Seafood permits the transmission of many bacterial pathogens. In order to reconcile consumer demands with important safety standards, traditional means of regulating microbial spoilage and safety hazards in foods are combined with novel technologies. These include biological antimicrobial systems, such as the use of lactic acid bacteria (LAB) and/or their bacteriocins, such as Carnobacterium maltaromaticum CS526 and its bacteriocin piscicocin CS526. The aims of this study were to investigate the presence of Listeria monocytogenes in temperate seafood, namely fresh and smoked salmon, fresh and smoked haddock, and fresh mussels and oysters. Additionally, there was an aim to recover, characterise and use bacteriocin-like-substance to control Listeria monocytogenes in cold smoked haddock. Vibrio spp., Enterobacteriaceae representatives, total aerobic heterotrophic counts and Listeria monocytogenes were isolated from commercially prepared smoked and fresh Atlantic salmon, smoked and fresh haddock, live mussels and oysters using selective media and tryptone soya agar (TSA). Vibrio spp. occurred in high densities (>106 CFU gˉ1) in mussels and Enterobacteriaceae representatives were recorded at >106 CFU gˉ1 in fresh salmon. Total aerobic heterotrophic counts in fresh salmon, live mussels and oysters reached 107, > 107, and > 106 CFU gˉ1, respectively. Listeria monocytogenes was recorded at 5.0 x 104 CFU gˉ1 in mussels. In total sixty one bacterial isolates were recovered from the seafood examined. The results revealed 19 genera of bacteria, i.e. Acinetobacter, Aerococcus, Aeromonas, Bacillus, Brochothrix, Carnobacterium, Citrobacter, Corynebacterium, Enterobacter, Escherichia coli, Moraxella, Micrococcus, Pseudomonas, Psychrobacter, Serratia, Shewanella, Staphylococcus, Vibrio and Listeria. The prominent characteristics of fish spoilage isolates were demonstrated by the ability of the isolates to reduce trimethylamine oxide (TMAO) to trimethylamine, and to produce H₂S. Sh. baltica OS185, Aeromonas spp. HB-6, Sh. baltica, Sh. putrefaciens, A. hydrophila HX201006-3, A. salmonicida subsp. achromogenes, A. hydrophila, C. freundii, Enterobacter cloacae were strong producers of TMA and H₂S. The spoilage microorganisms were tested for potential pathogenicity. The result revealed that 6/15 of the spoilage microorganisms produced proteolytic, lecithinase, blood (β and α haemolysin) and elastinase activity, respectively, whereas 7/15 of the spoilage microorganisms showed lipolytic activity. Cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances of Carnobacterium maltaromaticum MMF-32 and KOPRI 25789 producing strains isolated from commercially prepared smoked salmon were investigated for their potential antimicrobial activity against potentially pathogenic and food spoilage microorganisms. Generally, a broad spectrum of activity was revealed against potentially pathogenic and food spoilage microorganisms in vitro. Cold-smoked haddock treated with bacteriocin producing C. maltaromaticum MMF-32, C. piscicola A9b bacˉ phenotype nonbacteriocin producing strain a mutant of C. piscicola A9b bac+, cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances was challenged with L. monocytogenes ATCC 19114 up to 103 CFU gˉ1, respectively. Samples were stored at 4 °C for 10 days. L. monocytogenes and total bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amines production as well as texture, colour and odour. Although the study on anti-listerial effects of C. maltaromaticum MMF-32 was not successful, this organism did have a positive effect on retention of firmness and sensory perception in cold smoked haddock.

No description available.

Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. These microorganisms are naturally present on the grapes and in the cellar from where they can be introduced to the winemaking process at any given time and consequently impart specific contributions to the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions under which they can proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and other wild yeasts (non-Saccharomyces spp.) are typically associated with the formation of off-flavours under poorly controlled winemaking conditions. In recent years, evidence from the wine industry has highlighted a specific group of non-Saccharomyces yeast species as a serious cause for wine spoilage that required more research investigations. Yeast of the genus Brettanomyces or its teleomorph Dekkera has been identified as one of the most controversial spoilage microorganisms during winemaking as they can produce several compounds that are detrimental to the organoleptic quality of wine. This has triggered the research initiative behind this doctoral study on the significance of Brettanomyces and Dekkera yeasts during winemaking. In this dissertation, various aspects of the detection, isolation and identification methods of Brettanomyces yeast from the winemaking environment were investigated. As a first objective, a culture collection of Brettanomyces bruxellensis wine isolates had to be established. This followed after the isolation of Brettanomyces yeasts from various red wine cultivars from South African wineries from different stages of the winemaking process. Different conventional microbiological methods such as plating on selective agar media and microscopy were investigated along with molecular identification techniques such as the polymerase chain reaction (PCR) in this regard. Other focus areas of this study aimed at performing genetic characterisation and differentiation studies of B. bruxellensis wine isolates. For this purpose, different intraspecific identification methods were investigated on several strains, including strains of European origin. The application of molecular techniques allowing strain identification aided in the selection of specific strains that were evaluated for volatile phenol production in synthetic media and wine. The results obtained from this work indicated that a large degree of genetic diversity exists among B. bruxellensis strains and that the volatile phenol production differed between the strains after evaluation in synthetic media and wine. In addition to the molecular intraspecific strain identification techniques that were investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an alternative approach for differentiating between B. bruxellensis strains. The two approaches of FTIR spectroscopy that were investigated involved the use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral fingerprints of spoiled wines by different B. bruxellensis strains; and secondly, Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole cells of B. bruxellensis on microbiological agar media. The results of this study illustrated the potential of FTIR spectroscopy to become a reliable alternative to molecular based methods for differentiating between B. bruxellensis strains and for characterisation studies. The formation of volatile phenols in wine by species of the genera Brettanomyces and Dekkera is one of the primary reasons for their classification as wine spoilage yeasts. The enzymatic activities of this reaction have been identified and involve a phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase (VPR). However, only a limited amount of information is available about these enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been described. It was therefore imperative that this dissertation should include a genetic investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the species B. bruxellensis involved in the formation of volatile phenols. Strategies that were investigated included various molecular DNA techniques and protein purification procedures to obtain either genetic or protein sequence data. The decarboxylase activity of this yeast species towards p-coumaric acid was demonstrated and substantial genetic sequence data was obtained. The results from this dissertation made a substantial contribution to the current available knowledge about Brettanomyces/Dekkera spp. and led to a better understanding of this wine spoilage yeast. This research developed a platform from which further investigations could follow and the knowledge gained will be invaluable for future Brettanomyces research projects at the Institute for Wine Biotechnology at Stellenbosch University.

A utilização de embalagens biodegradáveis, tais como os filmes e coberturas comestíveis, apresenta-se como alternativa ao uso de recursos não-renováveis como material de embalagem. A incorporação de substâncias antimicrobianas em embalagens tem como objetivo minimizar o problema da contaminação microbiana em alimentos e, entre elas, os óleos essenciais (OE) têm recebido atenção especial por serem substâncias naturais e atenderem à preferência dos consumidores. Porém, a utilização de OE como um agente antimicrobiano natural é limitada por critérios organolépticos, sendo necessário determinar a concentração mínima necessária para inibir o desenvolvimento de microrganismos sem afetar sensorialmente as características do alimento. Assim, os objetivos desta pesquisa foram: desenvolver um filme comestível à base de alginato com incorporação de agentes antimicrobianos naturais e avaliar a adição de diferentes concentrações de cloreto de cálcio (CaCl2) como agente crosslinking na formulação do filme e na etapa complementar de formação do filme; caracterizar o filme frente às propriedades mecânicas e propriedades de barreira; determinar a concentração mínima inibitória (CIM) de óleos essenciais para Pseudomonas spp., Salmonella spp. e Listeria monocytogenes presentes em carne de frango, e verificar a aceitação pelo consumidor, através da análise sensorial (aroma), de pedaços de peito de frango in natura embalado com o filme antimicrobiano O OE de cravo foi o que se apresentou mais eficiente para os microrganismos testados com CIM de 0,2% sendo este o limite mínimo estudado no planejamento experimental para o desenvolvimento do filme antimicrobiano. As variáveis independentes neste planejamento foram: CaCl2 na faixa de concentração de 0,02 a 0,1% e OE cravo na faixa de 0,2 a 1,0%. Valores acima de 0,0316% de CaCl2, independente da concentração de OE estudada, diminuiram a zona de inibição do crescimento microbiano em testes realizados in vitro, possivelmente devido a formação de um gel muito forte que pode ter dificultado a incorporação da emulsão de OE na matriz polimérica dos filmes. Os resultados de permeabilidade ao vapor de água mostraram que a adição de CaCl2 à formulação dos filmes diminuiu a permeabilidade enquanto a adição de OE cravo foi responsável pelo aumento dessa propriedade. Com relação às propriedades mecânicas, tanto a adição de CaCl2 como a de OE cravo à formulação dos filmes aumentou a resistência máxima à tração. Porém, com relação ao alongamento máximo na ruptura, valores menores foram obtidos com a adição de CaCl2, enquanto maiores valores foram encontrados com a adição de OE cravo à formulação dos filmes. A avaliação da atividade antimicrobiana dos filmes em carne de frango foi realizada somente com a formulação que apresentou os maiores valores de zona de inibição in vitro (CaCl2=0,0316% e OE cravo=0,884%). Após 5 dias de armazenagem a 7º C, observou-se que a utilização do filme adicionado de OE de cravo como embalagem primária em amostras de carne de peito de frango promoveu o controle da multiplicação de L. monocytogenes o mesmo não ocorrendo para as populações de Salmonella spp. e Pseudomonas spp. A análise sensorial relacionada ao aroma da carne de peito de frango mostrou que o uso do filme à base de alginato incorporado de OE cravo é viável. Porém, este filme poderá sofrer interferência da matriz alimentar caso esta matriz apresente exsudação. / The use of biodegradable packaging such as edible films and coatings are an alternative to the use of non-recyclable packaging. The incorporation of antimicrobial substances in packaging aims at reducing food microbial contamination among which, essential oils (EO) have received special attention being natural and attending consumer demand. However, the use of EO as a natural antimicrobial agent is limited by organoleptic criteria making it necessary to determine the minimum concentration to inhibit the multiplication of microorganisms without affecting the sensory characteristics of the food. Therefore, the aims of this research were: to develop an alginate based edible film with natural antimicrobial agents, evaluating the addition of different concentrations of calcium chloride as a crosslinking agent in the formulation of the film and in the complementary stage; to characterize the mechanical properties and barrier properties; to determine the minimum inhibitory concentration (MIC) of EO for Pseudomonas spp, Salmonella spp and Listeria monocytogenes found in chicken meat and to verify consumer acceptance of the product through sensorial analysis (aroma). Among the studied EO, the concentration of 0.2% of clove oil was effective in inhibiting the microorganisms tested, this concentration being the minimum limit used in the experimental design for film development. The independent variables studied in this design were calcium chloride in the range of 0.02 to 0.01% and clove EO in the range of 0.2 to 1.0%. Concentrations of CaCl2 above 0.0316%, independent of the EO concentration, reduced the inhibition zone of microbial growth in in vitro tests, possibly due to the formation of a very strong gel which could have made the incorporation of the EO emulsion in the polymeric matrix of the film very difficult. The results of water vapor permeability tests showed that the addition of CaCl2 to the formulation of the films reduced the permeability while the addition of clove EO increased this property. Regarding to mechanical properties, the addition of CaCl2 as well as clove EO to the film formulation increased the values of tensile strength. On the other hand, relating to elongation at the break, smaller values were obtained with the addition of the salt while the addition of EO provided higher values. The evaluation of antimicrobial activity of the films in chicken meat was performed only with the formulation that showed the highest inhibition values presented in vitro (CaCl2=0.0316% and clove EO=0.0884%). After five days of storage at 7° C, it was observed that the use of the film added by clove EO as primary packaging provided the control of L. monocytogenes growth in samples of chicken meat but not of Salmonella spp and Pseudomonas spp. The sensorial analysis - aroma - showed that the use of alginate based film incorporated with clove EO is viable in food. However, when the food matrix presents exudation, it can interfere in this film.

The brewing industry has considerable economic impact worldwide; therefore, demand exists for a better understanding of the organisms that cause beer-spoilage. Low nutrient levels, depleted oxygen levels, high alcohol levels, and the presence of antimicrobial hop compounds all play a role in making beer an inhospitable environment for most microorganisms. Nonetheless, there are bacteria that are resistant to all of these selective pressures. The most common beer-spoilage bacteria are the Gram-positive lactic acid bacteria <i>Lactobacillus</i> and <i>Pediococcus</i>. It is currently believed that hop-resistance is the key factor(s) permitting <i>Lactobacillus</i> and <i>Pediococcus</i> bacteria to grow in beer. However, it is likely that in addition, ethanol-tolerance and the ability to acquire nutrients also play roles in the beer-spoilage ability of <i>Lactobacillus</i> and <i>Pediococcus</i> isolates. The ability of <i>Lactobacillus</i> and <i>Pediococcus</i> to grow in beer was assessed and correlated to the presence of previously described beer-spoilage related genes, as well as with the presence of novel genes identified in this study. Molecular and culture-based techniques for detection and differentiation between <i>Lactobacillus</i> and <i>Pediococcus</i> isolates that can and cannot grow in beer were established and described in detail. Interestingly, beer-spoilage related proteins were often found to share homology with multi-drug transporters. As such, the presence of these beer-spoilage associated genes was also compared to the ability of isolates to grow in the presence of a variety of antibiotics and, unexpectedly, beer-spoiling bacteria were found to be more susceptible to antibiotics than were non beer-spoiling isolates of the same genus. Additionally, it was found that isolates of <i>Lactobacillus</i> and <i>Pediococcus</i> that can grow in beer do not group phylogenetically. In order to fully appreciate the relationship of speciation with beer-spoilage, phylogenetic and whole genome/proteome studies were conducted to clarify the taxonomy of the <i>Lactobacillus</i> and <i>Pediococcus</i> genera. Through the research in this thesis, a greater understanding of the mechanism(s) enabling bacteria to grow in beer has been gained and taxonomy of the genera <i>Lactobacillus</i> and <i>Pediococcus</i> has been clarified.

The brewing industry has considerable economic impact worldwide; therefore, demand exists for a better understanding of the organisms that cause beer-spoilage. Low nutrient levels, depleted oxygen levels, high alcohol levels, and the presence of antimicrobial hop compounds all play a role in making beer an inhospitable environment for most microorganisms. Nonetheless, there are bacteria that are resistant to all of these selective pressures. The most common beer-spoilage bacteria are the Gram-positive lactic acid bacteria <i>Lactobacillus</i> and <i>Pediococcus</i>. It is currently believed that hop-resistance is the key factor(s) permitting <i>Lactobacillus</i> and <i>Pediococcus</i> bacteria to grow in beer. However, it is likely that in addition, ethanol-tolerance and the ability to acquire nutrients also play roles in the beer-spoilage ability of <i>Lactobacillus</i> and <i>Pediococcus</i> isolates. The ability of <i>Lactobacillus</i> and <i>Pediococcus</i> to grow in beer was assessed and correlated to the presence of previously described beer-spoilage related genes, as well as with the presence of novel genes identified in this study. Molecular and culture-based techniques for detection and differentiation between <i>Lactobacillus</i> and <i>Pediococcus</i> isolates that can and cannot grow in beer were established and described in detail. Interestingly, beer-spoilage related proteins were often found to share homology with multi-drug transporters. As such, the presence of these beer-spoilage associated genes was also compared to the ability of isolates to grow in the presence of a variety of antibiotics and, unexpectedly, beer-spoiling bacteria were found to be more susceptible to antibiotics than were non beer-spoiling isolates of the same genus. Additionally, it was found that isolates of <i>Lactobacillus</i> and <i>Pediococcus</i> that can grow in beer do not group phylogenetically. In order to fully appreciate the relationship of speciation with beer-spoilage, phylogenetic and whole genome/proteome studies were conducted to clarify the taxonomy of the <i>Lactobacillus</i> and <i>Pediococcus</i> genera. Through the research in this thesis, a greater understanding of the mechanism(s) enabling bacteria to grow in beer has been gained and taxonomy of the genera <i>Lactobacillus</i> and <i>Pediococcus</i> has been clarified.

Bacterial responses to environmental stresses may be easily observed and predicted under controlled laboratory conditions. However, realistic conditions encountered during manufacturing, in retail or in households may cause unpredicted responses of spoilage or pathogenic bacteria. Therefore it is essential to identify and understand the microbial dynamics under such conditions. The overall aim of the present study was to simulate the most common environmental conditions and consumer-style practices during storage or preparation of Ready-to-Eat (RTE) and Ready-to-Cook (RTC) products in the domestic environment, and predict the microbial dynamics which may deteriorate the quality or compromise the safety of these foods. Aiming to develop a unified mathematical model for the prediction of the growth of the specific spoilage microorganisms (SSOs), the spoilage pattern of three RTE acidic spreads of low pH was described in relation to microbial, physicochemical and molecular changes during storage. Results showed that the spoilage profile of the products was primarily affected by the initial pH and the storage temperature, despite the differences in their formulation. These findings enabled the assessment of two unified models (polynomial and Ratkowsky) for the prediction of the growth of lactic acid bacteria (LAB; SSOs) in such acidic spreads, using only the initial pH, the concentration of undissociated acetic acid and the storage temperature. The models were validated under realistic conditions in household refrigerators. Despite the abrupt fluctuations of the temperature during validation procedure, they both were able to adequately predict the growth of LAB in the spreads. However, the initial contamination level was proved to be necessary and crucial for the accurate prediction of microbial dynamics. The time-temperature profiles of the validation procedure revealed that the suggested storage conditions were not followed promptly and, therefore, concerns were raised on the effect of such consumer mishandlings on the safety of foods. Therefore, the responses of Salmonella spp. and Escherichia coli O157:H7 to the stresses encountered during frozen storage, thawing and cooking of ground beef, simulating typical scenarios followed by the consumers, were evaluated. The results revealed that the guidelines issued by the food safety authorities lack of some specific points that may affect the safety of the final product, such as the duration of frozen storage and the method of cooking. In particular, it was found that the heat resistance of E. coli O157:H7 was likely increased after long term frozen storage, while cooking in pan-grill did not ensure the safety of the final product, even when cooked at the suggested temperature. As shown in the first study, the initial contamination level played a significant role on the predictions of the models and further on the shelf-life of the products. Therefore, the dynamics of realistically low initial populations of Listeria monocytogenes and Salmonella Typhimurium versus higher levels of the pathogens (such those used during in vitro trials) in RTE fresh-cut salads were compared. In addition, any potential uncertainty sources for the growth potential of the pathogens in broth-based simulations were investigated. Results showed that the growth variability of low inocula is highly affected by the marginal storage temperatures, the indigenous microflora and the availability of nutrients. Because of this, growth from low populations showed the likelihood to exceed the growth derived from unrealistically high inocula, suggesting that ―fail-dangerous‖ implications may derive from such challenge tests. Data derived from this part were compared with broth-based simulations and the results showed that high uncertainty should be expected when extrapolating such predictions from low initial populations in fresh-cut salads, due to the various factors affecting the microbial growth on a real food, which are (inevitably) ignored by broth-based models. Overall, the present Thesis highlights the significant impact of consumer mishandlings on the food safety and quality of foods and contributes to the identification of unpredicted potential risk origins in the domestic environment.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009. / Grape juice is transformed into wine through the complex processes of alcoholic and malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid bacteria. However, the microbes involved in these processes do not only take part in ensuring the successful production of wine, but also cause spoilage of the wine if their growth is not controlled. Conventional, culture-dependent methods of microbiology have been used as the main technique in detecting and identifying these spoilage microbes. Cultureindependent techniques of molecular biology have recently become more popular in detecting possible spoilage microbes present in must and wine, since it allows the detection and identification of viable, but non-culturable microbes and are not as timeconsuming as conventional microbiological methods. The aim of this study was to investigate the sustainability of polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in detecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85% (m/v) NaCl) and sterile white wine and red wine as single microbial species and as part of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white wine and sterile red wine inoculated with reference microbial strains were compared in terms of DNA concentration and purity, as well as simplicity of the technique. These three DNA isolation methods were the TZ-method, the proteinase K-method and the phenol extraction method. DNA could not successfully be isolated from red wine using any of the three DNA isolation methods. The TZ-method was the method of choice for the isolation of DNA from inoculated SSS and sterile white wine as this technique gave the best results in terms of simplicity, DNA concentration and purity. PCR and DGGE conditions were optimised for the universal primer pair, HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2, and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE detection limits were successfully determined when 106 cfu.ml-1 of the reference microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were separately inoculated into SSS and sterile white wine. It was possible to detect low concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, Grape juice is transformed into wine through the complex processes of alcoholic and malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid bacteria. However, the microbes involved in these processes do not only take part in ensuring the successful production of wine, but also cause spoilage of the wine if their growth is not controlled. Conventional, culture-dependent methods of microbiology have been used as the main technique in detecting and identifying these spoilage microbes. Cultureindependent techniques of molecular biology have recently become more popular in detecting possible spoilage microbes present in must and wine, since it allows the detection and identification of viable, but non-culturable microbes and are not as timeconsuming as conventional microbiological methods. The aim of this study was to investigate the sustainability of polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in detecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85% (m/v) NaCl) and sterile white wine and red wine as single microbial species and as part of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white wine and sterile red wine inoculated with reference microbial strains were compared in terms of DNA concentration and purity, as well as simplicity of the technique. These three DNA isolation methods were the TZ-method, the proteinase K-method and the phenol extraction method. DNA could not successfully be isolated from red wine using any of the three DNA isolation methods. The TZ-method was the method of choice for the isolation of DNA from inoculated SSS and sterile white wine as this technique gave the best results in terms of simplicity, DNA concentration and purity. PCR and DGGE conditions were optimised for the universal primer pair, HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2, and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE detection limits were successfully determined when 106 cfu.ml-1 of the reference microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were separately inoculated into SSS and sterile white wine. It was possible to detect low concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, iv Pd. pentosaceus, and B. bruxellensis in SSS when amplified with the HDA1-GC and HDA2 primer pair. A PCR detection limit of 102 cfu.ml-1 was determined in sterile white wine for Pd. pentosaceus and 103 cfu.ml-1 for B. bruxellensis using this primer pair. The results obtained from the PCR amplification with the WBAC1-GC and WBAC2 primer pair compared well with the results of the HDA1-GC and HDA2 primer pair. The results from the DGGE detection limits indicated that it was possible to detect lower concentrations (101 – 102 cfu.ml-1) of A. pasteurianus, Lb. plantarum and Pd. pentosaceus with the HDA1-GC and HDA2 primer pair than the WBAC-GC and WBAC2 primer pair (102 – 104 cfu.ml-1). Lower detection limits were also determined for B. bruxellensis amplified with the HDA1-GC and HDA2 primer pair (103 – 104 cfu.ml-1) than with the NL1-GC and LS2 primer pair (105 cfu.ml-1). PCR and DGGE detection limits for the inoculation of A. pasteurianus, Lb. plantarum and B. bruxellensis at an inoculum of 108 cfu.ml-1 as part of mixed populations in SSS and sterile white wine compared well with the results obtained from the reference microbes inoculated as single microbial species. PCR detection limits of 101 cfu.ml-1 were determined for all three reference microbes inoculated as part of mixed populations when amplified with the HDA1-GC and HDA2 and the WBAC1-GC and WBAC2 primer pairs. It was observed that similar or higher DGGE detection limits were obtained for the reference microbes inoculated in sterile white wine (101 – 107 cfu.ml-1) than when inoculated into SSS (101 – 105 cfu.ml-1). PCR-based DGGE analysis proved to be a technique that could be used successfully with the universal, wine-bacteria and yeast specific primer pairs for the detection of A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis. The culture-independent technique makes the early detection of possible spoilage microbes at low concentrations in wine possible.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formers that are able to spoil acidic food and beverage products through the production of guaiacol and other taint compounds, which causes a medicinal off-flavour and/or odour in the products. This thesis reports on the comparison of methods used for the isolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacol production of different strains isolated from the South African fruit processing environment. Two methods for guaiacol detection were also evaluated and compared. Three isolation methods frequently used by South African fruit processors were compared with regards to their ability to isolate a strain of A. acidoterrestris from diluted peach juice concentrate. Method 1, the International Federation of Fruit Juice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillus acidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidified potato dextrose agar (PDA); and Method 3 makes use of membrane filtration and incubation of the membrane on K agar. The IFU Method No. 12 was the most effective method for the isolation of A. acidoterrestris, with a recovery of 75.97%. These results support the use of the IFU Method No. 12 as a standard international method for the isolation and detection of species of Alicyclobacillus. Seven strains of Alicyclobacillus, including the type strains A. acidoterrestris DSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a South African fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A. acidocaldarius FB19, were analysed based on their growth characteristics and guaiacol production under optimum conditions. Strains were inoculated into BAT medium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for 7 d. All the strains had similar growth patterns, with cell concentrations increasing rapidly from 0-24 h, followed by a stabilisation around maximum cell concentrations of 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indication of spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating an increase in spores as the cell density and competition for resources increased. All the strains were able to produce guaiacol in detectable concentrations [as measured by the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess the potential to cause product spoilage. Bacteria belonging to the genus Alicyclobacillus are thermo-acidophilic spore-formers that are able to spoil acidic food and beverage products through the production of guaiacol and other taint compounds, which causes a medicinal off-flavour and/or odour in the products. This thesis reports on the comparison of methods used for the isolation of species of Alicyclobacillus, as well as the growth behaviour and guaiacol production of different strains isolated from the South African fruit processing environment. Two methods for guaiacol detection were also evaluated and compared. Three isolation methods frequently used by South African fruit processors were compared with regards to their ability to isolate a strain of A. acidoterrestris from diluted peach juice concentrate. Method 1, the International Federation of Fruit Juice Producers (IFU) Method No. 12, makes use of spread plating onto Bacillus acidoterrestris (BAT) agar plates; Method 2 involves pour plating using acidified potato dextrose agar (PDA); and Method 3 makes use of membrane filtration and incubation of the membrane on K agar. The IFU Method No. 12 was the most effective method for the isolation of A. acidoterrestris, with a recovery of 75.97%. These results support the use of the IFU Method No. 12 as a standard international method for the isolation and detection of species of Alicyclobacillus. Seven strains of Alicyclobacillus, including the type strains A. acidoterrestris DSM 3922T and A. acidocaldarius DSM 446T and five strains isolated from a South African fruit processing plant, A. acidoterrestris FB2, FB14, FB32, FB38 and A. acidocaldarius FB19, were analysed based on their growth characteristics and guaiacol production under optimum conditions. Strains were inoculated into BAT medium at pH 4.00, supplemented with 100 mg.L-1 vanillin, and incubated at 45°C for 7 d. All the strains had similar growth patterns, with cell concentrations increasing rapidly from 0-24 h, followed by a stabilisation around maximum cell concentrations of 105-107 cfu.mL-1. Cell concentrations after heat shock, measured as an indication of spore formation, increased to maximum values of 105-107 cfu.mL-1, indicating an increase in spores as the cell density and competition for resources increased. All the strains were able to produce guaiacol in detectable concentrations [as measured by the peroxidase enzyme colourimetric assay (PECA)], and, therefore, possess the potential to cause product spoilage. iv The influence of temperature on the growth and guaiacol production of the Alicyclobacillus strains was also investigated and two guaiacol detection methods, the PECA and headspace gas-chromatography mass-spectrometry (HS GC-MS), were compared with regards to their ability to detect guaiacol. The strains were incubated at 25°C and 45°C for 6 d and samples analysed every 24 h. Growth of the A. acidoterrestris strains was slower at 25°C, and maximum cell concentrations were lower than at 45°C. A decrease in cell concentrations was observed in the A. acidocaldarius strains at 25°C, as this temperature is below their growth temperature range. All the strains were able to produce guaiacol at 45°C, with guaiacol only being detected once a cell concentration of 104-105 cfu.mL-1 had been reached. The maximum guaiacol concentrations detected at 45°C in the samples containing A. acidoterrestris were significantly higher than those detected in the A. acidocaldarius samples. At 25°C there was a longer lag phase before guaiacol was detected in the A. acidoterrestris samples, while no guaiacol was detected in the samples containing A. acidocaldarius. Because guaiacol is produced at ambient temperatures, cooling of products is recommended to control spoilage by A. acidoterrestris. The sensitivity of the two guaiacol detection methods also differed significantly and, therefore, the PECA is recommended for presence/absence detection of guaiacol, while HS GCMS is recommended where accurate quantification of guaiacol is required. Alicyclobacillus acidoterrestris FB2 was investigated for its ability to grow and produce guaiacol in white grape juice supplemented with vanillin at different concentrations. Alicyclobacillus acidoterrestris FB2 was inoculated into white grape juice concentrate diluted 1:10 with distilled water containing 0-500 mg.L-1 vanillin and incubated at 45°C for 6 d. Similar growth patterns were observed in all the samples, except in the sample containing 500 mg.L-1 vanillin, which had a longer lag phase of growth. Guaiacol concentrations, detected using the PECA, increased as the vanillin concentration increased, with the exception of the sample containing 500 mg.L-1 vanillin, where less guaiacol was detected than in the sample containing 250 mg.L-1 vanillin, due to growth inhibition caused by the higher vanillin concentration. A number of conditions need to be favourable for detectable guaiacol production to occur and it could, therefore, be possible to minimise or prevent guaiacol production by controlling or eliminating some of these factors. Good manufacturing practices should be employed in order to minimise contamination and, therefore, spoilage, by Alicyclobacillus species. / AFRIKAANSE OPSOMMING: Bakterieë wat aan die genus Alicyclobacillus behoort, is termo-asidofiliese spoorvormers wat suur voedsel en drank produkte kan bederf deur die produksie van guaiakol en ander bederf verbindings, wat ‘n medisinale geur en/of reuk in die produkte veroorsaak. Hierdie tesis doen verslag oor die vergelyking van metodes wat vir die isolasie van spesies van Alicyclobacillus gebruik word, sowel as die groei kenmerke en guaiakol produksie van verskillende stamme wat uit die Suid- Afrikaanse vrugte prosesseringsomgewing geïsoleer is. Twee metodes vir die deteksie van guaiakol is ook geëvalueer en vergelyk. Drie isolasie metodes wat algemeen deur Suid-Afrikaanse vrugteprosesseerders gebruik word, is vergelyk ten opsigte van hul vermoë om H A. acidoterrestris stam uit verdunde perskesap konsentraat te isoleer. Metode 1, die Internasionale Federasie van Vrugtesap Produseerders (IFU) Metode No. 12, maak gebruik van spreiplating op Bacillus acidoterrestris (BAT) agar plate; Metode 2 behels gietplating met aartappel dekstrose agar (PDA) and Metode 3 maak gebruik van membraan filtrasie en inkubasie van die membraan op K agar. Die IFU Metode No. 12 was die mees effektiewe metode vir die isolasie van A. acidoterrestris, met H sel herwinning van 75.97%. Hierdie resultate ondersteun die gebruik van die IFU Metode No. 12 as H standaard internasionale metode vir die isolasie en deteksie van spesies van Alicyclobacillus. Sewe Alicyclobacillus stamme, insluitende die tipe stamme A. acidoterrestris DSM 3922T en A. acidocaldarius DSM 446T en vyf stamme geïsoleer uit ‘n Suid- Afrikaanse vrugte prosesseringsaanleg, A. acidoterrestris FB2, FB14, FB32, FB38 en A. acidocaldarius FB19, is geanaliseer met betrekking tot hul groei kenmerke en guaiakol produksie onder optimum toestande. Stamme is in BAT medium by pH 4.00, aangevul met 100 mg.L-1 vanillin, geïnokuleer en geïnkubeer teen 45°C vir 7 d. Al die stamme het soortgelyke groeipatrone getoon, met selgetalle wat vinnig toegeneem het van 0-24 h, gevolg deur ‘n stabilisering rondom maksimum selgetalle van 105-107 kve.mL-1. Selgetalle na hitte behandeling, gemeet as H aanduiding van spoorvorming, het toegeneem tot maksimum waardes van 105-107 kve.mL-1, wat aandui dat spore toegeneem het soos die seldigtheid en kompetisie vir voedingsbronne toegeneem het. Al die stamme kon guaiakol in bespeurbare konsentrasies produseer [soos gemeet deur die peroksidase ensiem kolorimetriese bepaling (PEKB)] en besit dus die potensiaal om produkte te bederf. Die invloed van temperatuur op groei en guaiakol produksie van die Alicyclobacillus stamme is ook ondersoek en twee guaiakol deteksie metodes, die PEKB en topspasie gas-kromatografie massa-spektrometrie (TS GK-MS) is vergelyk ten opsigte van hul vermoë om guaiakol op te spoor. Die stamme is geïnkubeer teen 25°C en 45°C vir 6 d en monsters is elke 24 h geanaliseer. Groei van die A. acidoterrestris stamme was stadiger by 25°C en maksimum selgetalle was laer as by 45°C. H Vermindering in selgetalle is waargeneem in die A. acidocaldarius stamme by 25°C, aangesien hierdie temperatuur buite hul groei temperatuur grense val. Al die stamme kon guaiakol produseer by 45°C, met guaiakol deteksie wat eers H aanvang geneem het nadat H sel konsentrasie van 104-105 kve.mL-1 bereik is. Die maksimum guaiakol konsentrasies wat by 45°C in die monsters met A. acidoterrestris opgespoor is, was beduidend hoër as die konsentrasies wat in die A. acidocaldarius monsters opgespoor is. By 25°C was daar H langer sloerfase voor guaiakol opgespoor is in die A. acidoterrestris monsters, terwyl geen guaiakol opgespoor is in die monsters wat A. acidocaldarius bevat het nie. Aangesien guaiakol by kamertemperatuur geproduseer word, word verkoeling van produkte aanbeveel ten einde bederf deur A. acidoterrestris te beheer. Die sensitiwiteit van die twee guaiakol deteksie metodes het ook beduidend verskil en dus word die gebruik van die PEKB aanbeveel vir teenwoordigheid/afwesigheid deteksie van guaiakol, terwyl TS GK-MS aanbeveel word waar akkurate kwantifisering van guaiakol vereis word. Ondersoek is ingestel na die vermoë van A. acidoterrestris FB2 om te groei en guaiakol te produseer in witdruiwesap aangevul met verskillende vanillin konsentrasies. Alicyclobacillus acidoterrestris FB2 is geïnokuleer in witdruiwesap konsentraat 1:10 verdun met gedistilleerde water wat 0-500 mg.L-1 vanillin bevat het en is geïnkubeer teen 45°C vir 6 d. Soortgelyke groeipatrone is waargeneem in al die monsters, behalwe die monster wat 500 mg.L-1 vanillin bevat het, wat H langer sloerfase van groei gehad het. Guaiakol konsentrasies, soos gemeet deur die PEKB, het toegeneem soos die vanillin konsentrasie toegeneem het, met die uitsondering van die monster wat 500 mg.L-1 vanillin bevat het, waar minder guaiakol opgespoor is as in die monster wat 250 mg.L-1 bevat het as gevolg van groei inhibisie veroorsaak deur die hoër vanillin konsentrasie. H Aantal toestande moet gunstig wees vir guaiakol produksie om plaas te vind en dit kan dus moontlik wees om guaiakol produksie te minimaliseer of te voorkom deur die beheer of uitskakeling van sommige van hierdie faktore. Goeie vervaardigingspraktyke moet in plek gestel word ten einde kontaminasie en bederf deur Alicyclobacillus spesies tot H minimum te beperk.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate, inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2 tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2 accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1. In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using 5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm whether these strains belonged to the Dekkera or Brettanomyces genus. A method to accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2 and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B. bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the same trends as previously found in literature whereas B. bruxellensis strains showed similar trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid, acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly monitored over the time course of each experiment. SO2 had the greatest impact on B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32 of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2 levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism to adapt to ensure survival. In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and metabolism and that SO2 tolerance is highly strain dependent and a far more complicated characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare, maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B. bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik. In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur 5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2- bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei. Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S. cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B. bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur, asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur. ‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer ingewikkelde kenmerk as wat tans verstaan word.