M.Tech. (Biomedical Technology) / A study was conducted to determine the extent of fungi and mycotoxins contamination of Rwandan selected food commodities. A total of one-hundred food samples including maize, rice, cassava, beans and peanuts were collected from all five provinces of Rwanda and analysed. Mycological data obtained revealed a high level of contamination of common toxigenic fungi belonging mainly to the Aspergillus, Penicillium and Fusarium genera. Accordingly, Aspergillus flavus was the most prevalent fungal contaminant in maize (90%), while A. carbonarius was mainly concentrated in peanuts at an incidence rate of 70%. Aspergillus fumigatus was mostly found in cassava (85%) in combination with Penicillium decumbens at the rate of 70%, meanwhile P. citrinum was found at an incidence rate of 80% in rice. The genus Fusarium was dominantly present with F. verticillioides and F. graminearum found in all analysed commodities. A toxigenicity study was also conducted to evaluate the capacity of these fungi recovered to produce their respective mycotoxins. Certain species such as A. flavus and A. parasiticus isolated from these commodities produced the aflatoxins (AFs). Other Aspergillus spp. such as A. carbonarius produced ochratoxin A (OTA) and F. verticillioides and F. graminearum also showed their capacity in producing different mycotoxins viz: zearalenone (ZEA), fumonisins (FBs) and deoxynivalenol (DON). The analysis of mycotoxins in these commodities was performed following thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Data obtained revealed that peanuts and maize were the most contaminated with mycotoxins at incidence rates of 85 and 80%, respectively, and at the highest contamination levels. The highest AF-contaminated commodity was maize from Western province (range: 1.3-3219.6 μg/kg; mean: 829.3 μg/kg) followed by peanut from the same region whose mean level found was 401.5 μg/kg (range: 3.2–1755.8 μg/kg). Ochratoxin A was also found in peanuts with a mean concentration of 302.6 μg/kg, while DON was found at the highest level of 419.6 μg/kg in a rice from Kigali-city. Maize was the main substrate for FBs (mean: 134 μg/kg; max: 4591 μg/kg). Zearalenone was also recovered from samples but at a low incidence rate of 40% with the highest level of 5.2 μg/kg recorded. It was also observed that 65% of samples analysed were contaminated with more than one mycotoxin.

Maize is the staple food of South Africa and its cultivation covers the largest area of farmland in the country. It plays an important role in the economy as South Africa is consuming 10 million tons of maize per annum. Most South Africans consume maize in some form or another. This commodity has regularly been associated with mycotoxigenic fungi and in some cases their respective mycotoxins. The problem is not only confined to the borders of South Africa but is a concern worldwide. Mycotoxins are known to affect both human and animal health. Some of the most well known mycotoxins are the fumonisins, deoxynivalenol and trichothecenes produced by Fusarium spp., aflatoxins and ochratoxin A produced by mostly Aspergillus spp., as well as patulin and citrinin that are produced by mainly Penicillium spp. Mycotoxins acting together and individually can be hepatotoxic, carcinogenic and teratogenic to humans and animals. The main objective of this study was to screen commercially produced maize cultivars in South Africa that would be either resistant to, or have a slower infection rate when inoculated with the ten selected mycotoxigenic fungi from South African maize. The objective was also to develop a method to detect and identify these mycotoxigenic fungi in the infected maize cultivars, using both basic microbiological and molecular means. These objectives were achieved by a series of experiments that are outlined in the individual chapters. The first part of this study evaluated the level of infestation of fungi in all the commercially produced maize cultivars in South Africa. A basic fungal enumeration and identification was carried out. This allowed the comparison of the in vivo ability of the selected cultivars to endure the natural invasion of mycotoxigenic fungi during cultivation in the same area namely Potchefstroom. As part of the maize evaluation, the cultivars were artificially inoculated with ten selected mycotoxigenic fungi, which consisted of five field fungi nl. Alternaria alternata, Fusarium graminearum, Fusarium verticillioides, Phoma sorghina and Stenocarpella maydis, and five storage fungi nl. Aspergillus flavus, Aspergillus ochraceus, Eurotium repens, Penicillium islandicum and Rhizopus oryzae under storage conditions. The ability of certain maize cultivars to resist the infestation by mycotoxigenic fungi under storage conditions was demonstrated. The second part of this study was to use basic molecular methods to detect and identify the ten mycotoxigenic fungi in the infected maize. This was done by making use of the sequence variations of the internal transcribed spacer (ITS) and D1/D2 regions of the fungal rRNA gene. Results showed that the ITS region gave better differentiation and in most cases allowed identification of the mycotoxigenic fungi. The application of the combined use of microbiological and molecular methodology for the detection and identification of mycotoxigenic fungi in maize by testing laboratories in South Africa were outlined in the final chapter. Implementation of a plan to evaluate maize cultivars in the pre-planting, harvesting and storage phases, can provide a holistic overview of the commodity and can be further implemented in the rural areas. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biological Sciences at Technikon Natal, 2000. / Spice oils are known to exhibit antifungal activity and therefore have the potential to control mycotoxin production. There is a need in the food industry to find measures to control mycotoxins that are frequently associated with grains that form the staple diet of the majority of the population in South Africa. Clove, cinnamon, oregano, tumeric, eucalyptus, neem, aniseed, mace and nutmeg oils were tested to determine their inhibitory potential against growth of Aspergillus parasiticus and Fusarium moniliforme using the agar overlay technique. Varying concentrations of the spice oils, ranging from 0.1 ppm to 2.0 ppm, were incorporated into broth cultures of A. parasiticus and maize patty cultures ofF. moniliforme. Levels of production of aflatoxins and fumonisin were determined using standard thin layer chromatography and highpressure liquid chromatography methods. In addition, the active component of the spice oils were isolated, characterised and tested. The inhibitory potential of these compounds for field use was tested by incorporating clove oil, whole cloves and ground cloves in samp / M

L'ochratoxine A (OTA) est un métabolite secondaire produit par des espèces appartenant aux genres Aspergillus et Penicillium qui a été liée à certaines conditions avec des effets néphrotoxiques, immunotoxiques, tératogènes et cancérogènes. La présence d'OTA dans le café vert a été détectée depuis 1974 et sa transmission à la boisson a été mise en évidence en 1989. La torréfaction du café est un procédé thermique qui peut avoir un effet sur la teneur en OTA, avant 1988, on pensait que l'OTA était détruite pendant la torréfaction, mais après plusieurs chercher sont des résultats contradictoires publiés dans % de réduction (de 0 à 100%). Plusieurs auteurs émis les hypothèses suivantes pour expliquer cette réduction : Isomérisation de la toxine dans la position C3 formant un diastéréoisomère moins toxique (Studer-Rohr et al, 1995 et Bruinink et al, 1997), protection de la dégradation d’OTA par l'humidité du grain (Boudra et al 1995; Blanc et al, 1998 et Stegen et al, 2001.), existence de réactions avec le café toxine parent ou réarrangements de la molécule OTA à températures de torréfaction (Suarez-Quiroz et al, 2005). Une autre étude sur la dégradation thermique de OTA pure a montré la formation de deux composés moins toxiques, 14-(R)-ochratoxine A et de la 14-descarboxi-ochratoxine A (Cramer et al, 2008). Parce qu'il n'y a pas de données concluantes sur l'effet de la torréfaction sur l'OTA dans le grain et le besoin de bases scientifiques pour établir des règles pour l'exportation de café vert, l'objectif de ce travail était d'étudier l'impact des différents types de torréfaction sur la stabilité thermique de l'OTA dans le café et l'élucidation chimique des produits de transformation. Deux niveaux de contamination ont été obtenus à partir de café contaminés artificiellement par Aspergillus westerdijkiae (5,3 et 57,2 ppb d'OTA). Ces lots sont grillés à 230° en utilisant deux méthodes : La torréfaction à tambour (TR) et à lit fluidisé (LF). Les échantillons ont été prélevés toutes les 3 min pour TR et chaque min 0,9 pour LF pour quantifier la valeur résiduelle d¡¯OTA. Les résultats ont montré que le procédé de torréfaction par TR (plus lent) était plus efficace que la FL dans l'élimination de l'OTA (67% et 36%, respectivement, pour une torréfaction moyenne). Nous avons déterminé le taux de dégradation thermique de OTA pure et de l'OTA mélangée avec les composants du café (5 sucres, 3 acides aminés, la caféine et les acides chlorogéniques), montrant que les interactions se déroulent en fonction des conditions de pH et de pKa des composants testés, dans ce cas, en influant sur la réactivité et la vitesse de dégradation de l'OTA. Un produit de transformation (PT) a été observé sur les chromatogrammes obtenus à partir de l'interaction de l'OTA avec les composants du café. Des tests d'alcalinisation et de chauffage de OTA pure ont confirmé que le PT provient de la modification structurale de la molécule d'OTA et n’est pas un produit de l'interaction avec les composants naturels du café. L'effet du pH et de la température sur l'extraction de l'OTA dans le café contaminé a été testé dans ce travail, les résultats ont montré une plus grande extraction de la toxine à un pH de 8,5 et 6°. Au même pH à 20° il y avait une plus importante formation d'un produit de transformation. Le PT a été purifié pour mener à bien sa caractérisation chimique. La nature chimique du produit de transformation, les données spectroscopiques telles que celles obtenus sous UV-Vis (max: 237nm), l'affinité avec la phase mobile de l'OTA, l'analyse de l'alcalinisation (phénomène de régénération de l'OTA et PT), l'analyse d’isotopes stables (SIDA’s) et le spectre de masse (ion moléculaire M+1: 420 m/z), suggèrent que la structure de le PT d'OTA durant le processus de torréfaction correspond à un analogue de l'OTA qui conserve son groupe carboxyle acide et conformément à la fragmentation correspond à la Hydroxy-ochratoxine A (OH-OTA), avec des quantités mineures d’ OTA et de ses isomères. / Ochratoxin A (OTA) is a secondary metabolite produced by species belonging to the genera Aspergillus and Penicillium and this toxin has been associated with certain illnesses within nephrotoxic effects, immunotoxic, teratogenic and carcinogenic. The presence of OTA in green coffee was detected in 1974 and its transmission into the beverage was made evident in 1989. Roasting coffee is a thermal process that have an effect on the OTA content. Before 1988 it was thought that the OTA was destroyed during roasting, but after several investigations, results published are contradictory results published in the % reduction (from 0 to 100%). Several authors have established hypothesis that try explain this reduction: Isomerization of the toxin in the C3 position forming a less toxic diastereomers (Studer-Rohr et al., 1995 and Bruinink et al., 1997), Protection of grain moisture degradation OTA (Boudra et al., 1995; Blanc et al., 1998 and Stegen et al., 2001), Existence of reactions with the parent toxin coffee or rearrangements of the OTA molecule roasting temperatures (200¡ã and 250¡ãC) (Su¨¢rez-Quiroz et al., 2005). Another study on thermal degradation of pure OTA showed the formation of two less toxic compounds 14 - (R)-ochratoxin A and the 14-descarboxi-Ochratoxin A (Cramer et al., 2008).Because there are no conclusive data regarding the effect of roasting on OTA in grain and the need for scientific bases for establishing regulations for export of green coffee, the objective of this work was to study the impact of different types of roasting on the thermal stability of OTA in coffee and chemical elucidation of the transformation products.Two levels of contamination were obtained by the contamination of coffee with a strain of A. westerdijkiae (5.3 and 57.2 ppb of OTA). These lots were roasted at 230 ¡ã C using two methods: Drum rotation (TR) and fluidized bed (LF). Samples were taken every 3 min from TR and every 0.9 min for LF to quantify the residual OTA. The results showed that in roasting process by TR (slower), it was more effective than with LF in the elimination of OTA (67% and 36%, respectively, for a medium roast). The thermal degradation rate of pure OTA and of OTA mixed with the components of coffee (5 sugars, 3 amino acids, caffeine and chlorogenic acids), were determined, showing that interactions took place dependent themselves on the conditions of pH and pKa values of the components tested, in this case by influencing by the reactivity and the rate of degradation of OTA. A transformation product (TP) was observed in the chromatograms obtained from the interaction of OTA with the components of coffee. A test of alkalinization and warming of pure OTA confirmed that the TP comes from the structural modification of the OTA molecule and is not a product of interaction with the natural components of coffee. The pH and temperature showed an effect in extraction of OTA in contaminated coffee, the results show better extraction of the toxin at pH 8.5 at 60 ¡ãC. At the same pH at 20 ¡ãC, it was shown a greater formation of the transformation product.The TP was purified to carry out its chemical characterization. The chemical nature of compound transformation and spectroscopic data such as UV-Vis (¦Ëmax: 237nm), the affinity with the mobile phase of the OTA, the analysis of alkalinization (OTA regeneration phenomenon and TP) analysis of stable isotopes (SIDA's) and the mass spectrum (molecular ion M +: 420 m / z), suggest that structurally the TP of OTA during the roasting process corresponds to an analogue of OTA which retains its acidic carboxyl group and in accordance to fragmentation corresponds to the Hydroxi- Ochratoxin A (OH-OTA), as well as minor amounts of OTA and its isomers.

No description available.

Thesis advisor: Babak Momeni / Thesis advisor: Charles Hoffman / Aflatoxins (AFs) are secondary fungal metabolites that contaminate common food crops and are harmful to humans and animals. The ability to remove AFs from feed commodities will improve health standards and counter the economic drain inflicted by AF contamination. Strategies to mitigate AF contamination fall into three categories: physical, chemical, and biological. In this thesis, I explore the identification of degraders and degradation mechanisms, as well as their enhancement, within the context of chemical and biological strategies. Known chemical strategies have used strong acids and bases to remove contaminating AF, but these methods often lead to ecological waste issues downstream. Chapter 3 investigates the application of weaker acidic and alkaline conditions to remove two types of AFs, AFB1 and AFG2. I find that a weakly alkaline environment is sufficient to degrade AF, providing an alternative solution for chemical decontamination. Biodetoxification is a promising solution to AF contamination because of its low cost and few undesired environmental side-effects. Microbes possess a rich potential for removing toxins and pollutants from the environment. Despite the fairly wide availability of this potential, identifying suitable candidates and improving them remain challenging. In Chapter 2, I explore the use of computational tools to discover strains and enzymes that detoxify harmful toxins. Of focus is the detoxification of mycotoxins by biological enzymes. Existing computational tools can be used to address questions in the discovery of new detoxification potential, the investigation the cellular processes that contribute to detoxification, and the improvement of detoxification potential in discovered enzymes. I showcase open bioremediation questions where computational researchers can contribute and highlight relevant existing and emerging computational tools that could benefit bioremediation researchers. In Chapter 4, I screen several environmental isolates for their AF detoxification ability, using AFG2. I used different carbon sources (glucose and starch) as isolation and culturing media to examine the effect of the environment on degradation ability. Overall, I find that starch medium expedites the screening process and generally improves the performance of isolates, making this a promising method for identifying new degraders and enhancing their performance. Chapter 5 highlights the characterization of degradation by two promising Rhodococcus species, R. erythropolis and R. pyridinivorans. While previous work has identified their degradation ability, further investigation into degradation mechanisms has been understudied. Here, I explore the characterization of degradation mechanisms toward enzyme identification. Finally, the appendix starts to broach the question of enhancing degradation of known degrading enzymes, the example here is laccase from the fungus Trametes versicolor. Using molecular dynamic and quantum mechanics simulations to identify mutations of interest in increasing the affinity of laccase toward AF, I create five mutants to test their degradation against the performance of wildtype. These mutants show a range of improvements against AF and showcase the efficacy of this approach to enhancement. Together, this body of work highlights the importance of understanding AF degradation for the creation of new strategies of AF mitigation. My thesis provides a framework for developing AF decontamination strategies, from identifying degraders and unlocking their mechanisms to enhancing their performance. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 88-99). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 88-99). Also available on the Internet.

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Mycotoxins have assumed worldwide importance due to the ubiquitous occurrence of toxigenic fungi, their infestation of plant-based foods and feeds and the subsequent economical and health impact it because of their contamination of commercial products. Ochratoxin A (OA) is a nephrotoxic mycotoxin produced by isolates of Aspergillus ochraceus and Penicillium verrucosum and occurs frequently in nature. The major target for toxicity of OA in mammalian species is the kidneys and it has been the major cause of Danish Porcine Nephropathy. OA has also been extensively implicated in the aetiology of Balkan Endemic Nephropathy and Chronic Interstitial Nephropathy in Northern-Africa. Furthermore, OA has been identified as a carcinogen, an immunosuppressant and a teratogen with respect to the foetal central nervous system. Although a large amount of research has been conducted into the chemical nature of the toxicity of OA, the exact molecular mechanism of action of OA is not yet conclusive. Numerous structure activity relationship studies have suggested that the toxicity of OA may be assigned to three major processes: (i) inhibition of ATP production; (ii) inhibition of protein synthesis; and (iii) the disruption of hepatic microsomal calcium homeostasis through the promotion of membrane lipid peroxidation. It is the aim of this thesis to gain a better understanding, through the synthesis ofOA analogues, of the chemical structure responsible for the toxic function of the ochratoxins. The halogen-group has extensively been implicated in the toxicity of the ochratoxins. This is evident in ochratoxin B (OB), the dechloro analogue of OA, which is approximately ten times less toxic than OA. Preliminary tests have indicated that bromo-ochratoxin B(BrOB), the bromo analogue of OA, is more toxic than ochratoxin A to renal cells. Fluoro-ochratoxin B and other analogues of OA, where other amino acids are incorporated, should provide invaluable information on the structure-activity relationships and the mode of action of the ochratoxins. Our research effort addresses both these aspects (i) fluorination of the dihydroisocoumarin moiety and (ii) the coupling of different amino acids and dipeptides to the non-toxic hydrolysed product of OA, ochratoxin a. Chapter one includes a review of the important biological aspects of OA that has served as a guideline to the synthesis of effective OA analogues. An overview of the relevant chemistry involved in the modification of OA will conclude the chapter. Chapter two entails a discussion of fluorine in bio-organic chemistry. This includes an overview of the impact that fluorine substitution has on the biological reactivity of molecules. A review on the synthesis of organofluorine compounds, which forms the emphasis of this study, concludes the chapter. Chapter three elaborates on the different methodologies used in our attempts to synthesise fluoro-ochratoxin B and other analogues. These included the direct electrophilic fluorination of OB and different analogous aromatic model compounds by xenon difluoride, N-fluorobenzenesulfonimides and Selectfluor™ as fluorinating agents. Also involved is an investigation into an alternative route for the synthesis of fluoro aromatic compounds from bromo and chloro analogues by means of palladium catalysed trimethyl- and tributylstannyl and trimethylsilylation which in tum may be substituted with fluorine by means of xenon difluoride. Efforts towards the direct catalytic fluorosubstitution of aryl halides are also investigated. The synthesis of a key intermediate, fluoroacetoacetaldehyde, in a de nova synthetic route to fluoroochratoxin B is also discussed. Furthermore, the synthesis of novel OA analogues with respect to the replacement of the L-phenylalanine moiety is addressed. This includes the conversion of OA to Oa, by acid hydrolysis, followed by the coupling of ortho-, meta- and para- substituted DL-fluorophenylalanine to the lactone acid. This is followed by the synthesis of histidylhistidine methyl ester and attempted coupling to Oa. The coupling of halosalicylic acids and salicylic acid to L-phenylalanine, for use as model aromatic substrates for fluorination, IS discussed. Peptide coupling by dicyclohexylcarbodiimide carboxyl activation, with reference to the protection of the phenolic hydroxyl group in 5-chlorosalicylic acid for application to Oa, concludes this work. / AFRIKAANSE OPSOMMING: Mikotoksiene is van wêreld-wye belang as gevolg van die alomteenwoordige voorkoms van toksigeniese fungi, hul besmetting van plantaardige kossoorte en voerstowwe en die gevolglike ekonomiese en gesondheidsimpak deur die besoedeling van kommersiële produkte. Ochratoksien A (OA) is 'n nefrotoksiese mikotoksien wat geproduseer word deur isolate van Aspergillus ochraceus en Penicillium verrucosum en kom algemeen in die natuur voor. Die niere is die hoof teiken vir vergifiting deur OA in soogdierspesies en is as die vername oorsaak van "Danish Porcine Nephropathy" aangewys. OA word verder aangedui as die oorsaak vir "Balkan Endemic Nephropathy" en "Chronic Interstitial Nephropathy" in Noord- Afrika. OA is verder geïdentifiseer as 'n karsinogeen, immuno-onderdrukker en is teratogenies ten opsigte van die sentrale senuweestelsel van fetusse. Alhoewel aansienlike navorsing alreeds gewei is aan die chemiese natuur van die toksisiteit van OA, is die presiese molekulêre meganisme van OA reaktiwiteit onbeslis. Verskeie struktuur-aktiwitweit verwantskaps studies dui daarop dat die toksisiteit van OA hoofsaaklik toegeskryf kan word aan drie hoof prosesse: (i) inhibisie van ATP produksie; (ii) inhibisie van proteïen sintese; en (iii) die ontwrigting van hepatiese mikrosomale kalsiumhomeostase deur die bevordering van membraanlipiedperoksidasie. Hierdie tesis het ten doel, deur die sintese van OA analoë, om 'n beter insig oor die chemiese struktuur wat verantwoordelik is vir die toksiese funksionaliteit van ochratoksiene te verkry. Die halogeen substituent is grootliks geïmpliseer in die toksisiteit van OA. 'n Bewys hiervan is ochratoksien B (OB), die dechlooranaloog van OA, wat ongeveer tien maal minder toksies is as OA. Voorlopige ondersoeke het aangetoon dat bromoochratoksien B (BrOB), die broomanaloog van OA, meer toksies is vir nierselle as OA. Fluoorochratoksien B en ander analoë van OA, waar ander aminosure geïnkorporeer word, behoort waardevolle inligting te voorsien met betrekking tot die struktuur-aktiwiteitsverwantskappe en die wyse waarop ochratoksiene funksioneer. Hierdie navorsingspoging spreek beide aspekte aan; (i) die fluorering van die dihidroïsokumarien gedeelte en, (ii) die koppeling van verskillende armnosure en dipeptiede aan die nie-toksiese hidrolieseproduk van OA, nl. ochratoksien a. Hoofstuk een vervat 'n oorsig van die belangrike biologiese aspekte van OA wat dien as riglyn vir die sintese van doeltreffende OA analoë. Die hoofstuk word afgesluit met 'n oorsig van die relevante chemie betrokke by die modifisering van die struktuur van OA. Hoofstuk twee bevat 'n bespreking van die aanwending van fluoor in bio-organiese chemie. Dit bevat 'n oorsig van die impak wat fluoorsubstitusie het op die biologiese reaktiwiteit van molekules. 'n Opsomming oor die sintese van organofluoorverbindings, wat die essensie van hierdie studie is, beëindig die hoofstuk. Hoofstuk drie handeloor die veskillende metodes wat toegepas is in pogings om fluoorochratoksien B en ander analoë te sintetiseer. Dit sluit in die direkte elektrofiliese fluorering van OB en ander verwante aromatiese modelverbindings deur gebruik te maak van xenondifluoried, N-fluoorbenseensulfonimied en Selectfluor™ as fluoreringsreagense. Dit behels verder ook 'n ondersoek na 'n alternatiewe roete tot die sintese van fluooraromatiese verbindings vanaf broom- en chlooranaloë. Vir die doel word palladiumgekataliseerde trimetiel- en tributielstannilering, en trimetielsililering wat vervolgens deur middel van xenondifluoried met fluoor gesubstitueer kan word, aangewend. Pogings tot die direkte katalitiese fluoorsubstitusie van arielhaliede word ook bespreek. Die sintese van 'n sleutelintermediêr, fluoroasetoasetaldehied, in 'n de nova sintese roete tot fluoorochratoksien B word bespreek. Die sintese van nuwe OA analoë, met betrekking to die vervangmg van die Lfenielalanien (L-Phe) groep word ondersoek. Dit bevat die omsetting van OA na Oa, deur suurhidrolise, gevolg deur die koppeling van orto-, meta- en paragesubstitueerde DL-fluoorfenielalanien aan die laktoonsuur, Oa. Daarna word die sintese van histidielhistidienmetielester en die verdere pogings aangaande koppeling met Oa bespreek. Die koppeling van halosalisielsure en salisielsuur aan L-Phe wat dien as model aromatiese verbindings vir fluorering, word behandel. Peptiedkoppeling met behulp van disikloheksielkarbodiimied-karboksielaktivering, met inbegrip van die beskerming van die fenoliese hidroksiel groep m 5-chloorsalisielsuur Vir die toepassing op Oa, beëindig hierdie werk.

Six lactating Holstein cows received ammonia-treated or untreated aflatoxin-contaminated whole cottonseed (AFWC) or pure AFB₁ with their regular ration. Treatments were: AFWC (5,010 ppb AFB₁), 4 kg/day; the same AFWC treated with 1.5% anhydrous ammonia and 10% water; pure AFB₁ (2.2 mg twice daily) given in capsules; same amount AFB₁ treated with 50% NH₄OH for 26 days at 29°C; same amount ammoniated AFB₁ acidified to final pH of 5.0; same treatment as first except concentration of AFB₁ was 5,511 ppb. Levels of aflatoxin M₁ (AFM₁) in milk were monitored before, during, and after each treatment, and conversion and feed-through ratios were calculated. Feed consumption and milk production were also measured. Mutagenicity of acetone extracts of spray-dried milk was determined using Salmonella/microsomal assay. Ammoniation of AFWC did not reduce concentration of AFB₁ to below FDA action level; however, when the seed was fed, the concentration of AFM₁ in milk was less than FDA action level (0.5 μg/L). Ammoniation of AFB₁ was very effective in reducing levels of AFM₁ in milk of treated cows to less than the FDA action level. The average AFB₁/AFM₁ conversion ratios for the steady-state period of AFM₁ excretion in milk while giving AFWC and AFB₁ was 1.06% and 1.18%, respectively. Ammoniation of AFWC reduced the average AFB₁/AFM₁ ratio to 0.20% during the constant-state period of AFM(,1) excretion in milk. The ration containing AFWC (5,010 or 5,511 ppb AFB₁) caused a highly significant decrease in total milk production and feed consumption; ammoniated AFB₁ decreased total milk production significantly. Complete disappearance of AFM₁ from milk after discontinuing Treatments 1-6 was 120, 48, 95, 72, 96, and 120 h, respectively. Under these laboratory conditions, significance of the results of the Ames test was questionable.