Lysogeny of Oenococcus oeni (syn. Leuconostoc oenos) and Study of Their Induced Bacteriophages

A large number of strains of Oenococcus oeni (formerly Leuconostoc oenos) that had been isolated from wines were checked for lysogeny with mitomycin C as inducer. As a result of this test, 45% of the strains proved to be lysogenic, suggesting that lysogeny is widespread among bacteria isolated from wines during malolactic fermentation. The sensitivity of bacteria to phages was very different, depending on the strain. All the lysogenic strains were resistant to infection by the temperate phage they released. Some phages infected none of the strains. Phages of Oenoc. oeni had a classical morphology, an isometric head, and a long striated tail. With the broadest host strain as an indicator, phages were detected in wines after malolactic fermentation. Received: 28 November 1997 / Accepted: 5 January 1998     

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines

Aims: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. Methods and Results: 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 wine‐like 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. Conclusion: 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. Significance and Impact of the Study: 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.     

Putrescine Accumulation in Wine: Role of Oenococcus oeni

Putrescine, the most abundant biogenic amine in wine, was proved to be produced by Oenococcus oeni strains in wine not only from ornithine but also from arginine. In this case, putrescine may originate from strains possessing the complete enzyme system to convert arginine to putrescine or by a metabiotic association, with an exchange of ornithine, between strains capable of metabolizing arginine to ornithine but unable to produce putrescine and strains capable of producing putrescine from ornithine but unable to degrade arginine. Putrescine production by this metabiotic association occurred once the malolactic fermentation was completed, whereas conversion of ornithine to putrescine by a single culture of the ornithine decarboxylating strain concurred with the degradation of malic acid. Moreover, in the former case, putrescine formation proceeded more slowly than in the latter. Metabiosis may play an important role in the accumulation of putrescine in wine, arginine being one of the major amino acids found in wine.     

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.     

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.     

Genomic DNA Fingerprinting of Oenococcus oeni Strains by Pulsed-Field Gel Electrophoresis and Randomly Amplified Polymorphic DNA-PCR

Genetic diversity of 60 Oenococcus oeni strains from different wines was evaluated by numerical analysis of (i) pulsed-field gel electrophoresis (PFGE) patterns with endonuclease ApaI and (ii) randomly amplified polymorphic DNA (RAPD)-PCR fingerprints with four oligonucleotide primers. Sixty-two percent of the strains could be distinguished by PFGE, whereas most strains were identified by distinct RAPD-PCR profiles and associated according to the geographical origin. Because of its rapidity and reliability, RAPD-PCR appeared to be a suitable method for typing and monitoring O. oeni strains in winemaking. Received: 3 November 1999 / Accepted: 8 December 1999     

Variety and variability of glycosidase activities in an Oenococcus oeni strain collection tested with synthetic and natural substrates

Aims: To evaluate the capacity of Oenococcus oeni strains to release aroma compounds from glycosylated precursors by measuring glycosidase activities with both synthetic and natural substrates. Methods and Results: Five glycosidase activities were investigated in 47 O. oeni strains using synthetic substrates. This screening revealed that activity levels vary considerably, not only for each strain (depending on the substrate tested), but also between strains. Fifteen strains exhibiting different activity profiles were further analysed using natural substrates extracted from both untoasted and toasted oak. In the latter, various amounts of aromatic compounds were measured, thus confirming the specific potentials of the selected strains, but the results were different from those obtained using synthetic substrates. In addition, the use of toasted wood extracts significantly increased the release of wood aromas, which minimized differences between strains. Conclusions: The capability of O. oeni to hydrolysate glycoconjugate aroma precursors is strain‐dependent and variable, depending on the substrate. Significance and Impact of the Study: Instead of synthetic substrates, natural aroma precursors should be used for an adequate evaluation of the glycosidase potential of O. oeni.     

Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking

Aims: To evaluate the ability of grapevine ecosystem fungi to degrade histamine, tyramine and putrescine in synthetic medium and in wines. Methods and Results: Grapevine and vineyard soil fungi were isolated from four locations of Spain and were subsequently identified by PCR. A total of 44 fungi were evaluated for in vitro amine degradation in a microfermentation system. Amine degradation by fungi was assayed by reversed‐phase (RP)‐HPLC. All fungi were able to degrade at least two different primary amines. Species of Pencillium citrinum, Alternaria sp., Phoma sp., Ulocladium chartarum and Epicoccum nigrum were found to exhibit the highest capacity for amine degradation. In a second experiment, cell‐free supernatants of P. citrinum CIAL‐274,760 (CECT 20782) grown in yeast carbon base with histamine, tyramine or putrescine, were tested for their ability to degrade amines in three different wines (red, white and synthetic). The highest levels of biogenic amine degradation were obtained with histamine‐induced enzymatic extract. Conclusion: The study highlighted the ability of grapevine ecosystem fungi to degrade biogenic amines and their potential application for biogenic amines removal in wine. Significance and Impact of Study: The fungi extracts described in this study may be useful in winemaking to reduce the biogenic amines content of wines, thereby preventing the possible adverse effects on health in sensitive individuals and the trade and export of wine.     

Biogenic amine formation by poultry-associated spoilage and pathogenic bacteria

The production of biogenic amines by 50 poultry-associated bacterial strains (25 Pseudomonas , 13 Salmonella and 12 Listeria ) was investigated on amine agar plates containing lysine, histidine, ornithine, phenylalanine, tryptophan and tyrosine. Seventy-four per cent of all the strains produced cadaverine and putrescine, while phenylethylamine, histamine, tyramine and tryptamine were produced by 72, 56, 34 and 24% of strains, respectively. Different patterns of biogenic amine production amongst the three bacterial genera tested were apparent as well as amongst strains of the same genus. This study highlighted a high incidence of biogenic amine-producing bacterial strains associated with poultry.     

Effect of Oleic Acid on Oenococcus oeni Strains and Malolactic Fermentation in Wine

A different capability to assimilate oleic acid from the culture medium has been demonstrated among malolactic Oenococcus oeni strains. Strains possessing higher percentages of oleic acid and its methylated derivative, dihydrosterculic acid, in their fatty acid profile showed higher cell viability and carried out a complete malolactic fermentation after their transfer into a wine lacking oleic acid. Wine supplementation with Tween 80 (polyoxyethylene-sorbitan-mono-oleate) enhanced cell survival of strains with lower capability to assimilate oleic acid and caused cell growth of strains with higher assimilative capacity, suggesting that oleic acid may act in wine as a survival factor for the former strains and as a growth factor for the latter strains. Practical consequences of these findings are also discussed. Received: 24 March 2001 / Accepted: 25 April 2001     

Correlation between indigenous <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strain resistance and the presence of genetic markers

This study reports on monitoring Oenococcus oeni intraspecific diversity evolution during winemaking. Three different wines were monitored. The proportion of O. oeni species was determined by species-specific PCR and O. oeni strains were distinguished by multiplex PCR-RAPD. Each strain was tested by PCR for 16 significant markers revealed by a previous genetic comparison between a strong oenological potential strain and one with poor oenological potential. Population levels and diversity changed according to winemaking stages, oenological practices and the chemical properties of the wine. In all situations, O. oeni was the best-adapted species. Within the O. oeni group, intraspecific strain diversity decreased and the malolactic fermentation was the result of the most resistant strains with the highest number of markers.     

Isolation and characterization of Oenococcus oeni from Aglianico wines

A technological characterization of Oenococcus oeni strains isolated from Aglianico wines was performed to select starter cultures for malolactic fermentation (MLF). One hundred and fifty six O. oeni isolates were extracted from Aglianico wines, and identified by using species-specific PCR. Malolactic activity (MLA), sulphur dioxide (SO₂) resistance, acetaldehyde metabolism and other technological characteristics were tested. Differences in the technologically relevant characteristics were observed. All O. oeni strains were able to grow at low temperature and none in presence of 14% of ethanol. About 80% of O. oeni degraded more than 80% of acetaldehyde, producing ethanol and acetic acid as final products. Among nine O. oeni chosen, four isolates were sensitive to 60 mg of SO₂ l⁻¹, while the other five had high resistance. Considering their technological characteristics, five O. oeni strains could be selected starter cultures for MLF in Aglianico.     

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.     

Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.     

Species attribution and distinguishing strains of Oenococcus oeni isolated from Chinese wines

Summary Twenty-four strains of Oenococcus oeni were isolated from different Chinese wines. Differentiation of isolates was carried out by analysis of total soluble cell protein patterns and random amplified polymorphic DNA (RAPD) patterns. The results indicated that the total soluble cell protein patterns could be used to distinguish different genera but fail to distinguish different strains. It was also found that strain RAPD pattern can successfully distinguish isolates by UPGMA analysis. The RAPD profiles (107 different prints) were strain specific and two main groups of strains were screened.     

A lytic enzyme cocktail from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. B578 for the control of lactic and acetic acid bacteria in wine

Beside yeasts, lactic acid bacteria (LAB) are the most abundant microbes in must during vinification. Whereas Oenococcos oeni is commercially used as a starter culture for the biological acid reduction in wines, other species are responsible for different types of wine spoilage. Members of the genera Pediococcus, Weissella, Leuconostoc, and Lactobacillus are producers of exopolysaccharide slimes, biogenic amines, acetic acid, and other off-flavors. In order to control microbial growth, different procedures such as heating of must and addition of sulfite or lysozyme from egg white are generally applied. Yet, because of health risks, the application of sulfite should be reduced and lysozyme is not effective against all LAB. In this study, we describe exoenzymes from a Streptomyces sp. strain B578 lysing nearly all wine-relevant strains of LAB and Gram-negative acetic acid bacteria. The lytic enzymes were active under wine-making conditions, such as the presence of sulfite and ethanol, low temperatures, and low pH values. The analysis of the exoenzyme composition revealed a synergistic action of different cell wall hydrolases. In conclusion, the lytic cocktail of Streptomyces sp. B578 is a promising tool for the control of wine-spoiling bacteria. This article is dedicated to Prof. Dr. Ferdinand Radler on the occasion of his 80th birthday.     

Biogenic amine production by lactic acid bacteria isolated from cider

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.     

Evolution of malolactic bacteria and biogenic amines during spontaneous malolactic fermentations in a Greek winery

AIMS: To study the population dynamics of indigenous malolactic bacteria in a Greek winery and to examine their potential to produce detrimental levels of biogenic amines (BA) under winemaking conditions. METHODS AND RESULTS: Although the wines studied were of different vintage, grape variety and enological characteristics, molecular typing of malolactic bacteria revealed only a low number of strains within the single-species populations of Oenococcus oeni that developed during spontaneous fermentations. Strain MF1, originating primarily from the vineyards surrounding the winery invariably predominated in almost all samples. HPLC analysis showed a slight increase in the BA, putrescine, tyramine and phenylethylamine after malolactic conversion, while histamine, methylamine and ethylamine remained unaffected. No correlation could be established between the BA profiles and the bacterial compositions or the amino acid concentrations in wine samples studied. CONCLUSIONS: A certain regional bacterial flora is established in the winery that prevails in spontaneous malolactic fermentations (MLF) irrespective of the wine characteristics. In all cases, the BA content of the wines after malolactic conversion was within enologically acceptable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the malolactic bacteria occurring naturally in spontaneous MLF in Greek red wines and a preliminary assessment of their impact on wine safety in relation to BA.     

A mathematical model of the link between growth and L-malic acid consumption for five strains of Oenococcus oeni

In winemaking, after the alcoholic fermentation of red wines and some white wines, L-malic acid must be converted into L-lactic acid to reduce the acidity. This malolactic fermentation (MLF) is usually carried out by the lactic acid bacteria Oenococcus oeni. Depending on the level of process control, selected O. oeni is inoculated or the natural microbiota of the cellar is used. This study considers the link between growth and MLF for five strains of O. oeni species. The kinetics of growth and L-malic acid consumption were followed in modified MRS medium (20 °C, pH 3.5, and 10 % ethanol) in anaerobic conditions. A large variability was found among the strains for both their growth and their consumption of L-malic acid. There was no direct link between biomass productivities and consumption of L-malic acid among strains but there was a link of proportionality between the specific growth of a strain and its specific consumption of L-malic acid. Experiments with and without malic acid clearly demonstrated that malic acid consumption improved the growth of strains. This link was quantified by a mathematical model comparing the intrinsic malic acid consumption capacity of the strains.     

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra

Aims: This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north‐west Spain. Methods and Results: Over three consecutive years (2007, 2008 and 2009), we examined musts and wines from four cellars in different zones of the region. Through biochemical and genetic tests, 459 isolates of lactic acid bacteria (LAB) were identified as the following species: Lactobacillus alvei (0·7%), Lactobacillus brevis (1·7%), Lactobacillus frumenti (0·9%), Lactobacillus kunkeei (12%), Lactobacillus plantarum (6·5%), Lactobacillus pentosus (0·9%), Lactococcus lactis ssp. lactis (3%), Leuconostoc citreum (0·7%), Leuconostoc fructosum (synon. Lactobacillus fructosum) (3·7%), Leuconostoc mesenteroides ssp. mesenteroides (2·8%), Leuconostoc pseudomesenteroides (0·2%), Oenococcus oeni (59%), Pediococcus parvulus (7%) and Weisella paramesenteroides (synon. Leuconostoc paramesenteroides) (0·9%). Of these species, O. oeni was the main one responsible for malolactic fermentation (MLF) in all cellars and years with the exception of Lact. plantarum, predominant in 2007, in one cellar, and Lact. brevis, Lact. frumenti and Ped. parvulus coexisting with O. oeni in one cellar in 2009. Different strains (84) of LAB species (14) were identified by biochemical techniques (API strips, the presence of plasmids, enzyme activities and MLF performance) and molecular techniques (PCR). All assays were carried out with every one of the 459 isolates. To select candidates for use as culture starters, we assessed malolactic, β‐glucosidase and tannase activities, the presence of genes involved in biogenic amine production and plasmid content. Conclusions: A high diversity of LAB is present in the grape musts of Ribeira Sacra but few species are responsible for MLF; however, different strains of such species are involved in the process. As far as we are aware, this is the first report of Lact. frumenti thriving in wine. Significance and Impact of the Study: Information on LAB populations in must and wine is presented. A large collection of well‐characterized strains of LAB are available as starter cultures to winemakers.