No abstract

In recent years, studies have reported the positive influence of non-Saccharomyces yeast on wine quality. Many grape varieties under mixed or sequential inoculation show an overall positive effect on aroma enhancement. A potential impact by non-Saccharomyces yeast on volatile and non-volatile compounds should benefit the flavor of Riesling wines. Following this trend, four separate sequential fermentations (using the non-Saccharomyces yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, and Lachancea thermotolerans with Saccharomyces cerevisiae) were carried out on Riesling must and compared to a pure culture of S. cerevisiae. Sequential fermentations influenced the final wine aroma. Significant differences were found in esters, acetates, higher alcohols, fatty acids, and low volatile sulfur compounds between the different trials. Other parameters, including the production of non-volatile compounds, showed significant differences. This fermentation process not only allows the modulation of wine aroma but also chemical parameters such as glycerol, ethanol, alcohol, acidity, or fermentation by-products. These potential benefits of wine diversity should be beneficial to the wine industry.

Most wine aroma compounds, including the varietal fraction, are produced or released during wine production and derived from microbial activity. Varietal aromas, typically defined as terpenes and thiols, have been described as derived from their non-volatile precursors, released during wine fermentation by different yeast hydrolytic enzymes. The perception of these minority aroma compounds depends on the chemical matrix of the wine, especially on the presence of majority aroma compounds, such as esters or higher alcohols. Strategies aiming to reduce the production of these masking flavors are on the spotlight of enology research as a way to better define varietal standard profiles for the global market. Using a natural white must from Verdejo variety (defined as a thiol grape variety), here we describe the analytical and sensorial impact of using, in sequential inoculations, a selected strain of Metschnikowia pulcherrima, in combination with two different Saccharomyces cerevisiae strains. An increase in the levels of the thiol 4-MSP (4-methyl-4-sulfanylpentan-2-one) over its sensory threshold, together with a decrease in higher alcohol production, was observed when M. pulcherrima was used. This has an important impact on these wines, making them fruitier and fresher, always preferred by the sensory panel.

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 ␤-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 ␣-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.Volatile sulfur compounds (VSC) are found in many cheese varieties and are important components of flavor (10). The sulfur compounds methanethiol, methional, and dimethyl trisulfide (DMTS) are key odorants of Swiss Gruyère cheese flavor and are presumably derived from the bacterial metabolism of methionine, which is present in caseins at higher concentrations than cysteine. Swiss Gruyère (place of designated origin) cheese is produced with thermophilic mixed-strain starters which usually contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. In addition, depending on the cheese factory, Lactobacillus helveticus can also be present. However, after ripening, Lactobacillus casei and Lactobacillus rhamnosus bacteria are found in high numbers (8). Interestingly, L. casei is also a constituent of the nonstarter lactic acid bacteria of many other cheese varieties (6). Therefore, this species is considered to play an important role during ripening and may be a key player in the formation of flavor compounds.The catabolic pathway(s) of methionine and cysteine in lactobacilli is not well characterized. Possible pathways involve transaminases, methionine ␥-lyases, cystathionine ␤-lyases (CBL), and cystathionine ␥-lyases (CGL) (13). The transamination of methionine by aminotransferases has been studied in lactococci (18,27,34). The results of these studies suggested that methionine is converted to 2-oxo-4-methylthiobutyric acid, which is then converted, either enzymatically or chemically, to methanethiol. In Brevibacterium linens, a methionine ␥-lyases which catalyzes the deamination of methionine and releases methanethiol has been identified and the corresponding gene has been cloned (2,14).CBL, which is encoded by the gene metC in Escherichia coli, is involved in the ␣,␤-elimination of cystathionine to form homocysteine, pyruvate, and ammonia. In comparison, CGL catalyzes the ␣,␥-elimination reaction of cystathionine, producing cysteine, ammonia, and ␣-ketobutyrate. CBL and CGL generally show catalytic promiscuity, which means that they catalyze different reactions. Thus, the CBL from Lactococcus lactis subsp. cremor...

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.