Metabolic profiling as a tool for revealingSaccharomycesinteractions during wine fermentation

The characterization of industrial yeast strains by examining their metabolic footprints (exometabolomes) was investigated and compared to genome-based discriminatory methods. A group of nine industrial brewing yeasts was studied by comparing their metabolic footprints, genetic fingerprints and comparative genomic hybridization profiles. Metabolic footprinting was carried out by both direct injection mass spectrometry (DIMS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), with data analysed by principal components analysis (PCA) and canonical variates analysis (CVA). The genomic profiles of the nine yeasts were compared by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis and microarray comparative genome hybridizations (CGH). Metabolomic and genomic analysis comparison of the nine brewing yeasts identified metabolomics as a powerful tool in separating genotypically and phenotypically similar strains. For some strains discrimination not achieved genomically was observed metabolomically.

Diploid cells of Saccharomyces cerevisiae were grown under controlled conditions with a Bioscreen instrument, which permitted the essentially continuous registration of their growth via optical density measurements. Some cultures were exposed to concentrations of a number of antifungal substances with different targets or modes of action (sterol biosynthesis, respiratory chain, amino acid synthesis, and the uncoupler). Culture supernatants were taken and analyzed for their "metabolic footprints" by using direct-injection mass spectrometry. Discriminant function analysis and hierarchical cluster analysis allowed these antifungal compounds to be distinguished and classified according to their modes of action. Genetic programming, a rule-evolving machine learning strategy, allowed respiratory inhibitors to be discriminated from others by using just two masses. Metabolic footprinting thus represents a rapid, convenient, and information-rich method for classifying the modes of action of antifungal substances.

Metabolic footprinting has been applied as a non-invasive approach to study the behaviour and responses of cultured cells to a range of genetic and environmental perturbations. Gas chromatography interfaced with time-of-flight mass spectrometry (GC-ToF-MS) has become a powerful tool for the analysis of metabolome-derived samples. Generally, two data analysis strategies are used to interrogate and understand the biological patterns within the multi-dimensional data. The first strategy, a commoner one, uses multivariate analysis after chromatographic and mass spectral deconvolution, and the second strategy directly employs multivariate analysis of non-deconvoluted data. Here, two strategies have been assessed for the separation and classification of metabolic footprints (exometabolomes) of two strains of Candida albicans grown on three different carbon sources (glycerol, glucose and galactose). We describe a semi-automated approach that simultaneously processes all samples using the chromatographic dimension data with principal components analysis (PCA), which can include data pre-processing before PCA analysis. The preprocessed and non-deconvoluted total ion chromatogram (TIC) data showed good separation of classes defined by growth on different carbon sources and when comparing the two strains grown on the same carbon source separation was achieved for strains grown on glucose and glycerol after preprocessing. The discrimination observed is greater for preprocessed and non-deconvoluted TIC data than for that of preprocessed and non-deconvoluted single ion chromatogram data. The results from the proposed approach with those produced by MZmine were compared. The results from MZmine data depicted separations in PCA space according to carbon source, but no separation was seen when studying strains grown on the same carbon source. Our research showed that the non-deconvoluted strategy is suitable for fast comparison of large sets of GC-MS data although it will not directly provide biological information. The non-deconvoluted strategy can avoid problems of analyzing complex samples using deconvolution software.

SummaryBasic analysis, electronic senses and HS‐SPME/GC‐MS were used to evaluate the effects of single and mixed cultures of Lactobacillus acidophilus‐26 and Lactobacillus plantarum‐56 on the quality of fermented red globe grape (RGG) juice. The results showed that mixed culture fermentation was superior to single strain fermentation in terms of viable bacteria counts, consumption of total sugar (TS), total soluble solids, pH, functionality, and antioxidant properties of RGG juice (RGGJ). Whereas the aroma profiles of developing juices were dominated by abundant esters, acids, alcohols, aldehydes, and ketones. Compared with the single inoculation, co‐inoculation of the two species induced lactic acid fermentations, leading to accumulation of acetic acid, ethyl acetate, and 2‐hexenol significantly. Moreover, higher concentrations of ethyl acetate, benzoic acid ethyl ester, octanoic acid, sorbic acid, 2‐phenylethanol, and 2‐hexenol represented the characteristic flavours of juices. To sum up, these findings suggested that L. acidophilus‐26 and L. plantarum‐56 could be used as fermented strains to ferment and improve the quality of RGGJ.

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D2 and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.

Effect of yeast cultures supplementation on live weight change, rumen fermentation, ciliate protozoa population, microbial hydrolytic enzymes status and slaughtering performance of growing lamb

British Food Journal Volume 44 Issue 11 1942

The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.

Comprehensive Multivariate Analysis of Red Wine Phenolic Composition, Color and Quality Components With Simultaneous Absorbance and Fluorescence Excitation Emission Mapping

Sixteen wine fermentations were examined for the presence of killer yeasts. Killer property and sensitivity to killer action were found in isolates of Saccharomyces cerevisiae but not in isolates of Kloeckera, Candida, Hansenula, and Torulaspora spp. Several killer and killer-sensitive strains of S. cerevisiae were differentiated by colony morphology, and this property was used to monitor their growth kinetics in mixed cultures in grape juice. Killer-sensitive strains died off within 24 to 48 h during mixed-strain fermentation. Killer action was demonstrated at pH 3.0 and pH 3.5 and over the range of 15 to 25 degrees C but depended on the proportion of killer to killer-sensitive cells at the commencement of fermentation. The dominance of killer strains in mixed-strain fermentations was reflected in the production of ethanol, acetic acid, and glycerol.

SUMMARYThirty‐four pairs of similar, commercially acceptable wines and a freshly prepared 50–50 blend of each pair were scored for quality by a small expert panel. The composite scores of the blends did not differ to even a low degree of significance from the score of the highest‐scoring member of the pair. The scores of the blends were very highly significantly better than the scores for the low‐scoring wines or the mean of the low‐ and high‐scoring wine in each pair. In 7 cases among the 34 the blend's composite score was higher than that of the highest‐scoring wine, and in no case was it lower than that of lowest‐scoring wine of the pair. Increased complexity of flavor is believed to be a major factor in the favorable effect of blending shown in this study; other possible effects are discussed.

In-situ carboxylate recovery and simultaneous pH control with tailor-configured bipolar membrane electrodialysis during continuous mixed culture fermentation

Globalization and free market dynamics have significantly impacted state economies, particularly in the wine industry. These forces have introduced greater diversity in wine products but have also heightened the risk of food fraud, especially in high-value commodities like wine. Due to its market value and the premium placed on quality, wine is frequently subject to adulteration. This issue is often addressed through regulatory trademarks on wine labels, such as Protected Designation of Origin (PDO) and Protected Geographic Indication (PGI). In this context, the metabolic profiles (organic acids, carbohydrates, and phenols) and stable isotope signatures (δ13C, δ18O, D/HI, and D/HII) of red and white wines from four agroclimatically similar regions were examined. The study explored how factors such as grape variety, harvest year, and geographical origin affect wine composition, with a particular focus on distinguishing samples from cross-border areas. Multivariate statistical analysis was used to assess the variability in wine composition and to identify distinct groups of samples. Preliminary results revealed that organic acids and volatile compounds were found in lower concentrations than carbohydrates but were significantly higher than phenols, with levels ranging between 1617 mg/L and 6258 mg/L. Carbohydrate content in the wines varied from 8285 mg/L to 14662 mg/L. Principal Component Analysis (PCA) indicated certain separation trends based on the variance in carbohydrates (e.g., fructose, glucose, galactose) and isotopic composition. However, Discriminant Analysis (DA) provided clear distinctions based on harvest year, variety, and geographical origin.

Assessing the effect of chitosan on pesticide removal in grape juice during clarification by gas chromatography with tandem mass spectrometry