Managing ethanol and sensory compounds by non-Saccharomyces yeasts

The pursuit of flavour and phenolic ripeness, augmented by climate warming and extreme weather events, often leads to excessive sugar accumulation in grapes translating to undesirably high ethanol content in wines. Other common characteristics of such grapes and wines are inadequate acidity and aroma profiles, which all together compromise the quality and marketability of the final product. To tackle these issues, research has devised a number of methods implemented across the entire grape and wine production chain. Among these, partial fermentations with non-Saccharomyces yeasts is of particular interest, as it represents an undemanding approach that can also impart ‘complexity’ and distinctness. However, the full potential of non-Saccharomyces yeasts in wine ethanol and flavour management remains elusive, and this work aimed to further explore it. Research has shown that mixed fermentations with non-Saccharomyces yeasts can lead to enhanced wine aroma and sensory properties, and albeit limited, the current range of non- Saccharomyces is indeed marketed for this purpose. The potential of commercially available (and thus readily implementable) non-Saccharomyces co-inocula was assessed in Shiraz fermentations at two maturity levels; earlier harvest (24 °Brix) and technical ripeness (29 °Brix). Eight yeast treatments trialled in pilot scale fermentations included sequential inoculations using three Torulaspora delbureckii strains, one strain each of Lachancea thermotolerans and Metschnikowia pulcherrima, a commercial blend of non-Saccharomyces and S. cerevisiae, and appropriate S. cerevisiae controls. Fermentation monitoring and comprehensive chemical and sensory analysis allowed for the comparison of the treatments. The results showed a pronounced matrix-derived modulation of wine profiles which was reflective of grape maturity levels. Within each harvest date, however, the yeast treatments had a significant impact on a range of compositional and wine sensory characters. At earlier harvest stage, certain non-Saccharomyces treatments, in particular T. delbrueckii, led to an increased wine sensory appeal (i.e. ‘floral’, ‘red fruit’, ‘aroma intensity’, ‘spice’) compared to the S. cerevisiae control (‘vegetal, ‘acidic’ and ‘bitter’). These treatments, however, were related to incomplete fermentations in higher ripeness conditions. Thus, some non-Saccharomyces yeast showed promise in enhancing the quality of wines produced from earlier harvested grapes and as such represent a complementary approach in managing wine ethanol concentrations. The following study addressed the selection of lower-ethanol producing non-Saccharomyces strain(s) for use in sequential cultures with Saccharomyces cerevisiae. Oenological performances of six M. pulcherrima strains were evaluated in fermentations with S. cerevisiae inoculated after seven days. The best-performing M. pulcherrima MP2 strain was further characterised in six sequential fermentations with different S. cerevisiae inoculation delays in both synthetic and white grape juice. The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in mixed culture fermentations. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae control, with even larger decreases achieved in the synthetic juice. The MP2 wines also had higher concentrations of glycerol and lower concentrations of acetic acid. The analysis of volatile compounds revealed compositional alterations arising from the S. cerevisiae inoculation delay, with increased acetate esters and higher alcohols detected in all analysed MP2 treatments. The concept of intra-specific variability was studied using L. thermotolerans as a model. This species harbours several metabolic traits that are of value in oenology, including lactic acid production, potential to decrease ethanol content and modulate flavour in wines. The relationships between 172 L. thermotolerans isolates, sourced from natural and anthropic habitats worldwide, were studied using a 14-microsatellite genotyping method. The resultant clustering revealed that the evolution of L. thermotolerans has been driven by the geography and ecological niche of the isolation sources. Isolates originating from anthropic, in particular oenological environments, were genetically close, thus suggesting domestication events within the species. The phenotypic performance of the strains, assessed using a number of agar platebased growth assays with different carbon sources and physicochemical conditions, provided further support for the observed clustering. To determine whether, and to what extent, L. thermotolerans strains differ in the traits of oenological importance, and harbour signatures of domestication and/or local divergence, 94 previously genotyped strains were trialled in Chardonnay fermentations. The strains and the genetic groups were compared for their fermentation performance, production of primary and secondary metabolites and pH modulation. The common traits of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. These observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Impact of <i>Lachancea thermotolerans</i> strain and lactic acid concentration on <i>Oenococcus oeni</i> and malolactic fermentation in wine

The yeast Lachancea thermotolerans can produce lactic acid during alcoholic fermentation (AF) and thereby acidify wines with insufficient acidity. However, little is known about the impact of L. thermotolerans on Oenococcus oeni, the primary lactic acid bacterium used in malolactic fermentation (MLF). This study explored the impact of sequential cultures of L. thermotolerans and Saccharomyces cerevisiae on MLF performance in white and red wines. Four L. thermotolerans strains were tested in Sauvignon blanc with sequential S. cerevisiae inoculation, compared to an S. cerevisiae control and the initially un-inoculated treatments. The L. thermotolerans wines showed large differences in acidification, and progression of MLF depended on lactic acid production, even at controlled pH. The highest and lowest lactic acid producing strains were tested further in Merlot fermentations with both co-inoculated and sequentially inoculated O. oeni. The low lactic acid producing strain enabled successful MLF, even when this failed in the S. cerevisiae treatment, with dramatically quicker malic acid depletion in O. oeni co-inoculation than in sequential inoculation. In contrast, a high lactic acid producing strain inhibited MLF irrespective of the O. oeni inoculation strategy. In a follow-up experiment, increasing concentrations of exogenously added lactic acid slowed MLF and reduced O. oeni growth across different matrices, with 6 g/L of lactic acid completely inhibiting MLF. The results confirm the inhibitory effect of lactic acid on O. oeni while highlighting the potential of some L. thermotolerans strains to promote MLF and the others to inhibit it

Beyond the Brain: Perinatal Exposure of Rats to Serotonin Enhancers Induces Long-Term Changes in the Jejunum and Liver

Serotonin (5-hydroxytryptamine, 5HT) homeostasis is essential for many physiological processes in the central nervous system and peripheral tissues. Hyperserotonemia, a measurable sign of 5HT homeostasis disruption, can be caused by 5HT-directed treatment of psychiatric and gastrointestinal diseases. Its impact on the long-term balance and function of 5HT in the peripheral compartment remains unresolved and requires further research due to possible effects on human health. We explored the effects of perinatal 5HT imbalance on the peripheral organs responsible for serotonin metabolism—the jejunum, a synthesis site, and the liver, a catabolism site—in adult rats. Hyperserotonemia was induced by subchronic treatment with serotonin precursor 5-hydroxytryptophan (5HTP) or serotonin degradation inhibitor tranylcypromine (TCP). The jejunum and liver were collected on postnatal day 70 and analyzed histomorphometrically. Relative mRNA levels of 5HT-regulating proteins were determined using qRT-PCR. Compared to controls, 5HTP- and TCP-treated rats had a reduced number of 5HT-producing cells and expression of the 5HT-synthesising enzyme in the jejunum, and an increased expression of 5HT-transporter accompanied by karyomegaly in hepatocytes, with these differences being more pronounced in the TCP-treated animals. Here, we report that perinatal 5HT disbalance induced long-term cellular and molecular changes in organs regulating 5HT-metabolism, which may have a negative impact on 5HT availability and function in the periphery. Our rat model demonstrates a link between the developmental abnormalities of serotonin homeostasis and 5HT-related changes in adult life and may be suitable for exploring the neurobiological substrates of vulnerability to behavioral and metabolic disorders, as well as for modeling the adverse effects of the prenatal exposure to 5HT enhancers in the human population

Geographic origin of the genotyped <i>L</i>. <i>thermotolerans</i> isolates obtained from different substrates.

<p>Isolates with unknown origin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184652#pone.0184652.s001" target="_blank">S1 Table</a>) are not represented on the map.</p

Genetic clustering of 172 <i>L</i>. <i>thermotolerans</i> isolates determined using 14 microsatellite makers.

<p>Each dot represents a genotype, with colours corresponding to determined genetic groups as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184652#pone.0184652.g002" target="_blank">Fig 2</a>. (A) Dendrogram constructed Bruvo’s distance and NJ clustering. (B) Reliability assessment of the nodes of the dendrogram constructed using Bruvo’s distance and NJ clustering. (C) Dendrogram constructed Bruvo’s distance and UPGMA clustering. (D) PCA of the allelic data.</p

Microsatellite loci for <i>L</i>. <i>thermotolerans</i> genotyping.

<p>Microsatellite loci for <i>L</i>. <i>thermotolerans</i> genotyping.</p

Phenotypic performance tested on plates using different carbon sources and physicochemical conditions.

<p><b>Dendrogram constructed with Euclidean distance and Ward clustering using normalised values of obtained growth of 132 <i>L</i>. <i>thermotolerans</i> and 11 non-<i>thermotolerans</i> strains in tested conditions, and/or a corresponding heatplot (left). Comparison of phenotypic performance at a genetic group level (right).</b> Glu–glucose, GF–equimolar mixture of glucose and fructose, Xyl–xylose, Fru–fructose, Gal–galactose, Man–mannose, Gly–glycerol; unless otherwise specified, carbon sources were supplemented in concentration of 2%, and incubation temperature was 24°C; numbers 3, 6 and 10 refer to the incubation duration. No quantifiable growth was observed for ‘GF-3-50%’, ‘G-3-8°’ and ‘G-6-8°’ modalities, thus not included graphical representation. Colours of the represented individuals/genetic groups correspond to Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184652#pone.0184652.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184652#pone.0184652.g003" target="_blank">3</a>. Dots and bars represent normalised growth means and ranges, respectively, and letters denote significance levels between genetic groups (KW tests; alpha = 0.05).</p

Pairwise F_ST distance matrix.

<p>F_ST values are given in the upper matrix, whereas the lower matrix indicates bootstrap values and, in brackets, associated confidence intervals.</p

Genetic relationships between 172 <i>L</i>. <i>thermotolerans</i> isolates determined using 14 microsatellite makers.

<p>Colour-coding of isolates corresponds to isolation substrate, as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184652#pone.0184652.g001" target="_blank">Fig 1</a>. (A) Dendrogram constructed using Bruvo’s distance and NJ clustering. (B) Barplot representing population structure (K = 8 and K = 12). The posterior probability (y-axis) of assignment of each isolate (vertical bar) to inferred ancestral populations is shown with different colours.</p

Impact of Lachancea thermotolerans on chemical composition and sensory profiles of Merlot wines

International audienceWines from warm(ing) climates often contain excessive ethanol but lack acidity. The yeast Lachancea thermotolerans can ameliorate such wines due to partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in two inoculation modalities (sequential and co-inoculation) to Saccharomyces cerevisiae and un-inoculated treatments in high sugar/low acidity Merlot fermentations. The pH and ethanol levels in mixed-culture dry wines were either comparable, or significantly lower than in controls (decrease of up to 0.5 units and 0.90% v/v, respectively). The analysis of volatile compounds revealed marked differences in major flavour-active yeast metabolites, including up to a thirty-fold increase in ethyl lactate in certain L. thermotolerans modalities. The wines significantly differed in acidity perception, alongside 18 other sensory attributes. Together, these results highlight the potential of some L. thermotolerans strains to produce ‘fresher’ wines with lower ethanol content and improved flavour/balance