The biological preparation of aflatoxins was accomplished by culturing the mold Aspergillus flavus on various media. Aflatoxin was isolated by chloroform extraction of the culture, and purified by column fractionation and crystallization. Rice, wheat, soya bean, peanut and YES media were found to give high yields of aflatoxin. It was also found that A. flavus could grow and produce toxin on fresh tomatoes and grapes. The pure aflatoxins B₁ and G₁ were treated separately with standardized NaOH and NH₄OH solutions. Changes in solvent system, exposure time and alkali concentration were found to affect the stability of aflatoxin. Solutions of aflatoxins B₁ and G₁ in ethanol and methanol were quite unstable when treated with strong base even after a short exposure period. The aflatoxins were quite stable, however, if treated in a chloroform solution at a lower base concentration. Several crude chloroform extracts containing aflatoxins were prepared from various culture media with different mold strains. These extracts were then shaken gently with an equal volume of 0.25 N NaOH solution for several seconds. This treatment produced final chloroform extracts which were lower in pigments, total solids, and lower R [subscript f]-value compounds than similar extracts which received no base treatment. The decolorizing ability of the alkali treatment was demonstrated by comparison of the ultraviolet absorption spectra of the treated samples and control samples. This base treatment shortened the preparation time and increased the yield of aflatoxins from crude chloroform extracts. Photodegradation of aflatoxins B₁ and G₁ was observed in both ethanol and chloroform solutions. After an induction period, the aflatoxin was degraded to low R [subscript f]-value compounds, which were then converted to non-fluorescent products. Aflatoxin G₁ was found to be more stable than aflatoxin B₁ to light exposure. Base treatment as described above was found suitable for purifying aflatoxin which contained photodegraded contaminants. / Graduation date: 1967

No description available.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 132-135). Also available on the World Wide Web.

Heterocyclic amines and Lipid oxidation products are important classes of toxins in food. Prevention of their formation in food might minimize their dietary exposure and lower their health risk to people. This research work is to identify the potential vitamins which may inhibit the formation of these toxins. Moreover, the possible inhibition mechanism for potent inhibitor(s) is also investigated. The first part was related to heterocyclic amines which are a class of important Maillard reaction products. The inhibitory activities of 11 water-soluble vitamins against heterocyclic amine formation were examined in a PhIP and a MeIQx producing chemical model. Six investigated vitamins (pyridoxiamine, pyridoxine, nicotinic acid, biotin, thiamine and L-ascorbic acid) out of eleven, exhibited significant inhibition (>40%) in both models. The activity of pyridoxamine, niacin, alpha-tocopherol, retinol acid and ascorbic acid was further investigated using fried beef. Moderate inhibition (20%) of the formation of PhIP, 4,8-DiMeIQx and MeIQx was found for most of them; whereas pyridoxamine reduced the levels of all three HAs by 40%. LC–MS analysis revealed that pyridoxamine directly reacts with phenylacetaldehyde to form an adduct which was characterized by LC-MS and NMR spectroscopy. The second part was related to cholesterol oxidation products (COPs). The capacities of 15 vitamins to inhibit the formation of 7α-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol was examined in beef patties. Among them, L-ascorbic acid, retinoic acid, and α-(±)-tocopherol were found to exert a potent inhibitory effect (30−50%) on the formation of three COPs. Pyridoxamine inhibited 7-ketocholesterol formation by 60% and it could directly react with 7-ketocholesterol via the addition reaction. Finally, the inhibitory activities of 15 vitamins against Malondialdehyde (MDA) formation were examined in beef patties samples. Solid Phase Extraction (SPE) and TBA test were performed followed by HPLC-DAD analysis. Five vitamins (pyridoxiamine, pyridoxine, retinoic acid, α-tocopherol and L-ascorbic acid) exhibited significant inhibition (>20%) in triplicate experiments. The fatty acid profile was examined by GC-MS and significant difference was only observed for three antioxidants (retinol acid, L-ascorbic acid and alpha-tocopherol) but not pyridoxamine. An alternative explanation for the inhibition mechanism against lipid oxidation was proposed for pyridoxmine. With the application of chromatographic and spectral analysis (LC–MS and NMR), it was demonstrated that pyridoxamine could directly react with Malondialdehyde via addition reaction. In summary, certain vitamins were found to be potential inhibitors against both heterocyclic amines and lipid oxidation products formation. Mechanistic studies showed that direct-trapping of these toxins and the intermediate compounds for their formation by pyridoxamine and other vitamins was an effective way to lower the concentration of these food toxins during high-temperature processing. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Biological toxins such as ricin and the Bacillus anthracis toxins have become an increasing bioterrorism threat due to the relative availability, extreme potency and ease of production of these agents. Extensive research has been conducted investigating methods for decontaminating spaces affected by biological agents such as bacteria and viruses, but little research has been done to determine the efficacy of these methods on toxins. The efficacy of gaseous chlorine dioxide (GCD) and vaporous hydrogen peroxide (VHP) at inactivating ricin and B. anthracis lethal factor and protective antigen was tested. The presence and activity of the toxins after exposure to the decontamination methods was detected using a cytotoxicity assay and protein gel electrophoresis. Both VHP and GCD were found to be effective at detoxifying the anthrax toxins within a short exposure, with close to complete inactivation observed during longer exposures. Ricin proved to be more resistant to inactivation, with longer exposures needed to achieve similar levels of detoxification to the anthrax toxins. Overall, GCD and VHP have great potential for use in inactivating biological toxins.

Biological toxins such as ricin and the Bacillus anthracis toxins have become an increasing bioterrorism threat due to the relative availability, extreme potency and ease of production of these agents. Extensive research has been conducted investigating methods for decontaminating spaces affected by biological agents such as bacteria and viruses, but little research has been done to determine the efficacy of these methods on toxins. The efficacy of gaseous chlorine dioxide (GCD) and vaporous hydrogen peroxide (VHP) at inactivating ricin and B. anthracis lethal factor and protective antigen was tested. The presence and activity of the toxins after exposure to the decontamination methods was detected using a cytotoxicity assay and protein gel electrophoresis. Both VHP and GCD were found to be effective at detoxifying the anthrax toxins within a short exposure, with close to complete inactivation observed during longer exposures. Ricin proved to be more resistant to inactivation, with longer exposures needed to achieve similar levels of detoxification to the anthrax toxins. Overall, GCD and VHP have great potential for use in inactivating biological toxins.

This thesis reports research undertaken which will lead to improved pretreatments and therapies for disease caused by Clostridium perfringens, Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. C. perfringens is thought to be the most widely distributed bacterial pathogen and is the most important Clostridial species associated with enteric disease in domesticated animals. During warfare C. perfringens has been a significant causes of mortality. Between 1 and 10% of wounded personnel developed gas gangrene during the 1st and 2nd world wars. The ability of the bacterium to cause a range of diseases is due largely to the differential production of toxins. The first reported cloning and nucleotide sequencing of three of the four major toxins (α, β and ε-toxins) is documented in this thesis. The regulation of expression of α-toxin in C. perfringens has been investigated and methods for the expression of recombinant proteins in E. coli have been devised This information has been used to develop improved PCR-based diagnostic tests, and to investigate structure-function relationships. A high resolution crystal structure of a-toxin (phospholipase C) is reported. Using molecular and biophysical techniques, the functions of the two domains of the protein have been determined. Residues that play roles in the interaction of the toxin with host cell membranes have been identified using site-directed mutagenesis. This work has also provided a major insight into the structures and functions of related phospholipases C (the zincmetallophospholipases C) from other bacterial pathogens. This pioneering work with α-toxin is recognised by invitations to write reviews and book chapters on this subject and on bacterial phospholipases C. C. perfringens β-toxin has been shown to be related to pore forming toxins such as Staphylococcus aureus α-toxin. This finding suggests, for the first time, the mode of action of β-toxin. The interaction of C. perfringens ε-toxin with host cells has been investigated and progress made in identifying the cell-surface receptor for the toxin. Genetically engineered toxoids have been devised which induce high-level protection against α and ε-toxins. These vaccines are currently being developed by industry for veterinary use. Similar approaches have been used to devise a recombinant vaccine against Clostridium botulinum toxin F. The wider applications of toxins as therapeutics have also been investigated, and a novel cancer drug delivery system based on targeted lysis of drug-containing liposomes by α-toxin has been devised and patented. F. tularensis is the etiological agent of tularemia, a disease of man that is found in most countries in the Northern hemisphere and most frequently in Scandinavia, N. America, Japan and N. Russia. In this thesis the efficacy of antibiotics for the prevention and treatment of experimental tularemia is documented. Two surface antigens (lipopolysaccharide and FopA) have been evaluated as sub-unit vaccines. Of these, lipopolysaccharide shows potential as a protective antigen. However, because of the paucity of information available on this bacterium, a wider approach to vaccine development, involving the determination of the genome sequence of a fully virulent strain of F. tularensis has been undertaken. A preliminary analysis of the genome sequence is reported here, which has allowed the identification of targets for the development of a rationally attenuated mutant for use as a live vaccine. Y. pestis is generally recognised to have caused three major pandemics of disease, and credible estimates indicate that together these resulted in 200 million deaths. WHO figures indicate that there is a continuing public health problem from plague, especially in Africa, Asia and South America. In this thesis existing vaccines and antibiotics have been evaluated for the prevention and treatment of plague and found to have limitations. A number of approaches to the development of an improved vaccine have been investigated including rationally attenuated strains of the bacterium and isolated surface antigens. A sub-unit vaccine against plague has been devised based on recombinant forms of the F1- and V-antigens. This vaccine provides high level protection against both bubonic and pneumonic plague. This recombinant sub-unit vaccine has been patented and is currently in phase I clinical trials in man. This vaccine has been formulated for single oral or intranasal delivery, using microencapsulated or Salmonella-based delivery systems. Methods for enhancing the stability and efficacy of these vaccines have been investigated. Reviews on plague and plague vaccines have been written, confirming the status of the author as a world leader in this field. The work to devise an improved vaccine has also provided insight into the molecular basis of pathogencity of Y. pestis. A phoP / phoQ regulatory system has been discovered in the bacterium, which plays a key role in survival of the bacterium within macrophages. The V-antigen has been shown to be surface located to play a key role in the translocation of effector proteins into host cells. The biogenesis of the F1-capsular antigen has been investigated at a genetic and biophysical level. In order to underpin future work with this pathogen, the genome sequence is currently being determined. This work has already provided major new insights into the evolution of this pathogen. B. pseudomallei (formerly Pseudomonas pseudomallei) is found primarily in S. E. Asia, N. Australia and other tropical areas of the world. Melioidosis has recently appeared in temperate zones, including mainland France and the UK possible as a consequence of increased international travel. Acute disease can be treated with antibiotics but the bacterium can persist in the host and subsequent disease episodes can occur. In this thesis ciprofloxacin and doxycyline have been are evaluated and shown to have significant limitations for the treatment of melioidodis. In the longer term there is a requirement for an effective vaccine against melioidosis, and work is reported here to devise the genetic tools which will be necessary for the genetic manipulation of the bacterium, with a view towards the identification of virulence determinants.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.