M. Du Toit

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces.

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by α‐chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS‐PAGE, was approximately 3·4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.

Oxygen in Must and Wine: A review

Oxygen can play an important role during the winemaking process. It can influence the composition and quality of the must and wine. Phenolic compounds are the main substrates for oxidation in must and wine. Oxygen addition leads to colour changes and the polymerisation of phenolic molecules in wine. Oxygen can, however, also influence the flavour and microbial composition of wine drastically, with certain off-flavours being formed and spoilage micro-organisms able to grow at too high oxygen additions to wine. A state-of-the-art, up-to-date review on the effects of oxygen in must and wine has, however, not been published recently. This review focuses on the effects of oxygen in must, during alcoholic fermentation, extended lees contact and during ageing of white and red wines. The effects it has on acetic acid bacteria and Brettanomyces are also discussed, as well as micro-oxygenation, a relative new technique used in wine production.

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

ABSTRACT The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, 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 andent1071B, 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 ofE. faecalis bacteriocin.

Significance of Brettanomyces and Dekkera during winemaking: a synoptic review.

Wine comprises a complex microbial ecology of opportunistic microorganisms, some of which could potentially induce spoilage and result in consequent economic losses under uncontrolled conditions. Yeasts of the genus Brettanomyces, or its teleomorph Dekkera, have been indicated to affect the chemical composition of the must and wine by producing various metabolites that are detrimental to the organoleptic properties of the final product. These yeasts can persist throughout the harsh winemaking process and have in recent years become a major oenological concern worldwide. This literature review summarises the main research focus areas on yeasts of the genera Brettanomyces and Dekkera in wine. Specific attention is given to the spoilage compounds produced, the methods of detection and isolation from the winemaking environment and the factors for controlling and managing Brettanomyces spoilage.

Biogenic Amines in Wine: Understanding the Headache

The presence of biogenic amines in wine is becoming increasingly important to consumers and producers alike, due to the potential threats of toxicity to humans and consequent trade implications. In the scientific field, biogenic amines have the potential to be applied as indicators of food spoilage and/or authenticity. Biogenic amines can be formed from their respective amino acid precursors by various microorganisms present in the wine, at any stage of production, ageing or storage. To understand the large number of factors that could influence the formation of biogenic amines, the chemical, biochemical, enzymatic and genetic properties relating to these compounds have to be considered. Analytical and molecular methods to detect biogenic amines in wine, as well as possibilities that could enable better control over their production levels in wine will also be explored in this review.

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

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 (Σ1278) 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.

Malolactic fermentation: the ABC's of MLF.

There are two main fermentations associated with the winemaking process. Alcoholic fermentation is conducted by the yeast culture and malolactic fermentation takes place as a result of the metabolic activity of lactic acid bacteria, specifically from the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Malolactic fermentation is defined as the conversion of malic acid to lactic acid and CO2 and besides deacidification also contributes to microbial stability and modification of the aroma profile. This paper aims to provide a comprehensive review discussing all the main aspects and factors related to malolactic fermentation, including practical considerations for monitoring and ensuring a successful fermentation.

Identification of lactic acid bacteria isolated from South African brandy base wines

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.

Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production.

The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS-PCR, PCR-DGGE and REA-PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.

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.