Construction of a recombinant autolytic wine yeast strain overexpressing the csc1‐1 allele

During the aging step of sparkling wines and wines aged on lees, yeast cells kept in contact with the wine finally die and undergo autolysis, releasing cellular compounds with a positive effect on the wine quality. In view of the interest of autolysis for wine properties, biotechnologists have tried to improve autolytic yield during winemaking. In this work we used genetic engineering techniques to construct an autolytic industrial strain by expressing the csc1‐1 allele from the RDN1 locus. The expression of this mutant allele, that causes a “constitutive in autophagy phenotype,” resulted in accelerated autolysis of the recombinant strain. Although autophagic phenotype due to csc1‐1 expression has been reported to require the mutant allele in multicopy, autolytic acceleration was achieved by expressing only one or two copies of the gene under the control of the constitutive promotor pTDH3. The acceleration of autolysis together with the unaltered fermentative capacity, strongly supported the overexpression of csc1‐1 allele as a strategy to obtain wines with aged‐like properties in a shortened time. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

陕西朱鹮黑米酒氨基酸成份分析

采用121MB型氨基酸分析仪测定了陕西朱鹮黑米酒中的氨基酸质量浓度。结果表明:朱鹮黑米酒中含有18种氨基酸和γ-氨基丁酸,氨基酸总质量浓度为9133.5 mg.L-1,必需氨基酸占氨基酸总量的51.7%。半必需氨基酸占氨基酸总量的10%。味觉氨基酸占氨基酸总量的73.8%。与文献中报道的发酵酒氨基酸总量相比是啤酒的5倍、红葡萄酒的5.4倍、黄酒的1.35倍、清酒2.18倍、沉缸酒的2.8倍,此外必需和半必需氨基酸的含量、味觉氨基酸含量与之比较也均为最高。     

Red wine decreases asymmetric dimethylarginine via SIRT1 induction in human endothelial cells

To investigate the effect of three red wines (RWs) from different growing areas and made from different grapes on asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, in young and senescent human endothelial cells (ECs). All RWs decreased ADMA levels, but 2-fold concentration of German RW was necessary to reach the same effect on ADMA compared to Italian RW and French RW without affecting the cell viability and morphology. The ADMA-lowering effect of RW was increased in senescent compared to young cells, accompanied by enhanced activity of the metabolizing enzyme: dimethylarginine dimethylaminohydrolase (DDAH) II, whereas the same amount in the upregulated protein expression of DDAH II and the downregulated protein expression of the synthesizing enzyme: protein arginine methyltransferase 1 was revealed. These effects were associated with decreased 8-iso-prostaglandin F_2α and peroxynitrite formation, enhanced protein expression of NAD⁺-dependent class III histone deacetylase sirtuin (SIRT) 1, and downregulated protein expression of histone senescence factor p53. Blockade of SIRT1 activity abolished the effect of red wine on ADMA. These data are the first demonstration that RW by activating SIRT1 impairs synthesis and increases metabolism of ADMA. This effect of RW is accentuated in senescent cells probably due to enhanced DDAH activity.     

Genetic improvement of thermo-tolerance in wine Saccharomyces cerevisiae strains by a backcross approach

During red wine fermentation, high temperatures may cause stuck fermentation by affecting the physiology of fermenting yeast. This deleterious effect is the result of the complex interaction of temperature with other physicochemical parameters of grape juice, such as sugar and lipid content. The genetic background of fermenting yeast also interacts with this complex matrix and some strains are more resistant to high temperatures than others. Here, the temperature tolerance of nine commercial starters was evaluated, demonstrating that, at high sugar concentrations, half of them are sensitive to temperature. Using a classical backcross approach, one thermo-sensitive commercial starter was genetically improved by introducing quantitative trait loci conferring resistance to temperature. With this breeding program it is possible to obtain a thermo-resistant strain sharing most of its genome with the initial commercial starter. The parental and improved strains were compared for population growth and fermentation ability in various conditions. Despite their common genetic background, these two strains showed slight physiological differences in response to environmental changes that enable identification of the key physiological parameters influencing stuck fermentation.     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non‐ropy wines

Aims: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine . Methods and Results: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with β‐glucan formation and ropiness were grown on grape juice medium, dialysed MRS‐derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol‐sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf?). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. Conclusion: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than β‐glucan. Significance and Impact of the Study: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.     

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability

Aims: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. Methods and Results: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real‐time RT‐PCR (qRT‐PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. Conclusions: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT‐PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. Significance and Impact of Study: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.     

Cork stoppers industry: defining appropriate mould colonization

AIMS: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. METHODS AND RESULTS: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. CONCLUSIONS: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.     

Yeasts present during wine fermentation: comparative analysis of conventional plating and PCR-TTGE

Yeasts isolated from must before and during fermentation at a wine cellar of La Mancha region in Spain were characterised using Polymerase Chain Reaction / Restriction Fragments Lengths Polymorphism and Polymerase Chain Reaction / Temporal Temperature Gradient Gel Electrophoresis. S. cerevisiae strains were differentiated using mtDNA restriction analysis. Direct PCR-TTGE was also used to study biodiversity during wine fermentation, and revealed the variations in the population. It was observed that isolation by conventional plating may afford a skewed view of the strains taking part in wine fermentation.