Comparative impact of exogenous phenylalanine on oenological isolates of Kluyveromyces marxianus and Saccharomyces cerevisiae.

AIMS: Kluyveromyces marxianus' high production of 2-phenylethyl acetate (2-PEA) via L-phenylalanine (Phe) catabolism makes it relevant for industries relying on the production of aroma compounds through fermentation processes. This study assessed the physiological impact of exogenous supplementation of Phe on cell viability, fermentation performance, and, by extension, on lipid and amino acid metabolism in a wine isolate of this yeast. METHODS AND RESULTS: The data showed that Phe exerted cytotoxic effects on K. marxianus IWBT Y885, which were minimal on Saccharomyces cerevisiae and impacted amino acid metabolism and aroma production. We demonstrated that K. marxianus strains fermented sugars more effectively in the absence of Phe. While lipid supplementation did not mitigate any deleterious effects of Phe, it supported viability maintenance and fermentation performance in the absence of Phe. Phe supplementation succeeded in augmenting the production of 2-PE and 2-PEA. CONCLUSIONS: The enhanced production of 2-PEA in K. marxianus suggests that this transesterification may be, at least in part, a compensatory detoxification mechanism for this yeast.

Thiamine: a key nutrient for yeasts during wine alcoholic fermentation.

Alcoholic fermentation is a crucial step of winemaking, during which yeasts convert sugars to alcohol and also produce or biotransform numerous flavour compounds. In this context, nutrients are essential compounds to support yeast growth and ultimately ensure complete fermentation, as well as optimized production of flavour compounds over that of off-flavour compounds. In particular, the vitamin thiamine not only plays an essential cofactor role for several enzymes involved in various metabolic pathways, including those leading to the production of wine-relevant flavour compounds, but also aids yeast survival via thiamine-dependent stress protection functions. Most yeast species are able to both assimilate exogenous thiamine into the cell and synthesize thiamine de novo. However, the mechanism and level of thiamine accumulation depend on several factors. This review provides an in-depth overview of thiamine utilization and metabolism in the model yeast species Saccharomyces cerevisiae, as well as the current knowledge on (1) the intracellular functions of thiamine, (2) the balance between and regulation of uptake and synthesis of thiamine and (3) the multitude of factors influencing thiamine availability and utilization. For the latter, a particular emphasis is placed on conditions occurring during wine fermentation. The adequacy of thiamine concentration in grape must to ensure successful fermentation is discussed together with the effect of thiamine concentration on fermentation kinetics and on wine sensory properties. This knowledge may serve as a resource to optimise thiamine concentrations for optimal industrial application of yeasts. KEY POINTS: • Thiamine uptake is preferred over biosynthesis and is transcriptionally repressed. • Multiple factors affect thiamine synthesis, availability and uptake for wine yeast. • Thiamine availability impacts fermentation kinetics and wine's sensory properties.

Combinatorial analysis of population dynamics, metabolite levels and malolactic fermentation in Saccharomyces cerevisiae/ Lachancea thermotolerans mixed fermentations.

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.

The expression, secretion and activity of the aspartic protease MpAPr1 in Metschnikowia pulcherrima IWBT Y1123.

Protease-secreting yeasts have broad biotechnological potential for application to various industrial processes, including winemaking. However, this activity is influenced by the yeast response to environmental factors such as nitrogen and protein sources, as are found in grape juice. In this study, the wine-relevant yeast Metschnikowia pulcherrima IWBT Y1123, with known protease-secreting ability, was subjected to different nitrogen-containing compounds to monitor their impact on protease secretion and activity. Protease activity increased above basal levels for haemoglobin-containing treatments, indicating an inductive influence of proteins. On the other hand, treatments containing both haemoglobin and assimilable nitrogen sources led to a delayed increase in protease activity and protein degradation, suggesting a nitrogen catabolite repression mechanism at work. Protease activity and expression were furthermore evaluated in grape juice, which revealed increased expression and activity levels over time as promising results for further investigations into the impact of this yeast on wine properties.

Understanding the regulation of extracellular protease gene expression in fungi: a key step towards their biotechnological applications.

The secretion of proteases by certain species of yeast and filamentous fungi is of importance not only for their biological function and survival, but also for their biotechnological application to various processes in the food, beverage, and bioprocessing industries. A key step towards understanding the role that these organisms play in their environment, and how their protease-secreting ability may be optimally utilised through industrial applications, involves an evaluation of those factors which influence protease production. The objective of this review is to provide an overview of the findings from investigations directed at elucidating the regulatory mechanisms underlying extracellular protease secretion in yeast and filamentous fungi, and the environmental stimuli that elicit these responses. The influence of nitrogen-, carbon-, and sulphur-containing compounds, as well as proteins, temperature, and pH, on extracellular protease regulation, which is frequently exerted at the transcriptional level, is discussed in particular depth. Protease-secreting organisms of biotechnological interest are also presented in this context, in an effort to explore the areas of industrial significance that could possibly benefit from such knowledge. In this way, the establishment of a platform of existing knowledge regarding fungal protease regulation is attempted, with the particular goal of aiding in the practical application of these organisms to processes that require secretion of this enzyme.

Candida pyralidae killer toxin disrupts the cell wall of Brettanomyces bruxellensis in red grape juice.

AIMS: The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2 ) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B. bruxellensis cells in red grape juice. METHODS AND RESULTS: Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B. bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of ß-glucan, suggesting a compensatory response of the sensitive cells. CONCLUSIONS: The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.

Optimization of carbon and nitrogen medium components for biomass production using non-Saccharomyces wine yeasts.

UNLABELLED: The impact of different nitrogen and carbon sources on biomass production of the non-Saccharomyces wine yeast species Lachancea thermotolerans, Metschnikowia pulcherrima and Issatchenkia orientalis was assessed. Using a molasses-based medium, yeast extract and corn steep liquor as well as ammonium sulphate and di-ammonium phosphate (DAP) as nitrogen sources were compared in shake-flask cultures. A medium with 20 g l⁻¹ sugar (diluted molasses) and 500 mg l⁻¹ total yeast assimilable nitrogen, from yeast extract, gave the highest biomass concentrations and yields. Invertase pretreatment was required for cultures of M. pulcherrima and I. orientalis, and respective biomass yields of 0.7 and 0.8 g g⁻¹ were achieved in aerobic bioreactor cultures. The absence of ethanol production suggested Crabtree-negative behaviour by these yeasts, whereas Crabtree-positive behaviour by L. thermotolerans resulted in ethanol and biomass concentrations of 5.5 and 11.1 g l⁻¹, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Recent studies demonstrate that non-Saccharomyces yeasts confer positive attributes to the final composition of wine. However, optimal process conditions for their biomass production have not been described, thereby limiting commercial application. In this study, industrial media and methods of yeast cultivation were investigated to develop protocols for biomass production of non-Saccharomyces yeast starter cultures for the wine industry.

Genetic screening of wine-related enzymes in Lactobacillus species isolated from South African wines.

AIMS: The objective of this study was to investigate the presence of genes coding for enzymes of oenological relevance in wine Lactobacillus strains isolated from South African grape and wine samples during the 2001 and 2002 harvest seasons. METHODS AND RESULTS: A total of 120 wine lactobacilli isolates belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus sakei and Lactobacillus paraplantarum were genetically screened for enzyme-encoding genes using PCR with primers specific for beta-glucosidase, protease, esterase, citrate lyase and phenolic acid decarboxylase. The results of PCR screening showed that the Lactobacillus strains possessed different combinations of enzymes and that some strains did not possess any of the enzymes tested. Confirmation analysis with gene sequencing also showed high similarity of genes with those available in GenBank database. CONCLUSION: In this study, we have demonstrated the existence of genes coding for wine-related enzymes in wine lactobacilli that could potentially hydrolyse wine precursors to positively influence wine aroma. SIGNIFICANCE AND IMPACT OF THE STUDY: An expansion of knowledge on the genetic diversity of wine-associated lactic acid bacteria will enable the selection of novel malolactic fermentation starter cultures with desired oenological traits for the improvement of the organoleptic quality of the wine, and hence wine aroma.

Genetic characterization of strains of Saccharomycescerevisiae responsible for 'refermentation' in Botrytis-affected wines.

AIMS: Saccharomyces cerevisiae is responsible for alcoholic fermentation of wines. However, some strains can also spoil sweet Botrytis-affected wines. Three 'refermentation' strains were isolated during maturation. Characterization of those strains in regards to their fingerprint, rDNA sequence and resistance to SO2, which constituted the main source of stress in Botrytis-affected wines, was carried out. METHODS AND RESULTS: Refermentation strains could be clearly discriminated by interdelta fingerprinting. However, they exhibited close relationships by karyotyping. A part of RDN1 locus sequence was examined by using PCR-RFLP and PCR-DGGE. The resistance of refermentation strains to SO2 was performed by using real time quantitative PCR focusing on SSU1 gene. CONCLUSIONS: Results suggested that refermentation strains were heterozygote in 26S rDNA and their ITS1-5.8S rDNA-ITS2 region sequence revealed relationships with 'flor' strains. As described in the literature for flor strain, two out of three refermentation strains constitutively developed a higher level of SSU1 expression than the reference strains, improving their putative tolerance to SO2. Therefore, refermentation strains of S. cerevisiae had developed many strategies to survive during maturing sweet wines. SIGNIFICANCE AND IMPACT OF THE STUDY: Singularities in rDNA sequence and SSU1 overexpression revealed a natural adaptation. Moreover, genomic relationship between flor and refermentation strains suggested that stress sources could induced selection of survivor strains.

Evidence for viable but nonculturable yeasts in botrytis-affected wine.

AIMS: In Botrytis-affected wine, high concentrations of SO2 are added to stop the alcoholic fermentation and to stabilize the wine. During maturing in barrels or bottle-ageing, an unwanted refermentation can sometimes occur. However, results of the usual plate count of wine samples at the beginning of maturing suggest wine microbial stability. The aim of this study was to investigate the mode of yeasts survival after the addition of SO2 and to identify surviving yeasts. METHODS AND RESULTS: Using direct epifluorescence technique, we observed the behaviour of cells after SO2 addition and compared the cell number determined by this method with the result of plate counts. The persistent yeast species were identified using two methods: polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-denaturing gradient gel electrophoresis. They were identified as Saccharomyces cerevisiae and Candida stellata, and after few months of maturing, other spoiling yeasts appeared, like Rhodotorula mucilaginosa or Zygosaccharomyces bailii. CONCLUSIONS: All characteristics of the cells lead to the conclusion that yeast persisted in wine in a viable but nonculturable-like state (VBNC). Suppression of the effect of free-SO2 did not lead to the resuscitation of the cells; however, another method proved the capacity of the cells to exit from the VBNC-like state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of the presence of VBNC-like yeasts in wine. The 'refermentation' phenomenon is probably due to the exit of the VBNC state.

Molecular characterization of Oenococcus oeni genes encoding proteins involved in arginine transport.

AIMS: This work was carried out to complete the sequence of the arc cluster involved in arginine catabolism in Oenococcus oeni, and particularly to characterize the genes encoding proteins involved in arginine transport. METHODS AND RESULTS: Using molecular cloning, two loci encoding proteins involved in the arginine-ornithine antiport were isolated. Their expression patterns were monitored by RT-PCR to study the influence of arginine on their transcription. Polycistronic mRNAs were detected. PCR performed directly on colonies with primer pairs specific of arc genes was used to discriminate strains able/unable to degrade arginine. CONCLUSIONS: Oenococcus oeni contains two arcD loci encoding similar proteins. Their expression is not influenced by arginine and polycistronic messengers were detected. The inability to use arginine is due to a lack of genetic information encoding proteins of the arginine deiminase pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: The constitutive expression of arcD genes points to the positive role of arginine on O. oeni cell growth. The occasional presence of all the arc ABCD genes together in O. oeni strains might provide insights into the growth rate variability within this species.