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.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Predominance of malolactic fermentation overd-glucose utilization inLactobacillus plantarum andLactobacillus curvatus wine strains

Summary Two selected wine strains of the genusLactobacillus (L. plantarum 197 andL. curvatus 783) were tested for their ability to complete malolactic fermentation (MLF) in a synthetic medium (PBM-broth) supplemented withL-malic acid (7.5–74.6 mM) andD-glucose (5.5–55 mM). The 24 directed fermentation assays, 12 for each bacterial strain, were carried out at 20°C and pH 3.5. MLF was completed (residualL-malic acid 0.2 mM) in eight days in 19 of the 24 fermentation assays, even in the presence of 74.6 mML-malic acid or 55.5 mMD-Glucose utilization was generally simultaneous to MLF but was completed (residual concentrations 0.2 mM) only in 6 of the 24 fermentation assays. These results support the use of these strains in directed MLF assays at the very differentL-malic acid andD-glucose concentrations tested.     

Electrogenic L-malate transport by Lactobacillus plantarum: a basis for energy derivation from malolactic fermentation.

L-Malate transport in Lactobacillus plantarum was inducible, and the pH optimum was 4.5. Malate uptake could be driven by an artificial proton gradient (delta pH) or an electroneutral lactate efflux. Because L-lactate efflux was unable to drive L-malate transport in the absence of a delta pH, it did not appear that the carrier was a malate-lactate exchanger. The kinetics of malate transport were, however, biphasic, suggesting that the external malate concentration was also serving as a driving force for low-affinity malate uptake. Because the electrical potential (delta psi, inside negative) inhibited malate transport, it appeared that the malate transport-lactate efflux couple was electrogenic (net negative) at high concentrations of malate. De-energized cells that were provided with malate only generated a large proton motive force (greater than 100 mV) when the malate concentration was greater than 5 mM, and malate only caused an increase in cell yield (glucose-limited chemostats) when malate accumulated in the culture vessel. The use of the malate gradient to drive malate transport (facilitated diffusion) explains how L. plantarum derives energy from malolactic fermentation, a process which does not involve substrate-level phosphorylation.     

Effect of l-malate,d-glucose and l-lactate on malolactic fermentation and growth of Lactobacillus plantarum and Lactobacillus curvatus wild strains isolated from wine

l-Malate fermentative rate in Lactobacillus plantarum and Lactobacillus curvatus was improved when l-malate was over 0.1% but decreased when exogenous d-glucose raised over 0.3% or in the presence of exogenous l-lactate. Significant increases in the growth were observed in the presence of exogenous d-glucose. l-malate residual concentrations were in all cases below 1%.     

Patagonian red wines: selection of Lactobacillus plantarum isolates as potential starter cultures for malolactic fermentation

The aim of this study was to evaluate fifty-three Lactobacillus plantarum isolates obtained from a Patagonian red wine, molecularly identified and typified using RAPD analysis, in order to select starter cultures for malolactic fermentation (MLF). The results obtained suggest a considerable genetic diversity, taking into account that all L. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentration of 14 % ethanol in MRS broth for 2 days, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase activities were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous Oenococcus oeni strain was included with comparative purposes. Differences in technologically relevant characteristics were observed among the eight L. plantarum selected isolates, revealing an isolate-dependent behavior. Detectable glucosidase and tannase activities were found in all isolates. The presence of genes encoding histidine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of L. plantarum isolates to grow and consume l-malic acid in simulated laboratory-scale vinifications revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterization.     

The inhibitory activity of wine yeast starters on malolactic bacteria

Malolactic fermentation is a process that is influenced by various factors that can inhibit the growth of the malolactic bacteria. Inhibitory metabolites produced by yeast may have an important role in the correct development of malolactic fermentation. For these reasons, we have investigated the effects of such metabolites on the growth of malolactic bacteria under different environmental conditions, to aid in our understanding of the significance of these interactions in the wine-making environment. Our screening methods to detect interactions between yeast and malolactic bacteria showed a variable and wide diffusion of yeast inhibitory activity on the growth of the malolactic bacteria. However, this first approach to determine this inhibitory activity of yeast gave an overestimation when compared to the results obtained under actual wine-making conditions. The evaluation of malic acid consumption indicated that under inhibitory conditions a partial L-malic acid degradation was seen, indicating that the malolactic activity continued without bacterial growth. However, these yeast-inhibiting effects in addition to other environmental factors could cause a complete failure of malolactic fermentation.     

一株植物乳酸杆菌的高密度发酵

目的研究一株植物乳酸杆菌的高密度发酵。方法通过单因素实验来确定此植物乳酸杆菌的最适生长温度、pH、碳源、氮源、缓冲盐等。结果最适生长温度是37℃、起始pH6.4、最适碳源为葡萄糖、最适氮源为牛肉膏,通过对缓冲盐的选择最终确定NaAc：CaCO3比为0.5：1.5;控制发酵条件,结合优化培养基,可使发酵后的培养液达到2.75×10^10CFU/ml。结论该缓冲盐能够有效的缓解发酵液pH的下降,延长对数生长期,使活菌数提高1倍,适合高密度发酵培养。     

Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR

AIMS: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. METHODS AND RESULTS: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. CONCLUSIONS: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.     

Leuconostoc oenos and malolactic fermentation in wine: a review

This review article summarizes the state of the art on Leuconostoc oenos, the bacteria responsible for malolactic fermentation in wine. Both basic and practical aspects related to the metabolism of this microorganism and malolactic fermentation in general are critically reviewed. The former examines the role of genetics for the identification and classification of L. oenos and energetic mechanisms on solute transport (malic and lactic acid). The latter includes practical information on biomass production, optimal growth conditions and stress factors, which are important in growth optimization of malolactic starter cultures. Extensive data and references on the effect of malolactic fermentation on wine composition and sensory analysis are also included. Received 06 May 1999/ Accepted in revised form 13 July 1999     

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise l-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.     

Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10⁶ CFU ml^?1) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R ² ≥ 0.97, white wine R ² ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R ² ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10⁴ CFU ml^?1 and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries.     

一种快速定量检测益生菌制品中植物乳杆菌的方法

目的建立快速定量检测植物乳杆菌的方法,排除近缘菌的干扰。方法利用SMM系统筛选植物乳杆菌种特异序列,根据特异序列设计引物进行实时荧光定量PCR反应。结果设计的引物具有良好的种特异性,检测限为2．26×10^2CFU／mL。结论该方法快速灵敏,可以在实际生产中应用。     

Development of a minimal growth medium for Lactobacillus plantarum

Aim:  A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum .
Methods and Results:  By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudominimal medium, supporting proper biomass formation of 350 mg l⁻¹ dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome-scale metabolic model of L. plantarum , it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum . This is because the biosynthesis of para- aminobenzoic acid ( p ABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for p ABA; a molecule which plays an important role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of p ABA. It was found that a 12 000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of p ABA.
Conclusion:  Largely reduced folate production pools do not have an effect on the growth of L. plantarum , showing that L. plantarum makes folate in a large excess.
Significance and Impact of the study:  These experiments illustrate the importance of combining genome-scale metabolic models with growth experiments on minimal media.     

Microsatellite PCR profiling of Saccharomyces cerevisiae strains during wine fermentation

AIMS: Use of microsatellite PCR to monitor populations of Saccharomyces cerevisiae strains during fermentation of grape juice. METHOD AND RESULTS: Six commercial wine strains of S. cerevisiae were screened for polymorphism at the SC8132X locus using a modified rapid PCR identification technique. The strains formed four distinct polymorphic groups that could be readily distinguished from one another. Fermentations inoculated with mixtures of three strains polymorphic at the SC8132X locus were monitored until sugar utilization was complete, and all exhibited a changing population structure throughout the fermentation. CONCLUSIONS: Rapid population quantification demonstrated that wine fermentations are dynamic and do not necessarily reflect the initial yeast population structure. One or more yeast strains were found to dominate at different stages of the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The population structure of S. cerevisiae during mixed culture wine fermentation is dynamic and could modify the chemical composition and flavour profile of wine.     

Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage

Bacteriophage present in wine can attack bacterial starter cultures and inhibit the malolactic fermentation. The possibility of starter culture failure due to phage attack was studied in a commercial dry red wine of pH 3·23, inoculated with a multiple strain starter culture. During two stages of malolactic fermentation, bacterial growth and malate degradation in the wine were inhibited. A phage capable of lysing isolates of Leuconostoc oenos was isolated from the wine. The isolated phage had an icosahedral head of 42–45 nm diameter and a flexible, regularly cross-striated tail 197–207 nm long with a small baseplate. The results confirm that phage can attack bacterial starter cultures in wine at low pH.     

Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11-12 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.     

The use of alternative technologies to develop malolactic fermentation in wine

The development of the malolactic fermentation, bioconversion of L-malic acid to L-lactic acid, is a difficult and time-consuming process that does not always proceed favorably under the natural conditions of wine. Traditional fermentations are used worldwide to produce high-quality wines, although delay or failure is not an unusual outcome. During recent years several technologies have been proposed to induce biological deacidification of wines by using malolactic bacteria, principally Oenococcus oeni and Lactobacillus sp. These alternative technologies usually involve the use of high densities of cells or enzymes, free or immobilized onto different matrices. Immobilization materials, several types of bioreactors, and the properties of many specific systems are discussed in this review.