Effect of 'loss of function' mutation in SER in wine yeast: fermentation outcomes in co-inoculation with non-Saccharomyces

Published: 20 April 2022In wine fermentation, improved wine complexity and sensorial properties can arise from the use of non-Saccharomyces yeast. Generally less alcohol tolerant, such strains often do not finish fermentation, therefore requiring a second inoculation with the more robust Saccharomyces cerevisiae, usually added on Day 3. This sequential approach affords non-Saccharomyces time to make an impact before being overtaken by S. cerevisiae. However, two inoculations are inconvenient; therefore the identification of a slow growing S. cerevisiae strain that can be used in a single co-inoculation with the non-Saccharomyces yeast is highly attractive. In this study we investigated the use of the naturally occurring ‘loss of function’ SER1 variant, identified in a Sake yeast, for the purposes of carrying out co-inoculated wine fermentations. The SER1-232(G > C; G78R) change was introduced into the commonly used wine strain, EC1118, via CRISPR/Cas9 editing. In a chemically defined grape juice medium, the SER1(G78R) mutant grew and fermented more slowly and increased acetic acid, succinic acid and glycerol concentrations. Simultaneous inoculation with the slower-growing mutant with a Metschnikowia pulcherrima or Lachancea thermotolerans strain in sterile Sauvignon blanc juice resulted in differences in sensorial compounds, most likely derived from the presence of non-Saccharomyces yeasts. The EC1118 SER1 (G78R) mutant completed fermentation with M. pulcherrima, MP2, and in fact improved the viability of MP2 compared to when it was used as a monoculture. The SER1 (G78R) mutant also promoted both the growth of the SO2-sensitive L. thermotolerans strain, Viniflora® Concerto™, in a juice high in SO2 and its subsequent dominance during fermentation. In co-fermentations with wild-type EC1118, the Concerto™ population was substantially reduced with no significant changes in wine properties. This research adds to our understanding of the use of a novel slow-growing S. cerevisiae yeast in wine fermentations co-inoculated with non-Saccharomyces strains.Tom A. Lang, Michelle E. Walker, Paul K. Boss and Vladimir Jirane

Grape Heterogeneity Index: Assessment of Overall Grape Heterogeneity Using an Aggregation of Multiple Indicators

Uniform grape maturity can be sought by producers to minimise underripe and/or overripe proportions of fruit and limit any undesirable effects on wine quality. Considering that grape heterogeneity is a multifaceted phenomenon, a composite index summarising overall grape heterogeneity was developed to benefit vineyard management and harvest date decisions. A grape heterogeneity index (GHI) was constructed by aggregating the sum of absolute residuals multiplied by the range of values from measurements of total soluble solids, pH, fresh weight, total tannins, absorbance at 520 nm (red colour), 3-isobutyl-2-methoxypyrazine, and malic acid. Management of grape heterogeneity was also studied, using Cabernet Sauvignon grapes grown under four viticultural regimes (normal/low crop load, full/deficit irrigation) during the 2019/2020 and 2020/2021 seasons. Comparisons of GHI scores showed grape variability decreased throughout ripening in both vintages, then significantly increased at the harvest time point in 2020, but plateaued on sample dates nearing the harvest date in 2021. Irrigation and crop load had no effect on grape heterogeneity by the time of harvest in both vintages. Larger vine yield, leaf area index, and pruning weight significantly increased GHI score early in ripening, but no significant relationship was found at the time of harvest. Differences in the Ravaz index, normalised difference vegetation index, and soil electrical conductivity did not significantly change the GHI score.Claire E. J. Armstrong, Pietro Previtali, Paul K. Boss, Vinay Pagay, Robert G. V. Bramley, and David W. Jeffer

Distribution of 3-Isobutyl-2-methoxypyrazine across Rachis Components of Vitis vinifera Shiraz and Cabernet Sauvignon

Published 18 April 2023. Special Issue - 18th Australian Wine Industry Technical Conference.Rootstock can significantly alter the concentration of methoxypyrazines (MPs) in the bunch stem (rachis) of Vitis vinifera L. cv. Cabernet Sauvignon and Shiraz, which has implications for winemaking and wine style. The distribution of MPs across the rachis is an important consideration, but such information was not available. This study aimed to address this research question by comparing MP concentrations in different rachis components throughout grape maturation and in the absence of ambient light. Shiraz and Cabernet Sauvignon bunches were sampled throughout development, segmented into four components (peduncle, top rachis, bottom rachis, and pedicel), and 3-isobutyl-2-methoxypyrazine (IBMP) was quantified in each. For both cultivars, IBMP showed a negative correlation with grape maturity, with concentrations in pedicel at harvest being significantly higher than other rachis components. Additionally, light exclusion significantly increased IBMP concentrations in all rachis segments. The concentration of IBMP varied significantly between different rachis components. The greatest concentrations were measured in the pedicel, which also contributed the largest proportion among the components to total rachis by weight. Due to elevated IBMP concentrations in rachis and the difficulties in excluding matter other than grape from a fermentor, the presence of pedicel during fermentation could produce Shiraz and Cabernet Sauvignon wines with higher concentrations of MPs, thereby potentially increasing vegetal sensory characteristics.Ross D. Sanders, Paul K. Boss, Dimitra L. Capone, Catherine M. Kidman, Emily L. Nicholson, and David W. Jeffer

Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Strange particle production in proton-proton collisions at s=0.9\sqrt{s}=0.9 TeV with ALICE at the LHC

The production of mesons containing strange quarks (K$^0_s$, $\phi$) and both singly and doubly strange baryons ($\Lambda$, Anti-$\Lambda$, and $\Xi$+Anti-$\Xi$) are measured at central rapidity in pp collisions at $\sqrt{s}$ = 0.9 TeV with the ALICE experiment at the LHC. The results are obtained from the analysis of about 250 k minimum bias events recorded in 2009. Measurements of yields (dN/dy) and transverse momentum spectra at central rapidities for inelastic pp collisions are presented. For mesons, we report yields () of 0.184 $\pm$ 0.002 stat. $\pm$ 0.006 syst. for K$^0_s$ and 0.021 $\pm$ 0.004 stat. $\pm$ 0.003 syst. for $\phi$. For baryons, we find = 0.048 $\pm$ 0.001 stat. $\pm$ 0.004 syst. for $\Lambda$, 0.047 $\pm$ 0.002 stat. $\pm$ 0.005 syst. for Anti-$\Lambda$ and 0.0101 $\pm$ 0.0020 stat. $\pm$ 0.0009 syst. for $\Xi$+Anti-$\Xi$. The results are also compared with predictions for identified particle spectra from QCD-inspired models and provide a baseline for comparisons with both future pp measurements at higher energies and heavy-ion collisions.Comment: 33 pages, 21 captioned figures, 10 tables, authors from page 28, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/387

Two-pion Bose-Einstein correlations in central Pb-Pb collisions at sNN\sqrt{s_{\rm NN}} = 2.76 TeV

The first measurement of two-pion Bose-Einstein correlations in central Pb-Pb collisions at $\sqrt{s_{\rm NN}} = 2.76$ TeV at the Large Hadron Collider is presented. We observe a growing trend with energy now not only for the longitudinal and the outward but also for the sideward pion source radius. The pion homogeneity volume and the decoupling time are significantly larger than those measured at RHIC.Comment: 17 pages, 5 captioned figures, 1 table, authors from page 12, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/388

Suppression of charged particle production at large transverse momentum in central Pb-Pb collisions at sNN=2.76\sqrt{s_{\rm NN}} = 2.76 TeV

Inclusive transverse momentum spectra of primary charged particles in Pb-Pb collisions at $\sqrt{s_{_{\rm NN}}}$ = 2.76 TeV have been measured by the ALICE Collaboration at the LHC. The data are presented for central and peripheral collisions, corresponding to 0-5% and 70-80% of the hadronic Pb-Pb cross section. The measured charged particle spectra in $|\eta|<0.8$ and $0.3 < p_T < 20$ GeV/$c$ are compared to the expectation in pp collisions at the same $\sqrt{s_{\rm NN}}$, scaled by the number of underlying nucleon-nucleon collisions. The comparison is expressed in terms of the nuclear modification factor $R_{\rm AA}$. The result indicates only weak medium effects ($R_{\rm AA} \approx$ 0.7) in peripheral collisions. In central collisions, $R_{\rm AA}$ reaches a minimum of about 0.14 at $p_{\rm T}=6$-7GeV/$c$ and increases significantly at larger $p_{\rm T}$. The measured suppression of high-$p_{\rm T}$ particles is stronger than that observed at lower collision energies, indicating that a very dense medium is formed in central Pb-Pb collisions at the LHC.Comment: 15 pages, 5 captioned figures, 3 tables, authors from page 10, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/98

Particle-yield modification in jet-like azimuthal di-hadron correlations in Pb-Pb collisions at sNN\sqrt{s_{\rm NN}} = 2.76 TeV

The yield of charged particles associated with high-$p_{\rm T}$ trigger particles ($8 < p_{\rm T} < 15$ GeV/$c$) is measured with the ALICE detector in Pb-Pb collisions at $\sqrt{s_{\rm NN}}$ = 2.76 TeV relative to proton-proton collisions at the same energy. The conditional per-trigger yields are extracted from the narrow jet-like correlation peaks in azimuthal di-hadron correlations. In the 5% most central collisions, we observe that the yield of associated charged particles with transverse momenta $p_{\rm T}> 3$ GeV/$c$ on the away-side drops to about 60% of that observed in pp collisions, while on the near-side a moderate enhancement of 20-30% is found.Comment: 15 pages, 2 captioned figures, 1 table, authors from page 10, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/350