Doris Rauhut

Cloning and Characterization of Two Lactobacillus casei Genes Encoding a Cystathionine Lyase

ABSTRACT Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine β-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of l-cystathionine, O-succinyl-l-homoserine, l-cysteine, l-serine, and l-methionine to form α-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.

Effect on quality and composition of Riesling wines fermented by sequential inoculation with non-Saccharomyces and Saccharomyces cerevisiae

Abstract The production of nonvolatile and volatile compounds by different commercial non-Saccharomyces yeast strains in Riesling grape musts was monitored during fermentation, and the related changes in wine quality were noted. Sequential fermentations of Saccharomyces cerevisiae with Pichia kluyveri, Lachancea thermotolerans, or Metschnikowia pulcherrima were compared to a single fermentation using S. cerevisiae alone. The results from all developed analyses showed significant differences in several parameters including population kinetics, nonvolatile and volatile compounds, and sensorial parameters.

Usage and Formation of Sulphur Compounds

Sulphur is important for the growth of all microorganisms due to the formation of sulphur-containing amino acids. Among the wine-related microorganisms, extensive data has been accumulated for yeast of the species Saccharomyces cerevisiae. In recent years, also more and more research work is focused on the sulphur metabolism of non-Saccharomyces yeasts and lactic acid bacteria.

Impact of Headspace Oxygen and Closure on Sulfur Dioxide, Color, and Hydrogen Sulfide Levels in a Riesling Wine

Following an experimental design replicating typical winery conditions, a Riesling wine was bottled with different headspace oxygen levels and sealed with either a coextruded closure or a screwcap to investigate the impact of headspace oxygen and closure oxygen transfer rate on wine evolution. Using luminescence technology, dissolved oxygen and headspace oxygen, as well as oxygen ingress through the closure, were monitored during 24 months of bottle storage. Under typical winery conditions, headspace oxygen introduced at bottling was found to be a major component of oxygen in bottled wine. Headspace oxygen at bottling influenced loss of sulfur dioxide during bottle storage, being the main cause of sulfur dioxide decline during the first four months after bottling in 375 mL bottles. The loss of sulfur dioxide was not correlated with the evolution of dissolved oxygen, but with the total amount of oxygen consumed by the wine. After 24 months in the bottle, color differences due to different headspace oxygen and closure oxygen transfer rate were generally minor. Conversely, differences in closure oxygen transfer rate were responsible for significant differences in the final concentration of the off-odor compound hydrogen sulfide, with screwcap generally associated with higher levels of this compound. Even if less significantly, the amount of oxygen present in the headspace at bottling also had an effect on final hydrogen sulfide, with higher concentrations observed in wines bottled with lower headspace oxygen.

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.

Development of a microwave assisted extraction method for the analysis of 2,4,6-trichloroanisole in cork stoppers by SIDA–SBSE–GC–MS

The aim of this research work was focused on the replacement of the time-consuming soaking of cork stoppers which is mainly used as screening method for cork lots in connection with sensory analysis and/or analytical methods to detect releasable 2,4,6-trichloroanisole (TCA) of natural cork stoppers. Releasable TCA from whole cork stoppers was analysed with the application of a microwave assisted extraction method (MAE) in combination with stir bar sorptive extraction (SBSE). The soaking of corks (SOAK) was used as a reference method to optimise MAE parameters. Cork lots of different quality and TCA contamination levels were used to adapt MAE. Pre-tests indicated that an MAE at 40 degrees C for 120 min with 90 min of cooling time are suitable conditions to avoid an over-extraction of TCA of low and medium tainted cork stoppers in comparison to SOAK. These MAE parameters allow the measuring of almost the same amounts of releasable TCA as with the application of the soaking procedure in the relevant range (<25 ng L(-1) releasable TCA from one cork) to evaluate the TCA level of cork stoppers. Stable isotope dilution assay (SIDA) was applied to optimise quantification of the released TCA with deuterium-labelled TCA (TCA-d(5)) using a time-saving GC-MS technique in single ion monitoring (SIM) mode. The developed MAE method allows the measuring of releasable TCA from the whole cork stopper under improved conditions and in connection with a low use of solvent and a higher sample throughput.

Cloning and characterisation of a cystathionine β/γ-lyase from two Oenococcus oeni oenological strains

Sulphur-containing compounds in wine have been extensively studied because of their effect on wine flavour and quality. In this study, an enzyme that degrades sulphur-containing amino acids was cloned and characterised from two Oenococcus oeni strains of oenological origins. The enzyme has features of a cystathionine-γ-lyase (EC 4.4.1.1), a pyridoxal-5-phosphate-dependent enzyme catalysing an α,γ-elimination reaction of l-cystathionine to produce l-cysteine, α-ketobutyrate and ammonia. Moreover, it was able to catalyse an α,β-elimination reaction producing homocysteine, pyruvate and ammonia from l-cystathionine. An elimination reaction of l-cysteine and dl-homocysteine was also efficiently catalysed by the enzyme, resulting in the formation of hydrogen sulphide. Furthermore, the ability to demethiolate methionine into methanethiol, an unfavourable volatile sulphur compound in terms of wine aroma, was observed. The findings of this work suggest that O. oeni seems to play a minor role in the production of volatile sulphur compounds during the vinification process as the optimal conditions were far from the harsh wine environment.

Identification and characterization of a strain-dependent cystathionine β/γ-lyase in Lactobacillus casei potentially involved in cysteine biosynthesis

The trans-sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans-sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans-sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei, which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli. The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds.

Influence of Torulaspora delbrueckii in varietal thiol (3-SH and 4-MSP) release in wine sequential fermentations

In last years, non-Saccharomyces yeasts have emerged as innovative tools to improve wine quality, being able to modify the concentration of sensory-impact compounds. Among them, varietal thiols released by yeasts, play a key role in the distinctive aroma of certain white wines. In this context, Torulaspora delbrueckii is in the spotlight because of its positive contribution to several wine quality parameters. This work studies the physiological properties of an industrial T. delbrueckii strain, for the production of wines with increased thiol concentrations. IRC7 gene, previously described in S. cerevisiae, has been identified in T. delbrueckii, establishing the genetics basis of its thiol-releasing capability. Fermentations involving T. delbrueckii showed improvements on several parameters (such as glycerol content, ethanol index, and major volatile compounds composition), but especially on thiols release. These results confirm the potential of T. delbrueckii on wine improvement, describing new metabolic features regarding the release of cysteinylated aroma precursors.

Effects of Botrytis cinerea and Erysiphe necator fungi on the aroma character of grape must: A comparative approach

Botrytis cinerea and Erysiphe necator are among the most relevant fungi in viticulture. In order to deepen our knowledge about their potential impact on wine quality, their effects on the aroma character of the initial stage of wine production, i.e. the must were studied. The main aroma compounds were determined by gas chromatography-olfactometry (GC-O) and ranked according to their relative intensities by means of aroma extract dilution analysis (AEDA). Clear differences between healthy and infected samples were observed. Botrytis cinerea had a positive impact on fruity and floral notes while several earthy smelling compounds were developed as result of the infection. Unlike in previous studies, however, we did not observe any clear differences in the quantities of earthy-mushroom-like smelling substances as result of the infection process with Erysiphe necator.