Domingo Marquina

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.

PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis

Pichia membranifaciens CYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 degrees C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibit Brettanomyces bruxellensis while Saccharomyces cerevisiae was fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well as P. membranifaciens CYC 1086, was able to inhibit B. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions.

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.

Dietary influence of kefir on microbial activities in the mouse bowel.

Aims: In this work the microflora present in kefir, a fermented milk product, was studied together with the effect of kefir administration on different groups of indigenous bacteria of mouse bowel.

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.

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 degrees C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.

(1→6)‐β‐D‐glucan as the cell wall binding site for Debaryomyces hansenii killer toxin

Aims: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin.

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.