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In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Wine is a processed beverage that offers high nutritional and health benefits. It is produced from grape must, which undergoes fermentation by yeasts (and sometimes lactic acid bacteria) to create a product that is highly appreciated by consumers worldwide. However, if only one type of yeast, specifically Saccharomyces cerevisiae, were used in the fermentation process, the resulting wine would lack aroma and flavor and may be rejected by consumers. To produce wine with a desirable taste and aroma, non-Saccharomyces yeasts are necessary. These yeasts contribute volatile aromatic compounds that significantly impact the wine’s final taste. They promote the release of primary aromatic compounds through a sequential hydrolysis mechanism involving several glycosidases unique to these yeasts. This review will discuss the unique characteristics of these yeasts (Schizosaccharomyces pombe, Pichia kluyveri, Torulaspora delbrueckii, Wickerhamomyces anomalus, Metschnikowia pulcherrima, Hanseniaspora vineae, Lachancea thermotolerans, Candida stellata, and others) and their impact on wine fermentations and co-fermentations. Their existence and the metabolites they produce enhance the complexity of wine flavor, resulting in a more enjoyable drinking experience.

Urban parks constitute one of the main leisure areas, especially for the most vulnerable people in our society, children, and the elderly. Contact with soils can pose a health risk. Microbiological testing is a key aspect in determining whether they are suitable for public use. The aim of this work is to map the spatial distribution of potential dangerous Enterobacteria but also bioremediation useful (lipase producers) isolates from soils in an urban park in the area of Valencia (Spain). To this end, our team has collected 25 samples of soil and isolated 500 microorganisms, using a mobile application to collect information of the soil samples (i.e. soil features, temperature, humidity, etc.) with geolocation. A combined protocol including matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing PCR has been established to characterize the isolates. The results have been processed using spatial statistical techniques (using Kriging method), taking into account the number of isolated strains, also proving the reactivity against standard pathogenic bacterial strains (Escherichia coli, Bacillus cereus, Salmonella, Pseudomonas and Staphylococcus aureus), and have increased the number of samples (to 896 samples) by interpolating spatially each parameter with this statistical method. The combined use of methods from biology and computer science allows the quality of the soil in urban parks to be predicted in an agile way, which can generate confidence in its use by citizens.

Cutinases (EC 3.1.1.74) are serin esterases that belong to the α/β hydrolases superfamily and present in the Ser-His-Asp catalytic triad. They show characteristics between esterases and lipases. These enzymes hydrolyze esters and triacylglycerols and catalyze esterification and transesterification reactions. Cutinases are synthesize by plant pathogenic fungi, but some bacteria and plants have been found to produce cutinases as well. In nature they facilitate a pathogen’s invasion by hydrolyzing the cuticle that protects plants, but can be also used for saprophytic fungi as a way to nourish themselves. Cutinases can hydrolyze a wide range of substrates like esters, polyesters, triacylglycerols and waxes and that makes this enzyme very attractive for industrial purposes. This work discusses techniques of industrial interest such as immobilization and purification, as well as some of the most important uses of cutinases in industries.

Agro-food industrial by-products signify a grave environmental problem. In addition to the conventional irrigation with wastewaters, the application of solids to the soil, and the extraction of worthwhile compounds for the pharmaceutical, cosmetics, and food industries, a microbiological strategy is also possible. In this review, the most promising microbiological methods for the valorization of wine and oil by-products are shown. Recently, there has been an upsurge in the exploitation of the waste materials generated by the wine and olive oil industries. Treatments by physical and chemical processes are generally costly, and they do not often provide entire solutions. Microbiological processes for the processing of olive and grape residues have seen worldwide applications, and they are considered to be environmentally respectful, reliable, and, in most cases, cost-effective. Production of added-value products such as edible mushrooms, biofuels, organic acids, polymers, and enzymes appear to be the new frontier in by-products valorization. Our chapter has been centered on two principal food industries in the Mediterranean area: wine and olive oil production.

The emergence of pathogenic bacteria that are multi-resistant to antibiotics lurks in today's society. In the golden age of the discovery of new antibiotic-producing microorganisms, each contribution was a step forward, but currently the progression is no longer so spectacular. The probability of finding new microorganisms and different antibiotics is lower and lower. The use of spatial statistical methods such as the Kriging technique has been shown to be suitable for this purpose, allowing us to determine areas with greater possibilities of locating these microorganisms. For a practical approach of our estimations, a total of 12 isolates capable of inhibiting the growth of several control strains (Escherichia coli, Bacillus cereus and at least one other) were analyzed. The isolates were preliminarily characterized, and subsequently identified at the species level by DNA sequence analyses (16S rDNA PCR) and protein analyses (MALDI-TOF MS). However, between soil pH values and the number of isolates capable of inhibiting the control strains. Geospatial mapping with RStudio software provide a satisfactory predictive tool for isolation of new microbial isolates.

Allergy is the most common chronic disease in Europe and about 40% of the population suffers from it. Microbiota are closely linked to the immune system, with some probiotics characterized as beneficial, contributing to reductions in allergenic symptomatology. To ensure the predominance of beneficial probiotics in the gut microbiota, the use of prebiotics is very important. Among the most important prebiotics are those of marine origin, particularly the fucoidan, a polysaccharide from brown algae (Phaeophyta). The effect of fucoidan on Lactobacillus reuteri in vitro growth behavior has been studied using turbidimetry, plate count and statistical analysis. The fucoidan revealed a significant prebiotic effect, increasing L. reuteri growth (≈2 Log10 cycles after 48 h exposure). The combination of the prebiotic effect of the fucoidan with the direct action that it exerts on allergy and other health problems make this molecule a promising nutraceutical for future use.

Scientific journals have played an essential role in the diffusion of research breakthroughs. For many years there was no competition between journals, but, in recent decades they have become categorized by a careful assessment of their published contents based on several metric parameters. Of greater note, the ‘prestige index’ has become an essential tool used by public and private institutions to develop their scientific policy. Thus, the evaluation of research staffs, the concession of grants or fellowships and even the scholarly reputation and academic positions are mainly founded on a given journal's ‘quality’. As a consequence, the prestige of some journals has gone up, based on the assumption that they publish cutting-edge science, while the reputation of others has gone down. Within the field of Microbiology, we have carried out a direct analysis by monitoring several representative classic journals according to customary metric parameters over 20 years. This analysis also covers another set of journals of recent appearance (novel journals). Although a direct comparison between both groups is not possible, this approach serves to perceive the trends of publication among microbiologists. Our preliminary conclusion is that the continued existence of many so-termed classic journals devoted to Microbiology is seriously threatened.

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (Km 0.85 mM, Vmax 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.

Background Trehalose is a non-reducing disaccharide highly conserved throughout evolution. In yeasts, trehalose hydrolysis is confined to the enzyme trehalase, an α-glucosidase specific for trehalose as sole substrate. Two kinds of trehalase activity exist in yeasts: neutral and acid enzymes. Scope of the review This review makes a comparative survey of the main biochemical and genetic parameters, regulatory systems, tridimensional structure and catalytic mechanism of the two yeast trehalases. Major conclusions The yeast neutral and acid trehalases display sharp differences in biochemical features (optimum pH, Mr or amino acid sequence) physiological roles, subcellular location (cytosol vs vacuoles or cell wall) and regulatory control (phosphorylation vs catabolite repression). However, their identical specificity for trehalose is based on the presence of an (α/α)6 toroid folding structure in the active centre and a catalytic mechanism of anomeric inversion. General significance This review expands our knowledge of the homology, functional features and catalytic mechanisms of α-glucosidases in yeasts. It provides a further analysis of the correlation between structures and predicted biological roles of macromolecules.

Fermentation, to obtain alcoholic beverages, is a process which implies the interaction of various microorganisms. This metabolic process is due to the action of yeasts, which can be divided into two groups, the Saccharomyces yeasts, particularly S. cerevisiae, and the non-Saccharomyces yeasts. Although they are considered spoilage microorganisms of the fruits, non-Saccharomyces yeasts possess some interesting applied characteristics and can be used in food industry. Some of the compounds produced by these microorganisms may be beneficial and improve the characteristics of the fermented beverages. In this chapter, recent advances in glycosidase, xylanase, lipase, esterase, protease, lyase and pectinase activities and their role in fermentation have been reviewed.

Candida albicans is a predominant cause of fungal infections in mucosal tissues as well as life-threatening bloodstream infections in immunocompromised patients. Within the human body, C. albicans is mostly embedded in biofilms, which provides increased resistance to antifungal drugs. The glyoxalase Glx3 is an abundant proteomic component of the biofilm extracellular matrix. Here, we document phenotypic studies of a glx3Δ null mutant concerning its role in biofilm formation, filamentation, antifungal drug resistance, cell wall integrity and virulence. First, consistent with its function as glyoxalase, the glx3 null mutant showed impaired growth on media containing glycerol as the carbon source and in the presence of low concentrations of hydrogen peroxide. Importantly, the glx3Δ mutant showed decreased fitness at 37°C and formed less biofilm as compared to wild type and a reintegrant strain. At the permissive temperature of 28°C, the glx3Δ mutant showed impaired filamentation as well as increased sensitivity to Calcofluor white, Congo red, sodium dodecyl sulfate and zymolyase, indicating subtle alterations in wall architecture even though gross quantitative compositional changes were not detected. Interestingly, and consistent with its impaired filamentation, biofilm formation and growth at 37°C, the glx3Δ mutant is avirulent. Our results underline the role of Glx3 in fungal pathogenesis and the involvement of the fungal wall in this process.

The dependence of plastic materials is an increasing problem. Although plastics are very useful for humankind many disadvantages derived from the difficulties linked to recycling and disposal are well known. A feasible alternative is the production and use of bioplastics. These compounds have multiple options at the end of his life that can ensure their safety and efficacy of reuse or recovery. For example, raw materials can be returned to the manufacturer for recycling. Bioplastics synthesized through biotechnology include mainly polyhydroxyalkanoates (PHAs), common lipoidic storage materials accumulated by prokaryotes. Some processes for producing PHAs by fermentation using microorganisms have been developed at a different extent. However, biopolymers (PHA) market is under development, and therefore cannot compete with traditional plastics since manufacturing is still more expensive. In this review we have focused on the study of the production processes of bioplastics where bacteria are present, also describing the scaling to industrial level aspects and future trends.

A double homozygous atc1Δ/atc1Δ/ntc1Δ/ntc1Δ mutant (atc1Δ/ntc1Δ KO) was constructed in the pathogen opportunistic yeast Candida parapsilosis by disruption of the two chromosomal alleles coding for NTC1 gene (encoding a neutral trehalase) in a Cpatc1Δ/atc1Δ background (atc1Δ KO strain, deficient in acid trehalase). The Cpatc1Δ/ntc1Δ KO mutant failed to counteract the inability of Cpatc1Δ cells to metabolize exogenous trehalose and showed a similar growth pattern on several monosaccharides and disaccharides. However, upon prolonged incubation in either rich medium (YPD) or nutrient-starved medium the viability of Cpatc1Δ cells exhibited a sensitive phenotype, which was augmented by further CpNTC1/NTC1 disruption. Furthermore, Cpatc1Δ/ntc1Δ KO cells had difficulty in resuming active growth in fresh YPD. This homozygous mutant also lacked any in vitro measurable trehalase activity, whether acid or neutral, suggesting that a single gene codes for each enzyme. By contrast, in Cpatc1Δ/ntc1Δ KO strain the resistance to oxidative and heat stress displayed by atc1Δ mutant was suppressed. Cpatc1Δ/ntc1Δ KO cells showed a significant decrease in virulence as well as in the capacity to form biofilms. These results point to a major role for acid trehalase (Atc1p) in the pathobiology of C. parapsilosis, whereas the activity of neutral trehalase can only partially counteract Atc1p deficiency. They also support the use of ATC1 and NTC1 genes as interesting antifungal targets.

Agro-food industrial wastes represent a serious environmental problem in producing countries. In addition to the traditional irrigation with wastewaters, application of solids to the land, and the extraction of valuable compounds for the pharmaceutical, cosmetics and food industries, a microbiological approach is also available. In this review, the most promising microbiological processes for the valorization of wine and oil by-products are presented. Recently, there has been an upsurge in the exploitation of the waste materials generated by the wine and olive oil industries. Treatments by physical and chemical methods are generally expensive, and they do not often provide complete solutions. Microbiological processes for the treatment of olive and grape residues have seen worldwide applications, and they are considered to be environmentally friendly, reliable and, in most cases, cost effective. Production of added-value products such as edible mushrooms, biofuels, organic acids, polymers and enzymes appear to be the new frontier in by-products valorization. Our study has been focused in these two major food industries in the Mediterranean area, engaged in wine and olive oil production.

The finding of new isolates of non-Saccharomyces yeasts, showing beneficial enzymes (such as β-glucosidase and β-xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non-Saccharomyces yeasts. Four isolates were selected because of their both high β-glucosidase and β-xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB-medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β-glucosidase and β-xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β-glucosidase and β-xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3-folds) when wines were treated with non-Saccharomyces isolates. In detail, terpineol, 4-vinyl-phenol and 2-methoxy-4-vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2-phenyl ethanol than those inoculated with other yeasts.

López-Goñi, Ignacio et al., Social networks are already being exploited for searching, storing, and sharing knowledge, demonstrating that they are an efficient vehicle for social learning. Consequently, they could be implemented as a competent tool for formal learning. Twitter is among the 10 most popular online social networks, integrating a community of over 500 million users around the world. Twitter has already been used in several educational programs and evaluated as a positive teaching experience with an outstanding potential in academic and educational environments (1–6). However, there are scarce examples available in the literature about its use in science teaching and communication. In this work, we present and analyze the application of Twitter to create an online space for communication and learning of basic microbiology. With this aim, a team of professionals in the field has imparted, to our knowledge, the first worldwide open access microbiology course via Twitter. Here we assess the results of our experience of using this social network as a tool for teaching, promoting, and communicating scientific knowledge to a wide audience.

The yeast biota associated to three eastern Spain oil mills has been evaluated. Each environment has associated a characteristic number and variability of yeasts. The D1/D2 rDNA sequencing protocol has been proved to be a useful method to identify 216 yeasts from oleic environments, showing us Candida (several species), Saccharomyces paradoxus, Citeromyces matritensis and Cryptococcus sp. as the most representative genera. Moreover, a further screening of biochemical activities has provided us with a collection of isolates able to metabolize different substrates. In particular, protease, β-glucosidase, pectinase, polygalacturonase, xylanase, lipase, esterase and catalase activities were investigated. The majority of the strains showed moderate or high β-glucosidase, lipase and pectinase activities, while protease and polygalacturonase activities are less frequent. In a second step, nutritional competition assays were carried out to investigate the antagonic effect of yeasts associated to olive oil mills with Aspergillus fungi. Yeasts can be an effective biocontrol tool as they can colonize olives and compete for space and nutrients with pathogenic fungi, avoiding the use of chemical fungicides.

Summary Twenty-six enological Hanseniaspora isolates were identified by ITS PCR-RFLP and D1/D2 domain of 26S rDNA sequencing and assayed for exocellular protease production. Based on qualitative data, six isolates, belonging to H. guilliermondii, H. valbyensis and H. occidentalis species, were selected to continue the study. Analytical procedure was optimised, and protease activities were quantified and characterised on the basis of different biotechnological factors. Protease activity was quite glucose, fructose and ethanol content independent, but divalent cation affects activity; these data support that they were aspartic proteases. The effect of 2-mercaptoethanol suggests the importance of disulphide bonds to maintain the structure of the active centre. Our results show these enzymes could be suitable for using in biotechnological processes at neutral pH, such as cheese, bread and meat industries. This is, to our knowledge, the first work showing the induction process to induce and characterise proteolytic activity in Hanseniaspora isolates.

A total of 17 Pichia ( Wickerhamomyces ) isolates obtained from enological ecosystems in the Utiel-Requena Spanish region were characterized by physiological (using API 20C AUX strips and ID Yeast Plus System miniaturized identification systems) and molecular (PCR-RFLP and sequencing) techniques as belonging to the species P. fermentans, P. membranifaciens , and W. anomalus . Data support the reclassification of P. anomala as Wickerhamomyces anomalus . In order to characterize their enzymatic abilities, xylanase, β-glucosidase, lipase, esterase, protease, and pectinase qualitative and quantitative assays were made. Wickerhamomyces anomalus and P. membranifaciens were the most interesting species as a source of enzymes for the winemaking industry. Glycosidase enzymes had a high degree of tolerance to high levels of glucose and ethanol, making them of great interest for enological use.

Aims: The purification and biochemical properties of the 1,4-β-xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol-tolerant 1,4-β-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS-PAGE. The activity of 1,4-β-xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48 ± 0·06 mmol l−1 and a Vmax of 7·4 ± 0·1 μmol min−1 mg−1 protein for p-nitrophenyl-β-d-xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4-β-xylosidase from P. membranifaciens to be used in the bioethanol production process.

Some non-Saccharomyces yeasts, including Hanseniaspora, participate in the first stages of wine fermentation. Besides their importance in the wine production process some of these yeasts have been described to be potential producers of hydrolytic enzymes to industrial level. In this work, we pretend to evaluate the technological abilities of the Hanseniaspora strains deposited in the Spanish Type Culture Collection (CECT). First of all, we considered verification of the correct identification of the strains using several miniaturized biochemical systems and molecular techniques (PCR, RFLP and sequencing of the ribosomal D1/D2 region). The results allowed us to verify the correct adscription of the 26 strains included in this study, which exhibited concordant profiles of restriction with one of the three species described in previous studies (H. occidentalis, H. osmophila and H. valbyensis). Some other strains assigned to the species (H. uvarum, H. vineae and H. guilliermondii) showed at least two different profiles. The other objective of this study was to perform an initial screening to characterize both at quantitative and qualitative levels, the ability of these yeasts to produce valuable enzymes for wine fermentation (increase of aroma) and other applications. The more important enzymatic activities detected were β-glucosidase, β-xylosidase, and protease. The HU7, HU8, HV1, HV3, HO2 and HOC1 strains showed high levels of β-glucosidase and β-xylosidase activity, whereas some strains (HG1, HG3, HVA1, HOC 3 and HOC4 were useful for protease production.

A total of 6047 open reading frames in theCandida albicansgenome were screened forZn(II)2C6-type zinc cluster proteins (or binuclear cluster proteins) involved in DNA recognition. These fungal proteins are transcription regulators of genes involved in a wide range of cellular processes, including metabolism of different compounds such as sugars or amino acids, as well as multi-drug resistance, control of meiosis, cell wall architecture, etc. The selection criteria used in the sequence analysis were the presence of theCysX2CysX6CysX5-16CysX2CysX6-8Cysmotif and a putative nuclear localization signal. Using this approach, 70 putativeZn(II)2C6transcription factors have been found in the genome ofC. albicans.

After screening a Candida albicans genome database the product of an open reading frame (ORF) (CA2880) with 49% homology to the product of Saccharomyces cerevisiae YPL133c, a putative transcriptional factor, was identified. The disruption of the C. albicans gene leads to a major sensitivity to calcofluor white and Congo red, a minor sensitivity to sodium dodecyl sulfate, a major resistance to zymolyase, and an alteration of the chemical composition of the cell wall. For these reasons we called it CaCWT1 (for C. albicans cell wall transcription factor). CaCwt1p contains a putative Zn(II) Cys(6) DNA binding domain characteristic of some transcriptional factors and a PAS domain. The CaCWT1 gene is more expressed in stationary phase cells than in cells growing exponentially. To our knowledge, this is the first Zn(II) Cys(6) transcriptional factor-encoding gene implicated in the cell wall architecture.

The finding of new isolates of non-Saccharomyces yeasts, showing beneficial enzymes (such as β-glucosidase and β-xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non-Saccharomyces yeasts. Four isolates were selected because of their both high β-glucosidase and β-xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB-medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β-glucosidase and β-xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β-glucosidase and β-xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3-folds) when wines were treated with non-Saccharomyces isolates. In detail, terpineol, 4-vinyl-phenol and 2-methoxy-4-vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2-phenyl ethanol than those inoculated with other yeasts.

The fermentation of grape must to produce wine is a biologically complex process, carried on by yeasts and malolactic bacteria. The yeasts present in spontaneous fermentation may be divided into two groups, the Saccharomyces yeasts, particularly S. cerevisiae, and the non-Saccharomyces yeasts which include members of the genera Rhodotorula, Pichia, Candida, Debaryomyces, Metschtnikowia, Hansenula and Hanseniaspora. S. cerevisiae yeasts are able to convert sugar into ethanol and CO2 via fermentation. They have been used for thousands of years by mankind for the production of fermented beverages and foods, including wine. Their enzymes provide interesting wine organoleptic characteristics. β-Glucosidase activity is involved in the release of terpenes to wine, thus contributing to varietal aroma. β-Xylosidase enzyme is also interesting in industry due to its involvement in the degradation of hemicellulose by hydrolyzing its main heteroglycan (xylan). The ability of yeasts to release proteases has been observed by many researchers because of their potential to degrade haze proteins in wine and to generate nutrient sources for microorganisms. Moreover, these enzymes are interesting in biotechnology, for use in food processing such as cheese, pickles or sausage.

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.

Summary The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation (MLF) in wine permits control over the timing of the process and the quality of the wine. Successful inoculation of bacterial starter cultures into wine depends on the selection of suitable strains and on the preparation and conservation of those cultures. Medium for Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cultures under laboratory controlled conditions for isolation and identification. However, this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine while cells grown in Medium of Preculture (MP) or wine, stored at −20 °C or freeze-dried retained the ability to induce MLF when inoculated in wine. Our results suggest that the use of freeze-dried cultures of O. oeni previously grown in MP is the best choice for industrial application.