Malic Acid Degradation Research Articles

Potential use of Starmerella bacillaris as fermentation starter for the production of low-alcohol beverages obtained from unripe grapes

To obtain beverages with reduced alcohol content, the use of unripe grapes, with low sugar and high malic acid concentration, was recently explored. Due to the low sugar, ethanol and glycerol production is limited during fermentation affecting important sensory aspects such as the palate fullness of these beverages. The high acidity influences their organoleptic quality, as well.So far, only S. cerevisiae starter, used in conventional fermentations, have been tested in this condition, and no selection has been performed to identify alternative yeasts suitable for unripe grape fermentation. S. bacillaris is known for the low ethanol tolerance, high glycerol and moderate volatile acidity production. Therefore, this non- Saccharomyces yeast have been investigated to improve the quality of low-alcohol beverages. Seven S. bacillaris strains were tested in synthetic musts with different sugar and malic acid levels, mimicking natural ripe and unripe grape musts. In all the tested conditions, S. bacillaris produced higher glycerol than S. cerevisiae. In single-strain fermentation at low sugar and high malic acid no S. bacillaris strains was able to transform all the sugars, although the produced ethanol was lower than that at high sugar condition. Therefore, sequential fermentations with S. cerevisiae were evaluated at low sugar and high malic acid. In this condition all the sugars were consumed and a significant glycerol increase was found. These results were confirmed when sequential fermentations were run in natural unripe grape must. Moreover, an increase in malic acid degradation, with respect to EC1118 single-strain fermentation, was observed.

The oenological interest of fumaric acid: Stop malolactic fermentation and preserve the freshness of wines

One of the problems related to the increase in average temperatures in the wine-growing regions is the lower accumulation of organic acids in the berries. Wine freshness depends to a great extent on its acidity. Herein, the effectiveness of fumaric acid to inhibit malolactic fermentation or to stop it once initiated is evaluated in order to preserve the malic acid content. Different doses of fumaric acid and SO2were tested. The ability of these compounds to inhibit bacterial development and stop the malic acid degradation was tested on a red wine of the varietyVitis viniferaL. cv. Tempranillo whose malic acid content was set at 1.5 g/L. The control wine inoculated with 6 log CFU/mL ofOenococcus oenifinished the malolactic fermentation in 12 days. However, the use of doses equal to or greater than 300 mg/L of fumaric acid delayed the onset of malolactic fermentation for more than 50 days with little degradation of malic acid. In addition, fumaric acid proved to be effective in stopping malolactic fermentation already started where the bacterial count was 7 log CFU/mL. Fumaric acid can be considered as a potent inhibitor of malolactic fermentation.

Schizosaccharomyces pombe Isolation Protocol.

This chapter describes a methodology to isolate yeast strains from Schizosaccharomyces pombe species. The method is based on a selective-differential medium that notably facilitates the isolation of S. pombe. The main difficulty in isolating microorganisms from this genus is their extremely low incidence in nature when they are compared to other microorganisms. The proposed methodology allows isolating and selecting strains from this species for industrial purposes. Methodologies allows detecting the presence of those yeasts when they are considered spoilage microorganisms. Several selective-differential agents based on the basic physiological characteristics of S. pombe species are exposed during the chapter introduction and the use is properly justified. Some of those representative characteristics are its extraordinary resistance to high sugar concentrations, sulfur dioxide, sorbic acid, benzoic acid, acetic acid, or their unique malo-ethanolic fermentation ability. The proposed selective medium is mainly based on S. pombe resistance to the antibiotic actidione and the unusual tolerance to the inhibitory agent benzoic acid compared to possible microorganisms that could produce false-positive results during an isolation process. In addition, malic acid is proposed as the main differential factor due to the exclusive ability of this species to metabolize malic acid into ethanol. This fact allows the detection of malic acid degradation. Cloramphenicol is used to inhibit bacteria growth and liquid media to avoid fungi development.

Biological Demalication and Deacetification of Musts and Wines: Can Wine Yeasts Make the Wine Taste Better?

Grape musts sometimes reveal excess acidity. An excessive amount of organic acids negatively affect wine yeasts and yeast fermentation, and the obtained wines are characterized by an inappropriate balance between sweetness, acidity or sourness, and flavor/aroma components. An appropriate acidity, pleasant to the palate is more difficult to achieve in wines that have high acidity due to an excess of malic acid, because the Saccharomyces species in general, cannot effectively degrade malic acid during alcoholic fermentation. One approach to solving this problem is biological deacidification by lactic acid bacteria or non-Saccharomyces yeasts, like Schizosaccharomyces pombe that show the ability to degrade L-malic acid. Excessive volatile acidity in wine is also a problem in the wine industry. The use of free or immobilized Saccharomyces cells has been studied to solve both these problems since these yeasts are wine yeasts that show a good balance between taste/flavor and aromatic compounds during alcoholic fermentation. The aim of this review is to give some insights into the use of Saccharomyces cerevisiae strains to perform biological demalication (malic acid degradation) and deacetification (reduction of volatile acidity) of wine in an attempt to better understand their biochemistry and enological features.

Characterisation of Non-Saccharomyces Yeasts Using Different Methodologies and Evaluation of their Compatibility with Malolactic Fermentation

Although Saccharomyces cerevisiae is the yeast species predominantly used for alcoholic fermentation, non-Saccharomyces yeast species are also important because they produce secondary metabolites that can contribute to the final flavour and taste of wines. In this study, 37 strains representing seven non-Saccharomyces species were characterised and evaluated for potential use in wine production, as well as for their effects on malolactic fermentation (MLF). Contour-clamped homogeneous electric field (CHEF) gel electrophoresis and matrix-assisted laser desorption ionisation using a time-of-flight mass spectrometer (MALDI-TOF MS) were used to verify species identity and to determine intra-species variation. Extracellular enzyme production, malic acid degradation and the fermentation kinetics of the yeasts were also investigated. CHEF karyotyping and MALDI-TOF MS were useful for identifying and typing Hanseniaspora uvarum, Lachancea thermotolerans, Candida zemplinina (synonym: Starmerella bacillaris) and Torulaspora delbrueckii strains. Only H. uvarum and Metschnikowia pulcherrima strains were found to have β-glucosidase activity. M. pulcherrima strains also had protease activity. Most of the strains showed limited malic acid degradation, and only Schizosaccharomyces pombe and the C. zemplinina strains showed mentionable degradation. In synthetic wine fermentations, C. stellata, C. zemplinina, H. uvarum, M. pulcherrima and Sc. pombe strains were shown to be slow to medium fermenters, whereas L. thermotolerans and T. delbrueckii strains were found to be medium to strong fermenters. The effect of the yeasts on MLF varied, but inhibition was strain dependent.

The effects of co- and sequential inoculation of Torulaspora delbrueckii and Pichia kluyveri on chemical compositions of durian wine.

This is a first study on using two non-Saccharomyces yeasts, Torulaspora delbrueckii Biodiva and Pichia kluyveri FrootZen to produce durian wine via co-inoculation (Co-I) and sequential inoculation (Seq-I). T. delbrueckii inhibited the growth of P. kluyveri and P. kluyveri also partly retarded the growth of T. delbrueckii in Co-I and Seq-I treatments. Co-I and Seq-I produced similar levels of ethanol to T. delbrueckii Biodiva monoculture. In addition, Seq-I increased malic acid degradation and higher succinic acid production. Compared with T. delbrueckii Biodiva, Co-I produced similar amounts of ethyl esters, higher alcohols and moderately increased levels of ethyl acetate. Seq-I 2th (T. delbrueckii inoculated after 2days fermentation with P. kluyveri) and Seq-I 5th produced excessive amounts of ethyl acetate (≥80mg/L) but relatively lower levels of higher alcohols. This study suggested that Co-I could complete alcoholic fermentation with more complex aromas and might be novel way for wine making.

Malic Acid Distribution and Degradation in Grape Must During Skin Contact: The Influence of Recombinant Malo-Ethanolic Wine Yeast Strains

Wine acidity plays an important role in determining wine quality and ensuring physiochemical and microbiological stability. In high-acid wines, the L-malic acid concentration is usually reduced through bacterial malolactic fermentation, while acidulation in low-acidity wines is usually done during final blending of the wine before bottling. This study showed that skin contact did not influence the relative concentration of L-malic acid in the pulp and juice fractions from Colombard, Ruby Cabernet and Cabernet Sauvignon grape musts, with 32%-44% of the L-malic acid present in the pulp fraction. Four recombinant malo-ethanolic (ME) Saccharomyces wine yeast strains containing the malic enzyme (mae2) and malate transporter (mael) genes of Schizasaccharomyces pombe, effectively degraded the L-malic acid in both the juice and pulp fractions of all three cultivars, with a complete degradation of malic acid in the juice fraction within 2 days.

Influence of the dominance of must fermentation by Torulaspora delbrueckii on the malolactic fermentation and organoleptic quality of red table wine

Torulaspora delbrueckii can improve wine aroma complexity, but its impact on wine quality is still far from being satisfactory at the winery level, mainly because it is easily replaced by S. cerevisiae yeasts during must fermentation. New T. delbrueckii killer strains were selected to overcome this problem. These strains killed S. cerevisiae yeasts and dominated fermentation better than T. delbrueckii non-killer strains when they were single-inoculated into crushed red grape must. All the T. delbrueckii wines, but none of the S. cerevisiae wines, underwent malolactic fermentation. Putative lactic acid bacteria were always found in the T. delbrueckii wines, but none or very few in the S. cerevisiae wines. Malic acid degradation was the greatest in the wines inoculated with the killer strains, and these strains reached the greatest dominance ratios and had the slowest fermentation kinetics. The T. delbrueckii wines had dried-fruit/pastry aromas, but low intensities of fresh-fruit aromas. The aroma differences between the T. delbrueckii and the S. cerevisiae wines can be explained by the differences that were found in the amounts of some fruity aroma compounds such as isoamyl acetate, ethyl hexanoate, ethyl octanoate, and some lactones. This T. delbrueckii effect significantly raised the organoleptic quality scores of full-bodied Cabernet-Sauvignon red wines inoculated with the killer strains. In particular, these wines were judged as having excellent aroma complexity, mouth-feel, and sweetness.

Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation.S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used.

Biotransformation of chemical constituents of durian wine with simultaneous alcoholic fermentation by Torulaspora delbrueckii and malolactic fermentation by Oenococcus oeni.

This work represents the first study on the biotransformation of chemical constituents of durian wine via simultaneous alcoholic fermentation (AF) and malolactic fermentation (MLF) with non-Saccharomyces yeast and lactic acid bacteria (LAB), namely, Torulaspora delbrueckii Biodiva and Oenococcus oeni PN4. The presence of PN4 improved the utilization of sugars but did not affect ethanol production. MLF resulted in the significant degradation of malic acid with corresponding increases in pH and lactic acid. The final concentrations of acetic acid (1.29g/L) and succinic acid (3.70g/L) in simultaneous AF and MLF were significantly higher than that in AF (1.05 and 1.31g/L) only. Compared with AF, simultaneous AF and MLF significantly elevated the levels of aroma compounds with higher levels of higher alcohols (isoamyl alcohol, active amyl alcohol, isobutyl alcohol, and 2-phenylethyl alcohol), acetate esters (ethyl acetate, isoamyl acetate), and ethyl esters (ethyl octanoate, ethyl dodecanoate). All the endogenous volatile sulfur compounds decreased to trace or undetectable levels at the end of fermentation. MLF accentuated the reduction of acetaldehyde and sulfides. The initially absent dipropyl disulfide was formed, then catabolized, especially in simultaneous AF and MLF. This study suggested that the simultaneous AF and MLF of non-Saccharomyces and LAB could modify the volatile compositions and potentially modulate the organoleptic properties of durian wine.

Assessment of the genetic polymorphism and physiological characterization of indigenous Oenococcus oeni strains isolated from Aglianico del Vulture red wine.

The aim of this study was a reliable intra-species discrimination and strain biodiversity in Oenococcus oeni populations of two different Aglianico wineries by molecular, biochemical, and physiological characterization. Pulsed field gel electrophoresis (PFGE) analysis revealed a high polymorphism related to the origin (winery) of strains, while differential display PCR (DD-PCR) allowed a further discrimination of strains from the same winery. Moreover, the heterogeneity of these natural populations was investigated by capillary electrophoresis and enzymatic assays. A variability related to a different surface charge distribution was observed among strains, linked to their origin. Malolactic activity study evidenced strain-specific differences in malic acid degradation, and then, only the presence of L(-)-malic acid in the medium induced the mle gene. This study provided evidences on the importance of intra-species biodiversity of malolactic bacterial populations in wine ecosystems, as each wine possess peculiar winemaking conditions and physical-chemical properties which make specific the bacterial survival and growth. This study highlighted a great biodiversity among O. oeni strains that can be also winery specific. Such biodiversity within a certain winery and winemaking area is important for selecting malolactic starters, and strain-specific trait identification is especially important to match individual strains to specific industrial process.

Autochthonous starter cultures and indigenous grape variety for regional wine production

To characterize Oenococcus oeni strains isolated from North-Apulian wines where malic acid degradation is usually achieved by spontaneous fermentations, and to determine the influence of bacterial inoculation time on the malolactic performances in 'Nero di Troia' wine using a complete autochthonous microbial regime. Oenococcus oeni strains from wines produced with the autochthonous (Apulia Region, southern Italy) grape variety 'Uva di Troia' were isolated, selected and characterized. Multilocus sequence typing and variable number tandem repeat analysis were used to investigate intraspecific diversity. Oenococcus oeni strains were tested in co-inoculation and in sequential inoculation, with two autochthonous yeast strains previously isolated from 'Nero di Troia' wine. After a preliminary screening using co-inoculation regime, the O.oeni strains were grouped in reason of the different behaviour in malic acid performances. Results suggested that the efficient degradation of malic acid in co-inoculation is a strain-dependent characteristic. Autochthonous yeast/bacterium combinations were identified as starter culture, and used in a co-inoculation approach, for vinification of regional wines. The 'microbial terroir' of typical fermented food and beverage production represents a dynamic sector of applied research in food microbiology. In this work, we propose the use of autochthonous bacteria and yeast for wine production from an indigenous grape variety.

Photocatalytic degradation of malic acid using a thin coated TiO2‐film: Insights on the mechanism of photocatalysis

Decontamination of opaque fluids using photocatalysts and near Ultraviolet (UV) irradiation involves major technical challenges. This study considers a thin TiO2 layer placed in a new Chemical Reactor Engineering Centre (CREC)‐photoreactor cell. This new photoreactor cell is used for the photocatalytic degradation of malic and malonic acids, typical apple juice components. Conversion of organic species can only proceed through the “dark side” of the TiO2 layer, which is in direct contact with the fluid. Under the selected operating conditions both external mass‐transfer limitations and photolysis are found to be negligible. Macroscopic radiation balance shows that 92% of near UV radiation is absorbed by the ‘back side” of the TiO2‐film. Photocatalytic degradation experiments with 10, 20, 30, and 40 ppm malic acid initial concentrations, show that malonic acid is a main intermediate. Complete malic acid conversion occurs after 5–8 h of irradiation. Kinetic modeling of malic and malonic acid photodegradation with kinetic parameter estimation is performed using both an “in series” and an “in series‐parallel” reaction networks. The “in series‐parallel” reaction network displays better ability for predicting CO2 formation, showing maximum quantum yields of 14.2%. Given that in the CREC‐photoreactor cell with a thin TiO2‐film, photocatalysis can only proceed via the transfer of mobile “h+” sites from the irradiated side to the “dark side', this study demonstrates the significance of this step on the overall photocatalysis mechanism. © 2014 American Institute of Chemical Engineers AIChE J, 60: 3286–3299, 2014

Selection of appropriate Schizosaccharomyces strains for winemaking

This paper describes the selection of Schizosaccharomyces yeasts with adequate oenological suitability and high capacity for the degradation of malic acid. Despite the almost non-existent number of commercial strains, the use of this yeast genus has recently been recommended by the International Organisation of Vine and Wine (OIV, in French). Thus, in the present study, a large number of Schizosaccharomyces strains were isolated using a selective differential medium. Initially, classic parameters of oenological interest for the use of fermentative strains of Saccharomyces cerevisiae (the most frequently used type of yeast) were assessed. Only five strains of moderate acetic acid production lower than 0.4 g/L were obtained at the end of fermentation. Other, more specific features of this yeast genus' physiology were also studied, including urease activity and the production of pyruvic acid and glycerol. Finally, oenological suitability was determined by comparing selected strains with other Schizosaccharomyces reference and S cerevisiae control strains. Schizosaccharomyces strains produced 80% less urea content, four times higher pyruvic acid levels and 1 g higher glycerol contents than the Saccharomyces reference strains. The results confirmed that it is possible to perform selective processes on microorganisms from the genus Schizosaccharomyces using methodology developed in this work to obtain strains of industrial interest.

Effects of chitosan and calcium chloride treatments on malic acid-metabolizing enzymes and the related gene expression in post-harvest pear cv. ‘Huang guan’

Malic acid in the ‘Huang guan’ pear (Pyrus pyrifolia Nakai) is the predominant organic acid associated with taste, flavor and juice quality. The effects of different treatments on malic acid-metabolizing enzymes and the related genes expression during storage have been studied. Malic acid content was higher in the peel than in the pulp. NAD-dependent malate dehydrogenase (NAD-MDH) and NADP-dependent malic enzyme (NADP-ME) correlated with gene expression during cold storage. NAD-MDH promoted malic acid synthesis, whereas NADP-ME promoted malic acid degradation. Chitosan treatment had a significantly positive effect on the activities of NAD-MDH and a negative effect on the activities of NADP-ME both in pulp and peel, and CaCl2 just significantly affected those in peel. The gene expressions of Mdh were not significantly impacted under both treatments, in addition to the gene expression of Me under CaCl2 treatment. An expression analysis of the proton pump gene revealed that vVAtp1 significantly negatively regulated the malic acid content, and there was a significant negative correlation between the vVpp gene and the accumulation of malic acid in the peel. Different treatments inhibited the gene expression of vVAtp2, as well as promoted gene expression of vVAtp1. Overall, both treatments attenuated enzyme activity and malic degradation of pear fruit during storage period.

Effects of co-fermentation by Saccharomyces cerevisiae and Issatchenkia orientalis on sea buckthorn juice

This work relates to the development of a co-fermented product of sea buckthorn (Hippophae rhamnoides L.) with Saccharomyces cerevisiae and Issatchenkia orientalis. Besides malic acid degradation, the parameters of present production technology were also standardized with emphasis on the retainability of total phenolic content (TPC) of sea buckthorn juice. The effect of co-fermentation on physico-chemical characteristics, organic acids, flavonoids, TPC and antioxidant activities was studied. The high-performance liquid chromatography (HPLC) analysis showed 55% reduction in malic acid content after the co-fermentation of sea buckthorn juice. The TPC of sea buckthorn product was 2.18 g gallic acid equivalent (GAE)/l. The estimated scavenging effect on 2,2-diphenyl-1-picrylhydrazyl free radicals was 2.63 Trolox equivalent (TE) mmol/l. Ferric-reducing antioxidant power and 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) assays also showed that sea buckthorn product was on a par with commercial wines (Cabernet Shiraz and Beaujolais). We conclude that the process of co-fermentation resulted in a significant antioxidant potential of sea buckthorn product.

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

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.

Simultaneous yeast–bacteria inoculum. A feasible solution for the management of oenological fermentation in red must with low nitrogen content

The simultaneous inoculum of yeasts and bacteria is a feasible solution for improving fermentation in wines with a harsh chemical composition, capable of inhibiting microbial activity. Considering the risk of wine spoilage due to lactic bacteria, co-inoculum is suggested in white wines with a low pH. However, climate change has also caused problems in achieving malolactic fermentation in red wines, due to the high concentration of ethanol and the low nutrient content. In this work, 5 pairs of commercial oenological starters were tested in simultaneous fermentation, using 4 red musts with a low nitrogen content, and compared with a traditional winemaking process. The simultaneous inoculum caused a slowdown in the activity of yeasts, although no problems in the accomplishment of alcoholic fermentations were observed. More reliable malolactic fermentation was performed in the co-inoculum trials, while, in traditional winemaking, some failures in the degradation of malic acid were observed. Microbiological analyses agreed with these observations. No differences were found in yeast density during alcoholic fermentation, demonstrating the absence of negative interaction between the yeast and the bacteria. However, simultaneous fermentation is not without risks; the highest increases of acetic acid were noted in the co-inoculum trials. The addition of yeast and bacteria to must with a serious lack of nutrients would appear to be a promising alternative to traditional fermentation; however, careful control of the chemical composition of must is mandatory to obtain reliable microbiological activity in the first stages of winemaking.

The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni

The effects of stress shocks on the freeze-drying viability, malolactic activity and membrane fatty acid composition of the Oenococcus oeni SD-2a cells were studied. O. oeni SD-2a cells after 2 h of stress exposure exhibited better freeze-drying viability and malolactic fermentation ability. A decrease in unsaturated fatty acids/saturated fatty acids (UFA/SFA) ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in C19cyc11 relative concentration were observed in all stress shocked cells. There was a significant negative correlation between C19cyc11 and C18:lcis11, C16:0 in all stress shocks. The freeze-drying viability exhibited a significant positive correlation with the levels of C19cyc11 in cold and acid shocks. The only significant positive correlation between the ability of O. oeni SD-2a to conduct malic acid degradation and membrane composition existed with C14:0 in ethanol shocks. In general, freeze-drying viabilities were maximum for cells with low UFA/SFA ratio and high CFA levels, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting stress shocked cells from lyophilization. However, changes observed in membrane fatty acid composition are not enough to explain the greater freeze-drying viability of cells shocked at 8% ethanol. Thus, other mechanisms could be responsible for this increase in the bacterial resistance to lyophilization.

Influence of inoculation time of an autochthonous selected malolactic bacterium on volatile and sensory profile of Tempranillo and Merlot wines

A study was carried out to determine the effect of the inoculation time of the lactic acid bacteria (LAB) on the kinetic of vinification and on chemical and sensory characteristics of Tempranillo and Merlot wines. Traditional vinifications, with LAB inoculated after completion of AF, were compared with vinifications where yeast and bacteria were co-inoculated. Two commercial yeast strains and an autochthonous Oenococcus oeni strain (C22L9) previously identified and selected at our laboratory were used. Monitoring of alcoholic and malolactic fermentations was carried out by yeast and lactic acid bacteria counts and by measuring contents of glucose+fructose, malic acid and lactic acid. The implantation rate of O. oeni C22L9 was calculated by typing isolates obtained from count plates using the RAPD-PCR (Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction) technique. Wines were chemically characterised and analysed for biogenic amine and volatile compound contents. A sensory analysis, consisting in a descriptive and a triangular test was also carried out. Results from this study showed that for both grape varieties, the concurrent yeast/bacteria inoculation of musts produced a significant reduction in duration of the process, without a pronounced degradation of malic acid during AF, nor an excessive increase in volatile acidity. Biogenic amine content was also lower in wines produced by co-inoculation. Important differences in volatile compound contents were observed, although there was little impact on the sensorial profile of wines. These results suggest that co-inoculation using O. oeni C22L9 is a worthwhile alternative compared to traditional post AF inoculation for Tempranillo and Merlot winemaking.