Continuous In-Flight Synthesis for On-Demand Delivery of Ligand-Free Colloidal Gold Nanoparticles.

We demonstrate an entirely new method of nanoparticle chemical synthesis based on liquid droplet irradiation with ultralow (<0.1 eV) energy electrons. While nanoparticle formation via high energy radiolysis or transmission electron microscopy-based electron bombardment is well-understood, we have developed a source of electrons with energies close to thermal which leads to a number of important and unique benefits. The charged species, including the growing nanoparticles, are held in an ultrathin surface reaction zone which enables extremely rapid precursor reduction. In a proof-of-principle demonstration, we obtain small-diameter Au nanoparticles (∼4 nm) with tight control of polydispersity, in under 150 µs. The precursor was almost completely reduced in this period, and the resultant nanoparticles were water-soluble and free of surfactant or additional ligand chemistry. Nanoparticle synthesis rates within the droplets were many orders of magnitude greater than equivalent rates reported for radiolysis, electron beam irradiation, or colloidal chemical synthesis where reaction times vary from seconds to hours. In our device, a stream of precursor loaded microdroplets, ∼15 µm in diameter, were transported rapidly through a cold atmospheric pressure plasma with a high charge concentration. A high electron flux, electron and nanoparticle confinement at the surface of the droplet, and the picoliter reactor volume are thought to be responsible for the remarkable enhancement in nanoparticle synthesis rates. While this approach exhibits considerable potential for scale-up of synthesis rates, it also offers the more immediate prospect of continuous on-demand delivery of high-quality nanomaterials directly to their point of use by avoiding the necessity of collection, recovery, and purification. A range of new applications can be envisaged, from theranostics and biomedical imaging in tissue to inline catalyst production for pollution remediation in automobiles.

Genomic diversity of Oenococcus oeni populations from Castilla La Mancha and La Rioja Tempranillo red wines.

The Oenococcus oeni populations of Tempranillo wines from Castilla La Mancha and La Rioja winemaking regions were analysed from one to three years and up to ten wineries. The objective was to evaluate the genetic variability and the O. oeni population structure. For this purpose a MLST scheme based on four loci (gyrB, purK, pgm and recP genes) and PFGE with SfiI restriction enzyme were developed for later combination. The results showed an O. oeni population completely adapted to winemaking regions. A purifying selection influenced the genes evolution, especially recP that along with purK were the most interesting loci to analyse the genetic variability of the isolates. In this way linkage disequilibrium and intergenic and intragenic recombination were determined between isolates. PFGE typing with UPGMA data were not coincident with the phylograms assessed for MLST by Maximum likelihood and combination of both techniques differentiated all the isolates as strains. Those results led the research to conclude that O. oeni population from CM and LR was a panmictic population with a slight clonal evolution, so subpopulations could not be described. A broader study including more winemaking regions with different grape varieties and distinct ways of elaborating would be interesting to complete the knowledge about O. oeni populations.

Sequential inoculation versus co-inoculation in Cabernet Franc wine fermentation.

A study has been carried out in order to determine the effect of the lactic acid bacteria inoculation time on the kinetic of vinification and on chemical and sensory characteristics of Cabernet Franc wines. Traditional vinifications, with lactic acid bacteria inoculated after completion of alcoholic fermentation were compared with vinifications where yeast and bacteria were co-inoculated at the beginning of vinification. One commercial yeast strain 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 l-malic acid concentration. Wines were chemically characterized and analysed for volatile compounds content. A sensory analysis, consisting of a descriptive and a triangular test, was also carried out. Results from this study showed that the concurrent yeast/bacteria inoculation of musts at the beginning of vinification produced a reduction in duration of the process without an excessive increase in volatile acidity. Differences in volatile compounds content and the corresponding impact on the sensorial profile of wines were also displayed. These results suggest that co-inoculation is a worthwhile alternative for winemaking of Cabernet Franc wines, if compared with traditional post-alcoholic fermentation lactic acid bacteria inoculation.

Microbial communities in air and wine of a winery at two consecutive vintages.

The aim of this study was to assess, both quantitatively and qualitatively, the populations of lactic acid bacteria (LAB) and yeasts in air and wine of a winery, in order to evaluate the possible exchange of microorganisms between them. Samples were taken in a winery located in Castilla-La Mancha (Spain) during the winemaking period of two consecutive vintages (2011 and 2012). The microbial composition was determined by using both a culture-dependent method and a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In addition, genetic characterization of isolates from plates was carried out. A high diversity of species was detected in air and wine samples from both vintages. Leuconostoc mesenteroides was the predominant lactic acid bacteria in air from both vintages while Oenococcus oeni was the predominant in wine. Saccharomyces cerevisiae was the most frequently isolated yeast in both air and wine. Typing of O. oeni and S. cerevisiae isolates from air and wine samples showed the presence of coincident genotypes in both samples, that would confirm the exchange of microorganisms between the two environments, air and wine, and furthermore some of these genotypes were also found at samples taken at different vintages, indicating that they would remain in the winery. The results display the influence of the activity taking place in the winery and the moment of fermentation of the wines in tanks, on the microorganisms present in the air and the role of the air for the dispersal of microorganisms within the winery.

Are Enterococcus populations present during malolactic fermentation of red wine safe?

The aim of this study was the genetic characterisation and safety evaluation of 129 Enterococcus isolates obtained from wine undergoing malolactic fermentation. Genetic characterisation by randomly amplified polymorphic DNA-PCR displayed 23 genotypes. 25 isolates representative of all genotypes were identified as Enterococcus faecium by species-specific PCR and assayed for antibiotic resistance, presence of virulence genes and aminobiogenic capacity, both in decarboxylase medium and wine. The aminobiogenic capacity in wine was analysed in presence (assay 1) and absence (assay 2) of Oenococcus oeni CECT 7621. Resistance to tetracycline, cotrimoxazol, vancomycin and teicoplanin was exhibited by 96% of the strains, but none of them harboured the assayed virulence genes. All of the strains harboured the tyrosine decarboxylase (tdc) gene, while 44% were positive for tyramine in decarboxylase medium. Only five out of 25 strains survived in wine after seven days of incubation, and when concentrations of biogenic amines in wines were determined by HPLC, only those wines in which the five surviving strains occurred contained biogenic amines. Histamine, putrescine and cadaverine were detected in wines from both assays, although concentrations were higher in assay 2. Tyramine and phenylethylamine were detected only in absence of O. oeni. This research contributes for the knowledge of safety aspects of enterococci related to winemaking.

Esterase activity of lactic acid bacteria isolated from malolactic fermentation of red wines.

The goal of this study was to examine the esterase activity of 243 lactic acid bacteria (LAB) strains from wines of different red grape varieties, belonging to the genera Oenococcus, Lactobacillus, Pediococcus and Enterococcus. p-Nitrophenyl octanoate was used as substrate. All strains presented esterase activity in the first screening, but only those showing higher activity were used in subsequent studies to determine the cellular location of this activity, the influence of pH, temperature and the presence of ethanol and the substrate specificity. For the thirteen selected strains, the highest activity was observed in the intracellular fraction. Responses to pH, temperature and ethanol were strain-dependent, but for all the strains, a marked decrease in activity in presence of ethanol was observed. When the influence of pH and ethanol acting together was studied at 25 °C and 37 °C, temperature-dependent differences were not observed for any of the strains except for Oen6. In the substrate specificity assay, the majority of strains of all genera displayed a trend to more readily hydrolyse ester substrates from C8 and longer.

Screening for glycosidase activities of lactic acid bacteria as a biotechnological tool in oenology.

The aim of this study was to evaluate the ability from a number of lactic acid bacteria isolated from different sources to produce glycosidase enzymes. Representative isolates (225) from clusters obtained after genotyping, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis, of 1,464 isolates, were screened for ß-D-glucosidase activity. Thirty-five of them were selected for subsequent analysis. These strains were able to hydrolyze α-D-glucopyranoside, ß-D-xylopyranoside and α-L-arabinofuranoside although ß-D-glucosidase activity was the predominant activity for 22 of the selected strains. Only some of them did so with α-L-rhamnopyranoside. All of these were from wine samples and were identified as belonging to the Oenococcus oeni species using Amplification and Restriction Analysis of 16S-rRNA gene (16S-ARDRA). When the influence of pH, temperature and ethanol or sugars content on ß-D-glucosidase activity was assayed, a strain-dependent response was observed. The ß-D-glucosidase activity occurred in both whole and sonicated cells but not in the supernatants from cultures or obtained after cell sonication. Strains 10, 17, 21, and 23 retained the most ß-D-glucosidase activity when they were assayed at the conditions of temperature, pH, ethanol and sugar content used in winemaking. These results suggest that these strains could be used as a source of glycosidase enzymes for use in winemaking.

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.