RESUMEN
Malolactic fermentation (MLF), usually induced by Oenococcus oeni (O. oeni), is an important process to improve wine quality. Acid acclimation has been proven to be useful for enhancing the viability of lyophilized O. oeni. To explain the involved mechanisms, cell integrity, morphology and protein patterns of lyophilized O. oeni SD-2a were investigated with acid acclimation. After lyophilization, improvement of cell integrity and more extracellular polymeric substances (EPS) were observed in acid acclimated cells. Combined with GO and KEGG analysis, different abundant proteins were noticeably enriched in the carbohydrate metabolism process, especially amino sugar and nucleotide sugar metabolism. The most significant result was the over-expression of proteins participating in cell wall biosynthesis, EPS production, ATP binding and the bacterial secretion system. This result indicated the important role of acid acclimation on cell envelope properties. In addition, protein response to stress and arginine deiminase pathway were also proven to be over-expressed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Liofilización , Oenococcus/química , Oenococcus/metabolismo , Aclimatación , Adenosina Trifosfato/metabolismo , Amino Azúcares/metabolismo , Proteínas Bacterianas/química , Metabolismo de los Hidratos de Carbono , Membrana Celular/metabolismo , Pared Celular/metabolismo , Electroforesis en Gel Bidimensional , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Citometría de Flujo , Microbiología de Alimentos/métodos , Microscopía Electrónica de Rastreo , Oenococcus/citología , Vino/microbiologíaRESUMEN
The aim of the present study was to evaluate the effects of freeze-drying in the presence of trehalose as a cryoprotectant, followed by incubation in synthetic wine, on surface damage, viability and l-malic acid consumption of the oenological strain Oenococcus oeni UNQOe 73.2. After freeze-drying, no significant differences were observed in the number of viable cells (for both acclimated and non-acclimated cultures) respect to the fresh culture. In contrast, loss of viability was observed after wine incubation for 24â¯h, being acclimated freeze-dried cells the best conditions for this. After the preservation process, small changes in cell morphology were observed by Atomic Force Microscopy (AFM). The Zeta potential and AFM showed that 24â¯h of wine incubation was enough to induce several cell surface modifications. Plate count data allowed us to establish that surface damage is an important factor for loss of viability, regardless of the acclimation treatment. Although the number of surviving O. oeni cells decreased dramatically after incubation in synthetic wine for 15 days, the consumption of l-malic acid was higher than 70%, with freeze-dried cells showing a better performance than fresh cultures. These results demonstrate that O. oeni freeze-dried cultures could be applied to direct wine inoculation, to conduct malolactic fermentation, maintaining its technological properties and reducing the time and costs of the winemaking process.
Asunto(s)
Membrana Celular/patología , Crioprotectores/farmacología , Liofilización/métodos , Malatos/metabolismo , Oenococcus/citología , Trehalosa/farmacología , Vino/microbiología , Aclimatación , Fermentación , Microscopía de Fuerza AtómicaRESUMEN
Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell-loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell-loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels.
Asunto(s)
Fermentación , Malatos/metabolismo , Oenococcus/metabolismo , Alcohol Polivinílico/metabolismo , Vino/microbiología , Células Inmovilizadas/química , Células Inmovilizadas/metabolismo , Hidrogeles/química , Hidrogeles/metabolismo , Oenococcus/química , Oenococcus/citología , Alcohol Polivinílico/química , Vino/análisisRESUMEN
Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.
Asunto(s)
Fermentación/fisiología , Ácido Láctico/metabolismo , Malatos/metabolismo , Oenococcus/genética , Oenococcus/fisiología , Plásmidos/genética , Vino/microbiología , Proteínas Bacterianas/metabolismo , Dosificación de Gen/genética , Genes Bacterianos/genética , Cinética , Datos de Secuencia Molecular , Oenococcus/citología , Oenococcus/crecimiento & desarrollo , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la PolimerasaRESUMEN
AIMS: To study the effect of ethanol on Oenococcus oeni activity at the single cell level. METHODS AND RESULTS: The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l-malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells. CONCLUSION: From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production.