the total protein synthesis level than strain F3, but both strains expressed Lo18 similarly. Evaluation of malolactic fermentation (MLF) performance by microvinification trials was carried out. Strain U1 was able to complete MLF, whereas strain F3 degraded malic acid partially when inoculated in Amarone wine. CONCLUSIONS: Considering its performances in microvinifications experiments, strain U1 could be a good candidate for malolactic starter as an alternative to deficient commercial starters.     

Lactic acid bacteria associated with wine grapes from several Australian vineyards

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.     

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.     

Evidence of distinct populations and specific subpopulations within the species Oenococcus oeni

Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni, an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.     

Lactic acid bacteria in the quality improvement and depreciation of wine

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 10⁶CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteri a, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet c ompletely elucidated . Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.