Benoit Divol

Surviving in the presence of sulphur dioxide: strategies developed by wine yeasts

Sulphur dioxide has been used as a common preservative in wine since at least the nineteenth century. Its use has even become essential to the making of quality wines because of its antioxidant, antioxidasic and antiseptic properties. The chemistry of SO2 in wine is fairly complex due to its dissociation into different species and its binding to other compounds produced by yeasts and bacteria during fermentation. The only antiseptic species is the minute part remaining as molecular SO2. The latter concentration is both dependent on pH and concentration of free bisulphite. However, certain yeast species have developed cellular and molecular mechanisms as a response to SO2 exposure. Some of these mechanisms are fairly complex and have only been investigated recently, at least for the molecular mechanisms. They include sulphite reduction, sulphite oxidation, acetaldehyde production, sulphite efflux and the entry into viable but not culturable state, as the ultimate response. In this review, the chemistry of SO2 in wine is explained together with the impact of SO2 on yeast cells. The different defence mechanisms are described and discussed, mostly based on current knowledge available for Saccharomyces cerevisiae.

Genetic screening of lactic acid bacteria of oenological origin for bacteriocin-encoding genes

A total of 330 lactic acid bacteria isolated from South African red wines during alcoholic and malolactic fermentations and 9 commercial malolactic bacteria starter cultures were screened for antimicrobial activity. Of the entire screened isolates, 26 strains, belonging to the species Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus hilgardii and Oenococcus oeni, showed activity towards various wine-related and non-wine-related indicator strains. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Furthermore, a co-culture experiment with Lb. plantarum and Enterococcus faecalis was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 h. Four putative bacteriocin-encoding genes in the genome of O. oeni were identified and sequenced.

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.

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.

Characterization of novel killer toxins secreted by wine-related non-Saccharomyces yeasts and their action on Brettanomyces spp.

Wine spoilage associated with Brettanomyces bruxellensis is a major concern for winemakers. An effective and reliable method to control the proliferation of this yeast is therefore of utmost importance. To achieve this purpose, sulphur dioxide (SO2) is commonly employed but the efficiency of this chemical compound is subject to wine composition and it can elicit allergic reactions in some consumers. Biological alternatives are therefore actively sought. The current study focused on identifying and characterizing killer toxins which are antimicrobial compounds that show potential in inhibiting B. bruxellensis in wine. Two killer toxins, CpKT1 and CpKT2, from the wine isolated yeast Candida pyralidae were identified and partially characterized. The two proteins had a molecular mass above 50kDa and exhibited killer activity against several B. bruxellensis strains especially in grape juice. They were active and stable at pH3.5-4.5, and temperatures between 15 and 25°C which are compatible with winemaking conditions. Furthermore, the activity of these killer toxins was not affected by the ethanol and sugar concentrations typically found in grape juice and wine. In addition, these killer toxins inhibited neither the Saccharomyces cerevisiae nor the lactic acid bacteria strains tested. These preliminary results indicated that the application of these toxins will have no effect on the main microbial agents that drive alcoholic and malolactic fermentations and further highlight the potential of using these toxins as agents to control the development of B. bruxellensis in grape juice or wine.

Microbial aspartic proteases: current and potential applications in industry

Aspartic proteases are a relatively small group of proteolytic enzymes that are active in acidic environments and are found across all forms of life. Certain microorganisms secrete such proteases as virulence agents and/or in order to break down proteins thereby liberating assimilable sources of nitrogen. Some of the earlier applications of these proteolytic enzymes are found in the manufacturing of cheese where they are used as milk-clotting agents. Over the last decade, they have received tremendous research interest because of their involvement in human diseases. Furthermore, there has also been a growing interest on these enzymes for their applications in several other industries. Recent research suggests in particular that they could be used in the wine industry to prevent the formation of protein haze while preserving the wines’ organoleptic properties. In this mini-review, the properties and mechanisms of action of aspartic proteases are summarized. Thereafter, a brief overview of the industrial applications of this specific class of proteases is provided. The use of aspartic proteases as alternatives to clarifying agents in various beverage industries is mentioned, and the potential applications in the wine industry are thoroughly discussed.

Untangling the wine metabolome by combining untargeted SPME–GCxGC-TOF-MS and sensory analysis to profile Sauvignon blanc co-fermented with seven different yeasts

Saccharomyces cerevisiae (SC) is the main driver of alcoholic fermentation, however for aroma and flavor formation in wine, non-Saccharomyces species can have a powerful effect. This study aimed to compare untargeted volatile compound profiles from SPME–GCxGC-TOF-MS and sensory analysis data of Sauvignon blanc wine inoculated with six different non-Saccharomyces yeasts followed by SC. Torulaspora delbrueckii (TD), Lachancea thermotolerans (LT), Pichia kluyveri (PK) and Metschnikowia pulcherrima (MP) where commercial starter strains, while Candida zemplinina (CZ) and Kazachstania aerobia (KA), were isolated from wine grape environments. Each wine showed a distinct profile both sensorially and chemically. SC and CZ wines were the most distinct in both of these cases. SC wine had guava, grapefruit, banana, and pineapple aromas while CZ wine was driven by fermented apple, dried peach/apricot, and stewed fruit as well as sour flavor. Chemically over 300 unique features were identified as significantly different across the fermentations. SC wine had the highest number of esters in the highest relative concentration but all the yeasts had distinct ester profiles. CZ wine displayed the highest number of terpenes in high concentration but also produced a large amount of acetic acid. KA wine was high in ethyl acetate. TD wine had fewer esters but three distinctly higher thiol compounds. LT wine showed a relatively high number of increased acetate esters and certain terpenes. PK wine had some off odor compounds while the MP wine had high levels of methyl butyl-, methyl propyl-, and phenethyl esters. Overall, this study gives a more detailed profile of these yeasts contribution to Sauvignon blanc wine than previously reported.

Quantitative PCR: An appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10cellsmL(-1). B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated.

Identification and partial characterization of extracellular aspartic protease genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384

ABSTRACT The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomyces cerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence.

Effect of alternative NAD(+)-regenerating pathways on the formation of primary and secondary aroma compounds in a Saccharomyces cerevisiae glycerol-defective mutant.

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD+-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD+ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD+/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.