Salvador Vallés

Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits.

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.

Screening and typing of Patagonian wine yeasts for glycosidase activities.

Aims:  The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in industrial production of Patagonian red wines.

Aroma improving in microvinification processes by the use of a recombinant wine yeast strain expressing the Aspergillus nidulans xlnA gene.

A recombinant wine yeast strain has been constructed expressing the gene coding for beta-(1,4)-endoxylanase from Aspergillus nidulans under the control of the yeast actin gene promoter. The resulting recombinant strain is able to secrete active xylanase enzyme into the culture medium. Wines obtained by microvinification with the control and the recombinant wine yeast strain did not differ in their physicochemical characteristics although an increase in fruity aroma was organoleptically detected in the wine produced by the recombinant yeast. Also, an increase in the concentration of some esters, higher alcohols and terpenes was observed in the case of the recombinant strain.

Monitoring a mixed starter of Hanseniaspora vineae-Saccharomyces cerevisiae in natural must: impact on 2-phenylethyl acetate production.

The effect of simultaneous or sequential inoculation of Hanseniaspora vineae CECT 1471 and Saccharomyces cerevisiae T73 in non-sterile must on 2-phenylethyl acetate production has been examined. In both treatments tested, no significant differences in Saccharomyces yeast growth were found, whereas non-Saccharomyces yeast growth was significantly different during all days of fermentation. Independently of the type of inoculation, S. cerevisiae was the predominant species from day 3 till the end of the fermentation. The dynamics of indigenous and inoculated yeast populations showed H. vineae to be the predominant non-Saccharomyces species at the beginning of fermentation in sequentially inoculated wines, whereas the simultaneous inoculation of S. cerevisiae did not permit any non-Saccharomyces species to become predominant. Differences found in non-Saccharomyces yeast growth in both fermentations influenced the analytical profiles of final wines and specifically 2-phenylethyl acetate concentration which was two-fold increased in sequentially inoculated wines in comparison to those co-inoculated. In conclusion we have shown that H. vineae inoculated as part of a sequential mixed starter is able to compete with native yeasts present in non-sterile must and modify the wine aroma profile.

Purification and characterization of an α- l-rhamnosidase from Aspergillus nidulans

An enzyme exhibiting α‐ l‐rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on l‐rhamnose as the sole carbon source. The α‐ l‐rhamnosidase was shown to be N‐glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4·5–6 and 60 °C, had an isoelectric point of 5. With ρ‐nitrophenyl‐α‐ l‐rhamnopyranoside as the substrate it showed Km and Vmax values of 0·27 mmol l−1 and 64·6 U mg−1, respectively. The enzyme was competitively inhibited by l‐rhamnose (Ki 0·3 mmol l−1). Ca2+ (2 mmol l−1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l−1) inhibited it by 63%. Substrate specificity studies showed the α‐ l‐rhamnosidase to be active both on α‐1,2 and α‐1,6 linkages to β‐ d‐glucosides.

Antihypertensive properties of lactoferricin B-derived peptides.

A set of eight lactoferricin B (LfcinB)-derived peptides was examined for inhibitory effects on angiotensin I-converting enzyme (ACE) activity and ACE-dependent vasoconstriction, and their hypotensive effect in spontaneously hypertensive rats (SHR). Peptides were derived from different elongations both at the C-terminal and N-terminal ends of the representative peptide LfcinB(20-25), which is known as the LfcinB antimicrobial core. All of the eight LfcinB-derived peptides showed in vitro inhibitory effects on ACE activity with different IC(50) values. Moreover, seven of them showed ex vivo inhibitory effects on ACE-dependent vasoconstriction. No clear correlation between in vitro and ex vivo inhibitory effects was found. Only LfcinB(20-25) and one of its fragments, F1, generated after a simulated gastrointestinal digestion, showed significant antihypertensive effects in SHR after oral administration. Remarkably, F1 did not show any effect on ACE-dependent vasoconstriction in contrast to the inhibitory effect showed by LfcinB(20-25). In conclusion, two LfcinB-derived peptides lower blood pressure and exhibit potential as orally effective antihypertensive compounds, yet a complete elucidation of the mechanism(s) involved deserves further ongoing research.

Fluidized bed anaerobic biodegradation of food industry wastewaters

Abstract Anaerobic fluidized bed reactors were used to reduce the COD of low-strength food industry wastewaters. Soluble organic removal efficiencies of 75%, 80% and 50% were obtained for hog slaughterhouse, dairy and brewery wastewaters, respectively, at 35°C and 8 h hydraulic retention time. Removal efficiencies decreased with decreasing temperature (35°C to 20°C); no detrimental effect of temperature was observed when treating the slaughterhouse wasterwater. Methane production rate was only relevant on brewery wastewater treatment.

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

Abstract Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X24 xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)-1. In this culture condition, X24 is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X24 enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X24 xylanase had a Michaelis constant, Km, of 12.43 mg oat spelt xylan ml-1 and a Vmax of 1639 μmol min-1 (mg protein)-1.

Selectivity and antimicrobial action of bovine lactoferrin derived peptides against wine lactic acid bacteria.

In this study, the antibacterial activities of a bovine Lactoferrin pepsin hydrolysate (LFH) and a synthetic peptide derived from bovine lactoferricin (LfcinB(17-31)) have been evaluated against Oenococcus oeni and three additional lactic acid bacteria (LAB) known to cause spoilage during winemaking processes. Inhibition of bacterial growth was demonstrated in vitro in synthetic broth media (MRS) for both LFH and LfcinB(17-31). The bactericidal activity of the synthetic peptide was also assayed and found to vary depending on the bacterial species and the matrix in which exposure to peptide occurred (either MRS broth or white must). Specificity of LfcinB(17-31) for Lactobacillus brevis, Pediococcus damnosus, and O. oeni was demonstrated in must fermentation experiments in which these three LAB co-existed with the winemaking Saccharomyces cerevisiae T73 in the presence of the peptide. Finally, fermentation experiments also showed that LfcinB(17-31) at inhibitory concentrations did not alter either fermentation kinetics or specific enological parameters.

Inhibition of the wine spoilage yeast Dekkera bruxellensis by bovine lactoferrin-derived peptides.

The antimicrobial action of lactoferrin (LF)-derived peptides against Dekkera bruxellensis strains isolated from spoiled wines has been examined. The study included a fifteen-residue peptide (LfcinB(17-31)) derived from bovine lactoferricin B and a bovine LF pepsin hydrolysate (LFH). In vitro assays showed the inhibitory properties of LfcinB(17-31) on D. bruxellensis growth with IC(50) and MIC values in the micromolar range. Strains tested showed different sensitivity to the peptide. LfcinB(17-31) showed fungicidal properties towards all strains tested in laboratory growth medium. However, the extent of fungicidal activity was strain-dependent in must and wine, confirming the different antimicrobial action of peptides depending on both the food matrix and the target micro-organism. The binding of LfcinB(17-31) to D. bruxellensis cells was visualized by fluorescence microscopy and correlated with the fungicidal activity in the different matrixes. LfcinB(17-31) and LFH showed growth inhibitory properties in wine suggesting their potential use for spoilage control.