A putative glucan synthase gene dps detected in exopolysaccharide-producing Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider

Some lactic acid bacteria can induce viscosity in wine, beer and cider by production of exopolysaccharides (EPS). A polymerase chain reaction (PCR) assay was previously described for the detection of ropy Pediococcus damnosus strains in wine [J. Appl. Microbiol. 90 (2001) 535]. The primers used in that study, PF5 and PF6, are investigated in addition to new primers which broaden the range of spoiling agents detectable by PCR. Primers PF1 and PF8 allow the amplification of DNA from ropy P. damnosus strains isolated from wine, as was observed with PF5 and PF6. In addition, PF1 and PF8, unlike PF5 and PF6, are able to generate an amplicon using template DNA from a ropy P. damnosus strain isolated from cider and a ropy Oenococcus oeni strain isolated from champagne. Two different ropy Lactobacillus species were also isolated, but their DNA was not amplified using primers PF1 and PF8. The new primers PF1 and PF8 were chosen from the sequence of gene dps, a putative glucan synthase gene, found across all the ropy P. damnosus strains isolated, from both wine or cider, and also in a ropy O. oeni strain. To our knowledge, this is the first time that an EPS-producing O. oeni strain is described. Glucan biosynthesis was assessed by agglutination tests done with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. The results show that there is a direct correlation between glucan production and detection of gene dps. Therefore, Dps is considered a candidate for the glucan synthase enzyme responsible for EPS production by ropy strains of P. damnosus and O. oeni.     

Restriction fragment length polymorphism analysis of 16S rRNA genes in lactic acid bacteria isolated from red wine

Various lactic acid bacteria isolated from wine and alcoholic drinks attempted to identified by restriction fragment length polymorphism (RFLP). Oenococcus oeni strains exhibited unique RFLP patterns by HaeIII-digestion of 12 reference strains. We performed RFLP analysis using the AccII or HaeIII enzyme for 44 strains isolated from red wine and the results indicated profiles identical to O. oeni type strain. O. oeni does not exhibit interspecific diversity. Thus, the use of RFLP analysis of 16S rDNA is useful in the identification of O. oeni strains isolated from wines.     

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.     

Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832

Pediocin PD-1 is a ribosomally synthesized antimicrobial peptide produced by Pediococcus damnosus NCFB1832. It inhibits the growth of several food spoilage bacteria, including malolactic bacteria isolated from wine. Pediocin PD-1 is 2866.87+/-0.4 Da in size, has an isoelectric point (pI) of ca. 9.0 and, on amino acid composition, has partial homology to the lantibiotic plantaricin C. The highest activity of pediocin PD-1 against cells of Oenococcus oeni was observed at an external pH of 5.0 and at 25 degrees C. The primary mode of action of pediocin PD-1 is most probably due to pore formation, as indicated by the efflux of K+ from metabolically active cells of O. oeni. In the presence of 10 mM gadolinium (Gd3+), pediocin PD-1 did not affect cells of O. oeni. This suggests that the mode of action of pediocin PD-1 relies on a net negatively charged cell surface. In comparison to nisin, pediocin PD-1 is less active against non-growing cells of O. oeni.     

Differentiation of Australian wine isolates of Oenococcus oeni using random amplified polymorphic DNA (RAPD)

Intraspecific variation of Oenococcus oeni , the preferred lactic acid bacteria species for inducing malolactic fermentation in wine, was studied using the randomly amplified polymorphic DNA (RAPD) strain fingerprinting technique. Ten of fifteen isolates of O. oeni from Australian wineries situated in different wine regions could be distinguished by the RAPD technique. Strains of O. oeni which originated from the same winery were either indistinguishable or closely related to each other. Six different commercially available O. oeni strains could be differentiated with the four RAPD primers used and their genetic similarity determined. Analysis of O. oeni present in wines from a single source of fruit (Cabernet Sauvignon, vintage 2002) that underwent spontaneous malolactic fermentation revealed wide genetic variation amongst the isolates. Each fermentor contained several different O. oeni strains, which were present throughout alcoholic and malolactic fermentation. These data highlight the sensitivity of RAPDs when suitable primers are applied to O. oeni of unknown genetic origin, thus enabling O. oeni strains with desirable sensory and fermentation properties to be genetically analysed.     

Use of a polymerase chain reaction for specific detection of the malolactic fermentation bacterium Oenococcus oeni (formerly Leuconostoc oenos) in grape juice and wine samples

A PCR method has been developed that enables rapid and direct identification of the malolactic bacterium Oenococcus oeni from grape must or wine samples. Two primers, based on unique, highly conserved regions within the 16S rRNA gene of O. oeni , were used to amplify a 995 bp fragment which is specific for O. oeni . Other species of bacteria from Lactobacillus, Pediococcus and Acetobacter which may be found in grape must or wine were not detected using this technique. This diagnostic test is able to specifically detect in the order of 10³colony forming units per mL of O. oeni in a wine sample, and can be used for monitoring bacterial growth during malolactic fermentation.     

Both arginine and fructose stimulate pH-independent resistance in the wine bacteria Oenococcus oeni

The wine bacteria Oenococcus oeni has to cope with harsh environmental conditions including an acidic pH, a high alcoholic content, and growth inhibitory compounds such as fatty acids, phenolic acids and tannins. So how can O. oeni bacteria naturally present on the surface of grape berries acquire a natural resistance that will alleviate the effect of wine stresses? One mechanism displayed by O. oeni and many other bacteria against the damaging effects of acid environments is arginine consumption through the arginine deiminase pathway. Various studies have shown that the bacterial protection conferred by arginine depends on the rise in pH associated with ammonia production. However, many experimental results disagree with this point of view. The aim of this study was to clarify the protective effect of arginine on O. oeni stress adaptation. Is it only by increasing the pH through ammonia production that this effect is triggered, or does stimulation of appropriate cellular responses play an additional role? This study shows that: (a) arginine in combination with fructose triggers the expression of a subset of genes which are also stress-responsive; (b) cultivation of O. oeni in a fructose- and arginine-supplemented medium prior to wine exposure protects bacteria against subsequent wine shock, and (c) this acquired stress resistance is independent of pH.     

Inhibition of malolactic fermentation by a peptide produced by Saccharomyces cerevisiae during alcoholic fermentation

The ability of Saccharomyces to inhibit Oenococcus oeni during the alcoholic fermentation by mechanisms other than SO(2) production was investigated. During fermentation in synthetic grape juice, S. cerevisiae strain RUBY.ferm inhibited the malolactic fermentation by O. oeni while strain EC1118 did not despite both strains producing similar amounts of SO(2). The bacterial inhibition exerted by RUBY.ferm was diminished when the wine was treated with proteases but not through the addition of nutrients. Wine fermented by RUBY.ferm was fractionated based on molecular weight and each fraction tested for the ability to inhibit the growth of O. oeni. The fraction containing compounds larger than 3 kDa was the sole inhibitory fraction. The inhibitory fraction was analyzed by SDS PAGE and showed a 5.9 kDa protein band present in wine fermented by RUBY.ferm that was not present in wine fermented by a non-antagonistic yeast, S. cerevisiae strain Saint Georges S101. The ability of the peptide to inhibit O. oeni seemed to be dependent on the presence of SO(2).     

Identification of lactic acid bacteria isolated from South African brandy base wines

In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria. As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production. The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers. The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently. Many of the O. oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF). The Lactobacillus spp. were isolated from all the different stages of brandy base wine production. Lb. plantarum was the dominant species in the juice, but disappeared during the later stages of production. However, Lactobacillus hilgardii, Lb. brevis and Lb. paracasei were also isolated from base wine after spontaneous MLF. Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed. Total soluble cell protein patterns grouped O. oeni strains into two phenotypic groups. Two phenotypic clusters have also been identified for the Lb. brevis isolates. The Lb. paracasei isolates all grouped in one cluster. This is the first report of the presence of Lb. paracasei and Lb. vermiforme in brandy base wines. The presence of the Lactobacillus spp. could be correlated to the decrease in quality of the base wine and distillate, while O. oeni strains were found to have a more favourable influence on the quality of base wine and distillates. These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy.     

Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.     

Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii

The aim of this work was to investigate the effect of wine phenolic aldehydes, flavonoids and tannins on growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Cultures were grown in ethanol-containing MRS/TJ medium supplemented with different concentrations of phenolic aldehydes or flavonoids and monitored spectrophotometrically. The effect of tannins was evaluated by monitoring the progressive inactivation of cells in ethanol-containing phosphate buffer supplemented with grape seed extracts with different molecular weight tannins. Of the phenolic aldehydes tested, sinapaldehyde, coniferaldehyde, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and 3,4,5-trihydroxybenzaldehyde significantly inhibited the growth of O. oeni VF, while vanillin and syringaldehyde had no effect at the concentrations tested. Lact. hilgardii 5 was only inhibited by sinapaldehyde and coniferaldehyde. Among the flavonoids, quercetin and kaempferol exerted an inhibitory effect especially on O. oeni VF. Myricetin and the flavan-3-ols studied (catechin and epicatechin) did not affect considerably the growth of both strains. Condensed tannins (particularly tetramers and pentamers) were found to strongly affect cell viability, especially in the case of O. oeni VF. In general, this strain was found to be more sensitive than Lact. hilgardii 5 to the phenolic compounds studied. This work contributes to the knowledge of the effect of different phenolic compounds on the activity of wine lactic acid bacteria, which, especially in the case of aldehydes and of different molecular weight fractions of tannins, is very scarce.     

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase

Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.     

A bacterial gene homologous to ABC transporters protect Oenococcus oeni from ethanol and other stress factors in wine

The wine lactic acid bacteria Oenococcus oeni has to cope with harsh environmental conditions including an acidic pH, a high alcoholic content, non-optimal growth temperatures, and growth inhibitory compounds such as fatty acids, phenolic acids and tannins. We here describe characterisation and cloning of the O. oeni omrA gene encoding a protein belonging to the ATP-binding cassette superfamily of transporters. The OmrA protein displays the highest sequence similarity with the subfamily of ATP-dependent multidrug resistance (MDR) proteins, most notably the bacterial Lactococcus lactis LmrA homologue of the human MDR1 P-glycoprotein. The omrA gene proved to be a stress-responsive gene since its expression was increased at high temperature or under osmotic shock. The OmrA protein function was tested in Escherichia coli, and consistent with the omrA gene expression pattern, OmrA conferred protection to bacteria grown on a high salt medium. OmrA also triggered bacterial resistance to sodium laurate, wine and ethanol toxicity. The homologous LmrA protein featured the same stress-protective pattern than OmrA when expressed in E. coli, and the contribution to resistance of both OmrA and LmrA transporters was decreased by verapamil, a well-known inhibitor of the human MDR1 protein. Genes homologous to omrA were detected in other wine lactic acid bacteria, suggesting that this type of genes might constitute a well-conserved stress-protective molecular device.     

Evaluation of exopolysaccharide production by Leuconostoc mesenteroides strains isolated from wine

ABSTRACT:  Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPS monosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFF-GTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beverages.     

发酵过程果酒成分对苹果酸乳酸发酵的影响

苹果酸-乳酸发酵（MLF）是果酒（葡萄酒、苹果酒）酿造中非常重要的二次发酵过程。本研究采用人工模拟果酒，研究果酒成分对MLF的影响。结果表明，葡萄糖抑制乳酸菌的MLF，果糖却有促进作用，而且果糖可解除葡萄糖对酿酒酒球菌MLF的抑制作用，果酒中葡萄糖和果糖的浓度及其比例是预测和控制MLF的重要参数；高浓度乙醇、低pH抑制乳酸菌的MLF，其抑制作用的大小与使用的菌种有关；酒中酚类物质（没食子酸和阿魏酸）对酿酒酒球菌的MLF几乎无任何显著影响；酒精发酵完成后果酒中营养的缺乏会抑制乳酸菌的MLF。     

Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of beta-D-glucan-producing bacteria in foods and beverages

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.     

酒类酒球菌β-葡萄糖苷酶研究进展

酒类酒球菌（Oenococcus oeni）是葡萄酒苹果酸乳酸发酵（MLF）中的主要微生物,糖苷物质是葡萄酒中的重要香气前体物,β-葡萄糖苷酶是降解糖苷物质的关键酶。酒类酒球菌β-葡萄糖苷酶对增加葡萄酒香气,提升葡萄酒整体品质具有重要作用。该文介绍了β-葡萄糖苷酶的定义、分类、作用机制和测定方法,阐述了酒类酒球菌β-葡萄糖苷酶活,探讨了pH值、发酵温度、乙醇浓度、糖含量和二氧化硫含量对酶活的影响,在分子生物学水平上研究了酒类酒球菌β-葡萄糖苷酶基因,并对酒类酒球菌葡萄糖苷酶未来的研究热点和研究方向进行了展望。这对深入认识葡萄酒生物增香机理和提高葡萄酒整体品质具有重要意义。     

The effects of freezing and freeze-drying of Oenococcus oeni upon induction of malolactic fermentation in red wine

Summary The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation (MLF) in wine permits control over the timing of the process and the quality of the wine. Successful inoculation of bacterial starter cultures into wine depends on the selection of suitable strains and on the preparation and conservation of those cultures. Medium for Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cultures under laboratory controlled conditions for isolation and identification. However, this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine while cells grown in Medium of Preculture (MP) or wine, stored at −20 °C or freeze-dried retained the ability to induce MLF when inoculated in wine. Our results suggest that the use of freeze-dried cultures of O. oeni previously grown in MP is the best choice for industrial application.     

Characterization of EprA, a major extracellular protein of Oenococcus oeni with protease activity

Extracellular proteins from Oenococcus oeni, a wine-making bacterium, were isolated during growth on media differing by their nitrogen content. Analysis by two-dimensional electrophoresis revealed a low number of protein signals. Among the main spots, one signal corresponded to a single protein, which contained a lysine repeat domain characteristic of cell-wall hydrolases. We demonstrated that this major protein, named EprA, was able to hydrolyse several proteins. The heterologous production of this protein in Escherichia coli confirmed the protease activity of EprA. With a MW of 21.3 kDa and a pI of 5.3, EprA presents optimal activity at pH 7.0 and 45 degrees C. This O. oeni protease differs from all lactic acid bacteria proteases so far identified, and thus this bacterium possesses at least three proteases for wine protein hydrolysis.     

Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: current knowledge and perspectives

Winemaking can be summarized as the biotransformation of must into wine, which is performed principally by Saccharomyces cerevisiae strains during the primary or alcoholic fermentation. A secondary fermentation, the so-called malolactic fermentation (MLF) is a biodeacidification that is often encouraged, since it improves wine stability and quality. Malolactic fermentation usually occurs either spontaneously or after inoculation with selected bacteria after alcoholic fermentation. The main organism responsible for MLF, the lactic acid bacterium Oenococcus oeni, develops in physicochemically harsh conditions, which may lead to MLF failure. Furthermore, yeast that ferment must before or together with O. oeni can prevent or stimulate the progress of MLF. These phenomena are part of the interactions observed between yeast and bacteria. The mechanisms that govern yeast bacteria interaction are reviewed and the consequences for winemaking are discussed. In the light of recent advances, future prospects are also presented.