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The by-products of the food industry are an economic alternative as a source of nutrients to obtain biomass. At the same time, theiruse could solve the environmental problem related to their disposal, which is highly polluting due to their elevated biochemical oxygen demand. In this work, we seek to optimize the production of cellular biomass of two native Patagonian strains of Lactiplantibacillus plantarum (UNQLp 11 and UNQLp155), selected for its oenological and technological properties, using apple pomace (AP), a residue from the juice and cider industry. The supplementation of AP with yeast extract, salts, and Tween 80 (sAP), proved to maintain the growth of the Lpb. plantarum strains, similar to the commercial medium used to grow LAB (De Man, Rogosa, Sharpe, MRS). Cultures grown in sAP medium showed good tolerance to wine conditions (high ethanol content and low pH), demonstrated by its ability to consume L-malic acid. The subsequent inoculation of these cultures in sterile wines (Merlot and Pinot noir) was carried out at laboratory scale, evaluating cell viability and L-malic acid consumption for 21 days at 21 °C. Cultures grown in sAP media showed a similar performance to MRS media. Thus, sAP media proved to be a suitable substrate to grow oenological Lpb. plantarum strains where cultures (with high size inoculums) were able to drive malolactic fermentation, with an L-malic acid consumption higher than 90%.

Spontaneous malolactic fermentation (MLF) in the North Patagonian region of Argentina may have to bear low environmental temperatures, an important stress factor for the lactic acid bacteria (LAB) that conduct this process. Thus, inducing MLF by inoculation of a native starter culture able to lead this biological process at low temperatures is encouraged because it would allow preserving the regional character of the wine. The goal of this work was to obtain autochthonous Oenococcus oeni strains able to consume L-malic acid when MLF takes place at temperatures lower than 15 °C. Among the psychrotrophic isolates obtained from a Pinot noir wine, sixteen O. oeni strains were identified by sequencing the 16S rRNA gene and typing by RAPD PCR, allowing the differentiation of five genotypic groups. After the evaluation of the viability and metabolic performance of the strains in sterile wine at low temperatures, strains UNQOe4 and UNQOe19 were selected to evaluate their behavior in a non-sterile wine at 4 and 10 °C. The strains selected showed good implantation capacity and were able to successfully conduct MLF, suggesting that their use as starter cultures is suitable to improve this biological process in a wine region where environmental temperatures can be low. Fil: Manera, Camila. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Olguin, Nair Temis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tymczyszyn, Emma Elizabeth. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Delfederico, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Bibiloni, Baltasar Horacio. No especifíca; Fil: Caballero, Adriana Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Valdés la Hens, Danay. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina

Malolactic fermentation (MLF) is a process in winemaking responsible for the conversion of L-malic acid to L-lactic acid and CO 2 , which reduces the total acidity, improves the biological stability, and modifies the aroma profile of wine. MLF takes place during or after alcoholic fermentation and is carried out by one or more species of lactic acid bacteria (LAB), which are either present in grapes and cellars or inoculated with malolactic starters during the winemaking process. Although the main bacterium among LAB used in commercial starter cultures for MLF has traditionally been Oenococcus oeni, in the last decade, Lactobacillus plantarum has also been reported as a malolactic starter, and many works have shown that this species can survive and even grow under harsh conditions of wine (i.e., high ethanol content and low pH values). Furthermore, it has been proved that some strains of L. plantarum are able to conduct MLF just as efficiently as O. oeni. In addition, L. plantarum exhibits a more diverse enzymatic profile than O. oeni, which could play an important role in the modification of the wine aroma profile. This enzymatic diversity allows obtaining several starter cultures composed of different L. plantarum biotypes, which could result in distinctive wines. In this context, this review focuses on showing the relevance of L. plantarum as a MLF starter culture in winemaking. Fil: Brizuela, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Tymczyszyn, Emma Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Valdes la Hens, Danay. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Delfederico, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Hollmann, Axel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentina Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

This article belongs to the Special Issue Food Wastes: Feedstock for Value-Added Products 2.0., Lactiplantibacillus plantarum strain UNQLp 11 is a lactic acid bacterium with the potential to carry out malolactic fermentation (MLF) in red wines. Recently, the complete genome of UNQLp 11 was sequenced and this strain possesses four loci of the enzyme β-glucosidase. In order to demonstrate that these glucosidase enzymes could be functional under harsh wine conditions, we evaluated the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (p-NPG) in synthetic wine with different ethanol contents (0%, 12%, and 14% v/v) and at different pH values (3.2, 3.5, and 3.8). Then, the hydrolysis of precursor n-octyl β-D-glucopyranoside was analyzed in sterile Pinot Noir wine (containing 14.5% v/v of ethanol, at different pH values) by headspace sorptive extraction gas chromatography-mass spectrometry (HSSE-GC/MS). The hydrolysis of p-NPG showed that β-glucosidase activity is very susceptible to low pH but induced in the presence of high ethanol content. Furthermore, UNQLp 11 was able to release the glycosilated precursor n-octyl, during MLF to a greater extent than a commercial enzyme. In conclusion, UNQLp 11 could improve the aromatic profile of the wine by the release of volatile precursors during MLF., This research was funded by Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, for the funding (COOPB20346 international cooperation Project), grants from Universidad Nacional de Quilmes (Buenos Aires, Argentina), Agencia Nacional de Promoción Científica y Técnica (ANPCyT; PICT 2016 N°3435), and CONICET (PIP-2017–11220170100898C O).

Encapsulation of bacterial cultures by freeze-drying enables the storage and distribution of microorganisms in a reduced volume, avoiding the necessity of using freezing temperatures. Here we describe the encapsulation by freeze-drying process of lactic acid bacteria in different sugar matrices (sucrose, trehalose, or maltodextrin) to be used in the food industry.

Lactobacillus plantarum UNQLp 11 strain was isolated from a Patagonian Pinot noir wine at the oldest commercial winery (110 years old) in General Roca, North Patagonia, Argentina, and has demonstrated its ability to survive during winemaking processes and successfully carry out malolactic fermentation. This work aimed to obtain the whole assembled genome of the UNQLp 11 strain, analysing its architecture and the possible functions of the predicted genes from the oenological properties of this strain. The genome size is 3 534 932 bp, with a mean GC content of 44.2%, 3 412 CDS, 80 transposons and 148 tandem repeats. A comparison between the genome size and gene content of 14 Lb. plantarum strains from different origins was performed, and UNQLp 11 exhibited the largest size. The in silico genome-wide analysis allowed us to confirm the existence of genes encoding enzymes involved in the synthesis of several metabolites of oenological interest, in addition to bacteriocins and exopolysaccharides. Furthermore, it is possible to speculate on this strain’s adaptation to different environments, as it is able to use diverse substrates for its growth. All these features suggest the potential of UNQLp 11 to be a good starter culture for malolactic fermentation. Fil: Iglesias, Nestor Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Instituto de Microbiología Básica y Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Brizuela, Natalia Soledad. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tymczyszyn, Emma Elizabeth. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hollmann, Axel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Centro de Investigación en Biofísica Aplicada y Alimentos. - Universidad Nacional de Santiago del Estero. Centro de Investigación en Biofísica Aplicada y Alimentos; Argentina Fil: Valdes la Hens, Danay. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The aim of this work was to study the protective effect of sucrose, trehalose and glutamate during freezing and freeze-drying of three oenological L. plantarum strains previously acclimated in the presence of ethanol. The efficiency of protective agents was assessed by analyses of membrane integrity and bacterial cultivability in a synthetic wine after the preservation processes. No significant differences in the cultivability, with respect to the controls cells, were observed after freezing at -80 ºC and -20 ºC, and pre-acclimated cells were more resistant to freeze-drying than non-acclimated ones. The results of multiparametric flow cytometry showed a significant level of membrane damage after freeze-drying in two of the three strains. The cultivability was determined after incubation in wine-like medium containing 13 or 14% v/v ethanol at 21 °C for 24 h and the results were interpreted using principal component analysis (PCA). Acclimation was the most important factor for preservation, increasing the bacterial resistance to ethanol after freezing and freeze-drying. Freeze-drying was the most drastic method of preservation, followed by freezing at -20 °C. The increase of ethanol concentration from 6 to 10% v/v in the acclimation medium improved the recovery of two of the three strains. In turn, the increase of ethanol content in the synthetic wine led to a dramatic decrease of viable cells in the three strains investigated. The results of this study indicate that a successful inoculation of dehydrated L. plantarum in wine depends not only on the use of protective agents, but also on the cell acclimation process prior to preservation, and on the ethanol content of wine. Fil: Bravo Ferrada, Barbara Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Brizuela, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Gerbino, Oscar Esteban. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Gomez Zavaglia, Andrea. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Tymczyszyn, Emma Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina

The production of malolactic starter cultures requires the obtention of suitably large biomass at low-cost. In this work it was possible to obtain a good amount of biomass, at laboratory scale, of two enological strains of Lb. plantarum, by formulating a culture medium based on whey permeate (WP), a by-product of the cheese industry usually disposed as waste, when this was supplemented with yeast extract (Y), salts (S) and Tween 80 (T) (WPYST). Bacteria grown in WPYST medium exhibited good tolerance to stress conditions of synthetic wine (pH 3.5, ethanol 13% vol/vol). However, when WPYST was added with 8% vol/vol ethanol, cultures inoculated in synthetic wine, showed a lower viability and capacity to consume L-malic acid than when they were cultured in WPYST without ethanol. Subsequently, strains grown in WPYST were inoculated in sterile wine samples (final stage of alcoholic fermentation) of the red varietals Merlot and Pinot noir, and incubated at laboratory scale. Cultures from WPYST, inoculated in Pinot noir wine, showed a better performance than bacteria grown in MRS broth, and exhibited a consumption of L-malic acid higher than 90%. However, cultures from WPYST or from MRS broth, inoculated in sterile Merlot wine, showed a lower survival. This study allowed the formulation of a low-cost culture medium, based on a by-product of the food industry, which showed to be adequate for the growth of two enological strains of Lb. plantarum, suggesting their potentiality for application in the elaboration of malolactic starter cultures.

Oenococcus oeni UNQOe19 is a native strain isolated from a Patagonian pinot noir wine undergoing spontaneous malolactic fermentation. Here, we present the 1.83-Mb genome sequence of O. oeni UNQOe19, the first fully assembled genome sequence of a psychrotrophic strain from an Argentinean wine.

© 2018 Elsevier Inc. All rights reserved., 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 24 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 24 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., This work was funded by grants from Universidad Nacional de Quilmes, Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT – MINCyT, Argentina, PICT PICT 2013-1481, 2014-1395 and PICT 2016-3435), Comisión Nacional de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-BA, Argentina), and Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal). NB has fellowships from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). BMBF, EET and AH are members of the Carrera del Investigador Científico y Tecnológico (CONICET, Argentina). LS is a member of the Research Career of Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-BA, Argentina).

The ability of Patagonian L. plantarum and O. oeni strains to change the volatile profile of a sterile Pinot noir wine was studied through fermentation assays, at laboratory scale. Two strains of each LAB species were selected based on data regarding to their ability to survive in wine and to consume L-malic acid. Both O. oeni strains but only one L. plantarum (UNQLp 11) strain were able to remain viable, consuming L-malic acid through the fermentation assay with a concomitant increase of L-lactic acid. The volatile profile of Pinot noir wine, before and after LAB inoculation, was measured by using HS-SPME gas chromatography technique. This analysis showed that alcohols were the main volatile compounds after alcoholic fermentation and that after fermentation with the selected LAB strains, a decrease in the volatile alcohols concentration and an increase in the volatile esters concentration could be observed. The O. oeni UNQOe 73.2 strain produced the most notable change in the volatile profile, with the production of some important odorant esters at higher concentration, compared to O. oeni UNQOe 31b strain. Although, L. plantarum UNQLp 11 strain showed a better performance in the consumption of L-malic acid, this strain had a low capacity to modify the volatile compounds profile after incubation in red wine. The results found in the present work showed that different strains selected as potential malolactic starters could have different behavior when are incubated in real wine. Although L. plantarum UNQLp 11 strain showed a good consumption of L-malic acid, the O. oeni UNQOe 73.2 strain exhibited superior capacity to improve the flavor of wine due to its esterase activity that produce an increase of fruity and creamy odorants., This work was funded by grants from Universidad Nacional de Quilmes (Programa Microbiología Molecular Básica y Aplicada res No. 954/17), Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-PBA), and ANPCyT (PICT 2014 No. 1395, PICT 2016 No. 3435). NB is fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). LS is a member of the Research Career of CIC-BA; BMBF, ET are members of the Research Career of CONICET.

The performance of Patagonian Lactobacillus plantarum and Oenococcus oeni strains as malolactic starter cultures was compared. Two autochthonous strains of each species were selected, based on the presence of aroma-related genes, and inoculated in sterile wine of high ethanol content. The effects of initial inoculum size and acclimation treatment on the efficiency of malolactic fermentation (MLF) were analyzed for each strain. O. oeni strains were able to successfully conduct the MLF only when the inoculum concentration was higher than 1.108 CFU/mL and cells were acclimated in sublethal ethanol concentrations. The increase of ethanol concentration in the acclimation medium also improved the kinetics of malic acid consumption. Successful MLF with L. plantarum strains required lower inocula and no acclimation treatment. In addition, these strains showed a better profile of aroma-related genes than O. oeni. L. plantarum strains appeared to be more efficient than O. oeni strains as candidates for malolactic starter cultures to be used in Patagonian red wines. Fil: Brizuela, Natalia Soledad. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Valdes la Hens, Danay. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hollmann, Axel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Delfederico, Lucrecia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caballero, Adriana Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina Fil: Tymczyszyn, Emma Elizabeth. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina

Background and Aims Information regarding the composition and dynamics of microbial communities throughout winemaking is always valuable to control the process and contributes to improving the quality of wine. The aims of this work were to analyse the diversity of lactic acid bacteria (LAB) species present during the spontaneous malolactic fermentation (MLF) of Patagonian red wines, and to isolate and identify native LAB species. Methods and Results Samples of Merlot and Pinot Noir wines were collected during three vintages at one commercial cellar. Through polymerase chain reaction (PCR)-rpoB/denaturing gradient gel electrophoresis (DGGE), it was possible to identify 15 LAB species and through PCR-16S rRNA V3 region/DGGE, 11 LAB species, during spontaneous MLF of these wines, mostly, Oenococcus oeni and Lactobacillus plantarum. These two species were also largely recovered by culture and analysed by the random amplified polymorphic DNA–PCR technique. Isolates of these two LAB species exhibited considerable genotypic heterogeneity. Conclusions The prevalence of O. oeni and Lb. plantarum in all samples suggests that both species are involved in leading the spontaneous MLF of these Patagonian wines. The analysis of the LAB microbiota in wine by PCR-DGGE using two gene regions enabled addition to the number of species that can usually be detected during MLF. Significance of the Study The study offered a more complete knowledge of the LAB community present in wines in a cellar in the Argentine Patagonia during the spontaneous MLF. Our findings are particularly relevant, because they contribute to the possibility of designing an MLF starter composed of native strains of Lb. plantarum and O. oeni.

The aim of this study was to evaluate fifty-three Lactobacillus plantarum isolates obtained from a Patagonian red wine, molecularly identified and typified using RAPD analysis, in order to select starter cultures for malolactic fermentation (MLF). The results obtained suggest a considerable genetic diversity, taking into account that all L. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentration of 14 % ethanol in MRS broth for 2 days, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase activities were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous Oenococcus oeni strain was included with comparative purposes. Differences in technologically relevant characteristics were observed among the eight L. plantarum selected isolates, revealing an isolate-dependent behavior. Detectable glucosidase and tannase activities were found in all isolates. The presence of genes encoding histidine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of L. plantarum isolates to grow and consume L-malic acid in simulated laboratory-scale vinifications revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterization.

During winemaking, complex microbial interactions take place and microorganisms showing a selective advantage emerge in a given period as the dominant populations. At the beginning, yeasts, responsible for the alcoholic fermentation (AF), consume sugars present in grapes to yield ethanol and carbon dioxide, leading the transformation of must into wine. The tolerance of lactic acid bacteria (LAB) to low pH and high ethanol are the main factors that select their occurrence in winery ecosystems. LAB guide a secondary biological process, the malolactic fermentation (MLF), which produces deacidification of wine, enhancing its microbial stability and modifying the wine aroma profile. When MLF takes place spontaneously, it is carried out by one or more species of indigenous LAB present in grapes and cellars, naturally adapted to the regional peculiarities of wine. Thus, it is highly advisable to study the indigenous microbiota, best adapted to the agro-ecological conditions of a specific wine-producing area, to select the most representative strains with terroir characteristics for their use as starter cultures. In this chapter, we summarize the studies conducted so far on LAB population diversity in some Patagonian wines, as well as the methods and criteria to select potential indigenous malolactic cultures for these wines, including the adaptation to processing conditions.

Pinot noir vinifications carried out at industrial scale during 2008 vintage were monitored in Patagonian region, Argentina, and several lactic acid bacteria were obtained. By analysis of a fragment of rpo B gene, species- specific PCR of malolactic enzyme and sequencing of 16S rRNA gene, three isolates were identified as Oenococcus oeni. Polyphasic typing made by carbohydrate fermentation behaviour and RAPD-PCR grouped the isolates and O. oeni reference strain in three different clusters. Additionally, the effect of ethanol on bacterial growth and malolactic activity was evaluated. In spite of some inhibitory effects were found, the three isolates were able to growth in all ethanol concentration tested. A clear correlation was found between the different clusters obtained and their ethanol tolerance. All indigenous O. oeni showed a strong malolactic activity at all ethanol concentrations assayed, indicating that this alcohol just affect the growing parameters of bacteria, but not the malolactic activity. These promissory results suggest a potential enological application of the O. oeni indigenous isolates as malolactic fermentation starters in winemaking. Keywords- Pinot noir wine, Argentinean North Patagonia, malolactic fermentation, Oenococcus oeni

Five Oenococcus oeni strains, selected from spontaneous malolactic fermentation (MLF) of Patagonic Pinot noir wine, were assessed for their use as MLF starter cultures. After the individual evaluation of tolerance to some stress conditions, usually found in wine (pH, ethanol, SO2, and lysozyme), the behavior of the strains was analyzed in MLO broth with 14 % ethanol and pH 3.5 in order to test for the synergistic effect of high ethanol level and low pH and, finally, in a wine-like medium. Although the five strains were able to grow in MLO broth under low pH and/or high ethanol, they must be acclimated to grow in a wine-like medium. Additionally, glycosidase and tannase activities were evaluated, showing differences among the strains. The potential of the strains to ferment citrate was tested and two of the five strains showed the ability to metabolize this substrate. We did not detect the presence of genes encoding histidine, tyrosine descarboxylase, and putrescine carbamoyltransferase. All the strains tested exhibited good growth capacity and ability to consume l-malic acid in a wine-like medium after cell acclimation, and each of them showed a particular enzyme profile, which might confer different organoleptic properties to the wine. Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hollmann, Axel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Brizuela, Natalia Soledad. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Valdes la Hens, Danay. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tymczyszyn, Emma Elizabeth. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina

© 2015 The Society for Applied Microbiology, Aims: In this work, we evaluated freeze-drying damage at the surface level of oenological strain Lactobacillus plantarum UNQLp155, as well as its ability to grow in a synthetic wine with and without pre-acclimation. Methods and Results: Damage on cell surface was studied by flow cytometry, zeta potential and atomic force microscopy, and cell survival was analysed by plate count. Results showed that beside cells acclimated at lower ethanol concentration (6% v/v) became more susceptible to drying than nonacclimated ones, after rehydration they maintain their increased ability to grow in a synthetic wine. Acclimation at a higher ethanol concentration (10% v/v) produces several damages on the cell surface losing its ability to grow in a synthetic wine. Conclusions: In this work, we showed for the first time that sublethal alterations on bacterial surface induced by a pre-acclimation with a low ethanol concentration (6%), upon a freeze-drying process, result in a better bacterial adaptation to the stress conditions of wine-like medium, as well as to the preservation process. Significance and Impact of the Study: Understanding the adaptation to ethanol of oenological strains and their effects on the preservation process has a strong impact on winemaking process and allows to define the most appropriate conditions to obtain malolactic starters cultures., This work has been funded by grants from Universidad Nacional de Quilmes (Programa Microbiología Molecular Básica y Aplicaciones Biotecnológicas, Argentina), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT – MINCyT, Argentina; PICTO-UNQ 2006 N-36474 and PICT SU 2012 N° 2804), Comisión Nacional de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-BA, Argentina) and Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCTMEC, Portugal). Bravo-Ferrada, B.M. has a fellowship from the Consejo Nacional de Investigaciones Científicasy Técnicas (CONICET). AH and EET are members of the Carrera del Investigador Científico y Tecnológico (CONICET, Argentina). LS is member of the Research Career of Comisión de Investigaciones Científicas de la Provinciade Buenos Aires (CIC-BA).

The aim of this work was to evaluate the changes due to acclimation to ethanol on the fatty acid composition of three oenological Lactobacillus plantarum strains and their effect on the resistance to ethanol and malic acid consumption (MAC). Lactobacillus plantarum UNQLp 133, UNQLp 65.3 and UNQLp 155 were acclimated in the presence of 6 or 10% v/v ethanol, for 48 h at 28°C. Lipids were extracted to obtain fatty acid methyl esters and analysed by gas chromatography interfaced with mass spectroscopy. The influence of change in fatty acid composition on the viability and MAC in synthetic wine was analysed by determining the Pearson correlation coefficient. Acclimated strains showed a significant change in the fatty composition with regard to the nonacclimated strains. Adaptation to ethanol led to a decrease in the unsaturated/saturated ratio, mainly resulting from an increase in the contribution of short-length fatty acid C12:0 and a decrease of C18:1. The content of C12:0 was related to a higher viability after inoculation of synthetic wine. The MAC increased at higher contents in saturated fatty acid, but its efficiency was strain dependent. Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gomez Zavaglia, Andrea. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Semorile, Liliana Carmen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina Fil: Tymczyszyn, Emma Elizabeth. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Aims: The aim of this work was to evaluate the effect of acclimation on the viability, membrane integrity and the ability to consume malic acid of three oenological strains of Lactobacillus plantarum. Methods and Results: Cultures in the stationary phase were inoculated in an acclimation medium (Accl.) containing 0, 6 or 10% v/v ethanol and incubated 48 h at 28°C. After incubation, cells were harvested by centrifugation and inoculated in a synthetic wine, containing 14% v/v ethanol and pH 3 5 at 28°C. Viability and membrane integrity were determined by flow cytometry (FC) using carboxyfluorescein diacetate (cFDA) and propidium iodide. Bacterial growth and malic acid consumption were monitored in a synthetic wine during 15 days. In nonacclimated strains, the damage of bacterial membranes produced a dramatic decrease in microbial viability in synthetic wine. In contrast, survival of strains previously acclimated in Accl. with 6 and 10% v/v ethanol was noticeable higher. Therefore, acclimation with ethanol increased the cultivability in synthetic wine and consequently, the consumption of L-malic acid after 15 days of growth. Conclusion: Acclimation of oenological strains in media containing ethanol prior to wine inoculation significantly decreases the membrane damage and improves viability in the harsh wine conditions. The role of membrane integrity is crucial to warrant the degradation of L-malic acid. Significance and Impact of the Study: The efficiency of multiparametric FC in monitoring viability and membrane damage along with the malic acid consumption has a strong impact on winemaking because it represents a useful tool for a quick and highly reliable evaluation of oenological parameters. Fil: Bravo Ferrada, Barbara Mercedes. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tymczyszyn, Emma Elizabeth. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Gomez Zavaglia, Andrea. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina Fil: Semorile, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Microbiología Molecular; Argentina