Ochratoxin A is the most potent chlorinated derivative of the ochratoxin group, consisting of a 5'-chlorinated dihydroisocoumarin moiety linked by an amide bond to l-phenylalanine. Produced as a secondary fungal metabolite by several species of Aspergillus and Penicillium, ochratoxin A has been shown to readily contaminate a large variety of commodities including cereals, groundnuts, dried fruit, spices and coffee. This has led to widespread contamination of ochratoxin in wine, beer, milk and meat products. As ochratoxin A is a potent nephrotoxin exhibiting teratogenic and carcinogenic properties, the development of a rapid screening platform for the cost effective control of ochratoxin A content in foodstuffs is therefore required. The evaluation of metallophthalocyanine and carbon nanotube electrode modification toward the development of a nanostructured biosensor capable of enhancing the electrochemical detection of ochratoxin A in complex media is presented. Cyclic voltammetry at a glassy carbon electrode allowed for the optimization of detection parameters including pH and type of supporting electrolyte. Britton-Robinson buffer was found to be the most suitable supporting electrolyte in terms of sensitivity and reproducibility obtaining a LOD of 0.28 μM as determined by differential pulse voltammetry. Subsequent analysis determined the dependence of OTA oxidation on pH in acidic media which proceeds with the transfer of two electrons to form a quinone/hydroquinone couple shown to adsorb to the electrode surface. Passivation of the electrode through adsorption of oxidation products was shown to severely limit the detection of OTA upon successive detection cycles. Comparison of various metallophthalocyanine modifiers showed an increase in sensitivity toward the detection of OTA at phthalocyanine complexes with metal based redox processes. However with the exception of NiPc and CoTCPc complexes, phthalocyanine modification was limited by the increase in deviation of current response and extent of fouling. NiPc modification showed an increase in sensitivity by two fold with fouling characteristics comparable to an unmodified electrode while low improvements in fouling was observed at CoTCPc modified electrodes with sensitivity in detection comparable to an unmodified electrode.Modification of the electrode with multi- and single walled carbon nanotubes produced a significant increase in sensitivity toward the detection of ochratoxin A. The electrocatalytic activity of nanotube modifiers was attributed to the increase in surface area and to the addition of oxygenated functional groups upon acid treatment as confirmed by Raman spectroscopy. Acid functionalization of the carbon nanotubes for a period of two hours produced the greatest increase in sensitivity obtaining a respective LOD of 0.09 μM and 0.03 μM for analysis of ochratoxin A at multi- and single walled carbon nanotube modified electrodes. Centrifugal purification of carbon nanotubes was deemed necessary to improve the electrocatalytic activity of the nanotube modifiers through the removal of carbonaceous impurities as visualized by atomic force microscopy. Furthermore, a crude lipase preparation, lipase A, was investigated as a potential biological recognition element for selective detection of ochratoxin A in complex media. Lipase A enabled the hydrolysis of ochratoxin A to the electroactive species ochratoxin α as confirmed by thin layer chromatography and voltammetric analysis. Additional isolation of a pure hydrolase from the lipase A preparation is required prior to utilization within a nanostructured biosensor platform capable of detecting ochratoxin A in complex media.

This thesis presents solid phase extraction (SPE) methodologies based on mixed-mode polymeric sorbents; a mixed mode strong anion exchanger (Agilent SampliQ SAX) and a mixed mode strong cation exchanger (Agilent SampliQ SCX). Furthermore, dispersive-SPE based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) method was assessed for applicability in the determination of drug residues. The mixed-mode polymeric sorbents were evaluated for the simultaneous fractionation of drugs that exhibit diverse polarities with acidic, basic and neutral functionalities in biological matrices (plasma and urine). The polymeric skeleton of these sorbents entails an exchanger group and therefore provides two retention mechanisms, strong cation or anion exchange retention mechanisms with hydrophobic interactions. It was demonstrated that with a sequential elution protocol for sample clean-up analytes were fractionated into acidic, basic and neutral classes. The SAX was employed for analysis of ketoprofen, naproxen (acidic drugs), nortriptyline (basic) and secobarbital (neutral) from urine sample. The SCX was used for fractionating phenobarbital, p-toluamide (acidic), amphetamine, m-toluidine (basic) and acetaminophen (neutral drug) from plasma sample. QuEChERS method was employed for quantitative determination of 16 polycyclic aromatic hydrocarbons (PAHs) from fish fillets and soil; 9 sulfonamides (SAs) from chicken muscles and acrylamide (AA) in cooking oil. The analyte recoveries ranged from 79.6 - 109% with RSDs ranging from 0.06 - 1.9% at three different fortification levels. Good linearity (r2 > 0.9990) was attained for most analytes. The limits of detection and quantification ranged from 0.03 - 0.84 μg/ml and 0.81 - 1.89 μg/ml respectively for analytes in biological samples. LODs and LOQs for analytes in food and environmental samples ranged from 0.02 to 0.39 and 0.25 to 1.30 ng/g respectively.

Two hybridoma clones, labelled 4D10 C1 and 2H4 H12, produced monoclonal antibodies which recognized the outer membrane of an enteropathogenic Escherichia coli (EPEC) 0142:K86:H6 in an enzyme-linked immunosorbent assay (ELISA) and the whole cell in an immunofluorescence assay. Large scale production of the monoclonal antibodies was accomplished through ascites production in balb/c mice. Purification of the ascites fluid was achieved by gel filtration and ion exchange chromatography. Isotyping of the purified fractions showed 4D10 C1 to be an IgG2 and 2H4 H12 an IgM. These monoclonal antibodies were screened by immunofluorescence assay against several pathogenic and nonpathogenic strains of E.coli in addition to other Enterobacteriaciae. Results of the screening showed these antibodies to be specific for the E.coli serotype to which they were raised. Minimal cross-reactivity with other Enterobacteriaceae was observed. In a separate and concurrent project, the use of an ELISA capable of detecting ß-N-acetylglucosaminidase (NAGase) was examined. White Leghorn hens were injected with commercially prepared bovine NAGase. Eggs were collected and the immunoglobulin fraction separated from the egg yolk by polyethylene glycol precipitation followed by ion exchange on a DEAE-Sephacel column. The use of the purified immunoglobulins was examined in a sandwich, double-sandwich and a competitive ELISA. A statistically significant standard curve for the detection of NAGase was successfully derived using a double-sandwich ELISA when rabbit immunoglobulin was used to coat the microwell plates. This assay was used to measure the NAGase concentration in press juice and fish extract of fresh and frozen salmon muscle samples. The ratio of the NAGase concentration in the press juice to the total NAGase concentration was compared. No significant difference was found between the calculated concentration ratios of the fresh muscle samples and samples frozen for 1 week at -20°C. / Land and Food Systems, Faculty of / Graduate

No presente trabalho foram realizadas análises de enterobactérias e Salmonella em cecos e papos de frangos de corte criados sem reaproveitamento de cama e com baixa densidade, submetidos a períodos de jejum de 3, 6, 9, 12, 15 e 18 horas; também foram feitas análises das mesmas bactérias, incluindo esporos de Bacillus subtilis e bactérias ácido lácticas em papos e cecos de frangos de corte alimentados com probióticos (bactérias lácticas, Bokashi e esporos de B. subtilis) e antibiótico (bacitracina de zinco) nas rações. Não houve diferença significativa entre as populações de enterobactérias nos períodos de jejum analisados, e não foram isoladas cepas de Salmonella nos diferentes tratamentos. Houve diminuição significativa na população de enterobactérias nos cecos em relação ao grupo controle quando os frangos foram alimentados com bactérias ácido lácticas nas rações. Com Bokashi e antibiótico nas rações, a população de enterobactérias nos cecos foi maior que no grupo controle. Nos papos, não houve diferença significativa entre as populações de enterobactérias para os diferentes tratamentos, mas o tratamento com Bokashi foi o que obteve menor população das enterobactérias nesse habitat. As contagens de bactérias ácido lácticas e esporos de B. subtilis dos cecos e papos analisados não sofreram alteração significativa devido aos diferentes aditivos presentes nas rações, quando comparadas ao grupo controle. Novamente não foram isoladas cepas de Salmonella nos cecos e papos dos frangos analisados. A ausência de Salmonella pode ter explicação no modo de criação dos frangos, com baixa densidade (12 aves/m2) e sem reaproveitamento de cama, pois foi um resultado encontrado nas duas fases do trabalho. A diminuição de enterobactérias nos cecos por parte das bactérias ácido lácticas administradas na ração pode ter sido devido à exclusão competitiva, apesar da população dessas bactérias não ter aumentado significativamente neste habitat. O tratamento com administração de Bokashi obteve menor contagem de enterobactérias no papo possivelmente devido ao fato de ser um produto fermentado, com pH baixo, que poderia agir prontamente no papo. A administração de esporos de B. subtilis não surtiu efeito nas populações das bactérias analisadas nos cecos e papos dos frangos de corte. / This study analyzed the population of enterobacteria and Salmonella in the crop and caeca of broiler chickens submitted to 3, 6, 9, 12, 15 and 18 hours of feed withdrawal, and raised without reutilization of the litter, in low density. The same bacteria were analyzed, plus Bacillus subtilis spores and lactic acid bacteria, in the crop and caeca of broiler chickens fed rations with probiotics (lactic acid bacteria, Bokashi and B. subtilis spores) and antibiotic (zinc bacitracin). There were no significant differences between enterobacteria populations on the analyzed withdrawal periods, and Salmonella and antibiotic (zinc bacitracin). There were no significant differences between enterobacteria populations on the analyzed withdrawal periods, and B. subtilis spores populations from the caeca and crops of analyzed broiler chickens did not differ from the control group when the different additives were fed through the ration. Again there were no Salmonella isolates in the analyzed crop and caeca. The absence of Salmonella may have an explanation on the way that chickens were raised, with low density (12 birds/m2) and no litter reutilization, because this result was found on the two phases of the study. The lower caeca enterobacteria population found when the broilers were fed lactic acid bacteria may have been due to competitive exclusion, even though the lactic acid bacteria counting did not increase significantly in this habitat. Treatment with Bokashi administration had lower counting of enterobactérias in the crop, possibly due to the fact that Bokashi is a fermented product, with low pH, that could act readily in the crop. The administration of B. subtilis spores did not have any effect on the populations of the analyzed bacteria, in the crop and caeca of broiler chickens.

No description available.

The purpose of the present work was three-fold: (1) to determine the number of microorganisms found in root beer ready for consumer used, (2) to study the effects of various temperatures and durations of incubation on this microbial population, and (3) to characterize the predominant species of contaminating bacteria.

Text in English / Fungal species and their mycotoxins are the most predorminant contaminants of dried agricultural products in sub-Saharan Africa (SSA) and the main species of fungi that can synthesize mycotoxins are Aspergillus, Fusarium and Penicillium. In Africa, aflatoxin is labelled as a great threat to human and animal health due to its high contamination levels reported of aflatoxins in foods. The aim of this study was to survey fungi and aflatoxin contamination of small-scale processed foodstuffs sold at informal retail outlets in the Johannesburg metropolis, South Africa. A total of 270 food samples (10 starch and legume based foods, 11 meat and fish based foods, 22 spices and local condiments, 14 dried fruits and vegetables) were collected from retailers; and analysed four (4) times in different seasons of spring, summer, autumn and winter. Out of the 270 samples analysed, only 27.8% were contaminated with fungal. Of all the six categories of foods analysed, roots and tubers (60.0%), nuts and seeds (40.0%), dried vegetables (37.1%), and the Meat and Insect foods (33.3%) respectively, had the most contaminated samples with fungal respectively. The least contaminated food groups were the fish foods (10.0%) and spices and local condiments (16.7%) respectively. Twenty percent of the 270 dried food analysed were contaminated by Aspergillus species out of which 61.1% of the contaminated samples had fungal counts above 103 cfu/g. Aspergillus niger was the most predominant Aspergillus species identified in all the categories of food samples analysed. Fruits and vegetables (24.4%) and the nuts and seeds (20.0%) food groups had the highest number of samples contaminated with aflatoxin. Peanut flour and Cardamom had the most incidence of aflatoxin. AFB1, AFB2 & AFG1 were the most prominent aflatoxin types recovered from the food samples. Almost all the food samples in which aflatoxin were identified had aflatoxin values above 10μg/ml. / Life and Consumer Sciences / M.Sc. (Life Science)

The Hazard Analysis and Critical Control Point (HACCP) system for studying the food preparation process was conducted in four households in an environmentally poor urban neighborhood of Guayaquil, Ecuador. The analysis consisted of observing all of the steps in the food preparation process, measuring food temperatures at each step, and collecting food and water samples. Food and water samples were tested for total aerobic microorganisms, molds and yeast, total coliforms, and fecal coliforms. Temperatures reached during the cooking process were high enough to kill vegetative forms of foodborne pathogens; however, heat-resistant spores could have survived. Leftover food was held at room temperature for long periods of time which allowed multiplication of vegetative forms from spores or from contaminated food utensils. Leftover food was eaten either cold or reheated to warm temperatures. Water samples were taken from water delivery trucks and from each family. Water was identified as one of the main hazards in food preparation. None of the water samples met the criteria of the Ecuadorean National Institute of Standards for human consumption. Fecal samples were taken from children in the families, and were analyzed for parasites. Parasites were found in all of the samples. After critical control points were identified, appropriate interventions were taken to improve safety at each step. Based on these HACCP observations, a food and water safety program was designed and taught to five mothers attending a Guayaquil Child Care Center. The program included a lesson on each of four topics: food and water safety, parasites, care during diarrhea with emphasis on oral rehydration therapy and the introduction of solid food, and nutrition. A control mother was identified for each group. Diarrhea occurred in children of both groups but none of the children was hospitalized. With the exception of nutrition concepts, the knowledge about food and water safety concepts, parasites and care during diarrhea were well understood by both groups. This was reflected in positive changes in families' behavior toward using improved food and water sanitation practices as observed during a visit to each family which followed the HACCP study and the educational program. / Graduation date: 1994

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The increased consumption of fresh produce has been shown to be related to increases in foodborne disease outbreaks and these have in many cases been ascribed directly to carry-over of pathogens from contaminated irrigation water. In South Africa, rivers are the main source of irrigation water but many have been found to be unsuitable for irrigation of fresh produce because of the unacceptably high levels of faecal contamination. The main aim of this study was to do a baseline evaluation of the microbiological quality of the Plankenburg and Eerste Rivers and to determine which bacterial contaminants are present. Two sampling sites were selected for the Plankenburg (Plank-1 and -3) and one for the Eerste River (Eerste-1). The microbiological analysis included aerobic colony count (ACC), aerobic and anaerobic sporeformers, Staphylococcus, Salmonella, Listeria, enterococci, coliforms, faecal coliforms and E. coli using standard methods. The faecal contamination levels for both rivers exceeded the DWAF and WHO guidelines of <1 000 E. coli per 100 mL water for irrigation of fresh produce intended to be consumed raw. The Plankenburg River sites always had higher coliform contamination levels (1 200 - 13 000 000 MPN per 100 mL water) than the Eerste River site (230 - 79 000 MPN per 100 mL water). There was also a high incidence of index organisms including Salmonella, Staphylococcus, Listeria and endosporeformers. The isolation of intestinal enterococci suggested the presence of potential pathogens that can cause disease outbreaks. The baseline data also showed large variations in microbial loads over the 15 month study with the faecal coliform counts ranging for Plank-1 from 1 200 to 7 000 000 MPN.100mL-1, Plank-3 from 10 to 460 000 MPN.100mL-1 and Eerste-1 from 28 to 79 000 MPN.100mL-1. The water temperatures at all three sites ranged from 12.1° to 21.7°C with COD values in most cases below 100 mg.L-1. As the baseline study showed large variations in microbial loads over the 15 month study period an assessment using the Colilert-18 system of the weekly, daily and hourly variations, for 6 weeks over a period of 4 months was conducted at site Plank-2. This site was specifically used as it is an irrigation source point for nearby fresh produce farmers and is about 2 km further downstream from an informal settlement. The weekly variation trend for total coliforms (TC) showed a decrease over the entire sampling period with the highest count of 3 200 000 MPN.100 mL-1 during the warmer period. The E.coli (Ec) counts showed a similar trend with the highest count of 440 000 MPN.100 mL-1 also in March. The daily variation trends were the same for both the TC and Ec and counts found to increase from Monday to Thursday followed by a decrease to Sunday. The highest counts were on Thursday with average TC and Ec counts of 1 900 000 and 160 000 MPN.100 mL-1, respectively. The hourly variation trends were similar for both TC and Ec with counts increasing from 06h00 to 12h00 followed by a decrease to 18h00. The increases in TC and Ec counts found during the weekly, daily and hourly variation trend studies clearly suggests that the 15 month sampling that was done once a month on Mondays at 08h00 could be considered an underestimation of the contamination levels of the Plankenburg and Eerste Rivers. The overall weekly variation trend for the water temperature showed a decrease over the sampling period while the daily and hourly variation trends showed an increase from 06h00 to 18h00. The overall weekly trend for pH differed from that of the temperature with an increase over the sampling period. The analysis of covariance showed no correlation (p < 0.05) between the physico-chemical (temperature and pH) and the microbial variables (TC and Ec). Therefore it was concluded that temperature and pH had no direct impact on either the total coliform or E. coli counts. Both the Plankenburg and Eerste Rivers were found to be unsuitable for the irrigation of fresh produce intended to be consumed raw due to the high levels of faecal contamination that exceeded DWAF and WHO guidelines. Irrigation with such water could pose a health risk because of presence of potential pathogens that could be carried-over to fresh produce. / AFRIKAANSE OPSOMMING: Die toenemende gebruik van vars produkte hou direk verband met die toename in voedseloordraagbare siektes. Alte dikwels kan dit toegeskryf word aan die teenwoordigheid van patogene in besproeiingswater. In Suid Afrika is riviere die hoofbron van besproeiingswater maar dit is al gevind dat meeste ongeskik is vir gebruik as besproeïngsbron as gevolg van die onaanvaarbare hoe vlakke van fekale besmetting. Die hoofdoel van hierdie studie was om ‘n basislyn evaluasie van die mikrobiologiese kwaliteit van die Plankenburg en Eerste Riviere te doen en ook vas te stel watter bakteriese kontaminante teenwoordig is. Twee bemonsteringpunte is geselekteer vir die Plankenburg (Plank- 1 en -3) en een vir die Eerste Rivier (Eerste-1). Mikrobiologiese analises met standaard metodes het die volgende ingesluit: aërobe kolonie telings (AKT), aërobe en anaërobe spoorevormers, Staphylococcus, Salmonella, Listeria, enterococci, koliforms, fekale koliforms en E. coli met gebruik van standaard metode. Die fekale besmettingsvlakke vir beide riviere het die DWAF en WHO leistreep van <1 000 E. coli per 100 mL water vir besproeiing van vars produkte wat rou geëet kan word oorskry. Die Plankenburg Rivier bemonsteringspunte het in alle gevalle ‘n hoër kolivorm besmettingsvlak (1 200 - 13 000 000 MPN per 100 mL water) as die Eerste Rivier punt (230 - 79 000 MPN per 100 mL water) gehad. Daar was ook ‘n hoër voorkoms van indeksorganismes insluitend Salmonella, Staphylococcus, Listeria en endosporevormers. Die voorkoms van ingewand enterococci was ‘n addisionele aanduiding van die voorkoms van patogene wat ernstige gesondheidsrisikos vir die verbruiker kan inhou. Die basislyn data het groot variasies in die mikrobe vlakke oor die 15 maand van studie getoon. Die faecal koliforms vir Plank- 1 het gewissel van 1 200 tot 7 000 000 MPN.100mL-1, vir Plank-3 van 10 tot 460 000 MPN.100mL-1 en vir Eerste-1 van 28 tot 79 000 MPN.100mL-1. Die water temperature het gewissel van 12.1° tot 21.7°C met die CSB waardes in meeste gevalle minder as 100 mg.L-1. Aangesien daar sulke groot variasies in mikrobe ladings oor die 15 maande tydperk voorgekom het, is die Colilert-18 sisteem gebruik om die weeklikse, daaglikse en uurlikse variasies vas te stel vir 6 weke oor ‘n periode van 4 maande by die Plank-2 bemonsteringspunt. Daar is spesifiek op die bemonsteringspunt gefokus omdat dit as ‘n besproeiingsbron gebruik word deur groente produsente. Dit is ook gelee ongeveer 2 km stroomaf van ‘n informele nedersetting. Die weeklikse variasies in totaal koliforms (TC) het ‘n afname oor die hele bemonsteringsperiode getoon, met die hoogstes telling van 3 200 000 MPN.100 mL-1 gedurende die warmer tydperk. Die E.coli (Ec) tellings het ‘n soortgelyke neiging getoon, met die hoogste telling van 440 000 MPN.100 mL-1 ook in Maart. Die daaglikse neigings was dieselfde vir beide die TC en Ec en die tellings het vermeerder van Maandag tot Donderdag, met ‘n afname tot Sondag. Die hoogste telling was op Donderdag met gemiddelde TC and Ec tellings van 1 900 000 and 160 000 MPN.100 mL-1, respektiewelik. Die uurlikse variasie profiel was soortgelyk vir beide TC and Ec met tellings wat vermeerder het van 06h00 tot 12h00 gevolg deur ‘n afname tot 18h00. Die toename in TC en Ec getalle soos vasgestel gedurende die weeklikse, daaglikse en uurlikse variasie het duidelik getoon dat die bemonsterings wat een maal per maand op Maandae om 08h00 gedurende die 15 maande tydperk uitgevoer is, tot ‘n erg onderskatting van die besmettings vlakke in die Plankenburg en Eerste Riviere gelei het. Die algehele weeklikse variasies vir die water temperatuur het ‘n verlaging oor die bemonsteringstydperk getoon terwyl die daaglikse en uurlikse variasie neigings ‘n verhoging van 06h00 tot 18h00 getoon het. Die weeklikse neigings vir pH het van die van die temperatuur verskil. Die analises van kovariante het geen korrelasie (p < 0.05) tussen die fisiese-chemiese (temperature en pH) parameters en die mikrobe veranderlikes (TC en Ec) getoon nie. Dus is daar afgelei dat temperatuur en pH geen direkte impak op die totale kolivorm of E. coli tellings gehad nie. Die data van die studie het duidelik getoon dat water van beide die Plankenburg en Eerste Riviere nie geskik is vir gebruik vir besproeiing van vars produkte wat rou geëet gaan word nie. In beide gevalle het die fekale besmettingsvlakke die DWAF en WHO leistreep oorskry. Besproeiing met sulke water hou ‘n gesondheidsgevaar in as gevolg van die teenwoordigheid van potensiële patogene wat oorgedra kan word na vars produkte.

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology