Browsing by Author "Divol, Benoit"

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    Altered fermentation performances, growth, and metabolic footprints reveal competition for nutrients between yeast species inoculated in synthetic grape juice-like medium
    (Frontiers Media, 2018) Rollero, Stephanie; Bloem, Audrey; Ortiz-Julien, Anne; Camarasa, Carole; Divol, Benoit
    The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance, fermentations performed with K. marxianus were characterized by the formation of phenylethanol and phenylethyl acetate, while those performed with P. burtonii or Z. meyerae displayed higher production of isoamyl alcohol and ethyl esters. When considering sequential inoculation of yeasts, the nutritional requirements of the yeasts used should be carefully considered and adjusted accordingly. Finally, our chemical data suggests that the organoleptic properties of the wine are altered in a species specific manner.
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    Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae
    (Public Library of Science, 2013-10-29) Salma, Mohammad; Rousseaux, Sandrine; Sequeira-Le Grand, Anabelle; Divol, Benoit; Alexandre, Herve
    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.
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    Comparative characterization of endo-polygalacturonase (Pgu1) from Saccharomyces cerevisiae and Saccharomyces paradoxus under winemaking conditions
    (2011) Eschstruth, A.; Divol, Benoit
    Wine strains of Saccharomyces cerevisiae have no to weak natural pectinase activity, despite their genetic ability to secrete an endo-polygalacturonase. The addition of external pectinase of fungal origin has therefore become a common step of winemaking in order to enhance the extraction of compounds located in the grape berry skins during maceration and to ease wine clarification after maturation. Recently, the strong pectinase activity of a wine strain of Saccharomyces paradoxus has been reported. In this study, the endo-polygalacturonase-encoding gene of S. paradoxus was sequenced and its activity was characterised, compared with that of S. cerevisiae and tested under winemaking conditions through overexpression of both genes individually in S. cerevisiae. A few differences in the amino acids sequences between the two proteins were revealed and the activity of the Pgu1 enzyme of S. paradoxus was shown to be weaker under winemaking conditions. Clear indicators of extracellular activity were observed in the wines made with both recombinant strains (i.e. enzyme activity in cell-free wine, higher methanol concentration and higher free-run wine), but the actual composition of the wines fermented with the mutants was only sparingly altered. Although unexpectedly found in lower concentrations in the latter wines, phenolic compounds were shown to be the most discriminatory components. Overexpressing the PGU1 gene of S. paradoxus or that of S. cerevisiae did not make much difference, showing that the higher activity of S. paradoxus strains under laboratory conditions could be due to a different regulation mechanism rather than to a different sequence of PGU1. © 2011 Springer-Verlag.
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    Effect of alternative NAD +-regenerating pathways on the formation of primary and secondary aroma compounds in a Saccharomyces cerevisiae glycerol-defective mutant
    (2012) Jain, V. K.; Divol, Benoit; Prior, B. A.; Bauer, Florian
    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. © 2011 Springer-Verlag.
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    Elimination of glycerol and replacement with alternative products in ethanol fermentation by Saccharomyces cerevisiae
    (2011) Jain, V. K.; Divol, Benoit; Prior, B. A.; Bauer, Florian
    Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2-3% of the sugar fermented is converted to glycerol. Replacing the NAD+-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD+ by transforming a double mutant (gpd1δgpd2δ) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD+ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains. © 2010 Society for Industrial Microbiology.
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    Epigenetic regulation of PGU1 transcription in Saccharomyces cerevisiae
    (2010) Louw, C.; Young, P. R.; Van Rensburg, P.; Divol, Benoit
    The PGU1 gene of Saccharomyces cerevisiae has been shown to encode a polygalacturonase. The polygalacturonase activity in S. cerevisiae is strain specific. There are no significant differences in the PGU1 promoter regions of strains with and without polygalacturonase activity. The PGU1 gene is subtelomeric because it is located within 25 kb of the right telomere of chromosome X. Expressions of genes located in subtelomeric regions in the yeast S. cerevisiae are inhibited compared with the rest of the genome. In this study, we showed that the deletion of genes involved in telomere silencing enhances polygalacturonase activity. PGU1 transcription and polygalacturonase activity are increased when PGU1 is shifted to a different location in the genome, away from the telomere located close to this gene, and the depletion of the histone H4 leads to an increase in PGU1 transcription. We concluded that PGU1 is silenced in strains without polygalacturonase activity due to an epigenetic effect. The results of this study suggest that PGU1 is silenced by being folded into a heterochromatin-like structure at its subtelomeric position on chromosome X. Formation of this silent structure is dependent on the Isw2p chromatin remodeling complex, its histone fold motif containing subunit Dls1p and the N-terminal tail of the H4 histone. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.
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    Evaluating Lactobacillus and Pediococcus strains for enzyme-encoding genes related to peptide and amino acid utilization in wine
    (INST MICROBIOLOGIA, VIA CELORIA 2, MILAN, ITALY, 20133, 2013) Mtshali, P. S.; Divol, Benoit; Du Toit, M.
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    Genetic screening of wine-related enzymes in Lactobacillus species isolated from South African wines
    (2010) Mtshali, P. S.; Divol, Benoit; Van Rensburg, P.; Du Toit, M.
    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 β-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. © 2009 The Authors.
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    Hanseniaspora uvarum from winemaking environments show spatial and temporal genetic clustering
    (Frontiers, 2016-01-20) Albertin, Warren; Setati, Mathabatha E.; Miot-Sertier, Cecile; Mostert, Talitha T.; Colonna-Ceccaldi, Benoit; Coulon, Joana; Girard, Patrick; Moine, Virginie; Pillet, Myriam; Salin, Franck; Bely, Marina; Divol, Benoit; Masneuf-Pomarede, Isabelle
    Hanseniaspora uvarum is one of the most abundant yeast species found on grapes and in grape must, at least before the onset of alcoholic fermentation (AF) which is usually performed by Saccharomyces species. The aim of this study was to characterize the genetic and phenotypic variability within the H. uvarum species. One hundred and fifteen strains isolated from winemaking environments in different geographical origins were analyzed using 11 microsatellite markers and a subset of 47 strains were analyzed by AFLP. H. uvarum isolates clustered mainly on the basis of their geographical localization as revealed by microsatellites. In addition, a strong clustering based on year of isolation was evidenced, indicating that the genetic diversity of H. uvarum isolates was related to both spatial and temporal variations. Conversely, clustering analysis based on AFLP data provided a different picture with groups showing no particular characteristics, but provided higher strain discrimination. This result indicated that AFLP approaches are inadequate to establish the genetic relationship between individuals, but allowed good strain discrimination. At the phenotypic level, several extracellular enzymatic activities of enological relevance (pectinase, chitinase, protease, β-glucosidase) were measured but showed low diversity. The impact of environmental factors of enological interest (temperature, anaerobia, and copper addition) on growth was also assessed and showed poor variation. Altogether, this work provided both new analytical tool (microsatellites) and new insights into the genetic and phenotypic diversity of H. uvarum, a yeast species that has previously been identified as a potential candidate for co-inoculation in grape must, but whose intraspecific variability had never been fully assessed.
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    Hanseniaspora uvarum from winemaking environments show spatial and temporal genetic clustering
    (Frontiers Media, 2016) Albertin, Warren; Setati, Mathabatha E.; Miot-Sertier, Cecile; Mostert, Talitha T.; Colonna-Ceccaldi, Benoit; Coulon, Joana; Girard, Patrick; Moine, Virginie; Pillet, Myriam; Salin, Franck; Bely, Marina; Divol, Benoit; Masneuf-Pomarede, Isabelle
    Hanseniaspora uvarum is one of the most abundant yeast species found on grapes and in grape must, at least before the onset of alcoholic fermentation (AF) which is usually performed by Saccharomyces species. The aim of this study was to characterize the genetic and phenotypic variability within the H. uvarum species. One hundred and fifteen strains isolated from winemaking environments in different geographical origins were analyzed using 11 microsatellite markers and a subset of 47 strains were analyzed by AFLP. H. uvarum isolates clustered mainly on the basis of their geographical localization as revealed by microsatellites. In addition, a strong clustering based on year of isolation was evidenced, indicating that the genetic diversity of H. uvarum isolates was related to both spatial and temporal variations. Conversely, clustering analysis based on AFLP data provided a different picture with groups showing no particular characteristics, but provided higher strain discrimination. This result indicated that AFLP approaches are inadequate to establish the genetic relationship between individuals, but allowed good strain discrimination. At the phenotypic level, several extracellular enzymatic activities of enological relevance (pectinase, chitinase, protease, β-glucosidase) were measured but showed low diversity. The impact of environmental factors of enological interest (temperature, anaerobia, and copper addition) on growth was also assessed and showed poor variation. Altogether, this work provided both new analytical tool (microsatellites) and new insights into the genetic and phenotypic diversity of H. uvarum, a yeast species that has previously been identified as a potential candidate for co-inoculation in grape must, but whose intraspecific variability had never been fully assessed.
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    Identification and characterization of Lactobacillus florum strains isolated from South African grape and wine samples
    (ELSEVIER SCIENCE BV, PO BOX 211, AMSTERDAM, NETHERLANDS, 1000 AE, 2012) Mtshali, P. S; Divol, Benoit; Du Toit, M.
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    Identification and characterization of Lactobacillus florum strains isolated from South African grape and wine samples
    (2012) Mtshali, P. S.; Divol, Benoit; Du Toit, M.
    A total of 213 strains of lactic acid bacteria were examined in this study. Among these, 30 strains previously isolated from South African grape and wine samples remained unidentified. The identification of these isolates was performed by BLAST and phylogenetic analyses of 16S rDNA gene sequences, which indicated that the isolates belonged to Lactobacillus florum. In this work, we also designed a discriminative species-specific primer FLOR targeting the 16S rDNA gene of Lb. florum. The validity and specificity of this primer was confirmed. Of particular interest in this study was to further evaluate the identified strains for the presence of genes encoding enzymes of oenological relevance. Reference strains included three flower-associated Lb. florum (F9-1 T, F9-2 and F17) and two Lactobacillus lindneri (AWRI B530 and DSM 20691) strains. Lb. lindneri strains were incorporated as being the closest relatives of Lb. florum. PCR detection results revealed that all Lb. florum strains and Lb. lindneri AWRI B530 (grape isolate) possessed the majority of the tested genes relative to DSM 20691 (beer isolate); these enzyme-encoding genes included malolactic enzyme, peptidases (PepC, PepI, PepN), citrate lyase (α- and β-subunits), phenolic acid decarboxylase and arginine deiminase pathway enzymes (arginine deiminase and ornithine transcarbamylase). Sequence verification of PCR-generated fragments was performed by sequencing. The sequence data were used to construct the phylogenetic trees, which indicated that our Lb. florum isolates cluster with other Lb. florum strains of flower origin but rather distinct from other LAB species, with Lb. lindneri being the next closest species. © 2011 Elsevier B.V.
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    Identification and partial characterization of extracellular aspartic protease genese from Metschnikowia pulcherrima IWBT Y1123 and candida apicola IWBT Y1384
    (AMER SOC MICROBIOLOGY, 1752 N ST NW, WASHINGTON, USA, DC, 20036-2904, 2012) Reid, V. J.; Theron, L. W.; Du Toit, M; Divol, Benoit
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    Non-saccharomyces killer toxins : possible biocontrol agents against brettanomyces in wine?
    (South African Society for Enology and Viticulture, 2015) Mehlomakulu, N. N.; Setati, M. E.; Divol, Benoit
    Red wine spoiled by the yeast Brettanomyces bruxellensis is characterised by off-odours commonly described as horse sweat, phenolic, varnish and band-aid. The growth of this yeast in wine is traditionally controlled by the use of sulphur dioxide (SO2). However, the concentration of SO2, the pH of the wine, the presence of SO2-binding chemical compounds in the wine, as well as the strain of B. bruxellensis, determine the effectiveness of SO2. Other chemical preservatives have been tested, but are not much more efficient than SO2, and methods used to clean barrels are only partially effective. Filtration of wine and the use of electric currents/fields are also reported to alter the physical and sensory properties of wine. In this context, alternative methods are currently sought to achieve full control of this yeast in wine. Killer toxins have recently been proposed to fulfil this purpose. They are antimicrobial compounds secreted by Saccharomyces and non-Saccharomyces yeasts, displaying killer activity against other yeasts and filamentous fungi. They are believed to play a role in yeast population dynamics, and this killer phenotype potentially could be exploited to inhibit the growth of undesired microorganisms within a microbial ecosystem such as that occurring in wine. In this review, non-Saccharomyces killer toxins are described and their potential application in inhibiting B. bruxellensis in wine is discussed in comparison to other tried methods and techniques.
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    Optimised extraction and preliminary characterisation of mannoproteins from non-saccharomyceswine yeasts
    (MDPI, 2021-04-22) Snyman, Carla; Nguela, Julie Mekoue; Sieczkowski, Nathalie; Marangon, Matteo; Divol, Benoit
    The exogenous application of yeast-derived mannoproteins presents many opportunities for the improvement of wine technological and oenological properties. Their isolation from the cell wall of Saccharomycescerevisiae has been well studied. However, investigations into the efficiency of extraction methods from non-Saccharomyces yeasts are necessary to explore the heterogeneity in structure and composition that varies between yeast species, which may influence wine properties such as clarity and mouthfeel. In this study, nine yeast strains were screened for cell wall mannoprotein content using fluorescence microscopy techniques. Four species were subsequently exposed to a combination of mechanical and enzymatic extraction methods to optimize mannoprotein yield. Yeast cells subjected to 4 min of ultrasound treatment applied at 80% of the maximum possible amplitude with a 50% duty cycle, followed by an enzymatic treatment of 4000 U lyticase per g dry cells weight, showed the highest mannoprotein-rich yield from all species. Furthermore, preliminary evaluation of the obtained extracts revealed differences in carbohydrate/protein ratios between species and with increased enzyme incubation time. The results obtained in this study form an important step towards further characterization of extraction treatment impact and yeast species effect on the isolated mannoproteins, and their subsequent influence on wine properties.
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    PCR detection of enzyme-encoding genes in Leuconostoc mesenteroides strains of wine origin
    (2012) Mtshali, P. S.; Divol, Benoit; Du Toit, M.
    Fifteen isolates of lactic acid bacteria originating from South African grape and wine samples were identified as Leuconostoc mesenteroides subsp. mesenteroides through the taxonomic analysis of their 16S rDNA gene sequences. These isolates were further tested for the presence of genes coding for enzymes of oenological relevance using PCR detection technique. A type strain of Leuc. mesenteroides (NCDO 529 T) was also incorporated for comparative analysis. From the PCR detection results, the estA, prtP, alsD, alsS, metK, metC and metB genes were present in all the strains tested. The bgl and gshR genes encoding β-glucosidase and glutathione reductase, respectively, were not detected in some strains. On the other hand, none of the tested strains possessed the genes encoding phenolic acid decarboxylase (padA), citrate permease (citP), citrate lyase (citD, citE and citF) and arginine deiminase pathway enzymes (arcA, arcB and arcC). The verification of PCR-generated fragments was performed by sequencing. GenBank database was used to search for homologous DNA sequences. Neighbour-joining trees based on nucleotide sequences of alsS, estA, metK and mleA genes were also constructed in order to study the phylogenetic relationship between Leuc. mesenteroides strains and closely related species. The phylogenetic analyses revealed that there are genetic heterogeneities between strains of Leuc. mesenteroides species. In conclusion, this study has improved our knowledge on the genetics of oenological strains of Leuc. mesenteroides and their genetic potential to contribute to certain wine aroma compounds. © 2011 Springer Science+Business Media B.V.
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    PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae
    (2007) Divol, Benoit; Van Rensburg, P.
    The PGU1 gene encodes an endo-polygalacturonase enzyme in Saccharomyces cerevisiae. The literature reports that most S. cerevisiae strains possess this gene, despite a wide range of enzyme activity levels. Nevertheless, a few wine strains lack the PGU1 gene. We investigated the PGU1 locus sequence in these strains. The results indicated that the gene had been replaced by a partial Ty mobile element, whereas the gene promoter was still at the expected location. As all the strains lacking the PGU1 gene experienced the same phenomenon, it was tempting to hypothesize a common phylogenetic origin. However, fingerprints only allowed grouping of a few of them within one cluster. © 2007 Federation of European Microbiological Societies.
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    The production of reduced-alcohol wines using Gluzyme Mono® 10.000 BG-treated grape juice
    (SASEV, 2009) Biyela, B. N. E.; Du Toit, W. J.; Divol, Benoit; Malherbe, D. F.; Van Rensburg, P.
    High alcohol wines have become a major challenge in the international wine trade. Several physical processes are used to produce wines with reduced-alcohol content, all of which involve the selective extraction of ethanol based on volatility or diffusion. In this study, the possibility of Gluzyme Mono® 10.000 BG (Gluzyme) (Novozymes, South Africa) to reduce the glucose content of synthetic grape juice before fermentation was investigated in order to produce wine with reduced-alcohol content. Gluzyme is a glucose oxidase preparation from Aspergillus oryzae, currently used in the baking industry. Glucose oxidase catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide(H2O2) in the presence of molecular oxygen. Gluzyme was initially used in synthetic grape juice, where different enzyme concentrations and factors influencing its efficiency were investigated under winemaking conditions. The results showed up to 0.5% v/v less alcohol at an enzyme concentration of 20 kU compared to the control samples. This reduction in alcohol was increased to 1 and 1.3% v/v alcohol at pH 3.5 and pH 5.5 respectively in aerated (8 mg/L O2) synthetic grape juice using 30 kU enzyme. Secondly, Gluzyme was used to treat Pinotage grape must before fermentation. Gluzyme-treated wines at 30 kU enzyme concentration after fermentation contained 0.68% v/v less alcohol than the control wines. A decrease in acetic acid concentration of the treated compared to control wines was also observed.