An evanescent wave fiber-optic biosensor was used in an effort to develop an assay for the rapid detection of two biological toxins: cholera toxin and botulinum toxin. The Analyte 2000 fiber-optic biosensor utilizes a sandwich immunoassay format. Gangliosides or liposomes are directly adsorbed to the surface of the fiber-optic waveguide through hydrophobic interactions. The waveguide is exposed to a sample containing the toxin of interest, then subsequently exposed to a polyclonal detection antibody conjugated to the fluorophore cyanine 5. Excitation light from a 635nm laser diode is propagated through the waveguide and fluorescent molecules within approximately 100nm of the waveguide are excited. The emission light from the excited cyanine 5 molecules reverberates into the waveguide and is quantitated in pico Amperes and displayed on a computer. The exotoxins of Vibrio cholerae and Clostridium botulinum, cholera and botulinum toxin, respectively, were used for pote ntial assay development. Assay development utilizing the biosensor was attempted for the detection of botulinum toxin in buffer. The limit of detection remained too high to generate a positive signal for the detection of botulinum toxin. Biosensor assays were developed to detect cholera toxin in buffer, oyster homogenate, pure culture and induction media. A cholera toxin standard curve was generated with a limit of detection of 1 ng/ml. The values were normalized by setting 100 ng/ml of cholera toxin to a value of 100. Signals were detected in oyster homogenate spiked at 5 ug/ml as well as unspiked oyster homogenate. A Western blot showed that there were cross reactive proteins in the oyster matrix at molecular weights different from those of the cholera toxin. Cholera toxin production by three strains of Vibrio cholerae with values estimated to range from 100 pg -- 100 ng was detected with the biosensor. Additionally, oysters were harvested from Tampa Bay and placed in a 10 gallon tank filled with different types of induction media. The tank was inoculated with Vibrio cholerae and the oysters and induction medium were analyzed at varying times for the presence of cholera toxin. Vibrio cholerae cells were viable through 24 hours but no toxin was detectable.

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.

No description available.

Several obstacles to widespread use of bacteriocins in food have been identified, including lack of specific, rapid quantitation methods, and little data on their efficacy in food systems. The first objective of this study was to develop a specific, rapid quantitation method for bacteriocins that did not rely on bioassays and their associated limitations. Phage display was chosen to reduce reliance on continued use of animals and produce antibodies to the bacteriocins leucocin A, piscicolin 126, and brochocin-A. Although the antibody libraries generated by phage display were not successful for antibody production, a strong immune response to leucocin A and piscicolin 126 was observed in mice. The second objective of the study was to determine the efficacy of brochocin-C against Clostridium botulinum in a model system and in a vacuum-packaged, chopped and formed pork product stored at refrigeration temperature. Group I Cl. botulinum was not controlled by brochocin-C, and was found to inactivate brochocin-C and several class IIa bacteriocins by proteolysis. Cell counts revealed that Group II Cl. botulinum was controlled by brochocin-C in a model meat system, but was not controlled in the chopped and formed pork product. Powdered smoke and NaCl in the pork product had a synergistic interaction against Group II Cl. botulinum, as shown by minimum inhibitory concentration testing. The choice of media for isolation of Cl. botulinum from the chopped and formed pork product was important, as the presence of background microflora isolated from the meat was found to impact growth of Group II Cl. botulinum on plating media. In the presence of the background microflora, which were identified by 16S rDNA sequencing as carnobacteria and staphylococci, inclusion of phosphate in the plating medium was found to allow growth of Cl. botulinum. Other nutrients such as magnesium, sulphur, or increased protein sources added to the medium had no effect on growth of Cl. botulinum. Two of the background microflora strains, Carnobacterium maltaromaticum MH3 and Staphylococcus pasteuri EIV-21, inhibited Cl. botulinum, while one strain, C. maltaromaticum MH2, stimulated growth of Cl. botulinum. / Food Science and Technology

Thesis (PhD) -- University of Western Sydney, 2003. / "A thesis submitted for degree of Doctor of Philosophy, Centre for Advanced Food Research, School of Science, Food & Horticulture, University of Western Sydney, Hawkesbury campus, Richmond, Australia, January 2003" Bibliography: leaves 199-227.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-30Bitstream added on 2014-06-13T20:04:34Z : No. of bitstreams: 1 ragazani_avf_dr_jabo.pdf: 399859 bytes, checksum: f93ad8981a9e4aaa051ed6bff03ccacc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium... / The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below)

Orientador: Ruben Pablo Schocken-Iturrino / Banca: Maria Luiza Poiatti / Banca: Alessandra Aparecida Medeiros / Banca: Hélio José Montassier / Banca: Antônio Carlos Monteiro / Resumo: Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below) / Doutor

The growth of Listeria monocytogenes and nonproteolytic Clostridium botulinum type B spores in cooked, uncured turkey was investigated separately under varying conditions of modified atmosphere packaging (MAP), refrigerated and temperature-abuse storage, and lactic acid bacteria (LAB) competition. L. monocytogenes (LM) growth was suppressed when initially outnumbered 3.5 logs:1 or 2.1 logs:1 by naturally-occurring LAB, but not when the initial LAB:LM population ratio was equivalent. Lowering storage temperature from 10 degrees to 4 degrees C enhanced the inhibitory effect of CO₂ in the packaging atmosphere, and extended the period of product olfactory acceptability. When the LAB:LM population ratio was equivalent, objectionable odors were not detected in most of the samples, despite LAB counts in excess of 10E⁸/g. This raises concerns with respect to public health, since high levels of L. monocytogenes can be present in MAP meat and poultry products without accompanying signs of overt spoilage. Cellular fatty acid (CFA) analysis was a valuable tool for distinguishing between phenotypically distinct isolates of LM inoculated into MAP turkey. Fatty acid composition of foodborne outbreak-associated (serotype 4) and non-outbreak-associated (serotype 1) strains of LM correlated with antigenic type (4 or 1) and agglutination reaction (granular or flocculent). Strain ATCC 43256 (serotype 4b) produced a consistently unique CFA profile, making it the easiest of the four test strains to be differentiated. Analysis of additional LM serotypes, as well as examination of existing clinical and environmental CFA databases for correlations between fatty acid profiles and diagnostic characteristics of LM, is necessary before CFA analysis can be effectively applied as an epidemiological tool for tracking the distribution of LM strains in food products and throughout the farm-to-table food chain. Reduced storage temperature significantly delayed botulinal toxin production and extended the period of olfactory acceptability of cooked turkey, but even strict refrigeration did not prevent growth and toxigenesis by nonproteolytic C. botulinum type B. Toxin was detected on day 7 for product stored at 15 degrees C and on day 14 for product stored at 10 degrees C, irrespective of packaging atmosphere. At 4 degrees C, toxin was detected on day 14 for product packaged without carbon dioxide, and on day 28 for product packaged with 30% carbon dioxide. At all three storage temperatures, toxin detection preceded or coincided with olfactory unacceptability, demonstrating the potential for consumption of toxic product when spoilage-signalling sensory cues are absent. / Ph. D.

Ziel der vorliegenden Arbeit war es, einen Beitrag zur Aufklärung der Ätiologie der Graskrankheit mit Hilfe der DGGE, besonders im Hinblick auf in vitro unkultivierbare Bakterien der Darmflora zu leisten. Es sollte ebenfalls geprüft werden, ob die DGGE die Diagnose der Graskrankheit erleichtern kann. Weiterhin sollte der Einfluß von C. botulinum auf die Erkrankung durch den Nachweis von Toxin, Bakterien und Antikörpern untersucht werden. Es standen zur Untersuchung Proben des Colons, Caecums und Kotes von erkrankten Pferden und Kontrolltieren, Kotproben von klinisch gesunden Pferden, die aus denselben Beständen wie die erkrankten Tiere stammen sowie Serum aller drei Gruppen zur Verfügung. Wegen der hohen individuellen Variabilität der Darmflora war kein eindeutiges Merkmal der Graskrankheit im Profil der mikrobiellen Gemeinschaft des Darmes oder Kotes nachweisbar. Allerdings ließ sich anhand der Clusteranalyse ein Abgrenzung der Flora des Caecums und besonders des Colons der erkrankten und gesunden Tiere erkennen. Für eine Diagnose der Graskrankheit am lebenden Tier anhand der Kotflora ist die DGGE jedoch wegen ihrer geringen Aussagekraft und methodischen Probleme nicht geeignet. Der Verdacht, dass C. botulinum an der Ätiologie der Graskrankheit beteiligt ist, konnte durch die Ergebnisse im Tierversuch und ELISA weiter untermauert werden.

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