Antonio Santos

The Antimicrobial Properties of Different Strains of Lactobacillus spp. Isolated from Kefir

The characteristics of 58 strains of Lactobacillus spp. isolated from kefir were studied. These strains were tested for adherence to human enterocyte-like Caco-2 cells, resistance to acidic pH and bile acid, antimicrobial activities against enteropathogenic bacteria and inhibition of Salmonella typhimurium attachment to Caco-2 cells. The best probiotic properties were observed in L. acidophilus CYC 10051 and L. kefiranofaciens CYC 10058. L. kefiranofaciens CYC 10058 produced an exopolysaccharide, which revealed that it was closely related to kefiran, a polysaccharide with antitumoral properties. This is the first in vitro study about the antimicrobial characteristics of the Lactobacillus population of kefir.

Dynamic analysis of physiological properties of Torulaspora delbrueckii in wine fermentations and its incidence on wine quality

This work examines the physiology of a new commercial strain of Torulaspora delbrueckii in the production of red wine following different combined fermentation strategies. For a detailed comparison, several yeast metabolites and the strains implantation were measured over the entire fermentation period. In all fermentations in which T. delbrueckii was involved, the ethanol concentration was reduced; some malic acid was consumed; more pyruvic acid was released, and fewer amounts of higher alcohols were produced. The sensorial properties of final wines varied widely, emphasising the structure of wine in sequential fermentations with T. delbrueckii. These wines presented the maximum overall impression and were preferred by tasters. Semi-industrial assays were carried out confirming these differences at a higher scale. No important differences were observed in volatile aroma composition between fermentations. However, differences in mouthfeel properties were observed in semi-industrial fermentations, which were correlated with an increase in the mannoprotein content of red wines fermented sequentially with T. delbrueckii.

Ion channel activity by Pichia membranifaciens killer toxin.

The cytocidal effect of Pichia membranifaciens killer toxin on Candida boidinii cells was studied. The halotolerant yeast P. membranifaciens CYC 1106 produces a unique 18 kDa killer toxin that exerts its killer activity against C. boidinii IGC 3430 only in the presence of NaCl. Metabolic events associated with the loss of C. boidinii IGC 3430 viability were quantitatively identical to those known to occur with K1 killer toxin‐treated sensitive strains of Saccharomyces cerevisiae. The death of sensitive cells was characterized by a leakage of potassium, an influx of sodium and a decrease in intracellular pH. These effects occurred prior to and concomitantly with cell death, indicating that they were primary effects of the action of the toxin. Here we report that this protein forms ion‐permeable channels in liposome membranes. These channels are freely permeable to common physiological ions. We suggest that channel formation is the cytotoxic mechanism of action of P. membranifaciens killer toxin. The channels described here are sufficiently non‐selective to mediate cell death through a discharge of cellular membrane potential and changes in ionic homeostasis. No specific effects against killer toxin‐treated sensitive cells were observed when the cell cycle was analysed. Copyright

Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis.

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.

Microbial Contribution to Wine Aroma and Its Intended Use for Wine Quality Improvement

Wine is a complex matrix that includes components with different chemical natures, the volatile compounds being responsible for wine aroma quality. The microbial ecosystem of grapes and wine, including Saccharomyces and non-Saccharomyces yeasts, as well as lactic acid bacteria, is considered by winemakers and oenologists as a decisive factor influencing wine aroma and consumer’s preferences. The challenges and opportunities emanating from the contribution of wine microbiome to the production of high quality wines are astounding. This review focuses on the current knowledge about the impact of microorganisms in wine aroma and flavour, and the biochemical reactions and pathways in which they participate, therefore contributing to both the quality and acceptability of wine. In this context, an overview of genetic and transcriptional studies to explain and interpret these effects is included, and new directions are proposed. It also considers the contribution of human oral microbiota to wine aroma conversion and perception during wine consumption. The potential use of wine yeasts and lactic acid bacteria as biological tools to enhance wine quality and the advent of promising advice allowed by pioneering -omics technologies on wine research are also discussed.

Unraveling the Enzymatic Basis of Wine “Flavorome”: A Phylo-Functional Study of Wine Related Yeast Species

Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early stages of wine fermentation. The high enzymatic potential of these yeasts makes them a useful tool for increasing the final organoleptic characteristics of wines in spite of their low fermentative power. Their physiology and contribution to wine quality are still poorly understood, with most current knowledge being acquired empirically and in most cases based in single species and strains. This work analyzed the metabolic potential of 770 yeast isolates from different enological origins and representing 15 different species, by studying their production of enzymes of enological interest and linking phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes related to terpene aroma release, the β-lyase activity responsible for the release of volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease, polygalacturonase and cellulase activities were also studied in the entire yeast collection, being related to the improvement of different technological and sensorial features of wines. In this context, and in terms of abundance, two different groups were established, with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant activities. By contrast, β-glucosidase and protease activities were widespread in the yeast collection studied. A classical phylogenetic study involving the partial sequencing of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast isolates for further typing, complementing the phylogenetic relationships established by using 26S rDNA. This has rendered it possible to foresee the contribution different yeast species make to wine quality and their potential applicability as pure inocula, establishing species-specific behavior. These consistent results allowed us to design future targeted studies on the impact different non-Saccharomyces yeast species have on wine quality, understanding intra and interspecific enzymatic odds and, therefore, aiming to predict the most suitable application for the current non-Saccharomyces strains, as well as the potential future applications of new strains. This work therefore contributes to a better understanding of the concept of wine microbiome and its potential consequences for wine quality, as well as to the knowledge of non-Saccharomyces yeasts for their use in the wine industry.

Application of Molecular Techniques to the Elucidation of the Microbial Community Structure of Antique Paintings

This paper uses molecular techniques to describe the microstructure and microbiological communities of sixteenth century artwork and their relationships. The microbiological populations, analysed by denaturing gradient gel electrophoresis (DGGE), were highly influenced by the chemical composition of the pictorial layers detected by energy-dispersive X-ray analysis. DGGE revealed that the diversity of microbial communities was lower in pictorial layers composed of pigments with metals, such as Pb, Cu and Hg, than in those found in pictorial layers without such compounds. The number of cultivable microorganisms, mainly fungi and bacteria, was very low in comparison to those found by DGGE, revealing the presence of both cultivable and as-yet-uncultivated (or not viable) species in the samples analysed. Both fungi and bacteria were present in a non-random spatial distribution. Environmental scanning electron microscopy and fluorescent in situ hybridisation analyses revealed that bacterial populations were usually found in close contact with the surface of the pictorial layers, and fungal populations were located on the bacterial biofilm. This work shows, for the first time, the correlation between the diversity of the microbial populations and the chemical composition of the pictorial layers of an artwork.

Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin

PMKT is a channel‐forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2Δ mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT‐affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring‐defective mutants were much less sensitive to PMKT, as were wild‐type protoplasts pretreated with phosphatidylinositol‐specific phospholipase C to remove GPI‐anchored proteins, indicating that the GPI‐anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein‐entrapped liposomes containing a purified GFP–Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein‐free liposomes. Cwp2p and its GPI‐anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.

The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.

25-hydroxycholesterol has a dual effect on the proliferation of cultured rat astrocytes

We examined the effects of 25-OH-cholesterol on the growth of cultured rat astrocytes in the presence of lipoprotein-deficient serum (LPDS). 25-OH-cholesterol at 0.5-8 microM induced an increase in DNA synthesis as measured by [3H]thymidine incorporation into DNA, staining the cells with crystal violet, or counting the number of cells in different phases of the cell cycle by flow cytometry; however, at higher doses, an inhibition of cell proliferation was produced. Similar dose-dependent effects were found in media containing albumin (alone or with added EGF, PDGF, IGF-I or insulin), fetal bovine serum (FBS), or cholesterol-enriched LPDS. Mevalonate, and partially 25-OH-cholesterol, reversed the decrease in cell viability caused by mevinolin (lovastatin). However, mevalonate did not have any effect on 25-OH-cholesterol-stimulated proliferation. Finally, in media with albumin alone or in the presence of fetal bovine serum, growth factors, insulin or forskolin, 25-OH-cholesterol did not affect the expression of either c-fos mRNA or c-fos protein, as measured by real-time quantitative PCR or by Western blot, respectively. These results suggest that 25-OH-cholesterol has a dual effect on the proliferation of cultured rat astrocytes through an AP-1-independent mechanism. This could be of interest for gaining a better knowledge of the pathophysiological processes occurring in these cells.