SUNScholar - Browsing by Author "Du Toit M."

Browsing by Author "Du Toit M."

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Anionic fischer-type carbene complexes as bidentate (N,O) ligands
(2004) Raubenheimer H.G.; Du Toit A.; Du Toit M.; An J.; Van Niekerk L.; Cronje S.; Esterhuysen C.; Crouch A.M.
New polynuclear complexes, (L1)3 M2{M 2 = Cr(III) (4a, 4b), Fe(III) (5), Co(III) (8)}, (L1) 2M2(L2)2 {M2 = Co(II)(7), Ni(II) (9)}, (L1)2M2(O)L2 {M 2 = V(IV) (6)} and L1M2Cp2 {M 2 = Ti(III) (10)} with L1 = (CO)M1=C{C=NC(CH 3)=CHS}O (M1 = Cr or W) and L2 = 4-methylthiazole or THF, are described. The molecular structures of these complexes determined by X-ray diffraction show that the Fischer-type carbene complexes act as bidentate ligands towards the second metal centre, coordinating through C(carbene)-attached O-atoms and imine N-atoms of the thiazolyl groups to form five-membered chelates with the oxygen atoms in the mer configuration. Isostructural complexes have similar characteristic band patterns in their far-IR spectra. Cyclic voltammetry of selected complexes reveals the oxidation of the carbene complex ligand between 1.01 and 1.29 V. Oxidation of the central metal (M2) takes place at 0.56 and 0.86 V for 7 and 9, respectively. Three stepwise reductions of Cr(III) to Cr(0) occur for 4a and 4b in the region -0.51 to -1.58 V. These new ligand types and other variants thereof should find application in ligand design with the first metal - and other ligands attached thereto - in the carbene complex ligand, playing an important role.
Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content
(1998) Du Toit M.; Franz C.M.A.P.; Dicks L.M.T.; Schillinger U.; Haberer P.; Warlies B.; Ahrens F.; Holzapfel W.H.
Three out of 297 Lactobacillus strains isolated from pig faeces were selected for a feeding trial on account of their high bile-salt hydrolase (BSH) activity, bile-salt resistance, low pH tolerance and the production of antimicrobial substances. Two strains were identified as Lactobacillus johnsonii and one as Lactobacillus reuteri by DNA-DNA hybridisation. L. johnsoniii BFE 1061 produced a bacteriocin active against a range of lactic acid bacteria (LAB) and nonrelated bacteria including Clostridium perfringens. Six minipigs were maintained on a high-fat, high-cholesterol ('Western Style') diet for 17 weeks after which the diet was supplemented with the 'probiotic mixture' containing the above mentioned three Lactobacillus strains at 2 x 1012 CFU per pig per day for five weeks. The mixture was given as a resuspended lyophilisate. During a two week follow-up period the minipigs received only the 'Western-style' diet without probiotic supplementation. A lowering effect on serum cholesterol levels was indicated after three weeks probiotic feeding, concomitant with an increase in the moisture content of the faeces and Lactobacillus cell numbers. Triglycerides, pH and number of lactic acid bacteria in faeces were not significantly influenced by probiotic supplementation.
Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071
(2000) Balla E.; Dicks L.M.T.; Du Toit M.; Van Der Merwe M.J.; Holzapfel W.H.
The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Gottingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2- 2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.
Cloning and characterisation of a cystathionine β/γ-lyase from two Oenococcus oeni oenological strains
(2011) Knoll C.; Du Toit M.; Schnell S.; Rauhut D.; Irmler S.
Sulphur-containing compounds in wine have been extensively studied because of their effect on wine flavour and quality. In this study, an enzyme that degrades sulphur-containing amino acids was cloned and characterised from two Oenococcus oeni strains of oenological origins. The enzyme has features of a cystathionine-γ-lyase (EC 4.4.1.1), a pyridoxal-5-phosphate-dependent enzyme catalysing an α,γ-elimination reaction of l-cystathionine to produce l-cysteine, α-ketobutyrate and ammonia. Moreover, it was able to catalyse an α,β-elimination reaction producing homocysteine, pyruvate and ammonia from l-cystathionine. An elimination reaction of l-cysteine and dl-homocysteine was also efficiently catalysed by the enzyme, resulting in the formation of hydrogen sulphide. Furthermore, the ability to demethiolate methionine into methanethiol, an unfavourable volatile sulphur compound in terms of wine aroma, was observed. The findings of this work suggest that O. oeni seems to play a minor role in the production of volatile sulphur compounds during the vinification process as the optimal conditions were far from the harsh wine environment. © 2011 Springer-Verlag.
Effect of potentially probiotic lactobacilli on faecal enzyme activity in minipigs on a high-fat, high-cholesterol diet - A preliminary in vivo trial
(2003) Haberer P.; Du Toit M.; Dicks L.M.T.; Ahrens F.; Holzapfel W.H.
Minipigs were fed a "Western-style", high-cholesterol diet for a baseline period, followed by the diet containing a mixture of three Lactobacillus strains with potential probiotic features, after which a normal pig diet was followed. The faecal enzyme activity for β-glucuronidase and azoreductase, which are commonly considered as markers for procarcinogenic activity, was significantly reduced during the 5 weeks of "probiotic" supplementation. During the period of Lactobacillus administration, the cell counts for total anaerobes increased, whereas the total number of aerobes showed no change. © 2003 Elsevier Science B.V. All rights reserved.
Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production
(2003) Malherbe D.F.; Du Toit M.; Cordero Otero R.R.; Van Rensburg P.; Pretorius I.S.
There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; β-D-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone α-factor secretion signal (MFα1S) and the phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T). The PGK1P-MFα1S-gox-PGK1T cassette (designated GOX1) was introduced into a laboratory strain (∑1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of D-glucono-δ-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.
Expression of the malolactic enzyme gene (mle) from Lactobacillus plantarum under winemaking conditions
(2011) Miller B.J.; Franz C.M.A.P.; Cho G.-S.; Du Toit M.
Malolactic fermentation (MLF) plays an important role in the production of wine, especially red wines, resulting in microbial stability, deacidification, as well as contributing to the aroma profile. MLF can be influenced by a number of factors. In this study, the influence of pH and ethanol on expression of the structural malolactic enzyme gene (mle) from Lactobacillus plantarum was investigated in a synthetic wine media, as well as in wine using quantitative PCR. Expression of mle was shown to be inducible by the presence of malic acid, with increased expression in the middle of MLF. Expression of mle was also shown to be increased at low pH values and decreased in the presence of ethanol. This indicates the role of MLF in acid tolerance and the negative impact of ethanol on the completion of MLF. The results therefore provide further evidence that L. plantarum should be applied as co-inoculation for MLF where alcohol will initially not have a negative impact on the malic acid degradation. © 2011 Springer Science+Business Media, LLC.
Identification of lactic acid bacteria isolated from South African brandy base wines
(2004) Du Plessis H.W.; Dicks L.M.T.; Pretorius I.S.; Lambrechts M.G.; Du Toit M.
In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria. As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production. The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers. The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently. Many of the O. oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF). The Lactobacillus spp. were isolated from all the different stages of brandy base wine production. Lb. plantarum was the dominant species in the juice, but disappeared during the later stages of production. However, Lactobacillus hilgardii, Lb. brevis and Lb. paracasei were also isolated from base wine after spontaneous MLF. Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed. Total soluble cell protein patterns grouped O. oeni strains into two phenotypic groups. Two phenotypic clusters have also been identified for the Lb. brevis isolates. The Lb. paracasei isolates all grouped in one cluster. This is the first report of the presence of Lb. paracasei and Lb. vermiforme in brandy base wines. The presence of the Lactobacillus spp. could be correlated to the decrease in quality of the base wine and distillate, while O. oeni strains were found to have a more favourable influence on the quality of base wine and distillates. These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy. © 2003 Elsevier B.V. All rights reserved.
Influence of pH and ethanol on malolactic fermentation and volatile aroma compound composition in white wines
(2011) Knoll C.; Fritsch S.; Schnell S.; Grossmann M.; Rauhut D.; Du Toit M.
The present study investigated the influences of pH and ethanol on malolactic fermentation (MLF) and the volatile aroma profile of the subsequent white wines from Riesling and Chardonnay inoculated with two different Oenococcus oeni strains. In all cases MLF was induced after completion of alcoholic fermentation (AF). Partial MLF occurred under low pH 3.2 and high alcohol (118.3 g/L) conditions. In the cases with complete MLF, the time required for each strain varied from 13 to 61 days and was dependent on bacterial culture, cultivar and wine parameter. Chemical properties of each wine were determined after AF, complete and partial MLF. The wines showed significant differences in total higher alcohols, esters and acids that are important for the sensory profile and quality of wine. This work demonstrated that the wine matrix as well as the pH and alcohol concentration affects MLF and the final volatile aroma profile. Results indicate that changes in volatile aroma composition are not necessarily related to complete MLF and that partial MLF already has distinct influences on the wine aroma profile of white wines. © 2011 Elsevier Ltd.
Investigation of the volatile composition of pinotage wines fermented with different malolactic starter cultures using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS)
(2011) Vestner, J.; Malherbe, S.; Du Toit M.; Nieuwoudt, Helene; Mostafa, A.; Goreck,i T.; Tredoux, A. G. J.; De Villiers, A.
Populations of surface-nesting seabirds at Marion Island, 1994/95-2002/03
(2003) Crawford R.J.M.; Cooper J.; Dyer B.M.; Greyling M.D.; Klages N.T.W.; Ryan P.G.; Petersen S.L.; Underbill L.G.; Upfold L.; Wilkinson W.; De Villiers M.S.; Du Plessis S.; Du Toit M.; Leshoro T.M.; Makhado A.B.; Mason M.S.; Merkle D.; Tshingana D.; Ward V.L.; Whittington P.A.
During the 1990s and early 2000s, populations of surface-nesting seabirds at Marion Island showed different trends, but for the majority of species numbers decreased. Reduced numbers of gentoo penguins Pygoscelis papua, eastern rockhopper penguins Eudyptes chrysocome filholi, Crozet shags Phalacrocorax [atriceps] melanogenis and probably macaroni penguins E. chrysolophus are most plausibly attributed to an altered availability of food. Decreases in numbers of dark-mantled sooty albatrosses Phoebetria fusca, light-mantled sooty albatrosses P. palpebrata, southern giant petrels Macronectes giganteus and possibly northern giant petrels M. halli may have resulted from mortality of birds in longline fisheries. However, populations of wandering Diomedea exulans and grey-headed Thalassarche chrysostoma albatrosses fluctuated around a stable level. Numbers of Subantarctic skuas Catharacta antarctica and kelp gulls Larus dominicanus breeding at Marion Island also decreased. Kerguelen Sterna virgata and Antarctic S. vittata terns remain scarce at the island. Trends for king penguins Aptenodytes patagonicus were not reliably gauged, but numbers probably remained stable or increased. There were large fluctuations in numbers of king penguin chicks surviving to the end of winter.
Preliminary evaluation of infrared spectroscopy for the differentiation of Brettanomyces bruxellensis strains isolated from red wines
(2010) Oelofse A.; Malherbe S.; Pretorius I.S.; Du Toit M.
The objective of this study was to evaluate different infrared spectroscopy methods in combination with chemometrics for the differentiation between Brettanomyces bruxellensis strains. These methods of discrimination were applied to intact yeast cells of B. bruxellensis strains and on wines spoiled by the same strains. Eleven wine isolates of B. bruxellensis were evaluated for volatile phenol production in red wine and their genetic diversity was determined by Restriction Endonuclease Analysis-Pulsed Field Gel Electrophoresis (REA-PFGE). Fourier transform mid-infrared (FTMIR) spectroscopy was used to obtain spectral fingerprints of the spoiled wines. Attenuated total reflectance (ATR) was used to obtain spectral fingerprints from the intact cells of the 11 B. bruxellensis strains. The groupings from the genetic fingerprints obtained with REA-PFGE were used as reference firstly for comparison with the groupings observed with the FTMIR spectral fingerprint of the wines and secondly for the FTIR-ATR spectral fingerprints from the whole cells. Results indicated that ATR-IR spectra obtained by scanning whole cells of B. bruxellensis could be useful for rapid strain typing in comparison or complementary to molecular techniques and FTMIR spectra from wines provide a useful resource for the discrimination between B. bruxellensis contaminated wines. © 2010 Elsevier B.V.
The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae
(1999) Schoeman H.; Vivier M.A.; Du Toit M.; Dicks L.M.T.; Pretorius I.S.
The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedC(D (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1(p)-MFa1(s)-pedA-ADH1(T), designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1(P)) and terminator (ADH1(T)). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone α-factor's secretion signal (MFa1(S)). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria.

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