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Thesis (M. Sc. ) - niversity of the Western Cape, 2009. / Includes bibliographical references (leaves 72-79)

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A total of twenty-eight Bacillus spp. isolated from value-added surimi nuggets and their raw ingredients, were tested against each other and selected reference strains of Bacillus and Clostridium for their production of inhibitory substances using the deferred antagonism assay plating method. The isolated Bacillus strains showed inhibitory activity against all Bacillus strains, with the exception of the producer strain, as well as being effective against various strains of C. botulinum (type A, B and E). Subsequent studies showed that the inhibitory activity was detected in the culture supernatant in the late stationary phase of growth prior to sporulation. The inhibitory activity of two Bacillus strains (FN2A and FN33) were selected for further study. The inhibitory substances produced by these two strains were proteinaceous in nature, heat stable (100°C for 15min) and unaffected by organic solvents. A comprehensive study was conducted on the structural characterization of the inhibitor produced by B. subtilis FN2A using FPLC, FTIR, MS and MS/MS. Structural analysis of the inhibitor produced by B. subtilis FN2A showed that it was similar in structure to Surfactin. / Preliminary studies have shown that the Surfactin-like-compound from B. subtilis FN2A was produced in significant amounts during growth in bread with maximum production occurring in the late stationary phase (72h), at 30--35°C and at pH 6.5--7.0. Optimization studies on the production of the Surfactin-like-compound by B. subtilis FN2A in bread using a response surface methodology approach showed that temperature (33--36°C); autoclaving time (30 min); inoculum level (4%), alkali pre-treatment (0.16%), water activity (0.995) and pH 6.66 enhanced the production of the Surfactin-like-compound in bread. The compound produced under these optimal conditions also maintained its activity when subjected to various processing treatments (autoclaving, freezing and freeze drying). / Initial studies showed that low levels (1% w/w) of the Surfactin-like-compound inhibited the growth of B. cereus and proteolytic and non-proteolytic strains of C. botulinum in a model agar system. However, it had no effect on non-proteolytic strains of C. botulinum when bread, or methanol extracts of bread (1--20%), were added to formulated value-added sterile trout nuggets, with all nuggets being toxic after 28 days at 12°C. Furthermore, inoculation of B. subtilis FN2A directly into nuggets also failed to inhibit growth of non-proteolytic strains of C. botulinum. Omitting certain ingredients in the formulation failed to enhance the anti-botulinal effect of the bread or methanol extracts of the Surfactin-like-compound in the value-added nuggets. However, reducing the pH of the nuggets to ~5.5 enhanced the anti-botulinal effect of the Surfactin-like-compound. Further research is required to improve the dispersibility of the Surfactin-like-compound to inhibit the growth of C. botulinum in food systems.

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