Estudi de lisogènia de leuconostoc oenos i incidència dels bacterioags sobre fermentació malolàctica del vi

Un gran nombre de soques de Oenococcus oeni aïllades de vins van ser analitzades per determinar si eren lisogèniques. Per això es va utilitzar la mitomicina C com agent mutagen inductor del cicle litic dels fags.Els resultats obtinguts demostres que quasi la mitat de le població de bactèries làctics analitzades son portadores d'un profag (lisogèniques) la qual cosa fa pensar que la lisogènia esta molt estesa entre les bactèries làctiques responsables de la fermentació Malolàctica. La sensibilitat de les diferents soques envers el fag es diferent en cada cas. Totes les soques son resistents a l'atac del seu propi fag. La morfologia dels fags es de una càpside isométrica, cua no contractil i sense especules. S'ha pogut identificar una soca indicatriu, sensible al 90% dels fags aïllats i que detecta la presència de fags en el vi durant la fermentació malolàctica.A large number of strains of Oenococcus oeni (formerly Leuconostoc oenos) that had been isolated from wines were checked for lisogeny with mitomicyn C as inducer. As a result of this test, half of the strains proved to be lisogenic, suggesting that lisogeny 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 O. 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

Acetic acid bacteria in oenology

Els bacteris de l'àcid acètic han estat sempre considerats perjudicials en els àmbits enològics, com a principal font de problemes en el vi (fonamentalment per ser una font d'acidesa volàtil). El desenvolupament de noves tècniques de biologia molecular ha permès que la taxonomia i el coneixement del metabolisme d'aquests bacteris que, per les seves exigències nutricionals, són molt difícils de cultivar evolucionin ràpidament. En l'àmbit taxonòmic, s'ha produït el canvi de dos gèneres i cinc espècies el 1984 a deu gèneres i més de quaranta espècies al moment actual. De totes maneres, les poderoses eines moleculars utilitzades en taxonomia no són apropiades per a l'ús rutinari en estudis ecològics, on s'ha d'analitzar un gran nombre de mostres. Per tant, s'han desenvolupat noves tècniques moleculars que han permès la millora del seu coneixement i control a Enologia. Així mateix, han permès un avenç considerable en la producció de vinagre, procés en el qual aquests bacteris són imprescindibles, com s'ha posat de manifest en el projecte europeu WINEGAR.Acetic acid bacteria have always been considered the bad microorganisms of oenology; responsible for wine spoiling (vinegary taint). The taxonomy and our knowledge of the metabolism of acetic acid bacteria are rapidly evolving, especially as new molecular biology techniques are applied to this fastidious group of microorganisms, which are still rather difficult to work with. The dramatic change that acetic acid bacteria taxonomy has undergone can be summarized by the transformation of 2 genera and 5 species in 1984 into 10 genera and over 40 species at present. The powerful molecular tools used in taxonomy are not appropriate for frequent use in identification and ecological studies; yet new molecular tools for routine analysis have also been developed. These provide new insights and means of controlling this microbial group. Furthermore, these advances have improved vinegar production; a process where the presence of acetic acid bacteria is essential. The WINEGAR European Project is evidence of these improvements in vinegar production

Acetic acid bacteria in oenology

Els bacteris de l'àcid acètic han estat sempre considerats perjudicials en els àmbits enològics, com a principal font de problemes en el vi (fonamentalment per ser una font d'acidesa volàtil). El desenvolupament de noves tècniques de biologia molecular ha permès que la taxonomia i el coneixement del metabolisme d'aquests bacteris que, per les seves exigències nutricionals, són molt difícils de cultivar evolucionin ràpidament. En l'àmbit taxonòmic, s'ha produït el canvi de dos gèneres i cinc espècies el 1984 a deu gèneres i més de quaranta espècies al moment actual. De totes maneres, les poderoses eines moleculars utilitzades en taxonomia no són apropiades per a l'ús rutinari en estudis ecològics, on s'ha d'analitzar un gran nombre de mostres. Per tant, s'han desenvolupat noves tècniques moleculars que han permès la millora del seu coneixement i control a Enologia. Així mateix, han permès un avenç considerable en la producció de vinagre, procés en el qual aquests bacteris són imprescindibles, com s'ha posat de manifest en el projecte europeu WINEGAR.Acetic acid bacteria have always been considered the bad microorganisms of oenology; responsible for wine spoiling (vinegary taint). The taxonomy and our knowledge of the metabolism of acetic acid bacteria are rapidly evolving, especially as new molecular biology techniques are applied to this fastidious group of microorganisms, which are still rather difficult to work with. The dramatic change that acetic acid bacteria taxonomy has undergone can be summarized by the transformation of 2 genera and 5 species in 1984 into 10 genera and over 40 species at present. The powerful molecular tools used in taxonomy are not appropriate for frequent use in identification and ecological studies; yet new molecular tools for routine analysis have also been developed. These provide new insights and means of controlling this microbial group. Furthermore, these advances have improved vinegar production; a process where the presence of acetic acid bacteria is essential. The WINEGAR European Project is evidence of these improvements in vinegar production

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

Characterization of a new virulent phage infecting the lactic acid bacterium Oenococcus oeni

International audiencePhi OE33PA is a new virulent siphophage infecting Oenococcus oeni that was isolated from a red wine collected in Pauillac (France). Although the phage could not lysogenize its host, a conserved sequence within the integrase genes harbored by oenophages could be detected, and corresponded to a B-type integrase. The phage host range encompassed ten out of the 38 O. oeni strains tested. One-step growth kinetics revealed latent and burst periods of 4 and 5 h, respectively, with a burst size of about 45 plaque-forming units per infected cell. The phage had a distinctive restriction profile when compared with Phi 10 MC, another B-type oenophage previously isolated in our laboratory. Incubation in wine could inactivate high-titer suspensions of FOE33PA in a short time at room temperature. However, encapsidated phage DNA could still be detected by real time PCR, and these non-infectious viruses dominated the wine samples showing that direct enumeration of phages in wine samples using the double-layer agar technique only informs about the quantity of residual infectious phages. Kinetics studies throughout the fermentation using both qPCR as well as plating should now provide reliable understanding of the phage dynamics during wine making

Bacteriophages of Leuconostoc, Oenococcus and Weissella

Leuconostoc (Ln.), Weissella and Oenococcus form a group of related genera of lactic acid bacteria, which once all shared the name Leuconostoc. They are associated with plants, fermented vegetable products, raw milk, dairy products, meat and fish. Most of industrially relevant Leuconostoc strains can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore bacteriophages attacking Leuconostoc strains may negatively influence the production process. Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using electron microscopy belong to the Siphoviridae family and differ in morphological details. Hybridization and comparative genomic studies of Leuconostoc phages suggest that they can be divided into several groups, however overall diversity of Leuconostoc phages is much lower as compared to e.g. lactococcal phages. Several fully sequenced genomes of Leuconostoc phages have been deposited in public databases. Lytic phages of Leuconostoc can be divided into two host species-specific groups with similarly organized genomes that shared very low nucleotide similarity. Phages of dairy Leuconostoc have rather limited host-ranges. The receptor binding proteins of two lytic Ln. pseudomesenteroides phages have been identified. Molecular tools for detection of dairy Leuconostoc phages have been developed. The rather limited data on phages of Oenococcus and Weissella show that i) lysogeny seems to be abundant in Oenococcus strains, and ii) several phages infecting Weissella cibaria are also able to productively infect strains of other Weissella species and even strains of the genus Lactobacillus