Masters Degrees (Food Science)

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Browsing Masters Degrees (Food Science) by browse.metadata.advisor "Cameron, M."

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    Detection and molecular subtyping of Listeria Monocytogenes isolated from a South African avocado processing facility
    (Stellenbosch : Stellenbosch University, 2011-12) Bester, Ingrid Muriel; Witthuhn, R. C.; Cameron, M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Listeria monocytogenes is a foodborne pathogen that has been isolated from a variety of food sources. It is the cause of the food-borne disease, listeriosis that shows symptoms such as meningitis, encephalitis and abortion. Different strains of L. monocytogenes exist and not all are thought to be pathogenic to humans. The aim of this study was to evaluate and compare conventional methods, culturing on selective (Oxford agar) and chromogenic (RAPID’L.mono agar) media, as well as speciesspecific and multiplex polymerase chain reaction (PCR) methods for the detection and identification of 94 L. monocytogenes isolates from various areas in an avocado processing facility, as well as the final product. To achieve a better understanding of the genetic diversity of the confirmed L. monocytogenes strains isolated from the avocado facility, two subtyping techniques, PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE), were employed. All of the isolates were identified as Listeria species on both Oxford and RAPID’L.mono agar. On the RAPID’L.mono agar, 76 of the 94 isolates produced colonies typical of L. monocytogenes, with the remaining 18 showing colonies typical of L. innocua (n=13) and L. ivanovii (n=5). The species-specific PCR successfully amplified a 730 base pairs region of the hly gene of 80 of the 94 isolates. For the same 80 isolates the multiplex PCR successfully amplified 800, 517 and 238 base pair (bp) fragments of the inlA, inlC and inlJ genes, respectively. The remaining 14 isolates included the 13 isolates identified as L. innocua, as well as an isolate identified as L. monocytogenes on RAPID’L.mono. The results obtained on the Oxford agar showed a 100 % positive correlation when compared to the PCR results in identifying Listeria species, while the RAPID’L.mono had a 4 % false negative result in identifying L. monocytogenes compared to the PCR results. Sixty-four of the confirmed L. monocytogenes isolates were subtyped using PCRRFLP and PFGE. For the PCR-RFLP analysis, a 733 bp fragment of the inlA gene was successfully amplified for all of the isolates, followed by digestion with the restriction enzymes, AluI and Tsp509I. AluI produced three different banding patterns and Tsp509I produced two different banding patterns. Subtyping of the isolates using PFGE was carried out by macrorestriction of the genomic DNA with ApaI and AscI. The restriction fragments were resolved by PFGE and the fingerprints were classified into four clusters. In the combined analyses, cluster I contained forty-eight isolates (n=48), cluster II 1 isolate (n=1), cluster III fifteen isolates (n=15) and cluster IV 1 isolate (n=1). The PCR-RFLP results had a 98 % correlation with the PFGE results. The results of this study indicated inconsistencies between the results obtained by conventional and molecular detection methods for the identification of L. monocytogenes. Species-specific and multiplex PCR, however, proved useful to accurately detect and identify L. monocytogenes in a shorter period of time and could replace the use of conventional agar during identification. Both PCR-RFLP and PFGE proved useful in the subtyping of L. monocytogenes isolates with the PCR-RFLP being less expensive and results obtainable in a shorter period of time.
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    Evaluation of the distribution and accumulation of species of Alicyclobacillus in the fruit concentrate processing environment
    (Stellenbosch : University of Stellenbosch, 2011-03) Steyn, Catharina Elizabeth; Witthuhn, R. C.; Cameron, M.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Alicyclobacillus species are thermo-acidophilic bacteria that produce highly resistant endospores able to survive the processing temperatures of fruit concentrate manufacturing, including evaporation and conventional pasteurisation (86 ° - 96 °C for ± 2 min). Alicyclobacillus endospores retain their viability in juice concentrates which, under favourable conditions, could germinate and multiply to numbers high enough to cause spoilage and product deterioration through the production of chemical taint compounds. This thesis reports on the distribution of Alicyclobacillus in the fruit concentrate processing environment and the effect of current manufacturing practices on the accumulation of Alicyclobacillus in fruit concentrates. These practices include the recirculation (recycling) of flume water as a means of water conservation, as well as continuous process running times when facilities operate at full capacity. This thesis also reports on the effect of fruit variety and skin type on the occurrence of Alicyclobacillus in fruit concentrates. Alicyclobacillus was monitored at nine processing stages of fruit concentrate manufacturing during the functioning of either a recirculating or a one-pass (not recirculated) flume water system. Significantly higher Alicyclobacillus levels were recovered in fruit mash, single strength juice, concentrate and the final pasteurised product (± 30 °Brix) during the functioning of a re circulating flume system, compared to when a one-pass flume system was functional (P < 0.05). Irrespective of the flume system, high Alicyclobacillus levels were recovered from the concentrate and condensate water (a by-product of juice concentration) from the evaporator, which makes this a point of concern during concentrate manufacturing. Manufacturing practices such as the recirculation of flume water and the recovery of condensate water for fruit washing purposes pose a potential risk of Alicyclobacillus contamination and accumulation in fruit concentrates and the processing environment. The effect of continuous process running times on Alicyclobacillus was monitored in a facility that was operating at full capacity. Sampling occurred every 12 h at four processing stages, during a processing tempo of 1.8 - 2.0 t h-1 for 108 h. Vegetative cells increased significantly (P < 0.05) in single strength juice and condensate water after 84 h of processing, with 3.15 and 3.85 log10 cfu mL-1 recovered, respectively. Similar accumulation patterns of vegetative cells were observed in concentrate and the final pasteurised product. Endospores in single strength juice, concentrate and the final product were also the highest after 84 h of processing with 1.32, 1.59 and 1.64 log10 cfu mL-1, respectively. When fruit concentrate manufacturing facilities process at full capacity, a restriction in the continuous process running time to under 84 h in between CIP procedures, along with good manufacturing practices, can minimise Alicyclobacillus accumulation in fruit concentrates. The effect of fruit skin type, specifically hairy-skinned stone fruits (peach and apricot) and smooth-skinned pome fruits (apple and pear) on the occurrence of Alicyclobacillus in concentrates were examined. Apple concentrate samples had the highest occurrence (average %) of vegetative Alicyclobacillus cells (50%), followed by apricot (40%), peach (15%) and pear (10%) concentrates. The occurrence of Alicyclobacillus endospores in fruit concentrate samples were also the highest in apple (50%), followed by pear (25%), apricot (20%), and peach (10%) concentrates. The occurrence of Alicyclobacillus vegetative cells and endospores did not differ significantly (P > 0.05) between concentrates from hairy-skin and smooth-skin fruit varieties. Thus it was concluded that fruit washing steps prior to processing was more critical for the control of Alicyclobacillus than the type of fruit skin being processed.
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    Identification of precursors present in fruit juice that lead to the production of guaiacol by Alicyclobacillus Acidoterrestris
    (Stellenbosch : University of Stellenbosch, 2011-03) Van der Merwe, Enette; Witthuhn, R. C.; Venter, P.; Cameron, M.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Alicyclobacilli are endospore-forming, thermophilic, acidophilic bacteria (TAB) that survive the pasteurisation process and spoil acidic fruit juices through the production of the taint compound guaiacol. Guaiacol causes an undesirable odour with an unpleasant smoky, medicinal or phenolic-like taste. This thesis reports on the precursors, vanillin and vanillic acid metabolised to guaiacol by Alicyclobacillus spp. in fruit juice, the pathway of guaiacol production and the spoilage potential of contaminated fruit juices supplemented with these precursors. A high performance liquid chromatography method with UV-diode array detection (HPLC-DAD) was developed for the simultaneous detection and quantification of guaiacol and its precursors. Alicyclobacillus acidoterrestris FB2 was incubated at 45 °C for 7 d in Bacillus acidoterrestris (BAT) broth supplemented with ferulic acid, vanillin or vanillic acid. The cell concentrations were determined every 24 h and the concentration of the precursors and the production of guaiacol was determined using HPLC-DAD. The guaiacol production was also determined using the peroxidase enzyme colourimetric assay (PECA). Alicyclobacillus acidoterrestris produced guaiacol from vanillin and vanillic acid, confirming both vanillin and vanillic acid as precursors for guaiacol production by A. acidoterrestris FB2. Furthermore, a metabolic pathway directly from vanillin to guaiacol was identified in this study. However, guaiacol was not produced by A. acidoterrestris FB2 in the samples supplemented with ferulic acid and it is, therefore, not considered a direct precursor for guaiacol production by A. acidoterrestris. The spoilage potential of apple juice supplemented with either 10 mg L-1 or 100 mg L-1 vanillin or vanillic acid by A. acidoterrestris FB2 (106 cfu mL-1) was also evaluated. The production of guaiacol increased with the increase in vanillin or vanillic acid concentrations (in BAT broth and apple juice) indicating that the concentration of vanillin and vanillic acid present in fruit juice will influence the spoilage potential of the juice. Guaiacol concentrations in apple juice well above the best estimated threshold value of guaiacol for taste (0.24 – 2.00 μg L-1) and odour (0.50 - 2.32 μg L-1) was produced by A. acidoterrestris FB2 in the apple juice supplemented with 10 mg L-1 vanillin or vanillic acid. This indicates that fruit juice with a vanillin or vanillic acid concentration as low as 10 mg L-1 has the potential to spoil if the juice is contaminated with A. acidoterrestris. The concentrations of vanillin and vanillic acid in different fruit juices can be used to indicate if a specific fruit juice is susceptible to guaiacol spoilage by Alicyclobacillus spp. In the development of juice products and different blends of fruit juices, special care must be taken not to concentrate the amount of vanillin and vanillic acid present in the fruit juices.
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    Phylogeny and molecular identification of Cronobacter strains isolated from south African food products
    (Stellenbosch : University of Stellenbosch, 2011-03) Strydom, Amy; Witthuhn, R. C.; Cameron, M.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia. Cronobacter infections have been reported in all age groups, however, immunocompromised infants are more susceptible to these infections. Furthermore, Cronobacter strains have been reported to show differences in sensitivity to antibiotics and virulence. These differences led to the reclassification of Cronobacter and currently the genus contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this reclassification was only accepted recently, there are not many typing methods optimised for differentiation between the five Cronobacter species. Typing of Cronobacter strains are important as the species may be diverse regarding their virulence. Cronobacter strains have been isolated from infant formula milk (IFM), the environment of an IFM processing facility and fresh produce in South Africa. However, little is known about the phylogeny and prevalence of these strains. The aim of this study was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii) and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide variety of isolation sources. Other studies have also found that irrespective of the isolation source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South African strains were found to be phylogenetically closely related. However, two distinct clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group based on the 16S rRNA gene analysis. Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr. sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %. Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not sufficient to distinguish between all the Cronobacter species. The sequence data of these two genes can be used to differentiate between the Cronobacter strains when used in combination with malonate utilisation analysis. A PCR-RFLP method was subsequently developed to facilitate the simultaneous differentiation between all five Cronobacter species. The PCR-RFLP assay was based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species were obtained and it was also possible to differentiate between Enterobacteriaceae and Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical analysis with malonate broth confirmed the identities of these two strains as Cr. malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures rapid differentiation between the five species of Cronobacter.