Proteomic Analysis of Listeria monocytogenes FBUNT During Biofilm Formation at 10°C in Response to Lactocin AL705.

Listeria monocytogenes is one of the major food-related pathogens and is able to survive and multiply under different stress conditions. Its persistence in industrial premises and foods is partially due to its ability to form biofilm. Thus, as a natural strategy to overcome L. monocytogenes biofilm formation, the treatment with lactocin AL705 using a sublethal dose (20AU/ml) was explored. The effect of the presence of the bacteriocin on the biofilm formation at 10°C of L. monocytogenes FBUNT was evaluated for its proteome and compared to the proteomes of planktonic and sessile cells grown at 10°C in the absence of lactocin. Compared to planktonic cells, adaptation of sessile cells during cold stress involved protein abundance shifts associated with ribosomes function and biogenesis, cell membrane functionality, carbohydrate and amino acid metabolism, and transport. When sessile cells were treated with lactocin AL705, proteins' up-regulation were mostly related to carbohydrate metabolism and nutrient transport in an attempt to compensate for impaired energy generation caused by bacteriocin interacting with the cytoplasmic membrane. Notably, transport systems such as ß-glucosidase IIABC (lmo0027), cellobiose (lmo2763), and trehalose (lmo1255) specific PTS proteins were highly overexpressed. In addition, mannose (lmo0098), a specific PTS protein indicating the adaptive response of sessile cells to the bacteriocin, was downregulated as this PTS system acts as a class IIa bacteriocin receptor. A sublethal dose of lactocin AL705 was able to reduce the biofilm formation in L. monocytogenes FBUNT and this bacteriocin induced adaptation mechanisms in treated sessile cells. These results constitute valuable data related to specific proteins targeting the control of L. monocytogenes biofilm upon bacteriocin treatment.

The killer yeast Wickerhamomyces anomalus Cf20 exerts a broad anti-Candida activity through the production of killer toxins and volatile compounds.

Candidiasis is a group of opportunistic infections caused by yeast of the genus Candida. The appearance of drug resistance and the adverse effects of current antifungal therapies require the search for new, more efficient therapeutic alternatives. Killer yeasts have aroused as suitable candidates for mining new antifungal compounds. Killer strains secrete antimicrobial proteins named killer toxins, with promissory antifungal activity. Here we found that the killer yeast Wickerhamomyces anomalus Cf20 and its cell-free supernatant (CFS) inhibited six pathogenic strains and one collection strain of Candida spp. The inhibition is mainly mediated by secreted killer toxins and, to a lesser extent, by volatile compounds such as acetic acid and ethyl acetate. A new large killer toxin (>180 kDa) was purified, which exerted 70-74% of the total CFS anti-Candida activity, and the previously described glucanase KTCf20 was inhibitory in a lesser extent as well. In addition, we demonstrated that Cf20 possesses the genes encoding for the ß-1,3-glucanases WaExg1 and WaExg2, proteins with extensively studied antifungal activity, particularly WaExg2. Finally, the 10-fold concentrated CFS exerted a high candidacidal effect at 37°C, completely inhibiting the fungal growth, although the nonconcentrated CFS (RCF 1) had very limited fungistatic activity at this temperature. In conclusion, W. anomalus Cf20 produces different low and high molecular weight compounds with anti-Candida activity that could be used to design new therapies for candidiasis and as a source for novel antimicrobial compounds as well.

Population and oenological characteristics of non-Saccharomyces yeasts associated with grapes of Northwestern Argentina.

Yeasts population associated with grapes from Northwest Argentina, a region with a significant vine-growing increase over the past years, was evaluated. Ten species of non-Saccharomyces yeasts were identified from four grape varieties (Malbec, Merlot, Syrah and Torrontes) being Hanseniaspora uvarum the dominant species. Typing of isolates revealed genetic variability within Hanseniaspora genus and also high variability was observed according to their oenological characteristics. Based on the oenological properties, the most adequate strains as starter cultures were H. uvarum HuT7, HuMe15, HuS16, H. vineae HvT-mc1 and Metschnikowia pulcherrima MpT2/MpT3. These selected yeasts exhibited moderate resistance to SO2, reduced values of volatile acidity, null or low production of H2S, high levels of enzymes related to aroma and did not produce killer toxins. Further studies using mixed cultures of these non-Saccharomyces strains and S. cerevisiae are needed to validate the contribution of selected indigenous yeasts on wine organoleptic characteristics.

Proteomic and genetics insights on the response of the bacteriocinogenic Lactobacillus sakei CRL1862 during biofilm formation on stainless steel surface at 10°C.

Some lactic acid bacteria have the ability to form biofilms on food-industry surfaces and this property could be used to control food pathogens colonization. Lactobacillus sakei CR1862 was selected considering its bacteriocinogenic nature and ability to adhere to abiotic surfaces at low temperatures. In this study, the proteome of L. sakei CRL1862 grown either under biofilm on stainless steel surface and planktonic modes of growth at 10°C, was investigated. Using two-dimensional gel electrophoresis, 29 out of 43 statistically significant spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ten proteins resulted up-regulated whereas 16 were down-regulated during biofilm formation. Differentially expressed proteins were found to belong to carbohydrate, nucleotide, aminoacid and lipid metabolisms as well as translation, peptide hydrolysis, cell envelope/cell wall biosynthesis, adaption to atypical conditions and protein secretion. Some proteins related to carbohydrate and nucleotide metabolisms, translation and peptide degradation were overexpressed whereas those associated to stress conditions were synthesized in lower amounts. It seems that conditions for biofilm development would not imply a stressful environment for L. sakei CRL1862 cells, directing its growth strategy towards glycolytic flux regulation and reinforcing protein synthesis. In addition, L. sakei CRL1862 showed to harbor nine out of ten assayed genes involved in biofilm formation and protein anchoring. By applying qRT-PCR analysis, four of these genes showed to be up regulated, srtA2 being the most remarkable. The results of this study contribute to the knowledge of the physiology of L. sakei CRL1862 growing in biofilm on a characteristic food contact surface. The use of this strain as green biocide preventing L. monocytogenes post-processing contamination on industrial surfaces may be considered.

Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis.

Draft Genome Sequence of Oenococcus oeni Strain X2L (CRL1947), Isolated from Red Wine of Northwest Argentina.

We report the draft genome sequence of Oenococcus oeni strain X2L, a potential starter culture of malolactic fermentation, isolated from Malbec wine of Argentina. Genes encoding for enzymes involved in the metabolism of malate, citrate, and nitrogen compounds, as well as aroma compounds, were found in this genome, showing its ability to improve the sensorial characteristics of wines.

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking.

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15-25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8-M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5-12 %) and SO2 (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.

Pectinolytic yeasts from viticultural and enological environments: novel finding of Filobasidium capsuligenum producing pectinases.

In this study indigenous yeasts associated with wineries, grapes and Malbec fermented must from San Rafael viticulture region (Argentina) were isolated to select pectinolytic strains for their potential use in enology. Pectinolytic yeasts were identified by physiological and molecular methods. Among 78 isolates, only nine were able to produce extracellular pectinases. Six isolated from berry surface were identified as Aureobasidium pullulans and the remaining isolates, recovered from wineries, belonged to Saccharomyces cerevisiae and Filobasidium capsuligenum species. Pectinase production was evaluated under vinification-related conditions: pH 3.5, 12 and 28 °C. A. pullulans U-12 produced the highest pectinolytic activity at low temperature (1.16 U ml(-1) ), while F. capsuligenum strains showed good activity at 12 and 28 °C (0.77 and 1.15 U ml(-1) , respectively) being this study the first report on the capacity of this species to produce pectinases. The pectinolytic activity of F. capsuligenum B-13 showed an optimum at pH 4.5 and two peaks at 20 and 50 °C. The enzyme half-life was 2 h at 40 °C and retained 65% of its activity at 40 °C after 1 h of incubation. This pectinolytic system displayed remarkable activity at pH and temperatures found in vinification, suggesting a potential candidate for applying to wine-making.

Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking.

Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.

Isolation and selection of yeasts from wine grape ecosystem secreting cold-active pectinolytic activity.

The present study was undertaken with the purpose of selecting yeasts from wine grapes that are able to produce extracellular cold-active pectinases. After two consecutive selections yeast isolates were identified by pheno- and genotyping, and pectinolytic activity was preliminarily characterised at proximate winemaking conditions. Out of 1023 indigenous microorganisms isolated from grape skins of D.O. San Rafael (Mendoza, Argentina) viticulture region, 565 (55%) showed pectinolytic activity on plates and, among them, 96 (17%) were chosen in a primary selection. Ten isolates were finally selected for exhibiting the greatest activity at low temperature (12 °C) and identified as Aureobasidium pullulans. GM-R-22 strain demonstrated the highest pectinolytic activity (0.751 U/mL) at pH 3.5 and 12 °C. Yeast pectinases were constitutively produced. This study is the first report about strains of A. pullulans producing pectinases which are able to show good activity at low temperature. These pectinolytic strains could be of interest in wine production.

Characterization of wines produced by mixed culture of autochthonous yeasts and Oenococcus oeni from the northwest region of Argentina.

Two autochthonous yeasts from the northwest region of Argentina, Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2, were used as pure or mixed starter cultures in microvinification trials conducted in Malbec red must. Also, the effect of Oenococcus oeni X(2)L was evaluated. S. cerevisiae mc2 showed adequate growth and fermentative activity in single and composite fermentations, producing standard concentration of ethanol. The amount of esters was higher in fermentations conducted using mixed yeast starters. Independent of the timing of inoculation of O. oeni, this malolactic bacterium completely depleted malic acid. Sensory evaluation indicated that young wines fermented with mixed yeast cultures and sequential inoculation of O. oeni were preferred, achieving the highest scores for positive descriptors and they allowed better control of the sensory quality. Consequently, this study proposes inclusion of autochthonous K. apiculata mc1 as an adjunct culture to S. cerevisiae mc2 during Malbec must fermentation to improve the organoleptic properties of red wines. Furthermore, sequential inoculation of O. oeni X(2)L should be carried out after completion of the alcoholic fermentation to enhance sensory characteristics.

Influence of wine-related physicochemical factors on the growth and metabolism of non-Saccharomyces and Saccharomyces yeasts in mixed culture.

The influence of two physicochemical factors involved in winemaking, temperature and SO(2), on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. Highest biomass was reached at 15 and 25 degrees C for K. apiculata and S. cerevisiae, respectively. Pure cultures of K. apiculata died off early with increasing temperature, but in co-culture with S. cerevisiae it showed higher viability and a change in the death curve from exponential to linear. Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. At temperatures from 15 to 30 degrees C the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture of K. apiculata. SO(2) addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. Statistical evaluation indicated that culture type and SO(2) addition significantly affected metabolite production, but the interaction between culture and SO(2) was not significant. These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.

Kinetics and metabolic behavior of a composite culture of Kloeckera apiculata and Saccharomyces cerevisiae wine related strains.

The kinetics and metabolic behavior of Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2 in composite culture was investigated. K. apiculata showed a higher viability through the fermentation; however the maximum cell density of both yeasts decreased. This behavior was not due to ethanol concentration, killer toxins production or competition for assimilable nitrogenous compounds between both yeasts. Despite the consistent production of secondary products by single culture of K. apiculata, an increase of these compounds was not observed in mixed culture. These results contribute to a better understanding of the behavior of non-Saccharomyces yeasts and their potential application in the wine industry.